Surai Livro Selenium 2006 Brazil

Selenium in Nutrition and Health
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SELENIUM IN NUTRITION AND

HEALTH
Peter F. Surai
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Nottingham University Press

Manor Farm, Main Street, Thrumpton
Nottingham NG11 0AX, United Kingdom
www.nup.com
NOTTINGHAM
First published 2006
PF Surai
All rights reserved. No part of this publication
may be reproduced in any material form
(including photocopying or storing in any
medium by electronic means and whether or not
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this publication) without the written permission
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the provisions of the Copyright, Designs and
Patents Act 1988. Applications for the copyright
holders written permission to reproduce any part
of this publication should be addressed to the publishers.
British Library Cataloguing in Publication Data
PF Surai
ISBN 1-904761-16-X
Disclaimer
Every reasonable effort has been made to ensure that the material in this book is true, correct, complete
and appropriate at the time of writing. Nevertheless, the publishers and authors do not accept
responsibility for any omission or error, or for any injury, damage, loss or financial consequences arising
from the use of the book.
Typeset by Nottingham University Press, Nottingham

Printed and bound by Cromwell Press, Trowbridge, Wiltshire
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CONTENTS
Preface
xix
1. Antioxidant systems in animal body

Introduction
Free radicals and reactive oxygen and nitrogen species
Three levels of antioxidant defence
Antioxidant-prooxidant balance in the body and oxidative stress
Conclusions
References
1
1
7
25
32
34
2. Molecular mechanisms of Se action: Selenoproteins

Introduction
Selenoprotein family
Glutathione peroxidase
Cytosolic glutathione peroxidase (cGSH-Px or GSH-Px1)
Negative effectors of GSH-Px activity
Upregulation of GSH-Px
Gender-specific expression of GSH-Px
Tissue-specific expression of GSH-Px
Response to dietary selenium
Alterations of GSH-Px expression
GSH-Px regulation and proteasomes
Phospholipid glutathione peroxidase (GSH-Px4, PH-GSH-Px)
PH-GSH-Px overexpression
Plasma glutathione peroxidase (pGSH-Px, GSH-Px3)
Gastrointestinal glutathione peroxidase (GI-GSH-Px, GSH-Px2)
Specific sperm nuclei glutathione peroxidase (sn-GSH-Px)
GSH-Px6
Glutathione peroxidases and their biological roles
Non-Se glutathione peroxidases
Thioredoxin reductase
Iodothyronine deiodinases (ID)
Other selenoproteins
Selenoprotein W
Selenoprotein P 10
Selenoprotein P12
Selenophosphate synthetase-2
15-kDa selenoprotein
18-kDa selenoprotein
45
45
51
51
56
57
58
58
59
60
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Contents
Mitochondrial capsular selenoprotein
Selenoprotein N
Selenoprotein R
Selenoprotein S
Selenoprotein U
Selenoprotein T and selenoprotein X
Selenium-vitamin E interactions: antioxidant properties and beyond
Prooxidant properties of selenite and possible antioxidant protection
by selenomethionine
References
116
3. Selenium in food and feed: selenomethionine and beyond

Introduction
Selenium in soils and plants
Plants as major sources of selenium for animals and human
Selenium absorption and metabolism
Selenium status and bioavailability
Effectors of selenium absorption, metabolism and bioavailability
Selenium and feeding behaviour
Selenium sources for animal and human consumption
Supplemental sources of selenium
References
151
151
156
161
171
181
184
184
191
198
4. Selenium and immunity

Introduction
Immune system and its evaluation
Phagocyte functions
Antibody production
Lymphocyte functions
In vitro effects of Se on immune cells
Disease resistance
Selenium deficiency and viruses
Mechanisms of immunomodulating properties of selenium
Immunocommunication, free radicals and selenium
Conclusions
References
213
214
224
228
233
240
243
251
252
257
260
262
5. Selenium and semen quality

Introduction
Fatty acid composition of avian and mammalian semen
Lipid peroxidation in semen
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Contents
Selenoproteins and their role in antioxidant protection in semen
Conclusions
References
286
304
305
6. Selenium and mycotoxins

Introduction
Mycotoxins of concern
Increased lipid peroxidation as a consequence of mycotoxicosis
Mycotoxins and apoptosis
Mycotoxins and immune system
Protective effect of selenium against mycotoxicosis
Conclusions
References
317
318
324
326
330
342
347
347
7. Selenium in poultry nutrition

Introduction
Selenium deficiency
Selenium and chicken embryo development
Effect of organic selenium in the maternal diet on antioxidant
defences of the developing chicks
Selenium and digestive and immune system development in birds
Selenium requirement for breeders
Selenium requirement for broilers
Se and meat quality
Se and egg quality
Practical approaches to improve selenium status of poultry
Conclusions
References
397
400
405
411
415
418
422
424
8. Selenium in pig nutrition

Introduction
Se-deficiency
Se requirement
Selenium applications in pig production
Maternal effect on the progeny: organic selenium vs selenite
Selenium nutrition of pigs at weaning period
Selenium in grower-finisher swine and meat quality
Selenium-vitamin E combinations
Selenium and iron injection
Conclusions
References
445
446
454
459
459
469
473
474
475
476
479
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363
364
374
385
Contents
9. Selenium in ruminant nutrition
Introduction
Selenium deficiency in ruminants
Selenium deficiency in dairy and beef industries
Important features of Se metabolism in ruminants
Meeting selenium requirements
Methods for Se status assessment
Routes of selenium supplementation
Practical applications of Se nutrition of ruminants
Selenized yeast vs selenite
Conclusions
References
487
488
518
521
528
531
537
538
546
559
561
10. Selenium in nutrition of other animals: horses, dogs, cats and fish
Selenium in horse nutrition
Introduction
Se deficiency
Se excess
Se requirement
Selenium and GSH-Px
Se metabolism and effective sources
Antioxidant defences and exercise
Se and maternal effect
Se and immunity
Conclusions
589
589
589
590
592
592
593
595
597
599
599
Se for companion animals

Introduction
Antioxidants and animal health
Food quality
Anorexia in companion animals and its connection to antioxidants
Antioxidant deficiencies in companion animals
Organic selenium vs sodium selenite
Selenium requirement
Taurine for cats: antioxidant properties and beyond
Antioxidants and immune system
Conclusions
601
601
601
602
603
604
605
607
608
612
615
Selenium in fish nutrition

Introduction
616
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Contents
Se deficiency
Se toxicity
Se requirement
Health and immunity
Selenium and fish quality
Effective forms of Se in fish diets
Se-fish as human food
Conclusions
References
616
618
620
621
622
624
626
627
628
11. Selenium and human health

Introduction
Nutrition and health
Are there ideal diets?
Omega-3 PUFAs
Selenium: global perspective
Se distribution and reserves in human body
Selenium deficiency
Se status assessment
Selenium and health
Cancer
Selenium and cancer
Epidemiological observations and prospective studies
Case-control studies of Se levels in tissues of cancer patients
Laboratory animal studies of anticancer effect of Se
Human intervention trials with selenium
Selenium and cancer treatment
Molecular mechanisms of anticancer effects of selenium
Selenium and cardiovascular diseases
Selenium and other diseases
Reproductive disorders
Selenium and asthma
Selenium and rheumatoid arthritis
Selenium and diabetes
Selenium and HIV
Selenium and pancreatitis
Selenium, brain function, mood and neurodegenerative diseases
Selenium and radiation
Selenium and ageing
Adverse health effects of excess selenium in humans
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643
643
644
651
654
657
658
662
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665
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671
672
682
687
694
705
708
720
729
729
731
732
733
735
736
738
741
746
751
Contents
Conclusions
References
754
755
12.Se-enriched eggs, milk and meat as functional foods and a solution to

selenium deficiency
Introduction
Different strategies to address Se deficiency in human
Selenium-enriched products
Improving the image of the egg
Selenium-enriched eggs as a route toward improving human
selenium status
Se-enriched meat and milk
Se-enriched eggs, meat and milk as functional food
Conclusions: back to nature?
References
13.Antioxidant-prooxidant balance in the intestine: from healthy gut to
general health
Introduction
The gastrointestinal tract (GIT) as a major site of antioxidant action
Prooxidants in the GIT
Peroxidized PUFAs
Oxysterols
Iron ions
Nitrites and nitrates
Heavy metals
Persistent organic pollutants
Mycotoxins
Alcohol
Immune system
Combinations of prooxidants in the GIT and their detrimental effects
Antioxidant defences in the GIT
Vitamin E
Coenzyme Q
Carotenoids
Vitamin A
Ascorbic acid (AA)
Glutathione
Flavonoids
Other poly(phenolics)
Spices and essential oils
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809
810
814
817
821
827
837
842
844
857
857
859
859
861
862
864
865
867
868
870
871
873
874
874
876
880
882
882
883
883
885
885
Contents
Selenomethionine
Synthetic antioxidants
Specific place for Se-dependent enzymes in antioxidant defence of GIT
Other antioxidant mechanisms
What should be changed to have a healthy diet?
Antioxidant-prooxidant balance in the intestine and animal health
Conclusions
References
14. Conclusions: Looking ahead
References
923
949
Index
955
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886
886
887
892
895
896
897
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To my wife Helen, my daughter Katie and my son Anton

who gave me an inspiration for writing this book
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ABBREVIATIONS
AA
Ab
ACS
AD
AFB1
ALA
Apo-AI
ApoB
ApoII
AR
BDI scores
BHT
BHA
CAT
CE
CH
CHD
CIND
CoQ
Con A
CPK
CRP
CSF
CVD
DAA
DAT
DHA
DM
DMBA
DON
DPA
DTA
DTH
ECs
Ascorbic acid
Antibody
Acute coronary syndromes
Alzheimers disease
Aflatoxin B1
Alpha-linolenic acid
Apoprotein AI
Apoprotein B
Apoprotein II
Androgen receptor
Beck Depression Inventory scores
Butylated hydroxytoluene
Butylated hydroxyanisole
Catalase
Cholesteryl esters
Cholesterol
Coronary heart disease
Cognitive impairment non-dementia
Coenzyme Q
Concanavalin A
Creatine phosphokinase
C-reactive protein
Cerebrospinal fluid
Cardiovascular diseases
Dehydroascorbic acid
Dementia of the Alzheimer type
Docosahexaenoic acid
Dry matter
7,12-Dimethylbenz[a]anthracene
Deoxynivalenol
Docosapentaenoic acid
Docosatetraenoic acid
Delayed-type hypersensitivity
Endothelial cells
ED
EPA
FB1
FCR
Exudative diathesis
Eicosapentaenoic acid
Fumonisin B1
Feed conversion ratio
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Abbreviations
FFA
GC-MS
GI-GSH-Px
GIT
GOT
GR
GSH
GSSG
GSH-Px
GST
HA
Hb
HCC
5-HETE
15-HPET
HI
HDL
HNE
HO-1
HPLC
HSP
ID
IFN
IL-1
IL-6
IL-2R
IMI
KD
LA
LE
LNA
LDL
LDH
5- LO
LOOH
LP
LPL
LPO
LPS
LTA
LTB
Free fatty acids

Gas Chromatography with Mass Spectrometry
Gastrointestinal glutathione peroxidase
Gastrointestinal tract
Glutamic oxalacetic transaminase
Glutathione reductase
Reduced glutathione
Oxidised glutathione
Glutathione S-transferase
Hemagglutination
Hemoglobin
Hepatocellular carcinoma
5-hydroxyeicosatetraenoic acid
15-hydroperoxy analogue
Hemagglutination inhibition
High density lipoprotein
4-hydroxynonenal
Heme oxygenase
High Performance Liquid Chromatography
Heat shock proteins
Iodothyronine deiodinase
Interferon
Interleukin 1
Interleukin 6
Interleukin 2 receptor
Intramammary infection
Keshans disease
Linoleic acid
Lymphedema
Alpha-linolenic acid
Low density lipoprotein
Lactate dehydrogenase
5-Lipoxigenase
Lipid hydroperoxides
Lipid peroxidation
Lipoprotein lipase
Lipid peroxidation
Lipopolysaccharide
Lymphocyte transformation assay
Leukotriene B
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Abbreviations
MAP
MCS
MDA
MD
Met
MODA
NDV
MIF
MIP-2
MCP
MSA
MsrA and MsrB
NE
NK cells
NMD
NPO
NRC
NRP
OA
OPN
OVA
PBMC
PC
PCA
PFC test
PGE2
pGSH-Px
PAM
PL
PLC
PMN
PG
PHA
PH-GSH-Px
PIH
PLA2
POPs
PRECISE
PSA
PUFA
PWM
Microangiopathy
Mitochondrial capsular selenoprotein
Malondialdehyde
Mareks disease
Methionine
Milan overall dementia assessment
Newcastle disease virus
Migration inhibitory factor
Macrophage inflammatory protein 2
Mitochondrial capsule protein
Methylseleninic acid
Methionine sulfoxide reductases
Nutritional encephalomalacia
Natural killer cells
Nutritional muscular distrophy
2'-(4-nitrophenoxy)oxirane
National Research Council
Nutrient Requirements of Poultry
Ochratoxin A
Osteopontin
Ovalbumin
Peripheral blood mononuclear cells
Phosphatidylcholine
Prostate cancer
Plaque-forming cell test
Prostaglandin E2
Plasma glutathione peroxidase
Prematurely aging mice
Phospholipid
Primary liver cancer
Polymorphonuclea cells, neutrophils
Prostaglandin
Phytohemagglutin
Phospholipid glutathione peroxidase
Pregnancy induced hypertension
Phospholipase A2
Persistent organic pollutants
Prevention of Cancer by Intervention with Se
Prostate-specific antigen
Polyunsaturated fatty acid
Pokeweed mitogen
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Abbreviations
RA
RBC
RDA
ROS
RNS
RP
RR
RSV
SCC
SCCs
SDG
SeCys
Sel-Plex
SECIS
SELECT
SeN
SeMet
SeP
SeR
SeS
SeW
SeRU
SFA
sn-GSH-Px
SOD
SPS
SRBC
SST
Sptrx-1
STZ
T3
T4
TBA
TBARS
Th
TG
TNF-a
TPA
Trx
TPx
TR
Rheumatoid arthritis
Red blood cells
Recommended Dietary Allowances
Reactive oxygen species
Reactive nitrogen species
Retained placenta
Relative risk
Respiratory syncytial virus
Somatic cell count
Ssquamous carcinoma cells
Selenodiglutathione
Selenocysteine
Selenium enriched yeast
Selenocysteine insertion sequence
The Selenium and Vitamin E Cancer Prevention Trial
Selenoprotein N
Selenomethionine
Selenoprotein P
Selenoprotein R
Selenoprotein S
Selenoprotein W
Se-responsive unthriftiness
Saturated fatty acids
Sperm nuclei glutathione peroxidase
Superoxide dismutase
Selenophosphate synthetase-2
Sheep red blood cells
Sperm storage tubules
Sperm-specific thioredoxin
Streptozotocin
Triiodthyronine
Thyroxine
Thiobarbituric acid
Thiobarbituric acid reactive substances
T helper
Triacylglycerol
Tumour necrosis factor a
12-O-tetradecanoylphorbol-13-acetate
Thioredoxin
Thioredoxin peroxidase
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Abbreviations
TRAIL
Txl-2
VEGF
VLDL
vMDV
WMD
p-XSC
YSM
Zea
Tumor necrosis factor-related apoptosis-inducing ligand

Thioredoxin-like protein 2
Vascular endothelial growth factor
Very low density lipoprotein
Virulent Mareks disease virus
White muscle disease
Xylene selenocyanate (1,4-phenylenebis(methylene)selenocyanate)
Yolk Sac Membrane
Zearalenone
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PREFACE
Among many minerals selenium has a special place being the most controversial trace
element. Indeed a narrow gap between essentiality and toxicity and environmental issues
from the one hand and global selenium deficiency from the other hand, fuel research in
this field. There were several breakthroughs in selenium research. The first one was a
discovery of Se essentiality in early 1960th. The second one was a discovery in 1973
that glutathione peroxidase is a selenoprotein. The third one came almost 30 years later
with characterisation of main selenoproteins in human and animal body and further
understanding the role of selenium in nutrition and health. Indeed, this third breakthrough
is really a selenium revolution creating many hypotheses, stimulating new research
and providing practical applications in medicine and agriculture. New insight in the
role of free radicals as signalling molecules, understanding the role of nutrients in gene
expression and maternal programming, tremendous progress in human and animal
genome work created new demands for further research related to biological roles of
selenium. For the last few years several comprehensive monographs and reviews have
been published addressing various Se-related issues. However, most of them were dealing
with Se roles in human health. However, animal food-producing industry is developing
very quickly and a great body of information was accumulated indicating importance
of Se in maintenance of animal health, productive and reproductive performance.
Unfortunately up to now there was no comprehensive review combining all major aspects
of selenium in relating to animal and human health. Indeed a chain: Se in soil - selenium
in plants-selenium in animals- selenium in human was not well characterised.
Therefore the goal of this volume is to provide up to date information about the
roles of Se in nutrition and health of human and farm animals, including poultry, pigs,
ruminants, horses, cats, dogs and fish. In the first chapter a special emphasis is given to
the role of selenium as an essential part of the integrated antioxidant system of the
body with regulatory functions providing necessary connections between different
antioxidants. In fact selenium is called the chief executive of the antioxidant defence.
The second chapter is addressing molecular mechanisms of Se action describing major
functions of the selenoproteins. Indeed, the family of selenoproteins includes 25
members and functions of many of them are still not well understood. Selenium in
food and feed is described in the third chapter. The main idea of this chapter is to
characterise main Se sources in relation to new knowledge about Se speciation in various
food and feed ingredients. It seems likely, that in grains and some other important food
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Preface
ingredients selenomethionine is the main Se form. The idea was put forward that during
evolution the digestive system of human and animals was adapted to natural form of
selenium consisting of SeMet and other organic selenocompounds. Therefore this form
of Se is more efficiently assimilated in the body than inorganic forms of selenium. In
fact SeMet is considered to be the storage form of selenium in the body. Accumulation
of the Se reserves in the body as a result of organic selenium consumption is considered
as an adaptive mechanism providing additional antioxidant defences in stress conditions.
The fourth chapter is devoted to the role of selenium in immunity. It is difficult to
overestimated immunomodulating properties of selenium and increased resistance to
various diseases of human and animals is a result of optimal Se status. The possibility
of virus mutation in the body of animals or human deficient in selenium is of great
importance for understanding mechanisms of spreading such diseases as AIDS, chicken
influenza, etc. New findings in the field of male reproduction in relation to Se status are
described in the chapter 5. From the data presented it is clear that Se has special important
roles in maintaining animal reproduction. It is well known that antioxidants play
important roles in preventing various stresses. Mycotoxins are considered to be the
biggest feed-related stress in farm animals. Therefore, possible protective role of Se in
mycotoxicosis is described in the chapter 6. Next four chapters are devoted to practical
applications of Se in animal nutrition. The data presented indicated importance of Se in
growth, development and reproduction of poultry, pigs, ruminants, horses, cats, dogs
and fish. The main idea of the chapters is to show possibilities of improvement of
productive and reproductive performance of animals and animal produce quality by
optimal Se supplementation. Indeed, organic selenium in the form of selenized yeast is
proven to be the most effective form of Se supplementation for poultry, farm animals,
companion animals and fish. Role of selenium in human health is described in the
chapter 11. Specific emphasis is given to anticancer properties of Se with detailed
analysis of possible molecular mechanisms of such Se action. Beneficial role of Se in
other diseases, including cardio-vascular diseases, diabetes, arthritis, pancreatitis,
reproductive disorders as well as protective role of selenium against radiation are also
described. The link between animal industry and human health is described in the
chapter 12 devoted to production of Se-enriched eggs, meat and milk. Indeed, production
of a range of Se-enriched products is considered as an important solution for global Se
deficiency. Se-enriched eggs are already on supermarket shelves in more than 25
countries worldwide with millions such eggs sold daily. The technologies of producing
Se-meat and milk are also developed and await practical applications. An important
issue of antioxidant-prooxidant balance in the digestive tract is considered in the chapter
13. It seems likely that this balance has been overlooked by scientists. However, from
the one hand this balance could explain a beneficial effect of fruit and vegetables in
human diet. On the other hand, this balance is a key for general health. In the conclusive
chapter of the book the general trends for future research in the field of the Se biology
are shown. Information provided in this book will be of practical importance to medical
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Preface
and pharmaceutical industry professionals, dieticians, egg, meat and milk producers,
animal scientists, feed formulators as well as for students of biological, medical,
veterinary and other related sciences at universities and colleges.
Much of the material in this book is based on ideas and concepts that in many
instances have been developed from work carried out in the Department of Biochemistry
and Nutrition and further in the Avian Science Research Centre at the Scottish
Agricultural College. I am, therefore, grateful to the SAC for providing an environment
to develop these ideas, and to the Scottish Executive Environment and Rural Affairs
Department for its generous support of this research. I understand that my views on
the role of selenium in nutrition and health are sometime different from those of other
scientists and therefore I would appreciate very much receiving any comments from
readers which will help me in my future research.
I would like to thank my colleagues with whom I have had a pleasure to collaborate
and share my ideas related to natural antioxidants and selenium in particular, who
helped me at various stages of this research by providing reprints of their recent
publications. I am also indebted to the Alltech Inc. for providing organic selenium for
experiments and some important information and ideas and to the Worlds Poultry
Science Association for the Research Award.
P.F. Surai
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Antioxidant Systems in Animal Body
ANTIOXIDANT SYSTEMS IN ANIMAL BODY

Self-preservation is the first law of nature
Introduction
For the majority of organisms on Earth, life without oxygen is impossible. Animals,
plants and many microorganisms rely on oxygen for the efficient production of
energy. However, the high oxygen concentration in the atmosphere is potentially
toxic for living organisms. It is interesting that oxygen toxicity was first described
in laboratory animals in 1878 (see Knight, 1998). For the last few years free
radical research has generated valuable information for further understanding not
only detrimental, but also beneficial role of free radicals in cell signaling and
other physiological processes. The benefit or harm of free radicals ultimately
depend on the level of their production and efficiency of antioxidant defence.
Free radicals and reactive oxygen and nitrogen species

Free radicals are atoms or molecules containing one or more unpaired electrons.
Free radicals are highly unstable and reactive and are capable of damaging
biologically relevant molecules such as DNA, proteins, lipids or carbohydrates.
The animal body is under constant attack from free radicals, formed as a natural
consequence of the bodys normal metabolic activity and as part of the immune
systems strategy for destroying invading microorganisms. The internal and
external sources of free radicals are shown in Table 1.1. Recently collective terms
reactive oxygen species (ROS) and reactive nitrogen species (RNS) have been
introduced (Halliwell and Gutteridge, 1999) including not only the oxygen or
nitrogen radicals, but also some non-radical reactive derivatives of oxygen and
nitrogen (Table 1.2).
Did you know that free radicals are produced as a result of
physiological metabolism in the body?
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Table 1.1 Internal and external sources of free radicals (Adapted from Furst, 1996 and Halliwell, 1996).
Internally generated
External sources
Mitochondria (ETC)
Phagocytes
Xanthine oxidase
Reactions with Fe2+ or Cu+
Arachidonate pathways
Peroxisomes
Inflammation
Biomolecule oxidation (adrenaline, dopamine,
tetrahydrofolates, ect.)
Cigarette smoke
Radiation
UV light
Pollution
Certain drugs
Chemical reagents
Industrial solvents
Table 1.2 Reactive oxygen and nitrogen species (Adapted from Halliwell and Gutteridge, 1999).
Radicals
Non-radicals
Alkoxyl, RO*
Hydroperoxyl, HOO*
Hydroxyl, *OH
Peroxyl, ROO*
Superoxide, O2*
Nitric oxide, NO*
Nitrogen dioxide, NO2*
Hydrogen peroxide, H2O2

Hypochlorous acid, HOCl
Ozone, O3
Singlet oxygen, 1O2
Peroxynitrite, ONOONitroxyl anion, NONitrous acid, HNO2
Superoxide (O2*-) is the main free radical produced in biological systems during
normal respiration in mitochondria and by autoxidation reactions with half-life at
37C in the range of 1 x 10-6 second. Superoxide can inactivate some enzymes
due to formation of unstable complexes with transition metals of enzyme prosthetic
groups, followed by oxidative self-destruction of the active site (Chaudiere and
Ferrari-Iliou, 1999). Depending on condition, superoxide can act as oxidizing or
a reducing agent. It is necessary to mention that superoxide, by itself, is not
extremely dangerous and does not rapidly cross lipid membrane bilayer (Kruidenier
and Verspaget, 2002). However, superoxide is a precursor of other, more powerful
ROS. For example it reacts with nitric oxide with a formation of peroxynitrite
(ONOO-), a strong oxidant, which lead to formation of reactive intermediates due
to spontaneous decomposition (Kontos, 2001; Mruk et al., 2002). In fact ONOOwas shown to damage a wide variety of biomolecules, including proteins (via
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nitration of tyrosine or triptophan residues or oxidation of methionine or

selenocysteine residues), DNA and lipids (Groives, 1999). Superoxide can also
participate in the production of more powerful radicals by donating an electron,
and thereby reducing Fe3+ and Cu2+ to Fe2+ and Cu+, as follows:
O2- + Fe3+/Cu2+
Fe2+/Cu+ + O2
Further reactions of Fe2+ and Cu+ with H2O2 are a source of the hydroxyl radical
(*OH) in the Fenton reaction:
H2O2 + Fe2+/Cu+
OH + OH- + Fe3+/Cu2+
The sum of reaction of superoxide radical with transition metals and transition
metals with hydrogen peroxide is known as the Haber-Weiss reaction. It is
necessary to underline that superoxide radical is a double-edged sword. It is
beneficial when produced by activated polymorphonuclea leukocytes and other
phagocytes as an essential component of their bactericidal activities but in excess
it may result in tissue damage associated with inflammation.
Did you know that superoxide radical is the major radical
produced in biological systems?
Hydroxyl radical is the most reactive species with an estimated half-life of only
about 10-9 second. It can damage any biological molecule it touches, however, its
diffusion capability is restricted to only about two molecular diameters before
reacting (Yu, 1994). Therefore, in most cases damaging effect of hydroxyl radical
is restricted to the site of its formation. In general, hydroxyl radical can be generated
in human/animal body as a result of radiation exposure from natural sources (radon
gas, cosmic radiation) and from man-made sources (electromagnetic radiation
and radionuclide contamination). In fact in many cases hydroxyl radical is a trigger
of chain reaction in lipid peroxidation.
Did you know that hydroxyl radical is short-lived but most
powerful radical in biological systems?
Therefore, ROS/RNS (Table 1.3) are constantly produced in vivo in the course of
the physiological metabolism in tissues. It is generally accepted that the electron-
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Table 1.3 Effect of free radicals on DNA damages (Adapted from Diplock, 1998).
Radical
Effect
ROO*
OH*
O2*-, H2O2
ONOO-
Guanine oxidized
All four bases are affected
No base changes
Xanthine, hypoxanthine, 8-nitroguanine are affected
transport chain in the mitochondria is responsible for major part of superoxide

production in the body (Halliwell and Gutteridge, 1999). Mitochondrial electron
transport system consumes more than 85% of all oxygen used by the cell and,
because the efficiency of electron transport is not 100%, about 1-3% of electrons
escape from the chain and the univalent reduction of molecular oxygen results in
superoxide anion formation (Halliwell, 1994; Singal et al., 1998; Chow et al.,
1999). About 1012 O2 molecules processed by each rat cell daily and if the leakage
of partially reduced oxygen molecules is about 2%, this will yield about 2 x1010
molecules of ROS per cell per day (Chance et al., 1979). An interesting calculation
has been made by Halliwell (1994), showing that in the human body about 1.72
kg/year of superoxide radical is produced. In stress condition it would be
substantially increased. Clearly, these calculations showed that free radical
production in the body is substantial and many thousand biological molecules
can be easily damaged if are not protected. Recently the role of mitochondria as
a permanent source of ROS has been questioned (Staniek and Nohl, 2000). The
activation of macrophages in stress conditions is another important source of free
radical generation. Immune cells produce ROS/RNS and use them as an important
weapon to destroy pathogens (Schwarz, 1996; Kettle and Winterbourn, 1997;
See Chapter 6).
Did you know that about 2 x1010 molecules of ROS
are produced per cell per day?
The most important effect of free radicals on the cellular metabolism is due to
their participation in lipid peroxidation reactions. The first step of this process is
called the initiation phase, during which carbon-centered free radicals are produced
from a precursor molecule, for example a polyunsaturated fatty acid (PUFA):
LH
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Initiator
L*
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The initiator in this reaction could by the hydroxyl radical, radiation or some
other events or compounds. In presence of oxygen these radicals (L*) react with
oxygen producing peroxyl radicals starting the next stage of lipid peroxidation
called the propagation phase:
LOO*
L* +O2
At this stage a relatively unreactive carbon-centered radical (L*) is converted to a

highly reactive peroxyl radical. A resulted peroxyl radical can attack any available
peroxidazable material producing hydroperoxide (LOOH) and new carboncentered radical (L*):
LOO* + LH
LOOH + L*
Therefore lipid peroxidation is a chain reaction and potentially large number of

cycles of peroxidation could cause substantial damage to cells. In membranes the
peroxidazable material is represented by PUFAs. It is generally accepted that PUFA
susceptibility to peroxidation is proportional to amount double bounds in the
molecules. In fact, docosahexaenoic acid (DHA, 22:6n-3) and arachidonic acid
(AA, 20:4n-6) are among major substrates of the peroxidation in the membrane.
It is necessary to underline that the same PUFAs are responsible for maintenance
of physiologically important membrane properties including fluidity and
permeability. Therefore as a result of lipid peroxidation within the biological
membranes their structure and functions are compromised. Proteins and DNA are
also important targets for ROS.
Did you know that in the human body about 1.72 kg/year of
superoxide radical is produced?
It has been shown that the DNA in each cell of a rat is hit by about 100,000 free
radicals a day and each cell sustains as many as 10,000 potentially mutagenic (if
not repaired) lesions per day arising from endogenous sources of DNA damage
(Ames and Gold, 1997; Helbock et al., 1998; Ames, 2003; Diplock, 1994).
Therefore, some oxidative lesions escape repair and the steady state level of
oxidative lesions increased with age, and an old rat has accumulated about 66,000
oxidative DNA lesions per cell (Ames, 2003). Oxidation, methylation, deamination
and depurination are four endogenous processes leading to significant DNA
damage with oxidation to be most significant one and approximately 20 types of
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oxidatively altered DNA molecules have been identified. The chemistry of attack
by ROS on DNA is very complex and lesions in chromatin include damage to
bases, sugar lesions, single strand-breaks, basic lesions and DNA-nucleoprotein
cross-links (Diplock, 1994).
Did you know that an old rat has accumulated about 66,000
oxidative DNA lesions per cell?
The complex structure of proteins and a variety of oxidizable functional groups
of the amino acids make them susceptible to oxidative damage. In fact, the
accumulation of oxidized proteins has been implicated in the aging process and
in other age-related pathologies. A range of oxidized proteins and amino acids
has been characterised in biological systems (Table 1.4; Kehrer, 2000; Dean et
al., 1997).
Table 1.4 Oxidized protein and amino acids found in biologic systems (Adapted from Kehrer, 2000;
Dean et al., 1997).
2-Oxohistidine
3-chlorotyrosine
3-Nitrotyrosine
5-Hydroxy-2-aminovaleric acid
Aminomalonic acid
Dimers of hydroxylated aromatic amino acids
Dopa
Hydro(pero)xyleucine
Hydro(pero)xyvaline
N-Formylkynureinine
Kynurenine
o- and m-tyrosine
p-Hydroxyphenylacetaldehyde
Protein carbonyls
In general the accumulation of oxidized proteins depends on the balance between

antioxidants, prooxidants and removal/repair mechanisms. Oxidation of proteins
leads to the formation of reversible disulfide bridges. More severe protein oxidation
causes a formation of chemically modified derivatives e.g. shiffs base (Tirosh
and Reznick, 2000). Nitric oxide, hydroxyl radical, alkoxyl and peroxyl radicals
as well as carbon-centered radicals, hydrogen peroxide, aldehydes or other
products of lipid peroxidation can attack protein molecules. Usually oxidative
modification of proteins occurs by two different mechanisms: a site-specific
formation of ROS via redox-active transition metals and non-metal-dependent
ROS-induced oxidation of amino acids (Tirosh and Reznick, 2000). The
modification of a protein occurs by either a direct oxidation of a specific amino
acid in the protein molecule or cleavage of the protein backbone. In both cases
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biological activity of the modified proteins would be compromised. The degree

of protein damage depends on many different factors (Grune et al., 1997):
the nature and relative location of the oxidant or free radical source;
nature and structure of protein;
the proximity of ROS to protein target ;
the nature and concentrations of available antioxidants.
Free radicals are implicated in the initiation or progression phase of various diseases,
including cardiovascular disease, some forms of cancer, cataracts, age-related
macular degeneration, rheumatoid arthritis and a variety of neuro-degenerative
diseases (Hogg, 1998; McCord, 2000; Table 1.5). In general, it is widely believed
that most human diseases at different stages of their development are associated
with free radical production and metabolism. Normally, there is a delicate balance
between the amount of free radicals generated in the body and the antioxidants to
protect against them. For the majority of organisms on Earth, life without oxygen
is impossible, animals, plants and many micro-organisms relying on oxygen for
the efficient production of energy. However, they pay a high price for pleasure of
living in an oxygenated atmosphere since high oxygen concentration in the
atmosphere is potentially toxic for living organisms.
Did you know that free radicals damage not only lipids
but also DNA and proteins?
Three levels of antioxidant defence

During evolution living organisms have developed specific antioxidant protective
mechanisms to deal with ROS and RNS (Halliwell and Gutteridge, 1999). Therefore
it is only the presence of natural antioxidants in living organisms which enable
them to survive in an oxygen-rich environment (Halliwell, 1994). These
mechanisms are described by the general term antioxidant system. It is diverse
and responsible for the protection of cells from the actions of free radicals. This
system includes:
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natural fat-soluble antioxidants (vitamins A, E, carotenoids, ubiquinones,

etc.);
water-soluble antioxidants (ascorbic acid, uric acid, taurine, etc.)
antioxidant enzymes: glutathione peroxidase (GSH-Px), catalase (CAT) and
superoxide dismutase (SOD).
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Table 1.5 Free radical involvement in the development of human diseases (adapted from Surai and
Sparks, 2001; McCord, 2000; Hogg, 1998 and references there).
Liver
Reperfusion
Toxic effects of chemicals: halogenated
hydrocarbons, quinones, iron,
acetaminophen, ethanol
Endotoxin
Eye
Retionopathy of prematurity
Photic retinopathy
Macular degeneration
Ocular hemorrhage
Cataracts
Kidney
Autoimmune nephrosis:inflammation
Toxic effects of chemicals: aminoglycosides, heavy metals
Muscle
Muscular dystrophy
Over-exercising
Lung
Normobaric hyperoxic injury
Bronchopulmonary displasia
Toxic effects of chemicals: paraquat,
bleomicin
Emphysema
Asbestosis
Idiopathic pulmonary fibrosis
Skin
Radiation (UV or ionising)
Thermal injury
Toxic effects of chemicals:
tetracyclines stimulating
photosensitization
Contact dermatitis
Porphyria
Heart and cardiovascular system

Atherosclerosis
Hemochromatosis
Reperfusion: after infraction or
transplant
Selenium deficiency (Keishan disease)
Toxic effects of chemicals: ethanol,
doxorubicin
Myocardial infarction
Brain and nervous system

Parkinsons disease
Alzheimers disease
Tardive dyskinesia
Neuronal ceroid lipofuscinosis
Neurotoxins
Hypertensive cerebrovascular injury
Allergic encephalomyelitis
Multiple sclerosis
Gastrointestinal tract
Inflammatory-immune system
Reperfusion
Glomerulonephritis
Toxic effects of chemicals: nonsteroidal
Vasculitis
and anti-inflammatory agents, alloxan, iron
Autoimmune disease
Pancreatitis, Colitis, Intestinal ischemia,
Lupus erythermatosus
Gastric ulcers
Reumatroid arthritis
Blood
Malaria
Various anemias
Protoporphyrin photooxidation
Toxic effects of chemicals: phenylhydrazine, primaquine and related drugs,
sulfonamides, lead etc.
Favism
Fanconis anemia
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Miscellaneous/general
Aging
AIDS, Cancer, Diabetes
Inflammation
Trauma
Ischemia/reperfusion
Radiation injury
Rheumatoid arthritis and lupus
Toxic effects of chemicals: alloxan
(diabetes), iron overload
Acute pancreatitis, Amyloidosis
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thiol redox system consisting of the glutathione system (glutathione/

glutathione reductase/glutaredoxin/glutathione peroxidase and a thioredoxin
system (thioredoxin/thioredoxin peroxidase/thioredoxin reductase) (for
details see Chapter 2).
The protective antioxidant compounds are located in organelles, subcellular

compartments or the extracellular space enabling maximum cellular protection to
occur. Thus antioxidant system of the living cell includes three major levels of
defence (Niki, 1996; Surai, 1999; Surai, 2002):
The first level of defence is responsible for prevention of free radical formation
by removing precursors of free radicals or by inactivating catalysts and consists
of three antioxidant enzymes namely SOD, GSH-Px and CAT plus metal-binding
proteins (Figure 1.1). Since the superoxide radical is the main free radical produced
in physiological conditions in the cell (Halliwell, 1994) superoxide dismutase
(EC 1.15.1.1) is considered to be the main element of the first level of antioxidant
defense in the cell (Surai, 1999). This enzyme dismutates the superoxide radical
in the following reaction:
SOD
2O2* +2H+
H2O2+O2
First level of defence:
Prevention of radical formation
Superoxide dismutase, glutathione peroxidase, catalse,

glutathione and thioredoxin systems and
Metal-binding proteins
Second level of defence:
Prevention and restrictition of chain formation
and propagation
Vitamins A, E, C, Carotenoids, Ubiquinols, Glutathione, Uric acid
Third level of defence:
Excision and repair of damaged parts of molecules
Lipases, Peptidases, Proteases, Transferases,
DNA-repair enzymes etc
Figure 1.1 Three lines of antioxidant defence in animal cells (adapted from Surai, 1999).
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Superoxide dismutase was discovered by McCord and Fridovich in 1969 as an

enzymatic activity in preparations of carbonic anhydrase or myoglobin that
inhibited the aerobic reduction of cytochrome c by xanthine oxidase. Therefore,
haemocuprein, which was discovered much earlier, became copper-zinc superoxide
dismutase (Bannister, 1988). This discovery opened new era in free radical research.
At present, three distinct isoforms of SOD have been identified in mammals, and
their genomic structure, cDNA, and proteins have been described (Zelko et al.,
2002). SOD1, or Cu,Zn-SOD, was the first enzyme of this family to be
characterised and is a copper and zinc-containing homodimer that is found almost
exclusively in intracellular cytoplasmic spaces. It exists as a 32 kDa homodimer
and is present in the cytoplasm and nucleus of every cell type examined (Zelko et
al., 2002).
Did you know that there are three different forms
of superoxide dismutase in mammalian cells?
The second member of the family (SOD2) has manganese (Mn) as a cofactor and
therefore called Mn-SOD. It was shown to be a 96 kDa homotetramer and located
exclusively in the mitochondrial matrix, a prime site of superoxide radical
production (Halliwell and Gutteridge, 1999). Therefore the expression of MnSOD is considered to be essential for the survival of aerobic life and the
development of cellular resistance to oxygen radical-mediated toxicity (Fridovich,
1995). Mn-SOD is inducable enzyme and its activity is affected by cytokines and
oxidative stress. In fact, Mn-SOD has been shown to play a major role in promoting
cellular differentiation and in protecting against hyperoxia-induced pulmonary
toxicity (Fridovich, 1995). The biological importance of Mn-SOD is illustrated in
Table 1.6. In 1982, a third SOD isozyme was discovered by Marklund and
coworkers and called extracellular superoxide dismutase (EC-SOD), due to its
exclusive extracellular location. EC-SOD is a glycoprotein with a molecular weight
of 135,000 kDa with high affinity for heparin. However, there are some speciesspecific variations in molecular weight. EC-SOD is present in various organisms
as a tetramer or, less commonly, as a dimer and contains one copper and one zinc
atom per subunit, which are required for enzymatic activity (Fattman et al., 2003).
The expression pattern of EC-SOD is highly restricted to the specific cell type and
tissues where its activity can exceed that of Cu,Zn-SOD or Mn- SOD. The fourth
form of the enzyme Fe-SOD was isolated from various bacteria but not found in
animal tissues (Michalski, 1992). Furthermore, a novel type of nickel-containing
SOD was purified to apparent homogeneity from the cytosolic fractions of
Streptomyces sp (Youn et al., 1996). The biosynthesis of SODs, in most biological
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11
systems, is well controlled. In fact, exposure to increased pO2, increased

intracellular fluxes of O2-, metal ions perturbation, and exposures to several
environmental oxidants have been shown to influence the rate of SOD synthesis
in both prokaryotic and eukaryotic organisms (Hassan, 1988).
Table 1.6 The biological importance of Mn-SOD (adapted from Mates et al., 1999).
N Mn-SOD manipulationm
Effects
1 Inactivation of Mn-SOD genes in

E. coli
2 Elimination of Mn-SOD gene in
Saccharomycetes cervisiae
3 Lack of expression in Mn-SOD
knock-out mice
4 Effect of TNF
Increases mutation frequency in aerobic

conditions
Increases its sencitivity to oxygen
5 Transfection of Mn-SOD cDNA into

cultural cells
6 Expression of human Mn-SOD
genes in transgenic mice
Dilated cardiomyopathy and neonatal lethality

Selectively induces Mn-SOD in various mouse
tissues and cultural cells
Rendered the cells resistant to paraquat, TNF and
adriamycin-induced cytotoxicity
Protects against oxygen-induced pulmonary
injury and adriamycin-induced cardiac toxicity
The hydrogen peroxide formed by SOD action can be detoxified by GSH-Px or

CAT which reduce it to water as follows:
GSH-Px
H2O2 + 2GSH
2H2O2
Catalase
GSSG+2H2O
2H2O + O2
Catalase (EC 1.11.1.6) is a tetrameric enzyme consisting of four identical subunits

of 60 kDa containing a single ferriprotoporphyrin group per subunit. It plays an
important role in the acquisition of tolerance to oxidative stress in the adaptive
response of cells (Mates et al., 1999). In mammalian cells, NADPH is bound to
catalase protecting it from inactivation by H2O2 (Chaudiere and Ferrari-Illiou,
1999). Since GSH-Px has a much higher affinity for H2O2 than CAT (Jones et al.,
1981) and wider distribution in the cell (catalase is located mainly in peroxisomes),
the H2O2 removal from the cell is very much dependent on GSH-Px. Details of
GSH-Px action are shown in Chapter 2. Recently it has been shown that thioredoxin
peroxidases are also capable of directly reducing hydrogen peroxide (Nordberg
and Arner, 2001). It is interesting that the levels of antioxidant enzymes are regulated
by gene expression as well as by post-translational modifications (Fugi and
Taniguchi, 1999).
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12

Did you know that as part of GSH-Px and Thioredoxin
reductase Se regulates the first and second levels of
antioxidant defence?
Transition metal ions also accelerate the decomposition of lipid hydroperoxides

into cytotoxic products such as aldehydes, alkoxyl radicals and peroxyl radicals:
LOOH + Fe2+
LOOH + Fe3+
LO* + Fe3+ + OHLOO* + Fe2+ + H+
Therefore, metal-binding proteins (transferrin, lactoferrin, haptoglobin, hemopexin,

metallothionenin, ceruloplasmin, ferritin, albumin, myoglobin, etc.) also belong
to the first level of defence. It is necessary to take into account that iron and
copper are powerful promoters of free radical reactions and therefore their
availability in catalytic forms is carefully regulated in vivo (Halliwell, 1999).
Therefore organisms have evolved to keep transition metal ions safely sequestered
in storage or transport proteins. In this way the metal-binding proteins prevent
formation of hydroxyl radical by preventing them from participation in radical
reactions. For example, transferrin binds the iron (about 0.1% of the total body
reserves), transports it in the plasma pool and attaches it to the transferrin receptor.
The important point is that iron associated with transferrin will not catalyse free
radical reaction. Ferritin is considered to be involved in iron storage (about 30%
of total body reserves) within the cytosol in various tissues including liver and
spleen. Major part of iron in the body (55-60%) is associated with hemoglobin
within red cells and about 10% with myoglobin in muscles (Galey, 1997). A range
of other iron-containing proteins (mainly enzymes) can be found in the body
including NADH dehydrogenase, cytochrome P450, ribonucleotide reductase,
proline hydroxylase, tyrosine hydroxylase, peroxidases, catalase, cyclooxygenase,
aconitase, succinate dehydrogenase, etc. (Galey, 1997). Despite an importance
of iron in various biochemical reactions, iron can be extremely dangerous when
not carefully handled by proteins. In fact, in many stress conditions a release of
free iron from its normal sites and its participation in Fenton chemistry mediate
damages to cells. For example superoxide radical can release iron from ferritin
and H2O2 degrades the heme of hemoglobin to liberate iron ions (Halliwell, 1987).
Did you know that metal-binding proteins bind Fe and Cu and
prevent their participation in free radical production?
Ceruloplasmin is another major protein mediating free radical metabolism being
a copper-binding protein. Under physiological conditions it binds six or seven
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13
copper ions per molecule preventing their participation in free radical generation.
About 5% of human plasma copper is bound to albumin or to amino acids and the
rest is bound to ceruloplasmin. Furthermore ceruloplasmin possesses antioxidant
properties itself being able to scavenge superoxide radical (Yu, 1994). Therefore,
it is now quite clear that metal sequestration is an important part of extracellular
antioxidant defence.
Unfortunately this first level of antioxidant defence in the cell is not sufficient
to completely prevent free radical formation and some radicals do escape through
the preventive first level of antioxidant safety screen initiating lipid peroxidation
and causing damage to DNA and proteins. Therefore the second level of defence
consists of chain-breaking antioxidants - vitamin E, ubiquinol, carotenoids, vitamin
A, ascorbic acid, uric acid and some other antioxidants. Glutathione and
thioredoxin systems also have a substantial role in the second level of antioxidant
defence (for details see Chapter 2). Chain-breaking antioxidants inhibit
peroxidation by keeping the chain length of the propagation reaction as small as
possible. Therefore, they prevent the propagation step of lipid peroxidation by
scavenging peroxyl radical intermediates in the chain reaction:
Toc* + LOOH
LOO* + Toc
(LOO* is lipid peroxyl radical; Toc - tocopherol, Toc* - tocopheroxyl radical,

LOOH lipid hydroperoxide )
Vitamin E, the most effective natural free radical scavenger identified to date, is
the main chain breaking antioxidant in the cell. However, hydroperoxides, produced
in the reaction of vitamin E with the peroxyl radical, are toxic and if not removed,
impair membrane structure and functions (Gutteridge and Halliwell, 1990). In
facts, lipid hydroperoxides are not stable and in the presence of transition metal
ions can decompose producing new free radicals and cytotoxic aldehydes (Diplock,
1994). Therefore hydroperoxides have to be removed from the cell in the same
way as H2O2, but catalase is not able to detoxify these compounds and only Sedependent GSH-Px can deal with them converting hydroperoxides into nonreactive products (Brigelius-Flohe, 1999) as follows:
GSH-Px
ROOH + 2GSH
ROH (non-toxic)+ H2O + GSSG
Thus, vitamin E performs only half the job in preventing lipid peroxidation by
scavenging free radicals and forming hydroperoxides. The second part of this
important process of antioxidant defence is due to Se-GSH-Px. It is necessary to
underline, that vitamin E and selenium work in a tandem; and even very high
doses of dietary vitamin E cannot replace Se which is needed (in the form of
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14
GSH-Px and thioredoxin reductase) to complete the second part of antioxidant

defence as mentioned above. Thus, Se as an integral part of the GSH-Px and
thioredoxin reductase belongs to the first and second levels of antioxidant defence.
Did you know that vitamin E performs only half the job in
preventing lipid peroxidation by scavenging free radicals
and forming hydroperoxides and Se-GSH-Px is absolutely
essential to complete antioxidant defence?
Coenzyme Q is considered to be an important antioxidant, which is synthesised
in vivo (see chapter 10) and is an important integral part of the antioxidant defence
system in the cell.
Carotenoids recently were included into family of natural antioxidants. They
exhibit their maximum antioxidant activity at low oxygen pressures, which prevail
in healthy tissues. It has been recently hypothesised that carotenoids are not the
major antioxidant players themselves but rather are an important part of the
antioxidant system (Surai, 2002). Therefore antioxidant interactions including
their recycling provide an effective and reliable system of defence from free
radicals and toxic products of their metabolism.
Vitamin C is a hydrophilic antioxidant functioning in an aqueous environment
and possessing high free-radical-scavenging activity (Yu, 1994). It directly reacts
with O2- and OH* and various lipid hydroperoxides and is taking part in the
vitamin E recycling (Yu, 1994; Halliwell, 1996). Ascorbic acid is protective against
a number of ROS (Carr and Frei, 1999; Halliwell, 1999a, 1996; Table 1.7).
The major advantages of ascorbate as an antioxidant have been described as
follows (Carr and Frei, 1999):
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Both ascorbate and ascorbyl radical have low reduction potentials and can
react with most other biologically relevant radicals and oxidants;
Ascorbyl radical has a low reactivity as a result of resonance stabilisation of
unpaired electron and readily dismutates to ascorbate and dehydroascorbic
acid (DAA);
Ascorbyl radical and DAA can be converted into active ascorbate form by
enzyme-dependent or independent pathways. In particular, ascorbyl radical
can be reduced by NADH-dependent semidehydroascorbate reductase or
by thioredoxin reductase. At the same time DAA can be reduced to AA by
GSH, lipoic acid or glutaredoxin.
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15
Table 1.7 Antioxidant properties of ascorbate (Adapted from Halliwell, 1996; 1999a).
Scavenges O2* and HO2*

Scavenges water-soluble peroxyl (ROO*) radicals. Lypophylic ascorbate esters can scavenge
lipid-soluble ROO* radicals
Scavenges thiyl and sulphenyl radicals
Prevents damage by radicals arising by attack of OH* or ROO* upon uric acid
Powerful scavenger of hypochlorous acid
Inhibits damage by peroxinitrite
Powerful scavenger and quencher of singlet oxygen
Scavenges OH* radicals
Scavenges nitroxide radicals
May regenerate -tocopherol from -tocopheryl radicals in membranes and lipoproteins
Protects plasma lipids against peroxidation induced by activated neutrophils
Protects membranes and lipoproteins against lipid peroxidation induced by cigarette smoke
Powerful scavenger of O3 and NOO* in human body fluids
Inhibits oxidative damage by drug-derived radicals
Scavenges free radicals on proteins
Glutathione (GSH) is the most abundant non-protein thiol in avian and mammalian
cells, and considered to be an active antioxidant in biological systems providing
cells with their reducing milieu (Meister, 1992). Cellular GSH plays a key role in
many biological processes (Sen and Packer, 2000):
the synthesis of DNA and proteins;

cell growth and proliferation;
regulation of programmed cell death;
immune regulation;
the transport of amino acids;
xenobiotic metabolism;
redox-sensitive signal transduction.
Furthermore, GSH thiolic group can react directly with (Lenzi et al., 2000; Meister
and Anderson, 1983):
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H 2O 2;
superoxide anion;
hydroxyl radicals;
alkoxyl radicals;
hydroperoxides
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16
Therefore, a crucial role for GSH is as free radical scavenger, particularly effective
against the hydroxyl radical (Bains and Shaw, 1997), since there are no enzymatic
defences against this species of radical. Usually decreased GSH concentration in
tissues is associated with increased lipid peroxidation (Thompson et al., 1992).
Furthermore in stress conditions GSH prevents the loss of protein thiols and vitamin
E (Palamanda and Kehrer, 1993) and plays an important role as a key modulator
of cell signaling (Elliott and Koliwad, 1997). Animals and human are able to
synthesise glutathione.
Taurine, a sulphur containing amino acid (2-aminoethane sulfonic acid) is
synthesised from methionine and cysteine in the presence of vitamin B6 and
found in almost all tissues in mammals. It is the most abundant intracellular amino
acid in humans which is not incorporated into proteins. Antioxidant properties of
taurine were shown in various model systems in vivo and in vitro (Cozzi et al.,
1995; Haber et al., 2003; Sethupathy et al., 2002). However, antioxidant effect of
taurine is not restricted to PUFAs. For example, Ogasawara et al. (1993; 1994)
showed an inhibiting effect of taurine against the modification of protein, as well
as an antioxidative effect through the reactions of taurine with aldehydes in vivo.
It seems likely that in many cases in biological systems taurine could interact with
other antioxidants being an important part of an integrated antioxidant system.
For example, taurine supplementation of rats restored kidney GSH content and
GSH-Px activity and reduced MDA production levels in the kidney tissue following
cisplatin treatment (Saad and Al-Rikabi, 2002). In streptozotocin-diabetic rats,
dietary taurine supplementation ameliorates MDA levels, GSSG/GSH, and NAD+/
NADH (Obrosova and Stevens, 1999). Taurine reduced significantly a decrease
of glutathione antioxidant system activity protecting tissues against GSH pool
depletion and preventing a decrease of glutathione reductase and glutathione
peroxidase activities in acute severe hypoxia (Mankovska et al., 1998).
Uric acid is traditionally considered to be a metabolically inert end-product of
purine metabolism in man, without any physiological value. However, this
ubiquitous compound has proven to be a selective antioxidant (Becker, 1993;
Maples and Mason, 1988) which can:
react with hydroxyl radicals and hypochlorous acid, itself being converted
to innocuous products
serve as an oxidisable cosubstrate for the enzyme cyclooxygenase
protect against reperfusion damage induced by activated granulocytes
prevents oxidative inactivation of endothelial enzymes in stress conditions
chelate transition metal ions and scavenging ROS
Some specific enzymes which hydrolyse oxidised bases preventing their

incorporation into DNA can also be considered as a part of the second level of
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antioxidant defence (Slupphaug et al., 2003). The role of ubiquinones and uric
acid in the farm animal and poultry development has not yet been studied.
However, even the second level of antioxidant defence in the cell is not able to
prevent damaging effects of ROS and RNS on lipids, proteins and DNA. In this
case, the third level of defence is based on systems that eliminate damaged
molecules or repair them. This level of antioxidant defence includes lipolytic
(lipases), proteolytic (peptidases or proteases) and other enzymes (DNA repair
enzymes, ligases, nucleases, polymerases, proteinases, phospholipases and various
transferases).
Since maintaining the integrity of the genome is of the vital importance, living
organisms have evolved a range of systems that can recognise, signal the presence
of, and repair the various forms of DNA damage. In fact, DNA repair is one of
the fundamental processes of life (130 human DNA repair genes have been
identified; Wood et al., 2001) and if the systems are compromised devastating
consequences would be expected. In order to deal with the deleterious effects of
such lesions, leading to genomic instability, cells have evolved a number of DNA
repair mechanisms. They include the direct reversal of the lesion, mismatch repair,
the base excision repair, nucleotide excision repair, nucleotide incision repair,
transcription-coupled repair, global genome repair, translesion synthesis,
homologous recombination and non-homologous end-joining (Gros et al., 2002;
Slupphaug et al., 2003). These repair pathways are universally present in living
cells and extremely well conserved.
Did you know that 130 human DNA repair genes
have been identified?
Therefore, DNA repair systems include a number of enzymatic processes ranging
from base recognition and excision to ligation of DNA strands. In particular, the
DNA glycosylases recognise damaged purines and pyrimidines and hydrolyse
the bond linking the abnormal base to the sugar-phosphate backbone (Halliwell
and Gutteridge, 1999); the 5I-apurinic endonucleases process strand breaks, sites
of base loss, and the products of DNA glycosylase/apurinic lyase action. DNA
polymerase fills in the one-nucleotide gap with the correct base. DNA ligases
complete the repair process by sealing the 3I end of the newly synthesised stretch
of DNA to the original portion of the DNA chain (Cardozo-Pelaez et al., 2000;
Wallace et al., 1997; Croteau and Bohr, 1997).
It is believed that most damaged or inappropriate bases in DNA are removed
by excision repair, while a minority are repaired by direct damage reversal (Krokan
et al., 2000). The importance of these DNA repair systems is confirmed by the
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fact that defects in these can result in cell death and hypersensitivity to endogenous
or environmental mutagens (Jackson, 1998). Therefore removing mutagenic
lesions in DNA is a vital task for repair systems. In general, the repair DNA damage
mechanisms in bacteria are well defined, whereas in higher eukaryotes the genes
and proteins responsible for repair await further investigation (Croteau and Bohr,
1997). It seems likely that DNA repair is integrated with cell cycle regulation,
transcription and replication and use some common factors (Slupphaug et al.,
2003). However, the enzymes of the third level of antioxidant defence do not
achieve complete repair or removal of damaged DNA molecules and this could
lead to arrest of cell cycle and cell death. In fact, programmed cell death (apoptosis)
is involved in maintenance of the genetic integrity by removing genetically altered
cells.
Selenium in an organic form can directly or indirectly effect DNA integrity
and repair. For example, elderly male dogs were fed on the diet supplemented
with selenium in the form of SeMet or high-selenium yeast at 3 g/kg or 6 g/kg
body weight per day for 7 months and a comparison was made with
unsupplemented control dogs. The extent of DNA damage in prostate cells and in
peripheral blood lymphocytes, as determined by the alkaline comet assay, was
lower among the selenium-supplemented dogs than among the control dogs
(Waters et al., 2003). Furthermore, Seo et al. (2002a) showed that selenium in the
form of SeMet induces a DNA repair response in normal human fibroblasts in
vitro, and protects cells from DNA damage. A possible mechanism for the inducible
DNA repair response was shown to be enhanced repair complex formation in
selenomethionine-treated cells. Similarly, treatment with SeMet either on initiation
or on selection/promotion, or during the entire experiment showed that SeMet
was most effective in regulating the cellular antioxidant defence systems, DNA
chain break control and reducing aberrant crypt foci in the colorectal tissues of
rats (Mukherjee et al., 2001).
Did you that SeMet can affect DNA-repair enzyme systems?
Protective effect of Se against DNA damage depends on the dose used. For
example, measurements in acinar cells in Syrian golden hamsters suggested a
more rapid repair of single-strand breaks DNA in hamsters prefed 2.5 ppm Se
than in those prefed 0.1 ppm Se (Birt et al., 1988). It has also been shown that
selenium in the form of SeMet can activate the p53 tumor suppressor protein by a
redox mechanism that requires the redox factor Ref1 (Seo et al., 2002). Specifically,
SeMet induced sequence-specific DNA binding and transactivation by p53 and
therefore the DNA repair branch of the p53 pathway was activated. Recognition
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and signalling of DNA damage is an important event for the induction of subsequent
cellular responses such as increased repair, cell cycle arrest and apoptosis.
Recognition of DNA breaks is accomplished by a group of phosphatidylinositol3-kinases. These kinases are ATM (ataxia telangiectasia mutated), ATR (ataxia
telangiectasia related) and the catalytic subunit of DNAPK (Christmann et al.,
2003). ATR and ATM can bind to DNA ends of damaged DNA, which results in
activation of the kinase activity. It is interesting that treatment with SeMet was
shown to enhance ATR and CHK2 gene expression in cultured human thyroid
epithelial cells (Kennedy et al., 2004). The authors suggested that SeMet may
prevent radiation-induced adverse biological effects by enhancing the DNA repair
machinery in irradiated cells. Therefore it is clear that SeMet has a unique specific
effect on the DNA repair system as well as provides a protection against DNA
damage and this effect is not the case or even can be opposite when selenite is
used.
In spite of important roles of protein oxidation in pathogenesis of the
development of various diseases, mechanisms for the control of protein oxidation
and their repair have not been well studied and this has been a topic of great
interest for the last few years. The oxidative damage to proteins is associated with
alteration of transport proteins and ion dis-balance, disruption to the receptors
and impair signal transduction, enzyme inactivation etc. It is believed that
conversion of SH groups into disulphides and other oxidized species (e.g.
oxyradicals) is one of the earliest events during the radical-mediated oxidation of
proteins. Therefore, thioredoxin plus thioredoxin reductase deal with these changes
by reducing protein disulphides to thiols and regulating redox-sensitive
transcription factors (Dean et al., 1997).
It is interesting that reversible oxidation of cysteine could be an important
cellular redox sensor in some proteins (Finkel, 2000). Methionine residues in
proteins are also very susceptible to oxidation with methionine sulfoxide formation,
which was detected in native proteins (Gao et al., 1998). This could affect activity
of various proteins (Table 1.8). In fact, almost all forms of ROS oxidize methionine
residues of proteins to a mixture of the R- and S-isomers of methionine sulfoxide
(Stadtman et al., 2002). Methionine sulfoxide reductase (Msr) can reduce either
the free or the protein-bound methionine sulfoxide back to methionine. Therefore
Msr is considered a repair mechanism for dealing with the product of reaction of
oxidants with methionine residues (Levine et al., 1996). The authors hypothesized
that methionine residues function as a last chance antioxidant defence system
for proteins. It was shown that in bacterial glutamine synthetase surface-exposed
methionine residues surrounding the entrance to the active site are preferentially
oxidised and other residues (e.g. cystein) within the critical regions of the protein
are protected without loss of catalytic activity of the protein (Levine et al., 1996).
Indeed, due to Msr activity the methionine-methionine sulfoxide pair can function
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Table 1.8 Proteins and peptides with activity altered by methionine oxidation (adapted from Levine et al.,
2000).
Adrenocorticotropic hormone
-1-antitrypsin
-2-antitrypsin
-2-macroglobulin
Amyloid beta peptide
Antiflammin
Antitrombin
Apolipoprotein
Bombesin
Bugarotoxin
Calcitonin
Calmodulin
Chemotactic peptide f-Met-Leu-Phe
Cholecystokinin
Chorionic somatomammotropin
Chymotrypsin
Complement C5
Cytochrom c peroxidase
Cytochrome c
Echistatin
Enkephalin
Factor VII
Fibronectin
Glucagon
Glutamine synthetase
Growth hormone
Hemoglobin
HIV-2 protease
Interferon -2b
Interferon
Interleukin 6
Keratinocyte growth hormone
Lypoxygenase
Lutropin
Lysozyme
Mucus proteinase inhibitor
Neuropeptide Y
Ovoinhibitor
Parathyroid hormone
Pepsin
Phosphoglucomutase
Plasminogen activator inhibitor
Potassium channel
Prolactin
Ribonuclease
Ribosomal protein L12
Secretory leukocyte proteinase inhibitor
Small heat shork protein
Snake venon cardiotoxin
Stem cell factor
Subtilisin
Thrombomodulin
Tissue plasminogen activator
Triptophanase
Vasoactive intestinal peptide
catalytically. MsrA is present in most living organisms, is encoded by a single

gene and the mammalian enzyme has been detected in all tissues studied. In
particular it is found in the cytosol and mitochondria of rat liver cells (Vougier et
al., 2003). Msr is considered to have at least three important function in cellular
metabolism including antioxidant defence, repair enzyme and a regulator of certain
enzyme function and possibly participation in signal transduction (Bar-Noy and
Miskovitz, 2002; Stadtman et al., 2002 ). In particular, mouse that lacks the
MsrA gene (Moskovitz et al., 2001):
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exhibits enhanced sensitivity to oxidative stress

has a shorter lifespan
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develops an atypical walking pattern

accumulates higher tissue levels of oxidised protein under oxidative stress
is less able to up-regulate expression of thioredoxin reductase under
oxidative stress
On the other hand, overexpretion of the MsrA gene in the nervous system markedly
extends the lifespan of the fruit fly Drosophila (Ruan et al., 2002), human T-cells
(Moscovitz et al., 1998) and mouse (Moscovitz et al., 2001). Furthermore, the
authors showed that MsrA transgenic flies are more resistant to paraquat-induced
oxidative stress. In fact Msr is also considered to be a potential missing link in the
post-translational modification cycle involved in the specific oxidation and
reduction of methionine residues in cellular signalling proteins which can change
cellular excitability (Hoshi and Heinemann, 2001). This could well be the regulatory
mechanism similar to protein phosphorylation. The general scheme of antioxidant
function of Msr is shown in Figure 1.2.
CO2+
Pentose
G-6-P
Thioredoxin
NADPH
-ox
NADP+
Thioredoxinred
Msr -red
Msr -ox
ProteinMetSO
RSox
ProteinMet
RSred
Figure 1.2 Function of methionine sulfoxide reductase (adapted from Levine et al., 2000).
RSox represents a reactive species which is reduced with concomitant oxidation of methionine residue in
the protein. Thioredoxin-red and ox- are reduced and oxidised thioredoxin. Msr-ox and red- are
methionine sulfoxide reductase.
MsrA has been known for a long time, and its repairing function is well
characterised, however, recently, a new methionine sulfoxide reductase was
characterised (Grimaud et al., 2001). It was referred to as MsrB and it was shown
that the gene of MsrB is present in genomes of eubacteria, archaebacteria, and
eucaryotes. Therefore, in mammals two methionine sulfoxide reductases, MsrA
and MsrB, are expressed with different substrate specificity (Grimaud et al., 2001).
They catalyze the thioredoxin-dependent reduction of the S-isomer and R-isomer
of methionine sulfoxide to methionine, respectively.
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Did you know that mammalian methionine sulfoxide reductase B
has been identified as a selenoprotein, previously known as
selenoprotein R?
Recently the major mammalian MsrB has been identified as a selenoprotein

(Maskovitz et al., 2002; Kryukov et al., 2002). In fact it has been found that
selenoprotein R is a zinc-containing sterteo-specific Msr (Kryukov et al., 2002).
Furthermore, it has been shown that there was a loss of MsrB activity in the MsrA
/
mouse in parallel with losses in the levels of MsrB mRNA and MsrB protein
(Moskovitz and Stadtman, 2003). Therefore, the author suggested that MsrA might
have a role in MsrB transcription. Moreover, Se deficiency in mouse was associated
with a substantial decrease in the levels of MsrB-catalytic activity, MsrB protein,
and MsrB mRNA in liver and kidney tissues (Moskovitz and Stadtman, 2003). It
has been reported that human and mouse genomes possess three MsrB genes
responsible for synthesis of the following protein products: MsrB1, MsrB2 and
MsrB3 (Kim and Gladyshev, 2003). In particular, MsrB1 (Selenoprotein R) was
present in the cytosol and nucleus and exhibited the highest methionine-Rsulfoxide reductase activity due to presence of selenocysteine (Sec) in its active
site. Other mammalian MsrBs are not selenoproteins and contain cysteine in place
of Sec and were less catalytically efficient (Kim and Gladyshev, 2003).
Did you know that Se in the form SeMet is involved in
regulation of DNA-repair enzymes and as a part of MsrB
regulates protein repairing system and therefore regulates
the third level of antioxidant defence?
The reduced glutathione itself can also participate in maintenance of protein SH
groups. At the same time the thioredoxin system has alkyl hydroperoxide reductase
activity. Protein disulphide isomerase is also involved in re-pairing of SH groups
in proteins (Dean et al., 1997). Furthermore, the cells can generally remove
oxidized proteins by proteolysis. In fact, damaged proteins are degraded by the
proteasome, multicatalytic proteinase (an intracellular, nonlysosomal threonine
type protease, EC 3.4.99.46), which is responsible for degradation of the majority
of cytosolic proteins (Rock et al., 1994).
It is well recognised now that the proteasome is the major enzymatic system in
charge of cellular cleansing and plays a key role in the degradation of damaged
proteins controlling the level of altered proteins in eukaryotic cells (Friguet et al.,
2000). It is suggested that enhanced susceptibility to degradation by proteinases
is employed as a criterion of unfolding (Dean et al., 1997), however, heavily
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oxidized proteins are characterised by an increased resistance to proteolytic attack

by most proteinases. The proteasome complex recognises hydrophobic amino
acid residues, aromatic residues, and bulky aliphatic residues that are modified
during the oxidative stress and catalyse the selective removal of oxidatively
modified cell proteins (Grune et al., 1997). By minimising protein aggregation
and cross-linking and by removing potentially toxic protein fragments proteasome
is an active part of the cellular defence system against oxidative stress. The selective
degradation of oxidatively damaged proteins enables cells to restore vital proteins
including enzymes during physiological metabolism and during moderate stress
conditions (Grune et al., 1997). Oxidized proteins may also be recognised as
foreign by the immune system with corresponding antibody formation (Halliwell
and Gutteridge, 1999). Clearly, further work is needed to clarify molecular
mechanisms of the third level of antioxidant defence.
All these antioxidants are operating in the body in association with each other
forming an integrated antioxidant system. The co-operative interactions between
antioxidants in the cell are vital for maximum protection from the deleterious
effects of free radicals and toxic products of their metabolism. For-example, it is
well established that vitamin E is the major antioxidant in biological membranes,
a head quarter of antioxidant network. However it is usually present there in
low molar ratios (one molecule per 2000-3000 phospholipids) but vitamin E
deficiency is difficult to induce in adult animals. It is probably due to the fact that
oxidised vitamin E can be converted back into the active reduced form by reacting
with other antioxidants: ascorbic acid, glutathione, ubiquinols or carotenoids
(Figure 1.3). This figure demonstrates a connection of antioxidant defence to the
general body metabolism (the pentose phosphate cycle is the major producer of
reducing equivalents in the form of NADPH) and shows involvement of other
nutrients in this process. For example, dietary protein is a source of essential
amino acids for glutathione synthesis, riboflavin is an essential part of glutathione
reductase, niacin is a part of NADPH and Se is an integral part of thioredoxin
reductase. At the same time thiamine is required for transketolase in the pentose
phosphate pathway.
Thus, a major finding in recent years is the possibility of direct or indirect
vitamin E recycling from its oxidised radical form by means of ascorbate (Chan
et al., 1991; Chan, 1993), glutathione (Niki et al., 1982; Chan, 1993), cysteine
(Motoyama et al., 1989), ubiquinols (Freisleben and Packer, 1993; Chan, 1993),
lipoic acid (Packer, 1998), estrogens (Mukai et al., 1990), carotenoids (Palozza
and Krinsky, 1992; Bohm et al., 1997) and potentially quercetin and catechins
(Pietta, 2000), anthocyanins (Frank et al., 2002) and rosemary extracts (Madsen
et al., 1997). Enzymatic regeneration of -tocopherol has been also described
(Maguire et al., 1989; Kagan et al., 1998). The rate of reduction of phenoxyl
radical in the membrane decreased in the order of ascorbic
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acid>cysteine>glutathione (Niki, 1996). The rate of regeneration, or recycling,

of the vitamin E radicals that form during their antioxidant action may affect both
its efficiency in antioxidant action and its lifetime in biological systems and the
greater recycling activity is associated with increased efficiency of inhibition of
lipid peroxidation (Packer, 1995). Whether all these regeneration reactions take
place in vivo await investigation. Due to incomplete regeneration (the efficiency
of recycling is usually less than 100%) in biological systems, the antioxidants
have to be obtained from the diet (e.g. vitamin E and carotenoids) or synthesised
in the tissues (glutathione).
Niacin
Membrane Se
Transport
Riboflavin
Vit .E quinone
Loss
Loss
Vit .Eradical ROOH
CO2+
Pentose NADPH GSSH AA
2
G-6-P NADP+ 2GSH DAA Vitamin E ROO*

Thiamin
Glucose
Figure 1.2
Loss
Diketo-L- gulonic
acid
Redox cycle of vitamin E (Adapted from Winkler et al., 1994 ; Surai, 1999)
As a result of antioxidant action of vitamin E, tocopheroxyl radical is formed. This radical can be reduced
back to an active form of -tocopherol by coupling with ascorbic acid oxidation. Ascorbic acid can be
regenerated back from the oxidised form by recycling with glutathione which can receive a reducing
potential from NADPH, synthesised in the pentose phosphate cycle of carbohydrate metabolism. Enzymes
involved in vitamin E recycling are as follows: 1. Thioredoxin reductase; 2. Glutathione reductase; 3.
Glucose-6-phosphate dehydrogenase. Due to incomplete regeneration (the efficiency of recycling is usually
less than 100%) in biological systems, the antioxidants have to be obtained from the diet (vitamin E and
carotenoids) or synthesised in the tissues (ascorbic acid and glutathione).
Therefore the antioxidant protection in the cell depends not only on vitamin E
concentration and location, but also relies on the effective recycling. Indeed, if
the recycling is effective then even low vitamin E concentrations are able to
maintain high antioxidant protection in physiological conditions. For example,
this could be demonstrated using chicken brain as a model system. Indeed, our
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25
data (Surai, 2002) indicate that the brain is characterised by extremely high
concentrations of long chain polyunsaturated fatty acids predisposing this tissue
to lipid peroxidation. Furthermore, brain contains much lower levels of vitamin E
than other body tissues. However, in fresh chicken brain, levels of products of
lipid peroxidation are very low, which could be a reflection of an effective vitamin
E recycling by ascorbic acid which is present in this tissue in comparatively high
concentrations. Antioxidant recycling is the most important element in
understanding mechanisms involved in antioxidant protection against oxidative
stress. The rate of regeneration, or recycling, of the vitamin E radicals may affect
both its antioxidant efficiency and its lifetime in biological systems. As can be
seen from data presented above the antioxidant defence includes several options
(Surai, 2002):
Decrease localised oxygen concentration;

Prevention of first-chain initiation by scavenging initial radicals (SOD, GSHPx and catalase);
Binding metal ions (metal-binding proteins);
Decomposition of peroxides by converting them to non-radical, non-toxic
products (Se-GSH-Px);
Chain-breaking by scavenging intermediate radicals such as peroxyl and
alkoxyl radicals (vitamins E, C, glutathione, uric acid, ubiquinol, bilirubin
etc.);
Repair and removal of damaged molecules.
Additional defensive mechanisms responsible for maintenance of physiological

metabolism in stress conditions include (Surai, 2002):
Antioxidant recycling mechanisms;

Redox-signalling and gene expression with an additional synthesis of
important antioxidant molecules ;
Stress-protein synthesis (e.g. heat shock proteins);
Apoptosis (can remove damaged cells and restrict mutagenesis).
Antioxidant-prooxidant balance in the body and oxidative stress

In the body a delicate critical balance exists between antioxidant defence and
repair systems and free radical generation (Figure 1.4). In physiological conditions
the right and left parts of the so-called balances are in equilibrium i.e. free
radical generation is neutralised by the antioxidant system. Exogenous factors
are among the most important elements, which increase an efficiency of the
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antioxidant system of the organism. Natural and synthetic antioxidants in the

feed as well as optimal levels of Mn, Cu, Zn and Se help to maintain the efficient
levels of endogenous antioxidants in the tissues. Optimal diet composition allows
the antioxidants of the food to be efficiently absorbed and metabolised. Optimal
temperature, humidity and other environmental conditions are also required for
the effective protection against free radical production. The prevention of different
diseases by antibiotics and other drugs is an integral part of the efficient antioxidant
defence as well.
Increase of antioxidant defence
Natural antioxidants in the feed
(Vitamins A, E, C, carotenoids, flavonoids,
essential oils); Synthetic antioxidants in
the feed (BHT, ethoxiquine etc.)
Stress conditions
Se,Mn
Zn, Cu
Nutritional
Toxins, high PUFA,

deficiencies of vitamin E, Se,
Mn , Zn, Fe-overload
Diet optimization
Environmental condition
optimization
Disease prevention and
treatment by antibiotics and
other drugs
Environmental
Internal
Temperature, humidity,
hyperoxia,
radiation, UV,
microwave etc.
Diseases: bacterial, viral,
allergy etc.
Antioxidant systems of the organism
Fre e ra d ic a l g e n e ra tio n
Vitamins A, E, C, carotenoids, glutathione,

ubiquinol , uric acid, antioxidant enzymes
(SOD, GSH-Px, Catalase)
Electron-transport chain,
Phagocytes,
Xanthine oxidase etc.
Meat quality and

shelf life reduction
Milk taste deterioration
Immune incompetence
Lipid p eroxidation,
d amag es to lipids,
p rotein s, DN A
Injuries to heart -vascular, brain

and nervous and muscle systems
Membrane damage
Nutrient absorption
decrease
Nutrient imbalance
Decrease of productive
and reproductive
performances
Figure 1.4 Antioxidant-prooxidant balance in the organism (adapted from Surai, 1999).
Different stress conditions are associated with overproduction of free radicals

and cause oxidative stress i.e. a disturbance in the prooxidant-antioxidant balance
leading to potential tissue damage (Jaeschke, 1995). Stress conditions can be
generally divided into three main categories. The most important part is nutritional
stress conditions including high levels of PUFAs, deficiencies of vitamin E, Se,
Zn or Mn, Fe-overload, hypervitaminosis A and presence of different toxins and
toxic compounds (see chapter 10). A second group of stress factors includes
environmental conditions: increased temperature or humidity, hyperoxia, radiation
etc. Internal stress factors include various bacterial or viral diseases as well as
allergy. All the above-mentioned conditions stimulate free radical generation by a
decrease of coupling of oxidation and phosphorylation in the mitochondria that
results in an increased electron leakage and overproduction of superoxide radical.
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Living cells permanently balance the process of formation and inactivation of

ROS and as a result ROS level remains low but still above zero. Adverse
environmental conditions initiate attempts of organisms to resist the environment
that became more aggressive (Skulachev, 1998). Cells can usually tolerate mild
oxidative stress by additional synthesis of various antioxidants (glutathione,
antioxidant enzymes, etc.) in an attempt to restore antioxidant/oxidant balance.
At the same time, energy expenditures increased and respiration activated leading
to the increased yield of ROS (Skulachev, 1998). However these adaptive
mechanisms have limited ability. Once the free radical production exceeds the
ability of antioxidant system to neutralise them, lipid peroxidation develops and
causes damage to unsaturated lipids in cell membranes, amino acids in proteins
and nucleotides in DNA and as a result, membrane and cell integrity is disrupted.
Membrane damage is associated with a decreased efficiency of absorption of
different nutrients and leads to an imbalance of vitamins, amino acids, inorganic
elements and other nutrients in the organism. All these events result in decreased
productive and reproductive performances of animals. Immunity incompetence
and unfavourable changes in the cardio-vascular system, brain and neurones and
muscle system due to increased lipid peroxidation make the situation even worse.
As it was shown above all antioxidants in the body are working as a team
responsible for antioxidant defence and we call this team the antioxidant system.
In this team one member helps another one working efficiently. Therefore if
relationships in this team are effective, which happens only in the case of balanced
diet and sufficient provision of dietary antioxidant nutrients, then even low doses
of such antioxidants as vitamin E could be effective. On the other hand when this
team is subjected to high stress conditions, free radical production is increased
dramatically. During these times, without external help it is difficult to prevent
damage to major organs and systems. This external help is dietary
supplementation with increased concentrations of natural antioxidants. For
nutritionist or feed formulator it is a great challenge to understand when the internal
antioxidant team in the body requires help, how much of this help to provide and
what the economic return will be. Again, it is necessary to remember about
essentiality of keeping right balance between free radical production and
antioxidant defence. Indeed, ROS and RNS have another more attractive face
participating in a regulation of varieties of physiological functions (Table 1.9).
Did you know that all antioxidants in the body are working as a
team responsible for antioxidant defence and we call this
team the antioxidant system?
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Table 1.9 Some regulatory functions of free radicals (adapted from McCord, 2000; Droge, 2002; Yoon
et al., 2002; Thundathil et al., 2003; de Lamirande and Gagnon, 2003).
Type of radical
Source of radical
Nitric oxide (NO*) Nitric oxide synthase
Superoxide (O2-*) NAD(P)H oxidase

and related ROS
Superoxide and
related ROS
Any source
ROS
Any source
Physiological process
Smooth muscle relaxation (control of
vascular tone) and various other cGMPdependent functions; specific role in signal
transduction events leading to sperm
capacitation
Control of ventilation; Control of
erythropoitenin production and other
hypoxia-inductible functions; Smooth
muscle relaxation; Signal transduction from
various membrane receptors/enhancement of
immunological functions
Oxidative stress responses and the
maintenance of redox homeostasis; An
increased intracellular ROS level stimulates
cell proliferation, apoptosis and
differentiation depending on the relative
concentration of oxidants in the cell;
Promotes physiological capacitation in
sperm allowing the acquisition of fertilizing
capacity
Activation of a variety of kinases including
Src kinase family, preotein kinase C,
mitogen-activated protein kinase (MAPK),
receptor tyrosine kinases and transriptional
factors such as AP-1 and NF-kB;
Modification of redox-sensitive proteins
(e.g. thioredoxin) affecting stress kinases
In commercial animal and poultry production there is a range of stressors and

stress-conditions. For example, stress conditions related to overproduction of free
radicals in poultry production can be summarised as follows:
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Increased interval between an egg being laid and its cooling down for
storage. Eggs should be collected more frequently on warm days. In such
conditions free radical damages to lipids and proteins could occur and
antioxidant protection would be beneficial.
Egg storage before incubation could be associated with lipid peroxidation

within the egg membranes containing high levels of PUFAs. Increased Se
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concentration in combination with other antioxidants (vitamin E and

carotenoids) could be an effective means to prevent damaging effects of
free radicals produced within the egg.
Temperature, humidity and carbon dioxide concentration fluctuations during

incubation. They also can affect embryonic development, oxidation and
phosphorylation in the embryonic tissues leading to free radical production.
For example, high carbon dioxide concentrations during the incubation
period can jeopardise the liveability of the embryo (Decuypere et al., 2001).
Day 19 of embryonic development is an important point when risk of lipid

peroxidation is very high. At this stage chick tissues are characterised by
comparatively high levels of PUFAs (Speake et al., 1998). At the same
time, concentrations of natural antioxidants (vitamin E and carotenoids)
have not reached maximum (Surai, 1999). At this stage of development
piping occurs; and oxygen availability for tissues increases. Low antioxidant
status in combination with high temperature, humidity, and PUFAs could
increase susceptibility to lipid peroxidation.
Did you know that in commercial animal production there is a
range of stress-conditions associated with increased free
radical production?
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Hatching time is considered as an environmental stress for the chick. At this

point natural antioxidant concentrations have reached maximum (Surai,
1999), but high levels of lipid unsaturation in tissues, decreasing
concentration of ascorbic acid (can limit vitamin E recycling) and high
temperature and humidity increase risk of lipid peroxidation.
Delay in collecting chicks from incubator. Since not all chicks are hatched
at the same time because of heterogeneity of the starting material (eggs
from older breeders hatch earlier than those from young flocks and chicks
from smaller eggs hatch earlier than those from large eggs; Decuypere et
al., 2001), some would be in the incubator for 2-12 hours longer than others.
This puts pressure on antioxidant defence capacity. Furthermore, any delay
in food and/or water intake after hatching usually negatively affect a number
of performance parameters and a delay occurs in the maturation of the
enzymatic systems that control metabolism (Decuypere et al., 2001) and
free radical production and protection against them.
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Untitled-2
Transportation from hatchery to farm is another source of stress. For breeding

companies where chicken transportation could involve several thousand
miles, a very high degree of stress is associated with temperature fluctuation
and dehydration.
Suboptimal temperatures in the poultry house. Cold tolerance as well as

feather cover is influenced by thyroid hormone activity, which is Sedependent.
High levels of ammonia and CO2 in poultry house as a result of inadequate

ventilation. This could substantially decrease antioxidant system efficiency.
Disease challenge. Phagocytic immune cells themselves produce free

radicals in the process of killing internalised pathogens. Without adequate
antioxidant nutrient reserves, cellular machinery can be damaged by the
free radicals thereby reducing the effectiveness of the immune cell. In
addition, Se is considered to have a specific role in immune system
regulation, which could be independent on its antioxidant functions.
Vaccination is also a substantial stress; and in some cases using vitamin E,

for example, as a vaccine adjuvant can help improve vaccination efficiency.
Induced molting with feed withdrawal is an important stress condition when

decreased efficiency of heterophil function increases bird susceptibility to
various infections (Kogut et al., 1999). For example, colonic inflammation,
consisting of heterophils infiltrating lamina propria and epithelium, occurred
more often in molted infected chickens than in unmolted infected chickens
(Porter and Holt, 1993).
Mycotoxins in the feed can substantially decrease antioxidant assimilation

from the feed and increase their requirement to prevent damaging effects of
free radicals and toxic products of their metabolism, produced as a result of
mycotoxin exposure. It is now increasingly recognised that at least 25% of
worlds grains are contaminated with mycotoxins.
Heavy metals and other toxicants (dioxine, pesticides, fungicides,

herbicides, etc.) in the feed can also cause an oxidative stress, decreasing
immunocompetence, productive and reproductive performances and
increasing a requirement for antioxidants. For example, thiram a widely
used dithiocarbamate fungicide, in cultured human skin fibroblasts induced
GSH depletion, leading to oxidative stress, lipid peroxidation and finally
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31
cell death (Cereser et al., 2001). Similarly, in a recent study forty-one male,
healthy agricultural sprayers, exposed to pesticides for 5 years, were
compared with twenty one controls matched for age and economic status
with respect to free radical generation, lipid peroxidation, antioxidant status
and concentration of cellular enzymes were determined (Prakasam et al.,
2001). Significantly increased TBARS levels and decreased concentrations
of antioxidants such as GSH, alpha-tocopherol, ascorbic acid and
ceruloplasmin were observed in sprayer populations, when compared to
controls. When Mallard eggs were exposed to diquat dibromide, a commonly
used aquatic herbicide, significant manifestations of oxidative stress were
apparent in hatchlings and included increased hepatic lipid peroxidation
and decreased brain reduced glutathione concentration (Sewalk et al., 2001).
Another herbicide ANITEN I caused an oxidative stress in pheasant kidney
and liver by decreasing activities of antioxidant enzymes (Holovska et al.,
1998).
Untitled-2
Environmental pollutants can cause an oxidative stress in birds. For example

polychlorinated biphenyls (PCBs) increased a GSSG/GSH ratio in the liver
of American kestrels (Hoffman et al., 1996). When chicken eggs were
injected into the air cell with 0.4-1.6 mg PCB/kg egg in corn oil prior to
incubation lipid peroxidation was significantly increased in the embryonic
liver and adipose tissue simultaneously with significant decreases in GSHPx activity in these tissues (Jin et al., 2001). Another pollutant dioxin also
caused dose-dependent increases in the production of superoxide anion,
lipid peroxidation and DNA single-strand breaks rat liver and brain (Hassoun
et al., 2001).
Oxidized fat in the diet can cause oxidative stress in the intestine increasing
antioxidant requirement to prevent damages. For example the intake of
oxidized oil caused a growth depression and increased TBARS concentrations
in plasma and decreased concentrations of tocopherols, lutein, beta-carotene
and retinol in plasma and tissues of broilers (Enberg et al., 1996).
Furthermore, toxic products of lipid peroxidation may damage the brush
border membrane in the intestine (Kimura et al., 1984) decreasing absorption
of antioxidants. When a chicken diet includes spent fat after its high
temperature treatment, the fat usually contains peroxides and hydroperoxides
which can contribute substantially to oxidative stress. Therefore it is
necessary to evaluate a benefit and disadvantages of using such fat sources.
Extensive preventive medication (coccidiostats or other veterinary drugs in

the diet) can decrease antioxidant assimilation from the diet or increase
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32

their requirement to deal with generated stress conditions. For example,
monensin can stimulate lipid peroxidation in the chicken liver (Salyi et al.,
1990). Similarly, oral furazolidone treatment of chickens was associated
with a decreased vitamin E concentration and increased lipid peroxidation
in their liver (Sas, 1993). Other drugs, for example allopurinol, can also
cause an oxidative stress in broilers and decrease their body weight (Klandorf
et al., 2001).
Vitamin A excess in the diet is shown (Surai et al., 1998; 2000) to cause an
oxidative stress decreasing vitamin E and carotenoid concentrations in
tissues and increasing tissue susceptibility to lipid peroxidation.
The list of potential stresses can vary from one poultry farm to another, but
overproduction of free radicals and the critical need for antioxidant protection are
common factors. It is interesting, that in wild, birds are often exposed to
electromagnetic fields, which cause oxidative stress and suppressed plasma total
protein, hematocrit and carotenoids in kestrels (Fernie and Bird, 2001). Similar
stresses can be attributed to farm animal production. Some potential stressconditions, related to the production of free radicals in the digestive tract of human
and animals are presented in Chapter 10.
Did you know that antioxidant-prooxidant balance in the cell
is an important determinant of various physiological functions?
Conclusions
Antioxidant-prooxidant balance in the cell is an important determinant of various
physiological functions. Indeed, oxidative stress occurs when this balance is
disturbed due to overproduction of free radicals or compromised antioxidant
defences. Free radical overproduction and oxidative stress are considered as a
pathobiochemical mechanism involved in the initiation or progression phase of
various diseases.. In animal production free radial generation and lipid peroxidation
are responsible for the development of various diseases as well as for the decrease
of animal productivity and product quality (Hurley and Doane, 1989; McDowell,
2000). Dietary antioxidants may be especially important in protecting against the
development of the oxidative stress.
In general, ingestion of excessive amounts of antioxidants is presumed to
shift the oxidant-antioxidant balance toward the antioxidant side. This is supposed
to be beneficial; however, this may also adversely affect key physiological
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processes that are dependent on free radicals, including prostaglandin production,

cell division and differentiation (Azzi and Stocker, 2000). Recent evidence suggests
that cellular oxidation can induce changes in gene expression during normal
development. Conversely, antioxidants such as ascorbate, glutathione, tocopherol or carotenoids are inhibitory to differentiation in many types of cells
(Allen and Venkatraj, 1992).
Did you know that free radicals have a pleasant face being
essentials for a regulation of various physiological processes?
Free radicals are now considered to take part in signal transduction in the cell and
at least two well-defined transcription factors, nuclear factor kB and activator
protein-1 have been identified to be regulated by the intracellular redox state (Sen
and Packer, 1996). The regulation of gene expression by oxidants, antioxidants,
and redox state has emerged as a novel subdiscipline in molecular biology that
has promising implications for the feed industry and animal production. Thus the
redox state of the cell, which reflects antioxidant/prooxidant balance, can be
considered as an important element of gene regulation (Bowie and ONeill, 2000).
Therefore the effect of antioxidants on animal health is much deeper then one
could expect several years ago. The mechanisms by which natural antioxidants
act at the molecular and cellular level include roles in gene expression and
regulation, apoptosis, and signal transduction (Frei, 1999) and antioxidants are
involved in fundamental metabolic and homeostatic processes. However, there
are still many gaps in our knowledge of the basic molecular mechanisms of
oxidative damage and antioxidant defences (Frei, 1999).
Indeed, a range selenoproteins (see chapter 2) is involved in a regulation of
key elements of antioxidant defences. By maintaining optimal activities of such
antioxidant enzymes as GSH-Px, TR and MsrB as well as directly participating in
regulation of DNA-repair enzymes Se belongs to all three major levels of
antioxidant defences. Furthermore, Se also interacts with other antioxidants such
as vitamin E, ascorbic acid, glutathione and some others. That is why I would call
selenium chief executive of antioxidant defences. In the next chapters
information will be presented proving that Se is a key to effective antioxidant
defences and to maintaining animal and human health. Indeed Diet cures more
than lancet
Did you know that selenium is a chief executive of antioxidant
system responsible for regulation of most important antioxidant
defences in the body?
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Molecular Mechanisms of Se Action: Selenoproteins 45

2
MOLECULAR MECHANISMS OF SE ACTION: SELENOPROTEINS
A chain is no stronger than its weakest link
Introduction
It is generally accepted that in biological systems Se participates in various
physiological functions as an integral part of a range selenoproteins. In fact, it is
well known that sulphur and selenium occur in proteins as constituents of the
amino acids cysteine, methionine and selenocysteine, and selenomethionine,
respectively. The redox activity of those amino acids under physiological conditions
allows a wide variety of posttranslational protein modifications, metal free redox
pathways, and unusual chalcogen redox states (Jacob et al., 2003). For example,
unlike any other amino acid, cysteine and SeCys can participate in several distinct
redox pathways, including exchange and radical reactions, as well as atom-,
electron-, and hydride-transfer reactions. Furthermore, the position of selenium
in the periodic table between the metals and the non-metals makes selenoproteins
effective catalysts for many biological redox transformations (Jacob et al., 2003).
Selenoprotein family
The human selenoproteome consists of 25 selenoproteins (Kryukov et al., 2003;
Table 2.1) and about 35 Se-containing proteins or protein subunits can be distinguished
by two-dimensional electrophoresis after in vivo labelling with 75Se (Behne at al.,
2000; Table 2.2). Recently, 24 Se-containing proteins have been found in subcellular
fractions of normal human liver (Chen et al., 2002). The molecular weights of the
subunits were mostly in the range 20-30 kDa and 50-80 kDa. In accordance with
another estimation the number of selenoproteins in mammals could reach 30-50
(Kohrle et al, 2000). There is also a range of prokaryotic selenoproteins (Table 2.3).
Did you know that there are at least 25 selenoproteins
in the human body?
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Table 2.1 Characteristics of human selenoproteins (adapted from Kryukov, 2003)
Protein
Protein length
SeCys position
Cytosolic GSH-Px (GSH-Px1)

GI-GSH-Px (GSH-Px2)
p-GSH-Px (GSH-Px3)
PH-GSH-Px (GSH-Px4)
GSH-Px6
Cytosolic TR1
TR2 expressed in testis
Mitochondrial TR3
Iodothironine deiodinase I (ID1)
Iodothironine deiodinase II
Iodothironine deiodinase III
Selenoprotein H (SelH)
Selenoprotein I (SelI)
Selenoprotein K (SelK)
Selenoprotein M (SelM)
Selenoprotein N (SelN)
Selenoprotein O (SelO)
Selenoprotein P (SelP)
201
190
226
197
221
499
656
523
249
265
278
122
397
94
145
556
669
381
Selenoprotein R (SelR)
Selenoprotein S (SelS)
SPS2
Selenoprotein T (SelT)
Selenoprotein V (SelT)
Selenoprotein W (SelW)
15-kDa Selenoprotein (Sel15)
116
189
448
182
346
87
162
47
40
73
73
73
498
655
522
126
133
144
44
387
92
48
428
667
59, 300,318,330,345,
352,367,369,376,378
95
188
60
36
273
13
93
Table 2.2 Other eukaryotic selenoproteins (adapted from Kryukov and Gladyshev, 2002; Behne and
Kyriakopoulos, 2001)
Protein
Selenoprotein X3 (SelX)
Selenoprotein Z3 (SelZf1 and SelZf2)
Selenoprotein Pb1 (SelPb)
Selenoprotein W21 ) (SelW2)
Selenoprotein U4(SelU)
Selenoprotein T21 (SelT2)
Selenoprotein G-rich2 (SelG-rich)
Selenoprotein BthD2 (SelBthD)
18-kDa Selenoprotein5 (Sel18)
Protein length
SeCys position
265
94
165
109
249
162
64
13
19
110
37
93
Zebrafish selenoproteins, 2Drosophila melanogaster selenoproteins; 3Lescure et al. (1999);

Castellano et al. (2004), chicken and fish; 5rats
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Table 2.3 Selenoproteins in Prokaryotes (adapted from Kohrle et al., 2000)
Selenoprotein
Function
Glycine reductase
Glycine/sarcosine/betaine reductase
Selenoprotein A
Glycine reductase selenoprotein B
Sarcosine reductase selenoprotein B
Betaine reductase selenoprotein B
Proline reductase
Heterodisulfide reductase
Seleno-peroxiredoxin
Putative redox active selenoprotein
Formate dehydrogenase
Formylmethanofuran dehydrogenase
NiFeSe-hydrogenase
F420 non-reducing hydrogenase
F420 reducing hydrogenase
Selenophosphate synthetase
Formation of a selenoether
Redox function
Transfer of selenoether
Formation of selenoether
Redox function, formation of selenoether
Redox function
Redox function (peroxidase)
Redox function
Hydrogen donor
Redox function
Hydrogen donor
Redox function
Redox function
Formation of key metabolite for selenoprotein
synthesis
Formation of a carbon oxide selenide
Unknown
Unknown
CO dehydrogenase
Nicotinic acid hydroxylase
Xanthine dehydrogenase
The mammalian Se-containing selenoproteins are divided into three groups (Figure
2.1; Behne and Kyriakopoulos, 2001).
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1.
2.
3.
Proteins with non-specific incorporation of selenium

Specific selenium-binding proteins
Specific proteins containing Se in the form of genetically encoded
selenocysteine
When SeMet is available in the diet, non-specific incorporation of Se-Met

in place of methionine in various proteins is a way of preserving Se for
future use in the body. For example, these Se reserves can be used in stress
conditions, when requirement for antioxidant defence and Se is increased,
but food/feed consumption declined. The Se reserves can be built in muscles,
for example, and rate of protein turnover and amount of methionine residues
in proteins would determine the size of such a reserve. The selenium in the
form of SeMet becomes available for selenoprotein synthesis when those
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48

selenoproteins are catabolised in proteasomes and SeMet is catabolised to
H 2Se (Schrauzer, 2003). Furthermore, methionine deficient diet could
increase SeMet accumulation in tissues redirecting it from being used for
conversion to SeCys and further incorporation to the specific SeCys
containing selenoproteins.
Non-specific
incorporation
(Se-Met)
Se-Met
Se-Cys
Intermediary
Se pool
Specific
incorporation
(Se-Cys)
Selenate
Selenite
Specific
binding
Figure 2.1 Incorporation of Se into proteins (Adapted from Behne and Kyriakopoulos, 2001).
Some specific Se-binding proteins have been characterised where Se is not

incorporated but rather attached to the protein molecules including 14-kDa,
17-kDa and 56-kDa proteins in mouse (Behne and Ryriakopoulos, 2001).
The main effects of Se in animals including humans are due to specific
SeCys-containing selenoproteins. In fact, SeCys is considered to be the 21st
amino acid and understanding of selenoprotein synthesis substantially helped
in understanding of the genetic code. For example, when the code was
described in the middle 1960s, 20 amino acids were considered to be
incorporated in newly synthesised proteins. There were 64 possible codons
from which 3 codons were design to function as terminators for protein
synthesis. To a great surprise of many scientists it was found that UGA
serves as both a terminal codon and a SeCys codon (for details see Hatfield
and Gladyshev, 2002). Therefore for the incorporation of SeCys into protein,
the UGA codon is transformed from one that signals translation termination
to one specific for SeCys. The dual role of the UGA codon confounds the
identification of novel selenoprotein genes.
Synthesis of selenoproteins is associated with insertion SeCys into the
primary protein structure during translation (Burk, 2001) and SeCys
incorporation at UGA codons requires cis-acting mRNA secondary
structures and several specialized trans-acting factors. The latter include a
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selenocysteine-specific tRNA, an elongation factor specific for this tRNA
and a SECIS-binding protein, SBP2, which recruits the elongation factor to
the selenoprotein mRNA (Low et al., 2000). It is interesting that presence
of SeCys, rather than cysteine, in active site of selenoproteins increases
their enzymatic activity 100- to 1,000-fold (Burk, 2002).
Expression of individual eukaryotic selenoproteins is characterised by
high tissue specificity, depends on Se availability, can be regulated by
hormones, and if compromised contributes to various pathological conditions
(Kohrle et al., 2000). Selenoprotein synthesis includes (Lescure et al., 2002):
Charging of serine onto a specialized tRNA, tRNASec
Selenocysteine synthase catalyses the seryl- to selenocysteyl-conversion
on the tRNASec
SeCys cotranslationally incorporated into selenoprotein active centers
As mentioned above, the unusual feature of selenoprotein synthesis is that
selenocysteine insertion is specified by the stop UGA codon (Patching and
Gardiner, 1999). Most selenoprotein mRNAs (but not Selenoprotein P) contain a
single UGA codon encoding a single SeCys residue per polypeptide chain and a
single specific RNA secondary structure, termed a selenocysteine insertion
sequence (SECIS) element, which directs incorporation of SeCys (Chen and Berry,
2003). Therefore the SECIS element is an RNA hairpin in the 3UTR of
selenoprotein mRNAs required for decoding UGA selenocysteine codons.
Experimentally derived 2D structure model for the SECIS RNA revealed the
conservation of four consecutive non-Watson-Crick base pairs, with a central
G.A/A.G tandem (Walczak et al., 1998). The authors concluded that the SECIS
RNA is another member to be added to the growing list of RNAs containing
building blocks of non-Watson-Crick base pairs, required for structure and/or
function. In eukariotes, SeCys incorporation requires both an elongation factor
(eEFSeCys) and a separate SECIS binding protein (SPB2; Copeland, 2003). The
author suggested a model of SeCys incorporation into proteins in which SBP2/
SECIS complex interacts with the eEFSeCys/SeCys-tRNA complex to deliver SeCys
to SECIS containing mRNA. The discovery of the SECIS RNA binding protein
SBP2, which is not a translation factor, was an important step for the subsequent
isolation of mSelB/EFSec, the mammalian homolog of SelB and further
understanding selenoprotein synthesis mechanisms (Lescure et al., 2002). It has
been shown that SBP2 preferentially stimulates incorporation directed by the
selenoprotein P and PH-GSH-Px SECIS elements over those of other selenoproteins
(Low et al., 2000). Selenoprotein genes were completely sequenced in a range of
eukaryotic genomes (Table 2.4).
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50
Table 2.4 Selenoprotein genes in completely sequenced eukaryotic genomes (Adapted from Kryukov et
al., 2003)
Organism
Genome size
Homo sapiens
Mus musculus
Drosophila melangaster
Caenorhabditis elegans
Arabidopsis thaliana
Saccharomyces cerevisiae
3,400,000,000
3,454,200,000
137,000,000
97,000,000
100,000,000
12,067,280
Estimated number
Number of
of genes
selenoprotein genes
40,000
40,000
14,331
20,206
25,000
6,312
25
24
3
1
0
0
In general, there is a strong hierarchy in selenoprotein expression in various tissues

with brain being more conservative in terms of Se manipulation than other tissues.
The brain is followed by spinal marrow, pituitary, thyroid, ovaries and adrenals (Behne
and Kyriakopoulos, 2001). Furthermore, inside selenoprotein family there is also a
hierarchy in selenoprotein expression in various conditions, including Se deficiency.
In fact, the GI-GSH-Px and PH-GSH-Px are considered to be most preferentially
supplied with the Se in comparison to other members of GSH-Px family. This kind of
regulation has an important implication for the development of Se deficiency signs.
In fact, those selenoproteins which are less affected by Se-deficiency are considered
to be more physiologically important than others (Flohe and Brigelius-Flohe, 2002).
All known up to date specific selenoproteins can be divided into several distinct
groups depending on the location and functional properties of SeCys (Tapiero et al.,
2003):
GSH-Px group includes proteins in which SeCys is located in the N-terminal

portion of a functional domain. These selenoproteins contain a redox-active
SeCys residue that is separated from a conserved Cys by two other amino
acids. During catalytic reaction SeCys is oxidized to selenenic acid or forms
selenosulfide bonds. This group includes GSH-Px, BthD and selenoproteins
W, W2, T, T2, P and Pb (Kryukov and Gladyshev, 2002).
TR group is characterised by the presence of SeCys in C-terminal sequences
and includes TR and Drosophilae G-rich protein.
Third group includes other selenoproteins including three deiodinases, SeR,
SeN, SP2 and 15-kDa protein. In these selenoproteins SeCys and Cys are
separated by one amino acid.
Glutathione peroxidase (GSH-Px) and thioredoxin reductase (TR) are the most
abundant antioxidant Se-containing proteins in mammals (Gladyshev et al., 1998).
Major characteristics of GSH-Px and TR are shown in Table 2.5 and Figure 2.2.
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NADPH
Oxidixed ascorbate
NADP+
Reduced Thioredoxin
Reduced ascorbate
Oxidized Thioredoxin
Cell growth
Inhibited apoptosis
Thioredoxin
peroxidase
Ribonucleotide
reductase
Transcription
factors
Cellular sensitivity to
glucocorticoids
Immunomodulation
Pregnancy and birth
Antioxidant
DNA synthesis
Gene transcription
Neuronal survival
Figure 2.2 Thioredoxin reductase functions (Adapted from Mastacich and Powis, 2000; Arner and
Holmgren. 2000;Makino et al., 1996)
Glutathione peroxidase family includes several members from which there are at
least 6 different Se-dependent enzymes. They differ in molecular weight, substrate
specificity, cell distribution and perform a variety of functions.
Did you know that there are six Se-dependent glutathione
peroxidases?
CYTOSOLIC GLUTATHIONE PEROXIDASE (cGSH-PX OR GSH-Px1)
Glutathione peroxidase (glutathione H 2O 2 oxidoreductase E.C. 1.11.1.9) was
discovered by Mills in 1957, who showed that this enzyme had a protective effect
in erythrocytes against H2O2- or ascorbate-induced hemolysis. Sixteen years later
it became clear that GSH-Px was a selenoenzyme (Rotruck et al., 1973; Flohe et
al., 1973). In fact, Rotruck et al. (1973) were the first to show that in rat red cells
Se was tightly bound to the enzyme and demonstrated the uptake of 75Se by GSHPx (Ahrens, 1996). In the same year Flohe et al. (1973) reported that bovine
GSH-Px contained one Se atom per subunit. It is interesting that 1973 was a year
of Se discoveries, since in the same year selenoproteins have been described in
micro-organisms.
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GSH-Px5
Epididymal
GSH-Px
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Epididymal fluid,
epididymis
25.0
25.2
22.1
25.5
21.9
21.9
H2O2, organic
hydroperoxides,
Phospholipid
Hydroperoxides
Low activity
towards H2O2 and
organic hydroperoxides
GSH
GSH
GSH, DTT, 2-ME, Renal epithelial

L-Cys
cells and testes
H2O2, phospholipid hydroperoxides
Bovine ciliary body,

rat olfactory mucosa,
human and mouse
liver
Caput epididymis
Expressed in kidney
GSH,
thioredoxin,
glutaredoxin
H2O2, t-BHP,
phospholipid
hydroperoxides
Mucosal epithelial
cells in GIT
Erythrocytes,
kidney and liver
GSH
GSH
H2O2, t-BHP
H2O2, t-BHP
t-BHP- tret-Butylhydroperoxide; DTT- 1,4-Ditiothreitol; 2-ME 2 mercaptoethanol; L-Cys- L-Cysteine;
NonSeGSH-Px
GSH-Px4
Phospholipid
hydroperoxide
GSH-Px
Non-selenium
GSH-Px
Plasma
GSH-Px3
Extracellular
(plasma)
GSH-Px
Intracellular,
partly cytosolic,
partly mitochondrial, partly
membrane-bound
Intracellular,
cytosolic
Gastrointestinal GSH-Px2
GSH-Px
Intracellular,
cytosolic, partly
mitochondria
Other
characteristics
GSH-Px1
Electron
donors
Cytosolic
GSH-Px
Substrates
Nomenclature
Glutathione
peroxidase
GSH-Px
Se
Table 2.5 Glutathione peroxidase characteristics (Adapted from Hayes and McLellan, 1999; Singh and Shichi, 1998; Schwaab et al., 1998)
Subunit
size (kDa)
Localization
52

GSH-Px is responsible for detoxification of hydroperoxides and hydrogen
peroxide in the following reactions:
GSH-Px
ROOH + 2GSH
H2O2 + 2GSH
ROH +GSSG+H2O
GSSG + 2H2O
GSH-Px
These reactions employ a ping-pong mechanism. In particular, SeCys in the active

centre of the enzyme is oxidized with a selenenic acid formation, which is reduced
back by a reaction with 2 molecules of GSH (Figure 2.3). The Se atom in the
enzyme catalytic site undergoes a redox cycle involving the selenolate anion as
the active form which reduces H2O2 and organic peroxides (Mugesh and Singh,
2000). The selenolate, is oxidised to selenenic acid, which reacts with GSH to
form a selenosulfide adduct with the following reaction with GSH to regenerate
the active form of the enzyme.
ONOO-
ROOH
ROH
ONOE-Se-OH
E-Se-H
GSSG
GSH
GSH
H 2O
E-Se-SG
Figure 2.3 Catalytic mechanisms of GSH-Px (Adapted from Mugesah and Singh, 2000; Sies et al., 1997)
GSH-Px detoxifies peroxynitrite in the following reaction (Sies et al., 1997):

ONOO- + 2GSH
GSH-Px
ONO- +GSSG+H2O
Glutathione reductase (GR) is responsible for a reduction of oxidized glutathione

back to a reduced form:
GSSG + NADPH +H+
GR
2GSH + NADP+
GSH-Px is characterised by high specificity for GSH as a donor of a reducing

equivalent (substrate) and catalyses the reduction of a variety of hydroperoxides
(Tables 2.6-2.7). However, its activity is related only to free peroxides and it is not
able to reduce esterified fatty acid hydroperoxides. Therefore, in biological system
hydroperoxides in membranes have to be released by other enzymatic systems (e.g.
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phospholipases) or another member of GSH-Px (PH-GSH-Px) can deal with them.

General characteristics of various GSH-Px are shown in Table 2.5 and molecular
weights of GSH-Px from various species are presented in Table 2.8.
Table 2.6 Acceptor substrates for GSH-Px (adapted from Schamberger, 1983)
Substrate
Substrate
H2O2
Allopregnanolone 17-hydroperoxide
Cholesterol 7-hydroperoxide
Cholesteryl hydroperoxyoctadecadienoates
Cumene hydroperoxide
Ethyl hydroperoxide
Ethyl hydroperoxyacetadecatrienoate
Glyceryl 1-hydroxyoctadecadianoates
15-Hydroperoxyprostaglandine E1
1-linoleoyl lysophosphatidylcholine
hydroperoxide2
Linoleic acid hydroperoxide

Linolenic acid hydroperoxide
Methyl hydroperoxyoctadecadienoates
Peroxidized DNA
Pregnenolone 17-hydroperoxide
Prostaglandin G2
Tret-butyl hydroperoxide
Thymine hydroperoxide
Peptide peroxides1
Hydroperoxides of gamma linolenic, 11,14
eicosodienoic, homo gamma linolenic,
arachidonic, docosotetraenoic and
docosohexaenoic acids3
Peroxidized insulin and valine5
p-menthane hydroperoxide, diisopropylbenzene hydroperoxide, ethyl

hydroperoxide4
Peroxinitrite6
1
Morgan et al., 2004; 2Marinho et al., 1997; 3Kaplan and Ansari, 1984; 4Chaudiere and
Tappel, 1983; 5Gebicki et al., 2002; 6Sies et al., 1997
Table 2.7 Specificity of the antioxidant enzymes to peroxide substrates (as initial rate of reaction), mol/
min/mg protein (Adapted from Brigelius-Flohe et al., 2002)
Enzyme
cGSH-Px
eGSH-Px
PH-GSH-Px
SeP
t-BuOOH
H2O2
PC-OOH1
PC-OOH2
PC-OOH3
552
199
9.56
<0.02
793
373
34.8
<0.02
<0.02
54.1
2.65
<0.02
<0.02
0.03
64.8
1.11
<0.02
0.18
28.8
1.15
/7 mM sodium deoxycholate; 2/0.1% Triton X100 and 0.24 mm sodium depxycholate; 3/

0.025% Triton and 0.3 mM sodium deoxycholate.
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Table 2.8 Molecular weight of GSH-Px purified from various species
Species
Tissue
Native enzyme molecular weight
Rat*
Trout*
Japanese sea bass1
Black sea bass2
Liver
Lung
Erythrocytes
Lens
Erythrocytes
Liver
Aorta
Erythrocytes
Placenta
Liver
Liver
Liver
76,000
84,000
83,800-85,000
96.600
88,000-89,000
77,000-80,000
84,000
95,000-100,000
85,500
95,000
70,000
72,000
Human3
Placenta
85,000
Human4
Hamster5
Rabbit6
Human7
Human8
Rat9
Bovine10
Human11
Plasma
Liver
Lung macrophages
Platelet
Milk
Lung
Erythrocytes
Erythrocytes
92,000
92,000
80,000
92,000
92,000
80,000
83,800
95,000
Cow*
Sheep*
Pig*
Human*
*Adapted from Combs and Combs, 1986; 1Nagai et al., 2002; 2Braddon-Galloway and
Balthrop, 1985;3Awasthi et al., 1979; 4Broderick et al., 1987; 5Chaudiere and Tappel, 1983;
6
Chiba et al., 1999; 7Rey et al., 1994; 8Bhattacharya et al, 1988; 9Chiu et al., 1976; 11Flohe
et al., 1971;12 Awasthi et al., 1975;
GSH-Px activity is dependent on Se status of tissues. In fact, dietary Se

supplementation has been shown to be effective in increasing GSH-Px in a variety
of animal species including rat, mouse, chicken, quail, sheep, cattle, horse, pig,
deer, salmon, etc. (Brigelius-Flohe, 1999). On the other hand, there is a range of
nutritional means of decreasing GSH-Px activities in various tissues including
vitamin E excess, deficiencies of iron, zinc, riboflavin, vitamin B6 or copper as
well as consumption of silver, tri-o-cresyl phosphate or doxorubicin (Selenium in
Nutrition, 1983; Gomez et al., 2003). In fact, depending on concentration and
duration of exposure various chemical elements and compounds can either
decrease or increase GSH-Px activity in tissues. When Se is available, increased
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GSH-Px activity could be a compensatory mechanism to deal with stress conditions

and an oxygen-responsive element was identified in the 5I-flanking region of the
cGSH-Px gene (Flohe and Brigelius-Flohe, 2001).
Negative effectors of GSH-Px activity
High oxidative stress caused by various compounds could be responsible for

decreased GSH-Px activity in various tissues. For example, high ozone
concentrations were detrimental for GSH-Px activity (Lee et al., 2003). Exposure
to 605 mm Hg O2 for 48 h was inhibitory to GSH-Px activity in a cell culture
(Allen and Balin, 2003). Cd-treated cells (hepatic stellate cell line) increased lipid
peroxidation and decreased GSH-Px activity (del Carmen et al., 2002). Mercury
vapour exposure had no significant effects on GSH levels or GSH-Px activity in
the three brain regions, however, statistically significant decreases in GSH and
GSH-Px activity were detected in the kidneys of rats exposed to 2 mg/m3 (Goering
et al., 2002). Aluminium phosphide caused a significant decrease in myocardial
GSH-Px levels in rats (Azad et al., 2001). Nickel sulphate administration also
significantly increased the level of lipid peroxides and decreased GSH-Px activities
in rat liver (Das et al., 2001). The effect of various doses of sodium tellurite (0.4,
0.8, and 2.0 mg/kg body weight, orally) on the activity of antioxidant enzymes
(GSH-Px, glutathione reductase, glutathione-S-transferase, and catalase) and
content of GSH and TBARS in the cerebrum, cerebellum, and brainstem of male
albino mice was studied after 15 d of treatment. The consumption of sodium
tellurite (0.8-mg/kg, orally for 15 days) significantly decreased the activities of
GSH-Px in the cerebrum and brainstem of rats (Kaur et al., 2003). In rabbit liver
GSH-Px activity was significantly decreased by halothane anaesthesia
(Karamanlioglu et al., 2002). Chronic ingestion of ethanol resulted in a significant
decrease in GSH-Px activity in rat liver and kidney (Husain et al., 2001).
Rats were treated by gavage with a low dose (12.5 mg/kg body weight per
day) of cypermethrin in corn oil for 60 days. The increased oxidative stress
resulted in a significant decrease in the activity of GSH-Px in rat erythrocytes
(Gabbianelli et al., 2002). Treatment of mice with the different doses of
thymoquinone (25, 50 and 100 mg/kg/day orally) for 5 successive days, produced
significant reductions in hepatic SOD, CAT and GSH-Px activities (Mansour et
al., 2002). The potential mammary carcinogen 7,12-dimethylbenz(a)anthracene
was injected intraperitoneally (20 mg/kg/day) into 10-12 month old female mice.
After 21 days of application the mice were sacrificed and GSH-Px and Se levels
of liver homogenates were measured. Se and GSH-Px levels in 7,12-DMBA-treated
mice were significantly lower than those of controls (Batcioglu et al., 2002).
Subchronic doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin administered orally to
male Wistar strain rats for 45 days caused a significant decrease in GSH-Px activity
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in epididymal sperm (Latchoumycandane et al., 2002). 2,3,7,8tetrachlorodibenzo-p-dioxin injected into chicken eggs prior to incubation caused
a decrease in GSH-Px in the liver of newly hatched chicks (Hilscherova et al.,
2003). A decline in the activity of GSH-Px was observed in the small intestine of
streptozotocin-induced diabetic rats (Bhor et al., 2004). Nodularin (a hepatotoxin
from a cyanobacterium, Nodularia spumigena), administration (1, 5, and 10 g/
kg body weight) for 7 days decreased the activities of GSH-Px in the mouse liver
in a dose-dependent manner (Lankoff et al., 2002). Cisplatin-treated rats showed
a reduction in the kidney GSH-Px (Saad and Al-Rikabi, 2002). Significant dosedependent decreases in heart tissue selenium concentrations and Se-dependent
GSH-Px activity were observed in chronically iron-loaded mice in comparison
with placebo controls (Bartfay and Bartfay, 2002). In methoxychlor-incubated
goat sperm the activity of superoxide dismutase, glutathione reductase and GSHPx were decreased while lipid peroxidation was increased in a dose-dependent
manner (Gangadharan et al., 2001).
It is interesting that excessive consumption of some non-toxic compounds
could decrease GSH-Px activity. For example, -carotene supplementation (30
mg/day for 60 days) of the healthy adults was associated with a decreased GSHPx activity in their serum (McGill et al., 2003). GSH-Px activity was lower in the
erythrocytes of rats fed the fish oil-supplemented diet (Miret et al, 2003). The
products derived from lipid peroxidation were also higher in the FO groups. An
excessive vitamin E intake, when combined with salmon oil in the diet, lowers
the activity of GSH-Px in rat erythrocytes without affecting in vivo hemolysis
(Eder et al., 2002). High cholesterol or high-fat feeding rats led to markedly
decreased activities of hepatic GSH-Px (Lu and Chiang, 2001). Chronic proline
administration to rats provoked a significant decrease of GSH-Px activity in rat
brain (Delwing et al., 2003). A treatment of rats with growth hormone was also
associated with a decreased GSH-Px activity in liver and kidney (Brown-Borg
and Rakoczy, 2003).
Upregulation of GSH-Px
Chronic cold exposure resulted in a significant increase in GSH-Px activity in the

brain and intestine of stressed rats (Kaushik and Kaur, 2003). The results of various
studies showed that GSH-Px activity in kidney decreased with age in ad libitumfed rats, while calorie restricted rats consistently showed resistance to decreases
in this activity (Cho et al., 2003). The activities of GSH-Px in liver was elevated
significantly after ethanol administration to rats for durations of 2, 4 and 8 weeks
(Pathak et al., 2002). There was a significant increase in the activities of pulmonary
GSH-Px in rats exposed to cigarette smoke for 4, 6 or 8 weeks (Koul et al., 2001).
Increased activities of GSH-PX were found in heart and kidney of rats after CCl4
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intoxication (Szymonik-Lesiuk et al., 2003). In rat primary Leydig cells exposed

to cadmium the activity of GSH-Px was significantly increased (Yang et al., 2003).
Acute arsenic exposure significantly increased the GSH-Px activity in rat liver
(Maiti and Chatterjee, 2001). When male albino rats were treated with
sterigmatocystin-contaminated diet for 30 days the activity of GSH-Px in the liver
was increased (Sivakumar et al., 2001). When compared to a pair-fed control
group, d-amphetamine (a potential brain neurotoxic agent) treatment enhanced
the activity of GST in hypothalamus to 167%, GSH-Px in striatum to 127%, in
nucleus accumbens to 192%, and in medial prefrontal cortex to 139% (Carvalho
et al., 2001). Treatment of diabetic rats with melatonin increased brain and kidney
GSH-Px activity to the levels below that of control rats. Vitamin E was found to be
less effective on GSH-Px activity levels in brain and kidney than melatonin whereas
it was more potent than melatonin in liver (Baydas et al., 2002). Wistar-albino
rats fed a diet supplemented with cholesterol plus cholic acid and methionine for
six months. This diet was found to increase lipid peroxide levels in the liver of
rats. However, hepatic GSH-Px and catalase activities increased (Dogru-Abbasoglu
et al., 2002). Naringin, a citrus bioflavonoid, plays an important role in regulating
antioxidative capacities by increasing the SOD and catalase activities, up-regulating
the gene expressions of SOD, catalase, and GSH-Px in the liver, and protecting
the plasma in vitamin E high cholesterol-fed rabbits (Jeon et al., 2001). Blood
GSH-Px increased significantly in -carotene supplemented rats after hypoxiainduced stress (Sarada et al., 2002).
Gender-specific expression of GSH-Px
It is interesting that there is a gender-specific difference in GSH-Px activity in

various tissues. For example, throughout ageing, gender-related difference in GSHPx activity in mouse brain was observed, with higher level in females comparing
to males (Sobocanec et al., 2003). In young, healthy but not physically trained
subjects participated in a study, females had significantly higher serum GSH-Px3
concentration and serum GSH-Px activity than males; specific activity (U/mg)
was not different between genders (Rush and Sandiford, 2003). For both groups
(physically active and sedentary rats), liver GSH-Px activities in females were
significantly higher than in males (Yamamoto et al., 2002). Therefore, it seems
likely that GSH-Px is upregulated by estrogens (Flohe and Brigelius-Flohe, 2001).
Tissue-specific expression of GSH-Px
GSH-Px activity was shown to have species- and tissue-specificity. For example,
Chiaradia et al. (2002) determined lymphocyte GSH-Px activity, plasmatic GSH
levels and the effect of H2O2 on the responsiveness of lymphocytes to proliferative
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stimuli. Among the three species considered, sheep presented the lowest plasmatic
GSH and the highest lymphocyte GSH-Px activity. On the contrary, dogs showed
an inverted pattern (high GSH low GSH-Px). Horses displayed intermediate
values for both parameters analysed. In particular, high rate of peroxide production
in such tissues as liver, kidney or lung is associated with high GSH-Px activity.
This enzyme has a wide cell distribution and present in mitochondria and cytosol.
In fact, in rat liver 75% of the enzyme was fiound in cytosol and 25% in mitocondria
(Flohe and Brigelius-Flohe, 2002). It is interesting that GSH-Px was also detected
in peroxisomes (Singh et al., 1994). The ranking order of tissue-specific stability
of cGSH-Px is as follows (Brigelius-Flohe,1999): brain>>thymus>
thyroid>heart>liver, kidney, lung. In Japanese monkey (Macaca fuscata) tissues
GSH-Px activity was shown to be high in the liver, kidney, and adrenal gland
followed by the small intestine (Fukuhara and Kageyama, 2003). Expression of
GSH-Px-1 mRNA was found to be high in the liver, kidney, and adrenal gland, in
consistence with the tissue distribution of GSH-Px-1 activity.
Response to dietary selenium
GSH-Px activity is dependent of Se dietary supply. For example, compared with

Se-adequate rats, rats fed the basal amino acid diet retained only 1, 6, 4 and 9% of
the Se-adequate GSH-Px activity in liver, heart, kidney and lung respectively (Lei
et al., 1995). A highly significant correlation was found between Se-GSH-Px
activity and selenium concentration in selected rat tissues (r = 0.91; Gromadzinska
et al., 1988). GSH-Px has been purified from the tissues of cattle, humans, swine,
sheep and rats (Selenium in Nutrition, 1983) and was shown to be a tetrameric
protein with four identical subunits each with a molecular weight of ~23,000 Da
and each containing one Se atom (Sunde, 1993). In fact, there are species- and
tissue-specific differences in molecular weight of the enzyme (Table 2.8). The
enzyme has been cloned by many investigators from various tissues (liver, kidney,
placenta, pituitary, heart, blastocyst, epididimys, etc.) in humans, mice, rats,
bovines, rabbits, pigs and monkeys (Sunde, 1993). Amino acid analysis determined
that selenocysteine, identified as its carboxymethyl derivative, was the only form
of selenium (Chaudiere and Tappel, 1983). One residue of cysteine was found to
be present in each glutathione peroxidase subunit. The presence of tryptophan
was colorimetrically determined. An optimal pH of 8.0 at 37 degrees C and an
activation energy of 3 kcal/mol at pH 7.6 were found. A ter-uni-ping-pong
mechanism was shown by the use of an integrated-rate equation (Chaudiere and
Tappel, 1983). It was also demonstrated the amino acid composition of rat liver
GSH-Px with each subunit containing two cysteine and three methionine residues
out of total 153 amino acids (Selenium in Nutrition, 1983). Unlike the GSH-Px4
gene which encodes PH-GSH-Px with alternative transcription and translation
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start sites determining the subcellular localization of PH-GSH-Px, the GSH-Px1

gene appears to have a single translation start site and mitochondrial GSH-Px is
the product of the GSH-Px1 gene (Esworthy et al., 1997).
In liver, transcription of genes for cellular glutathione peroxidase, type I
iodothyronine 5'-deiodinase, and selenoprotein P was unaffected by selenium
intake (0.1 or 2.0 mg of selenium/kg of diet for 91 days; Christensen et al., 1995).
Steady-state levels of mRNA for glutathione peroxidase and selenoprotein P were
higher in liver than in kidney. For iodothyronine 5' deiodinase the opposite was
true. In liver, selenium deficiency reduced glutathione peroxidase mRNA by 89%
and virtually abolished enzyme activity. For iodothyronine 5' deiodinase, mRNA
and enzyme activity were reduced 69 and 70%, respectively. In kidney, selenium
deprivation decreased GSH-Px mRNA by 91% and reduced enzyme activity to
nearly zero. For iodothyronine 5'-deiodinase, decreases in mRNA and enzyme
activity were 19 and 62%, respectively. Reductions in selenoprotein P mRNA
were 50% in kidney but only 14% in liver (Christensen et al., 1995). Therefore,
GSH-Px activity ultimately depends on Se provision in the diet, however some
forms of GSH-Px are only synthesised when Se supply is optimal. There are
substantial differences among different forms of GSH-Px with regard to response
to Se deficiency (Brigelius-Floche, 1999). The selenoproteins retained in tissues
for longer periods during progressive Se deficiency are considered to have higher
physiological significance in comparison to those whose activities rapidly decline.
In this respect, the main GSH-Px forms rank as follows (Brigelius-Flohe, 1999):
GI-GSH-Px > PH-GSH-Px > Plasma GSH-Px = Cytosolic GSH-Px
Alterations of GSH-Px expression
To evaluate a role of GSH-Px in body metabolism two approaches have been

used namely over-expression of GSH-Px in various types of cells and animals
and GSH-Px knockout models. Therefore, a model of mice deficient in cellular
GSH-Px (GSH-Px knockout mice) was generated in various laboratories by genetargeting technology (Ho et al., 1997; Ho, 2002). It is interesting that alteration of
GSH-Px1 expression showed no impact on the expression of other selenoproteins
and antioxidant enzymes in unstressed mice. (Lei, 2001; Ho, 2002). To the surprise
of many scientists and to the disappointment of those who considered GSH-Px as
a wonder enzyme, the first experiments with GSH-Px knockout models showed
that the contribution of GSH-Px-1 to the cellular antioxidant mechanism under
normal animal development and physiological conditions is very limited. Indeed,
mice deficient in GSH-Px were apparently healthy. Their tissues exhibited neither
a retarded rate in consuming extracellular hydrogen peroxide nor an increased
content of protein carbonyl groups and lipid peroxidation compared with those
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of wild-type mice (Ho et al., 1997). Therefore these mice developed normally
and showed no marked pathologic changes under normal physiologic conditions
(Ho et al., 1998). Remarkably enough, a deficiency in these genes had no effects
on animal survival under hyperoxida. It was shown that kidney GSH-Px1 mRNA
levels and liver, kidney, lung, and testis total GSH-Px activities were affected by
the GSH-Px1 knockout and dietary Se concentrations (Cheng et al., 1998). In
contrast, kidney extracellular or plasma GSH-Px3 mRNA levels and PH-GSHPx4) activities in the four tissues were affected by only dietary Se concentrations.
Therefore, GSH-Px1 is expressed independently of GSH-Px3 or GSH-Px4 and
represents approximately 60% of the total hepatic Se in Se-adequate mice (Cheng
et al., 1997).
The gene knockout technique provides useful information in relation to possible
functions of GSH-Px (Table 2.9). One of the interesting model system includes
lenses from animals with lack of GSH-Px or with over-expression of this enzyme
incubated in a medium with H2O2. For example, following an exposure to H2O2,
lenses from normal animals showed some changes in the morphology of both the
epithelium and superficial cortex. The damage to these cells was much more
pronounced in lenses of GSH-Px knockout mice. In marked contrast, lenses of
transgenic mice with 5-fold increased activities of GSH-Px, were resistant to the
cytotoxic effects (Spector et al., 2001; 1998; Reddy et al, 1997). Furthermore,
similar changes were observed in relation to DNA damage. In fact, the extent of
DNA strand breaks was significantly decreased in H 2O 2-exposed lenses from
animals with GSH-Px overexpression as compared to normal lenses while DNA
damage in gene knockout lenses was 5 times greater than that of GSHPx-rich
transgenic lenses. In general, GSH-Px null mutant mice (GSH-Px1-/-) had elevated
H2O 2 as a consequence of the lack of its removal by GSH-Px1. For example,
fibroblasts derived from GSH-Px1 null mutant mice (Gpx1-/-) were characterised
by (de Haan et al., 2004):
reduced proliferative capacity and DNA synthesis

elevated levels of cell cycle inhibitory protein (Cip1);
increased NF-kappaB activation
morphological features of senescent cells
dose-dependent susceptibility to H2O2-induced apoptosis
It seems likely that GPX-1 provides protection against viral-induced damage in

vivo due to mutations in the viral genome of a benign virus. For example,
sequencing of viruses recovered from GSH-Px knockout-infected mice
demonstrated seven nucleotide changes in the viral genome, of which three
occurred at the G residue, the most easily oxidized base (Beck et al., 1998). No
changes were found in virus isolated from control-infected mice.
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Table 2.9 Comparison of GSH-Px knockout animals with wild type animals
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Effect
System
References
Reduced proliferative capacity and DNA

synthesis, elevated levels of cell cycle
inhibitory protein (Cip1), increased
NF-kappaB activation, increased
susceptibility to H2O2-induced apoptosis
Mice fibroblasts
De Haan et al., 2004
Increased cell-mediated LDL-oxidation

and oxLDL-induced apoptosis
Mice
Guo et al., 2001
Increased expression of genes encoding

inflammatory response, oxidative stress,
growth arrest and responses to DNA-damage
Galactosamine/
Li et al., 2003
endotoxin induced
toxicity in mice
Exacerbation of endothelial dysfunction
Hyperhomocysteinemic mice
Dayal et al., 2002
Marked sensitivity to numerous models of

oxidant tissue injury, but not hypersensitive
to hyperoxia
Mice
Ho, 2002
Enhanced susceptibility to secondary

necrosis or neutrophil-induced cell injury
Mice
Bajt et al., 2002
Increased age-related nuclear light

scattering, membrane damage and cataract
formation
Mice
Reddy et al., 2001
Increased 3-fold infract volume, increased

apoptosis, earlier activation of caspase-3
expression
Ischemia/
reperfusion mouse
model
Crack et al., 2001
Accelerated inflammatory response and

increased in brain cell death
Cold-induced
injury in mice
Flentjar et al., 2002
Increased loss of cell viability, high levels

of apoptotic cells, and severe DNA
fragmentation in diquat-treated animals
Mice
Fu et al., 2001a
Higher auditory brainstem response
Mice exposed to
Ohlemiller et al.,
2000
thresholds prior to noise exposure and

greater noise-induced hearing loss
broadband noise
Neurological disorders and enhanced

oxidative damage similar to those observed
in the patients with mitochondrial disease
Mice
62
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Table 2.9 Contd.
Effect
System
References
Less effective degradation of butyl

hydroperoxide by lenses which deteriorated
with age
Mice lenses
Spector et al., 2001;

1998
Increased levels of lipid peroxides in the liver,

increase mitochondrial susceptibility to H2O2,
decreased mitochondrial respiratory control
ratio and power output index
Mice
Esposito et al., 2000
Enhanced toxicity of N-methyl-4-phenyl1,2,3,6-tetrahydropyridine
Mice
Zhang et al., 2000
Increased vulnerability to malonate or

3-nitropropionic acid
Mice
Klivenyi et al., 2000
Decreased protection against lethality and

hepatic protein oxidation caused by diquat
Mice
Fu et al., 1999
Increased susceptibility to ischemia/reperfusion Mice

injury with increased apoptotic cell death
Maulik et al., 1999;

Ho et al., 1998;
Yoshida et al., 1997
Increased paraquat-induced pulmonary lipid

peroxidation and hepatic protein oxidation
Mice
Cheng et al., 1999a
Increased susceptibility of cortical neurons

to H2O2
Mice
De Haan et al., 1998
In general, studies of GSH-Px1 knockout mice lead De Haan et al. (2003) to

conclude that GSH-Px1 functions as the primary protection against acute oxidative
stress, particularly in neuropathological situations such as stroke and cold-induced
head trauma, where high levels of ROS occur during reperfusion or in response to
injury. The GSH-Px knock out model is not perfect, since it is unusual situation
when only one selenoprotein is affected. For example, in the case of Se-deficiency
a range of selenoproteins would be affected and therefore physiological changes
in the body would be quite different from those observed in this model system.
However, clearly, this model helped understanding a crucial role of classical GSHPx as an important antioxidant devise effective in various stress conditions.
A review of results based on over-expression GSH-Px is shown in Table 2.10.
It is clear that overexpression of GSH-Px is associated with an increased protection
against oxidative stress created as a result of various environmental or nutritional
manipulations. Indeed, GSH-Px is well regulated enzymes and its increased activity
can be considered as an additional protective mechanism in stress conditions. It
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64
is important to mention that GSH-Px overexpression approach has some drawbacks,

since the overexpression is unlikely to be physiologically relevant and may have
some detrimental effects on various systems in the body (Arthur, 2000).
Table 2.10 Effects of GSH-Px over-expression
Untitled-2
Effect
System
References
The survival rate during 4 weeks of

myocardial infarction was significantly
increased. This was associated with a
decrease in myocyte hypertrophy,
apoptosis, and interstitial fibrosis
Transgenic mice
Shiomi et al., 2004
Protected against experimental stroke and

the neuroprotective mechanism involved
attenuation of apoptosis-related events
Rats
Hoehn et al., 2003
Delayed cell growth and protected them

from GSH and H2O2 toxicity
Endothelial cells
Faucher et al., 2003
Decreased by 30% activity of the

chymotrypsin-like activity of the 20S
proteasome
Human cells
Kretz-Remy and
Arrigo, 2003
Enhanced the resistance of a rat beta cell

line to both ROS and RNS cytotoxicity
Rat beta cell line
Moriscot et al., 2003
Inhibited CD95-induced effector caspase

activation, DNA fragmentation, and apoptotic
cell death
Human breast
cancer T47D cells
Gouaze et al., 2002
Protected islets against adverse effects

of ribose
Human pancreatic Tanaka et al., 2002

beta cell line
Compensated for the adverse effects of

homocysteine on endothelial function
Mice
Increased two-fold the half-life of I kappa B

alpha in cells
Human T47D cells Kretz-Remy and

Arrigo, 2001
Attenuated NADPH and NADH oxidation,

protein carbonyl , F(2)-isoprostanes formation
and alanine transaminase release in various
tissues, detrimental consequences of moderate
oxidative stress induced by low levels of
paraquat or diquat
Mice
Lei, 2001
Increased resistance of cells to doxorubicin

toxicity
Human breast
cancer cells
Gouaze et al., 2001
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Table 2.10 Cont.
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Effect
System
References
Down-regulated NFkappaB DNA binding,

and transcriptional activation of an
NFkappaB-dependent luciferase reporter
MCF-7 cells
Li et al., 2001
Increased resistance to amyloid betapeptide-induced injury
PheochromocytBarkats et al., 2000

oma cells and rat
embryonic cultured
cortical neurons
Altered vascular permeability and production

of cytokines (interleukin-1 beta and tumor
necrosis factor-alpha) and NO, affected
arachidonic acid metabolism, and inhibited
leukocyte migration
Mice
Inhibited degradation of the inhibitory

subunit alpha of nuclear factor-KB
Human glioma cells Li et al., 2000
Increased protection against S-nitroso-
Insulin-producing
N-acetyl-D,L-penicillamine and
sodium nitroprusside, which generate both
NO and reactive oxygen species
RINm5F cells
Protected against a lethal dose of

acetaminophen.
Mice
Mirochnitchenko et
al., 1999
Partially protected dopaminergic neurons

against 6-hydroxydopamine-induced toxicity
Mice
Bensadoun et al.,
1998
Provided protection against cytotoxicity of
Insulin-producing
Tiedge et al., 1998
H2O2, hypoxanthine/xanthine oxidase or

menadione, combined expression of Gpx
plus Cat was more effective
RINm5F cells
The survival time of mice that received

50 or 125 mg paraquat/kg was solely a
function of tissue GPX1 activity
Mice
Cheng et al, 1998
Protection against ischemia/reperfusion

damage in the stroke model possibly
through direct scavenging of ROS or through
the influencing of signalling mechanisms
which lead to tissue damage
Mice
Weisbrot-Lefkowitz et
al., 1998
Increased the tolerance of the cells to

oxidative stress produced by H2O2 or by
the redox cycling of the quinone-containing
anticancer agent doxorubicin
Human MCF-7
breast cancer cells
Doroshow, 1995
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al., 2000
Tiedge et al., 1999
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66
Table 2.10 Cont.

Effect
System
References
Enhanced resistance to paraquat and to

peroxides
Breast carcinoma
cells
Kelner et al., 1995
Decreased susceptibility of dopamine

neurons to damage due to energy impairment
Mice
Zeevalk et al., 1997
Increased heart resistance to myocardial

ischemia reperfusion injury
Mice
Yoshida et al, 1996
GSH-Px regulation and proteasomes
It is well known that ATP- and ubiquitin-independent proteolysis by the 20S

proteasome is responsible for the selective degradation of oxidized proteins. In
particular, the 20S proteasome shows an increased proteolytic activity toward
oxidized polypeptides. On the other hand, it seems likely that increased GSH-Px1
activity can downregulate basal 20S proteasome activity. For example, it was
shown a 30% decreased activity of the chymotrypsin-like activity of the 20S
proteasome in human cells overexpressing GSH-Px1 (Kretz-Remy and Arrigo,
2003). This phenomenon was associated with a 2-fold increase in IkappaB alpha
half-life, a protein whose basal turnover is 20S proteasome-dependent. The author
suggested that, the intracellular redox status, is an important element activating or
down-regulating the 20S proteasome chymotrypsin-like activity in living cells.
This is a very important finding, which explains how Se-reserves in the body can
be used to improve antioxidant defences in stress conditions. The most probable
sequence of events is as follows:
Untitled-2
when organic Se is used in the diet Se reserves in the form of SeMet nonspecifically incorporated in various proteins, for-example in muscles, are
built
in stress conditions requirement for selenoproteins to prevent free-radical
related damages is increased, but selenium bioavailability decreased due to
decreased feed consumption
redox status of muscle cells decreased due to depletion of antioxidants and
probably some proteins are oxidised
proteosomes are activated and protein degradation is increased
SeMet is released from proteins and used for additional synthesis of
selenoproteins
Antioxidant defences are improved and redox status of the cells changed
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Proteosome activity decreased and antioxidant-prooxidant balance

maintained
PHOSPHOLIPID GLUTATHIONE PEROXIDASE (GSH-Px4, PH-GSH-Px)

In 1985 Ursini and co-workers reported that another form of GSH-Px, which
used a phosphatidyl choline hydroperoxide as a substrate (PH-GSH-Px), was Sedependent. They showed that the enzyme was a monomer of 23 kDa. It contained
one gatom Se/22 000 g protein. Se was found there in the selenol form. The
kinetic data were compatible with a tert-uni ping-pong mechanism, as in the case
of the classical glutathione peroxidase. The second-order rate constants (K1) for
the reaction of the enzyme with the hydroperoxide substrates indicated that, while
H2O2 is reduced faster by the cGSH-Px, linoleic acid hydroperoxide is reduced
faster by PH-GSH-Px. The authors suggested that this enzyme was active at the
interface of the membrane and the aqueous phase of the cell. PH-GSH-Px is
distinguished from classical GSH-Px as it is active in monomeric form and has a
different amino acid composition (Sunde, 1993). In particular, arginine residues
surrounding the reaction center, which are responsible for a productive binding
of GSH in GSH-Px1, are missing in PH-GSH-Px (Mauri et al., 2003).
Did you know that PH-GSH-Px is the only enzyme in the cell
able to detoxify lipid peroxides inside the membrane without
their preliminary release by phospholipases?
There are three different forms of PH-GSH-Px. It is synthesized as a long form (Lform; 23 kDa) and a short form (S-form, 20 kDa) from mRNA that is transcribed
from two initiation sites in exon Ia of PHGPx genomic DNA (Imai and Nakagawa,
2003). S-form PH-GSH-Px is the nonmitochondrial PH-GSHPx and L-form PHGSH-Px is the mitochondrial PH-GSH-Px. Recently, the third form of PH-GSHPx, a 34 kDa selenoprotein, was detected in rat sperm nuclei and called sperm
nuclei GSH-Px (snGSH-Px) and now it is considered to be a separate 5th member
of the GSH-Px family. The PH-GSH-Px is unique in its capability of reducing
ester lipid hydroperoxides even if they are incorporated in biomembranes or
lipoproteins. For other members of GSH-Px family, preliminary release of peroxides
from the membrane by such enzymes as phospolipase C is an essential part of
detoxification.
It is known that the PH-GSH-Px is encoded for by a joint sperm nucleus/PHGSH-Px gene (sn/PH-GSH-Px) and can be expressed as a mitochondrial or cytosolic
isoform (Ufer et al., 2003). In fact, mitochondrial and non-mitochondrial PHGSH-Px and sperm nuclei GSH-Px are transcribed from one gene by alternative
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68
transcription using different first exons Ia and Ib, respectively (Imai et al., 2003).
Recent data (Ufer et al., 2003) indicate that the basic PH-GSH-Px promoter
constitutes a 200 bp oligonucleotide, which is localized immediately upstream of
the 3'-ATG and involves functional stimulating proteins (SP1/SP3), nuclear factor
Y (NF-Y) and SMAD binding sites. The authors suggested that the corresponding
trans-regulatory proteins may contribute to differential expression regulation of
the mitochondrial and cytosolic PH-GSH-Px isoforms. Indeed, the genomic
sequence of rat PH-GSH-Px4 was established and investigated in respect to
expression into the cytosolic, mitochondrial, and nuclear forms of PH-GSH-Px.
In silico analysis suggested the presence of two distinct promoter regions, the
upstream one leading to transcripts translating into cPH-GSH-Px or mPH-GSHPx and the downstream one yielding nPH-GSH-Px (Maiorino et al., 2003). The
data suggested that the formation of nPH-GSH-Px is due to alternative transcription
and not to alternative splicing. In particular, transcripts encoding nPH-GSH-Px
were most abundant in testis although not restricted to this organ. This observation
points to a general role of the nuclear PH-GSH-Px variant in regulating cell division.
It is well known that PH-GSH-Px is widely expressed in normal tissue, and
especially high in testis (Imai et al., 1995), where it has an important role in
spermatogenesis and sperm function and is under gonadotropin control. In this
organ a relevant PH-GSH-Px activity is strongly linked to mitochondria of cells
undergoing differentiation to spermatozoa. In tested mitochondrial for of PHGSH-Px is electrostatically bound to the inner surfaces of the organelle. (Roveri
et al.,1994). In fact, there was no difference between the soluble and the
mitochondrial enzyme in terms of chromatographic properties, the electrophoretic
mobility, the reactivity to antibodies and the fragmentation patterns and substrate
specificity. However, two-dimensional electrophoresis followed by
immunostaining with monoclonal antibodies, showed the presence of multiple
isoforms with a different pattern between the soluble and the mitochondrial enzyme.
(Roveri et al., 1994)
The gene encoding for the porcine PH-GSH-Px was cloned (Brigelius-Flohe et
al., 1994) and the complete genomic sequences of the human (Kelner and Montoya,
1998) and the murine (Borchert et al., 1999) orthologs were reported. A full-length
cDNA for PH-GSH-Px including exon Ib from rat and mouse testis was also cloned
(Nakamura et al., 2003). In Western blot analysis of proteins extracted from testes of
mutant mice and from developing mouse testes, two signals at 19- and 22-kDa were
observed which confirm the existence of two PH-GSH-Px forms in testicular cells
(Nayernia et al., 2004). It is interesting that PH-GSH-Px is found to be localised in the
midpiece of spermatozoa in various species including Drosophila melanogaster, frog,
fish, cock, mouse, rat, pig, bull, and human (Nayernia et al., 2004). It is also important
to mentioned that PH-GSH-Px mRNA expression in the male reproductive organs is
under estrogen control (Nam et al., 2003).
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The most extraordinary discovery about PH-GSH-Px is related to its
polymerisation and conversion from active enzyme to the structural protein. Indeed,
PH-GSH-Px protein was identified as the major constituent of the keratin-like
material that embeds the helix of mitochondria in midpiece of spermatozoa (Ursini
et al., 1999; Foresta et al., 2002). Indeed failure of the expression of mitochondrial
PH-GSH-Px in spermatozoa is considered to be one of the causes of
oligoasthenospermia in infertile men (Imai et al., 2001). The evidence of chemical
polymerisation of PH-GSH-Px into structural protein in sperm was described as
follows (Mauri et al., 2003):
This process coincides with a complete loss of GSH in late sperm maturation
The aggregates could only be dissolved by drastic treatment with low nuclear
weight thiols
In vitro aggregation of solubilised capsular material by H2O2 depends on
the absence of GSH
It was postulated that an oxidised form of PH-GSH-Px reacts with surface
SH groups of itself and other proteins to create dead-end intermediates (Mauri
et al., 2003) building up mitochondrial capsule.
PH-GSH-Px specific activity in different rat organs was as follows: liver =

kidney>heart = lung = brain>muscle (Zhang et al., 1989). The authors also showed
that PH-GSH-Px activity was reasonably constant in different age groups, with a
lower specific activity observed only in kidney and liver of young animals. In
contrast, it has been shown that PH-GSH-Px specific activity in rat is age-dependent
with a maximum at 3 months of age in the testis germ cells and at 6 months of age
in the isolated epididymal sperm cells (Tramer et al., 2002). PH-GSH-Px has the
strong binding capacity to the sperm cell tails and to the sperm heads. The PHGSH-Px activities in tissues of rats fed the Se-deficient basal amino acid diet were
41, 50, 26 and 25% of the Se-adequate PH-GSH-Px activities in liver, heart, kidney,
and lung respectively (Lei et al., 1995). The authors showed that testis had a 15fold higher PH-GSH-Px activity than liver and kidney, and a 25-fold higher PHGSH-Px activity than heart and lung. Furthermore, it was shown that PH-GSH-Px
mRNA levels were not affected by Se deficiency.
PH-GSH-Px overexpression
Summary of the effects of PH-GSH-Px overexpression are shown in Table 2.11. It

was shown that PHGPx over-expression dampens expression of cell adhesion molecule
either by lowering stimulatory hydroperoxides or by using hydroperoxides for protein
modification (Banning et al., 2004). PH-GSH-Px overexpression protects against
ischemia/reoxygenation injury (Hollander et al., 2003) by decreasing:
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70
Table 2.11 Effects of PH-GSH-Px over-expression
Untitled-2
Effect
System
References
Induced a delay in cell proliferation due to

a slower progression from G1 to S was
observed
MCF-7 cells
Wang et al., 2003
Dampened expression of cell adhesion

molecule either by lowering stimulatory
hydroperoxides or by using hydroperoxides
for protein modification
Rabbit aortic
smooth muscle
cells
Banning et al., 2004
Protected against ischemia/reoxygenation

injury by decreasing: cell damage, DNA
laddering and histone-associated DNA
fragments, cytochrome c release from
mitochondria, damage to electron transport
chain complex IV function
Neonatal rat
cardiac myocytes
Hollander et al., 2003
Reduced arachidonate metabolism through

12(S)-lipoxygenase and cyclooxygenase 1
and arsenite-induced generation of reactive
oxygen species
Human epidermoid Chang, 2003

carcinoma A431
cells
Suppressed cell death induced by

actinomycin D and doxorubicin that induced
damage in the nucleolus
RBL2H3 cells
Nakamura et al., 2003
Protective effect against UV-induced lipid

peroxidation
Human dermal
fibroblasts
Dissemond et al.,
2003
Suppressed accumulation of cardiolipin

Rat basophil
hydroperoxide (CLOOH), effectively
leukaemia
prevented the inactivation of adenine
RBL2H3 cells
nucleotide translocator (ANT), the opening
of permeability transition pores and the release
of cyt c and apoptosis-inducing factor (AIF)
from mitochondria in hypoglycaemia-induced
apoptotic cells
Imai et al, 2003
Prevented the release of cytochrome c, the

activation of caspase-3, and apoptosis
RBL2H3 cells
Nomura et al., 1999
Suppressed synthesis of platelet-activating

factor
Stimulated
RBL-2H3 cells
Sakamoto et al., 2002
Prevented arsenite-induced increase of

intracellular peroxide levels, upregulation of
p21(WAF1/CIP1), and apoptosis
A431 cells
Huang et al., 2002
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Table 2.11 Contd.
Effect
System
References
Increased resistance to cholesterol

hydroperoxides-induced killing and
exhibited a striking hyper-resistance to
free radical-mediated lipid peroxidation
Tumor epithelial
cell line
Hurst et al., 2001
Cellular PH-GSH-Px activity had an inverse

linear correlation to the removal of lipid
hydroperoxides in living cells (r = -0.85), and
correlated positively with cell survival after
singlet oxygen exposure (r = 0.94)
Human breast
cancer MCF-7
cells
Wang et al., 2001
Inhibited linoleic acid hydroperoxide-induced Rabbit aortic

toxicity, dihydrorhodamine oxidation,
smooth muscle
NFkappaB activation and apoptosis
cells
Brigelius-Flohe et al.,
2000
Prevented cardiolipine peroxidation and is

considered to be an anti-apoptotic factor
rat basophile
leukaemia (RBL)
2H3 cells
Nomura et al., 2000
Decreased (by 8 fold) the rates of production

of leukotriene C4 and leukotriene B4
RBL-2H3 cells
Imai et al., 1998
Suppressed the peroxidation of membrane

lipids and, in particular, the production of
phosphatidylcholine hydroperoxide and
prevented cell death in response to extracellular attack by a lipid peroxide
RBL2H3 cells
Imai et al., 1996
Cell damage (creatine kinase and lactate dehydrogenase release)

DNA laddering and histone-associated DNA fragments
Cytochrome c release from mitochondria
Damage to electron transport chain complex IV function
Reduction of arachidonate metabolism through 12(S)-lipoxygenase and

cyclooxygenase 1 and that of the arsenite-induced generation of reactive oxygen
species are observed in cells overexpressing PH-GSH-Px (Chang, 2003). On the
other hand, enhancement of arachidonate metabolism and the arsenite-induced
generation of reactive oxygen species is detected in PH-GSH-Px-depleted cells.
Overexpression of 34kDa nucleolar PH-GSH-Px in RBL2H3 cells significantly
suppressed cell death induced by actinomycin D and doxorubicin that induced
damage in the nucleolus (Nakamura et al., 2003). PH-GSH-Px overexpression in
fibroblasts had the substantial protective effect against UV-induced lipid
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72
peroxidation (Dissemond et al., 2003). Suppression of the accumulation of

cardiolipin hydroperoxide by overexpression of PH-GSH-Px in mitochondria
effectively prevented the inactivation of adenine nucleotide translocator (ANT),
the opening of permeability transition pores and the release of cyt c and apoptosisinducing factor (AIF) from mitochondria in hypoglycaemia-induced apoptotic
cells (Imai et al, 2003). It was suggested that hydroperoxide, produced in
mitochondria, is a major factor in apoptosis and that mitochondrial PH-GSH-Px
might play a critical role as an anti-apoptotic agent in mitochondrial death
pathways. For example, overexpression of mitochondrial PH-GSH-Px prevented
the release of cytochrome c, the activation of caspase-3, and apoptosis (Nomura
et al., 1999). On the other hand, in the same experiment an ability to protect cells
from 2-deoxyglucose-induced apoptosis was abolished when the PH-GSH-Px
activity of M15 cells was inhibited by diethylmalate, indicating that the resistance
of the cells to apoptosis was indeed due to the overexpression of PH-GSH-Px in
the mitochondria. Recently it has been demonstrated that PH-GSH-Px is expressed
in early gastrulation stage at 7.5dpc and that the expression of PH-GSH-Px was
essential for normal mouse development. Imai et al. (2003) generated mice deficient
in PH-GSH-Px by a targeted disruption of all exons of the PH-GSH-Px gene.
Heterozygotes were viable, fertile, and appear normal, despite having decreased
levels of three types of PH-GSH-Px mRNA and protein. Embryos homozygous
for PH-GSH-Px-null die between 7.5 and 8.5 days post coitum (dpc), probably
developing distal apoptosis. The authors also showed that the expression of PHGSH-Px was detectable in the embryonic ectoderm and the yolk sac membrane at
7.5dpc. Reduced PH-GSH-Px expression in apoER2 knockout mice results in
the inability of the sperm to regulate the cell volume and in abnormal sperm
morphology and immotility (Andersen et al., 2003). GSH-Px4(-/-) embryos die
in utero by midgestation (E7.5) and are associated with a lack of normal structural
compartmentalization (Yant et al., 2003). The authors showed that cell lines derived
from GSH-Px4(+/-) mice were markedly sensitive to inducers of oxidative stress,
including gamma-irradiation, paraquat, tert-butylhydroperoxide, and hydrogen
peroxide, as compared to cell lines derived from wild-type control littermates.
GSH-Px4(+/-) mice also display reduced survival in response to gammairradiation. GSH-Px4(-/-) embryos die in utero by midgestation (E7.5) and are
associated with a lack of normal structural compartmentalization. GSH-Px4(+/-)
mice display reduced levels of GSH-Px4 mRNA and protein in various tissues.
Interestingly, cell lines derived from GSH-Px4(+/-) mice are markedly sensitive
to inducers of oxidative stress, including gamma-irradiation, paraquat, tertbutylhydroperoxide, and hydrogen peroxide, as compared to cell lines derived
from wild-type control littermates. GSH-Px4(+/-) mice also display reduced survival
in response to gamma-irradiation (Yant et al., 2003).
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PLASMA GLUTATHIONE PEROXIDASE (pGSH-PX, GSH-Px3)
GSH-Px from human plasma was purified to homogenity by Takahashi and coworkers in 1987. This enzyme is a glycoprotein and is synthesised in the kidney
and is extracellular enzyme found in blood plasma, chamber water of the eye or
amniotic fluid. In the same year, Maddipati et al. (1987) showed that the human
plasma glutathione peroxidase is a tetramer of identical subunits of 21.5 kDa
molecular mass as determined by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis. The plasma peroxidase is a selenoprotein containing one selenium
per subunit (Maddipati and Marnett, 1987). In general, the protein has a molecular
weight of approximately 92,000 Da with each subunit having molecular weight
of 23,000 Da and containing four Se atoms per molecule (Cohen and Avissar,
1993). Unlike several other glutathione peroxidases this enzyme exhibits saturation
kinetics with respect to glutathione (Km for glutathione = 4.3 mM). The peroxidase
exhibits high affinity for hydroperoxides with Km values ranging from 2.3 microM
for 13-hydroperoxy-9,11-octadecadienoic acid to 13.3 microM for hydrogen
peroxide at saturating glutathione concentration (Maddipati and Marnett, 1987).
Biological role of this enzyme remains speculative, since low concentrations
of extracellular GSH or reduced thioredoxin. pGSH-Px is considered to have
intermediate specificity to peroxides. It can reduce lipid hydroperoxides in LDL,
however, is not active against peroxidized cholesterol esters (for review see Flohe
and Brigelius-Flohe, 2001). Indeed, pGSH-Px is considered to be a redox buffer
involved in a regulation of inflammatory reactions.
A comparison between pGSH-Px and cGSH-Px showed (Esworthy et al., 1993)
that:
Untitled-2
the rate constants describing the reactivity of human p-GSHPx and human
GSHPx-1 with LinOOH and H2O2 are in the same range;
pGSH-Px is more reactive with LinOOH and GSHPx-1 is more reactive
with H2O2.
pGSHPx has a low level of reducing activity toward cholesterol 7 alphaOOH and no detectable activity with the 5 alpha-OOH isomer in contrast to
PH-GSPx which readily reduced both isomers. However, it catalyzed the
reduction of phosphatidylcholine hydroperoxides to the corresponding
hydroxy derivatives (Yamamoto et al., 1993). In fact, the reductions of
aromatic and small hydrophobic hydroperoxides (cumene hydroperoxide,
t-amyl hydroperoxide, t-butyl hydroperoxide, paramenthane hydroperoxide)
were better catalyzed by pGSH-Px than were reductions of the more
physiological substrates (linoleic acid hydroperoxide, hydrogen peroxide,
peroxidized plasma lipids, and oxidized cholesterol) (Howard and Hawkes,
1998);
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74

pGSHPx possesses catalytic activity toward phospholipid hydroperoxides
in the absence of detergents, enhanced at low concentrations by
deoxycholate, and strongly inhibited by Triton X-100 and incorporation
into liposomes
pGSHPx exhibits a smaller GSH rate constant than GSHPx-1.
GASTROINTESTINAL GLUTATHIONE PEROXIDASE (GI-GSH-PX, GSHPx2)

GI-GSH-Px was first described in 1993 (Chu et al., 1993). The enzymatic properties
of this enzyme are practically the same as those of cytosolic GSH-Px. The physical
properties are also similar; and activity of both enzymes depends on Se supply
(Chu et al., 1993). The authors showed similar substrate specificities for GSHPx1 and GSHPx-GI; they both catalyze the reduction of H 2 O 2 , tert-butyl
hydroperoxide, cumene hydroperoxide, and linoleic acid hydroperoxide with
glutathione, but not of phosphatidylcholine hydroperoxide. Furthermore, GSHPxGI mRNA was readily detected in human liver and colon, and occasionally in
human breast samples, but not other human tissues including kidney, heart, lung,
placenta, or uterus. On the other hand, in rodent tissues, GSHPx-GI mRNA was
only detected in the gastrointestinal tract, and not in other tissues including liver
(Chu et al., 1993). In fact, GSHPx-GI appeared to be the major glutathionedependent peroxidase activity in rodent GI tract.
Did you know that GI-GSH-Px is considered to be the main
defence in the gut against lipid peroxide absorption?
GI-GSH-Px activity was present in both the villus and crypt regions of rat mucosal
epithelium and its activity nearly equalled that of classical GSH-Px throughout
the small intestine and colorectal segments (Esworthy et al., 1998). GI-GSH-Px
could be considered to be a barrier against hydroperoxide resorption (BrigeliusFlohe, 1999). Furthermore, in the gastrointestinal tract there are at least three
more selenoproteins including plasma GSH-Px, selenoprotein P and thioredoxin
reductase (Mork et al., 1998). The data on GI-GSH-Px clearly indicate that this
enzyme should be considered as a major antioxidant defence in the intestine.
SPECIFIC SPERM NUCLEI GLUTATHIONE PEROXIDASE (SN-GSH-PX)

Specific sperm nuclei GSH-Px (sn-GSH-Px) has been recently identified in rat
testes (Behne et al., 2000) to be a 34-kDa selenoprotein. It is localised in the
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spermatid nuclei and found to comprise about 80% of total Se present. The nuclear
form of PH-GSH-Px differs from the mitochondrial and cytosolic forms by its
arginine-rich N-terminus that is encoded by alternative exon located in the first
intron of the PH-GSH-Px (Moreno et al., 2003). The authors suggested that the
Sperm nucleus specific N-terminus leads to nuclear localisation and binding of
snGSH-Px to chromatin via its polyarginine region.
It is interesting that 5 years earlier it was shown that, in rat testis nuclei the
activity of the PH-GSH-Px was much higher than in other tissues and subcellular
compartments, with the sole exception of mitochondria (Godeas et al., 1996). In
particular, in nuclei, the bound enzyme is solubilized by DNase I treatment, thus
suggesting a binding to chromatin. Furthermore, immunogold electron microscopy
indicated the association of PH-GSH-Px with chromatin structures in isolated nuclei.
The authors suggested possible role of the enzyme in protection against DNA
peroxidative damage. They also suspected a hypothetical thiol oxidase activity
toward specific thiol bearing proteins which could substitute for GSH as alternative
donor substrates. Such activity could be related to the enzyme new important
regulatory function in chromatin condensation (Godeas et al., 1996).
Immunochemical evidence and enzymatic activity revealed presence of PH-GSHPx in sperm heads and tail midpiece mitochondria. (Godeas et al., 1997).
Therefore snGSH-Px was identified as specific to the sperm nuclei GSH-Px
with similar properties to PH-GSH-Px (Pfeifer et al., 2001). The authors showed
that it differs from PH-GSH-Px in its N-terminal sequence. In rats, sn-GSH-Px is
highly expressed in the nuclei of the late spermatids where it is the only
selenoprotein present (Pfeifer et al., 2001). snGSH-Px was also detected in testis,
kidney and in human embryonic kidney cell line (Borchert et al., 2003). It has
also been shown by in situ hybridisation that snGSH-Px expression in testis, like
protamine expression, is restricted to late stages of spermatogenesis (Moreno et
al., 2003). In fact, in the nucleus, PH-GSH-Px was associated with electron-lucent
spots and with the nuclear envelope, and PH-GSH-Px in the latter region increased
after step 16. Furthermore, in early pachytene spermatids, PH-GSH-Px signals
were noted in the nuclear material exhibiting a very similar density to chromatoid
bodies and in the intermitochondrial cement (Haraguchi et al., 2003). In fact, the
PH-GSH-Px signals were detected in chromatoid bodies, clear nucleoplasm,
mitochondria-associated material, mitochondrial aggregates, granulated bodies,
and vesicles in residual bodies in addition to mitochondria, nuclei, and acrosomes.
It was shown that the nuclear signal sequence of the N terminal of rat nuclear
PH-GSH-Px was characterised by a different sequence from that previously
reported for rat sperm nuclei GSH-Px (snGSH-Px). Low level expression of
nucleolar PH-GSH-Px was detected in several tissues, but the expression of
nucleolar PH-GSH-Px was extensively high in testis. In fact, expression of nucleolar
PH-GSH-Px was observed in the nucleoli in the spermatogonia, spermatocyte,
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and spermatid (Nakamura et al., 2003). It seems likely that regulation of expression
of the PH/snGSH-Px gene involves transcriptional and post-transcriptional events.
Therefore, snGSH-Px has cytosolic localisation and has negative regulatyory
elements in the first intron of the PH/snGSH-Px gene with several protein-binding
sites (Borchert et al., 2003). In Se-depleted rats the concentration of sn-GSH-Px
decreased to a third of the normal level and chromatin condensation was severely
disturbed (Pfeifer et al., 2001). The authors suggested that sn-GSH-Px is a
protamine thiol peroxidase responsible for disulfide cross-linking by reduction of
reactive oxygen species. It seems likely that antioxidant protection of the DNA
could be another important role for this enzyme.
GSH-Px6
It is a close homolog to plasma GSH-Px3 and the gene encoding human and
porcine GSH-Px6 were cloned (Kryukov et al., 2003). However, in rodents SeCys
was replaced by Cys and therefore this enzyme is not a selenoprotein. The authors
suggested that in rats GSH-Px6 was previously described as odorant-metabolizing
protein. In fact, GSH-Px6 was only detected in embryos and olfactory epithelium
(Kryukov et al., 2003).
Glutathione peroxidases and their biological roles

The various GSH-Px are characterised by different tissue-specificity and are
expressed from different genes (Ursini et al., 1997; Brigelius-Flohe, 1999; Cheng
et al., 1998b). For example, GSH-Px1 is expressed independently of GPX3 or
GPX4 and represents approximately 60% of the total hepatic Se in Se-adequate
mice (Cheng et al., 1997). Furthermore, the overexpression of the GSH-Px1 gene
in mice was tissue specific and did not affect the expression of GSH-Px3, GSHPx4 or GST and plasma Se levels (Cheng et al., 1997a).
The major function of these peroxidases is considered to be the removal and
detoxification of hydrogen peroxide and lipid hydroperoxides (Mates and SanchezJimenez, 1999; Ursini et al., 1997). Maintenance of cellular redox state is another
important function of the GSH-Px enzymes; and GSH-Px forms are involved in
such physiological events as differentiation, signal transduction and regulation of
pro-inflammatory cytokine production (Ursini, 2000). Peroxynitrite scavenging
by GSH-Px (Sies et al., 1997) could also play a prominent role in cell signal
transduction events. Participation of GSH-Px enzymes in regulating biosynthesis
of leukotrienes, thromboxanes and prostaglandins is responsible for the modulation
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of inflammatory reactions, whereas PH-GSH-Px can bring about cytokine-induced
transcriptional gene activation (for review see Kohrle et al., 2000).
Since hydrogen peroxide is considered as intracellular messenger (Rhee, 1999),
and redox regulation can play a basic role in the activation of key transcription
factors (Jackson et al., 1998; Dalton et al., 1999), it has been suggested that
regulation of the delicate regional redox balance is one of the main functions of
glutathione peroxidases (Brigelius-Flohe, 1999). In contrast, the main function of
sn-GSH-Px is protamine thiol cross-linking during sperm maturation (Pfeifer et
al., 2001). Glutathione peroxidases are found in all mammalian tissues in which
oxidative processes occur (Kohrle et al., 2000). In general, the cytoplasmic GSHPx is considered an emergency enzyme (Kohrle et al., 2000) responsible for
prevention of detrimental effects of oxidative stress. GSH-Px enzymes are involved
in such physiological events as differentiation, signal transduction and regulation
of pro-inflammatory cytokine production (Ursini, 2000). Peroxynitrite scavenging
by GSH-Px (Sies et al., 1997) could also play a prominent role in cell signal
transduction events. Participation of GSH-Px enzymes in regulating biosynthesis
of leukotrienes, thromboxanes and prostaglandins is responsible for the modulation
of inflammatory reactions, whereas PH-GSH-Px can bring about cytokine-induced
transcriptional gene activation (for review see Kohrle et al., 2000).
Similar to other GSH-Px and sometimes in addition to their functions, PHGSH-Px has many various roles in animals and human (Maurt et al., 2003),
including:
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Antioxidant defence during spermiogenesis and embryonic development

(Imai et al., 2003)
Structural role in mature spermatozoa
Regulation of 15-lipoxigenase and cyclooxigenase and leukotriene synthesis
Prevention of Il-1 induced activation of NF-B
Inhibition of apoptosis
Supression of the production of platelet activating factor by stimulation
with A23187 (Sakamoto et al., 2002)
Driving chromatin compactation by oxidizing nucleoprotein thiols
Participation in redox regulation of signalling and differentiation by
eliminating hydrogen peroxide and lipid hydroperoxide as well as by
modifying specific protein thiols. In particular, PH-GSH-Px regulates cell
growth. For example, PH-GSH-Px induced a delay in cell proliferation due
to a slower progression from G1 to S was observed (Wang et al., 2003).
The authors suggested that PH-GSH-Px can lower the peroxide tone, which
might change the cellular redox environment resulting in a delay in G1
transit.
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78
In general, PH-GSH-Px is considered to be a key regullator of various

intracellular events including a modulation of the production of prostanoids
(Sakamoto et al., 2000) leading to inflammatory responses being a peroxidedependent thiol-modifying agent (Flohe and Brigelius-Flohe, 2001). Furthermore,
PH-GSH-Px activity is regulated by various environmental factors. For example,
lipids, cytokines and antioxidants modulate PH-GSH-Px in a complex manner,
providing physiological regulation of antioxidant defences in the cell (Sneddon
et al., 2003).
It is necessary to underline that, different forms of GSH-Px perform their
protective functions in concert, with each providing antioxidant protection at
different sites of the body. For example, gastrointestinal glutathione peroxidase
(GI-GSH-Px) could be considered to be a barrier against hydroperoxide resorption
(Brigelius-Flohe, 1999). Recently it has been suggested that digestive tract is a
major site of antioxidant-prooxidant interaction in the body (Surai, 2002a; Surai
et al., 2003; 2004). In this case a specific GI-GSH-Px is considered to be a major
protection against lipid hydroperoxides found in the food/feed. It is generally
accepted that during feed production and storage some polyunsaturated lipids are
oxidised and they can cause health-related problems, including decreased growth,
productive and reproductive traits of animals and immunocompetence (Kanazawa
and Ashida, 1998). It seems likely that this detrimental effect of feed peroxides
would ultimately depend on the activity of GI-GSH-Px. Indeed oxidised lipids
can react with transition metals which can be found in the feed as feed supplements
(usually in inorganic catalytic form) producing free radicals. Those free radicals
can react with natural or synthetic antioxidants present in the feed with formation
of lipid hydroperoxides. Therefore GI-GSH-Px would deal with those peroxides
preventing them entering blood circulation. Indeed, it has been shown that after
inclusion of lipid peroxides into the diet of the rats their concentration in plasma
was extremely low (Kanazawa and Ashida, 1998; 1998a). As mentioned above,
GSH-Px is an important antioxidant in plasma, which together with selenoprotein
P and other antioxidant compounds, maintain antioxidant protection. On the other
hand, PH-GSH-Px is an important antioxidant inside biological membranes where
lipid peroxidation occurs and lipid hydroperoxides are produced. Therefore, cGSHPx and PH-GSH-Px are found in cytosol, while eGSH-Px and SeP are located in
the extracellular fluids and working together they can provide antioxidant defence
to the biological molecules inside and outside the cell (Takebe et al., 2002). The
authors showed that cGSH-Px reduced t-BuOOH and H2O2 effectively but does
not reduce PC-OOH. Specific conditions required for the maximal enzymatic
activities were different for each enzyme, however, both PH-GSH-Px and eGSHPx showed very similar specific activities to PH-OOH (Table 7). These data suggest
that in different cellular and extracellular conditions a combination of various
selenoprotein can be effective in detoxifying H2O2 and lipid hydroperoxides.
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Despite great attention paid to the GSH-Px family, it seems likely, that
thioredoxin reductase, a relatively newly described as a selenoenzyme (Gladyshev
et al., 1996; Tamura and Stadtman, 1996), is going to outshine GSH-Px.
Non-Se glutathione peroxidases

It is generally accepted that two types of GSH-Px occur in the cell both of which
detoxify fatty acid hydroperoxides, thymine hydroperoxide and DNA
hydroperoxides. One is a Se-dependent enzyme which also detoxifies H2O2. The
other contains members of the GSH transferase supergene family (Ketterer and
Meyer, 1989). These non-Se-GSH -Px do not detoxify H2O2 and have substrate
specificities varying markedly with the isoenzyme. It was suggested that these
enzymes participate in the detoxication of lipid and DNA hydroperoxides they
may be important participants in mechanism for the repair of free-radical damage.
Activities of the two enzymes vary greatly among tissues and among animals.
The molecular weight of the enzyme with selenium-independent glutathione
peroxidase activity was estimated by gel filtration to be 35,000 and the subunit
molecular weight was estimated by dodecyl sulfate-polyacrylamide gel
electrophoresis to be 17,000 (Pierce and Tappel, 1978). The enzyme was inhibited
by N-ethylmaleimide, but not by iodoacetic acid. Inhibition by cyanide was
competitive with respect to glutathione and the Ki for cyanide was 0.95 mM. This
selenium-independent glutathione peroxidase also catalyzes the conjugation of
glutathione to 1-chloro-2,4-dinitrobenzene. Therefore, the data suggested that
the selenium-independent glutathione peroxidase and glutathione S-transferase
activities in rat liver are of the same enzyme. This was confirmed in another
study showing that glutathione S-transferase B contributes to the non-Se-dependent
glutathione peroxidase activity in rat liver and show that it increases in selenium
deficiency when the selenium-dependent glutathione peroxidase is decreased
(Lawrence et al., 1978). It could well be that increased activity on non-Se-GSHPx activity is a compensatory mechanism to deal with oxidative stress in Se
deficiency. Recently it has been shown that glutathione transferases not only
catalyse detoxication reactions, but also play an important role in signalling by
interacting with multiple proteins in pathways induced by cellular stress (Edalat
et al., 2003). Indeed, class GSTs are known to catalase conjugation of
electrophiles to glutathionone and catalyse GSH-Px reactions. It has been shown
that class GSTs possess glutathione peroxidase activity toward phospholipid
hydroperoxides residing in biological membranes without their release by
phospholipases C action (Yang et al., 2002). The authors showed that this GST
activity is responsible for about half for GSH-Px activity toward lipid
hydroperoxides in rat liver homogenate indicating an important role of GST in
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antioxidant defences. It is interesting that, in contrast with Se-GSH-Px, only GSH

could act as the reducing agent for GST (Hurst et al., 1998).
The distribution of various forms of GSH-Px has a tissue-specificity. For
example, Se-GSH-Px and non-Se-GSH-Px were determined in tissues of calves
(Scholz et al., 1981). Spleen, cardiac muscle, erythrocytes, brain, thymus, adipose
tissue, and striated muscles of calves were found to contain only the Se-dependent
GSH-Px. Tissues having both enzymes included liver, lungs, adrenal glands, testes,
kidney medulla, and kidney cortex. The liver contained the highest percentage of
non-Se-dependent GSH-Px activity of the calf tissues. In the rat liver and adrenal
gland non-Se-GSH-Px comprises about 35% of total GSH-Px activity. However
in the heart and lung all GSH-Px activity was related to Se-GSH-Px (Lawrence
and Burk, 1978). In contrast to other tissues, in testis non-Se-GSH-Px was the
major form of the enzyme. It seems likely that there is a species-specific expression
of non-Se-GSH-Px in animal tissues. In particular non-Se-GSH-Px was shown to
represent 100, 84, 81, 70 and 67% of total GSH-Px activity in guinea pig, human,
sheep, chicken and pig respectively (Lawrence and Burk, 1978).
Selenium-dependent and non-selenium-dependent glutathione peroxidases in
human tissue extracts were studied by Carmagnol et al. (1983) and they showed
hat:
the non-selenium-dependent glutathione peroxidase is predominant in liver,

in renal cortex and skeletal muscle;
non-selenium-dependent and selenium-dependent glutathione peroxidases
are in equal amounts in renal medulla;
the selenium-dependent glutathione peroxidase is predominant in adrenal
glands and platelets;
the selenium-dependent glutathione peroxidase represents 100% of the
glutathione peroxidase activity in the other organs, including heart and brain
There are several specific non-Se GSH-Px which have some unique tissue-specific
features of their expression. For example, a secreted 24-kDa sperm-bound
selenium-independent GSH-Px protein was characterized and cloned and called
GSH-Px5. It contains a 663 bp open-reading frame that, after conceptual translation,
shows extensive identity with proteins belonging to the GSH-Px family (Vernet et
al., 1996). The authors showed that GSH-Px5-expressing cells can metabolize
hydrogen peroxide in a manner that is consistent with a peroxidase activity.
However, GSH-Px5 is insensitive to a regular inhibitor of GSH-Px enzymes. The
expression of GSH-Px5 was found to be restricted to the mouse caput epididymidis
(Schwaab et al., 1998), but low levels of expression were shown in the kidney
and liver (Dufaure et al., 1996) and in other mouse tissues (Lahti et al., 2001). It
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has been suggested that the Se-independent GSH-Px5 could function as a backup system for Se-dependent GSH-Pxs (Vernet et al., 1999). For example, following
dietary Se deprivation it was shown that the epididymis is still efficiently protected
against increasing peroxidative conditions. In fact, the caput epididymis of
selenium-deficient animals showed a limited production of lipid peroxides, a total
GSH-Px activity which was not dramatically affected by the shortage in selenium
availability and an increase in GSH-Px5 mRNA and protein levels (Vernet et al.
1999). Therefore, in mice, GSH-Px5 is synthesized by the caput epididymidis.
The protein is secreted as early as the initial segment of the caput and is found
subsequently associated with the sperm plasma membrane in a sub-acrosomic
localization. In fact, GSH-Px5 is present in the caput and cauda epididymis lumens
in three different locations: either free as a soluble protein in the caput epididymal
fluid, weakly bound to caput sperm membranes, or, finally, associated to lipidcontaining structures conferring to the protein a protective effect against proteolytic
digestions (Rejraji et al., 2002). Within the cauda epididymidis, the amount of
free GSH-Px5 is low compared to the caput and the association with sperm
membranes proved to be more solid. In both caput and cauda sperm samples, the
association of GSH-Px5 with the sperm membrane protects GSH-Px5 from
proteolytic cleavages. Therefore, it was suggested that GSH-Px5 to play an
important role in sperm maturation from the early events up to the onset of
fertilization. During ontogenesis, GSH-Px5 appeared at 20 days postnatal, before
the completion of the morphological differentiation of the caput and concomitantly
with the appearance of spermatozoa within the epididymis (Vernet et al., 1997)
Furthermore, GSH-Px5 is probably under the hormonal control, since hormonal
privation by castration abolished the accumulation of the GSH-Px5 protein.
Another Se-independent GSH-Px was purified a glutathione peroxidase from
bovine ciliary body by Shichi and Demar (1990) and showed that:
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An apparent MW of 112 kDa by gel filtration and 29 kDa by SDSpolyacrylamide gel electrophoresis.
The enzyme consisted of four identical subunits.
The enzyme was active with a variety of peroxides including H2O2, cumene
hydroperoxide, t-butyl hydroperoxide, triphenylcarbinyl hydroperoxide,
linoleic hydroperoxide and 5-phenylpentenyl hydroperoxide
Glutathione was essential for the reaction, however, L-cysteine, dithiothreitol
and 2-mercaptoethanol were inactive.
No selenium was found in the enzyme by the fluorometric assay with 2.3diaminonaphthalene.
The enzyme demonstrated no glutathione S-transferase activity when tested
with 1-chloro-2,4-dinitrobenzene, and several other compounds.
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A partial sequence of the enzyme showed some similarities both to Seglutathione peroxidases and a glutathione S-transferase isozyme.
It seems likely that non-Se GSH-Px family is much bigger that it had been suggested
before. Recently a novel non-selenium GSH-Px has been identified from rat lung
(Power and Nicholas, 1999). In fact the same enzyme is known as 1-Cys
peroxiredoxin (a bifunctional enzyme with two distinct active sites) called
nonselenium GSH-Px (NSGP) and is characterised by Ca 2+ -independent
phospholipase A2 and GSH-Px activity and has a specific role in the regulation of
phospholipid turnover as well as in protection against oxidative injury (Chen et
al., 2000). Recombinant human NSGPx expressed in Escherichia coli from a
human cDNA clone (HA0683) showed GSH peroxidase activity with sn-2linolenoyl- or sn-2-arachidonoyl-phosphatidylcholine hydroperoxides as substrate;
NADPH or thioredoxin could not substitute for GSH (Fisher et al., 1999). It was
shown that the enzyme catabolised H 2O 2, as well as organic hydroperoxides
including phospholipid hydroperoxides. It is interesting that the enzyme had no
GSH S-transferase activity. Bovine cDNA encoding NSGPx showed >95%
similarity to previously published human, rat, and mouse sequences and does not
contain the TGA stop codon. The molecular mass of bovine NSGPx deduced
from the cDNA is 25,047 Da (Fisher et al., 1999). In normal human brain there
was a very low level of NSGP in astrocytes and this enzyme was upregulated in
pathological conditions including Parkinsons disease and demnia (Power et al.,
2002).
Clearly, the GSH-Px family is an important part of antioxidant defences in
human and animal bodies and specific roles of the enzymes in regulation of other
important functions warrant further investigation.
Did you know that first selenoprotein called cytosolic GSH-Px
was described in 1973 and the last member of this family
(GSH-Px6) was discovered 30 years later in 2003?
Redox status of the cell is a major determinant of many different pathways
including gene regulation (Morel and Barouki, 1999). A thiol redox system
consisting of the glutathione system (glutathione/glutathione reductase/
glutaredoxin/glutathione peroxidase, Holmgren, 1989; Cotgreave and Gerdes,
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1998) and a thioredoxin system (thioredoxin/thioredoxin peroxidase/thioredoxin
reductase) are believed to be the major players in this regulation (Holmgren, 2000,
2000a; Gromer et al., 2004). Together they supply electrons for
deoxyribonucleotide formation, antioxidant defence and redox regulation of signal
transduction, transcription, cell growth and apoptosis (Mustacich and Powis, 2000;
Arner and Holmgren, 2000; Figure 2.2). Interestingly, TR can reduce not only
thioredoxin, but also oxidized glutathione (Sun et al., 2001). Therefore these two
systems are linked much more closely than previously considered. Experiments
with yeast mutants lacking both the mitochondrial thioredoxin system and the
mitochondrial peroxyredoxin system suggest an important role for thioredoxin,
TR and peroxyredoxin in the protection against oxidative stress (Miranda-Vizuete
et al., 2000).
Did you know that TR participates in vitamin E recycling
and in regulation of redox status of the cell?
The mammalian thioredoxin system includes three major proteins (Sun et al.,
2001): thioredoxin, thioredoxin peroxidase and thioredoxin reductase (TR). As
TR is a Se-containing enzyme, it is worth noting some details of this system.
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Thioredoxin (Trx), an approximately 12 kDa thiol/disulfide oxidoreductase,

was first described in 1964 in E. coli and three years later it was described
in rat hepatoma cells (see Powis et al., 2000 for review). Only one
cytoplasmic Trx has been identified in human cells to date (Arner and
Holmgren, 2000) and Trx2 is located in mitochondria (Table 2.12).
Thioredoxin distribution in tissues of calf is shown in the Figure 2.4,
indicating the highest concentration of Trx1 in the liver. Thioredoxin with a
redox-active dithiol/disulfide is an electron donor for essential enzymes
including ribonucleotide reductase and a general protein disulfide reductase
(Holmgren, 2001). Therefore Trx is important for maintaining transcription
factors in active form during oxidative stress, thereby permitting the
expression of antioxidant or DNA repair genes (Favier, 2000). Thioredoxin
is reduced by NADPH and TR and in turn reduces oxidized cysteine groups
on proteins (Powis et al., 2000) and has an important role during
inflammation and tumour progression (Soderberg et al., 2000). Thioredoxins
(at least Trx1 and Trx2) are essential proteins in mammals since knockout
mice of each protein are embryonic lethal (Nakamura, 2004). Most, if not
all, of the functions of Trx depend on the activity of thioredoxin reductase
(Rundolf and Arner, 2004).
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Table 2.12 Classification of human thioredoxins and thioredoxin reductases (Adapted from MirandaVizuete et al., 2004)
Thioredoxins
Size, kDa
Name
Chromosomal
localization
Trx-1
9q31
11.71
Trx-2
Txl-1/Trp32
Erdj5/JDPI
22q13.1
18q21.2
2p22.1-23.1
11.87
32.25
91.08
Sptrx-1
18p11.2-11.31
53.27
Sptrx-2
7p14.1
67.27
Sptrx-3
Txl-2
Not dtermined
3q22.3-23
14.57
36.85
TrxR1
TrxR2
TGR
Thioredoxin
12q23-24.1
54.71
22q11.21
53.06
3p13-q13.33
63.63
Tissue
specificity
Subcellular
localization
Ubiquitous
Mainly cytosolic,
nuclear upon
certain stimuli
Ubiquitous
Mitochondrial
Ubiquitous
Cytosolic
Ubiquitous
Endoplasmic
reticulum
Testis/spermatid Sperm fibrous
sheath
Testis/spermatid Sperm fibrous
sheath
Testis/spermatid Golgi
Ubiquitous,
Associated with
especially in
microtubules in
testis and lung cilia and flagella
reductases
Ubiquitous
Cytosolic
Ubiquitous
Mitochondrial
Ubiquitous, but Cytosolic
highly expressed
in testis
10
8
6
4
2
0
Liver
Kidney
Thymus
Spleen
Lung
Brain
Heart
Erythrocytes
Figure 2.4 Concentrations of thioredoxins in tissues of 1-week-old calf (Adapted from Gromer et al.,
2004)
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Thioredoxin peroxidase (TPx) is a member of a newly discovered family of

proteins that are conserved from yeast to mammals. These proteins are
considered to be antioxidants that function as peroxidases associated with the
sulfhydryl reducing system. TPx protects against apoptosis, is able to inhibit
release of cytochrome c from mitochondria to cytosol, inhibits lipid peroxidation
in cells and could prevent H2O2 accumulation in cells (Zhang et al., 1997).
TPx consists of two 25 kDa subunits, contains two essential cysteines and
reduces H2O2 using electrons from Trx. There are at least three forms of this
enzyme (TPx-I, TPx-2 and TPx-3; Mitsumoto et al., 2001). A novel human
TPx (called AOE372) has been recently identified (Jin et al., 1997) and its
regulatory role in NF-kB activation has been suggested. TPx is part of an
important conserved sensing mechanism for redox conditions in eukaryotes
and is one of the main cellular enzymes for the detoxification of H2O2 through
the thioredoxin system (Ross et al., 2000). Mouse TPx was found to have
broad tissue distribution, but its expression was especially marked in cells that
metabolise high levels of oxygen molecules such as erythroid cells, renal tubular
cells, cardiac and skeletal muscle cells, and certain types of neurons (Ichimiya
et al., 1997).
Thioredoxin reductase (TrxR) was first characterised by Holmgren in 1977
from calf liver and thymus and purified in 1982 from rat liver cytosol by
Luthman and Holmgren. They showed that TrxR had a subunit molecular weight
of 58,000 and a native molecular weight of 116,000. The enzyme was highly
specific for NADPH with a Km of 6 M. It contained an FAD prosthetic group
and was sensitive to inhibition by arsenic. Fourteen years later it was shown
that human TrxR is a selenoenzyme (Gladyshev et al., 1996; Tamura and
Stadtman, 1996). Selenocysteine is required for the activity of this enzyme,
since the Cys mutant enzyme is inactive. Whereas H2O2 was a substrate for the
wild-type enzyme, all mutant enzymes lacked hydroperoxidase activity (Zhong
and Holmgren, 2000). Furthermore, radiolabelling of proteins by incubation
of the cDNA-transfected cells with sodium [75Se] selenite showed that 75Se was
incorporated into the expressed TrxR protein (Fujiwara et al., 1999) confirming
a requirement for Se for the formation of functional TrxR. Therefore, mammalian
TrxRs are a class of flavoproteins that use NADPH as a electron donor and
belong to the family of oxidoreductases (Ganther, 1999) that share sequence
identity and mechanistic similarity with glutathione reductases (Gasdaska et
al., 1995; Mustacich and Powis, 2000). These enzymes are involved in linking
the thioredoxin system to reduced glutathione and the nucleotide cofactors
(Holmgren and Bjornstedt, 1995).
Biological roles of the thioredoxin system are diverse and include (Gromer et al.,
2004; Das, 2004; Rundolf and Arner, 2004):
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Antioxidant defence: by direct catalysis of several antioxidant reactions
and by regeneration of other antioxidant enzymes such as peroxiredoxins
or methionine sulfoxide reductase inactivated by oxidative stress; recycling
dehydroascorbate to ascorbate and reduction of ubiquinone to ubiquinol.
In fact the thioredoxin system is a major line of cellular defence against
oxygen damage (Hirt et al., 2002). Indeed, cytochrome c is a substrate for
both TrxR1 and TrxR2 and cells overexpressing TrxR2 are more resistant to
impairment of complex III in the mitochondrial respiratory chain upon both
antimycin A and myxothiazol treatments, suggesting a complex III bypassing
function of TrxR2 (Nalvarte et al., 2004).
Redox regulation
Gene regulation by modulating several transcriptional factors, including
nuclear factor-B, Fos, Jun, Ref-1 and p53. The reducing activity of Trx for
transcriptional factors more than 100-fold higher than that of GSH
(Nakamura, 2004)
Modulation of protein phosphorylation: by affecting activity of mitogen
activating protein kinases and phosphoprotein phosphatases
Regulation of apoptosis: by controlling apoptosis signal-regulating kinase 1
Redox regulation of various cellular functions including cell proliferation,
differentiation and maintenance of viability. Indeed, overexpression of TrxR2
in mammalian cells reduced their growth (Attenuate et al., 2004).
Regulation of the synthesis of deoxyribonucleotides (DNA synthesis and
repair) by providing reducing equivalents to ribonucleotide reductase
Involvement in hormone action and cytokine function: Trx can act as an
autocrine growth-factor synergizing with IL-1 and Il-2; there is evidence
that Trx can act as ID activator
Protein biosynthesis: the Trx system is important to maintain high activity
of protein biosynthesis machinery in the cell
There are at least three forms of this enzyme (Table 13). TrxR1 is located
predominantly in the cytosol; TrxR2 is found in mitochondria (Miranda-Vizuete
et al., 1999; Powis et al., 2000). In fact, human mitochondrial TrxR consists of
521 amino acid residues with a calculated molecular mass of 56.2 Kda. It is also
highly homologous to the previously described cytosolic TrxR1. It is interesting
that TrxR2 has extra 33 amino acids in its molecule at the N-termins. It was
shown that mRNA for TrxR2 is highly expressed in prostate, testis and liver. TrxR2
gene consists of 18 exons spanning about 67 kb with a chromosomal localization
at position 22q11.2 (Miranda-Vizuele et al., 1999). One more TrxR3 is located in
the testes (Sun et al., 1999). Recently, Sun et al. (2001) demonstrated that testes
TR has broad substrate specificity and can reduce several components of the
thioredoxin and glutathione systems. It has been predicted that other members of
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Table 2.13 Reactions catalysed by cytosolic mammalian thioredoxin reductases (Adapted from
Holmgren, 2001)
Reactions
Reactions
H2O2 and lipid hydroperoxide reductase

Thioredoxin-S2 reduction
5,5-dithiobis-(2-nitrobenzoic acid) reduction
Electron donor to plasma GSH-Px

Reduction of alloxan and vitamin K
NK-lysin disulfide reduction and
inactivation of cytotoxic activity
Lipoic acid and lipoamide reduction
Reduction of dehydroascorbic acid
Reduction of ascorbyl free radical
Protein disulfide isomerase

Selenite and selenocysteine reduction
Selenodiglutathione reduction
Nitrosaglutathione reduction
TR family will be identified (Mustacich and Powis, 2000). Therefore it is called

thioredoxin and glutathione reductase (TGR). Recent findings indicate that TR is
a homodimer and a selenenylsulfide was identified as the active site of TR and a
structural model and mechanisms for the enzyme were proposed (Zhong et al.,
2000). The most striking feature of TR enzymes is their sensitivity to oxidising
conditions that cause changes in conformation (Gorlatov and Stadtman, 1998).
Such conformational changes are suggested as important with regard to triggering
cell signalling in response to oxidative stress (Ganther, 1999). In addition to
participation of TR in cell signalling and redox regulation of transcription factors,
reactivation of oxidatively inactivated proteins (Ganther, 1999) could be of great
importance in antioxidant defence in the cell. Therefore TRs are involved in protein
folding and critical protein-protein and protein-DNA interactions (Ebert-Duming
et al., 1999). TR can also directly reduce thioredoxin, hydrogen peroxide, lipid
hydroperoxides, ascorbyl free radical, dehydroascorbic acid, lipoic acid and
selenite (Arner and Holmgren, 2000; Holmgren, 2001; Rundolf and Arner, 2004,
Table 2.13) and may have a role in detoxification reactions (Holmgren and
Bjornstedt, 1995). The ability of mammalian TR to reduce dehydroascorbic acid
(May et al., 1997) could be an important link between Se, ascorbic acid and
vitamin E in general antioxidant recycling. Mammalian TRs show increased
activity with Se supplementation in the nutritional to supranutritional ranges
(Ganther, 1999; Holmgren, 2000). An additional unique property of TR is its
hydroperoxidase activity, which provides self-protection from inactivation by
hydroxyl radical (Zhong and Holmgren, 2000). A general scheme of reactions
and functions of thioredoxin reductase in the cell are shown in Figure 2.2 and
detailed information on this enzyme is presented by Nordberg and Arner (2001).
TR activity in cells is modulated by an intricate interplay, involving
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Regulation by Se availability: In rat liver and kidney TR activity increased

several fold as a result Se supplementation of the deficient diet (Berggren et
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88
al., 1999). However, there is a tissue-specificity in this regulation. For

example, after 12 month low Se diet consumption by rats TrxR activity
decreased in the heart, liver, and kidney, but increased in the arterial wall
(Wu and Huang, 2004)
Regulation of the promoter of TR: a housekeeping type promoter in
combination with alternative splice variants and transcriptional start sites,
Posttranscriptional regulation through AU-rich elements. Mammalian TR1
and TR2 exhibit alternative splicing around the first exon. Regulation via
Au-rich elements enables quick expression responses to various stimuli.
Posttranslational inactivation by ROS and electrophilic agents
(prostaglandin derivatives, lipid aldehydes, iodoacetic acid, arsenicals, gold
compounds, quinines, nitrosoureas, cisplantin, dinitrohalobenzenes),
Data on TR activity in various tissues obtained mainly with mammals, including

laboratory animals and hiumans. Recently, data on TR activity in chicken tissues
have been presented by Edens and Gowdy (2004). They showed that when Se
supplementation was low, the highest TR activity was found in kidney and barain
and lowest in the liver Table 2.14. After Se supplementation TR activity increased
practically in all tissues studied. Furthermore, Sel-Plex supplemnented at 00.3
ppm increased TR activity significantly more than selenite at the same dose in
heart and thymus. There was similar tendency of increased Se availability from
Sel-Plex for activation of TR in ther brain, breast muscle, bursa, thymus and spleen.
The authors also showed that the highest TR activity was found in nuclear pellet
and mitochondrial lysates and the lowest activity was seen in mitochondrial pellets
(Table 2.15; Edens and Gowdy (2004).
Table 2.14 Thioredoxin reductase activity in tissues of 3-week-old chickens (Adapted from Edens and
Gowdy, 2004)
Tissue
Liver
Lung
Heart
Kidney
Brain
Breast muscle
Bursa
Thymus
Spleen
RBC
Plasma
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Control,
(0 Se)
Selenite
(0.3 ppm)
Sel-Plex
(0.3 ppm)
Sel-Plex +Selenite
(0.3 ppm)
0.023
0.047
0.024
0.061
0.060
0.028
0.026
0.038
0.025
0.030
0.034
0.084
0.082
0.062
0.087
0.076
0.069
0.070
0.092
0.045
0.066
0.031
0.071
0.085
0.114
0.079
0.149
0.083
0.098
0.140
0.065
0.040
0.025
0.053
0.034
0.056
0.051
0.129
0.066
0.058
0.076
0.076
0.021
0.039
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Table 2.15 Subcellular distribution of chicken thioredoxin reductase activity (Adapted from Edens and
Gowdy, 2004)
Cellular distribution
mmol NADPH/min/mg total protein
Liver homogenate
Nuclear pellet
Post nuclear supernatant
Mitochondrial pellet
Post-mitochondrial supernatant
Mitochondrial pellet
Mitochondrial membranes
0.107
0.129
0.087
0.065
0.107
0.133
0.093
Iodothyronine deiodinases (ID)

It is well known that thyroid hormones play important roles in animals and human
by controlling growth, development, differentiation and general metabolism in
the body. More than 50 years have past since the first publication demonstrating
presence of triiodthyronine (T3) in the tissues of animals and humans given labelled
thyroxine (T4) (Gross and Pitt-Rivers, 1951). Synthesis of thyroid hormone takes
place in the thyroid gland mainly in the form of prohormone T4 (Kohrle, 2000).
In peripheral tissues, particularly liver and kidney, T4 is converted to T3 (catalysed
by ID) and it is believed that more than 80% of circulating T3 is derived from
deiodination of T4 in nonthyroidal tissue (Arthur and Beckett, 1994; Bianco et
al., 2002). There are three forms of ID. Activity of ID-I was found to be highest in
liver and kidney, ID-II highest in brain, brown adipose tissue and pituitary, and
ID-III is in highest concentrations in brain, skin and placenta (Arthur and Beckett,
1994). Several comprehensive reviews characterise these enzymes in details (Arthur
and Beckett, 1994; Kohrle, 2000; 2002; StGermain, 2001:Bianco et al., 2002).
Some important characteristics of these enzymes derived from aforementioned
reviews are shown in Table 2.16.
Did you know that thyroid hormone is synthesized in
non-active form (thyroxin) and a Se-dependent enzymes
called deiodinases are necessary to activate this hormone?
A major advance in the understanding of biochemical mechanisms of ID actions
began with the discovery that the activity of type one ID depends on selenium
status and therefore it was suspected that ID was a selenoenzyme. For example,
Se deficiency for periods of 5 or 6 weeks in rats produced an inhibition of T3
production from added T4 in brain, liver and kidney homogenate (Beckett et al.,
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90
RT3>>T4>T3
Homodimer of a 27-kDa subunit
Substrate specificity
Structure
Histidine, selenocysteine, cysteine,

phenylalanine
Essential amino acids

residues
Decreased
Human, rat, mouse, dog, chicken;

not expressed in rainbow trout
Cloned in species
Se deficiency
29,000
Molecular mass of
monomer, Da
T3, retinoids; TSH and cAMP in

thyroid only; testosterone (liver),
carbohydrate
Endoplasmic reticulum in liver,

inner plasma membrane in kidney
and thyroid
Subcellular location
Induction
Liver, kidney, thyroid, intestine,

pituitary, heart, brown adipose
tissue, placenta
Expression
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Heterotrimeric complex of appr.

200 kDa containing a 29-kDa subunit
T4>rT3
Decreased
cAMP, fibroblast growth factor ,

phorbolesters, atrial natriuretic peptide
and C-type natriuretic peptide in
glyal cells
Selenocysteine?
Human, rat, mouse, chicken, rainbow

trout
30,500
Inner plasma membrane; p29subunit

associate with F-actin and perinuclear
vesicles respectively;
Pituitary, brain, brown adipose tissue,

skeletal muscle, heart, skin, placenta,
thymus, pineal and harderian glands,
glial cells and tanycytes
Systemic>local T3 production,
Local>systemic T3 production;
degradation of rT3 and sulfated
provides intracellular T3 in specific
iodothyronines; source of plasma T3 tissues; souce of plasma T3 (50%)
Function
Type II
5I-deiodinase
Type I
5I-deiodinase
Property
Whole structure is not

known, contains a 32-kDa
subunit
T3>T4
Unknown
T3, fibroblast growth

factor, epidermal growth
factor
Selenocysteine
Human, rat, mouse,

chicken
31,500
Endoplasmic reticulum;
Placenta, brain; skin,

uterus, fetal tissues, many
tissues; not pituitary,
thyroid, kidney, adult liver
Inactivation of T3 and T4
Type III
5I-deiodinase
Table 2.16 Main mammalian deiodinases (Adapted from Kohrle, 2000; Arthur and Beckett, 1994; Germain, 2001:Bianco et al., 2002 ).
90

1989). Plasma T4 and T3 concentrations increased and decreased respectively in
selenium-deficient animals. Administration of selenium, as a single intraperitoneal
injection of selenite at 200 g/kg body weight completely reversed the effects of
selenium deficiency on thyroid-hormone metabolism. However, selenium
administration at 10 g/kg body weight had no significant effect on thyroid-hormone
metabolism (Beckett et al., 1989). Next year, it was suggested that hepatic ID-1 was
a selenoprotein (Arthur et al., 1990). Indeed, solubilized hepatic microsomes from
rats injected with 75Se-labelled Na2SeO3 four days before killing were found by
chromatography on agarose gels to contain a 75Se-containing fraction with ID-I activity.
It was shown that ID activity was related to a single 75Se-containing protein with
molecular weight of 27,400 Da. This protein could also be labelled with 125Ibromoacetyl reverse tri-iodothyronine, an affinity label for ID-I (Arthur et al., 1990).
In the same year, in a different laboratory the same conclusion that type I iodothyronine
5'-deiodinase is a selenoenzyme was made (Behne et al., 1990). Indeed, a 27.8 kDa
membrane selenoprotein was identified in rat thyroid, liver and kidney. That membrane
enzyme catalyzes the deiodination of L-thyroxine to the biologically active thyroid
hormone 3,3',5-triiodothyronine. The authors showed that a decrease in the activity
of this enzyme, observed in the liver of Se-deficient rats, was due to the absence of a
selenium-dependent membrane-bound component. Furthermore, by using various
biochemical techniques the identity of the 75Se-labeled selenoprotein and of the 27
kDa type I 5'-deiodinase subunit was confirmed and it was shown that the deiodinase
subunit contains one selenium atom per molecule in the form of selenocysteine (Behne
et al., 1990). Next year, in the paper published in Nature it was shown that the mRNA
for this enzyme contains a UGA codon for selenocysteine which is necessary for
maximal enzyme activity (Berry et al., 1991). Several years later, in 1994 it was
shown that type III deiodinadse is also selenoprotein (for details see Kohrle, 1999)
and finally type II deiodinase was also proven to be a selenoprotein (Curcio et al.,
2001; Bianco et al., 2002). Therefore, it was explained why conversion of T4 to T3 is
impaired in experimental selenium deficiency and an essential role for selenium in
thyroid hormone action was identified. In fact, Se deficiency altered both thyroid
hormone synthesis in the thyroid gland and conversion of T4 into T3 due to decreased
(by 10 times) activity of ID (Kohrle et al., 1992; Beckett et al., 1992; Table 2.17). For
example, Se deficiency in chickens caused a decrease in plasma T3 concentrations
and impaired growth of birds (Jianhua et al., 2000). In general, ID is ranked higher in
priority for available Se supply than is cytosolic GSH-Px and was similar in ranking
to that for PH-GSH-Px and SeP (Kohrle, 2000). In fact, selenium regulation of
selenoproteins in the Se-deficient rat liver model has four different patterns (Sunde,
2001; Table 2.18):
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GSH-Px is unique with a dramatic (90%) decrease of activity and mRNA

level
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Table 2.17 Effect of Se deficiency on thyroid hormone metabolism (Adapted from Germain, 2001)
Increased
Unchanged
Decreased
Serum T4
Serum free T4
Serum T3 (may be decreased)

Serum rT3
Serum T4 Sulfate
Serum T3 Sulfate
Serum TSH (may be increased)

D1 (thyroid, pituitary, gonads)
D3 (placenta, implantation site)
D1 (liver, kidney)
D2 (brain, pituitary, brown adipose
tissue)
D3 (brain, skin, uterus)
Tissue T3 levels (brain, liver)
cGSH-Px (liver, thyroid)
PH-GSH-Px (liver, thyroid)
Table 2.18 Downregulation of selenoproteins in selenium-deficient rats (adapted from Sunde, 2001)
Selenoprotein
Protein level (% decrease)
RNA level (% decrease)
99
59
95
90
90
90
<10
50
<10-33
<30
GSH-Px1
GSH-Px4
DI
SeP
TrxR1
GSH-Px4 represents the second pattern with modest (60%) decrease in

activity and little change in mRNA
TR and SelP show the third pattern with dramatic decrease of activity and
little change in mRNA
ID could be classified as having fourth pattern and characterised by dramatic
(95%) decrease in activity but modest (50%) decrease in mRNA
Other selenoproteins
In addition to the well-characterised selenoproteins mentioned above, there other
selenoproteins that are less well characterised and their functions are less obvious.
They include:
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Selenoprotein W (SeW) was purified and characterised by Vendelend et al.

(1993) and it consists of 87 amino acids and contains about 1 g atom Se as
selenocysteine per 1 g mol of protein (Ream et al., 2001). Four different
forms of this protein have been purified from rat muscle with molecular
masses ranging from 9.5 to 10 kDa (Allan et al., 1999). Therefore, SeW
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was originally isolated from muscle and now there is growing evidence
indicating presence of this protein in brain, testis and spleen (Yeh et al.,
1995). In sheeps, the highest levels of SeW were found in skeletal muscles
and heart and the lowest was found in liver (Yeh et al., 1997). Se-W mRNA
was highly expressed in the cortex, dentate gyrus, and hippocampus of
postnatal rat brains, and in the spinal cord and brain of developing embryos
(Jeong et al., 2004). The expression of SeW in cortex, cerebellum, and
thalamus was not significantly affected by Se depletion, but increased SeW
levels occurred only in thalamus with Se supplementation (Sun et al., 2001a).
The lowest dietary Se required for reaching normal expression of SeW in
rats was 0.05 mg/kg (Yang et al., 1999). Indeed Se deficiency leads to
decreased SeW concentration in the following sequence
muscle>spleen>testis>brain (Neve, 2000) and mRNA levels for SeW in
skeletal muscle also decreased (Vendeland et al., 1995). On the other hand,
SeW levels were significantly higher in muscle, spleen and testes of rats fed
0.1 mg Se/kg diet compared to those fed the deficient diet (Yeh et al., 1997;
Vendeland et al., 1995).
Structure and possible functions of SeW have been recently reviewed by Whanger
(2000) and Ream et al (2001) and major points from this review can be summarised
as follows:
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Selenoprotein W is found in muscle, spleen, testis and brain, but was not
detected in liver, kidney, intestinal mucosa, lungs, heart, plasma and
erythrocytes. It is necessary to take into account that SeW concentration in
tissues depends on Se dietary provision. For example, Se W was undetectable
in skeletal muscle of rats fed the basal diet, detectable in those fed 0.1 ppm
selenium in the diet, and much higher in muscle from rats fed 4 ppm
selenium diet. (Yeh et al., 1995).
SeW is believed to be involved in skeletal and cardiac muscle metabolism
(Rayman, 2002; Yeh et al., 1995).
In primates, SeW was found in several tissues with highest amounts in
skeletal muscle and heart and lowest levels in liver. Similarly, mRNA levels
were highest in monkey skeletal muscle and heart. However, as in the
monkey, selenium concentrations were highest in human kidney and lowest
in skeletal muscle and heart (Gu et al., 2000). Therefore SeW protein levels
correlated with SeW mRNA levels but not with tissue selenium
concentrations.
In contrast to primates and sheep, rodent cardiac does not affected by Se
deficiency. Furthermore, there are species-specific differences in SeW
structure and amino acid composition. For-example, SeW in rats and mouse
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contains four selenocysteine residues, but the primate SeW contains only
two cysteine residues (Whanger et al., 1997)
The tissue distribution of Se-W was similar in male and female rats. Se-W
protein level was high in testes of male rats but very low in ovaries of
female rats. Muscle and skin from female rats had significantly higher SeW levels than from male rats. Se-W mRNA levels from female skins were
significantly higher than from male rat (Yeh et al., 1998)
SeW contains a putative calcium-binding domain near its carboxy terminus
SeW is highly conserved (83% identical) in mammals including human,
monkey, rat, mouse, sheep and pig
The cDNAs encoding skeletal muscle SeW from human, rhesus monkey,
sheep, rat and mouse contained similar SECIS elements (Gu et al., 1997).
Similar to other selenoprotein encoding, rodent and sheep SeW mRNAs
used UGA as a stop codon and as SeCys codon;
Expression of this selenoprotein depends on dietary Se supplementation
and Se can stabilise SeW m-RNA, but Se levels do not affect the transcription
rate of the SeW gene. For example, the same rate of selenoprotein W mRNA
synthesis was shown in cells cultured in either low selenium or selenium
supplemented medium, suggesting that the transcription rate of the SeW
gene is independent of selenium (Gu et al., 2002). Indeed, the expression
of SeW mRNA increased significantly when L8 myotubes were cultured
with selenium (Yeh et al., 1997b). Therefore, it was shown that SeW mRNA
levels decreased over time with an estimated half-life of 57 h for cells grown
in low selenium medium. Selenium treatment increased the selenoprotein
W mRNA half-life 2-fold. (Gu et al., 2002)
Two forms of SeW isolated from rat muscle contained bound glutathione
(Belstein et al., 1996). In fact, GSH is bound to the 36th amino acid (cysteine)
of SeW (Gu et al., 1999)
Recently, the function of SeW has been investigated by cloning the
corresponding cDNA from mouse brain and expressing it in CHO cells and
H1299 human lung cancer cells (Jeong et al., 2002). Overexpression of SeW markedly reduced the sensitivity of cell lines to H 2O 2 cytotoxicity.
Furthermore, the intracellular peroxide concentration of the transfected cells
significantly decreased. It is interesting that, the resistance to oxidative stress
provided by Se-W was dependent on glutathione. It was also shown that
selenocysteine-13 and cysteine-37 residues in the selenoprotein are
responsible for the antioxidant activity of Se-W in vivo (Jeong et al., 2002).
SeW and its mRNA were over-expressed in C6 rat glial cells by 22- and 11fold (Sun et al., 2001b). There was a greater survival rate of overexpressed
than control cells when incubated with an inducer of free radical production
(AAPH), confirming an antioxidant function of SeW. However, the same
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cells with overexpressed SeW did not show any increase in resistance to
H 2O 2.
An important metabolic function of this protein is its involvement in
antioxidant defence, but clearly there is a need to further characterise this
selenoprotein
The human SeW gene maps to chromosome 19q13.3, spans approximately 6.3
kb and comprises six exons and SeW is expressed in all of the 22 tissues assayed,
and shows highest expression in skeletal muscle and heart (Bellingham et al.,
2003).
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Selenoprotein P 10 (SeP). The history of SeP discovery is related to

characterisation of plasma proteins after injection with 75Se-selenite. SeP
was distinguished from GSH-Px in plasma in 1977 by Herrman, and was
identified as a plasma selenoprotein in the early 1970s (for review see Hill
and Burk, 2001 and Mostert, 2000). Indeed, known at that time selenoprotein
(GSH-Px) was not the only one in the plasma (for review see Moschos,
2000). Finally, 9 years after GSH-Px characterisation as selenoprotein,
Motsenbocker and Tappel (1982) described the same plasma protein in
monkey and rat and called it Selenoprotein P. Indeed, SeP was the second,
after GSH-Px, selenoprotein identified in mammals and its name was given
to this protein because of its plasma location (Hill and Burk, 2001). SeP is a
glycoprotein representing from about one third of total plasma Se in humans
(Allan et al., 1999) to about 60-70% (Kohrle et al., 2000) or 60-80% of
total plasma Se in humans and rodents (Arthur and Beckett, 1994). It contains
up to 10 selenocysteine residues per 43 kDa polypeptide chain (Allan et
al., 1999). Indeed, the unique feature of SeP is that it is the only selenoprotein
known to have more than one Se atom per polypeptide chain. Furthermore,
it is the only known selenoprotein that uses a UGA codon in the open reading
frame of its mRNA alternatively for termination and for insertion of SeCys
(Hill and Burk, 2001). The gene for human SeP is located on chromosome
5q31 (Arteel et al., 2002). It was believed initially that this selenoprotein
was a major transport form of Se (Motsenbocker and Tappel, 1982) owing
to its comparatively high concentration of Se. In fact compared with other
Se-containing proteins, the addition of purified SeP to the SeP-depleted serum
or a serum-free medium was the most effective for the recovery of cellular
GSH-Px activity (Saito and Takahashi, 2002) confirming an idea that SeP
functions as Se-supply protein, delivering Se to the cells. Indeed SeP
participates in the distribution of Se from the liver to peripheral tissues such
as the brain (Burk et al., 2003). Furthermore, it has been suggested that SeP
might have an important role as an antioxidant in plasma (Burk and Hill,
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96

1993). Selenoprotein P can contribute to the decomposition of peroxynitrite
(Arteel et al., 1998; 1999, for review see Kohrle et al., 2000) and protection
of endothelial cells against damage from peroxynitrite, having peroxidase
activity in vitro (Sies et al., 1998). SeP of bovine serum acts as a survivalpromoting factor in neuronal cell culture (Mostert, 2000). Indeed, SeP reduces
neither H 2O2 nor tertiary butyl hydroperoxide, but reduces phospholipid
hydroperoxides using tert-uni ping-pong mechanism, similar to those
described for GSH-Px (Saito et al. 1999), therefore, SeP is considered to act
as an extracellular PH-GSH-Px (Saito et al., 1999; Brigelius-Flohe et al.,
2003). Thioredoxin is shown to be the preferred electron donor for SeP
(Takebe et al., 2002). This protein has been shown to bind heavy metals, is
considered as a negative acute phase protein and can promote neuron
survival in vitro (Kohrle et al., 2000). It has been suggested that SeP has
specific roles in protecting against Se toxicity and sequesting Cd2+ and
Hg 2+ ions (Yoneda and Suzuki, 1997). Clearly the antioxidant-related
functions of this protein could be a great advantage for animals. SePknockout mice were viable, but exhibited reduced growth and developed
ataxia (Schomburg et al., 2003).
Did you know that SeP is the major Se-transporting protein
possessing antioxidant activity?
SeP is expressed in various tissues including liver, heart, brain, kidney, testis and
muscle in rats and also in placenta and uterus in the mouse (Hill and Burk, 2001)
and associated with cell membranes (Brown and Arthur, 2001). It is interesting
that SeP can be synthesised in the brain (Yang et al., 2000). It has been suggested
that tissues like liver can take up small-molecule forms of selenium whereas
presence of the element in selenoprotein P facilitates uptake by tissues like brain
(Burk et al., 2003). Four isoforms of the protein have been identified in rat plasma,
including the full length protein containing 10 SeCys, and three shortened isoforms
(Chen and Berry, 2003). SeP genes in mammals include 10-12 UGA codons and
two SECIS elements in the 3I untranslated region (Chen and Berry, 2003). It is
interesting to note that in Se deficiency SeP mRNA and protein expression are
preferentially retained in comparison to other selenoproteins (Chen and Berry,
2002) showing high position of SeP in selenoprotein hierarchy.
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Selenoprotein P12 has been recently identified in the bovine cerebellar

cortex (Saijoh et al., 1995). The coding nucleotide sequence of its cDNA
insert displayed high homology with rat and human selenoprotein P cDNA
but contained 12 rather than 10 TGAs (12 rather than 10 selenocysteines in
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deduced amino acids). Moreover, mRNA from both this SeP-like protein
and SeP were expressed in all areas of the brain but most prominently in the
cerebellar cortex, hippocampus and olfactory bulb. The authors suggested
that this selenoprotein is a major Se carrier in the brain and has a role in the
morphological response of nerve or ganglial cells (Saijoh et al., 1995).
Selenophosphate synthetase-2 (SPS) is an enzyme involved in selenocysteine
biosynthesis providing an autoregulatory mechanism for selenoprotein
expression (Guimaraes et al., 1996). Selenophosphate is synthesised from
selenide and ATP by the selD gene product, selenophosphate synthetase,
and is required for selenocysteine synthesis and its subsequent incorporation
into selenoproteins (Low et al., 1995). There are two forms of SPS in
mammals with SPS-2 being a selenoprotein. For example, SPS was detected
using immunoblotting in extracts of rat brain, liver, kidney, and lung (Kim
and Stadtman, 1995). A magnesium ion and a monovalent cation, either
K+, NH 4+ or Rb +, are required for catalytic activity. Polyphosphates and
other common nucleotide triphosphates do not replace ATP as substrate
(Verez et al., 1994). The reaction catalysed by SPS proceeds as follows:
ATP+selenide+H2O selenophosphate+Pi+AMP
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15-kDa selenoprotein (Sep15) was discovered in 1998 when it was purified

from human T-cells; and its mRNA showed all the features known to be
necessary in other eukaryotic selenoprotein mRNAs to promote
selenocysteine insertion into proteins (Gladyshev et al., 1998). It contains a
single selenocysteine residue in the middle of a 162-amino acid open reading
frame and has no detectable homology to known proteins (Gladyshev et
al., 2001). The Sep15 gene spans 51 kb of the human genome and is
organised in five exons and four introns (Kumaraswamy et al., 2002). In
accordance with incidence of the human 15-kDa protein gene expression,
tissues can be placed in the following descending order: thyroid>parathyroid
tumor>prostate pre-cancerous cells>prostate>fetal lung>aortafetal
retina>retina>testis>fetal heart (Gladyshev et al., 1998). Sep15 is located in
the endoplasmic reticulum being bound to UDP-glucose:glycoprotein
glucosyltransferase (Gladyshev et al., 2001) suggesting Sep15 to participate
in the control of protein folding. Genes encoding Sep15 have also been
detected in mice and rat (Kumaraswamy et al., 2002). The role of this
selenoprotein is not clearly defined, but there is a possibility that it is
responsible for beneficial effect of selenium in certain human cancers.
18-kDa selenoprotein was characterised by combining methods for trace
element analysis, tracer techniques and various biochemical and
electrophoretical procedures (Kyriakopoulos et al., 2002). In particular,
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the authors showed that the 18 kDa-band was localized in the mitochondrial
and microsomal membranes. Furthermore, two-dimensional electrophoresis
showed that it consists of a single selenium-containing protein with an
isoelectric point of about 4.9-5.0. In addition, and six selenium-containing
peptides with molecular masses of 17, 16, 14, 12, 10, and 8 kDa were
detected. It was shown that selenium was present in the 18 kDa-protein in
form of selenocysteine which is a characteristic of a genetically encoded
selenoprotein (Kyriakopoulos et al., 2002).
Mitochondrial capsular selenoprotein (MCS) was identified in 1978 in
rats (Calvin, 1978). MCS is a major structural protein of the keratinous
mitochondrial capsule in mammalian sperm, a structure that functions in
shaping mitochondria into the helical sheath surrounding the flagellum.
The mouse MCS 54 codons contain three in-phase UGA codons, which
normally signify stop but encode selenocysteine in bacterial and mammalian
selenoproteins. The coding region of the mitochondrial capsule selenoprotein
gene is interrupted by a single intron (Karimpour et al., 1992). In mouse
testis, MCS mRNA first appeared in step 3 round spermatids, gradually
increased during early spermiogenesis, and persisted a high level until step
14 spermatids. After the step 14 spermatids, the signal began to decline and
was weakly detected in steps 15-16 spermatids (Nam et al., 1997). Therefore
MCS mRNA appears to be expressed only in haploid cells since low levels
could not be detected in Northern blots of RNA from pachytene primary
spermatocytes from 18 day prepubertal mice (Shih and Kleene, 1992). In
rats, the cDNA contains a reading frame for a 145-amino-acid protein and it
lacks the UGA codons, which have been found in the reading frame of the
mouse MCS cDNA and encodes the selenocysteine in the amino terminal
of the deduced mouse amino acid sequence (Adham et al., 1996). The rat
MCS gene contains two exons; the intron sequence interrupts the 5'
untranslated sequence at the same position as in the mouse MCS gene
(Adham et al., 1996). In human, the MCS is one of three proteins that are
important for the maintenance and stabilization of the crescent structure of
the sperm mitochondria (Aho et al., 1996). Nucleotide sequence analysis
of the human and mouse MCS cDNAs reveals that the 5'- and 3 untranslated sequences are more conserved (71%) than the coding sequences
(59%) (Aho et al., 1996). The open reading frame encodes a 116-aminoacid protein and lacks the UGA codons, which have been reported to encode
the selenocysteines in the N-terminal of the deduced mouse protein.
Northern blot and in situ hybridization experiments demonstrate that the
expression of the human MCS gene is restricted to haploid spermatids and
the human gene was assigned to q21 of chromosome 1 (Aho et al., 1996).
Hamster MCS cDNA was 820 bp long, including 24 bp of the 5-untranslated
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region (UTR) and 243 bp of the 3'-UTR, and showed identity of 75.6% and
73.9% with mouse and rat MCS (Nam et al., 1998). The authors also showed
that hamster MCS contained 2 in-frame UGA codons for selenocysteine
and MCS mRNA was expressed in various tissues as well as the testes.
During spermatogenesis, the hamster MCS mRNA first appeared in step 6
spermatids, gradually increased in round spermatids during spermiogenesis,
reached a peak in step 8 spermatids, and persisted a high level until step 13
spermatids. After step 14, the signal began to show a progressive decline in
the spermatids and was weakly detected in the tails of step 17 spermatids.
However, the signal was not observed in spermatogonia, spermatocytes,
Sertoli cells, peritubular myoid cells, and interstitial cells (Nam et al., 1998a).
It is interesting that hamster MCS mRNA was expressed in various tissues
as well as the testes (Nam et al., 1998a). Recently it has been suggested
that MCS could no longer be considered as essential selenoprotein (Kohrle
et al., 2000). Instead, PH-GSH-Px was found to contribute to the formation
of the mitochondrial capsule (Ursini et al., 1999). In fact, the authors showed
that PH-GSH-Px changed its physical characteristics and biological functions
during sperm maturation. It exists as a soluble peroxidase in spermatids but
persists in mature spermatozoa as an enzymatically inactive, oxidatively
cross-linked, insoluble protein (Ursini, 1999).
Did you know that sperm capsular selenoprotein and
PH-GSH-Px are the same selenoprotein?
In the midpiece of mature spermatozoa, PH-GSH-Px protein represents at least 50
percent of the capsule material that embeds the helix of mitochondria. It was
concluded that the protein thiol peroxidase activity of PH-GSH-Px is responsible
for cross-linking proteins in the mammalian sperm capsule and accounts for the
selenium dependency of spermatogenesis (Rovery, 2001). Therefore PH-GSHPx is considered to be the mitochondrial capsule selenoprotein of mammalian
sperm (Flohe et al., 2003). Indeed, further work is needed to clarify if PH-GSHPx is the same protein as MSC or it if it represents only part of it.
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Selenoprotein N was identified by Lescure et al. in 1999. As in other

selenoproteins incorporation of SeCys takes place at a redefined UGA codon
and requires the involvement of stemloop structure formed by the SeCIS
sequence. The human SEPN1 gene is located on chromosome 1p36 (RSMD1
locus) and contains 13 exons spanning 18.5 kb (Moghadaszadeh et al., 2001)
and it produces a 4.5 kb transcript encoding a 590 amino acid protein. The
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authors showed that mutations in SEPN1 caused congenital muscular dystrophy
with spinal rigidity and restrictive respiratory syndrome. Therefore, it was the
first description of a selenoprotein implicated in a human disease. Furthermore,
a relationship between classical multiminicore disease (MmD; an autosomal
recessive congenital myopathy characterized by the presence of multiple, short
core lesions in most muscle fibers) and the selenoprotein N gene (SEPN1) was
suggested (Ferreiro et al., 2002). Therefore, desmin-related myopathy with
mallory body-like inclusions was suggested to be caused by mutations of the
selenoprotein N gene (Ferreiro et al., 2004). The main SEPN1 gene product
corresponds to a 70 kDa protein, containing a singly SeCys residue (Petit et al.,
2003). The authors also showed that SeN is a glycoprotein localised within the
endoplasmic reticulum. SEPN1 was found to present in high levels in several
human fetal tissues and at much lower levels in adult tissues, including skeletal
muscle. This protein is also highly expressed in cultured myoblasts (Petit et al.,
2003). The authors suggested a role of SeN in regulation of early development
and in cell proliferation or regeneration.
Selenoprotein R recently, was identified as a zinc-containing stereo-specific
methionine sulfoxide reductase B (Kryukov et al., 2002; Moskovitz et al.,
2002). Furthermore, it has been shown that there was a loss of MsrB activity
in the MsrA/ mouse in parallel with losses in the levels of MsrB mRNA
and MsrB protein (Moskovitz and Stadtman, 2003). Se deficiency in mouse
was associated with a substantial decrease in the levels of MsrB-catalytic
activity, MsrB protein, and MsrB mRNA in liver and kidney tissues
(Moskovitz and Stadtman, 2003). It has been reported that human and mouse
genomes possess three MsrB genes responsible for synthesis of the following
protein products: MsrB1, MsrB2 and MsrB3 (Kim and Gladyshev, 2004).
Did you know that selenoprotein R (methionine sulfoxide
reductase B) is an enzyme of the third level of antixidant
defence responsible for prevention/repairing protein oxidation?
In particular, MsrB1 (Selenoprotein R) was present in the cytosol and nucleus

and exhibited the highest methionine-R-sulfoxide reductase activity due to
presence of selenocysteine (Sec) in its active site. Other mammalian MsrBs are
not selenoproteins and contain cysteine in place of Sec and were less catalytically
efficient (Kim and Gladyshev, 2004). Details of biochemical functions of MsrB
were described in Chapter 1.
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Selenoprotein S has been recently identified by Kryukov at al (2003). It is

considered to be a novel member of the glucose-regulated protein family
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and its function is related to the regulation of cellular redox balance. Recently
the induction of the SelS gene in HepG2 cells has been characterized and
its function examined as an antioxidant (Gao et al., 2004). Indeed SeS gene
expression was shown to be up-regulated in the liver of Psammomys obesus
after fasting. In fact SelS is regulated by glucose deprivation and endoplasmic
reticulum (ER) stress in HepG2 cells. For example, glucose deprivation and
the ER stress inducers tunicamycin and thapsigargin increased SelS gene
expression and protein content several-fold. The overexpression of SelS
increased Min6 cell resistance to oxidative stress-induced toxicity (Gao et
al., 2004). Therefore, this study widen existing knowledge about regulatory
functions of selenoproteins and showed for the first time that selenoprotein
can be induced by ER stress and glucose starvation.
Selenoprotein U was characterised by Castellano et al. (2004). They used a
comparative genomics approach that relies on the genome-wide prediction
of genes with in-frame TGA codons, and the subsequent comparison of
predictions from different genomes. They applied this method to various
genomes and identified a novel selenoprotein family, named SelU, which
present in fish, chicken and sea urchin. In particular, selenium incorporation
into chicken SelU was demonstrated and the SelU expression pattern in
zebrafish embryos was characterized (Castellano et al. (2004). In great
contrast, mammals, worms and land plants contained cysteine homologues.
Data of Castellano et al. (2004) indicate a scattered evolutionary distribution
of selenoproteins in eukaryotes, and suggest that other taxa-specific
selenoproteins probably exist.
Selenoprotein T and selenoprotein X have been recently identified as
members of the family of selenoproteins, however their functions are as yet
unclear (Lescure et al., 1999; Kryukov et al., 1999).
As shown above the a selenoprotein family is getting bigger every year and it
seems likely that selenoproteins are a key element in regulating antioxidant system
of the body. For example, important roles of selenoproteins in endothelial cell
function have been recently reviewed (Brigelius-Flohe et al., 2003) and the authors
showed that selenoproteins are involved in the regulation of :
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the vascular tone by affecting the superoxide/nitric oxide balance

cell adhesion by controlling cell adhesion molecule expression
apoptosis via regulation of apoptosis signal-regulating kinase-1
eicosanoid production by controlling the activity of cyclooxygenases and
lipoxygenases
inflammatory processes and atherogenesis by above mentioned mechanisms
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102
Molecular mechanisms of selenoprotein action are diverse (Brigelius-Flohe et al.,

2003) including :
scavenging hydroperoxides needed for direct enzyme activation and

regulating eicosanoid production and signalling events
repairing oxidized proteins (methionine sulfoxide reductase B) by reducing
SH groups into active form
prevention of oxidative inactivation of enzymes
modulation of protein phosphorylation by thioylation
release of Trx from complexes with kinases
regulation of hydroperoxide-dependent protein/protein interaction
A general scheme of selenoproteins related to animal production is shown in

Figure 2.5. Today we probably know only a limited number of the mechanisms
by which Se is involved in diverse systems such as redox signalling, regulation of
apoptosis, immunomodulation, spermatogenesis and embryonic development.
Recent findings associated with many various roles of thioredoxin and glutathione
systems in the cell could aid in explaining the effects of Se deficiency on animals,
particularly poultry. Indeed, lipid peroxidation is involved in the development of
diseases such as exudative diathesis, encephalomalacia, muscular and pancreatic
dystrophy. However an understanding of the molecular mechanisms of these wellknown diseases is still lacking. It seems likely that compromised glutathione/
thioredoxin redox systems in tissues as a result of Se and/or vitamin E deficiency
could be responsible for structural changes (as a result of damage to membranes)
leading to metabolic changes and development of clinical deficiency symptoms.
Biological systems contain a range of regulatory mechanisms to prevent damaging
effects of free radicals and toxic products of their metabolism. In particular, vitamin
E recycling, gene redox regulation and anti/pro-oxidant redox signalling should
be given increased attention in future studies. As mentioned previously, all the
antioxidants work together to create an integrated antioxidant system. For example,
in Se deficiency redox status of the cell could be maintained by the glutathione
system. However, the antioxidant system is dependent on the nutritional provision
of antioxidants (vitamin E and carotenoids) and cofactors (Se, Mn, Cu, Zn, Fe)
and has a limited ability to overcome dramatic stress conditions (high levels of
toxins or pro-oxidants in the feed, severe disease, etc.).
Taking into account a central role of selenoproteins in antioxidant system
regulation, it would be appropriate to call selenium chief-executive of antioxidant
system. Recent information on the effect of antioxidant-prooxidant balance in
the cell on various metabolic pathways as well as an importance of ROS in cell
signalling events stimulate re-consideration of antioxidant system regulation by
nutritional means. Indeed, excess of vitamin E or other such antioxidants as
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carotenoids is shown to have no immediate threat to animal/human health.
However, in long term, changes in antioxidant-prooxidant balance in the body/
cell resulted from overconsumption of antioxidants could be detrimental. On the
other hand, by providing body with optimal amounts of co-factors, such as Se in
right form and building Se reserves could be more safe option of affecting
antioxidant defences. Indeed, this could give an opportunity to existing regulatory
mechanisms to be in place and prevent possible damages. For example, in stress
conditions, the body can respond properly by additional synthesis of selenoproteins
to deal with overproduction of free radicals.
GSH -Px ( 6 forms)
Selenoprotein P
Selenoprotein W
Antioxidant
defence
Methionine sulfoxide
reductase B
Thioredoxin reductases
(3 forms)
Iodothyronine
5 -deiodinases
(3 forms)
Sperm capsule
selenoprotein,
PH -GSH -Px
Redox regulation
of gene
expression
Thyroid
metabolism
Sperm structure
integrity
Diseases:
Muscular dystrophy
Impaired fertility
Increased incidence of
mastitis,retained placenta and
cystic ovaries
Decreased: growth,
immunocompetence,
meat quality, feed efficiency
Elevated milk somatic cell
count
Poor thermoregulation and
Increased cold sensitivity
Compromised sperm motility,

viability and fertilizing capacity
Figure 2.5 Selenoproteins in animal production (Adapted from Surai, 2003)
Selenium-vitamin E interactions: antioxidant properties and beyond

From information presented above it is possible to conclude that selenium is a
key element in antioxidant defence in the body. What is more, selenium works in
close relation with other antioxidants, in particular with vitamin E. In fact nutritional
essentiality of Se was established on the basis of its interaction with vitamin E.
Beneficial effects of dietary Se were first seen in vitamin E-deficient rats (Schwarz
and Foltz, 1957). At that time vitamin E deficiency was well described and later it
was shown that Se could prevent some, but not all of the symptoms of vitamin E
deficiency in various animal species. Some selenium- and/or vitamin E-responsive
nutritional diseases in rats are shown in Table 2.19 (Levander et al., 1995). In
broader terms vitamin E and selenium deficiency is related to many different
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conditions in farm and laboratory animals where all major organs are affected
(Table 2.20). In many cases a combination of these two compounds is needed to
achieve the best effect in their treatment or prevention.
Table 2.19 Nutritional diseases of rats related to selenium or/and vitamin E deficiency (Adapted from
Levander et al., 1995)
Conditions responsive to dietary

Se
Vitamin E
Liver necrosis
IN second generation
Sparse hair coat
Poor growth
Poor sperm motility
Cataracts
Discoloration in body fat
Discoloration of uterus
Depigmentation of incisors
In vitro haemolysis
Impaired reproductive capacity of females
+
+
+
+
-
+
+
+
+
+
Did you know that selenium and vitamin E are working

in concert providing effective antioxidant defence?
Among various selenium-related disorders and diseases exudative diathesis (ED)
is the most studied one. ED is the disease which appears in chickens deficient in
both vitamin E and Se and is characterised by severe oedema as a result of an
increased permeability of the capillaries in combination with reduced levels of
blood proteins (Kristiansen, 1973). As a result of leakage of blood fluid through
the capillaries and from minor haemorrhages in muscles, in the area of the breast
under the skin an accumulation of an exudate with a protein pattern similar to
blood serum or plasma can be seen. Hemoglobin degeneration causes a bluishgreen colour of the exudates, which can be seen through the skin. Autopsy findings
and histopathological lesions were observed only in subcutaneous tissue and
skeletal muscle. In particular, the subcutaneous tissue was edematous with hyaline
vascular lesions and hemorrhages (Hassan et al, 1990). The thigh muscles were
more susceptible to deficiency lesions than were the breast muscles, and showed
in acute stages degenerative processes of the muscle fibers including calcium
deposits, vascular lesions and hemorrhages. In subacute and chronic cases,
reparative changes and muscle damage may develop independently of the hyaline
vasculosis (Hassan et al., 1990).
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Table 2.20 Diseases associated with selenium and vitamin E deficiency in animals (Adapted from
MacPherson, 1994; Surai, 2002)
Syndrome
Tissue or organ
affected
Species
Encephalomalacia
Cerebellum
Exudative diathesis
Microtic anaemia
Liver necrosis
Pancreatic fibrosis
Erythrocyte haemolisis
Muscular degeneration
Vascular
Blood, bone marrow
Liver
Pancreas
Erythrocytes
Skeletal muscle
Microangiopathy
Heart muscle
Kidney degeneration
Embryonic degeneration
Poor hatchability
Steatitis
Testicular degeneration
Kidney tubules
Vascular system
Egg embryo
Adipose tissue
Testes
Retained placenta
Impaired fertility
Ill-thrift
Placenta
Spermatozoa
Thyroid, pituitary
Chick, turkey, emus, partriges, quail,

pheasant and various zoo species
Chick, turkey, duck, salmon, catfish
Chick, monkey, pig, rat, salmon, catfish
Pig, rat, mouse
Chick, salmon, mouse
Chick, lamb, monkey, rat
Chick, duck, goose, ostrich, flamingo,
monkey, dog, rabbit, guinea pig, horse,
calf, lamb, kid, mink, antilope, pig,
rodents, salmon, catfish
Turkey, pigs, calf, lamb, rat, dog, rabbit,
guinea pig, cow, sheep, goat, baboon,
antelope, elephant, deer
Monkey, rat, mouse
Pig, rat, mouse
Chick, turkey
Pig, chick
Pig, calf, chick, pig, monkey, rat, rabbit,
guinea pig, hamster, dog
Cow
Sheep, cattle, poultry, pig, rat
Lamb, calf
The condition can occur at any age but is most prevalent in young growing chickens
and turkeys (Whitehead and Portsmouth, 1989). In the chicks, obtained from
laying hens depleted of vitamin E and selenium exudative diathesis was observed
at hatching, indicating that the deficiency lesions had developed during the
embryonic period, whereas these signs were not observed in chicks obtained
from commercial laying hens adequate in vitamin E and Se on the depletion diets
until they were 2 weeks old (Hassan et al., 1990).
ED will only occur if the diet is deficient in selenium and Se is 200 times more
effective than vitamin E in preventing this disease. Therefore this symptom is
primarily considered as a selenium deficiency (Machlin and Gordon, 1962). ED
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106
is also described in ducklings (Dean and Combs, 1981). ED develops in chickens

at age of 3-6 weeks and chicken mortality can be as high as 80% (for review see
Surai et al., 1994; Surai, 2002). ED was associated with low levels of muscle Se,
liver Se-GSH-Px and vitamin E and was also accompanied by a simultaneous
increase in the liver non-Se-GSH-Px (Hassan et al., 1990). It has been suggested
that the inflammatory response associated with an Se/vitamin E deficiency in a
chick may be responsible for ED (Bartholomew et al., 1998). The authors
suggested that cytokines produced by leukocytes could be responsible for
transparent fluid accumulation and hemorrhaging. It is well recognised that Se is
the major protection against ED. The vitamin E supplemental level of 15 mg/kg
was not adequate to provide a complete protection against ED (Hassan et al.,
1990). ED was inhibited by the rutin and silymarin treatments, but exacerbated
by quercetin, morin, and ferulic acid. Changes in concentrations of vitamin E in
plasma, liver, or muscle, caused by the various treatments (other than vitamin E),
were not related to protection against ED (Jenkins et al., 1992).
As mentioned above, molecular mechanisms of development of ED as well as
nutritional encephalomalacia (NE) and nutritional muscular distrophy (NMD) are
still not clear. However it is generally believed that oxidative stress is a crucial
factor in disease development. Indeed, lipid peroxidation, disrupting membrane
structure and function, is responsible for specific changes in the brain, muscles
and vascular systems. In this respect a great body of evidence indicates that vitamin
E is effective in preventing NE, NMD and ED. However, effective doses of this
vitamin for prevention of the diseases are different and its combinations with
other antioxidants such as selenium of cysteine are of great importance.
Furthermore protein oxidation and inactivation is another important factor involved
in those pathologies. Based on the concept of integrated antioxidant system (Surai,
2002) it is feasible to suggest that vitamin E recycling and its interactions with
other antioxidants, including selenium, ascorbic acid, glutathione etc. are driving
forces in prevention of vitamin E deficiency symptoms. For example, recent
understanding of the role of glutathione- and thioredoxin systems in general and
thioredoxin reductase in particular, helps explaining many interactions between
vitamin E and selenium in preventing NMD and ED. Indeed, thioredoxin system
is responsible for the maintenance of proteins in active reduced form and could
be considered as an important factor in the development of NMD. Furthermore
thioredoxin reductase takes part in vitamin E recycling via interactions with
ascorbic acid and could be a crucial point in prevention of NE and ED. Clearly
there is a need for further research in this fascinating field and chicken model
with specific diseases associated with vitamin E deficiency could be used for
further elucidation of molecular mechanisms of antioxidant interactions.
From information presented above it is clear that Se and vitamin E are important
elements in maintaining antioxidant system efficiency. An additive or co-operative
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effect of selenium and vitamin E is shown in chick embryonic development (Surai,
2000), in improvement of meat quality (Surai and Dvorska, 2002; 2002a), in
effect on semen quality (Surai et al., 1998), immunity, mycotoxicosis and in some
other cases (Surai, 2002). These interactions are mediated via GSH-Px, PH-GSHPx, GI-GSH-Px, thioredoxin reductase or SeMet itself (Figure 2.6).
Thioredoxin reductsase recycles ascorbic acid improving
vitamin E recycling, detoxifying peroxides decreasing vitamin E
requirement
GSH-Px removes H2O2 decreasing free radical formation

and decreasing vitamin E requirement for AO defence
Vitamin E
GSH-Px removed LOOH completing an antioxidant

protection started by vitamin E
PH-GSH-Px removes LOOH from membranes helping
vitamin E in antioxidant protection
Selenium
GI-GSH-Px deals with hydroperoxides in the GIT

decreasing vitamin E oxidation there
SeMet possess antioxidant properties dealing with NO
and peroxynitrile decreasing vitamin E requirement
Figure 2.6 Vitamin E-selenium interactions (Adapted from Surai, 2003).
It seems likely, that there are some specific stress conditions where protective
effect of selenium cannot be replaced by vitamin E. For example, GSH-Px provided
protection of the mice body against paraquat-induced lethality, while a-tocopherol
(20 mg/kg diet) was not effective (Cheng et al., 1998; 1998a). Using the GSHPx1 knockout mice it has been shown that even high vitamin E doses (750 or
7500 mg/kg diet) while inhibiting lipid peroxidation in the liver were not able to
replace the protection of GSH-Px against paraquat-induced lethality in mice
(Cheng et al., 1999). In the same study a linear increase in PHGSH-Px activity in
the liver as a result of increase in dietary vitamin E supplementation. Furthermore
it seems likely that normal GSH-Px expression is necessary to protect mice against
lethality and hepatic protein oxidation due to diquat (a herbicide generating
superoxide radical) toxicity (Fu et al, 1999). It is interesting that vitamin E can
affact GSH-Px activity in Se-deficient animals (Table 2.21). Futhermore, Se and
vitamin E deficiency affact the expression of a range of genes (Table 2.22). Indeed,
vitamin E deficiency alone did not induce any significant changes in expression
profile among the genes evaluated (Fisher et al., 2001). Se deficiency caused a
down-regulation of GSH-Px and induction of genes, encoding for detoxifying
enzymes in liver. However, combined vitamin E and Se deficiency affected much
more genes (Fisher et al., 2001). This means that a simplistic presentation of the
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Table 2.21 Effect of Se and vitamin E on antioxidant defences in rats (Adapted from Fisher et al., 2002)
Parameter
Selenium, g/kg
-Tocopherol, g/g
GSH-Px1, mU/mg protein
TrxR, mU/mg protein
GST, mU/mg protein
-Se, -vit.E
-Se, +vit.E
+Se, -Vit.E
+Se, +vit.E
25.8
1.03
5.45
1.32
209
31.8
29.5
10.9
1.16
226
892.3
1.15
154.2
9.46
141
881.5
32.9
161.0
9.25
162
Table 2.22 Effect of Se and vitamin E deficiencies on gene expression in rat liver (Adapted from Fisher
et al., 2002)
GenBank
-Se
Gene
Accession no. -Vit.E, fold
Function
X12367
J05181
18.8
3.4
PO4800
2.5
Stress response
GSH-Px1
Antioxidant protection
Glutamate-cysteine ligase GSH synthesis
catalytic subunit
Cytochrome P-450 3A1
Xenobiotic metabolism
J03969
D14014
2.9
3.1
Cell cycle
Nucleophosmin
G1/S-specific cyclin D1
Y13336
2.0
AF081503
2.6
U72350
3.2
Y22424
2.2
L49379
2.3
Apoptosis
Deefender against cell
death 12 protein
Inhibitor of apoptosis
protein I
Bcl2-L1
Inflammation
11--Hydroxysteroid
dehydrogenase 2
CMOT (canalicular multispecific organic anion
transporter)
Stimulation of normal cell growth

Inhibition of cell cycle, oncogene
Protection against apoptosis
Protection against apoptosis
Promotes cell survival
Conversion of corticosterone into
11-dehydrocortocosterone
Detoxification, export of
leukotriene C4
role of these two compounds as just antixidant components is not valid anymore.
Even though selenium and vitamin E are working together, there are various
functions for these two distinctive antioxidants where they cannot be replaced by
each other. For a nutritionist who is looking for the best productive and reproductive
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characteristics of farm animals this means that only an optimal combination of
these two elements is a valuable choice. Furthermore, organic selenium provides
extra benefits in comparison to traditional selenite and based on the recent findings
it seems likely that the full replacement of selenite by organic selenium is just the
matter of time.
It has been suggested that antioxidant/prooxidant balance in the body is
responsible for maintaining human and animal health, productive and reproductive
performances of farm animals (Surai, 2002). This balance can be adversely
modulated by sub-optimal diets and nutrient intakes or positively affected by
dietary supplementation. Therefore, feed components can modulate maintenance
of this balance and may thereby influence immune system and disease resistance
of the human and animals. Thus, the most important step in balancing oxidative
damage and antioxidant defence in the animal body would be to enhance the
antioxidant capacity by optimising the dietary intake of antioxidants. In this case
selenium and vitamin E could be considered as major players.
Prooxidant properties of selenite and possible antioxidant protection

by selenomethionine
It is somewhat surprising that the most commonly used inorganic selenocompound, sodium selenite, is capable of promoting superoxide radical formation
and oxidative stress through its reductive reaction with reduced glutathione.
Generation of superoxide radical by the reaction of selenite with reduced
glutathione was first reported in 1988 (Garberg et al., 1988; Kramer and Ames,
1988; Seko et al., 1989). The important insight on the mechanisms of ROS
formation came from the work of Yan and Spallholz (1993). In their experiment
sodium selenite, sodium selenate, selenocystine and SeMet were tested for their
abilities to generate superoxide by the oxidation of glutathione and other thiols in
the absence or presence of the human mammary tumor cell line HTB123/DU4475.
The data suggested that superoxide radical and H2O2 are produced from the reaction
of selenite and selenocystine with glutathione. For example, in the absence of
tumor cells, free radical generation (lucigenin-dependent chemiluminescence) was
observed from the reaction of selenite with the thiols glutathione, 2mercaptoethanol and L-cysteine, but not with SeMet. Superoxide dismutase,
catalase, and GSH-Px all suppressed the observed chemiluminescence; but when
these enzymes were heat-inactivated they had little suppressive inhibition on
chemiluminescence. The enhanced ROS production by selenite and selenocystine
in the presence of the tumor cells was also suppressed by superoxide dismutase,
catalase and GSH-Px.
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Did you know that selenite is a pro-oxidant and SeMet
possesses antioxidant activity?
Later the techniques used in those early experiments to detect ROS were questioned
(Seko and Imura, 1997). However, a great deal of evidence analysed by Spallholz
(1994) clearly showed that selenite Se is a pro-oxidant catalyst. Spallholz concluded
that Se compounds are toxic owing to their pro-oxidant catalytic activity to produce
superoxide (O2.-), hydrogen peroxide, and very likely other cascading oxyradicals.
The proposed scheme of superoxide radical generation was as follows (Seko et
al., 1989; Spallholz, 1997; Shen et al., 2000):
4GSH
SeO32-
GSSG
GSH GSSG
GSSeSG
GSH
GSSeH
GSSG
O2
H2Se
O2*Se0
Many Se compounds have been assessed in relation to their ability to produce

superoxide in vitro (Spallholz, 1997) and the results are summarised in the Table
2.23. Similarly, in their review of the prooxidant action of Se compounds Seiko and
Imura (1997) showed that it was possible to register superoxide radical and in some
cases hydroxyl radical formation as a result of selenite and glutathione reaction using
different techniques including luminol-dependent chemiluminescence, a salicylate
hydroxilation method, the decomposition of deoxyribose, single breakage of plasmid
DNA and an electron spin resonance method. Free radical production was shown to
be dependent on oxygen concentration and ultimately was responsible for Se toxicity
(Seiko and Imura, 1997). This was based on the results of several investigations
indicating increased thiobarbituric acid reactive species (TBARS) production and
other indexes of lipid peroxidation in the case of Se toxicity (Seiko and Imura, 1997;
Hoffman et al., 1991; Csallany et al., 1984). Therefore based on the in vitro results
and some in vivo findings, Seiko and Imura suggested that Se compounds are able to
generate ROS within tissues in vivo. In great contrast, in most experiments SeMet
was not able to produce ROS when added into incubation medium in a combination
with reduced glutathione.
Table 2.23 Ability of selenium compounds to generate superoxide in vitro* (Adapted from Spallholz,
1997)
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Superoxide produced in vitro
Superoxide not produced in vitro
Selenite
Selenium dioxide
Selenocystine
Diselenodipropionate
Diphenyldiselenide
Selenomethionine
Selenate
Elemental selenium
Selenobetaine
K-selenocyanate
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It has been confirmed that selenite generates ROS and causes cellular damage in
the presence of sulfhydryl compounds (Terada et al., 1999). When Se in the form
of selenite, selenate or SeMet were added to total parenteral nutrition and
intravenously administered, selenite generated ROS in the presence of clinical
concentrations of sulfhydryl compounds. This resulted in significant increases in
the [3H]-adenine and lactate dehydrogenase release rates from cells, a significant
decrease in the amount of cellular protein, and enhancement of cellular damage
as compared with after exposure to selenite alone.
The effects of selenite on DNA integrity, cell viability, and long-term proliferative
potential of mouse leukemic L1210 cells were examined by Lu et al. (1994).
Selenite treatment resulted in concentration-dependent increases in DNA singlestrand breaks and double-strand breaks. A time-course experiment showed that
DNA single-strand breaks preceded DNA double-strand breaks. Agarose gel
electrophoresis of DNA extracted from selenite-treated cells displayed a
nucleosomal fragmentation pattern that is characteristic of apoptotic cell death.
Therefore, selenite treatment of a mouse mammary tumor cell line rapidly induced
DNA damage and cell death (Lu et al., 1995; 1995a).
In experiments with human colonic carcinoma cells, selenite (>5 M) decreased
cell growth, increased cell detachment and decreased intracellular levels of reduced
glutathione (GSH), whereas >10 M selenite induced cell differentiation and
apoptosis (Stewart et al., 1997). When primary cultures of human keratinocytes,
melanocytes or the HaCaT cell line were preincubated with Se, it has been shown
that selenite was much more toxic to cells than was SeMet (Raferty et al., 1998).
For example, 1 M selenite killed approx. 25% of cells, and at a dose of 10 M
more than half the cells were destroyed. However, in the same experiment SeMet
showed no toxicity at the doses used in the study. In an experiment with human
promyelocytic leukemia HL-60 cells, the dose-response data of apoptosis induced
by selenite or selenodiglutathione were similar to those of cytotoxicity, implicating
a relationship between the induction of apoptosis and cytotoxicity (Cho et al.,
1999).
Did you know that selenite causes apoptosis and in the same
conditions SeMet is not related to apoptosis?
Therefore, superoxide radical formation and oxidative stress is related to the
induction of apoptosis in selenite-exposed cancer cells (Stewart et al., 1999; Shen
et al., 1999; 2000). It seems likely that selenite-induced apoptosis is related to the
ROS generation and specific factors activation. For example, in a model system
the liver cell apoptosis induced by selenite (10 microM) was confirmed by DNA
fragmentation and typical apoptotic nuclear changes (Kin et al., 2004). Treatment
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of selenite increased intracellular ROS level and c-Jun N-terminal kinase1 (JNK1)
phosphorylation. It is interesting that antioxidants such as GSH, N-acetyl cysteine,
curcumin, epigallocatechin gallat and epicatechin inhibited selenite-induced
intracellular ROS elevation and JNK1 phosphorylation (Kim et al., 2004).
The experimental results of Stewart et al. (1999) suggested that selenite and
selenocystamine generated 8-hydroxydeoxyguanosine DNA adducts, induced
apoptosis and were found to be cytotoxic in mouse keratinocytes. On the other
hand SeMet was not cytotoxic, did not generate 8-hydroxydeoxyguanosine adducts
and did not induce cellular apoptosis at any of the Se concentrations studied.
Therefore, in keratinocytes, apoptosis may be initiated by superoxide (O2*-) and
oxidative free radicals that are generated by selenite and selenocystamine, but
not by SeMet. Co-incubation of ascorbic acid or copper sulfate with selenite
appeared to protect primary human keratinocytes against selenite-induced
cytotoxicity. However, synergistic effects were observed between selenite and
trolox resulting in enhanced cytotoxicity (Shen et al., 2001).
Menter et al. (2000) showed that sodium selenite is a potent inducer of apoptosis
in normal and cancerous prostate cells. At the same time SeMet selectively induces
apoptosis in cancer but not primary cells of the human prostate. Similarly, Sundaram
et al. (2000) showed that selenite had a significant inhibitory effect on growth of
tumor cells but had little effect upon dermal fibroblasts that had been passaged
numerous times. Selenium also induced mitochondrial damage and high rates of
apoptosis in two brain tumor cell lines and in minimally passaged fibroblasts.
Therefore these results clearly showed the damaging effect of selenite on cells
and indicated that some types of cells after repeated passages can develop resistance
to Se damage.
Sodium selenite exerted clear cytotoxic effects on a human hepatoma cell line.
Shen et al. (1999) showed that Se-induced cell death occurs predominantly in the
form of apoptosis. The involvement of glutathione in selenite-induced oxidative
stress was further demonstrated by the concurrent decline of intracellular reduced
glutathione and an increase in oxidized glutathione content of Se-treated cells.
Moreover, the finding that selenite-induced oxidative stress and apoptosis was
significantly attenuated by superoxide dismutase, catalase and deferoxamine
provides additional evidence to suggest that Se-induced oxidative stress mediates
the induction of apoptosis. Recently, Shen et al. (2001) provided convincing
evidence that the intracellular O2- formed through the reaction of selenite with
GSH is a potent proapoptotic agent and mainly acts on mitochondria to trigger
the apoptotic signalling pathway.
The prooxidant effect is probably tissue-dependent, reflecting an antioxidant
composition and concentrations in each tissue studied. For example, the effect of
sodium selenite (0.05, 0.1, and 0.2 mg/kg body weight, i.p.) on the thiobarbituric
acid reactive substance (TBARS), and sulfhydryl group in the striatum and thalamus
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of a male Wistar rat was studied after 7 d of treatment (Zia and Islam, 2000). The
content of TBARS was elevated dose dependently in striatum, but its level was
depleted significantly with 0.1 mg/kg dose of sodium selenite in the thalamus. In
general, Se toxicity occurs as a result of increased thiol oxidation, redox cycling
and superoxide generation in a dose dependent manner (Stewart et al., 1999).
Vitamin E- and Se-deficient rats given selenite produced 15 times as much ethane
as did controls (Dougherty and Hoekstra, 1982). It was concluded that the increased
vulnerability of vitamin E- and Se-deficient rats to acute selenite toxicity might
involve peroxidation in vivo.
Selenium prooxidant action and cytotoxicity are important subjects in relation
to the anticancerogenic action of this trace element. In fact, it has been demonstrated
that cancer cells had higher sensitivity to Se cytotoxicity in comparison to normal
cells (Stewart et al., 1997; Medina and Oborn, 1981; Fico et al., 1986). This
difference in sensitivity to Se can reflect differences in glutathione concentration
in different cells. For example, recently Shen et al. (2000) demonstrated that GSH
has a dual role in Se cytotoxicity acting as a prooxidant to induce oxidative stress
or as an antioxidant to protect against Se-induced oxidative stress.
It is interesting to note that in most of the experiments reported above, ROS
generation and cellular damage were not observed after simultaneous
administration of various concentrations of selenate or SeMet with sulfhydryl
compounds (Terada et al., 1999). In contrast, selenomethionine (SeMet) is a
relatively non-toxic, non-catalytic and non-redoxing Se compound exhibiting low
toxicity and not producing superoxide (Stewart et al., 1999). In fact, when Se as
the selenite, SeMet, ebselen or Se-yeast were investigated in an in vitro LDL
oxidation model, it was shown that Se-yeast is a powerful in vitro and in vivo
antioxidant (Vinson et al., 1998). Similarly, treatment of lymphocytes with SeMet
prior to adding H2O2 caused an inhibition in peroxyl radical formation in a manner
dependent on SeMet concentration (Sun et al., 1997). Furthermore, SeMet is
considered as a powerful antioxidant protecting against damaging effects of
peroxynitrite. For example, it protected human fybroblast lysates from toxic effect
of peroxynitrite (Sies et al., 1998). SeMet also protected dihydrorhodamine 123
from oxidation and 4-hydroxyphenylacetate from nitration caused by peroxynitrite
while sodium selenite exhibited no effect (Briviba et al., 1996). It should also be
noted that the selenoxides can be effectively reduced by glutathione, establishing
a biological line of defence against peroxynitrite (Sies et al., 1998; Assmann et
al., 1998). The rapid and efficient reduction of the selenoxide to SeMet by
glutathione in a stoichiometric reaction utilizing two equivalents of thiol has been
described (Assmann et al., 1998). Probably radioprotective, and UV-protective
properties of SeMet (Schrauzer, 2000; 2003) could also be associated with its
antioxidant properties. SeMet has also been shown to have protective effect on
gap junctional communications diminished by peroxynitrite (Sharov et al., 1999).
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The antioxidant properties of SeMet were also demonstrated in a model system

based on olive oil oxidation process at 303 K and 333 K (Zalejska-Fiolka, 2000).
In addition to aforementioned beneficial effects of SeMet, it seems likely that
this selenoaminoacid is directly involved in the third line of antioxidant defence.
For example, recently it has been shown that SeMet induces a DNA repair response
and protects normal fibroblasts from DNA damage (Seo et al., 2002).
The induction of DNA repair occurred at a concentration (10 M) easily
attainable in vivo by dietary supplementation. Seo et al. (2002a) also showed
that selenium in the form of SeMet can activate the p53 tumor suppressor protein
by a redox mechanism that requires the redox factor Ref1. In fact, SeMet induced
sequence-specific DNA binding and transactivation by p53 indicating a stimulation
of DNA repair branch of the p53 pathway.
It is interesting that no evidence for enhancement of repair of methyl
methanesulfonate-, UV- or bleomycin-induced DNA damage was observed in
human fibroblasts treated with selenite (Snyder, 1987). In fact, selenite was shown
to induce DNA strand breaks as measured by two independent assays (Snyder,
1988). Sodium chromate induced DNA breaks in Chinese hamster V-79 cells
mediated by the formation of free radicals and/or cellular reductive metabolism
and pretreatment with sodium selenite resulted in an enhanced formation of DNA
breaks (Sugiyama et al., 1987). Selenate is also able to increase one-electron
oxidation of plasmid DNA (Milligan et al., 2002). Selenite is toxic and slightly
mutagenic for yeast leading to DNA damage (Pinson et al., 2000). Coincubation
of ascorbic acid or CuSO 4 with selenite appeared to protect primary human
keratinocytes against selenite-induced cytotoxicity. However, synergistic effects
were observed between selenite and trolox resulting in enhanced cytotoxicity. On
the other hand, SeMet alone or in combination with vitamin C, trolox or CuSO4
did not affect cell viability (Shen et al., 2001). Furthermore, selenium-containing
compounds can protect DNA from damage caused by peroxynitrite. For example,
SeMet and selenocystine protected DNA from single-strand breaks more effectively
than their sulfur analogs, methionine and cystine, and they also were more effective
than glutathione or the hydroxyl radical scavenger mannitol (Roussyn et al., 1996).
Furthermore, SeMet decreased DNA damage imposed by peroxynitrite more
effectively (by 77% at 0.1mM concentration) than selenocystine (by 42% at 1
mM concentration; Epe et al., 1996).
Did you know that SeMet decreases DNA damage imposed
by free radicals?
The extent of DNA damage in prostate cells and in peripheral blood lymphocytes,
as determined by the alkaline comet assay, was lower among the seleniumsupplemented (Se-Met or yeast-enriched with selenium) dogs than among the
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control dogs but was not associated with the activity of the antioxidant enzyme
GSH-Px in plasma (Waters et al., 2003).
Therefore it is clear that SeMet has a unique specific effect on the DNA repair
system as well as provides a protection against DNA damage and this effect is
not the case or even can be opposite when selenite is used. This effect of SeMet is
related to anticancer properties of this compound. In fact Mukherjee et al. (2001)
suggested several mechanisms related to this properties of SeMet:
acceleration of detoxification of carcinogenic compounds

altering hepatic drug metabolism
protection against chromosomal damage
enhancement of DNA-repair process
strengthen antioxidant protection
stimulation of immune defence system
All aforementioned mechanisms of beneficial effect of SeMet are relevant to animal/

poultry production and only some of them can be shared by vitamin E.
From the analysis the Se antioxidant/prooxidant properties it is possible to
draw the following general conclusions:
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Selenite is potentially highly toxic due to reactions with glutathione and

possibly other sulfhydryl compounds present in biological systems that
ultimately produce ROS. In contrast SeMet does not participate in pro-oxidant
reactions and in some cases possesses antioxidant properties.
Pro-oxidant properties of selenite and other Se compounds are an important
finding to aid in explaining some of the anticancer effects of Se. However,
in physiological context pro-oxidation effects can be detrimental. For
example, increased post-mortem drip loss from pork or broiler meat (Mahan,
1999; Edens, 2001) could be a result of the pro-oxidant action of selenite
inclusion in food animal diets.
Pro-oxidant actions of selenite should be studied in relation to possible
oxidative reactions in the digestive tract of animals associated with decreased
nutrient assimilation and development of various diseases. Indeed, feed
contains a range of antioxidants (tocopherols, tocotrienols, carotenoids,
flavonoids etc.) as well as pro-oxidants (Fe, Cu, various hydroperoxides
etc.). In such conditions the oxidizing potential of Se compounds can have
a substantial effect on lipid peroxidation. At present, oxidant and redox
control of pathophysiologic processes in the gastrointestinal tract is not well
understood (Aw, 1999). In general, enterocytes are characterised by
comparatively high turnover. For example in rats enterocytes turn over within
48-72 h (Miller et al., 1977). The intestinal mucosa is constantly challenged
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by diet-derived oxidants, toxic compounds and free radicals and toxic
products of their metabolism (Ames, 1983; Aw, 1999). In such conditions
antioxidant protective mechanisms in the intestine are the first line of defence
against all those toxic elements. Therefore, in order to maintain cellular
integrity and tissue homeostasis, the intestine has a range of defence
mechanisms (Aw, 1999), many of which are Se-dependent. For example,
gastrointestinal GSH-Px is the major protection against oxidative stress in
the intestine. Tocopherols, tocotrienols, carotenoids, ascorbic acid and
glutathione, which are released from the feed during digestion, are also of
great importance. There are other less studied nutrients with antioxidant
properties such as flavonoids, which also can contribute to antioxidant
defence. However, the most important defence in the intestine is provided
by up-regulation of antioxidant enzymes as well as by signalling mechanisms
responsible for cell death by apoptosis to dispose of injured or spent
enterocytes (Aw, 1999). Indeed intestinal glutathione is considered
responsible for maintenance of cellular redox balance and respective gene
regulation (Aw, 2003).
Prooxidant properties of selenite are proven, but molecular mechanisms of
this effect need further investigation. However, using organic Se in form of
SeMet could be a valuable alternative to avoid detrimental consequences of
the prooxidant properties of selenite.
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Selenium in Food and Feed: Selenomethionine and Beyond 151

3
SELENIUM IN FOOD AND FEED: SELENOMETHIONINE AND
BEYOND
Nature does nothing in vain
Introduction
Selenomethionine (SeMet) was first researched as a possible cause of toxicity of
seleniferous wheat in the 1950s and was later proven to be synthesised from
inorganic selenium sources by various plants, including yeast, marine algae,
Candida albicans, as well as by Escherichia coli and rumen bacteria (for review
see Schrauzer, 2000; 2003). Detailed analysis of the literature suggested that
organic selenium in the form of various selenoamino acids is a natural form of
selenium in animal and human diets and that the digestive system adapted to this
nutrient form during evolution, thereby explaining why there are principal
differences in assimilation and metabolism between organic and inorganic forms
of selenium (Surai, 2002). Therefore there is an inconsistency in common practise
of selenium supplementation of animal diets. On one hand, naturally occurring
organic selenium is represented by a mixture of selenoamino acids with SeMet
comprising more than 50% of total selenium in many feed ingredients, including
grains and forages, etc. On the other hand, until recently the supplemental form
of selenium for farm animals and poultry has been inorganic, either selenite or
selenate. Recent approval by the US Food and Drug Administration of organic
selenium in the form of selenized yeast (Sel-Plex, Alltech Inc.) for poultry, pigs
and cows is going to resolve the discrepancy between natural and supplemental
selenium sources.
Selenium in soils and plants

Selenium (Se) is a chemical element with atomic number 34 and atomic weight
78.96 belonging to group VI of the periodic table of elements. This group also
includes such non-metals as sulphur and oxygen (Figure 3.1). In nature Se exists
in two chemical forms, organic and inorganic. Elemental Se can be reduced to
the Se2 oxidation state (selenide) or oxidized to the Se+4 (SO3-2, selenite) or Se+6
(SO4-2, selenate). Therefore inorganic Se can be found in different minerals in the
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152
form of selenite, selenate and selenide as well as in the metallic (Se0) form. In
contrast, selenium in feed ingredients (forages, grains, oilseed meals, etc.) is an
integral part of the amino acids selenomethionine and selenocysteine and exists
in the Se-2 oxidation state. As a result, in nature animals receive Se mainly in the
form of selenomethionine (SeMet; Combs and Combs, 1986). Indeed SeMet is
considered a natural nutritional form of selenium for animals and human (Table
3.1).
15
16
P
39.974
33
As
74.922
17
Cl
32.06
35.453
34
35
Se
Br
79.904
78.96
51
52
Sb
121.75
53
Te
127.60
I
126.91
Figure 3.1 Selenium position in the periodic table of elements.
Did you know that Selenium in nature exists in various

oxidation states: +6 (selenate), +4 (selenite), 0 (metallic Se)
and 2 (selenide, SeMet, SeCys and others)?
The selenium cycle in the food chain of land animals and humans starts from
soils and includes plant and animal sources ultimately dependent on its assimilation
from the soil. Indeed, soils are the major source of Se for plants and therefore for
animals eating those plants and humans consuming plant and animal-derived
foods. Selenium concentration in soils varies significantly (Reilly, 1996). The Se
content of most soils ranges between 0.1 and 2 ppm; and soil Se exists in various
forms, including selenides, elemental Se, selenites, selenates and organic Se
compounds (Selenium in Nutrition, 1983). High concentrations of Se are found
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Table 3.1 Selenomethionine: a historic perspective (Adapted from Schrauzer, 2000;2003 and Surai,
2002).
Year
Se-Met-related events
1930
Suspected that SeMet is one of toxic compounds of seleniferous plants
1949
Evidence that SeMet presents in seleniferous wheat
1950-1960
SeMet is identified in plant proteins and produced by growing yeast
1970
Metabolic studies indicate that SeMet is well absorbed and metabolised in

animals
Commercial synthesis and marketing of L-Se-Met
1984
1980-2000
Numerous experiments proved that SeMet and Se-Yeast are suitable forms of
Supplemented Se
1990-2000
Results of a clinical trials published showing a protective effect of Se in the

form of Se-Yeast against cancer
1998-2001
Antioxidant properties of Se-Met were described
2001-2002
Se-Yeast is cleared by FDA for chicken, turkey and pig nutrition
2000-2003
Se-Yeast is used to produce Se-enriched eggs, pork, chicken and milk which
found their way on supermarket shelves in various countries
2001-2002
Two major cancer-preventing clinical trials (SELECT and PRECISE) started

using Se-Yeast and Se-Met as sources of selenium
2002
Se-Met is shown to stimulate DNA-repairing enzymes
2003
Se-Yeast is cleared by FDA for ruminants
2004
Se-enriched eggs produced by using Sel-Plex are on supermarket shelves in

more than 25 countries
mainly in sedimentary rocks and shales formed during the cretaceous period,
while lower concentrations of Se are characteristic for igneous (volcanic) rock,
sandstone, granite and limestone (Van Metre and Callan, 2001). Investigations
conducted in China indicated that soils developed under tropic and subtropic
conditions (laterite, yellow soil and red soil) are characterised by comparatively
high Se levels (>0.3 ppm; Tan et al., 2002). In contrast, the soils developed under
the temperate (warm) steppe and desert conditions (chernozem, chestnut soil,
calcic brown soil, desert soil and solonchak) have moderate Se concentrations
(0.14-0.30 ppm). Finally, such soils as brown earth, drab soil, dark brown soil,
loessial soils, purple soil, red drab soil, developed under the temperate (warm)
humid/sub-humid conditions are quite poor in Se (Tan et al., 2002). In particular,
low Se soils occur mainly in the northeast to the southwest of China.
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154

Did you know that Se in plants depends more on its availability
from soils than on actual soil Se concentration?
Furthermore, Se availability to plants depends on many factors including soil pH,

the oxidation-reduction potential and mineral composition of the soil, rate of
artificial fertilization and rainfall. In fact, the bioavailability of Se in soils for
plants depends more on its form than on its total concentration:
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In the case of acidic soils or poor soil aeration, Se can form insoluble
complexes with iron hydroxide and become poorly available. For example,
at pH 6, only 47% of labelled Se was transferred from soil to ryegrass leaves.
Increasing pH to 7 increased Se assimilation to 70% (Haygarth et al., 1995).
Indeed, Se in alkaline soils occurs in selenate form, where it is soluble and
easily available to plants.
Since sulfate competes with selenate for uptake by the sulfate transporter,
high soil sulfate decreases Se uptake by plants (Terry et al., 2002). This
explains low Se availability from soils following application of certain types
of fertilizers.
Selenium can also be leached from the topsoil in areas of high rainfall.
Therefore areas with higher rainfall have lower forage selenium content.
Solubility is the critical determinant of Se bioavailability to plants and the
amount of water-soluble Se in soils varies substantially and does not correlate
with total soil Se (Combs and Combs, 1986).
Selenite is strongly adsorbed by soils while selenate is only weakly absorbed
and leaches easily.
Selenide and elemental Se are usually found in reducing environments and
are unavailable to plants and animals.
Selenite is present in mildly oxidizing, neutral pH environments and typically
humid regions, while selenate is the predominant form under ordinary
alkaline and oxidized conditions (Goh and Lim, 2004). The authors also
showed that the adsorption of selenite and selenate by soils appeared to be
influenced by the variable pH-dependent charges on the soil particle surfaces.
In particular, phosphate had more profound effects than sulfate on Se
adsorption in the soil.
Application of gypsum (calcium sulfate) to soils decreased Se availability
for plants (Selenium in Nutrition, 1983)
Leaching during the soil development process and irrigation water decreased
Se level in plants (Selenium in Nutrition, 1983)
Forage Se is reported to be low on sandy soils and lower on mineral upland
soils than on organic moorland soils in the British Isles (MacPherson, 2000).
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The main chemical changes under long-term waterlogged conditions are

depletion of molecular oxygen, decrease of redox potential, and reduction
of Fe (III) to Fe (II) and SeO3-2 to Se0. This leads to low availability of Se in
soils, and subsequently low Se content (29 g/kg) in brown rice grain
produced in this Chinese region (Cao et al., 2001). Indeed, selenite binds
tightly to iron and aluminium oxides and thus is quite insoluble in soils
(Jonnalagadda and Rao, 1993).
Did you know that low soil pH, low aeration and presence of
sulphur either naturally or from certain fertilizers decreases Se
availability from soils?
Selenium is transported via the xylem to chloroplasts in leaves where it is processed

by the sulphur assimilation pathway into organic compounds. The, selenate form
is transported more easily from root to shoot than is selenite or organic Se (Terry
et al., 2002). Plants differ markedly in their ability to incorporate selenium from
soil into tissues; and based on this ability plants are divided into three major
categories:
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Selenium accumulators. Some species hyperaccumulate Se in leaves and

stems when grown on seleniferous soils. These plants stimulated the initial
Se research, since they caused Se toxicity in animals grazing them. Selenium
accumulator genera include species of Astragalus, Stanleya, Morinda,
Neptunia, Oonopsis, and Xylorhiza (Terry et al., 2000). These plants can
accumulate up to several mg Se per g of dry weight and are ultimately toxic
to animals. When these plants were grown hydroponically in the presence
of selanate, Se in the older leaves was predominantly inorganic while in
young leaves and roots was mainly (90-95%) in organic form (Ellis and
Salt, 2003). A specific odour causes grazing animals to avoid them on pasture
when other forages are available.
Secondary Se accumulators accumulate high Se concentrations even when
grown on soils with low or medium Se content. They include such species
of the genera Aster, Astragalus, Atriplex, Castilleja, Comandra, Grayia,
Grindelia, Gutierrezia, Machaeranthera and Brassica (Terry et al., 2000).
It is important to understand that in Se-accumulator plants, Se is not
incorporated into proteins. This contrasts with non-accumulator plants such
as typical forage and cereal grains where Se is found predominantly in
protein-bound form (Muth and Oldfield, 1967). This type of Se accumulator
plant is more an exception than the rule of Se metabolism in plants.
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156
Non-accumulator plants. The third category includes most forage, cereal

and oilmeal crop plants. Such plants contain less than 25 mg Se/kg dry
weight and do not accumulate Se in access of 100 mg/kg even when grown
on seleniferous soils (Terry et al., 2000). These plants typically have Se
concentrations in a range of 0.01 to 1.0 mg/kg dry weight. However, there
are species-specific differences in Se accumulation from the same soil. For
example, lucerne is shown to accumulate more Se than other grasses under
conditions of moderately low soil Se concentrations (Van Metre and Callan,
2001). Indeed, lucerne contains more selenium than in timothy, cocksfoot
or brome grass (Ehlig et al., 1968).
Did you know that there are 3 major categories of plants in
relation to Se accumulation: hyperaccumulators, secondary
accumulators and non-accumulators?
After absorption, the distribution of Se in various parts of the plant depends on

species, phase of development and physiological conditions. For example, Se
distribution was studied in Astragalus bisulcatus, an accumulator species capable
of accumulating up to 0.65% of its shoot dry biomass as Se (Pickering et al.,
2000). It was shown that plants exposed to 5 M selenate for 28 days contained
predominantly selenate in the mature leaf tissue, whereas the young leaves and
the roots contained exclusively organic Se. From this work it is clear that the fate
of selenate is differs with plant tissues and stage of growth. Therefore chemical
reduction of selenate to organic Se in plants is tissue-specific, inducible and
developmentally dependent. It is likely that selenate reduction is rate-limiting in
the conversion of Se to organic forms (Pickering et al., 2000).
Plants as major sources of selenium for animals and human

The plant absorbs Se from the soil in the form of selenite or selenate and synthesises
selenoamino acids with SeMet representing more than 50% of the Se in cereal
grains (Olson and Palmer, 1976) with Se-methyl-selenomethionine, selenocysteine
and Se-methyl-selenocysteine being the other seleno-compounds found in plants
(Brody, 1994). In general, plants can also take up from the soil organic forms of
selenium such as SeMet. At present, Se in any form has not been scientifically
demonstrated to be an essential nutrient for higher plants. Regardless, SeMet is
the major selenocompound in cereal grains, grassland legumes and soybeans
(Whanger, 2002; Table 3.2). For example, in corn, rice, wheat and soybeans,
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SeMet comprises 45.5-82%, 54.9-86.5%, 50.4-81.4% and 62.9-71.8% of total
Se, respectively (Yang et al., 1997). In human blood samples SeMet represented
56.1-74.4%. However, other data indicate that selenoprotein P (SeP) and
glutathione peroxidase (GSH-Px) comprise 70-80% total Se (Table 3.3). It is
possible that Se species in appearing in plasma depend on dietary Se sources.
Even in wheat grown on seleniferous soils Se (up to 31 ppm Se), almost half
occurred in the form of SeMet (Olson et al., 1970). The majority of the Se is
present as SeMet in both rice and corn (Beilstein et al., 1991). SeMet was the
main Se-containing amino acid identified in most of the extracts of Indian mustard
(Brassicaj uncea), sunflower (Helianthus annus), and white lupine (Lupinus albus;
Ximenez-Embun et al., 2004).
Table 3.2 Distribution of SeMet and selenocystine (SeCys) in various biological material, % of total
(Adapted from Whanger, 2002).
Biological materials
Rats injected with selenite
Rats injected with SeMet
1 day
5-35 days
Wheat grain
Corn
Rice
Soybeans
Phytoplankton
Astragalus
Se enriched garlic
Se enriched onions
Se enriched Broccoli Floretes
Se enriched Broccoli Sprouts
SeMet
SeCys
6-10
64-70
63
14-25
56-83
61-64
68-81
>80
3.2
37
1-6
1-4
5
12
22
46-57
4-12
15-16
6-10
1-13
7-38
11
-
Table 3.3 Distribution of Se in human plasma (Adapted from Whanger, 1998).
Plasma components
Se, ng/ml
GSH-Px, %
SeP, %
Albumin
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Controls
Se-enriched yeasta
127
15
67
18
214
17
52
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158

Did you know that the major part of selenium in grains and
forages (non-accumulator plants) is represented by organic Se
compounds with SeMet comprising more than 50% of total Se?
The variability in results of Se specification investigations of plant material reflects

analytical difficulties. For example, by using SeMet determination based on its
reaction with CNBr, it has been shown that wheat samples, though having a 30fold range in total Se content, all have about 45% of their total Se values in the
form of SeMet (Wolf and Goldschmidt, 2004). However, the authors suggested
that additional experiments were needed to verify that all selenomethionine in the
wheat samples had been accounted for. Even in seleniferous corn, wheat and
soybeans, SeMet represented more than 80% of total Se. SeMet is stored mainly
in the grain and the root, while lower concentrations of this amino acid are found
in the stems and leaves (Schrauzer, 2003).
It is interesting to note that the richest source of Se for human consumption,
Brazil nuts, also contains SeMet as the most abundant selenoamino acid
(Vonderheide et al., 2002). However, organic Se compounds can differ
substantially, depending on the plant material analysed, a range of
selenocompounds have been detected (Table 3.4). Analytical speciation studies
showed that the bulk of the Se in Se-garlic and Se-yeast is in the form of gammaglutamyl-Se-methylselenocysteine (73%) and SeMet (85%), respectively (Table
3.5; Ip et al., 2000). Se-methylselenocysteine is the major selenocompound in
Se-enriched plants such as garlic, onions, broccoli florets and sprouts, and wild
leeks (Whanger, 2002).
Table 3.4 Selenocompounds identified in plants (Adapted from Whanger, 2002; Birringer et al., 2002).
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Se-compound
Se-compound
Selenate
Selenite
SeCys
SeMet
Selenohomocysteine
Se-methylselenocysteine
-glutamyl-selenocystathionine
Selenomethionine selenoxide
-glutamyl-Se-methylselenocysteine
Se-adenosylselenohomocysteine
4-selenouridine
Selenosugars
Selenocysteineselenic acid
Se-proponylselenocysteine selenoxide
Se-methylselenomethionine
Selenocystathionine
Dimethyl diselenide
Selenosinigrin
Selenopeptide
Selenowax
Selenobiotin
Selenolanthionine
3-butenyl isoselenocyanate
-glutamyl-Se-methionine
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Table 3.5 Distribution of Se compounds in garlic and yeast (Adapted from Birringer et al., 2002).
Compound
Selenate
Selenite
selenocystine
Selenocystathione
Se-Methylselenocysteine
-Glutamyl-Se-methylselenocysteine
SeMet
-Glutamyl-selenomethionine
Se-Adenosylselenohomocysteine
Selenolanthionine
Total Se, %
Garlic (296 mg/kg Se)
Yeast (1922 mg/kg Se)
2
nd
0.5
0.5
3
73
13
4
Nd
Nd
96
Nd
1
0.5
1
0.5
0.5
85
Nd
3
1.5
93
Nd not detected
Selenium specification in various products is an important task for future research.

For example, mushrooms comprise a good source of Se (Table 3.6), however, Se
species in Se-accumulating mushrooms are yet to be determined. After proteolysis,
only a small fraction of the extractable Se could be identified as selenite (3.09.2%, Albatrellus pes-caprae), selenocystine (minor, Albatrellus pes-caprae; 7.5%,
Boletus edulis), or SeMet (1.0%, Boletus edulis), leaving the form of the bulk still
to be elucidated (Slejkovec et al., 2000). It seems likely that SeMet is not the
major form of Se in mushrooms, since SeMet accounts for only a minor (8.218.3%) amount of the selenocompounds present in mushroom (Ganoderma
lucidum) proteins (Zhao et al., 2004). It is interesting that the selenized shiitake
mushroom (Lentinula edodes) accumulated Se as SeMet, which is bound to the
water extractable high molecular mass proteins (Ogra et al., 2004). However,
analytical difficulties are the major obstacle for full identification of
selenocompounds in food and feed sources. For example, the sum of identified
Se species in the tuna and mussel samples was about 30% of the total Se present
in the enzymatic extract despite the fact that recoveries of total hydrolysed Se
were 93-102% (Quijano et al., 2000). Trimethylselenonium ion and SeMet were
found in both tuna and mussel samples and an unknown Se species was also
found in tuna samples.
Did you know that the major sources of variability of data on
Se species in plants and animal tissues are analytical
difficulties because of low stability of such compounds
under analytical conditions?
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Table 3.6 Selenium in mushrooms (Adapted from Golubkina et al., 2002)
Name
Boletus edulis
Rozites caperata
Agaricus campester
Clitocybe clavipes
Amanitopsis crocea
Clitocybe nebularis
Agaricus bispoirus
Suilus bovinus
Lactarius vellereus
Lycoperdon pyriforme
Lepista nuda
Leccinum aurantiacum
Lactarius torminosus
Tricholoma portentosum
Boletus erythropus
Suilus luteus
Verpa Bohemica
Russula fietens
Tricholomopsis rutilans
Lactarius scrobiculatus
Lactarius flexuosus
Se, mg/kg
Range of concentrations
21.06
10.9
6.5
5.1
3.9
3.4
3.0
3.1
2.9
2.7
2.2
2.1
1.8
1.5
1.5
1.4
1.3
1.3
1.2
1.2
1.1
14.1 27.9
1.25 14.5
1.1 11.5
3.8 6.0
2.1 5.7
2.0 4.2
1.9 4.1
1.9 4.0
1.1 3.6
1.2 3.1
1.3 3.0
0.9 3.4
1.7 2.0
0.7 2.4
0.8 2.1
0.1 2.4
1.0 1.5
0.8 - 1.7
0.9 1.4
0.9 1.3
0.6 1.2
It is generally accepted that environmental conditions and agricultural practises

have a major effect on the Se content of various plant feeds. Water extractable Se
accounted for 60.4-72.6% of the total Se in a plant (Stanleya pinnata) extract.
Among the soluble Se compounds in the plant extract, Se-amino acids comprised
73-85.5%, Se [VI] ranged from 7.5 to 19.5% and non-amino acid organic Se was
less than 7% (Zhang and Frankenberger, 2001). Selenium [IV] in most samples
was below the detection limit (1 g/g). This study showed that considerable
amounts of the accumulated Se [VI] in the plant was metabolized to Se-amino
acids during growth of the plant. The distribution of selenoamino acids in a Setolerant grassland legume species (Melilotus indica L.) grown in Se-laden soils
was studied using high-resolution gas chromatography and gas chromatography/
mass spectrometry (Guo and Wu, 1998). Five selenoamino acids including
selenocystine, selenomethionine, selenocysteine, Se-methylselenocysteine, and
gamma-glutamyl-Se-methylselenocysteine were identified and measured in plant
tissues. SeMet constituted more than 50% of the total selenoamino acid in the
plant. It seems likely that rate of Se accumulation in plants and its form depend on
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the Se form provided. For example, time-dependent kinetic studies in Indian
mustard (Brassica juncea) showed that selenate was taken up 2-fold faster than
selenite (de Souza et al., 1998). For both selenate- and selenite-supplied plants,
Se accumulation and volatilisation increased linearly with external Se
concentration. It is important to note that Se-volatilisation rates were 2- to 3-fold
higher in plants supplied with selenite compared with selenate. In fact, the
assimilation of selenate by plants appeared to be limited by its reduction, a step
that is thought to be mediated by ATP sulfurylase, which is rate limiting for selenate
uptake and assimilation (Pilon-Smits et al., 1999). Furthermore, there was a
difference in Se metabolism among plants supplemented with various forms of
Se. For example, selenite-supplied plants accumulated organic Se, most likely
SeMet, whereas selenate-supplied plants accumulated selenate (de Souza et al.,
1998). It seems likely that Se volatilisation from selenate is limited by the rate of
selenate reduction, as well as by the availability of Se in roots, as influenced by
uptake and translocation. On the other hand, Se volatilisation from selenite may
be limited by selenite uptake and by the conversion of SeMet to dimethylselenide
(de Souza et al., 1998).
Selenium volatilisation is also an important step of Se metabolism in algae. For
example, an axenically cultured isolate of single-celled freshwater microalgae
(Chlorella sp.) metabolized toxic selenate to volatile dimethylselenide at
exceptionally high rates when transferred from mineral-nutrient solution to water
for 24 h. The Se-volatilisation rates were orders of magnitude higher than those
similarly measured for wetland macroalgae and higher plants. Ninety percent of
20 M selenate supplied to the microalgae incubated without nutrients was removed
through accumulation and volatilisation. Additions of 1 mM sulphate, but not
nitrate, inhibited Se accumulation and volatilisation so that only 1.8% of the supplied
selenate was removed (Neumann et al., 2003).
Did you know that Se volatilisation is the protective mechanism
for plants against Se toxicity?
Selenium absorption and metabolism

Recent advances in Se biochemistry have provided a deeper understanding of the
principal differences in metabolism of the two forms of Se namely inorganic Se
(sodium selenite or selenate) and organic Se (mainly SeMet). Organic Se, which
can be found in grains, forages and other feed ingredients, is primarily in the
form of SeMet and is metabolised in the same way as methionine (Wolfram, 1999).
It is actively transported through intestinal membranes during absorption and
actively accumulated in such tissues as liver and muscle. It is well known that
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methionine is not synthesised by animals or humans and therefore it is an essential

amino acid. The same is true for SeMet, which is not synthesised in animals or
humans and must be derived from feed sources (Schrauzer, 2000; 2003; Figure
3.2). In contrast, inorganic Se is absorbed as a mineral and little is retained in
tissue reserves. Therefore, a large part of inorganic Se is excreted with faeces in
ruminants or with urine/urates in non-ruminants with little is stored in body
proteins (Wolfram, 1999).
Selenite (SeO3-2)
Selenate (SeO4-2)
+GSH
+ATP
Selenoglutathione
trisulfide
Adenosyl-phosphoselenate
Hydrogen selenide
(H2Se)
Non-acumulator
plants, marine algae,
bacteria and yeast
Plant proteins
Plant
selenoproteins
+Serine
Accumulator plants
Selenocysteine
+Homoserine
Selenomethylcysteine
Selenocystathionine
Selenohomocysteine
Selenomethionine
Figure 3.2 Selenomethionine biosynthesis in plants, marine algae and brewers yeast (Adapted from
Shchrauzer, 2000; 2003).
Did you know that selenite is absorbed as a mineral by passive

diffusion in the gut, but SeMet is absorbed similar to methionine
using active transport in the digestive tract?
A number of factors influence the bioavailability and distribution of selenium in
the body (Thomson, 1998) including
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chemical form of Se
other dietary components
selenium status
physiological status
species
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Most of the research in Se biochemistry and metabolism has been using inorganic
Se, namely selenite or selenate. For example, the concentrations of 82Se in organs
and body fluids and the distributions of their constituents depending on the dose
and time after the intravenous administration of 82Se-selenite and -selenate to rats
were investigated (Suzuki and Ogra, 2002). Selenite was taken up by red blood
cells within several minutes, reduced to selenide by glutathione, and then
transported to the plasma, bound selectively to albumin and transferred to the
liver. In contrast to selenite, intact selenate was either taken up directly by the
liver or excreted into the urine. In fact, the 82Se of selenite origin and that of
selenate origin were detected in the forms of the two Se peak materials in the
liver, A and B. The former was methylated to the latter in vivo and in vitro. The
latter was identical with the major urinary metabolite and was identified as Semethyl-N-acetyl-selenohexosamine (selenosugar). The chemical species-specific
metabolic pathway for Se was explained by the metabolic regulation through
selenide as the assumed common intermediate for the inorganic and organic Se
sources and as the checkpoint metabolite between utilization for selenoprotein
synthesis and methylation for the excretion of Se (Suzuki and Ogra, 2002).
Results of various in vitro and in vivo experiments with a variety of animal
species and model systems have demonstrated that SeMet is readily absorbed
through the gut. Indeed, SeMet is better absorbed than selenite (Daniels, 1996).
However, absorption is not a limiting factor to bioavailability. The specific role
of the chick duodenum in the assimilation of Se (selenate or selenite) was shown
by Apsite et al. (1993). Selenite is passively absorbed in the intestine with highest
concentrations found in duodenum, liver and kidneys (Apsite et al., 1994).
Absorption of Se was found to be greatest in the duodenum and anterior ileum of
the chicken (Pesti and Combs, 1976). Absorption of 75Se from selenite, selenate,
and SeMet was determined in ligated loops from duodena, jejuna, and ilea of Sedeficient rats (0.009 ppm Se) or rats fed selenite-supplemented diets (0.20 ppm
Se) (Vendeland et al., 1992). Selenium deficiency had no effect on absorption of
any selenocompound in any intestinal segment. SeMet was absorbed from all
segments. In contrast, selenate and selenite were most efficiently absorbed from
the ileum. In mice SeMet was absorbed in the entire intestinal tract, most rapidly
from the duodenum, with decreasing rate of absorption in the following intestinal
segments (Andersen et al., 1994).
Did you know that in many cases SeMet is better absorbed
than selenite?
Selenomethionine was more rapidly removed from the ligated intestinal segment
of chicken and more efficiently retained after oral or parenteral administration
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(Humaloja and Mykkanen, 1986). The percentage absorption of both Se

compounds was greatest from the duodenal segment of the small intestine. The
transport of these Se compounds does not appear to depend on the dietary level
of Se since the percentage absorption was not altered by feeding the birds diets
supplemented with 0.4 or 4.0 ppm Se prior to the measurement of absorption
(Humaloja and Mykkanen, 1986). Selenium absorption from the dog jejunum
(expressed as percent administered dose per centimetre) was 1.97 from D,L-SeMet,
1.15 from D,L-selenocystine, and 0.51 from sodium selenite (Reasbeck et al.,
1985). In separate studies in four anesthetized dogs, the jejunum was perfused
with L-[75Se] SeMet while concentrations of 75Se were measured in the portal
venous blood; these studies established that [75Se]SeMet disappearing from the
gut lumen corresponded quantitatively to 75Se appearing in the portal venous
effluent (74%) and incorporated into intestinal tissue (24%). These results are
consistent with the hypothesis that the absorption of amino acid-bound Se is
accelerated by the specific amino acid active transport mechanisms in the gut
mucosa. Sodium selenite is absorbed more slowly, possibly by simple diffusion
through the intestinal mucosa, than the amino acid-bound Se compounds.
Growing male birds were given orally 4 muCi of 75Se per kg live weight each
in the form of DL-SeMet, and were bled (via the wing vein) at fixed intervals
(Stanchev et al., 1979). It was found that the resorption of 75Se-SeMet through the
digestive tract of birds as registered by the radioactivity of the blood was most
intensive between the 3rd and the 6th hour, after which a decline was observed.
Most intensive was the metabolism of SeMet in the kidneys, spleen, testes, and
pancreas. Comparatively lower is the metabolism of this agent in the breast, bone
and in the brain. Radioactivity of SeMet was lowest in the femoral muscle. As
much as 18.2% of the introduced amount of SeMet was excreted for 72 hours
through the faeces and urine.
The relative efficiency patterns for uptake of different selenocompounds during
in vitro perfusion and in vivo ligated segments was SeMet > selenate > selenite. In
contrast, selenite was taken up most rapidly by brush border membrane vesicles,
followed by SeMet and selenate in decreasing order. Selenate and SeMet appeared
in the vascular effluent largely unchanged, but selenite was metabolized
extensively during absorption (Whanger et al., 1996). The uptake of selenite,
selenate and SeMet was studied using brush border membrane vesicles prepared
from rats fed Se-deficient and supplemented diets. The uptake of 75 Se from
[ 75 Se]selenite ranged from 16.5 to 18.9 nmol/mg protein with a curvilinear
relationship in the uptake of selenite over a concentration range of 10-1000 M
(Vendeland et al., 1994). In contrast, only about 2 nmol/mg protein was obtained
with SeMet, but there was a linear relationship in the initial uptake of SeMet over
a concentration range of 10-1000 M. On the other hand, the uptake of selenate
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was approximately 50-fold lower than selenite, reaching 350 pmol/mg protein.
However, dietary Se level had no effect on the rate of 75Se accumulation by brush
border membrane vesicles. Selenomethionine as well as methionine were
transported across the pig jejenual brush border membrane by a single, Na +dependent, carrier-mediated process common for both amino acids (Wolffram et
al., 1989). A study was undertaken to evaluate the in vitro availability of chemically
varying forms of Se supplemented in cows milk. Two inorganic (selenite and
selenate) and two organic (SeMet and SeCys) sources were evaluated. The in
vitro availability was estimated by the diffusibility of Se during simulated
gastrointestinal digestion. When the diffusibility was compared after adding a
constant amount of Se in various forms, SeMet and selenate were found to be
significantly more diffusible than SeCys and selenite under the simulated
gastrointestinal conditions (Shen et al., 1997).
The metabolism of 75Se-selenite and 75Se-SeMet in chick blood was studied in
vitro. 75 Se from selenite was rapidly taken up by erythrocytes and then
subsequently released into the plasma in a protein-bound form. However, 75SeSeMet showed a more gradual and continuous accumulation in erythrocytes over
the 12-hr incubation period, according to a hyperbolic type function. 75Se-selenite
was incorporated into GSH-Px, whereas 75Se from SeMet was mostly incorporated
into hemoglobin (Ilian and Whanger, 1989). In the same experiment binding of
75
Se from either selenite or SeMet to plasma proteins was dependent on the
presence of erythrocytes. Addition of reduced glutathione and glutathione reductase
to plasma produced the same effects as erythrocytes on binding of 75Se from
selenite, but not from SeMet, to plasma proteins. It was also shown that 75Se in
these Se-containing proteins is bound in selenotrisulfide bond.
When selenite or selenocystine was the dietary form of Se for rats, the majority
of the erythrocyte Se was present with GSH-Px, but when the Se was supplied
from either SeMet, yeast or wheat, it was deposited to a greater extent in Hb than
GSH-Px (Beilstein and Whanger, 1986). Injection of 75Se as either SeMet or selenite
gave results consistent with the feeding studies. 75Se-labeled selenite injection
resulted in labelling of primarily GSH-Px, but 75 Se-Met injection labelled
predominantly Hb. No differences were found in deposition of 75Se in liver, kidney,
testes, erythrocytes or plasma in rats injected with labelled selenite or SeMet, but
a significantly greater retention was found in muscle of SeMet-injected rats as
compared to those given selenite.
Did you know that SeMet can be non-specifically incorporated
into body proteins thereby building Se reserves in the body,
which can be used in stress conditions when the Se
requirement is increased but feed consumption is low?
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166
About 3% of total plasma Se of healthy adults was bound to lipoproteins, mainly

to the LDL fraction (Ducros et al., 2000). After solvent fractionation of LDL and
HDL, the major part of the Se was recovered in the protein extract, suggesting
that it may be incorporated in apolipoproteins. The exact form of Se is not yet
clearly established, but considering the different Se compounds found in proteins,
it was postulated to be SeMet. The distribution of Se in plasma fractions was
investigated in guinea pigs fed various levels (basal, 0.5, 1.0, 2.0, 4.0, 6.0 and 8.0
mg Se/kg) of dietary SeMet (Gu et al., 1998). There was a corresponding increase
of Se concentration in liver, kidney, brain, testis, spleen, heart and muscle with
each increase of dietary Se, but GSH-Px activity did not change in liver, brain,
testis, heart or muscle in pigs fed any of the Se levels as compared to controls fed
a basal commercial diet. There was a redistribution of Se between various fractions
in the blood. For example, on a percentage distribution basis, the Se in
selenoprotein P decreased, and that in the albumin fraction increased with increased
dietary intakes of Se as SeMet. Similarly, the greatest percentage of Se was in the
albumin fraction of Chinese people living in the high Se areas, whereas the greatest
amount was in the selenoprotein P fraction in subjects living in deficient and Seadequate areas of China (Gu et al., 1998). Increases in the ratios of Se:albumin in
either the plasma or the albumin fraction also occurred with increases of Se intake
of these subjects.
The majority of the Se was in the hemoglobin (Hb) fraction in women taking
supplemental SeMet, but was about equally distributed between GSH-Px and Hb
in women taking selenate (Butler et al., 1991). Therefore, the percentage of Se
associated with GSH-Px was found to be greater in RBCs and plasma of women
taking selenate than of those taking SeMet. About 68% of erythrocyte Se was
associated with GSH-Px in monkeys given selenite whereas only 34% was
associated with GSH-Px in those administered SeMet (Butler et al., 1990). Selenium
in breast milk occurs as GSH-Px (4-32 % total Se) >selenocystamine >
selenocystine > selenomethionine (Dorea, 2002).
The results of recent study (Okuno et al., 2002) indicated that in mouse liver
SeMet was directly metabolized to CH 3SeH by an alpha, gamma-elimination
enzyme analogous to bacterial L-methionine gamma-lyase, in addition to the
generally acceptable pathway via selenocysteine. It has been suggested that Lselenohomocysteine generated from SeMet metabolism can be efficiently recycled
to SeMet in mammals (Zhou et al., 2000). When sodium selenite was administered
to chickens, the kidney was the most responsive to additional dietary Se, while
skeletal (thigh) muscle showed no response to supplemental Se (Guenter and
Bragg, 1977). The total Se contents in the kidney and in the liver from rats treated
with SeMet, selenocysteine or sodium selenite were markedly higher than those
in other organs. The highest accumulation of the total Se was observed in these
two organs when rats were given SeMet (Nakamuro et al., 1997).
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The number of published studies in animals and man suggested that the
metabolic fate and physiological function of dietary selenite may differ from that
of SeMet or of food Se (Figure 3.3). It has been postulated that there are two
distinct metabolic pools of Se in the body (Daniels, 1996). The main exchangeable
metabolic pool includes all forms of Se derived from inorganic selenite/selenide,
including endogenously synthesized selenoproteins (e.g. GSH-Px, selenoprotein
P, etc.), excretory Se metabolites (trimethylselenium ion) and various other
intermediary products of selenite metabolism. This is an active Se pool providing
for synthesis of the primary functionally important selenocompounds (Daniels,
1996). The second Se pool consists of SeMet-containing proteins and potentially
can contribute to the first pool via participation in selenoprotein synthesis.
General body
proteins
Food
Selenomethionine
Selenocysteine
Supplements
Selenoproteins
Selenophosphate
Selenate
Selenite
GS -Se-SG
Hydrogen selenide: H2Se

Methylselenol: CH3SeH
Dimethylselenide: (CH3)2Se
Breath
Trimethylselenonium:(CH3)3Se+
Urine
Figure 3.3 Metabolism of selenomethionine, selenite and selenate (Adapted from Schrauzer, 2000;2003;
Combs, 2001; Meuillet et al., 2004).
In fact, Burk et al. (2001) demonstrated that Se from SeMet, but not that from
selenate or selenocysteine, can be incorporated into albumin, presumably as SeMet
in the methionine pool. In another study, albumin was purified from plasma of a
human before and after 28 days of supplementation with 400 g Se/day as SeMet.
It was shown that the albumin contained 1 Se atom, presumably as SeMet, per
8000 methionine residues before supplementation and 1 per 2800 after
supplementation (Hondal et al., 1999). These findings support the view that SeMet
is a non-specific form of Se that is metabolized as a constituent of the methionine
pool where it is randomly distributed and is unaffected by specific Se metabolic
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processes. Therefore SeMet can be considered as a storage form of Se in animals

and humans and it is metabolized as a constituent of the methionine pool and is
unaffected by specific Se metabolic processes. In contrast, no evidence was
obtained for non-specific incorporation of Se into plasma proteins when it was
administered as selenate or as selenocysteine. These forms of the element appear
to be metabolized by specific Se metabolic processes (Burk et al., 2001).
Did you know that SeMet incorporation into proteins is
metabolically regulated and because of protein turnover there
is no danger of Se over-accumulating in tissues in this form?
The skeletal muscles are the major Se-storage organ, accounting for about 46.9%
of the total Se in the human body, while kidney contains only 4% of Se reserves
(Oster et al., 1988). In humans, whole body Se depends on the regional location
and varies from 3-6 mg up to 13-20 mg (Daniels, 1996). GSH-Px activity and
deposition of Se were examined in tissues of rats given dietary Se for 7 weeks as
either selenite or SeMet with 75Se radiotracer of the same chemical form (Beilstein
and Whanger, 1988). The authors showed that the proportion of 75Se as SeMet
was higher in tissues of rats fed SeMet (highest in muscle and hemoglobin, 70%,
and lowest in testes, 16%). In contrast, selenocysteine was the predominant form
of Se present in tissues of rats given selenite. In fact, SeMet accumulated
nonspecifically in muscle proteins can build Se reserves, which can be used in
stress conditions when Se requirement is increased, but feed consumption usually
decreased. In stress conditions protein catabolism by proteasomes can release
SeMet, which could serve as a source of Se for newly synthesied selenoproteins,
such as GSH-Px, thioredoxin reductase and methionine sulphoxide reductase.
Those enzymes can deal with overproduction of free radicals and prevent decrease
in productive and reproductive performance of farm animals. It was proven that
Se from both selenite and SeMet is readily available for synthesis of the
selenoenzyme GSH peroxidase in rat tissues (Pierce and Tappel, 1977).
Did you know that the skeletal muscles are major Se-storage
reservoir, accounting for about 46.9% of the total Se
in the human body?
There are also several lines of evidence confirming the idea that Se accumulated
in tissues in the form of SeMet can be available for selenoprotein synthesis.
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First, studies in our laboratory (Surai, 2000) indicated that chicks hatched
from eggs enriched with Se by means of using Se yeast (Sel-Plex) had
higher liver GSH-Px activity not only at hatching, but more importantly,
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even at 5 days posthatch. More recent observations with quail and chickens
indicates that when organic Se in the form of Sel-Plex was included in the
maternal diet, Se concentration in the liver of the progeny was alleviated up
to 3 weeks posthatch (Surai et al., unpublished). This could be explained
by usage of SeMet accumulated in tissues as a result of Se transfer from the
egg during embryogenesis.
Secondly, the bioavailability of the Se pool in maintaining liver GSH-Px
activity during a period of Se deprivartion, following excess selenite or
SeMet loading was assessed in rats (Ip and Hayes, 1989). In this study halflife of decay of the enzyme was calculated to be 4.2 and 9.1 days,
respectively, in rats that had already been exposed to 3 ppm Se as either
selenite or SeMet.
Thirdly, in a human study Persson-Maschos et al. (1998) showed that in
individuals who had been supplemented with organic Se, the decline in the
level of selenoprotein P following a period of supplementation was slower
than in individuals who had been supplemented with selenite.
Fourthly, when wheat and selanate were used as Se sources in a
supplementation study in Finnish men it was shown that once the
supplements were withdrawn, platelet GSH-Px activity declined less in the
group given wheat Se (Levander et al., 1983).
Finally, after several weeks supplementation with high-Se bread, plasma
Se of New Zealand subjects increased from 50-70 ng/ml to 120-175 ng/ml
(Robinson et al., 1985). Plasma Se remained elevated when supplementation
ceased.
In addition, in SeMet or Se-yeast supplemented mice, liver GSH-Px activities
declined more slowly during Se depletion than in mice given selenite
(Spallholz and Rafferty, 1987 cited by Schrauzer, 2003).
Furthermore, in children the relative bioavailability of Se-yeast versus
selenite measured as GSH-Px activity was similar in plasma, red blood cells,
and platelets, however, Se-yeast provided a longer lasting body pool of Se
(Alfthan et al., 2000).
These data are in agreement with the suggestion that SeMet is the major
selenocompound found initially in animals given this selenoamino acid, but is
converted with time afterwards to selenocysteine when incorporated in functional
selenoproteins (Whanger, 2002). For example, the chemical forms of Se were
determined in erythrocyte and liver proteins after injection of 75Se as either sodium
selenite or SeMet in male weanling rats. Void volume proteins contained principally
selenocysteine (75Se-Cys) in [75Se]selenite-injected animals. This material contained
both 75Se-Met and 75Se-Cys 1 d postinjection in 75SeMet-injected animals, but
primarily 75Se-Cys at 20 d afterwards (Beilstein and Whange, 1986). This means
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that Se-Cys was synthesised from SeMet and with time all SeMet was converted
to Se-Cys. In acid hydrolysates of whole liver 75Se was recovered principally as
75
Se-Cys from animals injected with [75Se]selenite. However, for animals injected
with 75Se-Met, liver 75Se was present initially as 75SeMet, but after five days the
majority of liver 75Se was as Se-Cys. The long-term fate in rats of an oral dose of
[75Se]selenocystine was compared with that of an oral dose of [75Se]SeMet. It was
shown that intestinal absorption of [75Se]selenocystine was 81% of the administered
dose and that of [75Se]SeMet was 86% (Thomson et al., 1975). The initial utilization
of [75Se]selenocystine was different from that of [75Se]SeMet. However, after the
first week 75Se from both sources appeared to be metabolized similarly, suggesting
that dietary Se of both forms is ultimately incorporated into the same metabolic
pool (Thomson et al., 1975).
Weanling male rats were fed a basal Se-deficient diet or this diet plus 2 ppm Se
as either selenite, SeCys or SeMet for nine weeks (Deagen et al., 1987). Except
for the kidney, the tissue Se concentrations were similar in rats fed selenite or
SeCys, but the Se content in testis, muscle, pancreas, heart, spleen, whole blood,
erythrocytes and plasma was significantly higher in rats fed SeMet than in those
fed either selenite or SeCys. The greatest increase due to SeMet compared with
the selenite and SeCys treatments was about 10-fold in the muscle compared with
1.3- to 3.6-fold for the other tissues (Deagen et al., 1987). In general SeMet has
a slower whole body turnover in comparison to sodium selenite and there is greater
efficiency in the re-utilization of Se from SeMet (Swanson et al., 1991). Indeed,
the average whole body half-lives of SeMet and selenite in humans were shown
to be 252 and 102 days, respectively, confirming re-utilization of SeMet in the
body (Patterson et al., 1989). It should be noted that only a small proportion of
the methionine pool can be replaced by SeMet, since only part of methionine
could be replaced by SeMet in the diet. Furthermore, protein turnover prevents
accumulation of SeMet to toxic levels in the organism (Schrauzer, 2003).
In fact, rapid turnover of various selenoproteins and dependence of this process
on Se status were described. For example, the half-life of GSH-Px is approximately
3 days (Sunde et al., 1989), and 2-iodothyronine deiodinase has a half-life of
only 30 - 45 minutes (Kim et al., 2003; Botero et al., 2002; Curcio et al., 2001),
while that of selenoprotein P in plasma is 3-4 hrs (Burk and Hill, 1994). In growth
medium there was an increase in TR mRNA levels of 2-5-fold at 1 microM Se and
an increase in the stability of TR mRNA with a half-life for degradation of 21 hrs
compared to 10 hrs in the absence of Se (Gallegos et al., 1997). Similarly, the
selenoprotein W mRNA half-life in myoblasts is about 57 hrs for cells grown in a
low Se medium while Se treatment increased half-life by 2-fold (Gu et al., 2002).
Therefore, it is clear that Se reserve development could be an important regulatory
mechanism for maintaining effective antioxidant defence during periods of
increased demand. Therefore, from a nutritional viewpoint, SeMet is superior to
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selenite, especially with respect to maintenance of GSH-Px during periods of Se
inadequacy (Ip and Hayes, 1989).
Did you know that selenoproteins in the body are short-lived
and needed to be re-synthesised?
At physiological levels of Se intake, urine is the most important route of excretion
and regulates Se homeostasis (Daniels, 1996). For example, recently a study has
been conducted to evaluate the bioavailability of Se from pork in humans (Bugel,
2004). Twelve male volunteers (age 21-30 years) participated in a study with a
diet containing 170 g pig meat/10 MJ per day and 106 +/- 13 g Se/d for three
weeks. Complete faecal and urinary collections were made during the last week
of each period. The apparent absorption of Se was very high (94 +/- 2% ). Faecal
and urinary excretion were 7 +/- 1 g/day and 39 +/- 21 g/day, respectively,
resulting in a retention of 61 +/- 24 g/day (Bugel, 2004). However, at generous
intakes, faecal Se represents mainly unabsorbed dietary Se. Various Se metabolites
were found in urine, but only trimethylselenium was well characterised. There is
a great body of evidence indicating that urinary Se is lower when organic Se is
used in comparison to selenite.
Selenium status and bioavailability

To assess Se status of animals and humans various static or functional tests are
used. Static tests measure the total quantity of Se in various accessible tissues and
body fluids such as hair, nails, blood or its components, and urine. Functional
tests measure the activity of Se-dependent enzymes, or a physiological or
behavioural function dependent on Se (Gibson, 1989). There is no single test
that can describe Se status and a combination of various techniques is preferable.
For example, human Se status has been measured by a variety of methods with Se
concentration in plasma, serum or red blood cells (RBC) all widely used. When
Se status of young British people was assessed by Se analysis in plasma and RBC
and GSH-Px in whole blood (Bates et al., 2002), it was shown that following
factors are of great importance:
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Age
Gender
Social class
Parental smoking
Ethnic group
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Despite higher reported Se intakes, males and females had similar mean plasma
Se values. RBC Se and RBC GSH-Px activity were significantly higher in females
in comparison to males (Short et al., 1997).
There is a great discrepancy in results of comparative evaluation of Se
bioavailability from various sources. The problem is that it is difficult to choose
a proper end point for such evaluation. A range of techniques has been used for
this purpose:
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Prevention of specific Se-responsive disease in animals, including exudative

diathesis (ED) and pancreatic degeneration in chickens, hepatic necrosis in
rats and a variety of myopathies in pigs, lambs, calves and turkey poults
(Railly, 1996; Hakkarainen. 1993). However, the problems of test reliability
remain the same: ED is only one of many conditions affected by Se
deficiency. Another problem is related to the reference material used. In
most of studies sodium selenite was used for a comparison. However, as
mentioned above selenite is not natural form of dietary Se and its metabolism
differs from organic forms of Se. A combination of various tests should be
used to evaluate effects of Se source on Se-responsive disease.
Measurement of the uptake of Se in different tissues after ingestion of a

test compound (plasma, RBC, platelet, whole blood, liver, kidney, muscles,
hair and nails). In cattle, sheep and swine, Se concentration in kidney are
greater than in liver which is in turn greater than heart, followed by skeletal
muscle and lastly adipose tissue (Ullrey, 1987). Indeed, plasma or serum
Se reflects short-term status, erythrocyte Se reflects longer-term status and
hair or nail Se reflect long-term Se status (Thomson et al., 2004). The authors
concluded that plasma or serum Se is the favoured measure of Se status for
various comparisons. In fact, Se concentration in tissues might not always
be the best measure since Se availability from tissues for biological needs
may differ. Indeed, Se retention as percentages of Se intake may be helpful
in assessing Se status when facilities for metabolism studies are available
(Ullrey, 1987). It was shown that Se levels in toenails are highly reproducible
and reflected long-term Se exposure (Krogh et al, 2003). However age,
smoking status and acetone treatment are possible causes of misclassification.
Activity of GSH-Px (plasma, RBC, whole blood, liver and other tissues).
Since GSH-Px is only one of more than 20 selenoproteins described to date
in animal and human tissues, it is not clear at present if the requirement for
Se needed to maintain GSH-Px activity is higher than for the expression of
other important selenoproteins. The important limitations of this approach
are also related to variations in methodology of GSH-Px activity assessment
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(Thomson et al., 2004). In fact, inter-laboratory variation in GSH-Px values
is large, and it is doubtful that limits of normalcy developed in one laboratory
are applicable in others. The instability of GSH-Px should be considered so
that losses in activity during handling and storage may be minimized. It is
also necessary to distinguish between GSH-Px and glutathione-Stransferases, which can reduce organic hydroperoxides but which are not
Se-dependent (Ullrey, 1987).
Selenium excretion with urine. There is a strong positive correlation between

Se excretion and plasma Se and dietary intake in low Se populations (Griffiths
and Thomson, 1974). Indeed, a strong correlation has been established
between dietary daily urinary Se excretion in a wide range of populations
with different dietary Se intakes. Urinary Se excretion is decreased in
children, elderly people, and pregnant women. Workers exposed to heavy
metals, and cancer patients, have higher and lower urinary Se concentrations,
respectively, than control groups (Sanz Alaejos and Diaz Romero, 1993).
However, this test is dependent on kidney function. The 24 hr Se excretion
is dependent on the glomerular filtration rate of the kidney, which is
characterized by the creatinine clearance. The 24 hr Se excretion is further
influenced by the 24 hr urine volume and Se losses via urine may be
concomitant with protein losses in urine (Oster and Prellwitz, 1990).
Selenium availability from plant-based sources varies. For example, biological

availability of Se for prevention of ED was taken as 100% for sodium selenite. It
ranged in other forms of Se from 74% for sodium selenate to 7% for elemental Se;
in plant feedstuffs from 210% for lucerne meal to 60% for soybean meal and in
animal feedstuffs from 25% for herring meal to 8.5% for fish solubles (Table 3.7;
Cantor and Scott, 1974; Cantor et al., 1975). However, SeMet was four times as
effective than either selenite or selenocystine with respect to prevention of
pancreatic degeneration and increasing the relative weight and Se concentration
of the pancreas (Cantor et al., 1975). Cantor and Scott (1974a) observed that
protection against ED was closely related to plasma GSH-Px. It was concluded
that relative to selenite the retention of Se in fish meal or solubles was 43%, and
relative to SeMet, 31% (Miller et al., 1972).
Did you know that there is no universal method for Se

availability assessment and it is recommended to use a
combination of various approaches?
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Table 3.7 Bioavailability of Se in feedstuffs (Adapted from Cantor, 1997).
Feedstuff
Biological availability, %
Dehydrated alfalfa meal

Brewers yeast
Cottonseed meal
Corn
Brewers grains
Wheat
Distillers dried grains and solubles
Soybean meal
Herring meal
Tuna meal
Poultry by-product meal
Menhaden fish meal
Meat and bone meal
Fish solubles
210
89
86
86
80
71
65
60
25
22
18
16
15
9
Sodium selenite was used as standard and prevention of exudative diathesis in chicks was
used as an index of Se bioavailability.
Newly-hatched White Leghorn chicks with low-Se status were fed a low-Se basal
diet for two weeks post-hatch followed by either continued depletion or a repletion
period of four weeks with graded levels of Se (0.03, 0.06, 0.09 and 0.12 mg/kg)
provided via sodium selenite, wheat or fish meal. The bioavailability of Se in
wheat and fish meal in comparison to selenite for increasing the activity of GSHPx was 78% and 58%, respectively; and for increasing Se whole blood Se
concentration was 123% and 107%, respectively (Hassan et al, 1993). Based on
the activity of plasma GSH-Px, the biological availability of Se in soybean meal,
lucerne, fish meal and SeMet was 33, 85, 82 and 92%, respectively (Ikumo and
Yoshida, 1981). On the other hand, Se availability in fish meal, based on the
prevention of incidence of ED, was 74%. In contrast to previous data, Se in fish
meal was poorly able to prevent deficiency in chickens (Martello and Latshaw,
1982) and the results of Whitacre and Latshaw (1982) clearly showed that the
commercial preparation of fish meal significantly decreased Se utilization.
Availability of Se in feeds was estimated in relation to restoring blood serum
GSH-Px activity in Se-depleted chickens. The availability of the Se (relative to Se
in selenite) in capelin fish meal was 48.0 (38.5-60.0), mackerel fish meal, 34.1
(32.3-35.8), soybean meal, 17.5, maize gluten meal, 25.7, and SeMet, 78.3%
(Gabrielsen and Opstvedt, 1980). These data on Se availability from fish sources
are in line with others studied, but data on soybean meal are somewhat low. It is
possible that thermal treatment of processed soya could affect Se availability.
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In Se-deficient chicks, when providing Se at 10 g/kg diet, selenite and
selenocystine were about equal in promoting weight gain and preventing ED,
while SeMet was less effective. Tissues from chicks given the Se sources providing
60 g/kg diet for four weeks were analysed for Se. The content of tissues from
chicks given selenite or selenocystine was similar. Chicks given SeMet had higher
concentrations of Se in pancreas and breast muscle than the others, but lower
concentrations in kidney, liver and heart (Osman, and Latshaw, 1976).
Bioavailability of Se in oats, meat meal and SeMet based on efficacy in preventing
ED in Se-depleted chicks was 41, 30 and 77%, respectively, while based on Se
biopotency in elevating GSH-Px activity in plasma, it was 33, 21 and 77%,
respectively. When the increase in Se concentration in cardiac muscle was used
as the indicator of bioavailability, the Se bioavailabilities were 60, 42 and 114%
for oats, meat meal and SeMet, respectively. Thus, SeMet was superior to sodium
selenite in increasing Se concentration in tissues, but inferior to it in protecting
chicks against ED (Hassan, 1987). By taking both the Se level and the GSH-Px
activity into consideration, organic Se was judged to be more bioavailable than
selenite and selenate for humans (Clausen and Nielsen, 1988). Therefore Se from
yeast, wheat and lucerne is highly available while Se in most other plant sources
is moderately available. Selenium in fish meal is poorly available (Cantor, 1997),
despite the fact that Se content of fish meal can be quite high. Indeed Se availability
depends not only on feed ingredient but also on the methods used for evaluation
(Table 3.8).
Thirty-six New Zealand women between 18 and 23 years of age received 200
g Se as Se-enriched yeast (SeMet), or brewers yeast mixed with selenate, or no
added Se (placebo) daily for 32 weeks in a double-blind trial. Mean daily Se
excretion increased with both supplements. The selenate group excreted more
than the SeMet group, 123 v. 66 g/day, respectively, at week 2, equivalent to 57
v. 27% of the dose (Robinson et al., 1997). Six adults received a single oral 200
g dose of 74Se as L-SeMet. Average turnover time of the plasma Se compounds
varied from 0.01 to 1.1 days and the turnover time in the liver-pancreas subsystem
ranged from 1.6 to 3.1 days. On the other hand, turnover time ranged from 61 to
86 days in the peripheral tissues with the slowest turnover (Swanson et al., 1991).
The whole body residence time was approximately 5-fold greater than the turnover
time of the tissue pool with the slowest turnover, reflecting substantial reutilization
of labelled material.
Despite the high absorption and retention, plasma Se concentrations were only
moderately affected by an increase in Se intake of about 100 g/day in the chemical
forms found in shrimp (Bugel et al., 2001). After 9 wk of the dietary Se repletion,
relative activity of liver GSH-Px from the different dietary groups compared with
control rats (100%) was: flounder 106%, tuna 101%, pork 86%, sodium
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Table 3.8 Bioavailability of Se (%) in comparison to sodium selenite (100%) (Adapted from
Hakkarainen, 1993).
Source of Se
Induction of
GSH-Px
Plasma
Wheat
Barley
Oats
Fishmeal
Meatmeal
Selenomethionine
Bioassay
Se concentration
Whole Prevention Plasma

blood
of ED
79
83
90
71
77
80
33
37
62
66
61
73
21
20
26
77
80
83
46
56
21
-
100
83
41
80
30
77
109
107
151
102
90
107
85
86
40
47
-
Whole
blood
Liver
Heart
123
104
99
107
69
-
140
151
82
90
67
98
113
119
26
-31
-
108
87
60
100
42
114
selenite 81%, SeMet 80%, beef 80%, chicken 77%, veal 77%, and lamb 58%
(Wen et al., 1997). It seems likely that all cuts of beef are characterised by high
bioavailability of dietary Se when compared with selenite or L-SeMet. For example,
after eight weeks of dietary Se repletion, relative activity of liver GSH-Px from
the different dietary treatments compared with that of control animals (100%)
was (%): selenite 91, SeMet 122, beef liver 108, striploin 105, round 106, shoulder
106, brisket 103 (Shi and Spallholz, 1994). The authors also showed that Se
recovery for liver GSH-Px was generally highest from SeMet > beef muscle =
beef liver > selenite. Similar trends were seen for muscle tissue deposition of Se
which was highest from SeMet > beef muscle > selenite = beef liver. Furthermore,
the faecal excretion of Se was lowest from the SeMet dietary group and highest
from the selenite dietary group. The nutritional biopotency of Se in Brazil nuts
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was also evaluated by the repletion of two selenoenzymes, GSH-Px and type I 5'deiodinase, in Se-deficient rats. Supplementation with Brazil nuts as the sole source
of Se produced an efficient gradient of enzyme restoration at 0.05-0.2 g/g of
dietary Se. A parallel comparison with sodium selenite indicated that the Se in
Brazil nuts and selenite Se were equally bioactive (Ip and Lisk, 1994). The
bioavailability of Se in mushrooms, Boletus edulis, to young Finnish women was
studied by giving them 150 g Se as mushrooms for four weeks (Mutanen, 1986).
The indicators of body Se status were plasma and erythrocyte Se levels and plasma
and platelet GSH-Px activity. The Se level in erythrocytes increased significantly
(26%), while only slight enhancement was found in plasma Se and plasma or
platelet GSH-Px activity. The results indicate that bioavailability of mushroom Se
is reasonably low.
Did you know that Se is highly available from grains, but less
available from meat and fish by-products?
It is well known that fish is an important source of human dietary Se (Table 3.9).
The bioavailability of Se in the defatted dark muscle of tuna, as assessed by slope
ratio analysis in rats using selenite as the reference Se, was 87% for the liver Se
content and 168% for the GSH-Px activity (Yoshida et al., 2002). The
bioavailability of Se from raw and cured SeMet-enriched (Se-enriched) salmon
fillets was assessed in Se-deficient male albino rats. A higher bioavailability of Se
from raw and cured Se-enriched fish fillets compared with selenite was reported
(Ornsrud and Lorentzen, 2002). Apparent absorption of Se from fish was similar
to selenate, however, retention of fish Se was significantly higher than selenate
(Fox et al., 2004). However, high concentrations of heavy metals in some fish
species (Table 3.10) could be responsible for decreased availability of Se from
those sources.
The nutritional availability of Se to rats in two experimental Finnish milk sources
was compared to that in American milk naturally high in Se. Selenium content of
the experimental milks was increased by feeding cows either sodium selenite
(selenited milk) or selenited barley (selenited-barley milk). The bioavailability of
Se in weanling rats was calculated using the slope-ratio method. The Se in the
selenited-barley milk was significantly more available than that in the selenited
milk when the plasma Se level was the response criterion. On the other hand, the
bioavailability of Se from the various milks was not different when plasma or
liver GSH-Px activities were used as the response criteria (Mutanen et al., 1986).
Overall bioavailability for the selenited milk, selenited-barley milk and American
milk was similar to that for the standard (sodium selenite = 1.00), showing that
milk is a relatively readily available source of dietary Se.
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Table 3.9 Selenium concentration in seafood collected from Supermarkets in Taiwan, g/g wet weight
(Adapted from Chien et al., 2003).
Species
Mean
Range
Fish
Salmo salar Linnaeus
Scomberomorus commersoni
Sebastiscus albofasciatus
Eleutheronma tetradactylum Show
Liza macrolepis
Lateolabrax Japonicus Cuvier
Oreochromis sp.
Mola mola Linneaus
2.01
1.75
1.55
1.55
1.55
1.54
1.34
0.81
1.56 - 2.39
1.66 1.90
1.57 1.67
1.44 1.72
1.20 1.77
1.43 1.70
1.25 1.44
0.71 0.93
Crustaceans
Panulirus longipes
Ovalipes punctatus
Metapenaus ensis
Portunus sanguinolentus
1.30
1.28
1.27
1.09
0.87 1.76
1.15 1.35
1.00 1.38
0.91 1.33
Bivalve molluscs
Crassostrea gigas
Raphia amabilis
Rudiapes variegates
Meretrix lusoria
Amusium pleuronectes
1.18
1.33
0.87
0.73
0.63
0.77 1.59
1.00 1.49
0.67 1.20
0.53 1.00
0.56 0.70
The Se intake of a Japanese fisherman may be as high as 500 g/day and in Greenlanders it
was 652.7g/day (Chien et al., 2003).
Did you know Se is highly available from land animal-derived

products including meat, milk and eggs?
A study was conducted to evaluate the nutritional bioavailability of Se from Seenriched garlic with use of two liver selenoenzymes as biomarkers: GSH-Px and
type I 5'-deiodinase. Rats were fed a Se-deficient diet (0.01 ppm Se) for four
weeks after weaning and later were supplemented with nutritional levels of Se
(0.1-0.5 ppm) in the form of sodium selenite (positive control) or Se-enriched
garlic. The results showed that Se-enriched garlic was just as effective as selenite
in restoring the activity of both selenoenzymes (Ip and Lisk, 1993). It is well
established that both organic and inorganic forms of Se may be utilised by the
body, with the selenoamino acids showing greatest bioavailability (Patching and
Gardiner, 1999). The bioavailability of Se in Se yeast, as assessed by slope-ratio
analysis using selenite as a reference Se in rats, was 135% to 165% in the tissue
Se content and 105% to 197% in the GSH-Px activities (Yoshida et al., 1999).
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Table 3.10 Selenium and mercury contents in edible portions of fishes, mg/kg (Adapted from Plessi et
al., 2001)
Fish
Angler fish
Cod
Grayfish
Hake
Halibut
Salmon
Swordfish
Tuna
Whiting
Goby
Gurnard
Mackerel
Mullet
Ox-eye bream
Perch
Porgy
Redfish
Scorpion fish
Sole
Anchovy
Red banfish
Sardine
Selenium
Mercury
Se/Hg ratio
0.173
0.188
0.139
0.466
0.134
0.195
0.283
0.734
0.290
0.279
0.372
0.356
0.426
0.337
0.073
0.286
0.247
0.417
0.262
0.225
0.299
0.678
0.089
0.077
0.282
0.086
0.069
0.086
0.579
0.249
0.187
0.145
0.138
0.126
0.103
0.104
0.139
0.204
0.096
0.134
0.057
0.075
0.058
0.156
1.94
2.44
0.49
5.42
1.94
2.27
0.49
2.95
1.55
1.92
2.70
2.83
4.14
3.24
0.53
1.40
2.57
3.11
4.60
3.00
5.16
4.35
These results indicate that Se in Se yeast is more bioavailable than selenite Se,
and therefore is the preferred form for supplementation. The bioavailability of Se
in the form of yeast is higher than that of other Se compounds used for preterm
infants (Bogye et al., 1998). A daily supplement of 0.75 mg Se from the yeast
product maintained whole blood and milk Se concentrations at the same levels as
3.0 mg Se as sodium selenite. Further, 3.0 mg Se from the yeast product increased
whole blood Se by about 40% and that of milk by about 100%. The activity of
GSH-Px in erythrocytes of the group given selenite was not significantly different
from that in either of the groups given the yeast product. The concentrations of Se
in the tissues of two cows from each group were marginal to adequate, and there
was a trend for the concentrations to be higher in the tissues of the cows
supplemented with the yeast product (Ortman and Pehrson, 1997).
Supplementation of the feed either with 0.2 ppm organic Se or sodium selenite
for eight weeks increased the blood Se level within this period from a background
level of about 5.6 g/L to 167 (Se-yeast) and to 91 g/L (selenite; Malbe et al.,
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1995). The respective change in whole blood GSH-Px was from 0.22 to 3.0 (Se
yeast) and to 2.3 (selenite) microKat/g Hb. The bioavailability of yeast Se was
superior to selenite: the relative bioavailability (selenite = 1) of yeast Se was 1.4 if
blood GSH-Px, 1.9 if blood Se, and 2.7 if milk Se was used as the response
criterion (Malbe et al., 1995). Feeding organic Se from SeMet or selenized yeast
results in much higher tissue and milk selenium concentrations than are obtained
with selenite (Spears, 2003). Cows fed selenoyeast had greater whole blood Se
concentrations than cows fed sodium selenite. At birth, calves from cows fed
selenoyeast had greater whole blood Se concentrations and GSH-Px activities
than calves from cows fed sodium selenite (Gunter et al., 2003).
Utilization (absorption, retention and appearance in milk and blood) of two
different chemical forms of Se (selenite and SeMet) in lactating, non-lactating
and never pregnant women using stable isotope tracers was studied. It was shown
that significantly more Se from SeMet than from selenite was absorbed and
appeared in the plasma in all groups. Milk contained more Se from apparently
absorbed SeMet than from selenite. All groups retained significantly more Se
from SeMet than from selenite (Moser-Veillon et al., 1992). It has been shown
that SeMet-Se was more effective in raising plasma and RBC Se than was selenite
in men (Luo et al., 1985).
A purified, casein-based diet without added Se was fed to nine groups of rats
throughout pregnancy to produce a marginal Se deficiency. During lactation,
groups (n = 8) were fed experimental diets containing either 0.1, 0.25 or 0.5 ppm
Se as selenite, SeMet, or Se yeast. On day 18 of lactation, tissue Se and GSH-Px
activities of dams and pups were determined. Based on slope-ratio analyses, the
bioavailability of SeMet and Se yeast was greater than that of selenite in both
lactating dams and their nursing pups (Smith and Picciano, 1987). Weanling rats
were fed a basal diet or this diet plus 0.2, 1.0, 2.0 or 4.0 mg/kg Se as either
selenite or SeMet. Except at the 0.2 mg/kg Se level, Se accumulated in all tissues
at higher levels when SeMet was fed than when selenite was given, and the
magnitude of difference became more pronounced with increasing levels of dietary
Se (Whanger and Butler, 1988). This was particularly true for muscle and brain. It
appears that after saturation of activity of GSH-Px and other selenoproteins, Se in
inorganic form cannot be stored in the body and is released with urine and faeces.
In great contrast, SeMet was unspecifically incorporated in body proteins, especially
muscle, providing higher Se concentration in tissues. Although the tissue Se
concentrations differed markedly, there were no differences in the GSH-Px activity
in tissues of rats fed SeMet or selenite. This means that GSH-Px expression reached
maximum level at this high Se supplementation condition. The percentage of Se
associated with GSH-Px was lower in all tissues from rats fed SeMet than in those
from rats fed selenite, reflecting more Se accumulating in the form of SeMet
(Whanger and Butler, 1988).
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Did you know that selenized yeast is considered the optimal
form of Se supplementation for human and animals?
Effectors of selenium absorption, metabolism and bioavailability

Animal studies reveal that various factors influence Se bioavailability. Animals
cannot synthesize SeMet or distinguish it from methionine and as a result it is
non-specifically incorporated into a wide range of Se-containing proteins (Daniels,
1996). SeMet is retained in tissue proteins to a greater extent than selenocysteine
and the inorganic forms. However, a number of other factors besides chemical
form, may also influence the bioavailability and distribution of Se, including other
dietary components, Se status, physiological status and species (Thomson, 1998).
For example, Se is better absorbed from a high protein diet (Daniels, 1996).
Since SeMet is non-specifically incorporated into tissue proteins instead of
methionine (Met), their metabolism is interrelated. In particular, it has been
suggested that SeMet is preferentially incorporated into body protein when dietary
Met is limited. Once Met is supplemented, dietary SeMet would provide more Se
for the syntheses of GSH-Px and other selenoproteins. For example, in animals
given 0.06 mg/kg dietary Se, supplementing Met resulted in redistribution of Se
in tissues. Muscle Se content decreased, while liver and blood Se increased along
with GSH-Px activity in all tissues (Tian et al., 2001). On the other hand, in Metsupplemented animals at 0.50 mg/kg dietary Se, tissue Se content declined to
various extents, while GSH-Px activity remained unchanged. It seems likely that
methionine diverts SeMet from incorporation into general proteins and enhances
its conversion to selenocysteine for specific Se-requiring proteins, such as GSHPx. To determine the influence of methionine on SeMet metabolism, weanling
male rats were fed for eight weeks a basal diet marginally deficient in sulphur
amino acids containing 2.0 mg Se/g as DL-SeMet and supplemented with 0, 0.3,
0.6 or 1.2% DL-Met (Butler et al., 1989). Increased dietary Met caused decreased
Se deposition in all tissues examined, but increased GSH-Px activity in testes,
liver and lungs. A positive correlation was found between dietary Met and the
calculated percentage of Se associated with GSH-Px. In another experiment 75SeMet
was injected into weanling male rats fed the basal diet containing 2.0 mg Se as
DL-SeMet with or without the addition of 1.0% Met. The selenoamino acid content
of tissues and the distribution of 75Se in erythrocyte proteins were determined
(Butler et al., 1989). In comparison to the rats fed the basal diet without added
Met, significantly more 75Se-SeCys was found in liver and muscle, more 75Se was
found in erythrocyte GSH-Px and less 75Se was found in erythrocyte Hb of rats
fed 1.0% Met. In rats given 3 ppm SeMet, tissue Se was higher in those fed a diet
with a suboptimal amount of Met than in those with an adequate intake of Met
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(Ip, 1988). Altered Se metabolism at suboptimal dietary Met may occur because
more SeMet is incorporated into protein and thus less Se is available for GSH-Px
synthesis. Supplementation of cell culture with L-Met significantly reduced 75SeMet
incorporation, but significantly increased the proportion of cellular 75Se recovered
as SeCys (Beilstein and Whanger, 1987). More Se is incorporated into sheep wool
and plasma protein from feed supplemented with SeMet when dietary sulphur is
limiting than when it is not (White, 1980).
Did you know that selenium assimilation depends on dietary
mineral and vitamin balance?
Copper (Cu) (100 mg/kg) reduced the incorporation of selenium (when used at
level of 2 ppm) into protein fractions of egg white (Davis et al., 1996). Long-term
ingestion of moderate levels of Cu (10 mg /kg) influenced the metabolism of Se,
possibly through effects of chronic Cu toxicity on liver function. For example, at
19 weeks, ram lambs were allocated randomly to individual metabolism cages
and received a single oral dose of 75Se-SeMet. Sheep given Cu supplements had
reduced levels of 75Se activity in muscle compared with control animals (White et
al., 1989). This decrease in muscle 75Se in Cu-supplemented lambs was associated
with a nonsignificant increase in 75Se content of other tissues and a nonsignificant
increase in faecal excretion of 75Se. Marginal deficiency of zinc (Zn) (8.6 mg/kg
diet) in rats can decrease Se availability and a small excess of Zn increases Se
availability for hepatic GSH-Px activity. In fact, Se concentrations in plasma,
erythrocytes, muscle, heart, and liver were significantly elevated by Zn (Yin et
al., 1991). Furthermore, Zn had an antagonistic effect on Se absorption by Znadequate rats (House and Welch, 1989). Chronic magnesium (Mg) deficiency in
rats decreases Se absorption and retention and erythrocyte concentrations of this
mineral, and increases Se in plasma, kidney and heart (Jimenez et al., 1997).
There are also interactions between Se and heavy metals, which will be considered
separately.
Vitamin B6 is involved in the metabolism of selenium. For example, the addition
of pyridoxal phosphate to the media resulted in elevated GSH-PX activity in all
human lymphoblast cells regardless of the chemical form of Se used (Beilstein
and Whanger, 1992). The levels of Se and GSH-Px in erythrocytes and muscle
were significantly higher in vitamin B6-supplemented groups than in vitamin B6deficient groups (Yin et al., 1991). It seems likely that this vitamin is involved in
the transport and deliverance of Se in plasma to the other tissues and the
incorporation of Se from SeMet to GSH-Px in liver. Therefore the conversion of
SeMet to a form available for GSH-Px synthesis is reduced by vitamin B6 deficiency.
For example, tissue retention of 75Se provided as SeMet was increased in vitamin
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B6-deficient animals, but the proportion of 75Se retained in muscle and liver as
SeCys was significantly reduced (Beilstein and Whanger, 1989). In the absence
of vitamin B 6, SeMet was less effective in the liver and ineffective in the
erythrocytes while selenite was equally effective in both tissues and was as effective
as in the presence of vitamin B6 (Yasumoto et al., 1979).
It seems likely that riboflavin is also involved in regulation of Se metabolism.
Supplementation of diets fed weanling pigs with 10 mg vitamin B2/kg resulted in
increased kidney and muscle GSH-Px activity. Vitamin B 2 supplementation
weanling pig diets (10 mg vitamin B2/kg) increased kidney, muscle, heart and
brain GSH-Px activity when sodium selenite was the dietary Se source, but not
when SeMet was the dietary Se source (Parsons et al., 1985). Therefore, riboflavin
supplementation did not alter apparent Se absorption, but vitamin B 2
supplementation decreased urinary Se and thus increased Se retention. Absorption
studies with ligated duodenal loops or oral doses indicated that high vitamin A
intake (Combs, 1976) or dietary ascorbic acid (Combs and Pesti, 1976) promoted
the enteric absorption of Se. Ethoxyquin was effective in alleviating exudative
diathesis when fed separately from Se. It was also effective in promoting increases
in plasma GSH-Px (Combs, 1978).
Phytate increased Se in chicken tissues except muscle, however, it was not
clear if this resulted from increased absorption or increased retention (Shan and
Davis, 1994). In the same experiment phytate caused significant reductions in
chicken growth, food consumption and food conversion efficiency. Supplementary
Se was without effect on growth but significantly increased GSH-Px activity in all
tissues. Furthermore, phytate also increased GSH-Px activity in blood and heart,
and in muscle in the absence of supplementary Se, but decreased the GSH-Px
activity in kidney. Growth rate affects both the clinical and biochemical
manifestations of Se deficiency in the chick. For example, a low level of dietary
protein and a restricted level of feeding markedly reduced the incidence and
severity of ED (Zhou and Combs, 1984). The activity of Se-dependent GSH-Px
in plasma and liver varied inversely with the incidence of ED and was increased
by feed restriction. In Holstein cows increased dietary sulfate reduced Se balance:
linearly reduced plasma Se concentrations and apparent and estimated true Se
digestibility (Ivancic and Weiss, 2001).
It seems likely that Se can affect absorption of other nutrients. For example, in
studies investigating the relationship between vitamin E/Se and glucose absorption,
Se plus vitamin E or Se alone modified jejunal glucose absorption, depending on
the duration of feeding and age of chickens. In younger chickens, providing the
supplements for 11 days depressed glucose absorption, but when they were given
the supplements for 18 days glucose absorption was increased. In older chickens
Se alone or with vitamin E given for 13 days increased glucose absorption (Giurgea
and Roman, 1992).
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Selenium and feeding behaviour

Experiments were conducted to determine the initial effects of oral Se
administration on Se-deficient chicks. Administration of 5 g Se as seleno-DLMet increased voluntary feed consumption within 2-3 hrs, whereas selenite did
not have a significant effect until 3-4 hrs (Bunk and Combs, 1980). Spontaneous
activity, body weight gain and plasma glucose concentration increased 6-8 hrs
after Se administration. The earliest response in the specific activity of Se-dependent
GSH-Px occurred in plasma at 8 hrs and in liver at 24 hrs after Se administration.
The onset of pancreatic atrophy, however, was not affected by the level of feed
intake suggesting that the effect of Se upon appetite may be distinct from the
involvement of Se in nutritional pancreatic atrophy and fibrosis. Inorganic Se
compounds were shown to be anti-feed intake factors whereas the organic Se
compounds tested had little such activity.
An interesting study was conducted with an artificial diet to examine the feeding
responses of a generalist herbivore, Spodoptera exigua (Hubner) (Lepidoptera:
Noctuidae), to various forms and concentrations of Se (Vickerman and Trumble,
1999). Tests initiated with neonates showed larvae significantly preferred the
control diet over one containing sodium selenate, sodium selenite, or
selenocystine, but at most concentrations showed no preference between the SeMetcontaining and control diets. Choice tests initiated with third instars demonstrated
a preference for the control diet over sodium selenate and sodium selenite
treatments. In contrast, no significant responses were found in tests initiated with
third instars offered a choice between selenocystine or SeMet and untreated
controls. Similarly, when Se-enriched yeast was used as a source of organic Se,
young Se-deficient laying hens reduced their Se deficit by preferentially selecting
the high-Se diet (Zuberbuehler et al., 2002).
Did you know that animals have an ability to choose high
Se food when it is needed?
Selenium sources for animal and human consumption

As mentioned above, Se content of feed and food ingredients greatly varies
depending on many different factors. For example, Se concentration in corn and
rice grown in normal and high Se areas can vary 100-500fold (Table 3.11).
Indeed Se concentration in foods is significantly different across the world (Tables
3.12-3.14). Furthermore, even in the same country Se concentration in foods
significantly differs among regions, causing plasma Se to also vary (Table 3.15).
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Table 3.11 Selenium content in samples from control and high Se area in China (Adapted from Zhu et
al., 2002).
Substrate
Control area
Corn, g/g
Rice, g/g
Water, g/ml
Urine, g/ml
Hair, g/g
High Se area
0.096
0.029
0.05
0.38
1.37
46.52
6.53
0.352
3.27
43.88
Table 3.12 Average Se content (ppm, wet bases) of selected food reported in 1970th (Adapted from
Selenium in Nutrition, 1983).
Food
USA
Meats (no kidney, liver)

Kidney
Liver
Seafood (excluding fish)
Fish
Whole milk
Butter, cream
Cheese
Eggs
Cereal products
Vegetables
Fruits
0.22
1.62
0.43
0.53
0.38
0.012
0.006
0.082
0.098
0.38
0.01
0.006
Canada
0.14
2.77
0.43
1.25
1.08
0.015
0.003
0.08
0.039
0.52
0.027
0.007
Germany
UK
Ukraine
0.27
0.44
1.54
0.14
1.01
0.03
0.0.3
0.06
1.39
0.13
0.37
<0.01
<0.01
0.12
0.11
0.11
<0.01
<0.01
0.29
0.10
0.30
0.022
0.28
0.125
0.004
Table 3.13 Selenium contents (g/100g as consumed) in foods (Adapted from Combs, 2001).
USA,
1999
Red meats
Poultry
Fish
Milk
products
Eggs
Cereal
products
Vegetables
Fruits
England, Germany, New

China 1
1995
1986 Zealand,
1986
8- 50
5 - 14
1 - 26
5 - 15
13 -148 10 - 61
1 - 26
1-8
China2
13- 28
1- 4
1- 3
5- 25
5 - 15
5 - 10
2-6
5 - 10
24 - 53 3 - 31
3 - 20 10 - 60
1 - 10 0.3 - 2.5 0.2 - 1.0 1 - 3
6- 20
6 - 66
5- 20
2 - 53
5 - 20
3 - 88
24-98
4-9
2-6
0.5- 2
0.1- 14
0.5- 6
1- 9
0.5- 1
4- 0
0.2- 4
0.1- 2
0.1-0.4
0.2 - 2
0.1- 0.3
China3 Venezuela
17- 83
10 - 70
32 - 93
11-43
5 - 15
5 - 15
1.7 - 11 106- 690 12- 51
0.2- 9 34 - 4570 0.2- 298
0.5- 4
0.5 - 6
/before selenized fertilizers were introduced; b/after selenized fertilizers were introduced;
/Low Se-area; 2/ Moderate Se-area; 3/High Se-area
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186
Table 3.14 Selenium content in some common foods, g/100 g (Adapted from McNaughton and Mars,
2002).
Food item
Untitled-2
UK1991
UK1995
USA1999
New
Zealand2000
Australia2002
Cereals
Bread
28-35
Corn flakes
2.0
Muesli
Oats (cooked)
trace
Rice (white, cooked)
4.0
Pasta (white, cooked)
Trace-4.0
Wheat breakfast biscuits
4.0
4.3-9.2
4.7
4.2
3.1
13.0
4.8
2.3
28.2-36.6
5.1
17.3
8.1
7.5
21.3-21.7
4.7
3.2-5.9
1.8-2.0
2.2-2.5
2.0
0
0.4-5.5
2.4-12.5
9.3-12.5
6.3
12.8
11.0
2.5
3.9
17.8
Dairy
Milk
Cheese (cheddar)
Yoghurt
Ice-cream
Butter/margarine
1.0
12.0
1.0-2.0
trace
1.0-1.5
7.4
1.4-1.5
1.5-1.7
-
2.0-2.1
13.9
2.8-4.9
2.6
0-1.0
0.1-1.4
2.3
0.7-1.0
0.6
0.6-1.6
2.3-2.6
7.0-7.9
2.0
4.5
1.1-1.4
Eggs
Whole
Yolk
White
9.0-12.0
20.0
6.0
22.5-30.8
45.2
17.6
15.7-16.1
39.2
4.4
19.0-41.4
26.0
9.0
Fats
Butter/margarine
Oils
Fish
Fruit, fresh
trace
trace
20.-50
Trace-0.3
0.4
0-1.0
0
12.6-50.2
0.2-1.1
0.6-1.6
0
19.5-51.2
0-3.0
1.1-1.4
0.5-0.53
12.0-63.2
0.1-1.4
Meat products
Beef
Chicken
Lamb
Pork
Processed meat
Liver
3.0
6.0-7.0
1.0
14.0
20.0-22.0
7.0
7.6
3.8
14.0
42.0
13.4-19.0
19.0-27.6
26.4-27.4
14.4-45.0
57.0-116.0
2.2-8.3
13.7-14.5
3.7-5.6
1.9-15.0
0-18.0
8.7-42.0
7.2-12.1
11.6-28.0
13.0-22.0
9.4-20.5
4.7-23.3
30.0-37.9
Vegetables
Peeled, raw/cooked
canned
Mushrooms, raw
Trace-3.0
trace
9.0
0.2-3.0
-
0.2-1.9
0.5-0.7
8.8
0-1.1
0-0.25
7.7
0.05-3.3
0.7-3.1
25.5
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Table 3.15 Selenium content in wheat and blood serum in various regions of Russia (Adapted from
Tuteljan et al., 2002.
Region
Pskov
Gomel
Rjazan
Brjansk
Irkutsk
Altaiskij
Bashkortostan
Archangelsk
Vladimir
Karelia
Norilsk
Tula
Gorkiy
Perm
Chelabinsk
Sachalin
Se in wheat, ppb
Se in blood serum, ppb
40 2
68.0 25.1
77.7 26.8
84.9 26.1
88.0 26.6
91.3 40.0
99.6 21.3
100.0 52.9
108.0 61.5
110.0 81.3
119.0 39.0
126.0 42.1
146.0 40.4
161.0 68.0
161.0 65.2
257.0 114.0
72
79.3
83.0
84.0
75.0
87.0
90.0
93.0
101.0
90.0
102.0
89.0
108.0
103
101
137.5
Did you know that selenium concentration in feed ingredients

varies substantially and that usage of data in feed composition
tables to balance selenium is not appropriate approach?
Considering serving size and Se concentration in various foods in the USA (Table
3.16) and UK (Table 3.17), it is clear that fish, meat and eggs are important Se
sources in American diets. Estimated human dietary intake of Se from dietary
sources in various countries is shown in Table 3.18. Taking into account amounts
of various foods consumed in the UK (Table 3.19) it is important to stress that
meat, bread/cereals, fish and dairy products/eggs provide 15,5; 12.7; 12.2 and
10% of total Se in the diet.
The Se content of some frequently consumed foods from a commercial beef
packer, a road-side vendor, and local stores in Lubbock, Texas was determined
and compared to Se data for the same or similar foods in the United States and
from other countries. The comparison of Se in foods covered the period of time
between 1970 and 1993. The Se analyses of foods show that on a fresh weight
basis, the Se content of seafoods > commercial beef > pork > ground beef >
chicken. Cooking, air- or freeze-drying increased the Se content of all foods
significantly (Zhang et al., 1993).
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188
Table 3.16 Selenium content in American diet (Adapted from Holben and Smith, 1999).
Food
Grains and cereals
Bread, whole wheat
Bread, white
Spaghetti/macaroni,
cooked
Egg noodles, cooked
Rice, white, long
grain, cooked
Corn or wheat flakes
Crispy rice cereal
Oatmeal, cooked
Puffed wheat cereal
Serving
size, g
Selenium Food
content, g
28
25
140
10.2
7.1
29.8
160
158
37.4
11.9
28
28
234
12
1.4
4.3
19.0
14.8
Meats and meat alternatives

Beef, bottom round. 100
cooked
Beef ground
100
(27% fat), cooked
Beef, liver, cooked
100
Chicken, roasted
100
Lamb, cooked
100
Veal, cooked
100
Pork, leg (ham),
100
cooked
Pink salmon, canned 100
28.1
19.4
57.0
23.9
2.9
13.0
49.9
Serving
size, g
Tuna, light, canned 100

in water
Egg, cooked
50
Beans, great northern, 262
canned
Chickpeas, canned 240
Soybeans. Green,
cooked
180
Brazil nuts, dry
28
Almonds, dry roasted 28
Cashew nuts. Dry
roasted
28
Peanuts, dry roasted 28
Peanut butter
32
Dairy products
Milk, nonfat/skim
Selenium
content, g
80.4
15.4
10.7
6.7
2.5
839.2
1.4
3.3
2.1
2.4
245
5.1
Cheddar cheese
28
Mozzarella cheese,
part skim
28
Yogurt, plain, low fat 245
3.9
4.6
8.1
Vegetables
Garlic, raw
Mushrooms, raw
0.4
4.3
2.8
35
33.2
Selenium content in various feed ingredients is also highly variable (Table 3.20)
and average data on Se content in feedstuffs presented in various tables are not
suitable for diet balancing and Se supplementation is a routing practice in
commercial animal and poultry production. It has been shown that through usage
of Se in selenized fertilizers or by foliar application it is possible to substantially
increase Se content in various food and feed ingredients (Table 3.21). Data from
Finland are an example of successfully increasing Se status of the population by
using Se with fertilizers. However, environmental concern about washing out Se
from soil and contaminating water reservoirs is a limiting factor for the wide
application of such a technology. It seems likely that production of Se-enriched
animal-derived products is a safe and commercially valuable way of resolving Se
deficiency on a global scale.
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Table 3.17 Selenium concentrations in various UK food sources (Adapted from Brown and Arthur,
2001 and BNF, 2001).
Food
Selenium content, g/100 g fresh weight
Eggs
Beef
Pork
Lamb
Poultry
Turkey (white meat)
Fish
Liver
Kidney
Milk whole
Milk skimmed
Dairy produce
Cheese cheddar
Cheese parmesan
Cheese stilton
Bread white
Bread wholemeal
Cereals cornflakes
Cereals muesli
Cereals bran based
Rice brown
Rice white
Flour white
Flour brown
Flour wholemeal
Fruit
Vegetables
Mushrooms
Cashew nuts
Brazil nuts
11.0-19.4
7.6
14.0
3.8
18.5
10.0
36.0
42.0
145.0
1.5
1.0
3.2
7.4
12.0
16.0
6.0
9.0
4.7
4.2
3.6
10.0
13.0
2.3
4.4
10.0
1.0
2.0
9.0
27.0
254.0
Table 3.18 Estimated human dietary intake of selenium from dietary sources (Adapted from Selenium in
Nutrition, 1983).
Food
New Zealand USA
Total, g
Meat, fish, %
Cereals, %
Dairy products, %
Others, %
56.2
67.1
7.07
15.0
10.2
132.0
52.0
33.7
10.2
4.1
Canada
150.7
30.5
49.4
15.5
4.6
Japan Venezuela
88.3
63.0
27.1
2.6
7.3
325.8
46.8
27.1
21.6
4.5
UK1 Ireland2 Russia3

34
30.6
29.1
16.2
24.1
/Barclay et al., 1995; 2/Murphy et al., 2002; 3/Tutelyan et al, 2002
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50
41
24
9
26
30
50
10
30
190
Table 3.19 Estimated intake of Se from different food groups in the UK in 1997 (Adapted from BNF,
2001).
Group
Item
Bread/
cereals
Meat
Bread
Miscellaneous cereals
Carcass meat
Offal
Meat products
Poultry
Fish
Oils & fats
Milk
Dairy produce
Eggs
Green vegetables
Other vegetables
Canned vegetables
Fresh fruit
Fruit products
Nuts
Beverages
Sugars and preserves
Potatoes
Estimated total intake
Fish
Fats
Dairy
products/
eggs
Vegetables
Fruit
Other
Total
Selenium
Consumption
content
g/100 g fresh weight g/day
4.4
3.9
11.5
49.2
13.0
18.5
36.0
0.3
1.4
3.2
19.4
0.8
2.2
1.4
0.1
0.07
25.1
0.04
0.9
0.3
191.7
108
101
22
1
47
19
14.0
27
281
60
14
34
76
33
69
44
2
937
63
123
2075
Estimated contribution
to total Se intake
g/day
%
4.8
3.9
2.5
0.5
6.1
3.5
5.0
0.1
3.9
1.9
2.7
0.3
1.7
0.5
0.07
0.03
0.5
0.4
0.6
0.4
39
Table 3.20 Selenium concentrations in various feed ingredients, ppm (Adapted from Selenium in
Nutrition, 1983).
Ingredient
Alfalfa meal
Barley
Brewers grains
Corn
Fish meal
Linseed meal
Meat meal
Oats
Poultry by-product
Soybean meal
Wheat
Wheat bran
Wheat middling
Whole soybeans
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190
USA
Canada
0.01-2.00
0.05-0.5
0.15-1.00
0.01-1.00
1.0-5.0
0.5-1.2
0.08-0.5
0.01-1.00
0.5-.10
0.06-1.00
0.1-3.00
0.1-3.0
0.15-1.0
0.07-0.90
0.02-0.27
0.02-0.99
0.29-1.10
0.01-0.33
1.3-3.4
0.7-1.5
0.2-0.81
0.01-1.10
0.04-0.78
0.02-1.5
0.24-1.3
0.41-0.89
-
1/8/2007, 6:49 PM
12.2
10.0
6.3
1.3
15.5
8.9
12.7
0.3
10
4.8
6.9
0.8
4.3
1.3
0.2
0.1
1.3
1.0
1.5
1.0
100

Table 3.21 Effect of selenium by soil and foliar application on Se content in Soybean (g/g) (Adapted
from Yang et al., 2003).
Sample
Soil application
Soybean
Soybean protein
Soybean okra
Foliar application
Control
Selenite
Se fertilizer
Selenite
Se fertilizer
0.055
0.110
0.040
0.198
0.511
0.115
0.196
0.553
0.131
1.126
1.890
0.868
1.211
1.947
1.111
Did you know that animal-derived foods, including eggs, meat

and milk are major Se sources in the developing countries all
over the world?
Supplemental sources of selenium

Since Se levels in soils vary and Se availability to plants also depends on many
factors, the general agricultural practice in the world includes Se supplementation
of diets fed farm animals and poultry. In fact, FDA approved Se supplements for
poultry and swine in 1974 in the form of selenite or selenate.
Did you know that Sel-Plex, a source of organic selenium,
was approved by FDA for chickens, turkey, pigs and cows?
While Se form was not rigorously considered in the initial research into Se nutrition,
for the last 30 years information has accumulated indicating that the natural form
of Se in plant-based feed ingredients consists of various selenoanimo acids with
SeMet being major form of Se in grains, oil seeds and other important feed
ingredients. Therefore, organic Se is the natural form of Se to include in feed
formulations. However, sodium selenite remains in use in many animal feeds.
The limitations of using inorganic Se are well known and include toxicity,
interactions with other minerals and vitamins, low efficiency of transfer to milk,
meat and eggs and an inability to build and maintain Se reserves in the body. As
a result, a high proportion of the element consumed in inorganic form is simply
excreted. Further, a pro-oxidant effect of the selenite ion (Spallholz, 1997) is a
great disadvantage as well, particularly when shelf life of food animal products is
considered.
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192
Ingredient interactions should be carefully considered. When the premix contains

sodium selenite and ascorbic acid, the chemical reaction between them (Figure
3.4) causes selenite reduction to metallic Se, which is not adsorbed in the digestive
tract of animals, and ascorbic acid is also oxidized thereby loosing biological
activity. Therefore in such a situation, both nutrients are lost. Pink particles in the
premix very often represent metallic Se produced in a way mentioned above.
This could happen in the premix/feed during storage or in the digestive tract
during absorption. For example, transport of 75Se was inhibited when ascorbic
acid and selenite were injected directly into ligated duodenal loops of anaesthetized
chickens. The mechanism of the inhibition seemed to be precipitation of Se within
the intestinal lumen, since less 75Se was found in the supernatant fraction of the
luminal fluid when ascorbic acid was present (Mykknen et al., 1983). Therefore,
vitamin C in premixes is compatible with organic Se but incompatible with selenate
or selenite. This issue is extremely important for anti-stress premixes containing
increased levels of ascorbic acid. For example, an interaction of 0.5% vitamin C
with either selenite or seleno-DL-Met (SeMet, 3 ppm) was studied in rats (Ip,
1986). Results showed that the protective effect of selenite in tumorigenesis was
nullified by vitamin C, whereas the chemopreventive action of SeMet was not
affected. The authors suggested that selenite is reduced by vitamin C to elemental
Se and was not available for uptake by tissues. Similarly, the availability of Se
was reduced almost to zero when selenite and 1 g ascorbic acid were taken together
well before the meal (Robinson et al., 1985). This hypothesis was confirmed by
data indicating that 0.5 or 0.25% of vitamin C in the diet completely negated in
blood, liver and mammary gland the accumulation of Se induced by 3 ppm of
selenite supplementation. It is also interesting that other compounds in the premix
can similarly reduce Se in selenite to produce elemental Se, which is not absorbed
from the feed. For example, a reference from Michigan State University (Groce et
al., 1973) indicates changes in odour and/or colour when premixes containing
200 ppm Se were prepared with glucose monohydrate, corn starch or sucrose and
then stored at room temperature. The order was musty and sweetish in character,
while the colour changes involved appearance of pink to dark red particles in the
original white matrix. As can be seen in Table 3.23, Se retention was decreased
and Se excretion was increased as a result of old premix inclusion in pig diets. In
contrast, ascorbic acid enhances SeMet assimilation from the diet. Furthermore,
SeMet itself is considered to possess antioxidant properties (Schrauzer, 2000).
Furthermore, Se yeast is an effective in vitro and in vivo antioxidant (Vinson et
al., 1998). Thus, the use of sodium selenite in animal diets has recently been
questioned (Pehrson, 1993; Mahan, 1999; Surai, 2002). Indeed, recent discoveries
related to pro-oxidant properties of selenite and its interference with other dietary
compounds like ascorbic acid put pressure on feed manufactures to find new,
more effective sources of supplemental Se. Therefore, the simplest idea was to
use Se forms produced by plants.
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Selenite
Selenate
Seleno methionine
+ Ascorbate
Metallic Se
Not
absorbed and
not
assimilated
+ Ascorbate
Se-Met
Absorbed and
assimilated
Figure 3.4 Selenium-ascorbate interactions in premixes and digestive tract.
Table 3.22 Selenium content (mg/kg dry matter) of Finish retail-store foodstuff (Adapted from Varo et
al., 1994).
Foodstuffs
1984 a
1990b
1992 c
Wheat bread, French loaf

Rye bread, whole
Potato
Beef, steak
Pork, fillet
Milk, whole
Cheese, Edam type
Eggs
0.05
0.07
<0.01
0.17
0.35
0.06
0.09
0.69
0.23
0.06
0.11
0.64
1.09
0.23
0.42
1.27
0.14
0.04
0.07
0.54
0.84
0.17
0.29
1.16
/Before Se fertilization;
b
/The amount of sodium selenate in fertilizers used for grain production (16 mg/kg) and in
fertilizers used for fodder and hey (6 mg/kg);
c/ The amount of sodium selenate in all fertilizers (6 mg/kg) since 1991
Application of Se supplemented fertilizers was started in 1985, the amount of Se in
fertilizers was reduced in 1991
Table 3.23 Selenium balance in pigs as affected by premix (Adapted from Groce et al., 1973)
Item
Stored Se premix
Exp. 1
Exp. 2
Se intake, g/day
Se excretion, % of intake
Faecal
Urinary
Se retention, g/day
Se retention, % of intake
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193
Freshly prepared Se premix

Exp. 1
Exp. 2
53.2
132.2
52.0
129.5
51.7
29.8
9.8
18.6
25.9
47.7
34.9
26.5
41.0
12.7
23.7
46.2
22.5
41.7
46.5
35.9
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194

Did you know that selenite can react with ascorbic acid
in the premix, feed or digestive tract resulting
in loosing activities of both elements?
It is well known that chemical and physical properties of Se and sulphur are very
similar, reflecting similar outer-valence-shell electronic configurations and atomic
sizes (Combs and Combs, 1984). Therefore plants cannot distinguish between
these two elements when synthesising amino acids. As a result they can synthesize
SeMet when Se is available. This biological feature was the basis for development
of the commercial technology of organic Se production from yeast (Sel-Plex,
Alltech Inc., USA). Selenium composition in this product matches closely that
found in most grains with more than 50% of total Se being in the form of SeMet.
Analysis of the protein fraction of Se yeast has shown that Se is present in all
the major soluble proteins. SeMet was identified as the major Se-containing
compound in the protein fraction as well as in the whole cell (Korhola et al.,
1986). Therefore, yeast cells can take up Se in the form of selenite or selenate
from media and synthesise selenoamino acids. In particular certain strains of yeast
are capable of accumulating as much as 3000 ppm Se in organic form when in
the growth medium sulphur is replaced by selenium compounds and proper growth
conditions are provided (Gassner et al., 1999; Demirci et al., 1999). The influence
of various Se concentrations from organic (SeMet) and inorganic (sodium selenite)
Se compounds on growth pattern and cell viability and the alterations in the
antioxidant enzyme system of yeast were evaluated (Bansal and Kaur, 2002). A
continuous decrease in cell and colony-forming units counts was observed with
increasing concentrations of Se from either source. Increasing Se status of yeast
cells was found with increasing concentrations of Se with both forms, with much
greater uptake for organic Se at maximum Se concentrations. However, high
concentrations of sodium selenite in the culture medium have a strong inhibitory
effect on the growth of this yeast (Suhajda et al., 2000). Sodium selenite had
stronger inhibition on the yeast growth than sodium selenate and the ratio of
selenium to protein was higher with sodium selenate than with sodium selenite.
Did you know that a composition of selenocompounds in the
selenized yeast is similar to that of grains with more than 50% of
selenium in the form of SeMet?
As mentioned above, SeMet is the major selenocompound in Se-enriched yeast.
For example, SeMet in yeast and nuts comprised respectively 65% and 75% of
total Se (Wrobel et al., 2003). Similarly, a proteolytic enzyme extract of Se yeast
was found to contain Se as SeMet (74.8%), selenocystine (9.9%), selenite (5.1%)
and as at least three unknown Se compounds (10.2%, Yoshida et al., 2002). SeMet
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comprised about 85% of total Se compounds found in selenized yeast used for
human trials (Ip et al., 2000). Similarly selenized yeasts, which were used as a
source of Se in the PRECISE and other trials, contained SeMet at 54-60% of the
total selenium (Larsen et al., 2004). A commercial source of Se-enriched yeast
tablets containing 210 g Se/g was found to contain 73% of the total Se as SeMet
(Wolf et al., 2001). Recently it has been shown that the synthesis of SeMet in
yeast actively takes place in the growth phase (Ponce de Leon et al., 2002).
It seems likely that selenoamino acid composition of the yeast depends on
various factors, including yeast species, growth conditions as well as analytical
techniques used. For example, recently three different commercial yeast products
were analysed. Results showed (Table 3.24) that the proportion of water-soluble
Se varied from 11.5% up to 28.0% and water insoluble polysaccharide bound Se
proportion varied from 15.5% up to 72% (Encinar et al., 2003). This means that
not all yeast products are the same and results obtained in studies with one product
cannot be generalized to all yeasts.
Table 3.24 Selenium species in three selenized yeast products (Adapted from Encinar et al., 2003)
Selenium species in yeast fractions
A, %
B, %
C, %
Water soluble Se
Water insoluble polysaccharide-bound Se
Water insoluble protein-bound Se
Residual protein-bound Se
Residual hydrolysable Se
11.5
15.5
19.0
38.0
16.0
28.0
27.0
38.0
6.0
1.0
21.0
72.0
4.0
1.0
1.0
Did you know that not all Se-yeasts are the same and data
obtained with one product can not be automatically applied
for another product?
When selecting a Se supplement, another important consideration is composition
of organic Se compounds in the supplement. While SeMet represents the dominant
Se form in Se-enriched yeast, each yeast has a unique combination of organic Se
compounds which must be considered when beneficial effects of organic Se are
expected. This means that SeMet alone sometimes less effective that Se-enriched
yeast. For example, in mice high-Se yeast caused the largest increase of GSH-Px
activity followed by sodium selenite and SeMet (Bergman and Slanina, 1986).
Furthermore, SeMet in purified form is unstable and easily oxidised. For example,
recently it has been shown that in the freeze-dried samples of oyster total Se and
the Se species evaluated are stable for at least 12 months, under all the conditions
tested. However, after purification of Se species, including SeMet, in the enzymatic
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196
extracts they are only stable for 10 days if stored at 4C in Pyrex containers
(Moreno et al., 2002). In contrast, SeMet is quite stable in the yeast. Indeed,
analysis of high-Se yeast stored at room temperature for more than 10 years showed
SeMet as the major Se product (Block et al., 2004). Furthermore, the shelf life of
Se yeast at 25C predicted from the Arrhenius plot exceeded 1126 days (Szulc et
al., 2003).
Did you know that pure SeMet is unstable and easily oxidised?
In contrast, in the form of selenized yeast SeMet is quite stable.
The most common dietary supplement form of Se for human is Se-enriched yeast.
The development and commercial application of Sel-Plex with guaranteed
composition and evidence from research and commercial trials (which will be
presented in this book) opens a new era in animal nutrition providing opportunities
not only for improvement of animal health and productivity but also for production
of Se-enriched meat, milk, eggs and other foods considered to be important steps
in improvement of human diets. Indeed, a comparison between Sel-Plex and
selenite, based on published data, presented in this book in various chapters,
clearly showed advantages of the natural form of Se in comparison to selenite
(Table 3.25). Indeed sodium selenite has a range of various properties, which are
not shared by other forms of selenium (Table 3.26). Therefore, it seems appropriate
that selenite be considered as a drug, which should be used accordingly. For
example, when Se deficiency is diagnosed based on clinical signs, selenite would
be the preparation of the choice. Using it via feed, water or injection will solve the
short-term or acute Se deficiency, which has been demonstrated under various
experimental conditions with chickens, pigs and cattle. However, when the goal
is meeting physiological requirements of animals in order to maintain high
productive and reproductive performance, optimum food animal product quality
and immunocompetence, a Se supplement that allows tissue reserves is needed.
Did you know that based on the mode of action selenite should be considered
and used as a drug and Sel-Plex as a feed additive?
Did you know that based on the mode of action selenite
should be considered and used as a drug and Sel-Plex
as a feed additive?
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Table 3.25 Major differences between organic selenium (Sel-PlexTM) and selenite.
Organic selenium (Sel-PlexTM) Selenite
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Absorption
Similar to methionine with

active transport in the gut
Accumulation
Building Se reserves by non- Not accumulated in the body

specific incorporation of
SeMet into the proteins
Toxicity
At least 3 times less toxic

than selenite
Highly toxic, can penetrate via skin

causing problems
Bioavailability
Higher bioavailability in
comparison to selenite to
animals and human
Very low availability for ruminants

due to reduction by rumen microbes
Antioxidant activity
SeMet possesses antioxidant Possess pro-oxidant properties and

properties per se and could
could stimulate free radical
scavenge NO and other
production when reacting with GSH
radicals
Effect on DNA
SeMet stimulates DNA-repair Selenite can cause DNA damage

enzymes
Transfer to eggs,
milk and meat
Transferred to egg, milk and

meat giving a possibility to
produce designer/functional
food
Poorly transferred to eggs, milk and

meat
Transfer via placenta
Better transferred via

placenta than selenite
Poorly transferred via placenta
Reactions with other

elements
Neutral, ascorbic acid

Highly reactive, reduced to metallic,
promotes SeMet assimilation unavailable selenium by ascorbic
from the diet
acid
Protective effect in
stress conditions
Provides additional protection Cannot provide additional

due to Se reserves in the body protection due to absence of Se
reserves in the body
Effect on drip loss
Did not affect drip loss
Increases drip loss
Environmental issues
Better retention in tissues,

less released with faeces and
urine
Low retention in tissues and high

release with faeces and urine
Stability during
storage and feed
processing
Stable
Stable
Classification based
on the mode of action
Feed additive
Drug
197
Similar to other mineral with passive

transport in the gut
1/8/2007, 6:49 PM
198
Table 3.26 Reactions of selenite which are not shared by other forms of selenium (Adapted from
Whanger, 2002).
Reactions
Reactions
Reduction to elemental selenium by

ascorbate
Low diffusibility during gastrointestinal digestion
Conversion to insoluble selenium by tea Significantly greater binding to brush border

membrane vesicles
Reduction to insoluble selenium by
rumen microbes
Much greater variation in absorption
Causes nuclear cataract in young rats
Lucigenin-dependent chemilumiscence with GSH

(free radical production)
Significantly lower absorption in

humans with stable isotopes
Formation of selenotrisulfides with cysteine or

reduced GSH
Significantly less absorption in ligated

loops
Many metabolites in absorption during intestinal

perfusion
Lower absorption from infant formulas
A potent inducer of apoptosis
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Selenium and Immunity 213

4
SELENIUM AND IMMUNITY
If you give a man a fish, you feed him for a day,
If you teach him to fish, you feed him for a lifetime
(Confucius)
Introduction
Human and animal defence against various diseases depends on the efficacy of
the immune system responsible for elimination of foreign substances (e.g. parasites,
bacteria, moulds, yeast, fungi, viruses and various macromolecules) or the creation
of specific inhospitable conditions within the host for a wide range of pathogens.
This protective capacity is based on the effective immune system which is
considered to be an important determinant of animal health and well being. In
that sense, a remarkable ability of components of the immune system to distinguish
between self and non-self is a great achievement of animal evolution.
Commercial animal production is based on balanced feed, providing
requirements in major nutrients and optimised environmental conditions. However
it is very difficult to avoid various nutritional or environmental stresses which are
responsible for immunosuppression and increased susceptibility to various diseases
and as a result decreased productive and reproductive performance of animals.
For example, mycotoxins are among major immunosupressive agents in animal
diet. In such situations immunomodulating properties of certain macro- and
micronutrients are of great importance for the animal industry. In fact, almost all
nutrients in the diet play a crucial role in maintaining an optimal immune
response, and both insufficient and excessive intakes can have negative
consequences on the immune status and susceptibility to a variety of pathogens.
Information has been actively accumulated for the last 10 years indicating that
selenium is among major immunostimulating agents and its requirement for such
action is usually higher than that for animal growth and development. In fact,
selenium has been shown to have immunomodulatory effects in a variety of species
when administered in quantities in excess of established dietary requirements.
Therefore, selenium as an essential component of selenocysteine-containing
proteins is involved in most aspects of cell biochemistry and function and immune
cell activity is also Se-dependent. For example, Se deficiencies are found in chronic
renal failure, malnutrition malabsorption, long term parenteral nutrition, AIDS
and probably Se deficiency interferes with chronic infections which often go with
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these diseases (Dubois and Belleville, 1988). In particular, selenium concentration

showed significant depression in the acute stage of infection compared with the
values after the recovery (Sammalkorpi et al., 1988). Furthermore, Se deficiency
attenuates the host immune response, thereby increasing the risk of bacterial and
viral infections.
Immune system and its evaluation

There are two major types of immune function: natural and acquired immunity
(Figure 4.1). Natural immunity, called the innate immune system, includes physical
barriers (e.g. skin, mucus coat of the GI tract), specific molecules (e.g. agglutinins,
precipitins, acute-phase proteins, lysozyme), phagocytic function of phagocytes
(macrophages and neutrophils), and lysing activity of a class of lymphocytes
called natural killer (NK) cells (Table 4.1).
Immunity
Natural (Innate)
Physical
bariers
Phagocytes
Mast
cells
NK cells
Macrophages
ROS, RNS,
Small peptides
Kill pathogen
Acquired (adaptive, specific)

Specific
molecules
Humoral
Dendritic
cells
B-lymphocytes
T-lymphocytes
Immunoglobulins
(antibodies)
Direct contact
with target cells
Neutrophils
Eicosanoids
Cytokines
Complement,
Acute phase protein
Inflammatory
response
Cell-mediated
Pathogen removal and resistance to

pathogens
Figure 4.1 General scheme of the immune system (adapted from Surai 2002).
Macrophages perform a range of functions, including phagocytosis of foreign

particles, destruction of bacterial or tumor cells, secretion of prostaglandins and
cytokines (Table 4.2) and as a result regulating activity of lymphocytes and other
macrophages (Qureshi, 1998). In fact, phagocytosis is the major mechanism by
which microbes are removed from the body and is especially important for defence
against extracellular microbes. As a result of stimulation (for example by microbes)
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Table 4.1 Key elements of the immune system (adapted from Kolb, 1996; Lydyard et al., 2000).
Cells
Significance
Monocytes, macrophages
Phagocytosis, synthesis of interleukins 1,6, 8 and other

substances
Neutrophils
Phagocytosis of bacteria, viruses and toxins
Eosinophils
Destruction of parasites
Basophils
Initiation of inflammatory processes
Mast cells
Release of imflammatory mediators
B cells (B lymphocytes)
Production of plasma cells

(immunoglobulins=antibodies), antigen- specific; 10%
of total lymphocytes
T helper cells (Helper T

lymphocytes)
Antigen-specific, produce cytokines: IL 2, IL3,IL4, IL5,

IL9 and IL10; 55% of total lymphocytes
Cytotoxic T cells (T
lymphocytes)
Destruction of tumour cells and virus- Infected cells;

antigen-specific, 25% of total lymphocytes
Suppresser T cells (T
lymphocytes)
Inhibition of immune reactions (development of

autoimmune diseases)
Natural killer cells
Destruction of tumour cells and virus- Infected cells,

10% of total lymphocytes
Macromolecules
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Immunoglobulins
Binding of foreign cells and proteins; Promotion of their

ingestion by phagocytes
Interferons (IFN-; IFN-;IFN-)
Activation of macrophages (-interferon); Inhibition of

viral replication
Complement system: a set of

over 20 soluble glycoproteins
Destruction of foreign cells
Interleukins
Regulation of specific types of leukocytes
Leucotrienes
Lysozymes
Promotion of inflammatory process

Dissolution of bacterial membranes
Collectines- a group of carbohydrate- binding proteins
Act as opsonins in nonadaptive immune

Response to pathogen
Acute phase proteins- a group of

plasma proteins produced in the
liver in response to microbial
stimulus
Maximise activation of the comlement system
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Table 4.2 Macromolecules produced in immune cells (adapted from Kolb, 1996; Lydyard et al., 2000).
Name
Site of production
Principal effects
IL-1
MPH, epithelial cells
IL-2
T-helper cells, Mast cells
IL-3
T-helper cells
IL-4
T-helper cells, mast cells
IL-5
T-helper cells, mast cells
IL-6
IL-9
IL-10
MPH, T-helper cells,

fibroblasts, endothelial
cells; mast cells
Thymic epithelial cells,
bone marrow cells
Monocytes, MPH, fibroblasts, keratinocytes
T-helper cells
T-helper cells, MPH
Stimulation of T- and B-cell proliferation and

production of inflammation- modifying proteins
in the liver; induction of acute phase proteins;
increase of effector cell access; tissue destruction
Proliferation of T- and NK cells, increase in
NK cell activity; activation of production of
cytokines
Induction of proliferation and differentiation of
Stem cells in bone marrow
Stimulation of T- and B-cell proliferation and IgG
and IgE synthesis in plasma cells; increase in
macrophage cytotoxicity; induction of Th2 and
inhibition of Th1 responses
Stimulation of B-cell proliferation and activation
and IgA synthesis. In plasma cells stimulation of
eosinophil production and accumulation;
Induction of differentiation of B-cells and
production of inflammation-modifying proteins in
the liver; lymphocyte activation; fever
Stimulation of pre-T and pre-B-cell proliferation
IL-11
IL-12
Mesenchymal cells
B cells, MPH
INF-
T cells, NK cells
TNF-
MPH, T cells
IL-7
IL-8*
PlateletMPH
activating
factor (PAF)
Fibroblast
MPH
growth factor
Complement MPH
-I-Protease MPH
inhibitor
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Promotion of neutrophil activation, adhesion to

endothelial cells and migration to inflamed site
Stimulation of mast-cell proliferation
Inhibition of production of INF- and some ILs;
B-cell activation
Stimulates megakaryocyte production
Stimulation of production of T-helper cells type 1
and INF- synthesis by T cells and NK cells
MPH activation; induction of Th1 and inhibition
of Th2 responses
Activation of vascular endothelium; fever; shock;
mobilisation of metabolites
Promotes neutrophil migration and eosinophil
mobilisation
Induces division of connective tissue cells at sites
of injury
Lysis of foreign cells
Inhibits proteases and breakdown of healthy
tissues
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Table 4.2 Contd.
Name
Site of production
-2-Macro- MPH
globin
Leukotrienes MPH
Principal effects
Inhibits proteases and breakdown of healthy
tissues
Promote leukocyte accumulation at inflammatory
foci
Increases the production of granulocytes
(neutrophils) in the bone marrow
Granulocyte MPH
colonystimulating
factor
Granulocyte- MPH
macrophage
colonystimulating
factor
Increases granulocyte and monocyte production
*chemokine; MPH- macrophages
monocytes differentiate into macrophages that are more powerful in mediating

host defence (Klasing, 1998a). The phagocytic process includes several stages
(Lydyard et al., 2000):
Movement of phagocyte towards the microbe using chemotactic signals

Physical contact of the micro-organisms with macrophage and attachment
to the phagocyte surface with a nonspecific or receptor-mediated binding
Endocytosis (engulfment) of microbe resulting in a phagosome by
invagination of surface membrane
Fusion of the phagosome with a lysosome
Killing of microbes by bombarding them with oxidants (superoxide and
hydroxyl radicals, hydrogen peroxide, nitric oxide, hypochlorous acid etc.).
Macrophage activation and phagocytosis of foreign particles are regularly

accompanied by a so-called respiratory burst, an increase in the production of
reactive oxygen species (ROS), exerted by the enzyme complex NADPH oxidase
(Figure 4.2). Therefore macrophages as well as other phagocyte leukocytes (e.g.,
neutrophils, monocytes and eosinophils) can synthesize toxic oxygen metabolites
such as superoxide anion (O2-), hydroxyl radical (OH*), singlet oxygen ( 1O 2),
hydrogen peroxide (H2O2), nitric oxide (NO), peroxinitrite (ONOO-) hypochlorous
acid (HOCl) and chloramines during the respiratory burst (Zhao et al., 1998). For
example, a bacterium coming into contact with the plasma membrane is enclosed
in a plasma membrane vesicle containing NADPH oxidase, and exposed to an
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intensive flow of superoxide radical (Gille and Sigler, 1995). Superoxide radical
can disproportionate to H2O2 which penetrates into the bacterium with a production
of hydroxyl radical, which is ultimately a deadly weapon able to damage any
biological molecules. In general, the production of ROS and reactive nitrogen
species is a characteristic for both mammalian and avian macrophages (Qureshi
et al., 1998). It is interesting that, people whose phagocytes possess no functional
NADPH oxidase, are shown to suffer from chronic infections of the skin, lung,
liver and bones leading to a premature death (Halliwell and Gutteridge, 1999).
Microbe
Activation
O2
Nucleus
NADPH
oxidase
NADPH oxidase
O2*-
NADPH
oxidase
SOD
H 2O2
Fe2+
OH*
Mieloperoxidase
Granules
O2-
HOCl
OH* H O
2 2
O2* Microbe
O2* HOCl
Fe2+
OH*
Phagosome
Figure 4.2 Respiratory burst in neutrophils (adapted from Kettle and Winterbourn, 1997; Nordberg and
Arner, 2001).
In general ROS, RNS and eicosanoids (e.g. leukotrienes and prostaglandins) have
recently received substantial attention as major metabolites produced by
macrophages (Dietert and Golemboski, 1998). Because of this powerful weapon,
macrophages bind, internalize, and degrade foreign antigens (e.g. bacteria) quite
quickly. It takes only 15 minutes for chicken macrophages to kill more than 80%
of the internalized Salmonella (Qureshi et al., 1998). Therefore natural immunity
works rapidly, gives rise to the acute inflammatory response. They contain various
substances (including enzymes producing free radicals and small peptides with
an antibiotic activity) involving in microbial killing. They have also receptors for
chemoattractive factors released from microbes. In addition to ROS, RNS and
eicosanoids mentioned above, macrophages also synthesize and secrete a great
number of such communicational molecules as cytokines, including the proinflammatory cytokines called interleukin 1 (IL-1), interleukin 6 (IL-6), and tumour
necrosis factor- (TNF). They also produce cytokine inhibitors, endocrine
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hormones, neurotransmitters (Klasing, 1998a) which regulate specific immunity
(initiating and directing the immune and inflammatory responses) and many other
related physiological responses. Therefore these cells are important amplifiers of
the immune response, both by cytokine production and by serving to present
parasite derived peptides to T cells.
The purpose of immune cell products is to destroy invading organisms. However,
excessive or inappropriate production of these substances is associated with
mortality and morbidity after infection and trauma, and in inflammatory diseases.
Oxidants enhance IL-1, IL-8, and TNF production in response to inflammatory
stimuli by activating the nuclear transcription factor, NF kappa B. Therefore, they
also can cause a series of harmful effects, including killing host cells, injuring
cells and tissue directly via oxidative degradation of essential cellular components
and damaging cells indirectly by altering the protease/ antiprotease balance that
normally exists within the tissue interstitium (Conner and Grisham, 1996). They
also can cause cell mutations and sister chromatid exchanges (Weitzman and
Stossel, 1981).
Therefore, overproduction of ROS or impaired antioxidant defence can result
in oxidative damage to host macromolecules (McBride et al., 1991) and extensive
pathology and disease (Dietert and Golemboski, 1998). For example, in the case
of disease challenge, the overproduction of ROS may occur and they can leak
from fagolysosomes into the surrounding cytoplasm and intracellular space. A
number of antioxidant enzymes is expressed at the same time to protect the cells
from the cytotoxic effects of ROS directed against engulfed microorganisms.
However, low efficiency of these antioxidant mechanisms, for example due to
selenium deficiency, could damage cells microbicidal and metabolic functions
(Larsen, 1993). Indeed, macrophage function is disturbed, if there is a lack in
antioxidant enzyme activity (Ebert-Dumig et al., 1999). It was shown that TrxR
activity as well as GSH-Px activity in these cells can be upregulated by the addition
of selenium in vitro and ex vivo.
Once a pathogen enters to the host, the initial nonspecific response is an
inflammatory reaction. It results in a series of behavioral, immunologic, vascular
and metabolic responses (Korver and Klasing, 2001). As a result growth rate
could be slowed, appetite decreased, muscle protein degradation increased with
possible increased morbidity and mortality and decreased productivity. Therefore
a price for this defence against pathogens could be quite high.
Did you know that our immune system contains about 1012
lymphocytes?
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Acquired or specific immunity includes humoral immunity and cell-mediated

immunity (Figure 4.3). There are two major types of lymphocytes, B cells and T
cells. Humoral immunity is mediated by antibodies that are released by Blymphocytes into the bloodstream. This immunity is based on the production of
immunoglobulins (Table 4.3). They are responsible for specific recognition and
elimination of various antigens: they bind and remove from the host invading
organisms/substances.
Adaptive immune system
Cell-mediated
T Helper/Inducer
(CD4)
Th1
Th2
Th3
Th0
Humoral
T Supressor/Cytotoxic
(CD8)
Type 1
Plasma cells
(B-lymphocytes)
Type 2
IL-4, IL-5, IL-10, IL-13
Antibody production
IL-2
INF-
Delayed-Type hypersensitivity
(cell-mediated inflammatory response)
Figure 4.3 General scheme of adaptive immune system (adapted from Field et al., 2001).
Table 4.3 Main immunoglobulin classes (adapted from Lydyard et al., 2000).
Immunoglobulin
Characteristics
IgG1; IgG2;
IgG3; IgG4
Largest quantity; provide the bulk of immunity to the most blood

borne infectious agents; the only antibody class to cross the placenta
to provide humoral immunity to the infant; vaccination asks the
immune system to produce IgG specific for a particular antigen
A first line of defence against microbes entering through mucosal
surfaces directly communicating with the environment; synthesised
by plasma cells; prevents colonisation of mucosal surfaces by
pathogens
The first antibody produced in an immune response in large quantity
Present in humans, not documented in animals; functions as an
antigen receptor on B cells
Involved in allergy development and in Immediate hypersensitivity
syndromes such as hay fever and asthma
IgA
IgM
IgD
IgE
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Cell-mediated immunity is based on specific antigen recognition by thymus-derived
T-lymphocytes. Due to this immunity, cells infected with a foreign agent, for
example virus, are destroyed via a direct contact between an activated T-cell and
target (infected) cell (Qureshi et al., 1998). Cell-mediated immunity is responsible
for delayed-type hypersensitivity reactions, foreign graft rejections, resistance to
many pathogenic microorganisms and tumor immunosurveillance (Wu and
Maydani, 1998).
NK cells were originally described as a population of granular lymphocytes
with the ability to lyse tumour and virally-infected target cells. They differ from
classical lymphocytes being larger in size, containing more cytoplasm and having
electron dense granules. The mechanism of killing is mediated through release of
its granule contents (perforins and granzymes) onto the surface of the infected
cell (Lydyard et al., 2000). In human NK cells comprise about 10-15% of all
circulating lymphocytes. They produce cytokines and chemokines and can lyse
target cells without prior sensitisation being crucial components of the innate
immune system. They also exert cell cytotoxicity by recognising and inducing
lysis of antibody-coated target cells and can be activated non-specifically by several
cytokines via various receptors (Middleton et al., 2002).
In birds, both T cell and B cell precursors originate in the bone marrow. Actual
development of T cells takes place in the thymus and B cells develop in the bursa
Fabricius (Lahtala, 1998). Interactions between T and B-cells, as well as antigen
presenting cells, are responsible for the development of specific immunity. These
defence mechanisms of specific immunity are induced or stimulated by exposure
to foreign substances, are specific for distinct macromolecules and increase in
magnitude with each successive exposure to a particular macromolecule (Miles
and Calder, 1998). In comparison to natural immunity, specific immunity takes
longer to develop, but is highly specific for antigens and has memory (Table 4.4).
These two parts of the immune system work together through direct cell contact
and through interactions involving such chemical mediators as cytokines and
chemokines (Figure 4.4). Therefore the chicken immune system requires cooperation of macrophages, bursa-derived B-lymphocytes and thymus-derived Tlymphocytes with various other types of cells. Therefore, the immune response
involves cellular proliferation (T-lymphocytes), enhanced protein synthesis
(including immunoglobulin synthesis by B-lymphocytes and acute phase protein
synthesis by liver) and inflammatory mediator production. Physiological changes
resulting from stimulation of the immune system include fever, anorexia and loss
of tissue components (Grimble, 1997).
When analysing data related to immunomodulating properties of various
nutrients it is necessary to pay special attention to methods used to assess
immunological functions. For example, in vivo methods of immune function
assessment are based on two main approaches: antibody response to vaccine or
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Table 4.4 Key features of innate and adaptive immunity (adapted from Hansson et al., 2002; Calder,
2001).
Innate
Adaptive
Appearance in
evolution
Primitive organisms
Vertebrates
Induction time
Fast (hours to days)
Slow (days to decades)
Recognizes
Common pathogen-associated
microbial pstterns (PAMPs)
Unique epitopes on each pathogen/

antigen
Cellular
components
Phagocytes (macrophages and

T and B cells
neutrophils); NK cells; mast cells;
dendritic cells
Generation of
specificity
Encoded in germline; Has some

specificity, no memory
Effector
mechanisms
Complement (Alternative
Antibodies; cytotoxic T cells
pathway); cytokines; chemo(CTL); classical complement
kines; cell-mediated cytotoxicity activation; antibody-dependent
cell-mediated cytotoxicity;
cytokines; chemokines
Soluble mediators
Macrophage-derived cytokines
Lymphocyte-derived cytokines
Characteristic
transcription
factors
NF-B (+JNK/AP1)
Jak/STAT, NF-B, etc.
Physiological
barriers
Skin
Cutaneous and mucosal immune

systems
Antibodies in mucosal secretions
Mucosal membranes
Lysosyme
Stomach acid
Commensal bacteria
Somatic rearrangement; Highly

specific and has memory
delayed-type hypersensitivity (DTH) reaction. In the first method immunisation

with appropriate antigens (viral or bacterial) can elicit serum antibodies. So called
hemaglutination (HA) assay measures serum antibody concentration (titer) against
antigens. Sheep red blood cells (SRBC) are often used as antigens. This assay
provides information about humoral immunity (B-cell responsiveness) and its
association with cell-mediated immunity (T cell co-operation). The second (DTH)
method is used to assess cell-mediated immune function.
In vitro indices of immune function include (Wu and Meydani, 1999):
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Insult
(e.g. infection, trauma,

burns, surgery, etc.)
Antigen presentation
Cytokines
Macrophages
Endothelial cells
Cytokines
T-lymphocytes
B-Lymphocytes
Macrophages
Neutrophils
Cytokines
Inflamatory mediators
(TNF, IL-1, Il -6, Il -8, PGE2
ROS, RNS)
Tissue damage
Brain: reduced
appetite, fever
Antibodies
Skeletal muscle
Adipose tissue
Nutrient release
Liver
Altered metabolism
Acute phase proteins
Complement
Pathogen
destruction
Homeostasis
Immunological memory
Figure 4.4 The natural and adaptive immunity interactions (adapted from Calder, 2001).
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lymphocyte proliferation assay. Lymphocyte proliferation assay provides

information about cell-mediated immune response and includes measuring
the number of cells in culture with or without addition of stimulatory agent
(mitogen). In this assay, isolated lymphocytes are incubated with mitogens
which activate division of either T-or B-lymphocytes. Decreased proliferation
may indicate impaired cell-mediated immunity. Various mitogens are used
in such assays, but most often they include (Hayek et al., 1996):
- concanavalin A (conA, a type of protein found in the jack bean, T-cell
mitogen);
- phytohemagglutin (PHA, a protein obtained from plant seeds, T-cell
mitogen);
- lipopolysaccharide (LPS, obtained from the membrane of Gram-negative
bacteria, B-cell mitogen) and
- pokeweed mitogen (PWM, T- and B cell mitogen)
measuring cytokine production. T cells produce a range of protein mediators
called cytokines which regulate cell activation, growth, differentiation,
inflammation and immunity.
cytotoxicity assay. This assay assesses activity of cytotoxic T lymphocytes
(a group of T cells that kill other cells by recognising their cell-surface
antigens) and Natural killer (NK) cells (a group of non-T and non-B
lymphocytes that kill virus-infected and tumor cells).
flow cytometric analysis. The assay deals with identifying the cells with
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different surface markers. The results can be used for understanding the
cellular basis of immune response.
plaque-forming cell (PFC) test, which shows the number of antibodyproducing cells.
Effect and role of animal immune system in modern animal production systems is
difficult to overestimate. Banning feed grade antibiotics in Europe will make
immune system competence the major factor determining efficiency of poultry
production. Molecular immunology is developing very quickly and mechanisms
of immunocompetence recently have received substantial attention (McCorkle,
1998; Saif and Swayne, 1998) and nutritional modulation of resistance to infectious
diseases (Klasing, 1998) is a frontline for future research.
Did you know that the total number of lymphocytes represent a
little more than 1% of the body weight of an animal?
Among different nutrients playing an important role in modulating immune
response (Klassing, 1998) are natural antioxidants. Selenium (Turner and Finch,
1991; Larsen, 1993; MacPherson, 1994; McKenzie et al., 1998) is shown to be
very promising. However, antioxidant roles in animal and human immune system
modulation received only limited attention. Mechanisms of nutritional modulation
of resistance to infectious diseases were divided by Klasing (1998) into seven
categories. However, Selenium was not mentioned there.
It is well known that several indicators of immune responsiveness are depressed
when animals are selenium and/or vitamin E deficient. In particular, decreases in
both cellular and humoral immune function in man, laboratory and farm animals
and chickens are also observed (Combs and Combs, 1986). Since these nutrients
serve as antioxidants, membrane integrity may be affected by a deficiency.
However, cellular integrity is very important for receiving, and responding to the
messages needed to co-ordinate an immune response (Latshaw, 1991). Therefore
the antioxidant status of the host is a critical consideration in the optimal functioning
of the immune system. Furthermore, modulating immune responses of birds by
nutritional means in many cases targets specific immunity (Korver and Klasing,
2001).
Phagocyte functions
Phagocytosis is the process by which leukocytes and other cells ingest particulate
ligands whose size exceeds about 1 m. By ingesting microbial pathogens,
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phagocytes accomplish two important immune functions: initiate a microbial death
pathway and build a bridge (send specific signals) between the natural and adaptive
immune responses (Greenberg and Grinstein, 2002). As mentioned above
phagocytes play an important role in natural immunity. Among 6 major specific
rationales for modulating macrophage function in poultry analysed by Klasing
(1998a), mitigation of immunosuppression arising from infectious diseases, dietary
toxins or stress could be affected by dietary antioxidants. In fact the availability
of certain substrates and enzymatic cofactors can greatly influence the capacity
for metabolite production by macrophages.
Studies conducted in late 70th indicated that neutrophils that were from either
vitamin E-deficient or Se-deficient mice or humans had impaired bactericidal
activities (Hogan, 1999). In particular Boyne and Arthur (1979) reported that
polymorphonuclear neutrophil (PMN) function was compromised in Se-deficient
cattle. On the other hand, intracellular kill of S. aureus was greater in neutrophils
isolated from Se-supplemented cows than in neutrophils from cows without
supplemental Se (Hogan et al., 1990). Similarly, PMN of Se-vitamin E injected
cows killed the phagocytized bacteria significantly better than did PMN from Sedeficient cows (Gyang et al., 1984). Supplementation of cow macrophages in
vitro with vitamin E (1 mM) and selenium (0.5 mg/ml) enhanced the production
of neutrophil chemotaxins by the macrophages and enhanced the proliferation of
the lymphocytes in response to stimulation with Con A, but not to PHA or
pokeweed mitogen (Ndiweni and Finch, 1995). Neutrophils obtained from milk
of cows fed Se-supplemented diets killed a significantly higher percentage of
ingested bacteria than did neutrophils from cows fed the Se-deficient diet with
neutrophils from Se-deficient cows having lower viability (Grasso et al., 1990). It
is necessary to underline that PMN provide the major cellular defence mechanism
within the mammary gland and these results are relevant to mastitis development
in Se-deficient cows. When calves, aged 3 to 8 weeks, received (with an interval
of 2 weeks) two intramuscular injections, each of which contained 5.75 mg
selenium and 75 mg -tocopheryl acetate they showed higher blood leukocyte
counts (with less variation), a greater phagocytosis index and more NBT-positive
granulocytes than untreated animals (Bednarek et al., 1996). PMN from goats fed
selenium-deficient and selenium-adequate diets were tested for their ability to
perform random migration under agarose, leukotaxis toward serum chemotaxins
under agarose, and phagocytosis of opsonized zymosan by chemiluminescence
analysis (Aziz et al., 1984). Function of PMN from goats fed the selenium-deficient
diet was severely depressed, and incubation of these cells with selenium resulted
in marked functional enhancement. Furthermore, administration of selenium to
goats fed the selenium-deficient diet resulted in increased PMN functions.
Multifarious sows were fed ensiled, shelled corn-soybean meal-based diets without
supplemental Se during gestation and to d 4 of lactation. Blood was obtained on
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0, 30, 60, and 90 d of gestation and at parturition for serum Se assays.

Lymphocytes and PMN were isolated from the blood, colostrum, and 4-d milk
samples for immune studies. The Se-deficient diet reduced serum Se concentrations
and the phagocytic activity of PMN (Wuryastuti et al, 1993). Therefore, Se
deficiency compromises the microbicidal activity of phagocytes (Serfass and
Ganther, 1975). In particular, dietary deficiencies of Se and/or vitamin E have
been shown to impair neutrophil and macrophage activities in chickens including
decrease in peritoneal macrophages and decreased phagocytosis of red blood
cells (Dietert et al., 1990). The intracellular killing of yeast and bacteria by
neutrophils and macrophages from chicken with selenium deficiency is reduced
(Larsen, 1993). Similar effects were observed in rat, goat, cattle and fish (for
review see Larsen, 1993). For example, intracellular superoxide anion production
of channel catfish macrophages was higher in fish fed the diet fortified with Se
(0.8 vs 0.2 mg/kg) and vitamin E (240 vs 60 mg/kg feed) in comparison to the
fish fed the control diet (Wise et al., 1993).
The influence of a selenium deficient diet in mice and rats on secretory activities
of peritoneal macrophages and mitogenesis of spleen cells has also been studied
(Parnham et al., 1983). Macrophage GSH-Px activity was significantly reduced
from 9 weeks on the selenium deficient diet with enhancement of macrophage
H2O2 release on zymosan stimulation after 12 weeks on the diet. This could mean
that H 2O 2 over-production in macrophages could be dealt with by GSH-Px.
Increased cellular damage in macrophages due to Se deficiency could explain a
reduction in their H 2O 2 generation induced by phorbol myristic acetate. It is
interesting that Se-deficient macrophages are not able to produce a respiratoryburst reaction and as a result their antimicrobial function is compromised. However,
macrophage -glucuronidase release is not affected by Se. It seems likely that
peroxide-mediated cell injury would also account for the reduction in mitogenesis
of spleen cell cultures in response to T and B cell mitogens after 8 weeks on the
diet. Incubation of rat phagocyte cells in an H2O2-generating system resulted in a
time-dependent loss in their ability to ingest and caused a decreased degranulation.
Se-deficient phagocytes were affected to a greater degree than control once (Baker
and Cohen, 1984). Similarly, feeding Se-deficient diet for 75 days to rats resulted
in compromised neutrophil functions, including reduction in candidacidal activity,
superoxide production, oxygen consumption, glucose utilisation and GSH-Px
activity (Kukreja and Khan, 1994). Supplementing the diet with Se for 30 days
resulted in partial restoration of all the activities. Thus, selenoproteins appear to
be important in protecting those aspects of phagocytic function that are sensitive
to the destructive properties of exogenous H2O2. In fact, Se-deficient granulocytes
did not metabolise H2O2 as well as replete granulocytes and H2O2 caused damage
to the O2-generating system. For example, prolonged incubation with stimulants
and prior incubations with an H2O2-generating system caused loss of activity of
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the membrane-bound, NADPH-dependent, O -2-generating system in seleniumdeficient granulocytes with low GSH-Px activity (Baker and Cohen, 1983). It
seems likely that macrophage function is insufficient in the case of altering the
generation of a respiratory burst (e.g. hereditary chronic granulomatous disease)
or when there is a lack in antioxidant enzyme activity. Thioredoxin has been
identified as a lymphocyte growth factor and might therefore be involved in the
crosstalk between macrophages and lymphocytes (Ebert-Dumig et al., 1999).
Selenium deficiency was demonstrated to impair the ability of mouse neutrophils
to kill C. albicans in in vitro tests (Boyne and Arthur, 1986). As a result, deaths in
the selenium-deficient mice from i.v. injections of 0.1 ml suspensions of 1 X 105
or 5 X 104 C. albicans in 0.9% sterile saline, started after 2.5-3.5 d compared with
7-8.5 d in the selenium-supplemented animals. Furthermore significantly more of
the microorganisms were found in the kidneys, livers and spleens of the seleniumdeficient mice compared with the same organs of selenium-supplemented animals.
Did you know that , in extreme cases, macrophages can
internalise the equivalent of >100% of their surface area
within 30 minutes?
Regulatory effects of Se and vitamin E on phagocyte functions have been shown
in experiments with farm and laboratory animals as well as in various in vitro
systems. For example, both vitamin E and selenium enhanced the chemotactic
and random migration of PMN and increased the production of superoxide
following stimulation with phorbol myristate acetate (Ndiweni and Finch, 1996).
In vitro selenium supplementation of the J774.1 macrophage cell line enhanced
phagocytosis, degranulation by the release of beta-glucuronidase after N-formylmethionyl-leucyl-phenylalanine (FMLP) or cytochalasin B, and the production
of superoxide anion after phorbol myristate acetate stimulation of these cells (Safir
et al., 2003). Selenium supplementation also substantially increased the release
of tumor necrosis factor, interleukin-1 and interleukin-6 after lipopolysaccharide
stimulation compared to selenium-deficient cells.
The in vitro effects of an inorganic selenium salt on phagocytic functions of
human neutrophilic granulocytes from donors with a low activity of GSH-Px have
been investigated (Urban and Jarstrand, 1986). The phagocytic and bactericidal
activities were significantly increased in granulocytes exposed to selenium in
physiological concentrations (100 and 200 ng Se/ml). However, at 2000 ng Se/ml
these activities were found to be equal to or lower than control levels probably
reflecting Se toxicity. It is necessary to bear in mind that effect of supplemental
dietary Se on phagocyte function depends on many factors, including background
Se level, dose and form of Se used, age and sex of animals tested, etc. For example,
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ingestion of sodium selenite at 400 g/day (182.8 g pure selenium) by volunteers

resulted in a significant increase in plasma selenium levels (Greenman et al.,
1988). However, the phagocytic function of PMNs measured by ingestion of Oil
Red O paraffin droplets and chemiluminescence tests was not significantly affected.
Therefore, it was concluded that inorganic selenium was not an efficient
stimulating agent of phagocytosis in humans. Therefore, high selenium doses
could be detrimental for immune cells. For example, data reported by Shilo and
Tirosh (2003) showed that exposing Jurkat T cells or J774.2 macrophages to >5
M sodium selenite induced cell death. They suggested that selenite induces
changes in the balance between mitochondrial superoxide and hydrogen peroxide
production, which can facilitate cell death in immune system cells. The authors
considered these changes as a mechanism by which selenium down-regulates the
immune systems inflammatory response and protects against overproduction of
peroxides.
Since various mycotoxins are shown to reduce macrophage viability and effector
functions (Bondy and Pestka, 2000) a protective immunostimulating effect of
antioxidants, including selenium, could be especially important in commercial
conditions associated with mycotoxicoses (Hegazy and Adachi, 2000).
Furthermore, optimised management of macrophage metabolite production by
nutritional means is a crucial factor for bacterial resistance of poultry (Dietert and
Golemboski, 1998).A summary of Se effects on phagocyte functions is shown in
Table 4.5
Table 4.5 Effect of Se on phagocyte functions (adapted from Larsen, 1993; Turner and Finch, 1991;
Ndweni and Finch, 1996).
Species
Effect of Se
Cattle
High Se: Intracellular killing ability ; viability

Extracellular H2O2 ; chemotactic and random migration of PMN ;
superoxide production
Low Se: neutrophil candidacidal activity ; myeloperoxidase activity
Low Se: Neutrophil functions
Low Se: Peritoneal macrophages , viability , phagocytosis
Low Se: Killing of Candida by neutrophils/macrophages ,
Mortality from infection
Low Se: Killing of Candida by neutrophils/macrophages ,
Mortality from infection ; neutrophil chemotaxis
Goat
Chicken
Rat
Rat
Antibody production
Research data accumulated for the last 30 years clearly indicate that antioxidant
deficiency is associated with impaired immune system in human and laboratory
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and farm animals. In fact, effects of Se on immunity have been studied since
1972 when Bernstein showed that Se enhanced humoral immunity with following
a series of publications by Spallholz who tested immunomodulating properties of
Se in mice. His results showed that adding 2.8 ppm Se to the diet stimulated
antibody synthesis to SRBC while 1.25 ppm Se increased number of plaqueforming and concentration of IgM (Spallholz et al., 1973). It is interesting that
concentration of IgG was also affected by Se supplementation of mice at 1-3 ppm
(Spallholz et al., 1973a). Sodium selenite administered to mice i.p. (5 g Se)
enhanced the primary immune response to the sheep red blood cell antigen
(Spallholz et al., 1975). Enhancement of the primary immune response was greatest
when Se is administered prior to or simultaneously with the sheep red blood cell
antigen.
At the next stage of the research related to immunomodulating properties of Se
a chicken model was widely used. For example, when two-week-old chickens
were maintained on Se- or Se-vitamin E deficient diets from hatching, they had
reduced antibody titres to SRBC (Marsh et al., 1981). In the same experiment
three-week old chicks had decreased antibody production only in combined Sevitamin E deficiency. Therefore it was suggested that Se and vitamin E may be
important components of immune function, and their effects depend on antigen
concentration, sex, and the age (Larsen, 1993).
Did you know that man and mice, normally, have about 10 mg
of antibodies in a millilitre of their blood?
In contrast to selenium and/or vitamin E deficiency, antioxidant supplementation
of the diet is shown to have immunostimulating effect and the level of vitamin E
or selenium required for optimal immune function may be higher than that required
for growth and other physiological functions. The simplest tests with antibody
production against SRBC clearly showed a beneficial effect of vitamin E and
selenium.
Newcastle disease challenge is often used to assess immunostimulating
properties of various antioxidants. For example, the immune response of chicks
vaccinated with living Newcastle disease vaccines was significantly improved by
selenium and vitamin E. Diets were supplemented 14 days before vaccination, at
a ratio of 0.25 ppm and 300 mg/kg respectively and improvement in
haemagglutination inhibition (HI) antibody titres and protection rate against
challenge with velogenic Newcastle disease virus were observed (Bassiouni et
al., 1990). When Se was added to the feed of White Leghorn chickens (between
0.1 and 0.8 mg/kg) prior to challenge with either Escherichia coli or sheep
erythrocyte antigen an antibody titre against sheep erythrocytes increased by 77%
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(Larsen et al., 1997). Following chilling, antibody titre response was substantially
reduced and the titre reduction was prevented with dietary additions of Se between
0.1 and 1.2 mg/kg (Larsen et al., 1997). Furthermore, significantly higher antibody
titres at 10 d post-immunisation for Newcastle Disease Virus were attributed to
0.06 mg/kg and 150 IU/kg Se and vitamin E, respectively (Swain et al., 2000).
The antibody immune response in White Leghorn chicks was also significantly
improved for the Se enrichment diet in the Salmonella or Salmonella and aflatoxin
inoculated groups (Hegazy and Adachi, 2000). Selenium can also be used with
drinking water. For example, Deng et al. (1999) studied effects of sodium selenite
solution on the humoral immunity and erythrocyte immunity function against
Newcastle disease in chicks. Their results showed that the HI titre of the control
group was much lower than that of the 4 selenium-supplemented groups. At the
same time, the erythrocyte immune function of the control group was lower than
that of the tested groups. Similarly, when chicken were vaccinated with attenuated
strain C30-86 of Newcastle disease virus via drinking water at 3 and 21 days of
age supplementation with selenium through water increased HI antibody titre by
stimulating antibody production (Yang et al., 2000).
Immunomodulating properties of antioxidants were shown with a range of
animal species, including various farm and laboratory animals. For example,
increased selenium supplementation was beneficial to swine immunity. In particular,
the most consistent trend was seen in the humoral immune response to lysozyme,
where diets with Se 0.9 mg/kg and various concentrations of vitamin E invariably
produced higher titres than diets which contained Se 0.3 mg/kg (Blodgett, et al.,
1988). However, cell-mediated immunity as measured by the in vivo skin response
to phytohaemagglutinin was similar among diets. Injections of Se (5 mg) to sows
on d 100 of gestation resulted in higher IgM levels in colostrum and in serum
from pigs at birth (Hayek et al., 1989). However, concentrations of colostral and
serum IgA or IgG were not affected by treatment. Piglets receiving the higher
doses of vitamin E (200 ppm) and selenium (0.3 ppm) had more antibodies present
against Aujeszky vaccine in the third week post-weaning than piglets receiving
the lower doses (30 and 0 ppm) (Daza et al., 2000). Also in pigs, haemagglutination
assays indicated that vitamin E (220 IU/kg diet) and Se (0.5 mg/kg diet)
independently increased the immune response, particularly during the latter weeks
of the experiment (Peplowski et al., 1980). The combination of both nutrients
provided in the diet or by injection, resulted in a further increase in
haemagglutination titres, suggesting an additive response.It seems likely, that
immunomodulating properties of Se are observed at doses usually much higher
than commercially used. For example, the effect of dietary Se on the immune
response was evaluated in 96 crossbred weanling swine. Six groups of 16 pigs
were fed diets with Se supplemented as sodium selenite at 0, 0.3, 0.6, 0.9, 1.2,
and 1.5 mg/kg. The basal diet contained 0.068 mg of Se/kg. Whole blood
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concentrations of Se linearly increased as the dietary concentrations of Se
increased. The humoral response was monitored by immunoglobulin G titers to
lysozyme and ribonuclease, using an enzyme-linked immunosorbent assay. The
diet with 0.9 mg of added Se/kg produced the highest antibody response to both
antigens, whereas the diet with 0.3 mg of added Se/kg produced the lowest titres
for both antigens (Blodgett et al., 1986).
Calves fed 20 mg of Se/kg of mineral mixture ad libitum had lower antibody
responses (P< 0.02) to hen egg lysozyme inoculation, compared with calves fed
20 mg of Se/kg of mineral mixture and given 0.1 mg of Se and 0.22 IU of vitamin
E/kg of body weight, IM, or with calves fed 80, 120, 160, or 200 mg of Se/kg of
mineral mixture (Swecker et al., 1989). Selenium supplementation of calves (0.1
mg Se/kg feed) was associated with increased serum IgM concentrations on day
17 (Stabel et al., 1989). Similarly, humoral immunity was effected by Se
supplementation in calves challenged with infectious bovine rhinotracheitis virus
(IBRV). By d 49, IBRV antibody titers were higher for Se-supplemented calves
than in Se-deficient animals indicating that selenium deficiency may depress the
immune response of calves challenged with a foreign pathogen (Reffett et al.,
1988a). In stressed cattle serum antibody response (IgG titres) to P. haemolytica
vaccination was enhanced only when combination of Se and Vitamin E used with
single nutrients being non-effective (Droke and Loerch, 1989). Sixty clinically
healthy Holstein cows were randomly assigned to one of four groups according
to their age and parity and vaccinated in late pregnancy (day 190) with a multivalent
vaccine against Escherichia coli. The results of the study indicated that the
intramuscularly injection of sodium selenite at 0.1 mg Se/kg bodyweight either
alone or in combination with vitamin E significantly improved the production of
specific antibodies against E coli, and that the production of specific antibodies
was greater after the administration of selenium alone (Panousis et al., 2001). The
cattle with adequate -tocopherol and marginally deficient selenium status
manifested significantly lower anti-Haemophilus somnus antibody titer than the
cattle supplemented with Se in the later stage of an 8-week trial (Makimura et al.,
1993).
Improved antibody responses following selenium administration have also been
found in sheep (Suttle and Jones, 1989). The effect of dietary selenium (sodium
selenite, 0.2 mg Se/kg diet) on various blood characteristics and the primary and
secondary humoral immune response of lambs challenged with parainfluenza-3
virus were studied (Reffett et al., 1988). Serum IgM concentrations were enhanced
by Se supplementation on d 14, 35 and 49 of the study. Furthermore, titre levels
appeared to increase in Se-supplemented lambs after primary inoculation. The
effect of selenium (two injections of sodium selenite at 0.1 mg/kg b.w.) on antibody
production of sheep vaccinated against Chlamydia psittaci (ovis) was investigated
(Giadinis et al., 2000). The results indicated that Se alone led to a significant
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increase of Chlamydia antibody response but not when it was given in combination
with vitamin E.
Six groups of three six-month-old lambs were fed a basal diet containing 0.13
mg Se/kg supplemented with either 0.1, 0.5 or 1.0 mg Se/kg in the form of sodium
selenite or as selenomethionine. Experimental animals generally showed enhanced
antibody response to tetanus toxoid, parainfluenza-3 virus and Corynebacterium
pseudotuberculosis, and their total serum IgG concentrations were higher than in
unsupplemented control animals (Larsen et al., 1988a). In two field studies
significantly higher titres to tetanus toxoid were detected in ewes injected with
100 mg selenium as barium selenate. Lambs from selenium supplemented ewes
had significantly higher titres to tetanus toxoid than lambs from ewes in the control
group (Larsen et al., 1988a).
Fifteen Shetland ponies were used in a 7-wk trial to study the effect of
supplemental Se on humoral antibody production (Knight and Tyznik, 1990).
Before the experiment, the ponies were depleted of Se and assigned randomly to
either a low Se (0.02 ppm) or higher Se (0.22 ppm) diet. In comparison with those
ponies receiving the low Se concentrate, ponies receiving the Se-supplemented
diet had higher GSH-Px activities and blood Se concentrations during the later
weeks of the experiment. Se-supplemented ponies were also characterised by an
enhanced primary immune response as evidenced by increased hemagglutination
titers and higher IgG concentrations.
Eleven men were fed foods naturally high or low in selenium for 120 d. Selenium
intake was stabilised at 47 g /d for 21 d, then changed to either 13 or 297 g/d
for 99 d, leading to significantly different blood selenium and GSH-Px activity.
Antibody titers against diphtheria vaccine were 2.5-fold greater after reinoculation
in the high selenium group (Hawkes et al., 2001).
It is interesting to note that immune system can be adapted to low Sesupplementation and in some conditions antibody production can be less
compromised in Se-deficient animals in comparison to control once. For example,
the selenium-deficient mouse-trypanosome system was used to study the effects
of selenium deficiency in Swiss Webster mice infected with Trypanosoma musculi
(Ongele et al., 2002). The results indicated that there was a severe depression in
primary and secondary antibody responses to sheep red blood cells in all inoculated
mice. However, these responses were significantly less depressed in seleniumdeficient mice. In particular, in Se-deficient mice, a low parasitemia was observed
and infection was cleared by day 16 post-inoculation (PI), whereas control mice
sustained the parasitemia until day 24 PI.
Selenium is toxic at high doses so it is necessary to be careful choosing the
correct dose of this trace element for an experiment. For example, mice were
supplemented parenterally with Se 1.5-1.9 mg/kg body weight, vitamin E 500600 IU/kg body weight or Se plus vitamin E (25-100 g and 7.5-30 IU/mouse,
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respectively) 24 h before primary (day 1) and secondary (day 22) challenge with
ovalbumin (OVA) 0.2 ml (i.p.) (Jones and Stevenson, 1993). Treatment with Se
alone or the Se/vitamin E combination significantly decreased circulating antiOVA IgG antibody responses compared with those in unsupplemented controls.
Mice treated with vitamin E alone gave results comparable with controls.
Therefore, over-supplementation with Se, in the presence or absence of vitamin E
and in amounts insufficient to cause clinical toxicity, may impair vaccination
responses.
Therefore selenium and/or vitamin E deficiencies are associated with a
compromised humoral immune response not only in chicken but also in mouse,
horse, pig, cattle, calves, lambs and sheep (Larsen, 1993; Table 4.6) and inclusion
of increased doses of these nutrients into the diet is proven to improve the immune
response (Turner and Finch, 1991; Larsen, 1993; MacPherson, 1994).
Table 4.6 Effects of Se on the humoral immune response (adapted from Larsen, 1993).
Species
Effect of Se
Lamb
High Se: IgM ; IgG ; Primary AB to PIV-3, tetanus toxoid or C. pseudotbc.

; AB to Leptospira
High Se: Primary AB to Brucella abortus ; AB to RBC
High Se+E: AB to Pasteurella haemolitica
High Se: AB to egg lysozyme ; IgM ; AB to IBRV
High Se: Primary AB to RBC ; IgG
High Se+E: Primary AB to tetanus and to EIV
High Se: IgM (colostrum) , IgM, IgG (piglets)
Low Se: Lymphocyte response to mitogens
High Se: Primary AB to RBC ; IgG to RBC ; IgM to RBC
Sheep
Cattle
Calves
Horse
Pig
Chicken
Mouse
IBRV - infectious bovine rhinotracheitis virus
Did you know that the immune system maintains a repertoire

exceeding 10 million different proteins, called antibody
molecules?
Lymphocyte functions
It has been suggested that Se and vitamin E deficiencies affected T lymphocytes
to a greater extent than B lymphocytes (Larsen, 1993). This was explained to be
a result of higher level of PUFAs in T lymphocytes and higher membrane fluidity.
In fact, vitamin E and Se deficiencies may affect both the maturation of specific
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lymphocyte subpopulations and the functional and proliferative capabilities of

the peripheral lymphocytes. A combine Se- and vitamin E deficiency in chicks
showed a more severe depression of T-cell response than just Se-deficient chicks.
For example, in an experiment of Chang et al. (1994) the dietary deficiencies in
vitamin E and selenium (basal diet without vitamin E and Se supplementation
starting from hatching) resulted in a significant inhibition of T-lymphocyte
proliferation. In particular a decreased proportion of peripheral T cells and more
specifically a decreased the number of CD4(+) peripheral blood leukocytes were
observed. It is interesting to note that so-called regulatory CD4+ T cells are capable
of controlling the activity of other lymphocytes and they protect the integrity of
tissues and organs in vivo and also play a major role in the systemic homeostatic
mechanisms that control total lymphocyte numbers (Annacker et al., 2001). The
proliferative response to both ConA and PHA was impaired by the vitamin E and
Se dietary deficiencies. However, the proliferative response could be fully
reconstituted after vitamin E and Se supplementation for periods longer than 1
week.
Did you know that B lymphocytes carry about 105 identical
receptors per B cell?
Selenium deficiency in growing chickens was associated with impaired bursal
growth and Se-vitamin E deficiency caused inhibition in thymus growth. (Marsh
et al., 1986). In this experiment reduced number of lymphocytes were seen in
both the thymus and bursa in combined Se-vitamin E deficiency. On the other
hand, a significant increase in relative weight of the bursa of Fabricius was observed
in broiler chicks at a level of 0.10 mg of selenium and 150 IU of vitamin E (Swain
et al., 2000). It is interesting to note that bursectomy in chickens caused a small
but significant fall in spleen Se concentration, but not in that of other tissues
(Abdel-Ati et al., 1984). It has been shown that Se deficiency alone or in
combination with vitamin E is associated with depressed splenocyte ability to
proliferate in culture (Marsh et al., 1987). Such a depression was not due to
reduced lymphocyte viability in culture. Marsh et al. (1986) suggested that the
primary lymphoid organs are major targets of Se and vitamin E dietary deficiencies
and provide a possible mechanism by which immune function may be impaired.
In their experiment with chickens specific deficiencies of Se or vitamin E
significantly impaired bursal growth. Thymic growth was impaired only by the
combined vitamin E-selenium deficient diet. Severe histopathological changes in
the bursa resulted from the combined deficiency and these were detectable by
10-14 days after hatching (Marsh et al., 1986). These changes were characterised
by a gradual degeneration of the epithelium and an accompanying depletion of
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lymphocytes. On the other hand, chicks receiving Se (1 mg/kg) and vitamin E
(300 IU/kg) had significantly higher cellular immune responses in terms of per
cent leukocyte migration inhibition (Swain et al., 2000).
Inclusion of selenium in the chicken diet (0.3-0.6 mg/kg) for 2 months
significantly increased the response of peripheral blood lymphocytes to
phytohaemagglutinin and NK cell activities, accelerating the development of cellmediated immunity in chickens (Huang and Chen, 1999). Therefore, Se could
significantly increase the response of peripheral blood lymphocytes to
phytohaemagglutinin and NK cell activity, accelerating the development of cellmediated immunity in chickens (Huang and Chen, 1999). In general, splenic
Natural Killer cells activity is enhanced in selenium-supplemented, healthy animals
(Dhur et al., 1990). The results of Colnago et al. (1984) indicate that dietary Se
supplementation modifies the number of peripheral blood leukocytes in chickens
infected with coccidia. In particular selenium significantly increased blood
leukocyte number 8 hours after challenge in one experiment and produced
numerically higher leukocyte number in three other experiments. Immunization
of chickens against coccidiosis can be enhanced by Se supplementation (Colnago
et al., 1984a). Chicks receiving Se (1 mg/kg diet) and vitamin E (300 IU/kg diet)
had significantly higher cellular immune responses in terms of per cent leukocyte
migration inhibition (Swain et al., 2000).
A whole-blood lymphocyte transformation test was used to study the effect of
dietary vitamin E and selenium supplementation for 12 weeks on the PHA response
of peripheral pig lymphocytes. At the end of the experiment all groups given
additional vitamin E and, or, selenium showed increased PHA-response (Larsen
and Tollersrud, 1981). Usually combine deficiency of several antioxidants has a
more detrimental effect on immune system in comparison to a single antioxidant
deficiency. For example, compared with control, the vitamin E and selenium
deficient diet in sows during gestation until day 4 of lactation decreased the
mitogenic responses of lymphocytes of peripheral blood (PBL) and colostrum
(CL), phagocytic activity of blood and colostral PMN, and the microbiocidal activity
of blood, colostral, and milk PMN (Wuryastuti et al., 1993). The Se-deficient diet
reduced phagocytic activity of PMN. Six male weanling pigs were maintained
for 25 days on a torula yeast-based diet containing no measurable amount of tocopherol and less than 0.02 mg of Se per kilogram of feed. As a result, a marked
suppression of lymphocyte response to mitogens occurred in the pigs when the
cells were cultured in the presence of autologous serum (Lessard et al., 1991).
In an indoor experiment lambs were fed a basal diet containing 0.13 mg Se/kg,
and supplemented with, respectively, 0.1 or 0.5 mg Se/kg. Enhancement of the
proliferative response of lymphocytes to PHA, PWM and conA was found in
lambs following selenium supplementation at the lower levels. The highest dietary
selenium level, however, induced decreased mitogen response (Larsen et al.,
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1988). Three groups of lambs from a flock in which white muscle disease had
been diagnosed were used in another experiment (Ramos et al., 1998). Group 1
was inoculated with 6 mg of sodium selenite and 200 mg of vitamin E, group 2
was inoculated with 200 mg of vitamin E, and group 3 was the untreated control
group. Lambs in the group 1 treated with selenium and vitamin E showed a greater
response to the delayed hypersensitivity test compared to the other groups. It is
concluded that supplementation of lambs with selenium and vitamin E has a
positive effect especially on cell-mediated immunity. Similarly, the Con Astimulated lymphocyte proliferation was significantly lower in Se-deficient cows
(Cao et al., 1992). The effect of dietary selenium on leukocyte migration inhibitory
factor (LMIF) production was examined in vitro using lymphocytes from goats
fed a diet deficient in selenium. The ability of peripheral blood lymphocytes to
produce LMIF induced by Con A was significantly inhibited in Se-deficient cells
(Aziz and Klesius, 1985). Se deficiency in goats decreased the production of
leukotriene B4 (LTB4) by PMN and LTB4-mediated neutrophil chemotaxis (Aziz
and Klesius, 1986). Therefore dietary Se deficiency in goats is associated with
depressed PMN generation of factors that stimulate neutrophil chemotaxis and
neutrophil chemiluminescence (Aziz and Klesius, 1986a).
Dietary (2 ppm for 8 wk) or in in vitro (1 x 10-7M) supplementation with Se
results in a significant enhancement of the proliferative responses of spleen
lymphocytes from mice in response to stimulation with mitogen or antigen. In
contrast, Se deficiency (0.02 ppm for 8 wk) had the opposite effect. The alterations
in the ability of the cells to proliferate were related to the ability of Se to alter the
kinetics of expression of high-affinity IL2 receptors on the surface of activated
lymphocytes (Kiremidjian-Schumacheret et al., 1992). The results also suggested
that Se most likely affects processes in the cytoplasmic and/or nuclear
compartments of activated lymphocytes.
It seems likely that selenium can restore the age-related defects in cell
proliferation through an increase in the number of high-affinity IL-2 receptors.
For example, supplementation with selenium (2.00 ppm for 8 weeks) of aged
male mice resulted in a significant increase in the ability of spleen lymphocytes to
undergo blastogenesis (Roy et al., 1995). Furthermore, Se supplementation
restored the age-related deficiency of the cells to respond to stimulation by nuclear
DNA synthesis and cell proliferation. In the same experiment populations of in
vivo, alloantigen-activated lymphocytes from Se-supplemented aged animals
contained significantly higher numbers of cytotoxic lymphocytes than those from
Se-normal aged animals, which resulted in an enhanced capacity to destroy tumor
cells. The significant increase in the number of cytotoxic effector cells within
these activated T-lymphocyte populations was explained as the result of an
enhanced clonal proliferation of cytotoxic precursors cells, followed by the
differentiation of greater numbers of cytotoxic effector cells (Roy et al., 1995).
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It seems likely that Se alters the kinetics of expression of high affinity (p55/
p75) interleukin 2 receptors (IL-2R). For example, dietary (2 ppm for 8 weeks) or
in vitro (1 x 10-7 M) supplementation with Se results in a significant upregulation
of the expression of both the p55 and p70/75 IL-2 binding sites on the surface of
con A-stimulated lymphocytes from mice (Roy et al., 1993). This resulted in the
formation of significantly higher numbers of high affinity IL-2R/cell with
preservation of the normal ratio of high affinity to total IL-2 binding sites/cell.
The authors suggested that Se could accelerate clonal expansion of activated
lymphocytes.
Se deficiency (8 weeks at 0.02 ppm Se) in mice significantly inhibited the
ability of the lymphocytes to proliferate in response to allogeneic stimulation in
the mixed lymphocyte reaction or to mitogen stimulation by PHA, whereas Se
supplementation (2.00 ppm Se) significantly enhanced both responses
(Kiremidjian-Schumacher et al., 1990). It was also shown that the mechanism(s)
responsible for the observed effects of Se on lymphocyte proliferation are
independent of the levels of IL-2 or IL-1. Se supplementation or deficiency in
mice alters the kinetics of IL-2 receptor expression. Supplementation with Se in
vivo or in vitro resulted in an earlier expression of high affinity IL-2 receptors,
whereas Se deficiency resulted in a delayed expression of lower numbers of
receptors (Roy et al., 1992). There was a clear correlation between
supplementation with Se and enhanced 3H-thymidine incorporation into nuclear
DNA, preceded by enhanced expression of high affinity IL2-R (Roy et al., 1994).
The effects of dietary restriction of selenium and vitamin E were studied in
male weanling rats, which were maintained for 5-6 weeks on torula yeast-based
diets, with or without the addition of vitamin E (150 IU/kg) or selenium (0.5 mg/
kg). Selenium deficiency was associated with a decrease in lymphocyte
blastogenesis in response to mitogens. Furthermore, very marked suppression of
mitogen responses was seen in the doubly deficient group, as well as a greater
loss of viability during culture (Eskew et al., 1985). However, dietary deficiency
of vitamin E and selenium had no effect on alveolar macrophage function, as
measured by cell-mediated antibody-dependent cytolysis, killing of Staphylococcus
aureus or regulation of T-lymphocyte blastogenesis. NK cell-mediated cytotoxicity
in mouse was depressed after 8 wk on diets deficient in selenium and/or vitamin
E (Meeker et al., 1985). T-lymphocyte-mediated cytotoxicity was found to be
depressed by combined selenium-vitamin E deficiency after 7 weeks on diets. In
contrast, antibody-dependent cell-mediated cytotoxicity was not affected in such
conditions.
Similar effects of Se supplementation were seen in human. For example,
supplementation of patients with selenium (200 g/d of sodium selenite for 8
weeks) during therapy for squamous cell carcinoma of the head and neck resulted
in a significantly enhanced cell-mediated immune responsiveness. This was
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reflected by the ability of the patients lymphocytes to respond to stimulation with

mitogen, to generate cytotoxic lymphocytes, and to destroy tumor cells. The
enhanced responsiveness was evident during therapy and following conclusion
of therapy (Kiremidjian-Schumacher et al., 2000). In particular, Se
supplementation resulted in 118% increase in cytotoxic lymphocyte-mediated
tumor cytotoxicity and 82.3% increase in natural killer cell activity as compared
to baseline values. This apparently was related to the ability of the nutrient to
enhance the expression of receptors for the growth regulatory lymphokine IL-2,
and consequently, the rate of cell proliferation and differentiation into cytotoxic
cells (Kiremidjian-Schumacher et al, 1994).
The influence of selenium supplementation on the selected immune parameters
analysed from peripheral blood of corticoid-dependent asthmatics (Gazdik et al.,
2002). The results demonstrated the significant changes particularly in functional
parameters of both cellular and humoral types of immunity supporting the
immunomodulating effects of selenium. For example, in parameters of T cell
mediated immunity the relative number of CD3 HLADR+ T lymphocytes increased
after 24, 48 and 96 weeks of Se supplementation. However, proliferative activity
of T lymphocytes to mitogenes PHA and ConA in lymphocyte blastogenesis test
decreased significantly after 12, 48, 72 and 96 weeks of Se supplementation. In
humoral parameters activation of CP50 decreased after 24, 72 and 96 weeks of
supplementation to the reference range and AP50 after 96 weeks, respectively.
On the other hand, the levels of IgG elevated after 24 weeks, IgA after 24, 48
weeks, but the level of total IgE significantly decreased after 96 weeks of Se
supplementation. Dietary supplementation of psoriatic patients with Se-Yeast (400
micrograms Se/day for 6 weeks) modulated the immunological mechanism of
psoriatic lesions by increasing the number of CD4+ T-cells (Harvima et al., 1993).
It seems likely that the immune-enhancing properties of selenium in humans
are the result, at least in part, of improved activation and proliferation of Blymphocytes and enhanced T-cell function. For example, in men receiving Se at
297 g/d for 99 d in comparison to those having only 13 g/d increases in cytotoxic
T-lymphocytes and activated T-cells were observed (Hawkes et al., 2001).
Lymphocyte counts also increased on d 45 in the high-selenium group.
Furthermore in vitro proliferation of peripheral lymphocytes in autologous serum
in response to pokeweed mitogen was stimulated in the high-selenium group by
d 45 and remained elevated throughout the study, whereas proliferation in the
low selenium group did not increase until d 100. Similarly, in healthy aged humans,
Se supplementation (400 g/day for 6 months) enhanced immune function (NK
cell cytotoxicity) and phenotypic expression of T-cell subsets. In particular, it
caused an increase in total T cells by 27% and NK cell cytotoxicity over pretreatment levels by 58% (Wood et al., 2000). Many those effects were related to
the ability of Se to enhance the expression of the alpha (p55) and/or beta (p70/75)
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subunits of the interleukin-2 receptor (IL-2R) on the surface of activated
lymphocytes and NK cells. This resulted in a greater number of functional IL-2R/
cell and in enhanced proliferation and clonal expansion of cytotoxic precursor
cells (Kiremidjian-Schumacher and Roy, 1998).
Did you know that a stimulated B cell is excreting about
2000 antibody molecules per second?
It is necessary to underline that maternal nutrition can affect immune system
development in progeny. In fact, maternal selenium intake impacts neonatal
selenium status which in turn influences the neonatal immune system development.
For example, recently the impact of dietary selenium intake on neonatal immune
cell differentiation and function has been evaluated in pregnant rats (Dylewski et
al., 2002). A low selenium intake during pregnancy and lactation was associated
with reductions in maternal plasma selenium (by 33%), milk selenium (by 36%),
and corresponding neonatal plasma selenium (by 47%). This resulted in an
impaired thymocyte activation in neonates receiving low-selenium milk.
Furthermore, the percentages of CD8 cytotoxic T cells, CD2 T cells, panB cells,
and NK cells were all decreased in neonates nursed by mothers fed a low-selenium
diet. In fact neonates nursed by mothers fed a diet low in selenium showed reduced
T-cell and B-cell trafficking to the spleen. Thirty days before lambing, 36 Sardinian
ewes were assigned to one of three groups. Control group (C) was not treated; a
second group (Se-1) was given 5 mg of Se on day 30 before lambing; a third
group (Se-2) was given 2.5 mg of Se on day 30 before lambing and at lambing
(Lacetera et al., 1999). Ewes of group Se-1 had a significantly greater response to
PHA 6 h after injection than ewes of the control group. Lambs born to Se-1 and
Se-2 ewes had substantially improved response to PHA 24 h after injection.
Furthermore, responses of ewes and lambs to phytohaemagglutinin 24 h after
injection were positively related. The mean in-vitro responsiveness of peripheral
blood lymphocytes to stimulation with PHA in the high Se sheep (intraruminal Se
pellet) was significantly greater than that in sheep from the low Se group on day
22 (Jelinek et al., 1988). Therefore both, sheeps and their progeny can benefit
from Se supplementation. Clearly adequate maternal selenium is important for
cellular (T cytotoxic cells, NK cells) and humoral (B cells) immune system
development and function in the offspring.
Effect of Se on immune cells is dose-dependent and detrimental effects of Se
excess were shown in the experiment with fifteen pregnant cows fed diets
containing 0.25 (control), 6.0, and 12.0 ppm selenium beginning at 80-110 days
gestation (Yaeger et al., 1998). Elevated dietary selenium resulted in the depression
of several leukocyte function parameters in pregnant cows. For example, a
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depression in forced antibody response was found in both high seleniumsupplemented groups. A substantially diminished mitogenic response to Con A
and pokeweed mitogen was also observed in the 12-ppm selenium group. Similarly,
Se administered to female Sprague-Dawley rats for 10 weeks at 0.5 and 2.0 ppm
resulted in significant enhancement of splenic NK activity while the NK response
in the 5.0 ppm Se-treated rats was equivalent to the non-Se-treated controls.
Conversely, the DTH response was significantly suppressed at all three dosages,
while antibody synthesis and prostaglandin E2 activity were significantly reduced
compared to the controls at the highest dosage of Se (Koller et al., 1986).
As mentioned above, Se is proven to have an important function in maintaining
proliferative capacity of T- and B-cells (Turner and Finch, 1991). It has been
suggested that Se may modulate the expression of IL-2 receptors on the cell surface,
which could lead to the altered ability of lymphocytes from Se-deficient animals
to respond to mitogen and antigens (Larsen, 1993). Again this phenomenon is a
characteristic for various farmed and laboratory animals (Larsen, 1993; Turner
and Finch, 1991; MacPherson, 1994; Table 4.7).
Table 4.7 Effect of Se on Lymphocytes (adapted from Larsen, 1993; Turner and Finch, 1991; Roy et al.,
1990; Petrie et al., 1989).
Species
Effect of Se
Lamb
High Se: Lymphocyte response to mitogens

High Se: Lymphocyte response to mitogens
Low Se: PGE2 ; TXB2 ; LTB4
Low Se: Lymphokin (MIF)
High Se: Lymphocyte response to mitogens ,
T cell number
Low Se/E: Lymphocyte response to mitogens
Low Se and E: Lymphocyte response to mitogens
High Se: NK-activity
High Se: Lymphocyte response to mitogens ; ability to destroy tumour
cells ; production of IL-2 in the presence of lectine ; expression of specific
cytotoxic T lymphocytes in peritoneal exudate cells ; augumentation of the
response of T lymphoblasts to IL-2 and of thymocytes to IL-1
Sheep
Cattle
Goat
Pig
Chicken
Dog
Rat
Mouse
In vitro effects of Se on immune cells

Natural antioxidants are essential nutritional factors that affect the development
and expression of cell-mediated immune responses. Various in vitro models were
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used to assess effects of Se on lymphocyte functions. For example, lymphocytes
of lambs fed on a low selenium/vitamin E diet were isolated from peripheral blood,
and mitogenic responses to PHA tested in the presence of different doses of sodium
selenite added in vitro. An enhancing effect of selenium was observed at doses of
1 ng/ml or less, and reached a plateau at about 10 ng/ml. Toxic effects were
evident beyond 1 g/ml. (Finch and Turner, 1989). It has been shown that dietary
(2 ppm for 8 weeks) or in in vitro (1x10-7M ) supplementation with Se (as sodium
selenite) results in a significant enhancement of the proliferative responses of
spleen lymphocytes from C57B1/6J mice in response to mitogen stimulation. In
contrast, Se deficiency (0.02 ppm for 8 weeks) had an opposite effect. The
alterations in the ability of cells to proliferate were apparently related to the ability
of Se to alter the kinetics of expression of high-affinity IL-2 receptors on the
surface of activated lymphocytes. This was associated with enhanced or delayed
clonal expansion of the cells. The changes in tumour cytotoxicity were paralleled
by changes in the amounts of lymphotoxin produced by the activated cells. The
results also suggested that Se exerts its effect 8-24 h after stimulation, and that it
most likely affects processes in the cytoplasmic and/or nuclear compartments of
activated lymphocytes (Kiremidjian-Schumacher et al., 1992).
The impairment in lymphocyte proliferative response to antigens in Se and/or
vitamin E deficiency could be a result of lipid peroxidation and damages to
membrane structures and more importantly to membrane receptors (Larsen, 1993).
As a result cell-to-cell communication could be compromised. In this case a
protective effect of antioxidants could be a crucial factor in immune system
competence. For example, it was found that lipid peroxidation in lymphocytes
before Con A stimulation was lower than that after stimulation and that SOD
promoted lymphocyte proliferation dose dependently. The addition of Na2SeO3
to lymphocytes culture or supplementation in drinking water to mice decreased
the lipid peroxidation in lymphocytes stimulated by Con A. In the presence of Se,
there was an inverse correlation between the levels of LPO in lymphocyte and the
stimulated proliferation (Sun et al., 1995). In vitro vitamin E and selenium
supplementation was able to enhance significantly the depressed PMN-mediated
chemotaxis and phagocytosis or monocyte chemoattractant protein-1 (MCP-1)
production in elderly subjects (Ventura et al., 1994).
Thus, selenium appears to be capable of selectively regulating the generation
of functional lymphocyte subsets in vitro. For example, Se at physiologic
concentrations can inhibit human lymphocyte proliferation in response to irradiated
tumor cells in mixed lymphocyte/tumor cell cultures (MLTC; Petrie et al., 1989a).
Furthermore, the various lymphocyte functional activities generated in these
cultures exhibited different levels of sensitivity to the effects of selenium. In
particular, the generation of suppressor-cell activity in MLTC was strongly inhibited
by the presence of physiologic levels of Se, while the development of cytotoxic
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T-lymphocyte activity in identical cultures was not affected by Se. Production of

interleukin-2 in these cultures showed an intermediate sensitivity to the effects of
Se (Petrie et al., 1989a).
The effect of Se on NK and lymphokine-activated killer (LAK) cell activities
and proliferative responses of human lymphocytes was studied in vitro (Nair and
Schwartz, 1990). Direct addition of Se at 1.0 g/ml to the mixture of target and
effector cells significantly suppressed the NK activity of normal lymphocytes.
Similarly, when lymphocytes were preincubated with Se at concentrations as low
as 0.2 g/ml for a period of 48 h, a significant inhibitory effect on NK activity
was observed. Lymphocyte proliferative responses to T cell mitogens such as
PHA and Con A were also significantly suppressed by direct addition of Se at 0.51.0 g/ml. The inhibitory effect of Se was not due to non-specific toxicity of
effector cells as demonstrated by viability nor was the effect directed against
target cells. Therefore, although Se is an essential micronutrient for various immune
mechanisms, an excess of Se may have a deleterious effect on certain
immunological functions.
As mentioned above there are principal differences in metabolism of organic
and inorganic selenium. This could be a reason of differences in stimulating activity
of different forms of selenium on immune system. For example, sodium selenite
and SeMet were tested in parallel, and their capability to inhibit or to increase the
antibody production by human lymphocytes in vitro was investigated (Borella et
al., 1996). Low doses of Se (0.5-2.0 M) added as sodium selenite or SeMet did
not alter the secretion of antibodies. When Se was added at higher levels, instead,
an inhibitory effect was found using selenite, whereas a progressive increase in
immunoglobulin production was observed after exposure to SeMet. Therefore
there is an advantage in using organic selenium for immune system stimulation in
comparison to selenite.
In general, it is well known that selenium, especially in the form of selenite, is
clastogenic for cultured lymphocytes. For example, human lymphocyte cultures
were treated with increasing concentrations (from 8.0 x 10-8M to 8.0 x 10-5M) of
sodium selenite and SeMet 24 h after stimulation with PHA and were assessed for
chromosomal aberrations at 48 hours (Khalil, 1989). The yield of abnormal
metaphases was dependent on the dose and the form of selenium used. At 8.0 x
10-5M the proportion of aberrant cells reached 53.5% and 43.0% for selenite and
SeMet, respectively. The selenium-induced chromosomal aberrations were
primarily of the chromatid type and included breaks and fragments. In vitro
exposure of human peripheral blood lymphocytes to high concentration of two
inorganic salts of selenium- sodium selenite (2.9 x 10-5M) and sodium selenate
(2.65 x 10-5M) was found to be lethal. Lower concentrations of sodium selenite
(from 2.32 x 10-7 M to 2.9 x 10-6M) and sodium selenate (from 1.06 x 10-6M to
5.3 x 10 -6M) induced chromosomal aberrations and reduced cell division in
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proportions directly related to the dose (Biswas et al., 2000). Clearly, sodium
selenite was considerably more clastogenic than sodium selenate. Summary of
effects of selenium on immune cells is shown in Table 4.8.
Table 4.8 Effect of Se on animal immune cells (adapted from McKenzie et al., 1998).
Neutrophil migration and O22- activity (cow) Lymphokine activated killer cell activity
High-affinity IL-2 receptor (mouse)
Enhanced delayed-type response due to

better antigen presentation (mouse)
T-cell proliferation and function following

age-related decline (mouse)
Cell death following paraquat exposure

(rat)
Natural killer cell activity (mouse)
UV-induced skin cancers and mortality

(mouse)
Cititoxic T-cell activity (mouse)
Erythema following UV exposure

(mouse)
T-cell response to pokeweed mitogen (cow)
Vaccine-induced immunity to malaria

(mice)
Disease resistance
The final goal of the improvement of the immune system is to increase resistance
to various diseases. Indeed, this option was extensively studied with chickens.
For example, a combination of vitamin E with Se resulted in reduced mortality
and increased body weight gain in chickens infected with Eimeria tenella (Colnago
et al., 1984). The same authors showed that dietary supplementation with selenium
or vitamin E reduced mortality and increased body weight gain of nonimmunized
chickens infected with E. tenella in three of four experiments. When chicks were
inoculated with virulent Mareks disease (MD) virus at 10 days of age, selenium
(0.6 mg/kg) decreased the morbidity and mortality from MD. In particular, selenium
increased the ability of cells to remove ROS and lipid peroxides, and decreased
the degree of tissue damage caused by ROS (Huang and Chen, 1996). In another
experiment, from one day of age chicks were fed on a basal diet containing
selenium at 0.086 mg/kg (group I) or the basal diet supplemented with selenium
at 0.3 mg/kg (group II) or 0.6 mg/kg (group III). The chickens were infected with
infectious bursal disease virus at 39 days of age. Ten days later the mortality rates
in groups I, II and III were 33.3, 12.4 and 10.6%, respectively, and the infection
induced inhibition of T lymphocyte transformation was less in the selenium
supplemented birds (Bu et al., 1996). When Se was added to the feed of White
Leghorn chickens prior to challenge with either E. coli or sheep erythrocyte
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antigen, the incidence of death or lesions was reduced from 86 to 21% at the
optimal dose of Se (0.4 mg/kg feed; Larsen et al., 1997). Lower Se values were
measured in infected with Ascaridia galli chickens compared with controls, and
this was related to a lower degree of Se absorption and the regeneration of the
intestinal mucosa in infected birds (Damyanova et al., 1995).
Immunostimulating and disease-preventing effects of natural antioxidants are
not restricted to avian species, but obvious with other farm animals. For example,
vitamin E alone or in combination with Se was shown to have a protective effect
against swine dysentery infection, when challenged with Trioeponema
hyodysenteriae (Teige et al., 1982).
Se deficiency in cows is related to a range of various diseases including metritis,
mastitis, cystic ovaries, retained placenta, increased somatic cell count (SCC) and
some others. Decreased incidence of metritis in selenium-treated dairy cows
provides a good example of an association between selenium deficiency and
decreased disease resistance (Suttle and Jones, 1989). Incidence of metritis was
60% for cows injected with selenium and 84% for those not receiving selenium
(Harrison et al., 1984). Days to minimum uterine size were significantly less in
cows with metritis and selenium treated when compared with cows with metritis
and not selenium treated (32.9 vs. 35.8; Harrison et al., 1986).
Numerous studies have linked low Se and/or vitamin E status to increased
susceptibility of dairy cows to intramammary infections (Spears, 2000). An
increased Se status is protective against various infections. For example, in an
experiment, dairy cows were fed rations containing 0.05 or 0.35 mg Se/kg dry
matter and were inoculated intracisternally with Escherichia coli at 14-16 weeks
of lactation. Milk bacteria numbers were significantly higher between 16 and 24
h postinoculation in the Se-deficient group and three of the four cows in this
group required euthanasia, whereas all four cows in the Se-supplemented group
recovered without therapeutic intervention (Maddox et al., 1991). Seleniumsupplementation (0.2 ppm) showed a positive effect on udder health. The
percentage of quarters harbouring mastitis pathogens dropped from 22.9 to 13.0
in the Se-yeast group and from 18.4 to 7.4 in the selenite group during the
supplementation period. It is interesting that in cows supplemented with Se duration
of clinical symptoms was reduced by 46% and a combination of Se and vitamin E
was even more effective decreasing duration of mastitis by 62% (Smith et al.,
1984; Figure 4.5). These findings were confirmed in following studies from the
same department of the Ohio State University. A combine Se-vitamin E
supplementation of cows prior to calving and during lactation was associated
with a reduction of the following: in the prevalence of infected quarters at calving
(by 42.4%) and in quarter lactation days infected (by 59%), in clinical mastitis
over the entire lactation (by 32.1% and during the first four days of lactation (by
57.2%) and SCC (Smith and Conrad, 1987). Similarly, diets of first lactating
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heifers were supplemented with Se (0.3 ppm) and vitamin E (1,000 IU/day) and
they had significantly fewer quarters infected at calving, reduced prevalence of
infection during lactation, fewer cases of clinical mastitis, infections of shorter
duration and lower milk SCC in comparison with unsupplemented animals (Smith
et al., 1997). Similarly, bulk tank SCC decreased significantly as Se concentration
in plasma increased and plasma glutathione peroxidase was correlated positively
to Se intake but negatively to SCC (Weiss et al., 1990). Furthermore, Se
supplementation was associated with a decrease in the output of milk somatic
cells (Malbe et al., 1995). This was confirmed by Hemingway (1999) using Se
boluses and showing 2-fold decrease in SCC.
70
Mastitis reduction, %
Length of mastitis reduction, %
60
50
40
30
20
10
0
Se
Vitamin E
Se+vitamin E
Figure 4.5 Clinical cases of mastitis (adapted from Smith et al., 1984).
Rate of clinical mastitis was negatively related to plasma Se concentration (Weiss

et al., 1990). There was a significant negative correlation between the activity of
GSH-Px and the bulk milk cell counts in the herds with a low incidence of mastitis
suggesting that there was an association between the incidence of subclinical
mastitis or inflammation and the selenium status of these herds (Ndiweni et al.,
1991). Ten Holstein heifers were fed a selenium-deficient (SeD) diet (0.04 mg of
Se/kg on a total ration dry-matter basis) 3 months before calving and throughout
their first lactation. A selenium-supplemented (SeS) diet (2 mg of Se/head/d) was
fed to a group of 10 heifers. In about the 14th week of lactation, the cows were
challenge-exposed to Escherichia coli by administering 15 to 40 colony-forming
units (CFU) into 1 mammary gland. Results were as follows (Erskine et al., 1989;
1990):
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The frequency of quarter atrophy and agalactia, and reduction in wholeudder milk yield in the first 4 days after challenge exposure, were greater in
the SeD cows.
Log10 peak bacterial concentrations in milk were higher in SeD than in SeS
cows.
Mean log bacterial concentration was significantly higher from 12 to 20
hours after challenge exposure in SeD than in SeS cows.
Duration of infection was significantly greater in SeD than in SeS cows .
Milk somatic cell counts increased significantly more slowly in SeD than in
SeS cows from 8 to 16 hours after challenge exposure.
Ratios of milk somatic cells to bacteria in milk were significantly lower in
SeD than in SeS cows at 12 and 16 hours after challenge exposure.
Peak SCC were reached earlier in SeD than in SeS quarters.
Supplementing the feed of selenium-deficient diary cows with selenium (Se)yeast or selenite at a level of 0.2 ppm induced self-cure of subclinical mastitis; the
prevalence of quarters harbouring subclinical mastitis decreased to about one
half during the 8 week supplementation period. Possible mechanisms of such
protective effects of Se on mastitis include (Ali-Vehmas et al., 1997):
Improvement in the recruitment of phagocytes to the infected milk

compartment of the udder due to Se-supplementation;
Induction of an unspecified antibacterial activity in milk lactoserum (whey),
restricting in vitro growth of the mastitis pathogens Escherichia coli,
Staphylococcus aureus, Streptococcus agalactiae and Streptococcus uberis,
Regulation of redox activities and sulfhydryl activities in whey.
In 1999 Harrison and Hancock from the Washington State University conducted
a detailed meta-analyses of publications related to the effect of selenium and/or
vitamin E on mastitis in cows. They showed that from 19 studies conducted all
over the world including Mexico, Italy, UK, Israel, Canada, Portugal and Korea
and published in peer-review journals in 13 studies (68%) positive effect of
selenium/vitamin E supplementation was reported. On average, in control animals
mastitis was registered in 32% and in Se-supplemented cows mastitis was evident
only in 10% (Harrison and Hancock, 1999). The total costs associated with clinical
mastitis is about $107 per case (Hoblet et al., 1991).
The first report on relationship between retained placenta and selenium/vitamin
E status of cows was published by Trinder et al. (1969). Since then, this subject
has received substantial attention. It is interesting that Se-vitamin E combination
not only significantly reduced the incidence of retained placenta but also reduced
the days required for calving the first service (Kim et al., 1997). It is unclear why
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some cows fail to expel the placenta following calving. It was hypothesized that
impaired neutrophil function causes retained placenta (RP). For example,
neutrophils isolated from blood of cows with RP had significantly lower neutrophil
function before calving, and this impaired function lasted for 1 to 2 wk after
parturition (Kimura et al., 2002).
Cystic ovaries were diagnosed in 19% of cows injected with selenium, and
incidence was 47% for cows not treated with selenium (Harrison et al., 1984).
Cows received subcutaneous injection of a mixture of 30 mg Se (as sodium selenite)
and 408 IU vitamin E beginning 3 to 4 month prepartum and at 60-d intervals
throughout the 2-yr period. Calves born to Se-vitamin E treated cows were injected
with 5.5 mg Se and 75 IU E/100 kg body weight at 60-d intervals beginning at 1
month of age (Spears et al., 1986). Cows and calves receiving Se-vitamin E had
higher whole blood GSH-Px activity and plasma Se concentrations than controls.
Selenium-vitamin E injections reduced calf death losses from 15.3% to 4.2% and
slightly increased adjusted calf weaning weights.
Economic losses due to common health problems in dairy cattle were
investigated in 90 Friesian/Holstein herds (average size 152 cows), which calved
in England during the 1992/1993 season with an average annual yield of about
6000 litres per cow. By using only the direct costs of common production diseases
and other health problems, the cost of ill health in a 100 cow herd with average
rates of these problems (compared with target levels) was estimated at 6300
(from 1200 up to 13600) per year (Kossaibati and Esslemont, 1997). The main
losses were due to mastitis and lameness (38 and 27% of health cost, respectively).
Data derived from 340 dairy herds, mainly in southern England, between April
1998 and March 1999, showed that the average total culling rate was 22.1 per
cent, with 5.6 per cent for infertility and 3.6 per cent for mastitis (Whitaker et al.,
2000). The annual incidence of clinical mastitis was measured in 144 Holstein/
Friesian dairy herds in England (average size 132 cows) during 1994, 1995 and
1996 by means of carefully defined mastitis indices. The mean annual incidence
of the disease over the three-year period was 43.4 quarter-cases per 100 cows,
and the disease affected 25.9 per cent of the cows in the herds (Kossaibati et al.,
1998).
Medium wool ewes were injected with Se (subcutaneous monthly injections of
4 mg Se) over a 2-year period to evaluate the influence of these treatments on
reproduction. Selenium administration increased ewe blood Se concentrations
and preweaning survival of lambs was also increased (Kott et al., 1983) Selenium
can also increase the resistance of mice toward experimental Klebsiella
pneumoniae infection (Laschi-Loquerie et al., 1987). Selenium-deficient mice
were more sensitive to i.v. injections of suspensions of C. albicans with higher
mortality compared with the selenium-supplemented animals (Boyne and Arthur,
1986). In contrast, killing of Salmonella typhimurium and Staphylococcus aureus
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organisms and survival of rats after intraperitoneal injection of 8 X 107 S. aureus

organisms was not affected by Se deficiency. However a 5-fold increase in the
dose 4 X 108 S. aureus organisms led to a significantly greater mortality in the Sedeficient rats (Boyne et al., 1986). Summary of effects of Se on the immune
system of ruminants is shown in Table 4.9.
Low-dose supplementation of selenium and zinc provided significant
improvement in elderly patients by increasing the humoral response after
vaccination (Girodon et al., 1999). In particular, antibody titers after influenza
vaccine were higher in groups that received trace elements and the number of
patients without respiratory tract infections during the study was higher in groups
that received trace elements. An intervention study was conducted in China with
75 young children under one year hospitalized with pneumonia or bronchiolitis
caused by respiratory syncytial virus (RSV) to evaluate therapeutic effectiveness
of Se supplement on acute respiratory lower tract infection caused by RSV with
randomly controlled and double-masked method (Liu et al., 1997). Sodium selenite
was supplemented orally with 1 mg on the second day of hospitalization. Results
showed that days needed for their relief of symptoms and signs were fewer in Se
supplement group than that in controls and recovery in indicators of cell immune
was better in the former than that in the latter. In general, Tables 4.10 and 4.11
summarise effects of Se on human immune system.
Therefore, the results presented above clearly showed that various components
of the immune system as well as general animal health have been improved when
Se and/or vitamin E have been added to deficient diets or supplemented at levels
far above those required for growth. The benefit of Se and/or vitamin E
supplementation would be greatest in situations when animals are infected with a
particular pathogen. In this case clinical signs of infection could be reduced.
Furthermore, for optimising the animals resistance to disease, Se and vitamin E
requirements are higher than that for adequate growth, feed efficiency, egg
production or even reproduction (Nockels, 1988). The optimal doses of Se and
vitamin E for maximum disease protection depends on many factors and need
further elucidation (Tengerdy, 1990).
It is well known that Se and other antioxidants are able to protect cells from
free radical damage, reduce the detrimental effects of certain eicosanoids, and
enhance humoral and cellular immune responses in disease (Nockels, 1988).
Improved immune system could ultimately lead to higher resistance of animals to
various diseases. It is important to remember that during disease challenge nutrient
assimilation from the diet could be further compromised as a result of absorption
impairment or decreased feed consumption. This could lead to decreased efficiency
of antioxidant system leading to immunocompetence declining. For example Hao
et al. (1999) studied the activities of Se-GSH-PX and lipid peroxidation in central
and peripheral immune organs and main visceral organs of broilers experimentally
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Table 4.9 Effect of Se on immune response in ruminants.
Species
Immunological response
References
Cattle
Intracellular killing
Extracellular hydrogen peroxide
Metritis, Cystic ovaries
Se+vit.E: AB (IgG ) to Pasterella haemolitica
Antibody titres
Serum and colostrum IgG
Neutrophil kill
Lymphocyte proliferation
Mastitis
IgM
Antibody titres after vaccination
Incidence and duration of mastitis by 46%
Colostrum IgG
Virus titer after vaccination
Limphocyte proliferation
Antibody titres after vaccination
Se+vit.E: prevalence of infected quarters at
calving by 42%; Quarter lactation days infected
by 59%; clinical mastitis by 32%; somatic cell
numbers
AB to egg lysozyme
IgM , IgG by stress , AB to Pasterella
haemolitica ; AB to IBRV
IgM , IgG, AB to IBRV
In vitro lymphocyte killing ability
Antibody titers , IgG
Cell-mediated immunity
AB to Leptospira
IgM , primary AB to PIV-3
IgG , AB to tetanus tox.
Lymphocyte response to PWM, PHA, ConA
Lymphocyte response to PHA and PWM
Se+VitE: response to PHA, ConA, PWM
Primary AB to Brucella abortus, AB to RBC
Chlamydia antibody response
Neutrophil activity
Low Se: lymphokin (MIF)
Grasso et al., 1990
Cattle
Cattle
Beef cattle
Beef cows
Dairy cow
Dairy cows
Dairy cows
Dairy cows
Dairy cows
Dairy cows
Dairy cows
Dairy cows
Heifers
Heifers
Heifers
Calves
Calves
Calves
Calves
Lambs
Lambs
Lambs
Lambs
Lambs
Lambs
Lambs
Lambs
Lambs
Sheep
Sheep
Sheep
Ewes
Goat
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Harrison et al., 1984

Droke &Loerch, 1989
Nicholson et al., 1993
Awadeh et al., 1998
Hogan et al., 1990
Maddox et al, 1991
Cao et al., 1992
Stabel et al., 1991
Panousis et al., 2001
Smith et al., 1984
Lacetera et al., 1996
Ellis et al., 1997
Sordillo et al., 1993
Stabel et al., 1990
Nemec et al., 1990
Smith and Conrad,
1987
Swecker et al., 1989

Stabel et al., 1989
Reffett et al., 1988a
Pollock et al., 1994
Finch and Turner, 1989
Reffett et al., 1988
Turner and Finch, 1990
Lacetera et al., 1999
Ellis et al., 1990
Reffett et al., 1988
Larsen et al., 1988
Larsen et al., 1988a
Turner et al., 1985
Finch and Turner, 1989
Jelinek et all., 1988
Giadinis et al., 2000
Morgante et al., 1999
Aziz and Klesius, 1985
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250
Table 4.10 Effect of Se supplementation on humans (adapted from Hawkes et al., 2001; Larsen, 1993;
McKenzie et al., 1998).
Proliferation of peripheral blood

lymphocytes in response to mitogens
Cell death, IL6, IL8 and TNF m RNA

following UV irradiation of skin cells
The expression of high-affinity IL-2R
Cell death following paraquat exposure
The incidence of hepatitis B virus

infection and primary liver cancer
Apoptosis in tumours ; Apoptosis induced

by UV in normal skin cells
Cytotoxic lymphocyte-mediated tumor

cytotoxicity and NK cell activity
Killing by macrophages
Antibody titres against diphteria vaccine
Reduction in gastrointestinal, prostate and

lung cancers
Cytotoxic T-lymphocytes and activated

T-cells
Depressed neutrophil activity and increased

monocyte chemoattractant protein in the
elderly
Lymphocyte counts
Protection against hepatitis B-induced

hepatoma
IgG ; IgA
Improved sperm motility in subfertile men
NF-kB activation
B-cell lipoxigenase activity
Table 4.11 Effect of Se deficiency on human (adapted from Hawkes et al., 2001; Larsen, 1993;
McKenzie et al, 1998).
Keshan disease (dilated endemic

cardiomyopathy)
Kashin-Beck disease (endemic

deforming arthritis) Osteoarthropathy;
Spontaneous abortion
Skeletal myopathy
Psoriasis-severity
Increased mortality in HIV-positive

drug- users and HIV-infected children
Atopic asthma low platelet GSH-Px
NK-cytotoxic activity ; IgG and IgM

titres ; Platelet aggregation and
leukotriene synthesis
Myxodematous cretinism low serum Se
infected at one day of age with virulent Mareks disease virus (vMDV). They
showed that in infected birds the Se-GSH-Px activity was significantly decreased
and lipid peroxidation was enhanced. Therefore preventing these changes in
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antioxidant defence system is believed to be an effective means to maintain immune
system efficiency and this could be associated with better survival infected chicks.
A summary of the effects of Se on disease resistance in animals is shown in Table
4.12.
Table 4.12 Effect of Se on disease resistance (adapted from Larsen, 1993; Turner and Finch, 1991).
Species
Effect of Se
Cattle
High Se: Mastitis ; clinical symptoms of mastitis ;

Metritis ; Cystic ovaries
Chicken
High Se: resistance to E. tenella

High Se+E: resistance to ND ; AB to ND virus
Pig
High Se: Resistance to Treponema hyodysenteriae
Mouse
Low Se: Survival of experimentally infected animals with Diplococcus

pneumoniae Type 1 ; Candida albicans
Rat
Low Se: Survival of experimentally infected animals with Staphylococcus

auerus
Selenium deficiency and viruses

It is generally accepted that the nutritional status of the host is associated with
both severity and susceptibility to infectious disease. For example, inadequate
nutrition impairs the functioning of the immune system, thus resulting in increased
susceptibility to infection. Recently, it has been suggested (Beck and Levander,
2000) that the nutritional status of the host affect not only the immune response,
but it can also substantially affect the viral pathogen. In particular, in a mouse
model, a benign strain of coxsackievirus B3 became virulent and caused
myocarditis in selenium-and vitamin E-deficient mice (Beck and Levander, 2000).
The change in pathogenicity was a result of mutations in the viral genome, which
changed an avirulent virus into a virulent one. It is interesting that six nucleotide
changes were found in the virus that replicated in the deficient mice, and once
these mutations occurred, even mice with normal nutrition became susceptible to
disease (Beck, 1999). Therefore mice deficient in Se were more susceptible to
infection with coxsackievirus, as well as with influenza virus.. It was suggested
that the immune system was altered in the Se-deficient animals, as was the viral
pathogen itself. Mice fed a diet deficient in Se suffer much more severe lung
pathology after infection with the influenza virus than Se-adequate controls (Beck
et al., 2001).
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Sequencing of viral isolates recovered from Se-deficient mice demonstrated

mutations in the viral genome of both coxsackievirus and influenza virus (Beck,
2001). These changes in the viral genome are associated with the increased
pathogenesis of the virus. The effects of 10 week of Se supplementation (5 ppm)
in drinking water on immune responses and resistance to a myocarditic Coxsackie
virus B3 (CB3) infection were studied in female Balb/c mice (Ilback et al., 1998).
Se supplementation reduced CB3-induced mortality: at day 14 post-inoculation,
survival was 58% in the Se-treated group as compared to 25% in the untreated
group. A general scheme of Se-virus interaction is shown in Figure 4.6.
Host Se deficiency
Increased oxidative stress
Decreased immunity
Altered viral genome
Increased susceptibility to viral pathogen
Figure 4.6 Effect of Se on viral diseases (adapted from Beck, 1999).
Mechanisms of immunomodulating properties of selenium

It is believed that several mechanisms are involved in antioxidant-stimulation of
immune system (Wu and Meydani, 1998; Surai, 2002):
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Protection of cell membranes and receptors. It is well recognised that

sophisticated antioxidant defences directly and indirectly protect the host
against the damaging effects of cytokines and oxidants. In particular, indirect
protection is afforded by antioxidants, which reduce activation of NF-kappa
B, thereby preventing up-regulation of cytokine production by oxidants.
On the other hand, cytokines increase both oxidant production and
antioxidant defences, thus minimising damage to the host. Antioxidants
prevent oxidative stress-induced damage to immune cells. It is necessary to
take into account that cellular integrity is very important for receiving, and
responding to the messages needed to coordinate an immune response. As
mentioned above, phagocytosis is the major mechanism for microbe
removal from the body. The immune system generates ROS as part of its
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defence function and these ROS are an important weapon to kill pathogens.
However, chronic overproduction of ROS can cause damage to immune
cells and compromise their function (Wu and Meydani, 1998). In fact
immune cells are rich in PUFAs which are very susceptible to free radical
attack. It is well recognised that many immunological functions are
membrane-dependent. These are antigen recognition, receptor expression,
secretion of antibodies and cytokines, lymphocyte transformation, and
contact cell lysis (Wu and Meydani, 1998). In particular, the receptors are
important for antigen recognition and the secretion of various chemical
mediators such as interferon, tumor necrosis factor, prostaglandins and
interleukins. Therefore lipid peroxidation can change membrane structure
and properties (e.g. fluidity, permeability, flexibility etc.) which would affect
immune cell functions. In contrast antioxidants are able to prevent those
damaging effects of ROS and maintain immune function. For example, H2O2
depressed lymphocyte proliferation (Metzger et al., 1980), while vitamin E
decreased H2O2 formation by PMN (Baehner et al., 1977). It was found that
H2O2 injured lymphocytes immunocompetence deeply and administration
of Se counteracts this damage (Sun et al., 1995). It is well known that
macrophage activation and phagocytosis of foreign particles are regularly
accompanied by a so-called respiratory burst, an increase in the production
of ROS, exerted by the enzyme complex NADPH oxidase. A number of
selenoproteins is expressed at the same time to protect the cells from the
cytotoxic effects of ROS directed against engulfed microorganisms.
Selenoproteins participating in antioxidant defences (GSH-Px, thioredoxin
reductases, methionine sulfoxide reductase B) are able to protect neutrophils
from oxygen-derived radicals that are produced to kill ingested foreign
organisms (Ebert-Dumig et al., 1999). Therefore, as a constituent of
selenoproteins, selenium is needed for the proper functioning of neutrophils,
macrophages, NK cells, T lymphocytes and some other immune mechanisms
(Ferencik and Ebringer, 2003).
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Effect on immunomodulator production. There is a range of regulatory

molecules produced by immune cells. For example, IL-2, a lymphocyte
growth factor, is recognised as an important immunomodulatory molecule.
Oxidative stress suppresses IL-2 production and antioxidants can help to
overcome this suppression. Therefore, selenium up-regulates the expression
of the T-cell high affinity IL-2 receptor and provides a vehicle for enhanced
T-cell responses. Binding of IL-2 by the IL-2 receptor induces proliferation
of T-lymphocytes. For example, recently it has been suggested that Se can
increase the inducibility of IL-2 receptor whereas vitamin E may counteract
the down-regulatory effect of cAMP on IL-2 activity (McCarthy, 1997).
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254

The mRNA for IFN- was much less abundant in Se-deficient vs. Se
adequate mice and mRNA for IL-2 was also lower in the Se-deficient mice
(Beck et al., 2001).
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Prostanoid synthesis regulation. Antioxidants alter the production of

immunomodulatory molecules such as prostaglandins and leukotrienes
altering a ratio between immunosuppressive and immunostimulating
eicosanoids. The relationship between antioxidants and inflammatory
reactions are shown in Figure 4.7. From the figure it is clear that poor
antioxidant defence is associated with enhanced inflammation,
overproduction PGE 2 resulting in suppression of lymphocyte activity
(Grimble, 1997). For example, at low concentrations, PGE2 is essential for
cellular immunity; however, increased PGE2 concentration is associated
with a suppression of cellular and humoral immunity, including antibody
formation, DTH, lymphocyte proliferation, and cytokine production.
Lymphocytes from Se-deficient rats stimulated by an ionophore produced
significantly less prostaglandins in comparison to Se-supplemented animals
and characterised by decreased activation of phospholipase D (Cao et al.,
2002). Similarly, when stimulated by calcium ionophore A23187, the
lymphocytes derived from Se-deficient cows produced significantly less 5hydroxyeicosatetraenoic acid (5-HETE) and leukotriene B4 (LTB4- essential
for neutrophil chemotaxis) than those obtained from Se-supplemented cows
(Cao et al., 1992). Dairy cows were fed rations containing 0.05 mg Se/kg
dry matter or 0.35 mg Se/kg dry matter. Cows were inoculated intracisternally
with 30 colony-forming-units of Escherichia coli at 14-16 weeks of lactation.
Milk from cows fed the Se-depleted diet had significantly higher
concentrations of TXB2, 6-keto-PGF1, PGE2 and LTB4 in comparison to
the milk from the nonsupplemented animals (Maddox et al., 1991). It was
postulated that dietary Se status, which in turn determines tissue Se
concentration, plays an important role in the regulation of arachidonate
metabolism affecting the 5-lipoxygenase pathway. This may be one of the
biochemical mechanisms underlying the inhibition of lymphocyte
proliferation and the decrease in resistance to infectious diseases observed
in Se-deficient animals (Cao et al., 1992). 5-Lipoxigenase (5-LO), a key
enzyme in the biosynthesis of proinflammatory leukotrienes, is regulated
by the cellular redox status with a requirement in hydroperoxides (Werz et
al., 2000). In fact, granulocyte-derived ROS can activate B-lymphocyte 5LO. Since leukotrienes are involved in regulation of cell proliferation,
activation and maturation, they regulate immune responses.
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Inflammatory stimulus
Vitamin E, Se,
carotenoids,
ascorbate
Antioxidant status
Poor Good
Increased NFB
activity
Enhanced
inflammation
Cytokines
Oxidants
Sulphur
amino acids
GSH
Normal NFB
activity
+
Lymphocyte
activity
Inflammation
PGE2
Figure 4.7 Antioxidants and inflammation (adapted from Grimble, 1997).
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Effect on signal transduction. Natural antioxidants such as Se and vitamin

E may protect against oxidant-mediated inflammation and tissue damage
by virtue of their ability to scavenge free radicals and by their ability to
inhibit the activation of NF-B (and possibly other oxidant-sensitive
transcription factors). In vitro Se supplementation at low levels increased
production of interferon by human peripheral lymphocytes and high Se
doses were detrimental for interferon production (Watson et al., 1986). When
13 synthetic seleno-organic compounds, were studied, seven of them were
found to be inducers of IFN- and/or TNF- in human peripheral blood
leukocytes cultures (Piasecki et al., 1992). In fact, NF-B is required for
maximal transcription of many inflammatory cytokines and adhesion
molecules (Hughes, 1999). Furthermore, Se at physiological levels mediates
inhibition of the activation of the transcription factor NF-B which regulates
genes that encode inflammatory cytokines (Maehira et al., 2003). Thus,
maintaining adequate antioxidant status may provide a useful approach in
attenuating the cellular injury and dysfunction observed in some
inflammatory disorders. (Conner and Grisham, 1996). In fact, there is an
inverse relationship between cellular Se status and inducible form of nitric
oxide synthase expression in LPS-stimulated cells (Prabhu et al., 2002).
Following LPS stimulation, the nuclear localization of NF-B was
significantly increased in Se-deficient macrophages, thereby leading to
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256

increased expression of pro-inflammatory enzyme cyclooxygenase-2
(Zamamiri-Davis et al., 2002). It is necessary to underline that non-toxic
concentrations of reactive metabolites of oxygen and nitrogen play an
important role in regulating the expression of genes involved in the
inflammatory response and in modulating apoptosis (Jourdheuil et al.,
1997). At the same time an immune response requires extensive
communication between a wide range of cell types (Klasing, 1998) and
special cell receptors are of great importance in this communication.
Therefore protective effect of antioxidants in prevention of membrane and
receptor damages due to peroxidation could provide an important way of
enhancing the immune system.
Apoptosis regulation. Antioxidants are considered to prevent apoptosis

caused by oxidative stress. This could have a great effect on immune cell
apoptosis preventing immunosuppression. It has been shown that in vitro
selenium deficiency in a subset of hepatocellular carcinoma-derived cell
lines causes oxidative stress and cytochrome c release with subsequent cell
death by apoptosis (Irmak et al., 2003). New techniques are available and
they can be used to re-evaluate old data on Se-deficiency. For example,
selenium and vitamin E deficiency in chickens significantly increased
caspase-like activity suggesting that cell death associated with exudative
diathesis can follow the apoptotic pathway (Nunes et al., 2003).
Immunomodulating properties of Se could be mediated via prevention of
apoptosis of immune cells in the case of mycotoxicoses. Indeed, many
mycotoxins are immunosupressive and they can cause apoptosis (Surai,
2002), therefore Se supplementation of the mycotoxin-contaminated feed
could potentially have a beneficial effect. Since more than 25% of the worlds
grain production is contaminated with mycotoxins, sub-clinical
mycotoxicoses seem to be a real problem.
Did you know that mycotoxins are immunosuppressive due to
apoptosis stimulation and Se-supplementation potentially could
decrease those detrimental effects?
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Adhesion molecules expression and production of soluble mediators of

the immune response. Recently it has been shown that Se is able to affect
the adhesion molecules expressions that are crucial in the inflammatory
process (Jahnova et al., 2002). The inhibitory effect of Se on the adhesion
molecule expression has been studied in cultured endothelial cells after
interferon-gamma stimulation (Horvathova et al., 1999). The data suggest
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that Se affects the expression of P-selectin, ICAM-1, VCAM-1, and ELAM1 in a dose-dependent manner.
Immunocommunication, free radicals and selenium

As mentioned above the immune system of the animal is based on natural and
adaptive immunity. Natural immunity is dependent on the efficient function of
phagocytic cells, namely neutrophils and macrophages. These cells are equipped
with an array of microbicidal weapons, such as proteases, enzymes that hydrolyse
proteins disrupting membranes. This weaponry is stored in granules in the
cytoplasm. Furthermore, these cells have a powerful system for generating large
amounts of ROS and they use them as an effective weapon to kill pathogen.
However, on escape from the phagosome the same free radicals become dangerous
and can damage biological molecules, which compromises phagocyte function
and reduces adaptive immunity. Phagocytes also produce communication
molecules (eicosanoids, cytokines, etc.), that are used for effective communications
between various immune cells.
Invading pathogens are controlled by the natural and adaptive branches of the
immune system. It seems likely that the establishment of the adaptive immunity is
not sufficiently quick to eradicate microorganisms and natural immunity is
responsible for recognition of invading pathogens by specific receptors. Binding
of pathogen to those receptors induces the production of ROS and RNS, proinflammatory cytokines as well as communicating molecules which are responsible
for sending regulatory signals to the adaptive immunity (Werling and Jungi, 2003).
As mentioned above, adaptive immunity is based on activity of B- and Tlymphocytes, which produce antibodies to specific non-self substances (Blymphocytes) or directly attach to them (T-lymphocyte) and remove them from
the cell. Specific adaptive immune responses rely on the major histocompatibility
complexs restricted recognition of peptide antigens derived from pathogen to
activate a variety of effector T cells (helper and cytotoxic cells) that interact with
B cells that produce effector proteins called antibodies (Castle, 2000). The adaptive
immunity is characterised by high plasticity to recognise up to 10 11 distinct
structures and is tightly regulated to turn on or off a response aiming in eradication
of pathogens but not destruction of self. In the healthy animal or human resistance
to infection relies on a balance between the natural and adaptive immunity.
Regulation of the immune system is extremely complex. We are only starting to
understand how the immune system co-ordinates the bodys response to a disease
or invading pathogen. It seems likely, that communication between immune cells
is a crucial factor of immunocompetence. Interaction between the different
immune cell types that make up these components of host defence is carried out
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258
by the relative mix of cytokines, hormone-like proteins, as well as other

communicating molecules (Castle, 2000). The innate response, including its
inflammatory component, reacts initially to the stimulus, acting directly to eliminate
it by the activities of complement or phagocytosis. Cytokines produced by
monocytes and macrophages regulate this response and also act on the liver,
skeletal muscle, adipose and brain changing their metabolism and stimulating
various responses. The cytokines also interact with T-lymphocytes (Figure 4.4;
Calder, 2001).
Did you know that T lymphocytes carry receptor molecules on
their surface and they have been discovered only in 1980th?
If we imagine that immune system is an army fighting against invaders
(microorganisms, viruses, etc) than we would expect them to have something
like mobile phones to receive and send signals to each other. Remarkably enough,
major immune cells (macrophages, neutrophils, T- and B-lymphocytes) have on
their surface something like mobile phones called receptors. Those receptors
are extremely sensitive to communicating molecules, but they are also sensitive
to free radicals and can be easily damaged. In such a situation without proper
communication all those huge armies of immune cells would become useless.
They also can start fighting each other as well as eventually destroying
immunocompetence and causing autoimmune reactions. If we imagine that immune
cells are soldiers using chemical weapon to kill enemy, than special ammunition
protecting them from their own weapon would be a crucial for effective battle. In
the case of immune cells such ammunition is represented by natural antioxidants
with Se-GSH-Px and thioredoxin reductase being major defences. Indeed if not
properly protected, macrophage functions could be compromised including initial
overproduction of free radicals with consecutive damages to specific enzymatic
systems resulting in decreasing efficiency of oxidative burst and apoptosis. Based
on the presented model it is clear that antioxidant defence is a crucial factor of
immune defence in the body.
There is a great body of information confirming this idea. In fact Se- and vitamin
E deficiencies are associated with compromised functions of natural and adaptive
immunity. In particular phagocytic functions, lymphocyte proliferation and
antibody production are compromised (Surai, 2002). On the other hand, Se
supplementation is shown to improve immunocompetence and increase resistance
to various diseases. This is true for variety of farm and companion animals
including poultry, cows, sheep, horses, pigs, fish, cats and dogs. A summary of
the effect of compromised antioxidant system on immune system is shown in
Figure 4.8.
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Apoptosis of
immune cells
Damages to lymphocyte
receptors and disruption
of communications
between immune cells
and compromised
recognition
of target cells
Disbalancein eicosanoid
production by phagocytes
and inflammation
Phagocyte functions
compromised
Compromised AO system
and oxidative stress
Decreased activity
of NK cells
Damages to healthy
tissues by free
radicals
produced by
phagocytes
Compromised antibody
production by
B-lymphocytes
Figure 4.8 Oxidative stress and the immune system (adapted from Surai 2002).
Did you know that organic Se in the maternal diet can

provide newly born animals or newly-hatched chicks with
increased Se reserves for future antioxidant system
development and immunocompetence formation?
The importance of a delicate turning of the immune system is reflected by various
observations showing that over-reacting immune system has as detrimental
consequences as immunosuppression. For example, in some individuals, the
immune system recognises host antigens as non-self, attacking them and
producing tissue damage leading to chronic inflammatory or autoimmune diseases.
The immune system can also become sensitised to usually benign antigens from
the environment causing allergies (Calder, 2001). It seems likely, that in these
immune system miscommunication between immune cells plays a crucial role.
The immune system is functionally immature at birth. Therefore postnatal
development of the immune system is associated with accumulation of
polyunsaturated fatty acids and a desperate need for antioxidant protection.
Therefore, expression of selenoproteins in immune cells in early development
would be a crucial factor in a regulation of the immunocompetence development.
However, selenium reserves in the newly born animals or newly hatched chicks
are very limited when inorganic selenium is used in the maternal diet. In great
contrast, organic selenium, for example in the form of Sel-Plex, is shown in a
number of studies to be able to significantly increase Se concentration in colostrum,
milk and egg. This selenium is absolutely essential for the formation of effective
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260
antioxidant defences resulting in effective immune system maturation and

immunocompetence building.
Conclusions
Selenium affects all components of the immune system, including the development
and expression of nonspecific, humoral, and cell-mediated responses. In general,
a deficiency in Se appears to result in immunosuppression, whereas
supplementation with low doses of Se appears to result in augmentation and/or
restoration of immunologic functions. On the one hand, a deficiency of Se has
been shown (Kiremidjian-Schumacher and Stotzky, 1987) to inhibit
resistance to microbial and viral infections,

neutrophil function,
antibody production,
proliferation of T and B lymphocytes in response to mitogens, and
cytodestruction by T lymphocytes and NK cells.
On the other hand, Se supplementation has been shown (Kiremidjian-Schumacher

and Stotzky, 1987) to stimulate:
the function of neutrophils,

production of antibodies,
proliferation of T and B lymphocytes in response to mitogens,
production of lymphokines,
NK cell-mediated cytodestruction,
delayed-type hypersensitivity reactions and
allograft rejection, and
the ability of a host to reject transplanted malignant tumors.
When considering immuno-facilitating properties of selenium it is necessary to

take into account several points:
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Individual antioxidants in the body interacts with each other (vitamin E, C,

carotenoids, Se) and prooxidants (iron, high level of PUFAs, mycotoxins)
which themselves have immunostimulating or immunosuppressive effects.
Therefore in every experiment the results reflect a sum of all these interactions
and if background dietary concentrations of those nutrients differ, results
could be completely different. This could explain inconsistency of some
results published for the last 20 years. Furthermore antioxidants can suppress
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respiratory burst, however, it is not clear at present if there is a limit of this
suppression after which the phagocyte anti-microbial activity would be
compromised.
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Immunostimulating effect of antioxidants is shown to be maximal at their

supplementation usually above the requirement for maximal growth and
maintenance of reproduction. It could well be that the Se and vitamin E
doses that are adequate for maximal productivity in healthy, unchallenged
birds are not optimal for immunocompetence and disease resistance.
There is no data available on the effect of antioxidants on intestinal immune

structures, which could be crucial barriers to external pathogens (Qureshi
et al., 1998). Since free radicals can be damaging to intestinal structures
(Hoerr, 1998) antioxidant functions of selenium are responsible for
prevention damages to intestinal lymphoid structure as well as damages to
intestinal enterocyte membranes. This could be especially important in
relation to digestive immunosuppression caused by toxins/mycotoxins,
nutritional deficiencies and infectious agents (Hoerr, 1998). The evidence
is accumulating indicating a non-immunological protective effect of
selenium from various toxic agents including cadmium (Zasadowski et al.,
1997), monensin (Yarsan, 1998), salinomicin (Zarski et al., 1995) and
mercury (Maretta et al., 1995) in chickens.
Selenium source (organic vs inorganic) seems to be an important element

of its immunostimulating properties. Organic selenium appears to be at an
advantage because it is better assimilated from feed and better accumulated
in tissues. Indeed with the same dose of supplementation organic selenium
can deliver more element to the target tissues and because of toxicity of
high selenium levels this could be a solution to avoid adverse effect of
selenium overdose. Recent results presented by Johnson et al. (2000)
indicated that splenic macrophages and lymphocytes are sensitive to Se
intoxication and there is a disparity in the immune system toxicity of
inorganic and organic forms of Se administered via the drinking water,
inorganic Se being more toxic. In fact, sodium selenite at 9 ppm in the
drinking water for 14 days increased the production of proinflammatory
cytokines, tumor necrosis factor alpha and interleukin-1 beta, in LPSstimulated splenic macrophages. However, mice exposed to Se as selenoL-methionine in the drinking water did not display any effects on the
parameters examined at the dose range in the study. Furthermore, selenium
supplementation in organic form could have a beneficial effect during acute
phase response (APR) in many infections diseases. The APR is characterized
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by the synthesis of acute phase proteins, fever, accelerated whole-body
protein turnover, high rates of hepatic gluconeogenesis (Klasing, 1998) and
decreased selenium concentration in plasma (Sattar et al., 1997). It is
interesting that acute infections decrease serum selenium levels regardless
of the infective agent (Sammalkorpi et al., 1988). Therefore if the body has
selenium reserves in the form of SeMet in muscles, during acute phase
response it would be liberated as a result of skeletal muscle protein catabolism
by proteosome action and would be used for re-synthesis of new
selenoproteins which could decrease oxidative stress.
First two weeks posthatch represent most important period of immune system
development and maternal diet is shown (Klasing, 1998; Surai and Sparks,
2001) to have a profound effect on this process. In particular, the first week
of chick life is a period of rapid expansion of leukocyte population, seeding
of lymphoid organs and other events ultimately leading to the production
of unique clones of lymphocytes that will mediate immunity in postnatal
development (Klasing, 1998). In this respect effects of various combinations
of natural antioxidants and n-3 PUFA await investigation.
Because of complexity of regulation of the immune response and lack of

understanding of molecular mechanisms involved in such a regulation there
is a reasonable suggestion (Klasing, 1998a) to direct immunomodulation
firstly toward the correction of dysfunctional situation created by immune
system immaturity, stress, immunosuppressive disease, or genetics. In this
respect natural antioxidants, especially selenium, could have a prominent
role.
As a result of antioxidant (selenium) deficiency increased oxidative stress

of a host can lead to increased virus mutation rate and change in a viral
pathogen (Beck, 1999) resulting in emerging viral pathogens with new
pathogenic properties. Therefore, Se deficiency was associated with a
change to the viral genotype, converting the virus from a benign to a virulent
strain (Beck, 1998). This possibility was not exploited in animal/poultry
production, but seems important to study more extensively.
There is a need to study antioxidant composition and fatty acid profile of
immunocompetent tissues depending on chicken age and nutritional
supplementation of antioxidants and n-3 PUFAs.
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5
SELENIUM AND SEMEN QUALITY
We never know the worth of water till the well is dry
Introduction
Animal and human spermatozoa are unique in structure and chemical composition
and are characterized by high proportions of polyunsaturated fatty acids (PUFAs)
in the phospholipid fraction of their membranes (Surai, 2002). This feature of
these highly specialized cells is a reflection of the specific needs of their membranes
for high levels of fluidity and flexibility, which are necessary for sperm motility
and fusion with the egg. This functional advantage conferred by PUFAs is, however,
associated with disadvantages in terms of the susceptibility of sperm to free radical
attack and lipid peroxidation. Therefore antioxidant protection is a vital element
in maintaining sperm membrane integrity, motility and fertilizing ability. It has
been suggested (Surai, 2002) that natural antioxidants (vitamin E, ascorbic acid
and glutathione) together with antioxidant enzymes (superoxide dismutase and
glutathione peroxidase) build an integrated antioxidant system in avian semen
capable of protecting it against free radicals and toxic products of their metabolism.
The delicate balance between free radical production and antioxidant defense is
considered to be an important determinant of semen quality and in particular its
fertilising ability.
Fatty acid composition of avian and mammalian semen

Lipids are important constituents of the mammalian and avian semen. They serve
as structural compounds of the spermatozoa membranes, are precursors of different
biologically active compounds (eicosanoids) and can be used for energy
production. The fatty acid composition of spermatozoa lipids of 5 avian species
are shown in Table 5.1. As can be seen from the data PUFA comprise a major part
(46.0-54.4%) of the total fatty acids and there are species-specific differences in
the fatty acid profiles (Surai et al., 1998). For example, duck spermatozoa contained
the highest proportion of PUFA and goose and turkey spermatozoa were
characterised by much lower proportions of PUFA. As a result of those very high
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proportions of PUFA, duck and chicken spermatozoan lipids were characterised

by the highest peroxidability index compared to the other avian species.
Table 5.1 Polyunsaturated fatty acids in avian spermatozoa (adapted from Surai et al. 1998).
18:2n-6
20:2n-6
20:3n-6
20:3n-9
20:4n-6
22:3n-9
22:4n-6
22:5n-3
22:6n-3
Total PUFA
Peroxidability Index
Chicken
Turkey
Guinea fowl
Duck
2.6a
1.5a
1.6
<0.5
11.7 a
3.8a
27.9 a
0.5
2.1a
52.1
215.0
4.6b
4.0ab
0.9
2.7a
9.4b
9.2b
12.6b
0.7
2.7a
46.8
184.0
3.8abc
3.3ab
<0.5
1.0b
15.8c
4.3a
19.4c
<0.5
1.7a
49.3
195.1
3.8abc
1.6a
1.0
<0.5
18.9d
0.6c
19.9c
0.6
8.0b
54.4
231.3
Goose
5.7bd
3.9b
0.5
0.9b
13.3 ac
1.4c
17.5cd
<0.5
2.8a
46.0
185.6
*/Values within a row that do not share a common letter are significantly different (P<0.05).
Phospholipids are the major lipid fraction in avian spermatozoa comprising 66.470.7% of total sperm lipid in the case of the chicken (Cerolini et al., 1997; Kelso
et al., 1997a; Surai et al., 2000) or 59.5% for the turkey (Cerolini et al., 1997). It
seems that the phospholipid proportion in the chicken spermatozoa increases with
aging reaching 72.1% of total lipid at 60 weeks of age (Kelso et al., 1996), but
decreasing by 72 weeks of age down to 51% (Kelso et al., 1997). In some cases
the phospholipid proportion is reported to be 84.5% of the total lipids in the cockerel
spermatozoa (Blesbois et al., 1997), but the authors did not detect any
triacylglycerol in the spermatozoa, which could comprise up to 4.2% (Cerolini et
al., 1997) or 7.3% (Kelso et al., 1997) depending on the age of cockerels.
Our data indicated that docosatetraenoic (DTA; 22:4n-6) and arachidonic acid
(AA; 20:4n-6) are the major PUFAs in the phospholipid fraction of the chicken
spermatozoa (Surai, 2002). Their proportions varied depending on the cockerels
age. DTA is the characteristic PUFA of avian spermatozoa in contrast to mammalian
spermatozoa where docosahexaenoic acid (DHA; 22:6n-3) is the major PUFA
(Poulos et al., 1973). In fact, DHA is generally the most important spermatozoan
PUFA in mammals, including man (Nissen and Kreysel, 1983), bull (Kelso et al.,
1997), monkey, (Lin et al., 1993), ram and boar (Poulos et al., 1973). However,
in dog and rabbit spermatozoa, docosapentaenoic acid (DPA; 22:5n-3) is the main
PUFA (Poulos et al., 1973). The importance of C22 polyunsaturates in relation to
male fertility has been shown in humans where the amount of DHA in spermatozoa
is positively correlated with sperm motility (Nissen and Kreysel, 1983; Zalata et
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al., 1998; Conquer et al., 1999) and with the normal morphology of sperm cells
(Lenzi et al., 2000). Therefore the best morphological pattern corresponded to
the highest DHA concentration in the human semen (Lenzi et al., 2000a). In
general, in mammalian spermatozoa long chain PUFAs containing 20-22 carbon
atoms comprise more than 50% of total fatty acids in the phospholipid fraction
and the level of DHA in semen depends on stage of sperm maturation (Ollero et
al., 2000). In mammals, long chain PUFAs are actively produced during the
maturation of spermatozoa after testicular release (Lenzi et al., 2000a). In contrast
to mammals, avian spermatozoa are less unsaturated and are characterised by the
presence of DTA and AA which comprise more than 30% of total PUFA (Surai et
al., 1998b).
The biological reason for these species-specific differences in the PUFA profiles
of spermatozoa is not clear at present. However, there is a growing body of
evidence indicating that the fatty acid composition of sperm membranes, especially
levels of PUFA, determine their biophysical characteristics such as fluidity and
flexibility as appropriate for their specific functions, including sperm motility and
fertilising capacity (Ladha, 1998). For example, increased PUFA concentrations
in human spermatozoa were associated with increased sperm membrane fluidity
(Comhaire et al., 2000). It is also necessary to appreciate that phospholipids
containing DHA are not evenly distributed throughout the membranes of
mammalian spermatozoa, being located mainly in the tail (Connor et al., 1998).
Whether this is the case for avian spermatozoa, with DTA being confined to the
tail, awaits investigation. However, the very high proportion of long chain PUFA
in the avian spermatozoa predisposes them to lipid peroxidation and it seems
reasonable to suggest that antioxidant protection plays a crucial role in the
maintenance of spermatozoan membrane integrity and their fertilising ability.
Clearly, avian and mammalian spermatozoa are rich in PUFAs (Figure 5.1) and
are vulnerable to lipid peroxidation.
Did you know that the fatty acid composition of sperm
membranes, especially levels of PUFA, determine their
biophysical characteristics as appropriate for their specific
functions, including sperm motility and fertilising capacity
Lipid peroxidation is semen

It is somewhat surprising that toxicity of oxygen free radicals to human
spermatozoa was reported more than 60 years ago (McLeod, 1943) and the toxic
effect of H2O2 on chicken semen was shown by Wales et al. (1959). However,
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60
Boar
Ram
Chicken
50
40
30
20
10
0
20:4n-6
22:4n-6
22:5n-6
22:6n-3
PUFAs
Figure 5.1 PUFAs in spermatozoa phospholipids, % (adapted from Surai, 2002)
20:4n-6 arachidonic acid; 22:4n-6- docosatetraenoic acid; 22:5n-6-docosapentaenoic acid; 22:6n-3
docosahexaenoic acid.
major attention to this subject came in 1970 after publication of several milestone
papers by Jones and Mann based on the results of experiments conducted in the
Agricultural Research Councils Unit of Reproductive Physiology and
Biochemistry, University of Cambridge (Jones and Mann, 1973; 1976; 1977;
1977a; Jones et al., 1978; 1979). These publications clearly showed that lipid
peroxidation:
Takes place in mammalian spermatozoa

Caused decline in motility of spermatozoa
Irreversibly abolished the fructolytic and respiratory activity of spermatozoa
Increased release of intracellular enzymes from spermatozoa into medium
Is the major biochemical cause of sperm senescence under storage conditions
in vitro
Caused predominant oxidation of 22:6n-3 and 20:4n-6 fatty acids
Furthermore, those authors also showed that the susceptibility of spermatozoa to

peroxidation was increased in cells damaged prior to incubation and that
peroxidized PUFAs added to a washed sperm suspension immobilised the
spermatozoa rapidly and permanently. Those publications presented results
obtained with ram and human semen. However results on lipid peroxidation in
other mammalian species have also been published, including boar (Mrotek et
al., 1966; Smutna and Synek, 1979; Brzezinska-Slebodzinska et al., 1995; Cerolini
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et al., 2000), bull (Dawra et al., 1983; Beconi et al., 1991; Slaweta et al., 1988;
OFlaherty et al., 1997; Beorlegui et al., 1997), buffalo (Singh et al., 1989), rabbit
(Castellini et al., 2000) and horse (Baumber et al., 2000; Ball and Vo, 2002).
Furthermore, lipid peroxidation in human semen has been studied further in detail
and several comprehensive reviews have discussed their findings (Aitken 1994;
1995; 1999; Lenzi, 2000, 2000a). The conclusion is that lipid peroxidation in
mammalian semen is considered to be one of the most important factors causing
infertility in man as well as causing decreased sperm quality during the storage of
semen from farm animals.
Research on lipid peroxidation in avian semen started much later in comparison
to mammalian species, This is probably because such research has largely been
driven by a practical need to understand molecular mechanisms of semen
deterioration during storage for further artificial insemination. In poultry production,
however, artificial insemination was introduced several decades later than for
mammalian species, and its practical usage is related mainly to turkey production.
Therefore, a publication by Fujihara and Howarth showing that, during incubation
at 41C, chicken spermatozoa produced a thiobarbituric acid-reactive product
and that susceptibility to peroxidation was enhanced by the addition of ascorbate
was a starting point in research related to lipid peroxidation in avian semen (Fujihara
and Howarth, 1978). The authors concluded that chicken spermatozoa, following
ejaculation and exposure to air, could undergo peroxidation with a consecutive
decrease in their viability. The conclusion was very important for future
improvement of techniques for avian semen storage in vitro. Further evidence for
lipid peroxidation in avian semen was presented by Wishart (1984), who found
that the formation of high concentrations of malondialdehyde (MDA) during a 5hour aerobic incubation of chicken semen was associated with a partial or complete
loss of fertilizing ability. Most importantly, the fertilizing ability of samples that
produced low or negligible concentrations of MDA remained unimpaired (Wishart,
1984). Semen samples incubated under anaerobic conditions produced only a
negligible amount of MDA. There was a considerable (70-fold) variability between
individual males in relation to MDA production (Wishart, 1984). This could be
explained as a result of compositional and functional differences in sperm
membranes among individual male chickens (Fujihara and Koga, 1992).
Did you know that lipid peroxidation in semen is considered
to be one of the most important factors causing infertility in
man as well as causing decreased sperm quality during the
storage of semen from farm animals
Independently of the work conducted in the UK by Wishart (1984), investigation
of lipid peroxidation in turkey semen was started approximately at the same time
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in Ukraine (Surai, 1983; 1984). In particular an induced (by Fe2+) lipid peroxidation
in turkey semen was used to assess semen susceptibility to lipid peroxidation,
since the initial level of peroxides in fresh or even stored semen was shown to be
comparatively low. At that time an idea of the possible protective effects of natural
antioxidants in the male diet on the lipid peroxidation in the semen was developed
(Surai, 1984). In particular, it was shown that dietary supplementation of male
turkeys with vitamin E was associated with a significant decrease in semen
susceptibility to lipid peroxidation. This work was further developed by Cecil
and Bakst (1993) who showed that during aerobic storage of turkey spermatozoa,
lipid peroxidation was time and temperature-dependent. The authors also suggested
that turkey spermatozoa are more sensitive to lipid peroxidation than semen from
other species. It seems likely that lipid peroxidation in semen is also age-dependent.
For example, Donoghue and Donoghue (1997) reported that MDA concentrations
were 10-fold higher in semen from older turkey males (56 wk of age) than for
younger ones (30 wk of age). Turkey male ageing (between 31 and 52 weeks)
was accompanied by a 30% increase in lipid peroxidation at 47 and 52 weeks of
age. Furthermore in vitro storage did not cause lipid peroxidation in sperm obtained
during the first half of the reproductive period but MDA levels significantly
increased in sperm obtained during the second half of this period (Douard et al.,
2003). In general lipid peroxidation is a significant factor affecting the fertility of
stored turkey sperm and males varied in production of MDA during in vitro storage,
with most pairs exhibiting a threefold increase (Long and Kramer, 2003). This is
in agreement with the data of Kelso et al. (1996) indicating a fall in the antioxidant
defence (activity of GSH-Px) of chicken spermatozoa between 25 and 60 weeks
of age. Accumulation of TBARS in duck semen as a result of lipid peroxidation
has also been recently described (Surai et al., 2000). It seems likely that MDA
accumulation is associated with mid-piece abnormality in human spermatozoa
(Aitken et al., 1993) and with a decrease in the fertilising capacity of chicken
(Wishart, 1984) and turkey (Cecil and Bakst, 1993) spermatozoa. It is interesting
that lipid peroxidation was particularly strong in the midpiece and tail of frozen/
thawed bovine spermatozoa and significantly less intense in the head (Brouwers
and Gadella, 2003).
The molecular mechanisms of lipid peroxidation in avian semen have received
little attention. Recently it has been shown that during sperm storage, lipid
peroxidation is associated with a significant decrease in PUFA concentration in
spermatozoa. In particular, the main PUFA in the chicken semen (22:4n-6) was
most susceptible to peroxidation. Its proportion in the phospholipid fraction was
significantly decreased as a result of incubation of chicken sperm for 12 hours at
20C (Surai et al., 1998b). The inclusion of a promoter of lipid peroxidation
(Fe2+) in the incubation medium further increased the rate of lipid peroxidation,
significantly decreasing the proportions of not only 22:4n-6, but also of 20:4n-6,
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22:5n-3 and 22:6n-3 in the phospholipid fraction of the spermatozoa. The

confirmation of the suggestion that the loss of PUFA was due to peroxidation
came from the data showing simultaneous accumulation of TBARS in the semen
(Surai et al., 1998b). Recently, it has been shown that the total lipid content, the
proportion of total phospholipids, and the levels of phosphatidylcholine (PC),
phosphatidylethanolamine (PE) and sphingomyelin (Sph) were significantly
decreased in chicken semen during in vitro storage and this was associated with a
reduction in the proportion of motile, viable and morphologically normal cells
(Blesbois et al., 1999). Similarly, in turkey spermatozoa incubated at 37C in the
presence of exogenous Fe2+, a significant decrease in PS (by 47%) and PE (by
35%), the two most unsaturated fractions of avian spermatozoa, was observed
(Surai et al., 1998a; Maldjian et al., 1998). Storage of diluted turkey semen for
48h at 4C was also associated with a decrease in total phospholipid content and
of PC and, to lesser extent, of Sph, phosphatidylserine (PS) and phosphatidylinositol
(PI) (Douard et al., 2000). The significance of the decreased concentration of
these phospholipids needs further investigation. However PS appears to be an
important phospholipid fraction in avian spermatozoa, having the highest degree
of unsaturation (Surai et al., 1997a; Surai et al., 2000), decreasing during ageing
(Kelso et al., 1997), and showing a significant positive correlation with the
fertilizing ability of chicken semen during the reproductive cycle (Cerolini et al.,
1997).
Therefore, the mechanisms by which ROS disrupt sperm function probably
involve the peroxidation of PUFA in the sperm plasma membrane. For example,
it has been shown that in human spermatozoa, lipid peroxidation damages the
cell plasma membrane, leading to loss of cytoplasmic components and hence to
cell death - a process that is considered to play an important role in the
pathophysiology of male infertility (Aitken et al., 1993). A negative correlation
between the MDA production and sperm motility was observed in human semen
(Huang et al., 2000). However, sperm storage at refrigeration temperature is not
always associated with lipid peroxidation. For example, in contrast to the abovementioned observations, storing turkey semen for 48 h at 4C did not significantly
affect the fatty acid profile nor the level of free cholesterol, but the motility, viability
and morphological integrity of spermatozoa significantly decreased (Douard et
al., 2000).
It is necessary to note, that lipid peroxidation is not restricted to the damage to
membranes but other detrimental consequences for cell metabolism have been
described as well (for review Surai, 2002):
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chromatin destabilisation
marked alterations in the DNA-protein complex
changes in the activities of various enzymes, including cytochrome oxidase,
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lactate dehydrogenase and glucose-6-phosphate dehydrogenase

disruption of mitochondria functions
inhibition of the synthesis of DNA, RNA and proteins
increase of DNA fragmentation
modification of the cytoskeleton
alteration in the sperm axoneme
inhibition sperm-oocyte fusion
It is necessary to stress that most of the studies on mechanisms and consequences

of lipid peroxidation have been associated with mammalian (mainly human)
spermatozoa with much less emphasis on avian semen. Nevertheless, lipid
peroxidation in avian semen without doubt would have similar consequences.
For example, H2O2 and organic hydroperoxides had toxic effects on avian sperm
motility (Surai et al., 1998b). On a molar basis, H2O2 was about 3 times more
powerful in terms of decreasing spermatozoa motility than cumene hydroperoxide.
The difference in the toxicity is probably due to higher permeability of plasma
membranes for H2O 2 than for organic hydroperoxides. At the same time, the
susceptibility of chicken spermatozoa to H2O2 toxicity was much higher than that
in mammalian spermatozoa (Wales et al., 1959).
Selenoproteins and their role in antioxidant protection in semen

The antioxidant protection in semen is poorly characterised. Recently, it has been
suggested that the antioxidant system of the spermatozoa includes three major
levels of antioxidant defence (Surai, 1999; 2002) responsible for maintenance of
spermatozoan functions in various stress conditions including sperm dilution,
storage and deep freezing. Antioxidant enzymes (SOD and GSH-Px) and metalbinding proteins comprise the first level of antioxidant defence responsible for
prevention and restriction of free radical formation (Surai, 2002). Natural
antioxidants (vitamin E, ascorbic acid, glutathione), together with additional actions
of GSH-Px, build the second level of antioxidant defence dealing with prevention
and restriction of chain formation and propagation. Lipid peroxidation can be
kept under control until these antioxidants are used up and the chain reaction
becomes uncontrolled, resulting in damage to cellular constituents and structures.
The third level of defence is based on the enzymatic system responsible for repair
or/and removal of damaged molecules from the cell. It seems likely (Hammerstedt,
1993) that this level of antioxidant defence in the spermatozoa is either not present
or is very inefficient.
The essentiality of selenium for mail fertility was shown in the early 1980s
(Behne et al., 1982; Calvin et al., 1987; Wu et al., 1979; Hansen and Deguchi,
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1996). This conclusion was based on the results of a range of different experiments
with mammals showing that:
in mild deficiency, Se is preferentially retained in rat testes (Behne et al.,

1982)
mammalian semen is considered to contain the highest selenium
concentration of all other body tissues (Behne et al., 1986).
in the human, a significant positive correlation in the selenium concentration
was demonstrated between the different reproductive organs with the testis
having the highest concentrations of this element (Oldereid et al., 1998).
after 75Se intravenous injection, the highest levels of 75Se were found in the
kidney followed by seminal vesicles and testicles (Vanha-Perttula and Remes,
1990). The seminal vesicle secretion was particularly rich in 75Se and its
fractionation resembled that of the seminal plasma.
progressive selenium deficiency was associated with morphological
alterations of spermatids and spermatozoa (Calvin et al., 1987) with
subsequent complete disappearance of mature germinal cells (Behne et al.,
1996).
in selenium-deficient mice, the proportion of abnormal sperm ranged from
6.8% to 49.6%, while in the control group it was only 4.0-15.0%. The most
frequently occurring abnormalities in sperm shape were found in the sperm
head. However there was also a tendency of increasing abnormalities in
other spermatozoa regions, including neck, mid-piece and tail (Watanabe
and Endo, 1991).
in human semen, selenium was found mainly (more than 85%) in the seminal
plasma and sperm motility was maximal when semen Se levels were between
50 and 69 ng/ml (Bleau, 1984).
It is generally accepted that Se participates in various physiological functions as

an integral part of a range of selenoproteins. The selenoprotein family includes at
least 25 eukaryotic proteins (Kohrle et al., 2000, see Chapter 2). Expression of
individual eukaryotic selenoproteins is characterised by high tissue specificity,
depends on Se availability, can be regulated by hormones, and if compromised
contributes to various pathological conditions (Kohrle et al., 2000).
Did you know that there are sperm-specific selenoproteins
responsible for structural integrity of spermatozoa
For more than 10 years major textbooks referred to Sperm Capsular Selenoprotein
(SCS) as an important selenoprotein involved in maintenance sperm functions.
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Indeed, SCS was suggested to be localised in the midpiece of the spermatozoa

stabilising the sperm flagella. Initially, it was shown that the sperm selenoprotein
was identical with mitochondrial capsule protein (MCP). It is interesting that
selenium-free MCP was found in immature germ cells and sperm selenoprotein
first appeared in very low concentration during late meiosis and its concentration
increased sharply during early spermatogenesis (Calvin et al., 1987). However,
in following years this protein created a lot of controversy. In particular, SCS was
considered not to be a selenoprotein because: (1) it is not labelled with 75Seselenite in seminiferous tubule culture; (2) cleavage with cyanogen bromide and
translation of T7 RNA polymerase transcripts in vitro indicate that the translation
start site is located downstream of potential UGA selenocysteine codons in the
mouse cysteine-rich mRNA; (3) the reading frame encoding the cysteine-rich
protein in rat lacks inphase UGA selenocysteine codons (Cataldo et al., 1996).
Therefore, it was concluded that the pertinent genes of rats and mice did not
contain any TGA codons within the translated regions and, as a result, the inclusion
of SCS into selenoprotein family was questioned (Kohrle et al., 2000). Recently
SCS has been identified as PH-GSH-Px (Ursini et al., 1999).
Therefore renewed interest to PH-GSH-Px appears to be associated with its
structural role in spermatozoa. It is well established that PH-GSH-Px (E.C.
1.11.1.12) is a monomeric seleno-enzyme present in different mammalian tissues
in soluble and bound form. Its function, like the other GSH-Px, was originally
viewed as a protective role against hydroperoxides, but it seems likely that it has
additional regulatory roles. It was reported that the relative PH-GSH-Px mRNA
levels in rats were much higher in the testis than in the other tissues (Mizuno et
al., 2000). Expression of PH-GSH-Px activity in rat testis and seminal vesicles
was high and regulated by dietary Se and vitamin E differently from that in liver
(Lei et al., 1997). For example, testes and seminal vesicles had substantially higher
(5- to 20-fold) PH-GSH-Px activity than liver. Dietary Se deficiency did not affect
PH-GSH-Px activities in the reproductive tissues, but reduced it in liver by 28%.
On the other hand, dietary vitamin E supplementation did not affect PH-GSH-Px
activity in liver, whereas it raised PH-GSH-Px activity in seminal vesicles by 43%
(Lei et al., 1997). Therefore, PH-GSH-Px is present in testis cells and sperm cells,
and its appearance is hormone regulated (Tramer et al., 2002). Furthermore PHGSH-Px specific activity in rat is age-dependent during the life-span monitored
(from 36 to 365 days), with a maximum at 3 months of age in the testis germ cells
and at 6 months of age in the isolated epididymal sperm cells (Tramer et al.,
2002). Clearly, this selenoprotein involves in sperm maturation. The age or
gonadotropin-dependent expression of PH-GSH-Px in testis does not result from
direct transcriptional gene activation by testosterone, but is due to differentiation
stage-specific expression in late spermatids, which are under the control of Leydig
cell-derived testosterone (Maiorino et al., 1998).
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It seems likely that the selenoprotein PH-GSH-Px changes its physical

characteristics and biological functions during sperm maturation. It exists as a
soluble peroxidase in spermatids but persists in mature spermatozoa as an
enzymatically inactive, oxidatively cross-linked, insoluble protein (Ursini et al.,
1999). Specifically, in the midpiece of mature spermatozoa, PH-GSH-Px protein
represents at least 50 percent of the capsule material that embeds the helix of
mitochondria. Therefore PH-GSH-Px of rat sperm mitochondrial capsule is
inactive and its thiol peroxidase activity is responsible for cross-linking proteins
and accounts for the selenium dependency of spermatogenesis (Roveri et al.,
2001). In fact PH-GSH-Px accounts for almost the entire selenium content of
mammalian testis and abundantly expressed in spermatids as active peroxidase
but is transformed to an oxidatively inactivated protein in mature sperm, where it
is a major constituent of the mitochondrial capsule in the midpiece (Foresta et al.,
2002). In human sperm, the inactive form accounted for more than 95% of total
PH-GSH-Px and low PH-GSH-Px content of sperm was associated with impaired
male fertility (Flohe et al., 2003). Therefore, male infertility in selenium-deficient
animals, which is characterised by morphological midpiece alterations, is
considered to result from insufficient PH-GSH-Px content and PH-GSH-Px appears
to be indispensable for structural integrity of spermatozoa and to codetermine
sperm motility and viability. (Foresta et al., 2002). Indeed, the role of PH-GSHPx as a structural protein may explain the mechanical instability of the
mitochondrial midpiece that is observed in selenium deficiency.
Recently a fifth member of Se-dependent glutathione peroxidases: a specific
sperm nuclei GSH-Px (sn-GSH-Px), has been characterised (Behne et al., 2000;
Pfeifer et al., 2001). In particular, this selenoenzyme has been identified in rat
testes (Behne et al., 2000) to be a 34-kDa selenoprotein. It was localised in the
spermatid nuclei and found to comprise about 80% of total Se present. It was
identified as specific to the sperm nuclei GSH-Px with similar properties to PHGSH-Px (Pfeifer et al., 2001). The authors showed that it differs from PH-GSHPx in its N-terminal sequence. In rats, sn-GSH-Px is highly expressed in the nuclei
of the late spermatids where it is the only selenoprotein present (Pfeifer et al.,
2001). In Se-depleted rats the concentration of sn-GSH-Px decreased to a third of
the normal level and chromatin condensation was severely disturbed and it seems
likely that the main function of sn-GSH-Px is protamine thiol cross-linking during
sperm maturation (Pfeifer et al., 2001).
A family of Se-dependent GSH-Px is considered to be responsible for protection
of spermatozoa from oxidative stress and loss of motility in the female genital
tract following ejaculation. However, antioxidant defences of spermatozoa also
include Se-independent GSH-Px. For example, the mammalian epididymis is the
site of expression and secretion of an abundant, tissue-specific, androgen-regulated,
selenium-independent, glutathione peroxidase isoenzyme (GSH-Px5). GSH-Px5
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has been proposed to play a role in protecting the membranes of spermatozoa

from the damaging effects of lipid peroxidation and/or preventing premature
acrosome reaction and maintains sperm fertilizing ability in the epididymis
(Williams et al., 1998; Okamura et al., 1997). In particular, this protein was found
to bind to the acrosomal region of the epididymal sperm and to disappear during
the acrosome reaction and significantly retarded the acrosome reaction induced
in vitro. Similarly, in mouse the secreted GSH-Px5 binds to spermatozoa and is
found predominantly on the sperm acrosomic region (Vernet et al., 1997). It seems
likely that the GSH-Px5 could function as a back-up system for Se-dependent
GSH-Px. This enzyme was found to be express exclusively in the mouse caput
epididymidis and an increase in GSH-Px5 mRNA and protein levels was seen in
epididymidis of selenium-deficient animals (Vernet et al., 1999). However, the
activities of the purified porcine GSH-Px5 toward hydrogen peroxide or organic
hydroperoxides were by far lower than the activity of cytosolic selenium-dependent
glutathione peroxidase (less than 0.1%; Okamura et al., 1997).
As mentioned above, a range of other selenoproteins is expressed in animal
tissues but their functions are less obvious (Holben and Smith, 1999; Burk and
Hill, 1999). In relation to the antioxidant defence in semen provided by
selenoproteins, TR is of great importance. However, roles of TR in animal and
human semen await investigation. It is well known that thioredoxins compose a
growing family of proteins that participate in different cellular processes via redoxmediated reactions. Therefore, it is interesting that sperm-specific thioredoxin
(Sptrx-1), has been recently identified and characterised in humans (MierandaVizuete et al., 2001). The authors showed that Sptrx-1 mRNA to be only expressed
in round and elongating spermatids, while the Sptrx protein is located in the
cytoplasmic droplets of ejaculated sperm, suggesting that it might be an important
factor in regulating critical steps of human spermatogenesis. Therefore Sptrx-1 is
the first member of the thioredoxin family of proteins with a tissue-specific
expression pattern, found exclusively in the tail of elongating spermatids and
spermatozoa. Functionally, Sptrx-1 behaves as an oxidant in vitro when using
selenite suggesting that this protein might govern the stabilisation (by disulfide
cross-linking) of the different structures in the developing tail of spermatids and
spermatozoa (Jimenez et al., 2002). Mouse Sptrx-1 has been cloned and
characterised (Jimenez et al., 2002a), being similar to that described for the human
and showing protein disulphide reducing activity in an enzymatic assay coupled
to NADPH and TR. The expression pattern of Sptrx-1 during rat spermatogenesis
suggests that it could be part of a nucleation center for the formation of the
longitudinal columns and transverse ribs that bridge the latter (Yu et al., 2002).
Furthermore, a second member of this family, called Sptrx-2 and specifically
expressed in human sperm cells, has been identified and characterised (Sadek et
al., 2001) and considered to be a novel component of the human sperm axonemal
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organisation. Mouse and rat Sptrx-2 proteins display a high homology to their
human ortholog and the coding genes are located at syntenic positions (MirandaVizuete et al., 2003). The authors also showed that Sptrx-2 is required during the
final stages of sperm tail maturation in the testis and/or epididymis, where extensive
disulfide bonding of the fibrous sheath proteins occurs.
It seems likely that it is just the beginning of deeper understanding of roles of
new proteins in sperm function. Recently a novel member of the thioredoxin
family, thioredoxin-like protein 2 (Txl-2) has been cloned and characterised (Sadek
et al., 2003). In the study light and electron microscopy analyses showed that the
protein was associated with microtubular structures such as lung airway epithelium
cilia and the manchette and axoneme of spermatids. In fact full-length Txl-2 weakly
associates with microtubules and the author suggested that this thioredoxin might
be a novel regulator of microtubule physiology. Therefore it is just a matter of
time before TR in the sperm is characterised and its essential role in sperm
antioxidant protection and structural integrity maintenance is shown.
There are species-specific differences in the Se level in semen. For example,
the level of Se in seminal plasma was lowest in the human and the stallion, higher
in ram and boar, with the highest levels in the bull (Saaranen et al., 1989). In
bulls, 75Se appeared in the epididymal caput within 5 days and passed through
the epididymis in 20 days (Vanha-Perttula and Remes, 1990). Furthermore, in
semen 75Se was first mainly found in seminal plasma, where a plateau level was
reached at 5 d followed by a gradual decline after 12 d. The total semen level,
however, increased after 14 d and this increase was due to a rapid appearance of
the label in spermatozoa. In the sperm 75Se level reached a plateau at 20 d and
remained high until 40 days, after which a gradual decline ensued (Vanha-Perttula
and Remes, 1990). Selenocysteine is shown to be the main form of Se in rat
sperm and selenocysteine and selenomethionine were found in ovine sperm (Alabi
et al., 2000).
Since hydrogen peroxide and lipid peroxides are toxic for the spermatozoa
(Alvarez et al., 1987; De Lamirande and Gagnon, 1992), GSH-Px plays an
important role in protecting cell membrane lipid from peroxidation, thus
maintaining the integrity of the cell (Flohe and Zimmermann, 1970). In fact,
GSH-Px in the sperm is considered to be the main enzyme which removes peroxides
and thereby protects cells against damage caused by free radicals and the products
of lipid peroxidation in vivo (Griveau et al., 1995).
There is species and tissue-specificity in GSH-Px expression. For example,
GSH-Px activity has been found to be expressed in the semen of several mammalian
species including ram, dog, human, goat, bull (Li, 1975; Kantola et al., 1988;
Kelso et al., 1997b). However, there are species-specific differences in expression
of this enzyme in semen. In bulls, for example, GSH-Px is exclusively associated
with the seminal plasma and not found in spermatozoa (Brown et al., 1977; Smith
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et al., 1979). In contrast, GSH-Px activity in seminal plasma was low in man and
ram and not detectable in boar and stallion (Saaranen et al., 1989). Bull and ram
seminal plasma GSH-Px activities per mg protein were comparable, but when
expressed per ml seminal plasma, activity of the bull was more than 7 times the
highest activity of ram seminal plasma (Pond et al., 1983). It has been shown that
approximately two thirds of GSH-Px activity in bull semen was non-Se-GSH-Px
(Slaweta et al., 1988). In the same experiment it was found that the MDA level
was negatively correlated with Se-GSH-Px activity and it has been suggested that
Se-GSH-Px plays a role in protecting the acrosome membranes against disruption.
GSH-Px has been found to be expressed in chicken seminal plasma and
spermatozoa (Figure 5.2; Surai et al., 1998, 1998b). There are species-specific
differences in activity and distribution of GSH-Px in avian semen. For example,
in seminal plasma total GSH-Px activity was the highest in turkey and lowest in
duck and goose (Figure 5.3; Surai et al., 1998). In spermatozoa, on the other
hand, the highest GSH-Px activities were found for goose and duck and much
lower GSH-Px activity was recorded for guinea fowl, turkey or chicken (Figure
5.4). Recently, it has been shown that despite a high proportion of PUFAs and a
low level of vitamin E, duck spermatozoa have the same susceptibility to lipid
peroxidation as chicken spermatozoa (Surai et al., 2000). It has been suggested
that an increased activity of Se-GSH-Px in duck semen compensates for the
relatively low concentrations of other antioxidants.
180
Se-GSH-Px
Activity (U/ml initial semen)
160
Non-Se-GSH-Px
140
120
100
80
60
40
20
0
Senimal plasma
Spermatozoa
Figure 5.2 Distribution of GSH-Px in chicken spermatozoa (adapted from Surai et al., 1998).
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250
Se-GSH-Px
Non-Se-GSH-Px
200
Units
150
100
50
0
Chicken
Turkey
Guinea fowl
Duck
Goose
Figure 5.3 GSH-Px activity in avian seminal plasma (adapted from Surai et al., 1998a).
160
Se-GSH-Px
140
Non-Se-GSH-Px
120
Units
100
80
60
40
20
0
Chicken
Turkey
Guinea fow l
Duck
Goose
Figure 5.4 GSH-Px activity in avian spermatozoa (adapted from Surai et al., 1998a.
If selenium is limiting in the diet (which is the case in many countries in the
world), then dietary supplementation of this trace element should have a beneficial
effect on the antioxidant defense in various tissues including sperm. This was
confirmed in our studies. Inclusion of Se in the diet of male chickens significantly
increased Se-GSH-Px activity in the liver, testes, spermatozoa and seminal plasma
(Figures 5.5 and 5.6; Surai et al., 1998c). As a result, a significant decrease in the
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sperms and tissue susceptibility to lipid peroxidation was observed (Figures 5.7
and 5.8). This protective effect was more expressed in stored semen as compared
to fresh. In this respect, it is extremely important that an inducible form of the
enzyme (Se-GSH-Px) represents more than 75% of the total enzymatic activity in
chicken spermatozoa and more than 60% in the testes and liver of cockerels.
25
Liver
Testes
GSH-Px, Units
20
15
10
0
20E
20E+Se
200E
200E+Se
Dietary supplementation
Figure 5.5 Effect of selenium and vitamin E on GSH-Px activity in chicken liver and testes (adapted from
Surai et al., 1998c).
700
Seminal plasma
600
Spermatozoa
GSH-Px, Units
500
400
300
200
100
0
20E
20E+Se
200E
200E+Se
Figure 5.6 Effect of selenium and vitamin E on GSH-Px activity in chicken semen (adapted from Surai et
al., 1998c).
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60
Fresh semen
Stored semen
MDA (nmol/g)
50
40
30
20
10
0
1
Groups
Figure 5.7 Effect of Se and vitamin E on lipid peroxidation in chicken spermatozoa (Adapted from Surai
et al., 1998c). Vitamin E and Se were supplemented in the feed (mg/kg) as follows: group 1, no
supplementation; group 2, vitamin E 20 and Se 0; group 3, vitamin E 200 and Se 0; group 4, vitamin E 20
and Se 0.3; group 5, vitamin E 200 and Se 0.3.
25
L
MDA (nmol/g)
20
15
10
0
1
Groups
Figure 5.8 Effect of Se and vitamin E on lipid peroxidation in chicken liver (L) and testes (T) (adapted
from Surai et al., 1998c). Dietary groupd are the same as in Figure 7.
An extensive work on effects of selenium on boar semen quality has been

conducted at the Columbus State University. For example, Marin-Guzman et al.
(2000) showed that Se involved in a regulation of spermatozoa maturation in the
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epididymis. They used 10 mature boars which were fed from weaning to 18 month
of age diets fortified with two levels of supplemental Se (0 or 0.5 ppm) or vitamin
E (0 or 220 IU/kg diet). The low-Se diet caused changes in spermatozoa: the
mitochondria in the tail midpiece were more oval with wider gaps between
organelles and the plasma membrane connection to the tail midpiece was not
tightly bound as when boars were fed Se. Furthermore, sperm ATP concentration
was decreased and percentage of immature spermatozoa with cytoplasmic droplets
increased when boars were fed the low-Se diet (Marin-Guzman et al, 2000). It
seems likely that, Se has a role in establishing the number of boar spermatozoal
reserves and Sertoli cells (Table 5.2). For example, when boars diet was
supplemented with Se 0 or 0.5 ppm for 18 months, testicular sperm reserves were
higher in boars fed on the high Se diet (Marin-Guzman et al., 2000a). In addition,
the boars fed dietary Se had also a greater number of Sertoli cells and round
spermatids at 6.2 month of age and by 18 month of age they also had more
secondary spermatocytes. It is well known that both Se and vitamin E are involved
in a regulation of animal reproduction. However, it seems likely that low Se in the
diet had a greater detrimental effect on semen quality that diets inadequate in
vitamin E. In particular, boars fed the nonfortified Se diets had sperm with lower
motility and a higher percentage of sperm cells with bent and shoehook tails
(Marin-Guzman et al., 1997). Therefore Se-supplementation improved sperm
motility and prevented its decline over the 16 week collection period and the
percentage of normal sperm was approximately 3-fold higher when the Se-fortified
diet was fed to boars (Mahan et al., 2002). At the same time the semen from boars
fed the nonfortified Se diet had a lower fertilisation rate of oocytes with fewer
accessory sperm penetrating the zona pellucida.
Did you know that low Se in the diet had a greater detrimental
effect on semen quality than diets inadequate in vitamin E
Positive effect of Se on semen quality was also shown with rams. For example,
thirty-three 8-month-old ram lambs were kept at grass and fed a supplement of
barley and peas, with ad libitum access to grass silage when grazing became
restricted. On day 0, the rams were allocated to two groups by restricted
randomisation of live weight. One group had a zinc, cobalt and selenium soluble
glass bolus administered with the other group not receiving a bolus to act as a
control (Kendall et al., 2000). Semen was collected once a week between days 44
and 86. The bolused lambs had a significantly increased erythrocyte GSH-Px on
all samplings after bolusing and had significant increases in motility, proportion
of live sperm and proportion of intact membranes.
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Table 5.2 Effect of Se on boar (adapted from Mahan et al., 2002)
Item
Dietary selenium
0.0
0.5
Liver Se, mg/kg

Liver GSH-Px, U/g
Testis Se, mg/kg
Testis GSH-Px, U/g
0.54
2.3
0.30
1.19
1.15
13.9
0.80
1.24
Semen
Volume, ml
Sperm concentration, no. x 109
Total sperm, no. x109
GSH-Px, U/ml
Se, mg/kg
160
290
43.5
33
0.031
163
253
43.5
71
0.134
Seminal plasma
Se, mg/kg
GSH-Px, U/ml
0.024
12.3
0.060
37.7
Sperm
Se, mg/kg
GSH-Px, U/g
0.42
579
0.94
977
Sperm production/g testis, no x 106

5.4 mo
6.2 mo
9 mo
18 mo
39.4
65.9
64.0
92.4
50.7
73.0
89.8
163.8
Semen quality
ATP concentration, nmoles ATP/106 spermatozoa
Sperm motility, %
Normal sperm, %
Fertilization rate, % of eggs
Accessory sperm, no./oocyte
1.15
60.4
24.2
73
14
1.55
87.9
61.9
99
60
Did you know that Se supplementation of infertile men

could help improving semen quality
Evidence has been provided indicating that Se supplementation enhances the in
vitro motility and oxygen uptake of human sperm in sub-fertile males (MacPherson
et al., 1993; Scott et al., 1998). In particular, 69 subfertile patients were recruited
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in Scotland and received placebo, selenium alone or selenium plus vitamins A, C

and E daily for 3 months. Selenium treatment significantly increased plasma
selenium concentrations and sperm motility but sperm density was unaffected.
Five men (11%) achieved paternity in the treatment group, in contrast to none in
the placebo group (Scott et al., 1998). In order to verify the hypothesis that
selenium and vitamin E could improve male fertility, nine
oligoasthenoteratozoospermic men were supplemented for a period of 6 months
with Se and vitamin E. Compared to the baseline period (pre-supplementation) of
4 months, statistically significant increases were observed for Se and vitamin E
levels, sperm motility, percent live, and percent normal spermatozoa. These
improvements are likely to be supplementation-dependent, since all of the
parameters returned to baseline values during the post-treatment period (Vezina
et al., 1996). Similarly, subfertile men in Russia were given 3 courses of selenium
(3.5 g/kg/day for 30 days) with a positive effects on semen quality (Nikolaev et
al., 1999). In Tunisia 28 men were supplemented daily by vitamin E (400 mg)
and selenium (225 g), during 3 months. Vitamin E and selenium supplementation
produced a significant decrease in MDA concentrations and an improvement of
sperm motility (Keskes-Ammar et al., 2003). Recently a significantly positive
correlation was observed between human semen concentration of Se and sperm
density, as well as sperm count, sperm motility and viability (Xu et al., 2001).
Moreover, 8-OHdG levels in sperm DNA inversely correlated with semen Se
concentrations in fertile and infertile subjects (Xu et al., 2003).
Results of Se supplementation depend on the diet used, background Se
concentration and some other factors. For example, Se concentration was measured
in 211 semen samples from 211 normozoospermic, oligozoospermic,
asthenozoospermic, and azoospermic men in Singapore (Roy et al., 1990). The
authors did not find any significant correlation between Se level in the seminal
plasma and sperm count or motility. Eleven healthy men were fed a controlled
diet of foods naturally high or low in selenium for 120 days while confined in a
metabolic research unit (Hawkes and Turek, 2001). Dietary selenium was 47 g/
d for the first 21 days, then either 13 g /d or 297 g /d for 99 days, resulting in
significant changes in selenium concentrations in blood and semen. Seminal
plasma selenium concentration increased 50% with high selenium and decreased
40% with low selenium. The fraction of motile sperm in the high-selenium group
decreased by 32% by week 13 and ended 18% lower than baseline (Hawkes and
Turek, 2001). In another study conducted in Poland, 33 subfertile men were
supplemented for 12 weeks with 200 micrograms Se/day in the form of yeast-rich
Se or sodium selenite (Iwanier and Zachara, 1995). The supplementation of
subfertile men with yeast-rich Se showed a more pronounced effect on Se
concentrations and GSH-Px activities in blood components and seminal fluid than
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selenite did. However, Se supplementation did not improve the spermatozoal

quality characteristics of sperm count, motility and, morphology. Furthermore,
effects of dietary -tocopheryl acetate (50 or 0 mg/kg diet) and selenium (Se, 0 or
0.5 ppm) supplementation on motion characteristics, oxidative stability and
fertilizing ability of rabbit spermatozoa were studied (Castellini et al., 2002).
Dietary Se increased GSH-Px activity in erythrocytes, seminal plasma and
spermatozoa, however, the semen quality parameters were not affected.
It is important to stress that the reproductive system of males is quite sensitive
to selenium excess, especially when selenite is used as a source of supplemental
selenium. For example, ingestion of 2 ppm and 4 ppm selenium by the house rat,
Rattus rattus, for 5 weeks caused a dose-dependent reduction in its body weight,
testicular and cauda epididymis weights, concentration, motility and percentage
of live spermatozoa with a simultaneous increase in the percentage of their
abnormal forms (Kaur and Parshad, 1994). Similarly, adult male albino rats were
fed 6 and 8 ppm Se in diet for 6 and 9 weeks. Excess of dietary Se caused dosetime-dependent reduction in body weight and reproductive organ weights but
increase in number of morphologically abnormal spermatozoa (Kaur and Kaur,
2000). Se-rich diets caused also dose-time-dependent reduction in tubular diameter,
epithelial height, number of spermatogenic cells and disintegration of cellular
associations in the seminiferous tubules of testes along with reduction in the
diameter of cauda epididymal tubules and pseudostratification of their epithelial
lining (Kaur and Kaur, 2000).
In this respect a choice of Se sources in the male diet is of great importance.
Recently it has been shown that Se is assimilated from organic sources much
more efficiently compared to commonly used selenite (Mahan, 1999). This can
be translated into higher Se accumulation in the animal tissues and building a
selenium reserve, which can be effectively used in stress conditions. For example,
replacing selenite by Se-Yeast and simultaneously increasing vitamin E
supplementation from 30 to 60 ppm was associated with: (Jacyno et al., 2002;
Figure 5.9-5.11):
improved boar libido,

increased concentration and total number of spermatozoa in an ejaculate,
decreased percentage of semen with morphological changes and
increased value of osmotic resistance test of acrosome membranes
Did you know that organic selenium can improve semen
quality in boars and cockerels
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300

30
Inorganic Se
Organic Se + vitamin E
No. of sperm (nx109)
25
20
15
10
5
0
Summer
Winter
Figure 5.9 Selenium source and sperm number in boars (adapted from Jacyno et al, 2002).
250
Inorganic Se
No. of sperm (nx106/cm3)
Organic Se + vitamin E
200
150
100
50
0
Summer
Winter
Figure 5.10 Selenium source and boar sperm concentration (adapted from Jacyno et al, 2002).
50
Inorganic Se
Organic Se
40
30
20
10
0
Summer
Winter
Figure 5.11 Selenium source and defective sperm in boars (adapted from Jacyno et al, 2002).
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301
More detailed studies on comparison between organic and inorganic selenium in

the diet of cockerels were conducted at the North Carolina State University. In
particular, Edens (2002) showed that, when cockerels were fed on a basal diet
containing 0.28 ppm Se without additional dietary supplementation of this trace
element, the percentage of normal spermatozoa was only 57.9% (Table 5.3) and
two major abnormalities seen were bent midpiece (18.7%) and corkscrew head
(15.4%). When this diet was supplemented with an additional 0.2 ppm Se in the
form of selenite, the percentage of normal spermatozoa increased to 89.4% and
abnormalities in the form of bent midpiece and corkscrew head were decreased
down to 6.2 and 1.8% respectively. However, when organic selenium was included
in the cockerels diet in the same amount, semen quality was further improved
and those abnormalities decreased down to 0.7 and 0.2% and the percentage of
normal spermatozoa increased up to 98.7%. These results clearly showed that the
form of dietary Se supplementation is a crucial factor of its efficiency, with organic
selenium being much more effective in comparison to selenite. Therefore Se
deficiency is associated with midpiece damage to spermatozoa (Figure 5.12). It is
clear that the midpiece of spermatozoa of the Se-deficient male is broken. In such
conditions sperm motility and fertilising capacity would be compromised.
Table 5.3 Effect of selenite or Sel-Plex on spermatozoal abnormalities (%) in semen from cockerels
(adapted from Edens, 2002).
Sperm morphology
Basal diet
Selenite
Sel-Plex
Normal sperm
Bent midpiece
Swollen midpiece
Ruptured midpiece
Swollen head
Corkscrew head
Coiled
Fragment/other
57.9
18.7
1.6
0.9
1.3
15.4
3.2
1.0
89.4
6.2
0.4
0.1
0.2
1.8
0.8
1.1
98.7
0.7
0.1
0.0
0.2
0.2
0.0
0.1
Did you know that Se deficiency is associated with midpiece

damage in spermatozoa
Additional data from Edens (2002) indicated that selenomethionine (0.3 ppm
from 21 weeks of age) in the diet of Hubbard roosters improved semen quality to
a greater extent than achieved by selenite at the same dose (Tables 5.4-5.5). The
sperm quality index significantly increased as well as the percentage of normal
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302
Figure 5.12 Image analysis of normal and Se-deficient chicken spermatozoa (adapted from Surai, 2002).
Table 5.4 Effect of selenium on semen quality (Hubbard Ultra-Yield Roosters (adapted from Edens,
2002)).
Variable
Selenite, 0.3 ppm

1st collection 2nd collection
Sperm Quality Index

Normal Sperm, %
Dead Sperm,%
Bent Midpiece, %
Corkscrew, %
Other, %
254
91.6
1.3
3.8
1.3
2.0
254
85.4
1.9
6.2
3.2
3.3
Sel-Plex, 0.3 ppm

1st collection 2nd collection
300
98.4
0.4
0.5
0.4
0.2
300
97.7
0.6
0.8
0.6
0.4
Table 5.5 Effects of sodium selenite or selenomethionine of productive parameters of Hubbard breeder
hens (adapted from Edens, 2002).
Farm 1
Variable
Egg production, %
Fertility, %
Daily settable eggs
Hatchability, %
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Farm 2
Selenite
Sel-Plex
Selenite
Sel-Plex
82.87
96.12
8799
83.10
85.46
96.68
9111
84.40
84.22
96.23
8379
82.40
84.74
97.26
8647
82.64
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303
spermatozoa. At the same time, the proportions of various abnormalities in semen

decreased. There was also a positive effect on fertility, which was improved by
0.56-1.03% by Sel-Plex dietary supplementation. These experimental results
confirmed the importance of selenium in maintaining chicken semen quality and
specifically showed the advantages of organic selenium. Therefore organic
selenium can improve fertility and, more importantly, increase a duration of fertility
(Agate et al., 2000). Preliminary observations in female chickens have also
revealed the effectiveness of dietary supplementation with vitamin E, organic
selenium or both to sustain fertility in aging flocks (Breque et al., 2003). This was
probably due to organic selenium effect on antioxidant system in sperm storage
tubular (SST, Surai et al., 1998). One of the unique features of avian reproduction
is the storage of spermatozoa within oviducal sperm storage tubular which unable
the hen to produce fertile eggs during the fertile period of 1-6 weeks, depending
on the species. Thus avian spermatozoa might be expected to have systems which
will maintain stability throughout this period. Indeed, recent results have confirmed
the existence of a complex antioxidant system in the utero-vaginal portion of the
fowl oviduct (Breque and Brillard, 2002). In particular GSH-Px activity in the
utero-vaginal junction was 12-fold higher than in the liver.
Considering antioxidant-prooxidant balance in mammalian and avian semen
it is necessary to take into account that very low and controlled concentrations of
ROS participate in signal transduction mechanisms in the spermatozoa (De
Lamirande et al., 1997). In low concentrations they act as mediators of normal
sperm function, including capacitation (Storey, 1997), whereas whenever they
produced in excess, they prove highly toxic to the cell (Griveau and Le Lannou,
1997) and are related to male infertility (De Lamirande et al., 1997). The data of
Dalvit et al. (1998) indicating adverse effect of inclusion of high vitamin E
concentrations in the capacitation medium confirm these suggestions. It seems
likely that sulfhydryl-disulfide status may be important for the regulation of sperm
capacitation (de Lamirande and Gagnon, 1998) and as mentioned above GSH-Px
and TR are involved in regulation of this balance. Therefore selenoprotein
expression in spermatozoa and seminal plasma are considered to be a natural
way of regulating sperm functions.
Did you know that in low concentrations ROS act as mediators
of normal sperm function, including capacitation
The results presented above clearly showed the importance of selenium in male
nutrition. In particular, Se supplementation of the male diet is needed to maintain
sperm membrane integrity during in vitro sperm manipulation including artificial
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insemination as well as during sperm transport and storage (avian species) in the
female reproductive tract. However, the form of dietary selenium is a key for
success in optimising Se supplementation. In fact organic selenium can help
maintaining reproductive performance in farm animals and poultry. The general
relationship between selenium and male fertility is shown in Figure 5.13.
Se in the feed
Biosynthesis of >25 selenoproteins
Testes: classical GSH-Px,

PH-GSH-Px,
selenoprotein P,
other selenoproteins
Mild Se deficiency: Se is preferentially retained in testis

Progressive deficiency: morphological alterations of
spermatids and spermatozoa
Extreme deficiency: complete disappearance of
mature germinal cells
Spermatozoa:
High levels of PUFAs (20:4n-6, 22:4n-6,22:5n-3, 22:6n-3) +

GSH-Px, PH-GSH-Px, mitochondrial capsule selenoprotein
Stress conditions of sperm

manipulation (dilution,
storage, deep freezing) and
free radical production
H2O2+Fe = OH*
O2*+SOD=H2O2 (toxic) + GSH-Px=H2O
ROO* +AO (vit.E) = ROOH (toxic)

ROOH +GSH-Px = ROH (nontoxic)
Second line of antioxidant defence
First line of antioxidant defence

Lipid peroxidation
Sperm membrane damage
Sperm function compromised
Fertilizing capacity decreased
Figure 5.13 Selenium and male fertility (adapted from Surai, 2002).
Conclusions
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Mammalian and avian spermatozoa are rich in PUFAs that make them
vulnerable to lipid peroxidation which is considered as an important
mechanism of impaired sperm quality and reduced fertilizing ability.
Antioxidant systems of semen play an important role protecting spermatozoa
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305
membranes against the damaging effects of free radicals and toxic products
of their metabolism.
GSH-Px and other selenoproteins are considered to be the major antioxidant
protection in mammalian and avian semen
Organic selenium supplementation is shown to be effective means in
improving semen quality
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6
SELENIUM AND MYCOTOXINS
Prevention is better than cure
Introduction
Silent killers, invisible thieves, unavoidable contaminants, natural toxicants all these names have been given to a group of fungal metabolites called mycotoxins
and their negative effects on animal production are incalculable. In general
mycotoxins are considered to be unavoidable contaminants in foods and feeds
and are a major problem all over the world (Wood, 1992). Interest in these naturally
occurring chemical compounds is intense due to their detrimental effect on human
health (carcinogenicity) and animal productive and reproductive traits. More than
300 mycotoxins have been shown to induce signs of toxicity in mammalian and
avian species (Fink-Gremmels, 1999; Leeson et al., 1995) and this number is
increasing. It has been estimated that 25% of the worlds crop production is
contaminated with mycotoxins (Fink-Gremmels, 1999). The most significant
mycotoxins in naturally-contaminated foods and feeds are aflatoxins, ochratoxins,
zearalenone, T-2 toxin, vomitoxin and fumonisins (Devegowda et al., 1998). In
many cases these mycotoxins can be found in combination in contaminated feed.
Mycotoxins appear at different stages of grain production. For example,
Fusarium species are known to invade grains during the growth of the plant and
they produce so-called field mycotoxins. On the other hand, Aspergillus and
Penicillium species generally develop during grain storage and so may be called
storage mycotoxins. This simple classification tends to over-simplify the situation.
However, two facts are clear: mycotoxin contamination depends on moisture
content of grain which should be less than 15% and drought stress can also increase
fungal contamination of grain. In practice, a range of mycotoxins can be found in
contaminated feeds, the type and level depending on climatic and storage
conditions. Temperate climates with high moisture conditions, e.g. Canada, USA
and Europe, encourage the growth of Fusarium and Penicillium species, producing
DON, zearalenone, ochratoxin A and T-2 toxin that are of concern for human
health. On the other hand, warm and humid climatic conditions, e.g. Latin America,
Asian countries and some parts of Australia, are ideal for the growth of Aspergillus
and the production of aflatoxin, considered to be a carcinogen. The winter season
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in these countries favours the development of zearalenone, DON, T-2 toxin,

ochratoxin A, etc. Worldwide trade in feed ingredients leads to a wide distribution
of the mycotoxins.
Among all mycotoxins, those from Fusarium species are considered to be
important contaminants of poultry feed. Trichothecenes, zearalenone, fumonisins,
moniliformin and fusaric acid are the major Fusarium mycotoxins occurring on a
worldwide basis in cereal grains, animal feeds and forages (DMello et al., 1999).
Furthermore, the trichothecene mycotoxins themselves comprise a vast group of
over 100 fungal compounds with the same basic structure (Leeson et al., 1995).
Acute mycotoxicosis outbreaks are rare events in modern poultry production,
however, low mycotoxin doses which very often are not detected are responsible
for reduced efficiency of production and increased susceptibility to infectious
disease. Biochemical changes in mycotoxicosis vary greatly and lipid peroxidation
and membrane disruption, apoptosis and compromised synthesis of DNA/protein
are regarded as the most important consequences of mycotoxicosis (Surai, 2002).
In this reagard, immunotoxicity is considered to be the most common consequence
of major mycotoxicosis (Bondy and Pestka, 2000).
Mycotoxins of concern
Three major economically important genera of mycotoxin-producing fungi are
Aspergillus, Penicillium and Fusarium. It is necessary to take into account that
there are many different species of each of the mentioned genera. For example,
Aspergillus species comprise a group of more than 150 members. About 45 of
them, 75 Penicillium species and 25 Fusarium species are known to produce
mycotoxins. Furthermore, mycotoxins can be toxic at very low concentrations.
When 19 mycotoxins were injected to chicken embryos, the maximum toxic effect
was exerted by T-2 toxin, which produced 100% embryonic mortality at doses as
low as 0.01mg.
The most significant mycotoxins in feeds are:
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Aflatoxins (Task Force Report, 2003):

- Aflatoxins are highly oxygenated secondary metabolites produced by
certain toxigenic strains of Asprgillus flavus and Aspergillus parasiticus
growing on a variety of feedstuff, including maize, wheat, rice and
cottonseed (Jayashree and Subramanyam, 2000).
- There are four naturally occurring aflatoxins B1, B2, G1 and G2 and a
range of their active metabolites with AFB1 being the most toxic and
carcinogenic compound of this group of mycotoxins (Smith, 1997).
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- They are unavoidable natural contaminants produced by specific moulds

that invade a number of feedstuffs and basic foods.
- They affect a range of avian (chicken, turkey, duck, pheasant, quail)
and mammalian (pigs, cattle, sheep, dog, cat, monkey and human) species
as well as fish
- This group of mycotoxins possesses high carcinogenic, teratogenic,
mutagenic and immunosuppressive activities (Ellis et al., 1991).
- Molecular mechanisms of cellular damages caused by AFB1 have not
been fully elucidated. Nevertheless, lipid peroxidation is one of the main
manifestations of oxidative damage and has been found to play an
important role in the toxicity of many different compounds, including
mycotoxins.
Did you know that aflatoxin was discovered as the causative
agent in Turkey X disease that killed 100,000 turkeys
in the UK in 1960?
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Ochratoxin A (Task Force Report, 2003):

- The widespread mycotoxin OA is a secondary fungal metabolite
produced by Penicillium and Aspergillus species of fungi during the
storage of cereals, cereal products and other plant-derived products
(Petzinger and Ziegler, 2000); and as a result it is found in various
compounded feeds.
- OA contamination can be found in cereal grains (wheat, barley, corn,
oats) and dry beans
- OA affects pig, chicken, duck, dog, rat and human
- In poultry, a number of outbreaks of acute ochratoxicosis have been
reported from the United States (for review see Fink-Gremmels, 1999).
- Ochratoxin is immunosuppressive, genotoxic, teratogenic and
carcinogenic to monogastric animals.
- There is a number of toxic effects of OA, the most prominent being
nephrotoxicity (Baudrimont et al., 1994).
- Feed contamination with OA typically results in increased mortality, poor
feed conversion, poor growth rates and feed refusal. Post mortem
examination revealed various detrimental changes including renal
carcinogenity (Gautier et al., 2001) and hepatotoxicity (Huff et al., 1988).
- Three distinct mechanisms of OA toxicity have been proposed by
Marquardt and Frohlich (1992) and they include:
- Competition with phenylalanine in the phenylalanine-tRNA
aminoacylation reaction and as a result inhibition of protein synthesis;
- Alteration of mitochondrial functions and inhibition of ATP synthesis;
- Stimulation of lipid peroxidation.
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- Among mycotoxins studied, OA is probably characterised as the one with
the most pronounced pro-oxidant properties. Several studies have reported
that OA induces oxidative stress.
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T-2 toxin (Task Force Report, 2003):

- The trichothecene group of mycotoxins accounts for over one hundred
fungal metabolites, of which T-2 toxin , produced by the Fusarium fungus,
was the first to be studied (Leeson et al., 1995; Bondy and Pestka, 2000).
- It contaminates corn, wheat, barley and oat
- It affects a range of animal species including swine, cattle, chicken, turkey,
horse, dog, cat, mouse, rat and human
- The adverse effects of trichothecene toxins on animal health is expressed in
a diverse range of symptoms including skin lesions, immunosuppression,
hepatotoxicity, nephrotoxicity, neurotoxicity, genotoxicity and even death
(Hollinger and Ekperigin, 1999).
- The damage caused by trichothecene mycotoxins results primarily from
the toxins interruption of cell division in bone marrow, immunocompetent
organs and intestinal mucosa (Bondy and Pestka, 2000), resulting in a serious
immunosuppressive effect (Bondy and Pestka, 2000; Pitt, 2000).
- T-2 toxin also has a strong inhibitory effect on protein synthesis, which in
turn results in the inhibition of DNA and RNA synthesis (Leeson et al.,
1995).
- The mechanisms of T-2 toxicity include effects on other metabolic functions
of the body. For example, the chemical structure of T-2 toxin makes it fat
soluble, which allows it to be incorporated into cell membranes, potentially
changing membrane structural and functional properties (Coulombe, 1993).
- Lipid peroxidation by T-2 toxin in the liver has also been identified as an
important underlying mechanism of T-2 toxin-induced cell injury (Leeson
et al., 1995; Hoehler and Marquardt, 1996) and DNA damage (Atroshi et
al., 1997). Therefore, oxidative damage caused by T-2 toxin may be one of
the underlying mechanisms for T-2 toxin-induced cell injury and DNA
damage, which eventually lead to tumourigenesis.
Vomitoxin (Deoxynivalenol, DON) (Rotter et al., 1996; Task Force Report,

2003):
- DON is produced world-wide by the Fusarium genus (Fusarium
graminearum and Fusarium culmorum)
- DON contaminates different cereal crops (wheat, maize and barley) used
for food and feed production.
- It is one of the least acutely toxic trichothecenes
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- The main toxic effect is associated with inhibition of protein synthesis via
binding to the ribosome.
- DON in moderate to low doses can cause a number of detrimental effects
associated with reduced performance and immune function
- The main effect at low dietary doses is a reduction in food consumption
(anorexia), while higher doses induce vomiting (emesis).
- It alters brain neurochemicals
- Animals fed low to moderate doses are able to recover from initial weight
losses, while higher doses induce more long-term changes in feeding
behaviour.
- Swine are more sensitive to DON than mice, poultry, and ruminants, with
males being more sensitive than females.
It seems likely that DON widely contaminates feed ingredients. For
example, wheat and barley from the 1993 crop year in the USA (630
samples collected in 25 states) were analysed for DON. The mycotoxin
contamination in the 483 wheat samples averaged 2.0 g/g and ranged
from < 0.5 to 18 g/g . DON contamination in the 147 barley samples
averaged 4.2 g/g and ranged from < 0.5 to 26 g/g. About 40% for the
wheat samples and 57% of the barley samples contained DON levels
that were greater than the U.S. Food and Drug Administration 1982
advisory level of 2 g/g for DON in wheat designated for milling (human
consumption; Trucksess et al., 1995). In the period September 1998January 2000, the average DON concentration in wheat was 0.446 g/g
(n= 219) in The Netherlands (Pieters et al., 2002).
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Fumonisin B1 (Task Force Report, 2003)

- The fumonisins are the most recently discovered family of
aminopolycarboxylic acids produced predominantly by Fusarium
moniliforme, but also by F. proliferatum and F. napiforme.
- Fumonisins are detected mainly in maize and maize-based diets
- Several different derivatives of this mycotoxin have been described of
which FB1 is recognised as the most toxic.
- Toxic effects of FB1 are associated with the fact that it resembles the
structure of cellular sphingolipids and therefore impairs ceramide
synthesis by inhibiting ceramide synthetase (Fink-Gremmels, 1999).
- FB1 is highly toxic and has been shown to be responsible for some
major toxicological effects in animals, including pigs and equine species
but has comparatively low toxicity for poultry (Leeson et al., 1995). In
particular, FB1 causes porcine pulmonary oedema with severe lung
oedema and hydrothorax and equine leukoencephalomalacia.
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Zearalenone (Zea) (Gajecki, 2002; Task Force Report, 2003):

- It contaminates corn, hay and pelleted commercial feed
- It affects swine, dairy cattle, chicken, turkey, lamb, rat, mouse and guinea
pigs
- Zea causes a variety of toxic effects in both experimental animals and
livestock, and possibly, in humans.
- It is a stable compound, both during storage/milling and the processing/
cooking of food
- It is fairly rapidly absorbed following oral administration
- It competitively binds to estrogen receptors in a number of in vitro systems
and in the uterus, mammary gland, liver and hypothalamus of different
species.
- Zea alters various immunological parameters
- It causes alterations in the reproductive tract of laboratory animals and
domestic animals causing decreased fertility, increased embryolethal
resorptions, reduced litter size, changed weight of adrenal, thyroid and
pituitary glands and change in serum levels of progesterone and estradiol
- Teratogenic effects of Zea were found in pigs and sheep.
World-wide, the most economic losses are due to the aflatoxins, followed by
ochratoxin A. A summary of the main effects of mycotoxins on poultry are shown
in Table 6.1.
In general, lipid peroxidation and stimulation of apoptosis seemed to be
important mechanisms of mycotoxin action. The wide range of mycotoxins that
can contaminate poultry feed and their different chemical compositions make
protection against mycotoxin-related toxicity a difficult task. There are several
problems that complicate mycotoxin prevention issues:
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In many cases, the low levels of mycotoxins remain undetected in feed

ingredients and their effects may also go unseen. For example, a decrease
in hatchability by 0.5% would be difficult to notice. Detrimental effects on
the immune system would be even more difficult to assess. However, the
immunosuppressive activities of the mycotoxins become of particular
concern in EU countries after a ban on feed-grade antibiotics.
Very often, a combination of various mycotoxins is present in the feed
because the various fungal species can produce several toxins. A
combination of several mycotoxins in low doses can have a bigger
detrimental effect than a single mycotoxin at a higher dose.
Mycotoxins can contaminate practically all feed ingredients. For example,
Fusarium species have been found in wheat, maize, barley, oats and rye.
On the other hand, aflatoxins can also contaminate oilseeds and other feed
ingredients.
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323
Table 6.1 Effects of common mycotoxins (adapted from Yaroshenko et al., 2003).
Mycotoxin
Effects on poultry
Aflatoxin B1
Ochratoxin A
Fumonisin B1
T-2 toxin, DON
Zearalenone
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Decreased performance
Reduced egg production and hatchability
Decreased serum proteins
Increased liver and kidney weight
Liver and kidney lesions
Decreased semen volume and testes weights
Disruption of germinal epithelium
Decreased hatchability
Enlarged, fatty livers and enlarged spleens
Reduced weight gain and feed consumption
Impaired feed efficiency
Reduced egg production
Reduced egg quality
Induced immunosuppression
Kidney lesions
Rickets like deformities in chickens with leg weakness
Hepatocellular hyperplasia
Increased kidney and proventriculus weights
Liver lesions
Immunotoxicity
Reductions in feed consumption and weight gain
Severe oral lesions
Abnormal behaviour
Altered feathering
Decreased resistance to pathogens
Decreased egg production
Impaired shell quality
Decreased egg production
There are no safe doses of mycotoxins. A dose that does not affect animal
at short exposure could be toxic at longer consumption. Doses that may be
safe under laboratory conditions can have detrimental effects on growth
and reproduction under conditions of commercial poultry production.
International trade of feed ingredients, e.g. maize and soybeans, especially
long shipments from Latin America to European and Asian countries, is
another important risk factor.
Most of mycotoxins are stable compounds that do not degrade during
storage, milling or high-temperature feed manufacturing processes.
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324

Did you know that adverse effects of various mycotoxins include
decreased feed intake and compromised production
characteristics of farm animals and poultry; immunosuppression and increased susceptibility to diseases; poor
reproductive performance and damages to various organs?
Increased lipid peroxidation as a consequence of mycotoxicoses

A delicate balance between antioxidants and pro-oxidants in the body in general
and specifically in the cell is responsible for regulation of various metabolic
pathways leading to maintenance of immunocompetence, growth and development
and protection against stress conditions associated with commercial poultry
production (Surai, 2002). This balance can be regulated by dietary antioxidants,
including vitamin E (Surai et al., 1999), carotenoids (Surai and Speake, 1998;
Surai et al., 2001) and selenium (Surai, 2000; 2002). On the other hand, nutritional
stress factors have a negative impact on this antioxidant/pro-oxidant balance. In
this respect mycotoxins are considered to be among most important feed-born
stress factors. It is not clear at present if mycotoxins stimulate lipid peroxidation
directly by enhancing free radical production or the increased tissue susceptibility
to lipid peroxidation is a result of compromised antioxidant system. It seems likely
that both processes are involved in this stimulation. In most cases lipid peroxidation
in tissues caused by mycotoxins was associated with decreased concentrations of
natural antioxidants.
As illustrated in Table 6.2, OA has a stimulating effect on lipid peroxidation. In
most of cases, thiobarbituric acid reactive substances (TBARS) accumulation was
used as a measurement of lipid peroxidation. Furthermore, ethane exhalation,
EPR registered free radicals, hydroxyl radical formation, single-strand cleavage
DNA, DNA adduct formation as well as LDH release were also used to confirm
pro-oxidant properties of OA. Various in vitro and in vivo systems were also used
including liver microsomes, phospholipid vesicles, primary cell cultures, whole
organs and whole body.
T-2 toxin was also shown to have pro-oxidant properties (Table 6.3). Those
properties were confirmed with rat, mouse and quail liver tissue and yeast. TBARS
accumulation was a method of choice for most of the studies, however, data on
conjugate diene formation and DNA fragmentation also showed those effects.
Effect of AFB1 on lipid peroxidation has been studied in rat liver and kidney as
well as in cultured hepatocytes and in an in vitro model system (Table 6.4). Similar
to the examples above, TBARS accumulation was substantially increased as well
as conjugate diene production. At the same time GSH concentration and activities
of antioxidant enzymes significantly declined as a result of AFB1 action.
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1/8/2007, 6:52 PM
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325
Rat liver microsomes
Rats
Rats
Phospholipid vesicles
OA in vitro
OA in feed
OA in feed
OA in vitro
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DNA adduct formation

TBARS
OA in vitro
OA in feed
OA in feed
OA in vitro
Rats
Rat kidney microsomes
Rat liver
TBARS
A model oxidation system OH* single-strand
cleavage DNA
Astrocytes and neurones
TBARS , LDH release
Mouse and rat kidney
DNA adduct formation
OA in diet
OA in vitro
TBARS
Vero cells in culture
OA in vitro
Ethane exhalation
MDA in serum,
liver, kidney
TBARS ,
oxygen uptake
Free radical generation
(EPR)
TBARS
Chicken liver
TBARS
Hoehler and Marquardt, 1996;

Hoehler et al., 1997
Baudrimont et al., 1997,
1997a
Gillman et al., 1999
Omar et el., 1990
Rahimtula et al., 1988,

Gautier et al., 2001;
Khan et al., 1989
Rahmitula et al., 1988
Meki and Hussein, 2001
References
Belmadani et al., 1999

Retinol, ascorbic acid, Grosse et al., 1997
vitamin E
Aspartame
Creppy et al., 1998
Gautier et al., 2001
SOD, catalase
aspartame
-
Vitamin E
Protective effect of
antioxidants
Lipid peroxidation
measurement
OA in feed
OA and its analogs Bacillus brevis bacteria
Tissue
Mycotoxin
Table 6.2 Stimulation of lipid peroxidation by ochratoxin A

325
326
Table 6.3 Stimulation of lipid peroxidation by T-2 toxin
Mycotoxin
Tissue
Lipid
Protective
peroxidation effect of
measurement antioxidants
References
T-2 in feed
Rat liver
TBARS
T-2 in feed
T-2 in feed
T-2 in feed
Rat liver nuclei TBARS

Mouse liver
TBARS -
Mouse
MDA in liver
after 24-48 h
after supplementation
Mouse liver
DNA fragmentation
Quail liver
TBARS
Rat tissues
Conjugate
diene
Chicken
Liver MDA
Chicken, duck, Liver MDA
goose
Tsuchida et al., 1984;

Suneja et al., 1989;
Schuster et al., 1987;
Rizzo et al., 1994
Ahmed and Ram, 1986
Karppanen et al., 1989
Vila et al., 2002
T-2 in feed
T-2 in feed
T-2 in feed
T-2 in feed
T-2 in feed
Se, ascorbic
acid, vitamin E
CoQ10 and
vitamin E
Lycopene
Atroshi et al., 1997

Dvorska and Surai, 2001
Chang and Mar, 1988
Leal et al., 1999
Mezes et al., 1999
FB1 also stimulated lipid peroxidation in rat liver, rat liver nuclei fraction, primary
rat hepatocytes, Vero cells in culture and PC bilayers. In those systems TBARS
accumulation and DNA strand breaks were increased (Table 6.5). DON increased
TBARS formation in rat and mice liver and decreased GSH in rat brain and spleen.
There are also data available indicating pro-oxidant properties of zearalenone
(Karagezyan et al., 1995; Ghedira-Chekir et al., 1999) and citrinin (Ribeiro et al.,
1997). Aurofusarin is shown to decrease antioxidant defences and stimulate lipid
peroxidation in quail egg and tissues of newly hatched quail (Dvorska et al.,
2002; 2003; Dvorska and Surai, 2004).
It is clear from the data that mycotoxins strongly promote lipid peroxidation in
various in vitro and in vivo systems. This effect was obvious no matter which
measurement was used to assess the process of lipid peroxidation.
Mycotoxins and apoptosis

The maintenance of tissue homeostasis involves the removal of superfluous and
damaged cells. This process is often referred to as programmed cell death or
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1/8/2007, 6:52 PM
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327
Rat liver
Rat testis
Cultured rat hepatocytes
Rat liver
Cultured primary rat

hepatocytes
Rat liver
AFB1 in feed
AFB1 in feed
AFB1
AFB1 in diet
AFB1 in vitro
AFB1
intraperitoneally
Aflatoxin B1 in feed Rat liver
AFB1
Rat liver and kidney
intraperitoneally
AFB1
Rat liver
intraperitoneally
AFB1 in vitro
Primary rat hepatocytes
Rat liver
Rat liver
AFB1 in feed
AFB1 in feed
Tissue
Mycotoxin
Table 6.4 Stimulation of lipid peroxidation by aflatoxin
Shen et al., 1994;
Rastogi et al., 2001a

Yang et al., 2000
Se, vitamin E
Picroliv
-
GSH-Px
TBARS
GSH, SOD, Catalase,
GSH-Px
TBARS , GSH ,
ROS generation
Choi et al., 1995

Rastogi et al., 2001
Souza et al., 1999
Vitamin E, ternatin
TBARS , ROS
formation
TBARS
MDA , NO
MDA
TBARS , LDH release
TBARS
Se , vitamin E
TBARS ,
conjugated dienes
MDA , NO
References
melatonin El-Gibaly et al., 2003

loaded chitosan
melatonin
Meki et al., 2001
Vitamin E
Verma and Nair, 2001
SOD, catalase
Shen et al., 1995
Semecarpus anaPremalatha et al., 1997
cardium nut extract
Silvia miltorrhiza extract
Liu et al., 1999
antioxidants
Lipid peroxidation
measurement
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327
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328
Rat liver nuclei
Primary rat hepatocytes

Rat liver
Phosphatidylcholine
bilayers
Vero cells
Glioma cells, mouse
fibroblasts
Macrophage cell line
Rat liver
FB1
FB1
FB1
FB1
FB1
FB1
Aurofusarin in feed Liver of newly hatched

quails
Zeralenone injected Rat liver
intravenuosly
Zeralenone in vitro Vero cells
Citrinin in feed
Rat liver
DON + 3-AcDON
Mice liver
in feed
Aurofusarin in feed Quail egg yolk
FB1
DON in feed
Tissue
Mycotoxin
Vitamin E
-
TBARS
TBARS
TBARS
TBARS
Se, ascorbic acid,

vitamin E
-
TBARS
TBARS
Rate of peroxidation ,
free radical formation ,
accelaration of chain
formation
TBARS
TBARS ,
DNA fragmentation
MDA
TBARS
Catalase, mannitol
TBARS , DNA strand

breaks
TBARS
TBARS
Vitamin E
-
antioxidants
Lipid peroxidation
measurement
Table 6.5 Stimulation of lipid peroxidation by some mycotoxins
1/8/2007, 6:52 PM
Ghedira-Chekir et al., 1999

Ribeiro et al., 1997
Dvorska et al., 2001,

Dvorska, 2001
Dvorska et al., 2002; Dvorska
and Surai, 2004
Karagezyan et al., 1995
Karppanen et al., 1989
Ferrante et al., 2002

Rizzo et al., 1994
Abado-Becognee et al., 1998

Mobio et al., 2003
Abel and Gelderblom, 1998

Abel and Gelderblom, 1998;
Lemmer et al., 1999
Yin et al., 1998
Sahu et al., 1998
References
328
329
apoptosis (Sastre et al., 1996) since it is thought that cells activate an intrinsic
death program contributing to their own demise. Several processes, such as
initiation of death signals at the plasma membrane, expression of pro-apoptotic
oncoproteins, activation of death proteases, endonucleases etc., ultimately coalesce
to a common irreversible execution phase leading to cell demise. A balance
between cell death and cell survival factors plays a major role in the decision
making process as to whether a cell should die or must live (Ray et al., 2000).
Apoptosis is distinguishable from necrosis. When cell death is induced by
osmotic, physical or chemical damage, early disruption of external and internal
membranes takes place with subsequent liberation of denatured proteins into the
cellular space and induction of an inflammatory response in the vicinity of the
dying cell (Sastre et al., 1996). In contrast, apoptosis is characterised by cell
shrinkage, nuclear pyknosis, chromatin condensation, DNA cleavage into
fragments of regular sizes and activation of the cystein-proteases called caspases
(Dare et al., 2001). Reactive oxygen species are thought to play a major role in
apoptosis (Hockenbery et al., 1993; Ratan et al., 1994), being involved in the
initiation as well as execution of apoptosis (Herdener et al., 2000). GSH depletion
increases the percentage of apoptotic cells in a given population; and increased
GSH concentration is shown to decrease the percentage of apoptosis in fibroblasts
(Sastre et al., 1996). In fact, GSH depletion sensitised cells for intracellular
induction of apoptosis (Zucker and Bauer, 1997). Therefore, a decrease in GSH
concentration, or an increase in GSSG concentration or perhaps a change in the
ratio of GSH/GSSG constitutes a trigger for apoptosis (Beaver and Waring, 1995).
For example, ROS from mitochondria can cause apoptosis after GSH depletion
(Zucker et al., 1997). Therefore, apoptosis is induced by oxidative damage either
directly from oxygen free radicals or hydrogen peroxide or from their generation
in cells by injurious agents. For example, H2O2 is considered a common mediator
for the apoptosis induced by various anticancer drugs (Simizu et al., 1998). In
line with those findings there are data showing protective effects of catalase and
SOD from different inducers of apoptosis (Sandstrom and Buttke, 1993; Herdener
et al., 2000). Indeed, intracellular induction of apoptosis depends on ROS
production and can be efficiently blocked by antioxidants (Langer et al., 1996;
Schaefer et al., 1995).
In many cases mycotoxins decreased cellular level of GSH, which can trigger
apoptosis. In general, T-2 toxin is a most potent apoptotic agent. However, there
are also reports indicating apoptosis caused by FB1, OA and AFB1 (for review
see Surai, 2002; Surai and Dvorska, 2005). For example, based on the DNA
fragmentation profile in gel electrophoresis and the morphological changes in
electron microscopy, the induction of apoptotic nuclear changes by various
mycotoxins was investigated in HL-60 human promyelotic leukemia cells (Ueno
et al., 1995). The results showed that T-2 toxin, nivalenol, deoxynivalenol, OA,
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330
citrinin, AFB1 and some other mycotoxins were positive for the induction of
DNA fragmentation. However, fumonisin B1 was not active in DNA fragmentation.
The toxicity of the mycotoxins nivalenol (NIV), DON and FB1 were studied in
the K562 human erythroleukemia cell line (Minervini et al., 2004). Morphological
evidence of apoptosis was related to the toxicity of the mycotoxins and more
toxic NIV and DON resulted in more late stage apoptotic events than FB1. The
results suggested that DNA damage and apoptosis rather than plasma membrane
damage and necrosis may be responsible for the observed cytotoxicity. Fumonisin
B1, T-2 toxin, Fusarenon-X and Deoxynivalenol could induce apoptosis of liver
cell, kidney cell, gastrointestinal epithelial cell, immunological cells as well as
several cell lines of human and animals. Furthermore the rank order of the potency
of trichothecene mycotoxins to induce internucleosomal DNA fragmentation was
found to be T-2, satratoxin G, roridin A >> diacetoxyscirpenol > baccharin B-5 >>
nivalenol, deoxynivalenol, 3-acetyldeoxynivalenol, fusarenon-X, baccharin B-4
vehicle control (Nagase et al., 2001). The apoptosis-stimulating effect of FB1 has
been clearly demonstrated and stimulation of lipid peroxidation by FB1 and
decreased antioxidant concentrations including GSH in tissues could lead to
changes in redox status of the cell and trigger a cascade of apoptotic changes.
In general, apoptosis is considered a common mechanism of toxicity of various
mycotoxins. Since antioxidant-prooxidant balance in the cell (redox status) is
responsible for regulation of apoptosis it seems likely that selenoproteins such as
GSH-Px, TR and MSR could be potentially involved in prevention of mycotoxinrelated apioptosis. Therefore, Se status of the animals could be an important factor
in their resistance to mycotoxicoses.
Mycotoxins and immune system

The clinical toxicological syndromes caused by ingestion of mycotoxins have
been characterised in domestic animals, poultry and laboratory animals and range
from acute mortality to decreased production and immunosuppression. In fact,
consumption of some mycotoxins, at levels that do not cause overt clinical
mycotoxicosis, suppress immune functions and may decrease resistance to
infectious disease (Corrier, 1991). The effects of several mycotoxins on the immune
responses have been investigated (Table 6.6-6.10). However, most data were
obtained with laboratory animals and only in some instances, farm animals and
cells derived from livestock species have been employed to evaluate the
immunotoxicity of mycotoxins (Sharma, 1993; Bondy and Pestka, 2000).
Did you know that immunosuppression is a common result of
mycotoxin contamination of the feed?
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331
Immunosupressive potency of various mycotoxins differs substantially. Effects

of DON, 3-acetyldeoxynivalenol, fusarenon-X, T-2 toxin, zearalenone, alphazearalenol, beta-zearalenol and nivalenol on T and B cells in a proliferation assay,
antibody-dependent cellular cytotoxicity NK cell activity on human peripheral
blood mononuclear cells were studied (Berek et al., 2001). The mycotoxin
concentrations used in the experiments were comparable with those, which can
be found in normal human peripheral blood system (0.2-1800 ng/ml). Among the
mycotoxins tested T-2 toxin, fusarenon X, nivalenol and DON showed the highest
immunosuppressing effect in vitro. In particular, mycotoxin-induced
immunosuppression was related to depressed T or B lymphocyte activity.
Furthermore, they also inhibited NK cell activity (Berek et al., 2001).
As can be seem from Table 6.6 immunosuppression caused by aflatoxin B1
has been demonstrated in various livestock species (e.g., chickens, turkeys, pigs
and lambs) and also in laboratory animals (mice and rats) and in various in vitro
systems. Aflatoxin is an immunomodulating agent that acts primarily on cellmediated immunity and phagocytic cell function. Therefore AFB1 mainly
decreases lymphocyte functions and may also affect macrophages assisting
lymphocyte functions. The immunomodulatory effects of ochratoxins have also
been considered for many years and a summary is shown in Table 6.7. OA has
been shown to affect mainly humoral immune function at the level of antibody
synthesis in chickens and rats and mice. However, number and phagocytic activity
of macrophages were also decreased in growing gilts receiving OA for 35 days
(Harvey et al, 1992). IL production was also compromised. It has been
demonstrated that exposure of purified human lymphocyte populations and
subpopulations to the toxin will abrogate the cells ability to respond to activating
stimuli in vitro (Lea et al., 1989). Thus, both IL-2 production and IL-2 receptor
expression of activated T lymphocytes were severely impaired. The results strongly
suggest that the toxin caused its immunosuppression through interference with
essential processes in cell metabolism (Lea et al., 1989). In particular, OA appears
to suppress NK cell activity by inhibiting production of basal interferon (Luster et
al., 1987).
Fumonisin toxicity has been characterized relatively recently in comparison to
aflatoxin and ochratoxin (Table 6.8), and fumonisin-induced immunotoxicity is
an area of active research (Bondy and Pestka, 2000). In fact, FB1 has diverse
effects on the immune system, causing both stimulation and suppression of the
response to foreign antigens, and apparently inducing an antigenic response to
FB1. Similar to FB1, the trichothecenes can both suppress and stimulate immune
function (Table 6.9). Most of research on T-2 toxin effects on immunity was
performed with laboratory animals and only a few publications addressing
immunomodulating properties of T-2 toxin in farm animals. Furthermore, the
molecular basis of immune function modulation by mycotoxins remains mainly
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332
Table 6.6 Effects of aflatoxin B1 on immunity
Species
Dose
Effects on immune system
Reference
Broiler
chicks
Broiler
chicks
1 ppm,
7-49 days
2.5 mg/kg
diet for 21
days
2.5 mg/kg
diet for 21
days
AB after vaccination against

NK
Peripheral T-lymphocyte
counts, splenic plasma cell
counts
Percentage and mean of
phagocytic activities;
immune response measured
by HI test
In progeny, anti-Brucella
abortus AB in 5 mg/kg AF
group; phagocytosis of SRBC
and ROS production by macrophages from AF progeny
chicks in 5 mg/kg AF group
Secondary AB production
against IBD
Cell-mediated immunity as
measured by a delayed hypersensitive skin test ; humoral
immunity;development of
the acquired immunity to ND
or fowl cholera vaccination
Total complement activity
Shivachandra et al., 2003
Broiler
chicks
Broiler
breeder
hens
0.2; 1; 5 or
10 mg/kg
Broiler
chicks
Broiler
chicks
5 ppm
Broiler
chickens
2.5 g/g of
feed for 42
days
2.5 g/g diet
from hatching
to 4 weeks of
age
Chickens
Chickens
400 ppb of
AFB1+AFB2
for 5 weeks
0.3 ppm
from 0 to 6
weeks
14-day-old 200 ppb

turkeys and
chicks
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332
Celik et al., 2000
Ibrahim et al., 2000
Qureshi et al., 1998
Okotie-Eboh et al., 1997

Giambrone et al., 1985a
Stewart et al., 1985
Giambrone et al., 1978
(graft-versus-host reaction) ;
DTH skin reactions to
tuberculin ; Humoral immunity
(AB to rabbit red blood) ;
serum IgG and IgA; IgM
Cell mediated immune
Kadian et al., 1988
response (delayed type hypersensitivity reaction) suppressed
at all the three periods tested
(30, 45 and 60 days of age).
Giambrone et al., 1985
measured by a delayed hypersensitive skin test ; humoral
immunity; development of
the acquired immunity to ND
or fowl cholera vaccination
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333
Table 6.6 Contd.
Species
Dose
Reference
4-month-old
wild
turkeys
Chick
embryo
100-400g/kg
feed for 2 wk
Lymphoblast transformation
Quist et al., 2000
6-day chick
embryos
6-day chick
embryos
Weanling
piglets
Weaned pigs
Pigs
Female
lambs
White-tailed
deer
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1.09-17.4 g/g
Mitotic index B cells ;
Potchinsky and Bloom,
embryo at day 18 Mitotic index T cells;
1993
of incubation
sister chromatid exchanges in
B lymphocytes (up to 8-fold);
sister chromatid exchanges in
T lymphocytes (up to 2 fold)
0.1, 0.5, and
Macrophage recruited in the
Neldon-Ortiz and
1 g/per embryo peritoneal cavity after i.p.
Qureshi, 1992
Sephadex elicitation, substrate
adherence potential of peritoneal
exudate cells at 1 mg AFB1;
macrophage phagocytic
potential at 0.5-1.0 mg AFB1
0.1 g
Incidence of sister chromatid
Dietert et al., 1985
exchanges in blood cells
(5-fold); cell-mediated immunity
(graft vs host and cutaneous
basophil hypersensitivity
reactions)
140-280 ppb,
AF decreased proinflammatory Marin et al., 2002
4 weeks
(IL-1beta, TNF-alpha) and
increased anti-inflammatory
(IL-10) cytokine mRNA expression
140, or 280 ppb, Skin thickness (PHA skin test) van Heugten et al., 1994
for 3 weeks
linearly with increasing dietary
AB1
300 or 500 mg/ The humoral immune response Panangala et al., 1986
kg of feed
to Erysipelothrix rhusiopathiae,
measured by enzyme-linked
immunosorbent assay; the
proliferative responses of
lymphocytes to mitogens;
complement titers; serum
immunoglobulin G and M values
2 ppm for 37 d
Response to intradermal
Fernandez et al., 2000
injection of PHA
0.8 ppm for 8
Lymphocyte proliferation and Quist et al., 1997
weeks
delayed type hypersensitivity
reactions
333
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334
Table 6.6 Contd.

Species
Dose
Weanling
rats
60, 300 and

600 g /kg BW
Reference
Cell mediated immunity, as

Raisuddin et al., 1993
measured by DTH response
assay, at the 300 and 600 g
dose levels
Weanling
350 and
Population and phagocytic
Raisuddin et al., 1990
rats
700 g/kgBW
capacity of macrophages
Rats and
Aerosol inhalAlveolar macrophage
Jakab et al., 1994
mice
ation or intraphagocytosis at 16.8 g/kg
tracheal instillwith the effect persisting for
ation to AFB1
approximately 2 weeks
Mice
0.03, 0.145 or
[3H]thymidine uptake in
Reddy et al., 1987
0.70 mg/kg orally lymphocyte cultures;
every other day Synthesis of DNA in mixed
for 2 weeks
lymphocyte; primary antibody
production by splenic cells,
from animals challenged with
SRBC; DTH reaction to
hemocyanin
Murine
10 or 50 M
NO production stimulated by
Moon et al., 1998
peritoneal
LPS, mediated by the reduction
macrophages
of iNOS activity, mRNA, and
protein
Human
0.1-1 pg/ml
Phagocytosis and microbicidal Cusumano et al., 1996
monocytes
activity, intrinsic antiviral
activity or superoxide
production
Rat Kupffer 0.01 pg/ml
Phagocytosis, intracellular
Cusumano et al., 1995
cells in vitro
killing of Candida albicans,
intrinsic anti-Herpes virus
activity
Table 6.7 Effect of Ochratoxin A on immunity
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Species
Dose
Reference
Broiler
chicks
2 ppm
Santin et al., 2002
Chicks
5 ppm
Broiler
chicks
130, 305 and

790 ppb
AB after vaccination against

NK , mitotic cells in the
Bursa
Weight of lymphoid organs,
humoral immune responce
HI antibody titers to vaccine
against ND
334
Stoev et al., 2002

Stoev et al., 2000
1/8/2007, 6:52 PM
335
Table 6.7 Contd.
Species
Dose
Broiler
chicks
Up to 4 ppm for
20 days from
hatch
0.5-8g/g from
day-old to 3
weeks of age
Reference
Immunoglobulin-containing
Dwivedi and Burns, 1984
cells in lymphoid organs;
total Ig levels at 2-4 ppm
Broiler
Total circulating lymphocytes Chang et al., 1979
chickens
of blood ; monocytes at
2.0 g/g; circulating
heterophils
Salmonella- 3.0 mg/kg
PHA- and ConA-stimulated
Elissalde et al., 1994
challenged
blastogenesis. Salmonella
broiler
typhimurium alone had no
chicks
affect on the variables measured
Growing
2.5 mg/kg of
Cutaneous basophil hyperHarvey et al., 1992
gilts
feed for 35 days sensitivity response to PHA,
delayed hypersensitivity to
tuberculin, stimulation index
for lymphoblastogenesis,
interleukin-2 production when
lymphocytes were stimulated
with concanavalin A, number and
phagocytic activity of
macrophages
Rats
Gavage with 0,
Thickening of the basement
Dortant et al., 2001
0.07, 0.34 or
membrane and reduction in
1.68 mg OTA/kg splenic T-cell fraction. IgG at
body weight for 0.34-1.68 mg/kg OTA
4 weeks
Rats
Dams exposure The proliferative response of
Thuvander et al., 1996b
to a single dose splenocytes to the T-cell
of OA (0, 10, 50 mitogen Con A in pups from
or 250 g/kg
dams given 10 or 50 g OA/kg
BW) on day 11
body weight; proliferation of
of lactation
thymocytes in response to Con
A in pups from dams exposed
to 50 g OA/kg body weight
Mice
Dams, OA, 200
In offspring on days 14 and 28 Thuvander et al., 1996
g/kg before and postpartum percentages of
during gestation splenic CD4+ and CD8+ cells
Mice
Dams exposure Proliferation of splenic and
Thuvander et al., 1996a
to OA (500g/kg thymic lymphocytes in
BW) on day 16
response to mitogens in the
of gestation
pups at 15 days of age;
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Table 6.7 Contd.
Species
Dose
Mice (contd)
percentages of mature CD4+

cells and percentages of
immature, double-positive
(CD4+CD8+) cells in the
exposed pups
Dams exposure Proliferative responsiveness
Thuvander et al., 1996a
to OA (500g/kg of lymphocytes in the
BW) on day 10 offspring when stimulated
postpartum
with B or T cell mitogens
3 days after the exposure
250 or 2600
After 28 days, AB production
Thuvander et al., 1995
g/kg diet, 28
and plague-forming cells ;
days
decrease in thymocyte cell
counts in the 250-g/kg
group; after 90 days, proportion
of mature CD4+ or CD8+ cells.
0.005 g/kg BW Immune response to SRBC
Haubeck et al., 1981
Mice
Mice
Mice
Reference
Table 6.8 Effects of Fumonisins on immunity
Species
Dose
White
Leghorn
chicks
6.1, 10.5, and

42.7 ppm from
day 7 to 42
Reference
Splenic, thymic, and liver

Qureshi et al., 1995
weights, normalized for body
weight, bursa of Fabricius;
total Ig and IgG; Macrophage
phagocytic activity; NK cell
activity
Turkey
75 mg/kg feed
Primary immune response to
Weibking et al., 1994
poults
and AB1, 200
SRBC (combination of FB1
g/kg feed, from and AF); PHA delayed hyperday 1 to 21
sensitivity response
Weaned pigs 1-100 mg/animal AB tires , lymphocyte
Tornyos et al., 2003
/day
stimulation by PHA, ConA,
LPS
Rat
Injected at 7.5 or Thymus weight, serum
Bondy et al., 1995
10 mg/kg BW/
immunoglobulin M;circulating
day for 4 days
phagocytic cell numbers
Mice
50 (LD), FM1+
After challenge with T. cruzi,
Dresden et al., 2002
FM2, 6 weeks
NO production by MG after
14 days
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Table 6.8 Contd.
Species
Dose
Reference
Mice
Five daily s.c.

injections of
2.25 mg/kg/day
of FB1
Johnson and Sharma,

2001
Mice
I.p. with SRBC,

at 5- 100 g
I.p daily, at
1 to 50 g
Relative spleen and thymus

weights in F, no effect on
organ weights in M; splenic
cellularity and the basal rate
of lymphocyte proliferation
in F, PHA-induced T-lymphocyte and LPS-induced
B-lymphocyte proliferation
in F, expression of IL-2 mRNA
in Splenocytes in F
Number of plaque-forming cells
produced against SRBC
4 to 12-fold increase in the
number of of plaque-forming
cells after SRBC injection
Membrane fluidity , membrane
peroxidative damage
LPS-induced production of NO
and PGE2- at 0.1-10M
Mice
Macrophage 1-10M
cell line
Murine
1-100 M
macrophage
cell line
Murine
1, 10, and
cell
100 M
macrophage
Rat splenic 1-100 g/ml
macrophages
and lymphocytes
Martinova et al., 1995

Martinova et al., 1995
Ferrante et al., 2002

Meli et al., 2000
LPS-induced
NO production
Rotter and Oh, 1996
NO production; ConA induced proliferation of

splenic cells in the presence
of the nitric oxide synthase
inhibitor
Dombrink-Kurtzman et
al.,. 2000
Table 6.9 Effects of T-2 toxin on immunity
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Species
Dose
Reference
Young
poults
7-week-old
pigs
Up to 1 ppm
for 32 days
0.5, 1.0, 2.0 or
3.0 mg/kg diet
for 3 weeks
AB to antigens
Sklan et al., 2003
337
Leucocyte count; proportion Rafai et al., 1995

of T lymphocytes; antibody
formation; blastogenic
transformation of lymphocytes
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Table 6.9 Contd.
Species
Dose
Reference
Aerosol, 390 g/L Lymphocyte count , neutrophil Pang et al., 1988

count ; mitogen-induced (Con A,
PHA, and PWM) blastogenic
responses of peripheral blood
mononuclear cells and hemagglutination titers to SRBC
9-10-week 15 mg/kg
The responses peripheral
Pang et al., 1987
old pigs
blood mononuclear cells to
mitogens (ConA, PHA and
PWM) at early (3 to 5 days) and
late (20 to 28 days) postdosing
intervals; HA titer to SRBC
Calves
0.6 mg/kg/day
Lymphocyte responses to
Buening et al., 1982
mitogens; chemotaxic migration
of neutrophils
Calves
0.6 mg/kg per
IgM and IgA ; IgG
Mann et al, 1982
day for 43 days
Monkeys
Gastric intubation Leucocyte counts at the end
Jagadeesan et al., 1982
at a 100 g/kg
of wk 4; bactericidal activity
BW/day for
of neutrophils, T-cell number ;
4-5 wk
lymphocyte transformation,
B-cell number; IgG and IgM
levels
Rats
2 ppm
In progeny, one day after birth Lafarge-Frayssinet et al.,
the cortex was atrophic while
1990
the medulla was proliferative.
In the spleen, both B and T cells
were impaired and their
responsiveness to PHA and LPS
decreased
Mice
3 mg/kg BW
Virulence of both E. coli and
Cooray and Jonsson,
by gavage
S. aureus
1990
Mice
1 mg/kg every
Markedly larger and more
Tai and Pestka, 1990
challenged other day for
bacterial-related lesions in the
with S.
10 d
spleens, kidneys, and livers than
typhimurium
animals challenged with S.
typhimurium alone
Mice
20 ng
Immune response to the THolt and DeLoach, 1988
dependent antigen sheep RBC
Mice
1.5 or 3.0 ppm
T-lymphocyte-dependent
Schiefer et al., 1987
in the diet for
humoral immunity was not
16 months
affected
9-10-weekold pigs
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Table 6.9 Contd.
Species
Dose
Mice
4.0 mg/ kg of
exposed to BW
Listeria
infection
Mice
20 ppm for
1- 4 weeks
Human
1 -5 ng/ml
lymphocytes
Reference
Growth of Listeria; mortality

because of listeriosis
Corrier and Ziprin, 1986
Spleen proliferative responses

to Con A;
50% inhibition in cell
proliferation
Friend et al., 1983

Meky et al., 2001
Table 6.10 Effects of DON on immunity
Species
Dose
Growing
pigs
0.6, 1.8 and

4.7 mg/kg
Young pigs
Mice
Mice
Mice
Mice
Weanling
mice
Weanling
mice
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Reference
Secondary antibody response

to tetanus toxoid dosedependently
0.75- 3.00 mg/kg Alpha-globulin levels, and
diet for 28 days antibody titers to SRBC
5 or 25 mg/kg
mRNAs for the proinflammatory
BW
cytokines IL-1beta, IL-6, and
TNF-alpha; the T helper 1
cytokines IFN-gamma and IL-2;
and the T helper 2 cytokines
IL-4 and IL-10
25 ppm for 8 or Serum IgA and microhematuria
24 weeks
index after 4 to 8 weeks and
continued to increase with
further vomitoxin exposure
25 ppm for 4
Total IgA; total IgG and IgM
and 8 wk
in serum
10-100 ppm in
Serum levels of anti-SRBC
the diet
antibodies ; stimulation of B
and T cells by mitogens
0.5- 25.0 ppm
Total circulating white blood
over 8 wk
cells;PMN neutrophils;
serum IgM; IgA
0.75, 2.5 or
Serum IgM to SRBC; plaque7.5 mg/kg BW
forming cell numbers
by gavage
339
Overnes et al., 1997
Rotter et al., 1994

Zhou et al., 1997
Dong and Pestka, 1993
Rasooly ands Pestka,

1992
Robbana-Barnat et al.,
1988
Forsell et al., 1986
Tryphonas et al., 1984
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340
Table 6.10 Contd.
Species
Dose
Murine
0.1 ng/mlperitoneal 1 g/ml
macrophages
Lymphocytes 0.005in vitro

100 ng/ml
Human
216 ng/ml
lymphocytes
Murine
25-100 ng/ml
macrophage
cell line
Reference
Phagocytosis, microbicidal
Ayral et al., 1992
activity; superoxide anion
production at 1 ng/ml; phagosome-lysosome fusion at
100 ng/nl
PHA-induced lymphocyte
Miller and Atkinson,
proliferation at 0.005- 0.5 ng 1986
DON/ml; at 50-100 ng/ml
lymphocyte proliferation
50% inhibition in cell
Meky et al., 2001
proliferation
Ji et al., 1998
Production of H2O2 after LPS
stimulation, but at 250 ng/m;
NO production at 25-250 ng/ml
unknown and continue to be a frontier for future research (Bondy and Pestka,
2000). It seems likely that trichothecenes are potent immunosuppressive agents
that directly affect immune cells and modify immune responses because of tissue
damage elsewhere. Immunomodulating properties of DON have been studied
mainly with rodents (Table 6.10). The evidence is quickly accumulating showing
that DON can be immunosuppressive or immunostimulatory, depending upon
the dose and duration of exposure. While immunosuppression is probably related
to the inhibition of translation, immunostimulation can be a result of interference
with various cellular regulatory mechanisms (Rotter et al., 1996).
Mycotoxin-induced immunosuppression is related to both natural and adaptive
immunity (Table 6.11):
depressed T or B lymphocyte activity

depressed NK cell activity
suppressed immunoglobulin and antibody production
reduced complement or interferon activity
impaired macrophage functions.
The sensitivity of the immune system to mycotoxin-induced immunosuppression

arises from the vulnerability of the continually proliferating and differentiating
cells that participate in immunomediated activities and regulate the complex
communication network between cellular and humoral components (Corrier, 1991).
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Table 6.11 Summary of effects of selected mycotoxins on the immune system (adapted from
Yaroshenko et al., 2003)
Mycotoxin
Aflatoxin B1
Ochratoxin A
Fumonisins
T-2 toxin
DON

- Immune system is compromised; detrimental effects on cell-mediated
immunity and phagocytic cell functions including decreased peripheral
T-lymphocyte counts
- Decreased antibody response to injected sheep red blood cells and
decreased weight of the Bursa of Fabricius and thymus
- Aflatoxin carry-over via the egg to the embryo compromises immune
system of the progeny, including cell-mediated, humoral immunity and
phagocytic functions. Progeny are more susceptible to various
pathogens.
- Inhibition of humoral, cellular and innate immune responses
- Cellular depletion of lymphoid organs
- Depressed delayed hypersensitivity responses
- Depressed blood monocyte phagocytic activity
- Increased susceptibility to infectious agents
- Depressed antibody responses to various mitogens and decreased serum
globulins
- Diffuse thymic cortical thinning, mild bursal follicular atrophy and
mild splenic lymphocyte depletion
- Decreased peritoneal macrophage numbers and compromised
phagocytic functional activity
- Reduced white blood cell counts
- Increased susceptibility to a range of pathogens
- Inhibition of lymphocyte proliferation and NK cell activity
- Alteration in interleukin metabolism
- Inhibition of lymphocyte proliferation
- Alteration in interleukin metabolism
- Inhibition of NK cell activity
In fact, high levels of polyunsaturated fatty acids in the immune cells and presence
of sensitive receptors on their surface make them an important target for free
radical attack (Surai, 2002). As mentioned above, oxidative stress caused by
mycotoxin consumption could be responsible for breaking effective
communications between immune cells which ultimately would misregulate
immune system and cause immunosuppression. Although the molecular basis for
many of the specific immunosuppressive effects of mycotoxins are presently
unclear, inhibition of DNA, RNA and protein synthesis via a variety of different
mechanisms appears to be directly or indirectly responsible for the immunosuppressive action of many mycotoxins (Corrier, 1991). Furthermore, detrimental
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effects on antioxidant defences, stimulation of lipid peroxidation and apoptosis

and damages to immune cell receptors seem to be involved in immonomodulation
properties of mycotoxins.
Did you know that four major mechanisms of mycotoxin
action include:
inhibition of protein, DNA and DNA synthesis and DNA adduct
formation;
membrane structure alteration and induction of lipid peroxidation;
induction of programmed cell death (apoptosis)
gene expression alteration?
Protective effect of selenium against mycotoxicoses

The range of mycotoxins that can contaminate poultry feed and their different
chemical compositions make protection against mycotoxin-related toxicity a
difficult task. There are various approaches to control or combat mycotoxin
problems. The simplest strategy is based on the prevention of the formation of
mycotoxins in feeds by special management programmes including storage at
low moisture levels and prevention of grain damage during processing (Dawson,
2001). However, modern agronomic technology is not able to eliminate pre-harvest
infection of susceptible crops by fungi (Wood, 1992). Therefore this strategy can
only partially be effective; and in countries with warm and humid conditions, this
strategy could be quite costly.
Other strategies based on use microbial or thermal inactivation of toxins,
physical separation of contaminated feedstuffs, irradiation, ammoniation and
ozone degradation have not been developed to the industrial level because they
are either time-consuming or comparatively expensive (Dawson, 2001a). In recent
years, nutritional manipulation has been actively used to improve animal selfdefence against mycotoxins or to decrease detrimental consequences of mycotoxin
consumption.
Did you know that in order to prevent detrimental effects of
mycotoxicosis some farmers use mycotoxin adsorbents as an
integral part of the feed on regular base?
Since lipid peroxidation plays an important role in mycotoxin toxicity, a protective
effect of antioxidants is expected (Galvano et al., 2001; Surai, 2002). Indeed, in
several experiments with various animal species, protective effects of antioxidants
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against toxic effects of mycotoxins were observed. For example, Burguera et al.
(1983) indicated that selenium (Se) has a protective effect against AFB1 toxicity
in turkey poults. To investigate the biochemical mechanism of the protective effect
of dietary Se against aflatoxin toxicity, hepatic metabolism of AFB1 in turkey
poults was examined at various dietary Se concentrations (0.2, 2.0 or 4.0 ppm).
The experimental results provided clear evidence of Se-induced enhancement of
aflatoxin detoxification (Gregory and Edds, 1984). It was suggested that the
protective action of Se was not mediated by an increase in glutathione availability
for aflatoxin conjugation or by effects on the activities of these enzymes as
measured in vitro. Therefore dietary supplements such as Se are considered effective
in the reduction of aflatoxicosis in poultry (Dalvi, 1986). Recently the protective
antioxidant effect of organic Se in combination with vitamin E has been shown in
chicks exposed to cold stress and aflatoxin-contaminated feed (Stanley, 1998).
Similar protective effects of Se against aflatoxicosis have been shown with
mammalian species. Pigs fed a diet containing 2.5 mg Se/kg of feed were protected
from toxic effects of AFB1 (Davila et al., 1983). The effects were measured by
alteration of clinical responses and hematologic (prothrombin times),
electrophoretic and clinical chemistry values. Effects of a single intramuscular
injection of Se-vitamin E (5 mg of Se + 68 IU of -tocopherol/60 kg of BW) as a
pretreatment 14 days before an oral dose of AFB1 (1.0 mg/kg) were studied in 24
dairy calves. Although aflatoxin exposure caused a significant decrease in body
weight and feed intake, Se was demonstrated to interact significantly with AFB1
for feed intake, causing an improvement of this parameter (Brucato et al., 1986).
Milks et al. (1985) showed that Se is able to protect against the
hepatocarcinogenic effects of AFB1 in the rat. Moreover inhibitory activity of Se
on mutagenesis induced by AFB1 in the presence of a rat liver microsomal
activation system has been shown in Salmonella typhimurium tests (Francis et al.,
1988). The results of another experiment showed that Se could effectively protect
cells from AFB1 cytotoxicity in cultured cells but had no effect on aflatoxin B1DNA adduct formation or mutagenesis (Shi et al., 1995). In a different study, the
same authors reported that Se could effectively inhibit AFB1-induced DNA damage
(Shi et al., 1994).
It has been revealed that Se can inhibit the formation of hyperplastic foci and
enzyme-altered foci as well as hepatocarcinogenesis induced by AFB1, but Se
can neither prevent the enlargement nor accelerate the regression of the foci already
developed after administration of carcinogens (Wang, 1990). Therefore Lei et al.
(1990) concluded that Se had an inhibitory effect on the initiation and promotion
stages of AFB1-induced preneoplastic foci and nodules. Selenium also prevented
progression of these nodules to hepatocellular carcinoma even after cessation of
AFB1 administration. Additional experiments were conducted to verify the effect
of Se on the mutagenic activity of AFB1. After 14 days of Se administration to
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Chinese hamsters, the incidence of chromosomal aberrations in bone marrow

cells due to a single administration of AFB1 (5 mg /kg BW) was significantly
reduced (Petr et al., 1990). A significant decrease in the frequency of aberrant
cells, breaks and gaps was observed throughout the investigation.
A protective effect of Se is not restricted to aflatoxins, but is obvious with T-2
toxin as well. For example when male Wistar rats were fed diets supplemented
with Se (0.5 and 2.5 mg/kg) for 6 weeks, signs of intoxication from T-2 toxin
were less distinct and mortality caused by T-2 toxin was two times (Kravchenko
et al., 1990) or 3-5 times (Tutelyan et al., 1990) lower compared to the
unsupplemented group. The acute lethal toxicity of T-2 toxin was reduced by
administration of sodium selenite (Yazdanpanah et al., 1997).
In summary, Se is shown to have protective effects against T-2 toxicity (Rizzo
et al., 1994; Shokri et al., 2000; Kravchenko et al., 1990; Tutelyan et al., 1990;
Yazdanpanah et al., 1997), to decrease damaging effects of AFB1 (Shen et al.,
1994; Choi et al., 1995; Shi et al., 1994; Shi et al., 1995) and to prevent DON
toxicosis (Rizzo et al., 1994; Atroshi et al., 1997). A synthetic seleno-organic
compound (ebselen) showed a potent protective effect against AFB1-induced
cytotoxicity (Yang et al., 2000). Indeed, as can be seen from data presented in
Tables 6.1-6.4, protective effects against lipid peroxidation caused by mycotoxins
were attributed to various antioxidant compounds including vitamins A and E,
ascorbic acid, CoQ10, selenium, antioxidant enzymes as well as synthetic
antioxidants and various plant extracts (Surai, 2002).
In spite of positive effects of natural antioxidants on animals fed mycotoxin
contaminated diets, the most promising and practical approach has been the
addition of adsorbents to contaminated feed (Ledoux and Rottinghaus, 2000).
Mycotoxins can be bound to the adsorbent and pass harmlessly through the
digestive tract. Many compounds have been tested for adsorbent effects, however
comparatively few have proven successful and still fewer (mainly bentonites,
zeolites and, alumosilicates and glucomannans) are used commercially
(Devegowda et al., 1998). The extent to which various compounds bind specific
toxins varies considerably. Many products only bind aflatoxin, leaving such
mycotoxins as T-2 in the intestinal tract without alteration. In addition to the various
clays and zeolites, a yeast cell wall-derived glucomannan (Mycosorb) has been
shown to be effective against a wide range of mycotoxins (Devegowda et al.,
1998).
Mycotoxin binders can substantially improve antioxidant systems of the
mycotoxin-fed animals. This effect depends on the mycotoxin-binding activity
of adsorbents. For example, inclusion of zeolite in the quail diet at 3% had a
minor protective effect on the antioxidants in the quail liver, however changes
were not statistically significant (Figure 6.1; Dvorska and Surai, 2001). Only the
concentration of retinyl-linoleate in the liver of quail exposed to T-2 toxin
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simultaneously with zeolite was significantly higher compared with birds fed the
diet containing T-2 toxin alone. Therefore, zeolites alone were not effective in
prevention of toxic effects of T-2 toxin. These data are in agreement with
observations of Kubena et al. (1990; 1998) indicating absence of protective effects
of aluminosilicate sorbents against T-2 toxicosis. Superactivated charcoal
(Edrington et al., 1997) and organic sorbents (Bailey et al., 1998) were also
ineffective against T-2 toxicosis. Therefore, zeolite probably was not able to bind
a substantial amount of T-2 toxin in the digestive tract; and as a result did not
interfere with pro-oxidant properties of this mycotoxin.
25
alpha-tocopherol
gamma-tocopherol
carotenoids
ascorbate
20
15
10
0
Control
T-2
T-2+Zeolite
T-2+Mycosorb
Figure 6.1 Effect of T-2 toxin on antioxidant concentrations in quail liver, g/g tissue (Adapted from
Dvorska and Surai, 2001).
In marked contrast, inclusion of yeast glucomannans (Mycosorb) in T-2 toxincontaining diets fed quail significantly slowed the depletion of natural antioxidants
and vitamin A in the liver (Dvorska and Surai, 2001). This protective effect can
be attributed to the high adsorbent capability that esterified glucomannans have
for T-2 (Dawson, 2001). It could well be that mycotoxin binding by Mycosorb
also prevents T-2 toxin participation in development of oxidative stress in the
intestine. As a result, damage to the enterocytes is prevented thereby maintaining
effective antioxidant absorption, assimilation and delivery to the target tissues.
Did you know that a combination of several mycotoxins in low
doses could have higher detrimental effects on animal health
than one mycotoxin in higher doses?
It seems likely that consumption of mycotoxins could compromise Se metabolism
in animals. Therefore, a combined usage of adsorbents and antioxidants would
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show beneficial effects against mycotoxicosis. In fact, Se concentration in the

quail liver was significantly reduced due to T-2 toxin consumption (Figure 6.2;
Dvorska and Surai, unpublished). Mycosorb inclusion into the diet showed a
significant protective effect against Se depletion. However, a combination of
Mycosorb and Sel-Plex was shown even more effective. Mycosorb also showed
protective effects against vitamin E, carotenoids and reduced glutathione depletion
in quail liver due to T-2 toxin consumption. Furthermore, lipid peroxidation in
the liver of quail fed on T-2-supplemented diet was significantly reduced due to
Mycosorb inclusion in the diet and a combination of Mycosorb and Sel-Plex was
shown to give extra protection (Figure 6.3).
1000
900
800
700
600
500
400
300
200
100
0
Control
T-2 toxin
T-2+Mycosorb
T-2+Mycosorb
+ Sel-Plex
Figure 6.2 Effect T-2 toxin, Mycosorb and Sel-Plex on Se level in chicken liver, ng/g
160
140
120
100
80
60
40
20
0
Control
T-2 toxin
T-2+Mycosorb
T-2+Mycosorb
+ Sel-Plex
Figure 6.3 Effect T-2 toxin, Mycosorb and Sel-Plex on lipid peroxidation in chicken liver, (MDA, g/g)
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Did you know that a combination of mycotoxin adsorbents

with natural antioxidants (organic selenium) could
substantially improve protection against mycotoxicoses in
poultry production?
However, despite positive effects on the birds, inclusion of Mycosorb in the quail or
chicken diet was unable to completely prevent the adverse effects of T-2 toxin on the
antioxidant systems of the liver of the growing quail; indicating that not all T-2 toxin
was bound and released from the intestine. Therefore a combination of mycotoxin
binders with natural antioxidants, in particular with Se and vitamin E, could be the
next step in preventing damaging effects of mycotoxins on animals including poultry.
Conclusions
Recent results show that in many cases membrane-active properties of various
mycotoxins determine their toxicity. Indeed, incorporation of mycotoxins into
membrane structures causes various detrimental changes. These changes are associated
with alteration of fatty acid composition of the membrane structures and with
peroxidation of long chain PUFAs inside membranes. This ultimately damages
membrane receptors, causing alterations in second messenger systems; then to
inactivation of a range of membrane-binding enzymes responsible for regulation of
important pathways. Finally, this causes alterations in membrane permeability, flexibility
and other important characteristics determining membrane function. Detrimental effects
of mycotoxins on DNA, RNA and protein synthesis together with pro-apoptotic action
further compromise important metabolic pathways. Consequently, changes in
physiological functions including growth, development, reproduction etc. occur. An
importance of lipid peroxidation in all these processes is confirmed by protective
effects of natural antioxidants against mycotoxin toxicity. As a part of a range of
selenoproteins selenium is involved in prevention of lipid peroxidation, membrane
alteration and apoptosis and could decrease mycotoxin toxicity. However, protective
effects of antioxidants including selenium are of limited value and a combination of
mycotoxin binders with organic selenium could be the next step in preventing
damaging effects of mycotoxins in animal and poultry production.
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7
SELENIUM IN POULTRY NUTRITION
Dont count your chickens before they are hatched
Introduction
After long heated debates about eggs, cholesterol and human health it seems
likely that the storm has at last calmed and today we can say, An egg a day is
OK and start thinking about egg improvement. The last 20-40 years have been
associated with rapid progress in the poultry industry through out the world. Indeed
between 1961 and 2002, world chicken egg output increased more than threefold,
reaching 53.8 million tones and the output is predicted to grow further in the next
25-30 years. Similar achievements have been seen in poultry meat production
reaching 72.5 million tons in 2003. Indeed genetic progress, together with
substantial achievements in poultry nutrition, is responsible for those successes.
However increased productive parameters are often associated with a decrease of
reproductive characteristics and more importantly with increased susceptibility to
various stresses. Therefore an optimal balance of various antioxidants in the diet
is considered to be an important way forward in modern poultry nutrition. Among
many compounds with antioxidant activities found in poultry feed, selenium is
considered to be a chief executive of the antioxidant system regulating whole
antioxidant network in the body. Indeed, Se deficiency is related to decreased
productive and reproductive performances of poultry. Commercial poultry
production is associated with various stresses and Se as a part of various
selenoproteins can help maintaining antioxidant defences preventing damages to
various tissues. In accordance with NRC (1994) Se requirement is quite low,
however, those data are not related to commercial conditions and it seems likely
Se requirement for optimal poultry health is much higher. In fact,
immunomodulating properties of Se are shown with doses which are substantially
higher than those needed for chicken growth and development. For the last few
years a great body of information was accumulated indicating preferences of
organic Se in comparison to selenite or selenate. In fact, in poultry production
organic Se is proven to be an effective feed supplement to meet chicken requirement
in this element in various commercial conditions. Important features of Se
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metabolism and its practical applications in poultry will be presented in this chapter
with a special emphasis to optimal Se forms for the dietary supplementation.
Selenium deficiency
Se deficiency in the chicken, especially in combination with low vitamin E supply,
is responsible for the development of a range of diseases. Indeed, vitamin E and
selenium deficiency is related to many different conditions in farm and laboratory
animals where all major organs are affected (Table 2.20, Chapter 2).
In general selenium and selenium+vitamin E deficiency are associated with
development of a variety of pathological conditions, which target major body
systems including muscles, heart-vascular, reproductive and nervous systems.
Various animal species are shown to have different susceptibility to selenium
deficiency and different tissues show various degrees of severity upon a selenium
deficient diet. In general, some signs of selenium and Se+vitamin E deficiency
(nutritional muscle dystrophy) are similar in various species; others
(encephalomalacia) are species-specific.
Did you know that Se deficiency in poultry is associated with
decreased productive and reproductive performances and
development of various diseases targeting major body systems
including muscles, heart-vascular and nervous systems?
In many cases a combination of these two compounds is needed to achieve the
best effect in their treatment or prevention. In poultry Se- or Se + vitamin E
deficiencies are related to the development of:
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Exudative diathesis (Nouguchi et al., 1973; Barthlomew et al., 1998).

Among various selenium-related disorders and diseases exudative diathesis
(ED) is the most studied one. ED is the disease, which appears in chickens
deficient in both vitamin E and Se and is characterised by severe oedema as
a result of an increased permeability of the capillaries in combination with
reduced levels of blood proteins (Kristiansen, 1973). As a result of leakage
of blood fluid through the capillaries and from minor haemorrhages in
muscles, in the area of the breast under the skin an accumulation of an
exudate with a protein pattern similar to blood serum or plasma can be
seen.
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Hemoglobin degradation causes a bluish-green colour of the exudates, which

can be seen through the skin. Autopsy findings and histopathological lesions
were observed only in subcutaneous tissue and skeletal muscle. In particular,
the subcutaneous tissue was edematous with hyaline vascular lesions and
hemorrhages (Hassan et al, 1990). The thigh muscles were more susceptible
to deficiency lesions than were the breast muscles, and showed in acute
stages degenerative processes of the muscle fibers including calcium
deposits, vascular lesions and hemorrhages. In subacute and chronic cases,
reparative changes and muscle damage may develop independently of the
hyaline vasculosis (Hassan et al., 1990).
Did you know that exudative diathesis is considered to be
mainly dependent on Se status of chickens?
The condition can occur at any age but is most prevalent in young growing
chickens and turkeys (Whitehead and Portsmouth, 1989). In the chicks,
obtained from laying hens depleted of vitamin E and selenium exudative
diathesis was observed at hatching, indicating that the deficiency lesions
had developed during the embryonic period, whereas these signs were not
observed in chicks obtained from commercial laying hens adequate in
vitamin E and Se on the depletion diets until they were 2 weeks old (Hassan
et al., 1990). ED will only occur if the diet is deficient in selenium and Se is
200 times more effective than vitamin E in preventing this disease. Therefore
this symptom is primarily considered as a selenium deficiency (Machlin
and Gordon, 1962). ED is also described in ducklings (Dean and Combs,
1981). ED develops in chickens at age of 3-6 weeks and chicken mortality
can be as high as 80% (for review see Surai et al., 1994; Surai, 2002). ED
was associated with low levels of muscle Se, liver Se-GSH-Px and vitamin
E and was also accompanied by a simultaneous increase in the liver nonSe-GSH-Px (Hassan et al., 1990).
It has been suggested that the inflammatory response associated with a Se/
vitamin E deficiency in a chick may be responsible for ED (Bartholomew et
al., 1998). The authors suggested that cytokines produced by leukocytes
could be responsible for transparent fluid accumulation and hemorrhaging.
It is well recognised that Se is the major protection against ED. The vitamin
E supplemental level of 15 mg/kg was not adequate to provide a complete
protection against ED (Hassan et al., 1990). ED was inhibited by the rutin
and silymarin treatments, but exacerbated by quercetin, morin, and ferulic
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acid. Changes in concentrations of vitamin E in plasma, liver, or muscle,
caused by the various treatments (other than vitamin E), were not related to
protection against ED (Jenkins et al., 1992).
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Nutritional pancreatic atrophy (Thompson and Scott, 1969; 1970; Cantor

et al., 1975). It seems likely that nutritional pancreatic atrophy (NPA) is the
only clearly defined Se deficiency syndrome uncomplicated by deficiencies
of other antioxidants (Combs, 1994). For example, Se deficiency
uncomplicated by vitamin E deficiency was produced in chickens by an
amino acid diet complete in all known nutrients except Se (Noguchi et al.,
1973; Gries and Scott, 1972);
Nutritional Muscular Dystrophy (NMD) appears in chickens at about four

weeks of age, is a result of simultaneous deficiency of vitamin E and sulfur
amino acids (e.g. cysteine; Machlin and Gordon, 1962) and characterised
by degeneration of the muscle fibers of the breast and sometimes leg muscles.
An exact role of selenium deficiency in the development of NMD in chickens
needs further clarification. Histological examination revealed Zenkers
degeneration, with perivascular infiltration, and marked accumulation of
infiltrated eosinophils, lymphocytes and histocytes (Leeson and Summers,
2001). Light and electron microscopic study of affected muscles revealed
fibers with hyaline and granular degeneration (Van Vleet and Ferrans, 1976).
In hyalinized fibers, the initial ultrastructural alterations included increased
density of the sarcoplasm and myofibrils, dilatation of sarcoplasmic
reticulum, formation of subsarcolemmal vacuoles, and disruption of
mitochondrial membranes. In later stages, alterations in these fibers included
myofibrillar disruption and lysis, nuclear pyknosis and lysis, disruption of
the plasma membrane with persistence of basal lamina and scattered adhering
satellite cells, and eventual invasion by macrophages (Van Vleet and Ferrans,
1976). In fibers with granular degeneration, the ultrastructural observations
included decreased density of the sarcoplasm, prominent mitochondrial
swelling and distortion, and multiple foci of myofibrillar lysis that eventually
coalesced to produce generalized lysis (Van Vleet and Ferrans, 1976).
Ultrastructural deterioration of the myopathic muscle included disintegration
of blood vessel walls, transverse tubules, and mitochondrial membranes as
well as the obvious disruption of the myofibrillar components (Gill et al.,
1980). NMD is often developed in goslings and ducklings (Dvinskaya and
Shubin, 1986). Usually it starts at 3-5 weeks of age, but sometimes it can be
observed earlier. In chickens and turkey poults muscles of gizzard and crop
are affected and as a result feed remains in the crop and mortality is expected
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3-10 days after disease started. In turkey poults muscle degeneration is

registered at 5-30 days of age and turkey poult mortality could be as high
as 70% (for review see Surai et al., 1994). Myopathy of the gizzard and of
the heart in turkeys is also a result of vitamin E and Se deficiency. NMD is
most noticeable in breast and thigh muscles, which develop pale streaks of
dystrophic degenerated fibres. When ducklings from time of hatch were
fed on the peroxidized diet first signs of NMD were seen at day 10 and at
day 14 mortality started and reached 22.5% at day 20 of the postnatal
development (Shemet, 1977). In the gizzard and heart of died birds
degenerative changes were observed. After 30 days of the experimental
period NMD was registered more than in a half ducklings. There is a
possibility for the development of NMD during first 7-15 days of postnatal
growth if chickens are hatched from the eggs low in vitamin E and maternal
diet was high in peroxides (German, 1981). Peroxidative damage to
membranes has been hypothesized as a mechanism of tissue damage in
muscular dystrophy.
Did you know that Se deficiency in poultry is associated with
development of exudative diathesis, nutritional pancreatic
atrophy, nutritional muscular dystrophy and impaired
immunocompetence?
Lameness associated with skeletal muscular degeneration was reported in
ostrich chicks and flamingos (Vorster, 1984; Dierenfeld and Traber, 1993).
It is interesting that skeletal muscle degeneration, encephalomalacia,
exudative diathesis, and reduced hatchability and fertility have been reported
in exotic birds with vitamin E deficiency (Dierenfeld, 1989). Vitamin E
deficiency among captive avifauna was mainly associated with piscovorous
birds and raptors fed on meat or fish rich in PUFA and poor in vitamin E. In
such birds pathological lesions of adipose tissues were described including
accumulation of ceroid pigment, necrosis and steatitis as well as
degeneration of cardiac and skeletal muscles are primarily signs of vitamin
E deficiency (Dierenfeld, 1989). In zoo birds vitamin E deficiency can also
cause degeneration of the so called hatching muscle (musculus complexus)
in embryos that failed to pip (Dierenfeld and Traber, 1993).
The contents of - and -tocopherol and their oxidation products, the
tocopheryl quinones, were measured at 1 to 4 weeks after hatching in the
muscle and other tissues of chickens with inherited muscular dystrophy
(Murphy and Kehrer, 1989). The affected muscle (pectoralis major) of
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dystrophic birds contained significantly higher levels of -tocopheryl
quinone and a decreased ratio of - to -tocopherol. These changes in the
tocopherols are suggestive of oxidative stress in dystrophic muscle
membranes. A disturbance of Ca2+ active transport and an increase in the
membrane permeability for Ca2+ in the membranes of sarcoplasmic reticulum
of the dystrophic muscles have been shown (Kurskii et al., 1978). In
particular the level of ATP-dependent consumption of Ca2+ and activity of
Mg 2+,Ca2+ -ATPase decreased.
It has been suggested that the oxidative deterioration of proteins in nutritional
muscular dystrophy due to vitamin E deficiency is of great importance for
the development of NMD (Shih et al., 1977). In accordance with this
suggestion vitamin E plays a specific role in maintaining a proper redox
state of the sulfur-containing amino acid in the proteins. In dystrophic
muscles, the ratio of protein-bound disulfide to sulfhydryl content increased
two- to three-fold as compared to that in the control muscle proteins. Proteins
of low molecular weight, supposedly derived from proteolysis, were also
found in the dystrophic muscles. Muscles of animals fed diets deficient in
vitamin E and selenium displayed the lowest reduced glutathione level and
GSH-Px activity (Avanzo et al., 2001). The decreased levels of reduced
glutathione were not due to a defective activity of glutathione reductase,
which was increased in both mitochondria and cytosol. The absence of
vitamin E was linked to lowering of mitochondrial thiol levels. Catalase
activity increased in an attempt to counteract the decrease in glutathione
peroxidase activity. The results obtained showed that -tocopherol and Se
deficiencies caused multiple alterations in the antioxidant system and
adversely affected the redox state of chicken superficial pectoralis muscle.
These data are not consistent with previous observations indicating that
GSH concentration and GSH-Px activity in dystrophic muscles were
significantly increased (Hull and Scott, 1976) suggesting the redistribution
or impaired utilization of various antioxidants in chicken tissues as a result
of vitamin E deficiency. A specific need for cysteine in NMD can be explain
as a result of reduction in transsulfuration of methionine to cysteine in the
vitamin E deficient chick (Hathcock and Scott, 1966). It seems likely that
selenoprotein methionine sulfoxide reductase B could be also involved in
regulation of thiol metabolism and prevention from NMD.
Vitamin E and its combination with other antioxidants is considered to be
effective in NMD prevention. For example a mixture of vitamin E (10 ppm),
santoquin (125 ppm), methionine (400 ppm), sodium selenite (1 ppm) and
ascorbic acid (50 ppm) is shown (for review see Surai et al., 1994) to be
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effective. Similarly for prevention of NMD in turkey poults and ducklings a

mixture of vitamin E (40 ppm), ascorbic acid (100 ppm), vitamin B12 (30
ppb) and sodium selenite (0,5 ppm) is recommended (Shemet, 1977).
It is interesting that alpha-tocopheryl quinone was the most active substance
in preventing chicken experimental muscular dystrophy (Donchenko et al.,
1981). Various flavonoids or simple phenolics at a dietary concentration of
1,000 ppm completely prevented nutritional muscular dystrophy NMD but
quercetin reduced its incidence and quercetin, morin, and ferulic acid
reduced the severity of the disorder (Jenkins et al., 1992).
Impaired immunocompetence (see chapter 4);
Impaired thyroid hormone metabolism with increased plasma T4

concentrations and decreased plasma T3 and thymulin concentrations
(Jianhua et al., 2000; Chang et al., 2005). This could be related to impaired
immunity and other detrimental consequences with respect to chicken growth
and development.
Did you know that immunomodulating properties of Se are
usually seen at doses substantially exceeding those used in
commercial poultry production?
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Reduced fertility. Fertility and hatchability were low on the basal (low Se)
diet and were corrected partly by vitamin E and completely by Se (Latshaw
and Osman, 1974). Details of effects of Se on male reproduction are
presented in Chapter 5.
Reduced egg production and quality. Selenium deficiency is associated

with impaired immunocompetence, reduced egg production and increased
embryonic mortality (Combs and Combs, 1984). Egg production and fertility
were maintained at about 77% and 92%, respectively, by the Se diet and fell
to about 56% and almost 0 with the basal low Se diet (Cantor and Scott,
1974). Egg production was only 69% in Se-deficient birds against 81% in
controls (Latshaw et al., 1977). Weights of day-old chickens from hens
given the 0.05 and 0.1 mg/kg Se-supplemented diets were significantly
heavier than those from hens given no Se.
Decreased hatchability and increased embryonic mortality. Hatchability

of eggs was depressed by the low-Se diet, further depressed by peroxidized
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fat and was restored to normal level by supplementation of Se and vitamin
E (Combs and Scott, 1977). Similar decrease in egg hatchability (from 9293% down to 52%) as a result of Se deficiency in the breeders diet was
reported by Cantor and Scott (1974). Indeed, high incidence of dead embryos
early in incubation was related to Se deficiency. In fact, eggs from hens fed
very low levels of Se were more often infertile (12.6%), there were more
dead embryos (29%) and lower hatchability of fertile eggs (15%). Mean
respective values for controls were 4.1, 2.9 and 91% (Latshaw et al., 1977).
Moreover, Se is required in breeder turkey diets for optimum hatchability
and viability of offspring (Cantor et al., 1978).
Nutritional encephalomalacia (NE, Century and Hurwitt, 1964; Combs

and Hady, 1991). It is widely believed that NE is one of the most important
signs of antioxidant (mainly vitamin E) deficiency in chickens, fed on the
diet containing high amounts of linoleic or arachidonic acid (Pappenheimer
and Goettsch, 1931; Machlin and Gordon, 1962; Marthedal, 1973). The
role of selenium and particularly various selenoproteins in prevention of
NE is not clear at present, however, it seems likely, that improved antioxidant
defences by optimisation of Se supply could have a positive effects. The
major symptoms of NE include ataxia, sudden prostration, with legs
outstretched and toes flexed, and head retraction with lateral twisting. The
head becomes twisted either backwards or forwards and uncoordinated
muscular spasms affect the legs (Whitehead and Portsmouth, 1989). It is
believed that NE is associated with peroxidative dysfunction (Fuhramann
and Sallmann, 1995) leading to ataxia, prostration and death (Hassan et al.,
1990).
Gross and histological lesions are primarily found in cerebellum and in
chickens the lesions can be also seen in the cerebrum (NRP, 1977). In poultry,
gross lesions include a friable, swollen cerebellum with necrotic reddish or
brownish areas and microtrombosis (Jungherr et al., 1956; Pappenheimer
and Goettsch, 1931). Ischemic necrosis of the cerebellar cortex and white
matter, capillary thrombi, haemorrhages, malacia and edematous neutrophil
are observed in the cerebellum (Wolf and Pappenheimer, 1931).
Encephalomalacia is mainly described for chickens (Pappenheimer and
Goettsch, 1931), but in turkeys (Rigdon et al., 1961), emus (Aye et al.,
1998), game birds (partridges, quails and pheasants; Mutarov, 1979;
Swarbrick et al., 1986) as well as in zoo species (Dierenfeld and Traber,
1993) NE was described. In turkey poults major histopathological alterations
included congestion, hemorrhages, necrosis, and malacia associated with
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hyaline capillary thrombi affecting the cerebellar cortex and adjacent white
matter (Klein et al., 1994). NE in turkey poults leads to neurological signs
such as tremor, incoordination, and recumbency shortly after being moved
to new quarters. Associated lesions included ischemic necrosis of the
cerebellum and spinal cord (Jortner et al., 1985; Frank and Bergeland, 1988).
The disease usually occurs during the second or third week after hatching,
which may be related to the cerebellar PUFA accumulation that occurs at
that time (Budowski et al., 1987). In an experiment conducted by Fuhrmann
and Sallmann (1996) NE started at day 9. In other experiments of the same
authors (Sallmann et al., 1991; Fuhrmann et al., 1994) with similar type of
linoleic acid-rich diet and commercial chicks, the disease began after day
16 reflecting differences in the initial levels of vitamin E in the tissues of the
newly hatched chick. The lower the level of vitamin E in the incubation
eggs and chick tissues the earlier the disease appears. The effect of the
breeder diet on the severity of encephalopathy was observed in 4-week-old
chicks (Bartov and Bornstein, 1980).
As mentioned above, experimentally NE in chicks can be induced by diets
low in vitamin E and containing large amounts of linoleic acid (see Bartov
and Budowski, 1979). There are other nutritional and environmental factors
predisposing chickens to encephalomalacia, including deficiency in animal
protein and decreased temperature in hatcheries (Chen and Shi, 1985).
However, outbreaks of this disease were registered in commercial conditions
with diets containing sufficient levels of vitamin E and synthetic antioxidants
(Hislop and Whitte, 1967a,b; Marthedal, 1973).
It is well recognised that NE developed on the diet rich in linoleic acid, but
linolenic acid had a protective effect against this disease (Budowski and
Crawford, 1985). Therefore it has been postulated that n-6 fatty acids
especially arachidonic acid degradation products including pentane
(Fuhrmann and Sallmann, 1995) and hexanal (Hu et al., 1989) have a prooxidative toxic effect which could lead to encephalomalacia development
(Budowski et al., 1979; Fuhrmann and Sallmann, 1996). Indeed, an
overproduction of arachidonic acid-derived eicosanoids is considered as a
factor in the etiology of the cerebellar lesion and possibly a structural change
due to a loss of docosahexaenoic acid and gain of arachidonic acid
(Budowski et al., 1987).
Molecular mechanisms of NE are still not clear. In the vitamin E-deficient
cerebellum the cytosolic phospholipase A2 activity was increased (Fuhrmann
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and Sallmann, 1995a). Due to low content of vitamin E, the cerebellum is
the most susceptible tissue to oxidative stress during vitamin E deficiency.
Chicks hatched from eggs containing 16.5% linoleic acid of total yolk fatty
acids were more susceptible to NE than chicks hatched from eggs containing
7.5% linoleic acid. (Bartov and Bornstein, 1980).
NE was induced in young chicks using a diet low in vitamin E and containing
8% ethyl esters derived from safflower oil fatty acids. Chicks receiving
aerated linseed oil high in alpha-linolenic acid and low in -tocopherol did
not develop symptoms due to alterations in the AA metabolism and
thrombocyte function in young chicks (Vericel et al., 1991). In that
experiment tromboxane B2 synthesis was significantly decreased in the linolenic group in comparison to the ataxic chicks. Prostaglandin E2
production by aortal rings was also significantly influenced by the diet;
ataxic chicks yielded the highest value and -linolenic acid-supplemented
chicks had the lowest values. The lipoxygenase (LOX) oxygenation pathway
of arachidonic acid metabolism was investigated in the cerebellum and
cerebral hemispheres of young chicks (Greenberg-Levy et al., 1992).
Lipoxygenase products consisted mainly of 15-hydroxyeicosatetraenoic acid
(15-HETE), accompanied by the 15-hydroperoxy analogue (15-HPETE)
and the 5-HETE products. In the experiment, the yield of 15-HETE was 3
times greater in the cerebellum than in the cerebrum and phospolipase A2
activity of the cerebellum was 2-fold higher than that of the cerebrum. This
could lead to increased vulnerability of the cerebellum to oxidative
dysfunction.
In the liver of linoleic acid-fed vitamin E deficient chickens total aldehydes
were increased (Fuhrmann and Sallmann, 2000). In plasma, vitamin E
deficiency led to higher malondialdehyde and OH-nonenal concentrations.
However, in brain of vitamin E deficient chicks, aldehyde concentrations
was not affected. Therefore a direct role of unsaturated aldehydes for the
development of NE in the cerebellum was not confirmed (Fuhrmann and
Sallmann, 2000) and there was no increase in lipid peroxidation in the
cerebellum of the chickens fed the NE producing diet (Fuhrmann et al.,
1996). Rather, the liver seems to be affected by the oxidative stress. In this
respect our data (Surai et al., 1996) showed that even low levels of vitamin
E in the chicken embryonic brain are sufficient to prevent lipid peroxidation
in vivo, probably due to efficient vitamin E recycling by other antioxidants
including ascorbic acid. In comparison to other tissues studied chicken brain
contains increased ascorbic acid concentrations. It is necessary to underline
that in newly hatched chick cerebellum contains higher vitamin E and lower
ascorbic acids levels than cerebrum (Surai et al., 1999).
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For NE prevention and treatment, increased vitamin E doses and its

combinations with other antioxidants are shown to be effective (Surai et al.,
1994). Supplementation of selenium and methionine with or without
simultaneous supplementation of a low level of dl--tocopheryl acetate had
little effect on preventing NE. The preventive effect of other antioxidants
including ascorbic acid, methylene blue, ethoxyquin, 2,6-ditertiary-butylp-cresol and butylated hydroxyanisole was in proportion to their dietary
level, and a high level of any of them could almost completely protect the
chicks from NE, while diphenyl-p-phenylenediamine was not as effective
and the effect was not proportional to the dose (Yoshida and Hoshi, 1977).
When -tocopherylacetate, short-chain -tocopherylacetate, tocopherylquinone, short-chain -tocopherylquinone and tocopheronolactone were added to vitamin E deficient diets (Kovalenko et
al., 1979), -tocopherylacetate and -tocopheronolactone showed the
highest E-vitamin activity in preventing NE in chickens. The data confirmed
a nonspecific function of vitamin E in preventing alimentary
encephalomalacia.
High levels of lipid unsaturation and comparatively low antioxidant
protection make the brain vulnerable to free radical attack; this is of particular
importance in the chick in respect to the development of encephalomalacia
which is associated with an antioxidant system compromise (Pappenheimer,
Goettsch, 1931; Marthedal, 1973; Dror et al., 1976; Fuhrmann and
Sallmann, 1995). Nevertheless, the precise mechanisms of the disorder
development have remained unclear. For example, oxidative stress can
induce apoptosis in neurones (Ratan et al., 1994), which may have some
effect on the disease development. Involvement of Se deficiency and
antioxidant-prooxidant balance in the brain cells in the development of NE
need further clarification.
As mentioned above, molecular mechanisms of development of ED as well as
nutritional encephalomalacia (NE) and nutritional muscular distrophy (NMD) are
still not clear. However oxidative stress is a crucial factor in disease development
and lipid peroxidation, disrupting membrane structure and function, is responsible
for specific changes in the brain, muscles and vascular systems (Fraga et al.,
1987). For example, nutritional pancreatic atrophy in chicks may be overcome
by feeding vitamin E at 15-20-fold excess over the levels normally regarded as
nutritionally adequate (Whitacre et al., 1987). Selenium supplementation can
also decrease incidence of nutritional muscular dystrophy in the chick (Jonsson,
1993).
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Did you know that lipid peroxidation is considered to be
the major factor in aetiology of Se-deficiency related diseases?
Recent understanding of the roles of various selenoproteins as integral elements

of the antioxidant systems with complex relationships among individual
antioxidants in the biological system could help explaining some clinical signs of
diseases. For example, recent results show that liver cells can boost endogenous
ubiquinone-dependent protective mechanisms in response to deficiency in vitamin
E and Se (Navarro et al., 1999). Therefore in the absence of vitamin E and Se,
enhancement of ubiquinone-dependent reductase systems can protect the
membrane against peroxidation (Navarro et al., 1998). Similarly, Se participation
in a regulation of redox status of the cell can be crucial for explaining some signs
of its deficiency. In this respect, the selenoenzyme thioredoxin reductase, which
is involved in regulation of many metabolic reactions in the cell, could be the
major target for the future research. Furthermore, it seems likely that apoptosis
plays an important role in formation of clinical signs of Se deficiency (Nunes et
al., 2003).
Selenium and chicken embryo development

Chick embryo tissues contain a high proportion of highly polyunsaturated fatty
acids in the lipid fraction (Speake et al., 1998) and therefore need antioxidant
defence (Surai, 1999).
Did you know that chicken embryonic development is associated
with a progressive accumulation of polyunsaturated fatty
acids in various tissues and increased susceptibility
to lipid peroxidation?
It is well accepted that maternal diet composition is a major determinant of
antioxidant system development during embryogenesis and in early postnatal
development (Surai, 1999). The antioxidant system of the newly hatched chick
includes the fat-soluble antioxidants vitamin E and carotenoids (Surai et al., 1996),
water-soluble antioxidants ascorbic acid (Surai et al., 1996) and glutathione (Surai,
1999a) as well as antioxidant enzymes SOD, GSH-Px, catalase (Surai, 1999a)
and selenium (Surai, 2000; 2000a; 2000b). Vitamin E (Surai and Speake, 1998)
and carotenoids (Surai and Speake, 1998a; Surai et al., 2001; 2001a) are transferred
from feed into egg yolk and further to embryonic tissues. Selenium content of the
egg also depends on its concentration in the hens diet, and also on the form of
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dietary Se used, since organic Se is more efficiently deposited in the egg yolk
(Cantor, 1997; Paton et al., 2000; 2002).
Therefore, during egg incubation fat-soluble antioxidants are transferred to
the developing embryonic tissues mainly during the last week of incubation (Surai
et al., 1996; Surai, 1999). Only vitamin A transfer to the embryonic liver started
earlier than that of vitamin E or carotenoids. As for Se transfer, it is still not clear
from the present results at what stage of embryo development this trace element
is transferred to the embryonic tissues. It seems likely that Se from the albumin is
transferred to the embryo during first 2 weeks of the embryonic development,
while Se from egg yolk is delivered to the embryo during last week of incubation.
The form of selenium in the egg yolk and embryonic tissues needs further
investigation as well. It could well be that SeMet could represent a substantial
proportion of the total Se in the embryo when Se is supplied to laying hens in the
organic form. Water soluble antioxidant ascorbic acid started to be synthesised in
the yolk sac membrane (YSM) at early stages of incubation and its concentration
in this tissues progressively declined during embryonic development (Surai et
al., 1996; Surai et al., 1995). Reduced glutathione and antioxidant enzymes
glutathione peroxidase, superoxide dismutase and catalase are also expressed in
the embryonic tissues at various stages of their development (Surai, 1999a).
Did you know that selenoprotein synthesis in embryonic tissues
could be considered as major antioxidant defence strategy?
Our results indicate that there are tissue-specific features in antioxidant defence
strategy during embryonic development of the chicken. They can be summarised
as follows.
LIVER
At the time of hatching, liver phospholipids contain high levels of highly
polyunsaturated fatty acids, particularly arachidonic (20:4n-6) and
docosahexaenoic (22:6n-3) acids (Surai et al., 1999). It is clear that high levels of
PUFA require a considerable degree of antioxidant defence against peroxidation.
The three antioxidant enzyme activities in the liver changed in a different manner
during development. SOD activity increased between days 10 and 11 of
development, then significantly decreased in activity up to day 15 and remained
at the same level during the rest of the developmental period (Surai, 1999a).
Similarly there was no difference in SOD activity in the embryonic liver between
days 12 and 18 (Wilson et al., 1992). GSH-Px activity in the embryo liver increased
through the time of development: from 84.7 mU/mg protein on day 10 to 254.9
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mU/mg protein on day 22 (Surai, 2002) which is in agreement with Gaal et al.
(1995) and Wilson et al. (1992).
Comparison of GSH-Px activity in various tissues showed that the highest
activity was found in the embryo liver at all stages of development (Table 7.1). At
the early stages of embryo development (10-11 days) it was 1.2-1.3 times higher
compared to the yolk sac membrane and 2.5-2.6 times higher than that in the
brain. At day 17 of development it was higher by 4.0; 2.8; 2.0; 1.7; 1.3 and 1.2
times compared to the brain, skeletal muscle, heart, YSM, kidney and lung
respectively. In general, changes in GSH-Px activity patterns during the
development were very similar to those found for vitamin E, carotenoids (Surai et
al., 1996) and vitamin A (Gaal et al., 1995). Probably GSH-Px plays a primary
role in antioxidant defence of the liver by effectively removing hydrogen peroxide
and lipid hydroperoxides from cells whereas catalase may be less important,
decreasing throughout the development. Nevertheless, the concentration of GSH,
a primary cell water-soluble antioxidant, gradually decreases throughout the
development.
Table 7.1 GSH-Px activity in the chick embryo tissues, mU/mg protein (Adapted from Surai, 1999a)
10
Liver
84.7
Brain
34.4
YSM
70.1
Kidney
Lung
Heart
Leg Muscles
Days of embryo development

15
17
19
11
13
106.9
40.2
83.6
162.7
41.4
126.2
206.6
47.3
153.0
131.4
200.7
109.3
86.0
211.3
53.3
124.4
158.5
179.4
105.6
74.9
226.8
55.2
123.2
173.2
148.4
106.8
68.9
21
22
229.3
48.9
105.2
207.8
206.6
126.1
64.1
254.9
42.0
100.4
205.8
210.7
114.1
60.2
CAT had two peaks of activity at day 10 of embryonic development and in day
old chicks (Surai, 2002). After hatching the liver displayed CAT activity similar to
that in the kidney but much higher compared to other tissues. In rat postnatal
development CAT activity significantly decreased with ageing (Matsuo, 1993).
Did you know that a cooperative action of selenoproteins with
vitamin E provides an effective antioxidant defence in the
developing chicken liver?
An accumulation of natural antioxidants vitamins A, E and carotenoids,
comparatively high vitamin C concentration and an increased GSH-Px specific
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activity correspond with the extensive metabolic and detoxifying functions of

this organ, which are associated with free radical formation, and protect liver
lipids against peroxidation (Surai et al., 1996). The high levels of endogenous
antioxidants within the liver can clearly serve as a major adaptive mechanism for
the protection of the tissue during the oxidative stress experienced at hatching.
Thus the susceptibility of the liver lipids to peroxidation in the newly hatched
chick and immediately following hatching is low (Surai, 2002).
BRAIN
It may be envisaged that the brain of the chick embryo may be at particular risk
from peroxidative damage due to presence of very high levels of C20 and C22
polyunsaturated fatty acids in neuronal phospholipids (Surai et al., 1999). The
antioxidant system of the developing brain is poorly understood. It includes
natural antioxidants such as vitamins E and C, ubiquinols, glutathione, antioxidant
enzymes - superoxide dismutase, glutathione peroxidase and catalase and other
elements such as dopamine, noradrenaline, taurine, carnosine (Gaal et al., 1995,
Surai et al., 1996; Matsuo, 1993). The chick embryonic brain is characterised by
comparatively low level of vitamin E which is almost 100 fold lower compared to
the liver and remains practically constant during the development (Surai et al.,
1996) and this apparent deficiency in the brains antioxidant defence capacity is
further compounded by the relatively low levels of vitamin A, selenium, glutathione
peroxidase, and carotenoids (Speake et al., 1996). Our previous results (Surai et
al., 1993) also indicate that comparatively low levels of vitamin E persist in the
brain of adult chicken. Thus it is a characteristic of poultry brain to contain low
levels of vitamin E. At the same time, the brain generates especially high levels of
free radicals (Reiter, 1995) which are necessary for physiological responses
(Westermarck et al., 1993).
Did you know that during embryonic development and in
early postnatal development chicken brain is characterised by
low antioxidant defences and high levels of polyunsaturated
fatty acids?
Our data (Surai, 1999a) indicate that SOD is the main enzyme in antioxidant
defence of the brain, especially during the last days of incubation when SOD
activity in the brain was significantly higher compared to other tissues. However,
GSH-Px and CAT activities in the brain were low and it is unlikely that they are
the primary defence in this tissue.
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The antioxidant profile of the chicken brain is shown in Table 7.2. The
cerebellum had some distinctive features in antioxidant concentration compared
to the other brain regions, in particular the cerebrum. The cerebellum was
characterised by significantly increased alpha-tocopherol concentration and
catalase activity, but decreased ascorbic acid level and Mn-SOD activity compared
to that in the cerebrum. In general, the brain was characterised by low level of
vitamin E, low GSH-Px and catalase activity, but very high level of ascorbic acid
and high SOD activity. Furthermore, the brain was characterised by comparatively
low levels of Se (0.1870.01 g/g) and moderate levels of Zn (11.670.23 g/g)
and Cu (1.180.06 g/g) (Surai, 2002).
Table 7.2 Antioxidant composition of the brain of a newly hatched chick (Adapted from Surai et al.,
1999)
Antioxidants
-tocopherol, g/g tissue
Ascorbic acid, g/g tissue
Glutathione, nM/mg protein
Mn-SOD, U/mg protein
Cu-Zn-SOD, U/mg protein
Se-dependent GSH-Px,
mU/mg protein
Se-independent GSH-Px,
mU/mg protein
Catalase, U/ mg protein
Cerebrum
Cerebellum
Brain stem
Optic lobes
5.22
889.4
41.12
3.66
6.910
7.12
711.3
38.12
2.837
6.985
6.12
747.72
40.13
3.91
7.52
5.66
850.53
39.56
3.02
8.03
29.83
28.61
33.14
32.6
6.22
1.923
6.60
2.365
5.57
1.966
7.31
1.822
Although the basal levels of MDA in the brain were low (Surai et al., 1996), the
susceptibility of the brain homogenates to lipid peroxidation during incubation in
both the absence and the presence of Fe+ was very high in comparison with other
tissues. Thus the low levels of endogenous vitamin E in the chick embryo brain
may render the tissue homogenates highly susceptible to in vitro peroxidation
(Kornbrust and Mavis, 1980). In embryonic brain antioxidant defence is afforded
by high levels of ascorbic acid to effect the recycling of the low concentrations of
vitamin E in order to maintain physiological requirements (Surai et al., 1996).
From our results (Surai, 1999a) it is clear that SOD may be another important
protective element in the embryonic brain.
YOLK SAC MEMBRANE

During chick embryo development there is a preferential absorption by the yolk
sac membrane of yolk phosphatidyl ethanolamine species particularly rich in
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docosahexaenoic acid (Noble and Moore, 1967). The synthesis of arachidonic

acid in the YSM takes place as well (Noble and Shand, 1985). Thus the level of
lipid unsaturation in the YSM is quite high and requires an effective system of the
antioxidant defence.
In the YSM, maximum activity of GSH-Px was found on day 15 of incubation
and then decreased to the end of incubation. SOD activity increased from day 10
up to day 15 of the development with the following decrease and the minimal
activity just after hatching. The maximum CAT activity was found in the YSM on
day 15 of the development with consecutive decrease up to day 19 and following
increase at hatching time. GSH level in the YSM gradually decreased throughout
the development in such a way that at hatching time the concentration was 5-9
times lower compared to other tissues studied.
During the second half of incubation, YSM contains comparatively high
concentrations of vitamin E, carotenoids (Surai et al., 1996), vitamin A (Gaal et
al., 1995) but low levels of ascorbic acid (Surai et al., 1996). During this time the
susceptibility to peroxidation decreased (Surai et al., 1996). Probably increased
activity of GSH-Px and CAT at day 15 and a sharp increase in peroxide
accumulation at day 16 of development (Surai et al., 1996) indicate oxidative
stress at this stage. It seems that at the end of incubation when the YSM involution
begins, the protective mechanisms in this organ decreased as well.
LUNG
The lung of the chick is rich in phospholipids and there is a specific accumulation
of phospholipids at about the eighteenth day. The accumulation of
phosphatidylcholine is associated with the synthesis of the desaturated component,
dipalmitoyl phosphatidylcholine (Noble and Cocchi, 1990). At day 18 of the
development the lung contains a high level of iron (Richards et al., 1991). In the
lung GSH-Px activity decreased on day 19 and again increased just before hatching
but glutathione levels were on the same level during the development. In the
embryonic lung SOD activity significantly decreased from day 15 of the
development up to posthatching time. There was a dramatic decrease in CAT
activity between days 15 and 19. At hatching time the activity of this enzyme in
the lung increased again. The increased GSH-Px and CAT activities at hatching
time reflect the adaptation to the increased oxygen consumption and probably
free radical production.
Did you know that there are tissue-specific features in
antioxidant defences in the chicken embryo?
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At hatching time GSH-Px activity in the lung (206.6110.81) was similar to that
in the liver (229.3312.7) and kidney (207.8112.05) but significantly higher
compared to that of the heart, muscle or brain. GSH concentration in the lung was
half that of the liver or kidney and similar to that of the muscle (Surai, 2002). The
lungs are characterised by moderate levels of vitamin E and low levels of
carotenoids (Surai et al., 1996). In this respect GSH-Px and CAT probably play
an important role in the antioxidant defence of the tissue.
In general, comparatively low SOD activity, moderate levels of vitamin E and
carotenoids (Surai et al., 1996), together with high iron concentration (Richards
et al., 1991) and exposure to the oxygen at hatching time place the lung in a risk
situation associated with possible free radical formation, lipid peroxidation and
tissue injury. The importance of antioxidant/prooxidant balance in the lung is
exemplified by its involvement in the development of Pulmonary Hypertension
Syndrome in the chicken (Bottje and Wideman, 1995) and attenuation of PHS
mortality was achieved by vitamin E implanting to birds (Bottje et al., 1995).
HEART
The importance of the heart-vascular system for the posthatch development
determines the strategy of antioxidant defence. Phospholipids and triglycerides
of the heart contain high levels of docosahexaenoic (22:6n-3) and arachidonic
(20:4n-6) fatty acids (Surai et al., 1999; Noble and Cocchi, 1990). At day 18
heart contains a comparatively high level of iron (Richards et al., 1991). In the
heart the GSH-Px activity was half that of the liver or lung and comparatively
stable during embryo development with a slight increase before hatching. The
embryonic heart was characterised by a high SOD activity which significantly
decreased from day 15 of the development up to posthatching time but remains
higher than that in the liver, YSM or lung. In the heart CAT activity was low and
didnt change during the development. The embryonic heart is characterised by
moderate levels of vitamin E and low levels of carotenoids (Surai et al., 1996).
High proportions of arachidonic and docosahexaenoic acids in the heart
phospholipids are associated with moderate antioxidant protection and in stress
conditions the heart will be vulnerable to oxidative damage. For example, the
embryonic heart was characterised by comparatively high susceptibility to Leadinduced lipid peroxidation in vivo (Somashekaraiah et al., 1992).
KIDNEY
In the kidney GSH-Px activity increased during development in a very similar
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way compared to that of the liver reaching its maximum at hatching time. It was
comparable with GSH-Px activity in the liver and lung and higher than that in
other organs studied. GSH concentration significantly increased at hatching time
as well. On the other hand, SOD activity significantly and constantly decreased
during development. Nevertheless it was comparatively high and similar to that
in the brain, lung and heart and higher compared to the liver, muscles and YSM.
During development CAT activity was comparatively high and stable with
significant increase at hatching time.
Vitamin E accumulation in the embryonic kidney took place in the same manner
as in the liver (Surai et al., 1996). In the kidney the ascorbic acid levels were
comparatively high and didnt change during the development up to hatching
when there was a decrease in vitamin C concentration in this tissue (Surai et al.,
1996). Thus the antioxidant system of the embryonic kidney includes vitamins A,
E and C and comparatively high activities of antioxidant enzymes and probably
has an adequate protective potential at hatching to deal with potentially harmful
metabolic end products as a result of their functional activity.
SKELETAL MUSCLE
At day 16 of incubation, muscles contain 2.4% of total lipids. The major lipid
fractions of skeletal muscle of the embryo are phospholipid, triglycerides and
free cholesterol. Phosphatidylcholine and phosphatidylethanolamine, high in C20
and C22 PUFAs, are by far the major components of the phospholipid fraction
(Surai et al., 1999; Noble and Cocchi, 1990).
In the thigh muscle the activity of GSH-Px decreased on day 17 and then
remained on that level up to the end of the development. There were no significant
changes in the SOD activity throughout the development. In muscle a decrease
in CAT activity lasted from day 15 up to hatching time. Skeletal muscle is
characterised by a comparatively low and stable vitamin E concentration. On the
contrary, the ascorbic acid level significantly decreased during development (Surai
et al., 1996). Thus the antioxidant system of the muscle is not very potent and
imbalance of the natural antioxidants in postnatal development may cause such
diseases as muscle degeneration (Machlin and Shalkop, 1956).
The chicken tissues displayed a considerable variation in the Mn-SOD activity
with the heart having the highest value and lung the lowest. By contrast, the lung
was characterised by high Cu,Zn-SOD activity; in the heart activity of Cu,ZnSOD was comparable to the other tissues. Based on the total SOD activity the
tissues could be placed in the following descending order:
heart>muscle>YSM>kidney>lung>liver. As can be seen the tissues differed
markedly in the GSH-Px activities. In all the tissues Se-dependent GSH-Px was
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the main enzymic form, comprising from 65% (lung) up to 90% (heart) of the
total enzyme activity. The liver and kidney displayed the highest total GSH-Px
activity and the muscle the lowest. As in the case of GSH-Px, catalase activity was
also maximal in the liver and kidney. The liver contained the highest concentrations
of Zn, Cu and Mn. The kidney was also characterised by comparatively high
levels of the metals. The liver and kidney also contained high concentrations of
Se. Comparison between the tissues (excluding YSM) revealed a highly significant
positive correlation between the concentration of Se and the activity of Sedependent GSH-Px (r=0.95). There was also a positive correlation (r=0.93) between
Fe content and catalase activity in all the tissues other than YSM (Surai, 2002).
Antioxidant enzymes are a major cell defence against acute oxygen toxicity
(Harris, 1992) and they are responsible for the detoxification of reactive oxygen
species and preventing lipid peroxidation (Gille and Sigler, 1995). The expression
of their activities is regulated at the gene level (Tsan, 1997; Weiss et al., 1997)
with tissue-specific features (Bermano et al., 1995). They are considered as
important embryoprotective enzymes (Ozolins et al., 1996; Fantel, 1996) during
organogenesis when increased exposure to oxidant-derived free radicals or
inadequate systems for antioxidant defence could alter cellular response at critical
points in development (Fantel, 1996; Allen and Venkatraj, 1992).
The results of our studies (Surai, 1999a; Surai et al., 1999) indicate that different
tissues of the embryo display distinct development strategies with regard to the
acquisition of antioxidant capacity. A low oxygen pressure in the environment
surrounding embryos during development seems to have been retained in the
course of evolution to protect the vulnerable developing tissues from the damage
caused by the action of reactive oxygen species (Ar and Mover, 1994) as far as
the rate of free radical generation in cells is influenced by ambient oxygen
concentration (Turrens et al., 1982). The reactivity of oxygen radicals and their
derivatives implicate them as possible effectors of oxygen-mediated changes in
gene expression (Allen and Venkatraj, 1992).
Did you know that antioxidant enzymes, including a range of
selenoproteins, are considered a major cell defence against
oxygen toxicity?
Embryonic gene expression of antioxidant enzymes develops in proportion to
the degree of exposure to the reactive oxygen species in their environment and in
preparation for the oxygen-rich environment after birth (Ar and Mover, 1994).
Exposure of rat embryos with an immature antioxidant system to a high
concentration of oxygen (20%) during early neurulation significantly increased
the incidence of neural tube defects compared with control embryos exposed to a
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low O2 concentration. The concentration of GSH in 20% O 2 exposed embryos

was significantly reduced compared with that of control embryos and it was
suggested that impaired responsiveness of the GSH dependent antioxidant system
against oxidative stress plays a crucial role in oxygen-induced embryopathy
(Ishibashi et al., 1997). Yuan et al. (1995) studied species differences in the
resistance of human, rabbit, rat, mouse and chick embryo cells against oxygeninduced growth inhibition. The extent of the resistance of the cells was in the
following order: chick >rat >human >rabbit = mouse. Chick embryo cells, having
the highest resistance against oxygen-induced growth inhibition, were at the lowest
activity levels of antioxidant enzymes and at the highest concentration level of
reduced glutathione.
Did you know that hatching process is considered to be an
important oxidative stress for the developing chicks?
An accumulation of vitamin E in the liver and YSM during embryonic development
was associated with the decreased susceptibility of these tissues to lipid peroxidation
that reflects the protective effect of the natural antioxidant accumulation in the
embryonic tissues at hatching time (Surai, 2002). Such protection allows these
tissues to be quite resistant to oxidative stress of hatching process (Surai et al.,
1999). Nevertheless the susceptibility of lipids to peroxidation depends not only
on the level of natural antioxidants in tissues but also on the balance between the
level of antioxidants/prooxidants and the concentration of PUFAs. It is necessary
to underline that chicken egg yolk and embryo tissues are more unsaturated
compared to other domestic avian species (Noble and Cocchi, 1990; Surai et al.,
1999).
When chickens hatch, their immune system, digestive system, nervous system
and endocrine system are still not mature and are actively developing during at
least the first week posthatch. This development is associated with active lipid
metabolism and long chain PUFAs play a regulatory role in this development.
Therefore, proper antioxidant protection of the newly hatched chick is a crucial
factor of their success in the future. For example, if the antioxidant system is not
sufficient to prevent lipid peroxidation, the immune system development will be
compromised. As a result undeveloped immune system will be a major cause of
chicken mortality during first weeks of the postnatal development since efficiency
of vaccination will be low. Furthermore immune cells produce free radicals and
use them as an important weapon to destroy pathogens (Schwarz, 1996; Kettle
and Winterbourn, 1997). Therefore in the case of low antioxidant defence and
pathogen challenge, stimulated neutrophils will produce free radicals which will
be able not only to kill pathogens, but also to damage normal tissues which could
lead to the development of different disorders.
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The regulation of the antioxidant system during embryo development and in

early postnatal chick life is not clear at present, although, nutritional factors appear
to be important (Surai, 1999). Indeed, one of the most impressive features of
vitamin E metabolism in avian embryonic tissues is an abrupt decrease in the
concentration of this vitamin over the first two weeks of postnatal development.
As indicated previously the liver accumulates vitamin E during embryonic
development to supply chickens with this vitamin in the first days of life after
hatch (Surai et al., 1996). This reserve of vitamin E is used by chickens during
the first 2 weeks posthatch. During this period vitamin E concentration in the
liver decreased (Figure 7.1) by 10 times in chickens (Surai and Ionov, 1994),
goslings and ducklings (Surai et al., 1993) and more than 50 times in turkeys
(Soto-Salanova et al., 1993). Marusich et al. (1975) suggested that the low levels
of vitamin E in the liver of turkey poults resulted from the inefficient intestinal
absorption of vitamin E, which can be explained as a result of low pancreatic
lipase activity (Sell et al., 1991) and restricted bile production (Freeman, 1976) as
well as of greater (compared to chicken) production and excretion of tocopheryl
glucuronides (Sklan et al., 1982).
700
-tocopherol (g/g)
600
Chicken
Turkey
Duck
Goose
500
400
300
200
100
0
10.0
16/20/20/22
21/28/28/30
10
Days of embryo development
Figure 7.1 Vitamin E transfer from yolk to embryonic liver (Adapted from Surai, 1999).
The samples were collected in approximately equal stages of embryo development for each species. For
example day 20, 26, 26 and 28 for chicken, turkey, duck and goose respectively.
Did you know that vitamin E concentration in the chicken liver

decreased more than 20-fold for the first 2 weeks posthatch and
increased GSH-Px activity at the same time is considered as an
adaptive mechanism of antioxidant defences?
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During chick embryo development there is an antioxidant/prooxidant balance in

the tissues which is responsible for normal embryonic development and posthatch
chick viability. We suggest that an accumulation of natural antioxidants vitamins
A, E and carotenoids as well as an increase in GSH-Px activity in the embryonic
liver may have adaptive significance and were developed during evolution to
protect unsaturated lipids against peroxidation during hatching stress conditions.
On the other hand, an increased SOD activity and very high levels of ascorbic
acid in the brain may be considered as another tissue-specific adaptive mechanism
in embryo development. It seems likely, that in postnatal development there is a
different strategy in relation to antioxidant defence compared to embryonic chicken
(Surai, 2000; 2000a). For example, during mammalian prenatal development the
antioxidant system is considered as immature (Fantel, 1996; Allen and Venkatraj,
1992) with respective maturation in the postnatal life. Probably the same is true
in avian embryo development i.e. an embryo relies on natural antioxidants
accumulated in the egg yolk as a result of their transfer from the maternal diet and
after hatching antioxidant enzymes protect tissues against lipid peroxidation. It is
especially important because a lowered antioxidant activity has been reported in
tissues of young chickens which suffered from encephalomalacia, exudative
diathesis and muscular degeneration (Machlin and Gordon, 1962). In none of
these diseases the role of antioxidant enzymes has been studied yet. Nevertheless,
antioxidant protection at hatching time is considered to be an important determinant
of chick viability during first posthatch days (Surai, 1999; 2000).
Therefore activity of GSH-Px significantly increased during postnatal
development (Kalytka and Donchenko, 1995; Surai, 2000; 2000a). Thus, in
conditions when oxygen concentration in the tissues is higher, metabolic activity
and superoxide radical production are increased and tocopherol and carotenoid
concentrations are decreased, the required protection is afforded through the major
antioxidant enzymes SOD, GSH-Px and CAT. Therefore GSH-Px became the major
antioxidant protection for the tissues at this period of the development and to
maintain high GSH-Px activity in the chicken tissues during first days posthatch it
is necessary to use organic form of selenium in the maternal diet which allows
more selenium to be accumulated in the egg and transferred to the tissues of
newly hatched chick.
Effect of organic selenium in the maternal diet on antioxidant defences

of the developing chicks
The most important opportunity to regulate antioxidant system of the newly hatched
chick is by using organic selenium in the breeders diet. In an experiment conducted
at the Scottish Agricultural College, the effect of selenium and vitamin E
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supplementation of the maternal diet on their transfer to the egg yolk and their
concentrations in the tissues of the newly hatched chick was studied and the
effect of increased Se and vitamin E supply on the activity of Se-GSH-Px in the
chick liver in early postnatal development was determined (Surai, 2000). In this
experiment 90 Cobb broiler breeder hens were divided into nine equal groups
and housed in pens at 25 weeks of age. Each hen received one of the treatment
diets (Table 7.3). Selenium was supplemented in the form of selenium-enriched
yeast (Sel-Plex, Alltech, Inc). After six weeks, the hens were artificially inseminated
once a week. From week 8 eggs were collected and placed in an incubator.
Liver, yolk-sac membrane, brain and blood plasma were collected from chicks
for biochemical analysis.
Table 7.3 Selenium and vitamin E supplementation of the basal dietsa (Adapted from Surai, 2000)
Dietary Group
1
2
3
4
5
6
7
8
9
Diet
Vitamin E (mg/kg))
Semi-synthetic
Commercial (CD)
CD
CD
CD
CD
CD
CD
CD
No
No
No
No
40
100
200
40
100
Se (mg/kg)
No
No
0.2
0.4
No
No
No
0.2
0.4
The level of selenium in the semi-synthetic diet was 44 g/kg and in commercial diet 171
g/kg. Selenium was supplemented in the form of Sel-Plex. Semi-synthetic and commercial
diets contained 4.86 and 10.05 mg/kg -tocopherol. Both, commercial and semi-synthetic
diets were balanced in other nutrients.
Our results (Figure 7.2; Surai, 2000a; Surai and Dvorska, 2001) indicated that the
inclusion of organic Se into the commercial diet significantly increased the Se
concentration in the egg yolk and the albumin. The correlation between dietary
Se and egg yolk Se content was found to be very high. This relationship was
quantified through a regression equation:
y = 179.4 + 1.01x (r2=0.96, P<001),
where y is Se concentration in the egg yolk, ppb; and x is the Se concentration in
the feed, ppb).
Similarly, a significant correlation between Se in the egg white and feed was
found and an equation describing Se concentration in the egg white (y) is as
follows:
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y = -24.8 + 0.68x (r2=0.98; P<0.01).

Using these equations it is easy to predict Se concentration in the egg when organic
Se is used in the diet, because the Se content in the egg yolk and egg white reflect
its concentration in the feed.
1400
Yolk
White
Selenium in egg (ng/g)
1200
1000
800
600
400
200
0
0.0
0.1
0.2
0.3
0.4
0.5
0.6
Se in diet (mg/kg)
Figure 7.2 Effect of selenium supplementation on its concentration in egg (Adapted from Surai and
Dvorska, 2001).
Did you know that increasing selenite dose in the breeders

diet is not effective in increasing Se concentration in eggs?
Data presented by Paton et al. (2002) indicate that inclusion of sodium selenite in
the maternal diet has a limited potential in increasing Se concentration in eggs
(Table 7.4). Indeed, there was no difference in Se content of the egg when sodium
selenite was used at doses 0.1, 0.2 or 0.3 ppm. However, adding sodium selenite
to the basal diet increased Se content of the egg more than twice. It is not known
at present in which form Se is accumulated in the egg when selenite is its source
in the diet. In contrast, when organic Se was used in the form of Sel-Plex a gradual
increase in Se content in the egg (from 7.1 g up to 11.2 g/egg) was observed
(Paton et al., 2002). Similarly, increasing organic selenium content in the diet of
laying hens (from 0.1 up to 0.5 ppm) was associated with increased Se
concentration in the egg yolk (by 2.6-fold) and albumen (by 5.2-fold; Table 7.5).
The efficiency of Se transfer to the egg depends on many different factors and
probably analytical techniques used for Se analysis and form of Se in the diet
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could be important variables. For example, in experiments conducted in Croatia

it was shown that provision of organic selenium in concentrations ranging from
0.3 to 0.5 ppm in hens feed tends to accumulate only 30% more selenium in the
eggs when compared with same concentrations of inorganic selenium (Valentic
et al., 2003).
Table 7.4 Effect of dietary Se on its concentration in egg (Adapted from Paton et al., 2002)
Diet
Basal diet *(B)
B+0.1 Selenite
B+0.2 Selenite
B+0.3 Selenite
B+0.1 Sel-Plex
B+0.2 Sel-Plex
B+0.3 Sel-Plex
Egg, g
Egg yolk, g/g
Egg white, g/g
2.6
6.6
7.1
6.9
7.1
8.7
11.2
0.10
0.33
0.37
0.38
0.32
0.42
0.48
0.04
0.07
0.07
0.07
0.08
0.13
0.15
*/Se content in basal diet was 0.057 ppm

Table 7.5 Effect of Se supplementation of the maternal diet on the Se concentration (ng/g) of hatching
eggs and day-old chicks (Adapted from Pappas et al., 2004)
Tissue
0.1 Se
Yolk
Albumen
Breast
Liver
183
40
58
199
0.1 Se + FO
194
42
79
282
0.5 Se
0.5 Se + FO
505
209
235
684
496
205
217
698
*/ The diet contained 0.1 or 0.5 ppm organic selenium and also supplemented with fish oil
(FO)
Did you know that main form of selenium in the egg is SeMet
and this can explain why organic selenium is much
more effective than selenite in transferring to the egg?
Results of our experiments indicate that the Se concentration in egg yolk was
significantly higher than that in the albumen in all groups. However, it is interesting
to note that the albumen response to Se supplementation was higher in comparison
to egg yolk. For example, when chickens were fed a semi-synthetic diet, egg yolk
Se content was almost 5-fold higher in comparison to the albumen. When organic
Se was included in the diet at 0.4 ppm, this difference was reduced to 2-fold.
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Similar results were obtained in experiments with quail. For example, two groups
of quail (3 families in each group consisting of 4 females and 1 male) were formed
at the beginning of the reproductive period. They were fed a commercial maizebased diet containing 0.1 ppm feed-derived Se, supplemented with 0.2 ppm selenite
Se (control group) or 0.5 ppm organic selenium (Sel-PlexTM) for 6 months after
which eggs were analyzed and incubated in standard conditions (Karadas et al.,
2004). Selenium concentration in egg yolk and egg white significantly increased
as a result of organic selenium supplementation (Figure 7.3). The highest increase
(8.8-fold) was observed in egg albumin, while Se concentration in egg yolk
increased only 2-fold.
900
Control
800
Sel-Plex
700
600
500
400
300
200
100
0
White
Yolk
Shell
Figure 7.3 Selenium in quail eggs, ng/g (Karadas et al., 2004)
During incubation Se accumulated in the egg was transferred to the developing

embryo. As a result the liver Se concentration in day old chicks obtained from the
eggs enriched with this element was significantly higher compared to those from
the control eggs (Figure 7.4). Therefore, Se concentration in the egg yolk and in
the liver of the newly hatched chicks depends on the Se level in the maternal diet.
However, there was a high individual Se variation in the yolk and albumen. This
may account in part for the lack of significant difference in Se level in yolk and
albumen between the groups fed the semi-synthetic and commercial diets (Group
1 and 2) even though the level of Se in the diet differed substantially. It seems
likely that inclusion of fish oil in the parental diet can affect ability of the embryos
to assimilate selenium. Indeed, dietary fish oil did not change Se content of the
egg, however, Se concentration in the liver of the newly hatched chick increased
by 42% and by 36% in the breast muscle. However, at high dietary Se
supplementation, this effect of fish oil was not seen (Pappas et al., 2004: 2004a;
Table 7.5). In general, due to organic Se supplementation (0.4 ppm) of the maternal
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diet the Se concentration in the liver and breast muscle of the newly hatched
chicks increased by 3.6 and 4.1-fold respectively.
1800
Liver
1600
Brain
Selenium (ng/g)
1400
1200
1000
800
600
400
200
0
1
Dietary groups
Figure 7.4 Selenium concentration in the liver and brain of day old chicks (Adapted from Surai, 2000)
Did you know that increased Se concentration in the egg is

related to increased GSH-Px activity in tissues of the
chickens at hatch and in early postnatal development?
There is tissue specificity in selenium transfer from egg to the embryo. For
example, in contrast to the liver, there was only a trend (did not reach significance)
toward increased Se accumulation in the brain of chickens hatched from the egg
enriched in selenium (Figure 7.4). In contrast to chickens, Se enrichment of quail
eggs was associated with a significant increase in the Se concentration in the
brain of the newly hatched quail (Karadas et al., 2004). In comparison to the
control birds the highest increase in selenium level was observed for the liver
(3.5-fold) with a smaller (about 2-fold) increase in other tissues (Karadas et al.,
2004; 2004a; Table 7.6).
Our data indicate that selenium in maternal diet affects Se concentration in
tissues of postnatal quail. Indeed, when newly hatched quail from Se-enriched
eggs and normal quail eggs were placed on low Se-diet (0.1 ppm) the selenium
concentration in tissues dropped dramatically for the first 2 weeks posthatch (Table
7.6). This finding suggests that Se accumulated in the liver of newly hatched
quail is actively used during the first days post-hatch. It is possible to suggest that
Se absorption from the diet is not sufficient during the first few days of life and
the chick must rely on reserves of the element accumulated during embryogenesis.
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Table 7.6 Effect of Se in the maternal diet on the Se concentration in quail tissues, ng/g (Adapted from
Karadas et al., 2004).
Group
Liver
Breast
Leg
Brain
Control
Experimental
253.8
885.4
Day old quail

186.1
160.6
375.2
288.9
136.3
238.5
Control
Experimental
167.8
326.3
7 day old quail

101.0
95.5
147.4
165.3
151.9
250.6
Control
Experimental
79.0
149.7
14 day-old quail
37.3
34.4
53.6
52.5
105.5
152.8
*/ Basic diet contained 0.096 ppm Se. Control diet was supplemented with 0.2 ppm Se in the form of
sodium selenite and experimental diet was supplemented with 0.5 ppm Se in the form of Sel-Plex
However, the difference in selenium concentration between control and

experimental groups was significant at 2 weeks posthatch. These results clearly
indicated that the maternal diet affects not only newly hatched quails, but also
chicks in postnatal development. When similar experiment was conducted with
chicken breeders, it was shown that the maternal effect of Se was also significant
at 14 days posthatch (Table 7.7, Pappas et al., 2005). Indeed, pre-hatch maternal
nutrition affected the progenys Se status leading to significantly increased Se
concentration in the chicken liver two weeks posthatch and potentially could
affect antioxidant defences of the chickens.
Table 7.7 Effect of Se in the maternal diet on the Se concentration in chick tissues, ng/g (Adapted from
Pappas et al., 2005b).
Group
Liver
Control
Experimental
180
717
Control
Experimental
215
300
Control
Experimental
225
280
Brain
1 day-old chick
125
210
7 day-old chick
124
154
14 day-old chick
126
145
*/ Control chicks were hatched from laying hens fed the diet containing 0.1 ppm selenium and
experimental chicks were hatched from laying hens fed on 0.5 ppm Se. Both control and experimental
chicks were fed diets containing 0.2 ppm Se
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Recently a new experiment was conducted at SAC to address this question (Surai,
Karadas and Pappas, unpublished). Maternal diet was supplemented with 0.4 ppm
organic selenium in the form of Sel-Plex and comparison was made with a basic
diet containing 0.1 ppm feed-derived selenium. As a result of dietary Se
supplementation the Se concentration in the egg yolk, albumin, shell, shell
membrane and perivitelline membrane was significantly increased (Karadas et
al., 2005). The newly hatched chicks were placed on basal diet (0.1 ppm) without
Se supplementation for the next 4 weeks posthatch. After hatching, chickens fed
diets low in Se (0.1 ppm) but originating from parents fed diets high in Se (0.5
ppm) had, up to 4 weeks post-hatch, significantly higher blood Se levels than
those originated from parents fed diets low in Se (0.1 ppm; Pappas et al., 2005a).
Furthermore, our results indicate that maternal diet affected selenium concentration
in the liver up to 3 weeks posthatch and in the breast muscle and up to 4 weeks
posthatch. GSH-Px activity in the liver and muscles was also elevated. Taking
into account recent data presented by Koutsos et al. (2003) showing similar effect
of carotenoids in the maternal diet on the carotenoid concentration in 4-week-old
chickens, it is possible to suggest that Se and probably carotenoids could affect
gene expression during the embryonic development. As a result, Se/carotenoid
assimilation in postnatal development could be affected. Alternatively, antioxidant
system could be affected and less Se/carotenoids being used for metabolic needs
and higher concentrations of these compounds were observed in tissues. Indeed,
this hypothesis needs further clarification, however, it is clear that maternal effect
is seen beyond newly hatched chicks.
Did you know that increased Se concentration in the egg is
responsible for the increased Se levels in tissues of chickens
up to 4 weeks posthatch?
Vitamin E accumulation in the egg yolk reflected its level in the breeder diet and
varied with Se supplementation (Figure 7.5). Dietary organic Se significantly
increased vitamin E level in the yolk, but a combination of selenium and increased
vitamin E supplementation did not further increase vitamin E accumulation in the
egg yolk. Similarly, inclusion of Sel-Plex in fish oil-supplemented diet was
associated with a significant increase in vitamin E concentration in the egg yolk
(Narahari et al., 2004). Vitamin E in the liver (Figure 7.6), yolk sac membrane
(Figure 7.5), plasma and brain of day old chicks also reflected vitamin E levels in
egg yolk. Again there was a positive effect of selenium supplementation of the
maternal diet on the levels of vitamin E in the liver, brain and blood plasma of day
old chicks. Over the first 10 days of age, liver vitamin E rapidly decreased (Figure
7.6). A positive effect of Se and vitamin E supplementation of the maternal diet
was seen at day 5 and day 10 of age when vitamin E concentrations in the liver
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and plasma were significantly elevated compared with those of the control group
(Surai, 2000b).
2500
Egg yolk
YSM
-tocopherol (g/g)
2000
1500
1000
500
0
1
Dietary groups
Figure 7.5 Vitamin E concentration in the egg yolk and yolk sac membrane of day old chicks (Adapted
from Surai, 2000)
1800
Day old
5 day old
10 day old
1600
-tocopherol (g/g)
1400
1200
1000
800
600
400
200
0
1
Dietary groups
Figure 7.6 Vitamin E concentration in the chick liver (Adapted from Surai, 2000)
This study showed that one of the important features of chick postnatal
development is the depletion of vitamin E in the liver. Selenium supplementation
of the maternal diet increased the vitamin E levels in the liver and plasma of day
old chicks; and this difference was maintained through 10 days of postnatal
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development. Increased vitamin E supplementation of the maternal diet was even

more effective, delaying vitamin E depletion not only in the liver but in the brain as
well. These data explain why it is difficult to produce symptoms of vitamin E and Se
deficiency in chicks during postnatal development if the maternal diet contains
sufficient levels of vitamin E (Hassan et al., 1990).
Did you know that there is a suggestion that the increased Se
concentration in the egg could affect gene expression in the
developing chickens during embryogenesis?
The most striking finding of this work was the sparing effect of selenium on vitamin
E metabolism and transfer to the egg yolk and the developing tissues. This is in
agreement with previous reports indicating an increased vitamin E level in the plasma
of rats, chicken and ducklings as a result of Se supplementation (Scott et al., 1977;
Thompson and Scott, 1970; Dean and Combs, 1981). The mechanism for this sparing
is not clear. The effect could be related to Se antioxidant properties. One can also
speculate that Se can affect other aspects of vitamin E metabolism and transport to
target tissues. For example, vitamin E is metabolised more rapidly in selenium deficient
rats than in supplemented rats (Fisher and Whanger, 1977).
Another important finding in this study was the beneficial effect of organic Se
supplementation on the level of reduced glutathione in the liver of newly hatched
chick (Figure 7.7). The highest vitamin E dose in the maternal diet increased the
concentration of glutathione in the liver of newly hatched chicks as well. Similar
results were obtained with rats fed on a high level of vitamin E (Scott et al., 1977; Lii
et al., 1998).
1200
Day old
GSH (g/g)
1000
5 day old
800
600
400
200
0
1
Dietary groups
Figure 7.7 Effect of maternal diet on reduced glutathione concentration of chick liver (Adapted from
Surai, 2000)
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Did you know that a sparing effect of Se on vitamin E

metabolism was proven in the developing chickens?
GSH-Px activity in the liver of day old chicks depends on Se provision of the
maternal diet (Figure 7.8). Low dietary Se content was associated with decreased
Se in the egg yolk; and consequently liver Se-GSH-Px activity in newly hatched
chicks significantly decreased. Similarly, chicks produced from hens fed a low
Se/low vitamin E diet had low activities of GSH-Px in plasma and pancreas at
hatch (Bunk and Combs, 1981). On the other hand, dietary Se supplementation
increased Se-GSH-Px activity in the liver (Figure 7.8) and pancreas (Bunk and
Combs, 1981). An efficient carry-over of Se and vitamin E from hens to their
progeny was indicated by a significant increase in muscle Se, liver GSH-Px activity
and vitamin E content at hatch (Hassan et al., 1990). There was no difference in
Se-GSH-Px activity in chick liver in response to increasing dietary Se
supplementation from 0.2 to 0.4 mg/kg; which probably means that inclusion of
0.2 mg/kg Se in the maternal diet provides enough Se to the egg and embryonic
tissues to meet the requirement for maximum Se-GSH-Px activity. However, it is
necessary to take into account that the experiment was conducted in wellcontrolled laboratory conditions and therefore in commercial condition Se
requirement could be higher.
Day old
5 day old
10 day old
45
Se-GDH-Px activity (U/g)
40
35
30
25
20
15
10
5
0
1
Dietary groups
Figure 7.8 Glutathione peroxidase (Se-GSH-Px) activity in the chick liver (Adapted from Surai, 2000)
GSH-Px activity in the liver increased throughout embryonic development,

reaching its maximum at hatching (Surai, 1999a). In the liver of the newly hatched
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chick, Se-dependent GSH-Px is the major form of the enzyme comprising about
61% of total activity (Surai et al., 1999). In the majority of the tissues of the newly
hatched chick there was a highly significant correlation between Se level and the
activity of Se-GSH-Px (Surai et al., 1999). It is interesting to note that in chicken
liver about 28% of GSH-Px activity is represented by the monomeric form of the
enzyme (Miyazaki and Motoi, 1992). It has been suggested that the effect of Se
on the activity of GSH-Px is achieved through pretranslational mechanisms,
including Se-GSH-Px gene expression and cytosolic mRNA stabilisation
(Christinsen and Burgner, 1992). Further, dietary Se can also regulate the level of
GSH-Px mRNA in the post-transcriptional step (Toyoda et al., 1990). Therefore
GSH-Px mRNA is a primary target of the Se regulatory mechanism (Weiss et al.,
1997).
Tissue susceptibility to peroxidation significantly decreased between days 1
and 5 of age (Figure 7.9). MDA accumulation in the liver of day-old and 5-day
old chicks from antioxidant-supplemented hens was significantly reduced.
Therefore, liver susceptibility to lipid peroxidation substantially decreased in
postnatal development despite decreasing vitamin E and carotenoid concentrations.
30
Day old
5 day old
25
MDA (g/g)
10 day old
20
15
10
5
0
1
Dietary groups
Figure 7.9 Malondialdehyde (MDA) accumulation in the chick liver (Adapted from Surai, 2000)
This can be explained as a result of increased glutathione (Figure 7.7) and GSHPx activity (Figure 7.8) as well as of lipid composition changes (Noble and Cocchi,
1990). In fact, MDA accumulation in the chick liver in groups 3-6 was similar
and significantly lower than the controls (commercial diet). This means that
antioxidant protection afforded by increased GSH-Px activity is equal to dietary
inclusion of 40-100 mg/kg vitamin E. Similarly, in another experiment inclusion
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of 0.5 ppm Se or its combination with vitamin E for five weeks increased activities
of GSH-Px and SOD and decreased MDA in tissues, confirming the antioxidant
protective effects of Se (Huang et al., 1998). An inverse linear correlation was
found between lipid peroxide concentration and Se-GSH-Px activities in various
tissues of rats (Gromadzinska et al., 1988).
Did you know that increased Se concentration and activity of
GSH-Px in the embryonic tissues are associated with
decreased tissue susceptibility to lipid peroxidation?
The benefit of organic selenium use in breeder diets lies in its efficient absorption,
transport and accumulation in egg and embryonic tissues. This results in improved
antioxidant status of the newly hatched chick. As the levels of major natural
antioxidants (vitamin E and carotenoids) in chicken tissues progressively declined
after hatch, the antioxidant enzymes become a critical arm of antioxidant defence.
Therefore, enhanced GSH-Px activity in tissues as a result of organic Se
supplementation of the maternal diet may have a positive impact on chick viability
in the first few weeks post-hatch. An improved antioxidant system of the chick
may also enhance immune system function, which is extremely important at this
point in physiological development. The sparing effect of organic Se on vitamin
E also presents the possibility of further improvement of antioxidant defence of
the chick. Indeed, the source and level of Se has a large influence on the amount
of Se transferred to the developing embryo. It would also appear that the embryo
absorbs greater amounts of Se during days 10 to 15 of incubation than during
other periods. Improving the transfer of Se from the hens diet by using a Se-yeast
instead of inorganic sodium selenite is a useful strategy to improve the nutritional
status of the embryo as well as that of the newly hatched chick (Cantor et al.,
2003).
Selenium and digestive and immune system development in birds

The yolk sac content is the main source of nutrients during the first 2-3 days after
hatching and after this time the main energy source for the chick changes from
yolk-based lipid to dietary carbohydrate (Vieira and Moran, 1999). In fact, during
the first day after hatch, approximately 80% of residual yolk fat is transferred to
the tissues (Dibner, 2000). The digestive and absorptive activities of the intestine
are actively developing during first 2 weeks posthatch (Vieira and Moran, 1999)
and this development is also associated with polyunsaturated fatty acid metabolism
and possible lipid peroxidation. At the same time the ability to utilise carbohydrates
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is developing (Siddons, 1969) and by 4 days after hatch the ability to digest
starch can reach 85% complete (Noy and Sklan, 1995). In contrast pancreatic
lipase activity increases until 16 days after hatching (Vieira and Moran, 1999).
Ontogenic changes after hatching include increased levels of pancreatic and
intestinal enzymes, increases in gut surface area for absorption and changes in
nutrient transporters (Dibner, 2000). The intestine reaches a maximum growth
between 3 and 7 days posthatch (Murakami et al., 1992). In addition, early access
to food stimulates the growth of the intestine and its absorptive capacity (Moran,
1985). On the other hand, a delay in access to food and absence of stimulation
from food intake causes shortening and thinning of the villi (Michael and Hodges,
1973; Vieira and Moran, 1999) which would result in decreasing absorptive
efficiency of the intestine. Indeed, under commercial conditions, chicks may be
delayed access to feed for a considerable time after hatch and this increases the
likelihood of ketosis and dehydration (Vieira and Moran, 1999). The delays
between emergence and access to food and water resulted from the time spent in
the hatchery and transportation to the farm and could be considered as substantial
stress conditions. Chicks hatching early may be held 36 hours longer than those
hatching late. Because of the asynchrony of chick emergence eggs from older
breeders and smaller eggs tend to hatch earlier (Vieira and Moran, 1999). This
could result in dehydration and a shortage of available energy and lead to
subsequent reduction in the rate of nutrient absorption, growth rate and increase
early mortality. Immune system development is also compromised due to this
stress (Wyatt et al., 1986; Casteel et al., 1994).
Did you know that improved Se status of the chicks in early
postnatal development could improve chicken immunity and
the development of important physiological functions?
The delay in food and water intake is associated with a delay in the maturation of
the enzymatic systems that control metabolism (Decuypere et al., 2001), therefore,
holding chickens without feed (in commercial conditions many birds would have
an access to feed only 36-48 hours after hatching) leads to decreased body weight
and a decrease in the performance of the broiler (Noy and Sklan, 1999; Decuypere
et al., 2001). It is necessary to underline that any birds with restricted nutrition
early after hatching are not able to recover completely and do not reach the same
weight gain as those that are fed early (Vieira and Moran, 1999). Therefore, delayed
availability of feed and water is associated with an increased early mortality, lower
placement weight, poor four week and long term performance (Dibner, 2000). In
addition, these stress conditions put antioxidant defences under the pressure and
optimal dietary antioxidant supplementation of the maternal diet can help to
maintain high chicken viability.
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It is generally accepted that the ability to absorb dietary lipids is not well
developed in the newly hatched chick but improves with age and it is recommended
to avoid long chain saturated fatty acids and to use unsaturated fat during at least
the first week posthatch (Vieira and Moran, 1999; Noy and Sklan, 1995). It is
obvious that vitamin E is poorly assimilated from the diet during this period (at
least during first 5 days posthatch) when it is extremely important as a natural
antioxidant and as a result the chicken actively use tocopherol reserves accumulated
in the liver during embryonic development. At this period of the development
selenoproteins seem to provide essential antioxidant defences.
In the newly hatched chick, the immune system is immature and it is not
completely functional. At the time when a chicken is placed in poultry house it is
poorly protected from various infections, since its immune system is based mainly
on maternal antibodies (IgG and IgA) transferred from breeders via the egg. This
is a major immunological protection in the first week of chicken life. However,
the immune system is actively developing in postnatal life and this process involves
accumulation of PUFAs and increased susceptibility to lipid peroxidation. As a
result, natural antioxidants are considered to be absolutely essential for the
successful development of chicken immunity.
The first week of chicken life is a period of rapid expansion of leukocyte
populations, seeding of lymphoid organs, and educational events forming the
unique clones of lymphocytes mediating immunity at later stages of the growth
and development (Klasing, 1998). For example, the heterophils (the avian
counterpart to the mammalian neutrophil) of chickens are functionally immature
for the first 4-5 days posthatch and day old chicks are not able to activate them
(Kogut et al., 1998). Peripheral blood counts demonstrated no differences in the
percentages of heterophils during the first week post-hatch and the phagocytic
index of the heterophils did not change on day 1 or day 4, but doubled by day 7
(Wells et al., 1998). When heterophils (H) and lymphocytes (L) were measured in
embryos that had pipped into the air cell and through the shell, and in chicks from
1-d-old to 8 d of age, numbers of L in the perinatal stages were very low with H:L
ratios greater than 5.0. Posthatching H:L ratios decreased in a quadratic manner
from 1.76 at hatch to 0.39 on day 8 (Zulkifli and Siegel, 1994).
It is important to mention that in poultry, the heterophils are the primary cells
in the innate immune response to early bacterial invasion. Moreover, independently
of the efficiency of vaccination, at least 7-10 days are required for the stimulation
of the acquired immune system for protection (Kogut et al., 1998). In chickens
the bursa, producing B-cells, reaches maximum size and activity at about 2 weeks
after hatching. At the beginning the bursa is able to express only IgM and later
development of this lymphoid organ results in the appearance of circulating IgA
and IgG.
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Furthermore immune cells use free radicals as an effective weapons to kill pathogen
(Kettle and Winterbourn, 1997) and as a result the surrounding tissues could be
damaged if antioxidant protection is not appropriate (Schwarz, 1996). As described
in details in chapter 4, an immune response requires extensive communications
between wide variety of cell types and once cell receptors are damaged by free radical
attack communications are disrupted and immune competence compromised. In stress
conditions the requirement for antioxidant defence substantially increases and the
antioxidant system could be the crucial factor in chicken health. In absence of growth
promoters in chicken feed, the role of natural antioxidants in immune system
modulation is difficult to overestimate. It has been recently appreciated that nutrition
plays a vital role in reinforcing the innate immune system resulting in lower early
mortality and less variation in seven-day body weight and physiological development
(Postmouth, 2001). Therefore a strong immune system becomes a major player in
terms of health maintenance and disease prevention. At the same time it has long
been acknowledged that the first seven days growth determines a body weight at
marketing (Postmouth, 2001).
The generalised scheme of Se involvement in chick embryo development is shown
in Figure 7.10.
Selenium requirement for breeders

The Se requirement of poultry in physiological conditions is thought to be quite
low, varying from 0.06 ppm (laying hen) up to 0.2 ppm (turkey, duck; NRC,
1994; Table 7.8). However in commercial conditions associated with various
stresses the Se requirement increases substantially.
Did you know that Se requirement of chickens presented in NRC
represents only minimal requirement which could be substantially
increased in stress conditions of commercial poultry production?
Although there was no significant difference between 0.2 and 0.4 mg/kg Se
supplementation of the maternal diet in GSH-Px activity in the liver of day old
chicks in our experiment, 0.4 mg/kg Se gave more protection against peroxidation
due to higher levels of vitamin E and glutathione in the liver of day old and 5 dayold chicks (Surai, 2000). Indeed, 0.4 ppm Se supplementation in the breeders
diet significantly increased the Se concentration in all examined tissues (Table
7.17). The age of the breeders had a significant effect on the accumulation of Se
in the tissues. For example, at peak production when breeders where 27 wk old,
those that where supplemented with NRC Se levels in their diet exhibited lower
Se concentration in 3 out of 5 examined tissues compared to the levels that were
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Accumulation of PUFAs (20:4n-6;22:6n-3) in tissues during development
401
Se in the feed
Stress conditions of hatching

(poultry) or birth (mammals)
and free radical production
First line of antioxidant defence (preventive antioxidants)

O2*+SOD = H2O2 (Toxic)
H2O2+GSH-Px = H2O
Low GSH-Px activity may promote lipid peroxidation

via OH* formation from H2O2
Biosynthesis of
selenoproteins
and stabilisation
of their mRNA
Second line of antioxidant defence (chain-breaking antioxidants

ROO* + AO (vit.E) = ROOH (Toxic) ROOH + GSH-Px = ROH (nontoxic)
Low GSH-Px activity is associated with hydroperoxide

(ROOH) accumulation and its toxicity for cells
If antioxidant protection is not effective due to low Se in the diet and
compromised selenoprotein synthesis, lipid peroxidation takes place
Lipid peroxidation
Tissue membrane damages, enzyme inactivation etc.
Hormone, prostaglandin, enzyme synthesis is disrupted and their
levels are compromised
Growth and development are compromised
Viability in early postnatal life decreased
Figure 7.10 Relationship between selenium and embryonic postnatal development (Adapted from Surai,
2000)
Table 7.8 Selenium requirement of birds, mg/kg feed (Adapted from NRC, 1994)
White egg-laying
strains, 0-6 weeks
White egg-laying
strains, 6 weeksfirst egg
Brown egg-laying
strains, 0-6 weeksfirst egg
Brown egg-laying
strains, 6 weeksfirst egg
0.15
0.10
0.14
0.10
Broilers, all ages

0.2
Turkey, all ages

0.2
Japanese quail, all ages

0.2
Duck, 0-2 weeks

0.2
White egg layers with

feed intake 120 g
0.05
Brown egg layers with

feed intake 110 g
0.006*
White egg layers with White egg layers with

feed intake 80 g
feed intake 100 g
0.08
0.06
*/mg/hen/day
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noted when breeders where younger (Pappas et al., 2005b). Therefore, there was an
indication of depletion over time for the breeder hens that were fed NRC recommended
Se levels. On the other hand, hens fed the high Se diets (0.5 ppm total Se) were
supplied with enough Se to maintain their requirements as well as build Se reserves in
the tissues.
Since the process of Se transfer from feed to egg yolk, and subsequently to
embryonic tissues, has received limited attention (Cantor, 1997; Paton et al., 2002),
there is no clear answer as to which level of Se supplementation is optimal for broiler
breeders. However, an analysis of published research and commercial data indicates
that 0.3 ppm organic selenium in the form of Sel-Plex would be a recommended dose
of Se dietary supplementation for breeders. For example, at 21 weeks of age Hubbard
Ultra-Yield broiler breeders (11,600 pullets and 1530 roosters) were placed in each
of two floor barns on two separate farms (Sefton and Edens, 2004). They were reared
on Selenite until placement and after that fed on Selenite or Sel-Plex (0.3 ppm) for 33
weeks of production. Sel-Plex supplementation was associated with improvement of
fertility (by 0.4-4.5%), hatchability of fertile eggs (by 1-6%). As a result, the number
of settable eggs were greater in Sel-Plex group showing an advantage of over 67,000
on one farm and over 8,500 on the second farm at 27 and 30 weeks of production
respectively. An average 4.5 extra chicks per hen capitalized on Sel-Plex treatment
was realized during the field trial. When data were combined for the whole production
period (41 weeks on Farm 1 and 43 weeks on Farm 2) it was concluded that Sel-Plex
in broiler breeder diets was very beneficial from both production and economic
viewpoints (Edens and Sefton, 2003; Sefton and Edens, 2004c). Indeed, 5.63 extra
chicks per hen housed were obtained as a result of Sel-Plex supplementation and it
was calculated that the improved performance resulted in an increased potential revenue
of +$1.17 per hen housed.
Did you know that a great body of evidence indicates that Se
requirement of breeders is 0.3 ppm in the form of Sel-Plex?
From photostimulation (22 wk) Ross 508 pullets were fed a Se-free laying ration (Se), a standard Selenite-supplemented ration (0.3 ppm, Selenite) or Sel-Plexsupplemented (0.3 ppm) ration (Renema and Sefton, 2004). The egg production was
similar, with 175, 173 and 178 eggs produced by 58 wk of age. However, the rate of
lay was affected after 48 wk of age, when hen-housed production was 68% in SelPlex group and 60 and 61% in Se and Selenite groups respectively. In the Sel-Plex
supplemented group settable egg production from 40 weeks (87.4) was higher than
in Se (80.6) or Selenite (83.7) birds. Prior to 34 wk, hatchability averaged 88% in
Sel-Plex eggs compared to 80% in Selenite group and 77% in Se group. Sel-Plex
also reduced shell defects (Renema and Sefton, 2004a; Renema. 2003).
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Two experiments with broiler breeders have been recently conducted in Brazil. In
the first study, conducted at an integrated farm in the state Sao Paolo 42,000 Cobb
breeders were divided in two groups and allocated in four farms (Rutz et al., 2003).
The control group was fed on a corn-soybean meal basal diet containing 0.5 ppm Se
as sodium selenite, while in the experimental diet sodium selenite was replaced by
0.3 ppm Se from Sel-Plex and birds were on these diets throughout the laying period.
In the second trial, conducted in the state of Parana 16270 Cobb breeders were used.
The control diet were supplemented with 0.2 ppm Se in the form of sodium selenite
and the experimental diet sodium selenite was supplemented with 0.3 ppm selenium
as Sel-Plex in addition to 0.2 ppm Se as sodium selenite. The major results of the
experiments are shown in Table 7.9 (Rutz et al., 2003). As can be seen from the
results, replacement of sodium selenite by Sel-Plex gave 1-2 extra chicks per hen
housed. Similar results were reported by Renema (2004) showing increased egg
production at 49-58 weeks (Table 7.10) and chick produced per hen housed (Table
7.11). as a result of replacement of 0.3 ppm sodium selenite by the same amount of
Sel-Plex (Renema, 2004). Recently, it has been shown that embryonic mortality in
eggs laid by 23 wk old broiler breeders was higher in the first and last week of
incubation and significantly reduced as the age of the flock increased. In the 27 wk
old breeders mortality in week 3 of egg incubation was 3.53%, and 10.6% in the soya
oil and fish oils supplemented breeders (Pappas et al., 2005c). Inclusion of Sel-Plex
(0.4 ppm) in the diets decreased mortality to 3.06 and 6.22% respectively.
Table 7.9 Effect of selenium on broiler breeders (Adapted from Rutz et al., 2003)
Trail 1
Selenium
source
Selenite
Sel-Plex
Trail 2
Egg
Chicks/hen
production, % housed
82.28
83.52
81.66
82.61
Egg
Hatchability,
production, %
%
69.75
70.39
Chicks/hen
housed
84.19
84.22
71.09
73.04
Table 7.10 The effect of dietary selenium level and source on egg production traits of broiler breeders
(Adapted from Ranema, 2004)
Se supple- Egg production, Egg production, Total egg

Settable
Unsettable egg
mentation
39-48 wk, %
49-58 wk, % production, egg production, production,
No
No
% of total
No Se
Selenite
Sel-Plex
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75.6
73.1
75.5
403
60.2
60.1
67.7
174.5
172.7
177.7
168.5
168.6
174.6
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3.49
2.37
1.9
404
Table 7.11 The effect of dietary selenium level and source on reproduction traits of broiler breeders
(Adapted from Ranema, 2004)
Embryo mortality and culls
No Se
Selenite
Sel-Plex
Day 1-14,
%
Day 15hatch, %
5.33
3.72
3.52
3.66
3.85
3.14
Reproduction parameters
Fertility, Hatchability,
%
%
86.9
90.1
90.1
77.9
82.5
83.5
Hatch of
fertile, %
Chick
production, No
88.6
91.5
92.5
131.3
139.1
145.3
Did you know that an optimal solution for breeder nutrition

is a full replacement of sodium selenite by organic selenium
(Sel-Plex) in the diet?
It seems likely, that organic selenium at 0.3 ppm could also be effective in turkey
males. For example, sodium selenite (0.3 ppm) was replaced by organic selenium
(Sel-Plex) in the diet of turkey males (Dimitrov et al., 2004). After 30 weeks of
feeding the semen samples were collected and analyzed. After 6 hours of semen
storage, motility decreased in control group by 8.7%, while in semen from SelPlex-supplemented toms motility decreased much less (by 3.95%). The fertilizing
ability of stored semen was 88% in the control group and 90.5% in the Sel-Plexsupplemented group.
In addition, the Se requirement for different physiological functions could
substantially differ. For example, 0.05 ppm Se was adequate to maintain egg
production in laying hens, but at least 0.1 ppm Se was needed to maintain chicken
hatchability and post-hatching performance of progeny (Combs, 1994). When
considering the Se requirement of breeders, the main point to take into account is
its transfer from feed to egg and subsequently to the developing chick. Furthermore,
increased Se supplementation could have an immunomodulating effect.
Unfortunately there is no data available on Se requirement of egg-type breeders
in commercial conditions. Indeed, higher rate of egg production and lower feed
consumption in laying type of breeders indicate that their Se requirement could
be similar or even higher than that in broiler breeders. It seems likely that 0.3 ppm
Se in the form of Sel-Plex could also be effective in the egg-type breeder diet.
However, more research and commercial trials are needed to address this question.
It seems likely that effect of organic selenium on poultry depends on the
preparations used. For example, most of benefits of organic selenium reported
above were shown to be related to selenized yeast in the form of Sel-Plex. However,
other forms of organic selenium could give completely different results. For
example, selenized yeast produced in laboratory conditions in Poland was included
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into the chicken diet for 11 days at 0.5 ppm and comparison with the same amount
of sodium selenite was made (Dobrzanski et al., 2003). There was no difference
in Se availability from both sources and the Se concentration in the egg content
only slightly (by 10.5%) increased. Similarly selenium-malt produced in laboratory
conditions in China and clamed to be organic selenium and fed to laying hens at
0.51 ppm for 24 days was not different from that of sodium selenite in terms of
affecting Se content of the egg (Jiakui and Xialong, 2004). Similarly, inorganic
selenium supplementation in the feed showed no effect on its concentration in the
egg (Salobir et al., 2003).
Selenium requirement for broilers

Requirement of growing chickens in Se could substantially differ depending on
conditions. From the one hand, laboratory studies indicate that biochemical indexes
of Se status (e.g. GSH-Px activity) could be positively affected by inclusion into
the diet of various doses of selenium. For example, inclusion of 0.3 mg/kg Se
from bakers yeast to chickens from hatch to day 35 significantly increased GSHPx activity in erythrocytes, plasma and liver (Arai et al., 1994). Replacing sodium
selenite by Sel-Plex at 0.2 ppm in the diet of growing chicks was associated with
significantly increased GSH-Px activity in chicken blood at 2, 3 and 4 weeks of
age (Mahmoud and Edens, 2003; Figure 7.11). The difference in the GSH-Px
activity in blood was maintained after heat stress (Figure 7.12).
180
2 weeks
160
3 weeks
140
4 weeks
120
100
80
60
40
20
0
Basal (B)
B+0.2SN
B+0.2SP
Basal diet contained 0.26 ppm Se

Figure 7.11 Effect of dietary sodium selenite (SN) and Sel-Plex (SP) on GSH-Px activity in blood of
chickens, mU/mg Hb (Adapted from Mahmoud and Edens, 2003)
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200
180
Pre-stress
160
Post-stress
140
120
100
80
60
40
20
0
Basal (B)
B+0.2SN
B+0.2SP

Figure 7.12 Effect of dietary sodium selenite (SN) and Sel-Plex (SP) on GSH-Px activity in blood of 4
week old chickens, mU/mg Hb (Adapted from Mahmoud and Edens, 2003)
Did you know that when replacement of sodium selenite is

technically difficult, adding Sel-Plex at the top of the selenite
could have a beneficial effect for chickens?
On the other hand, commercial results clearly showed that sodium selenite is a
wrong form of selenium in the chicken diet. Indeed, for the last few years a great
body of evidence has been accumulated indicating that replacement of sodium
selenite by organic selenium in the form of Sel-Plex appears to beneficially affect
broiler performance. For example, body weight of broilers at 42 days of age was
2.38 kg in the control group (diet contained Se at level of 0.28 mg/kg without
additional Se supplementation), 2.43 kg after selenite (0.2 mg/kg) supplementation
and 2.45 kg after Sel-Plex (0.2 mg/kg) supplementation (Edens, 2001). Naylor et
al. (2000) fed diets containing inorganic or organic Se at 0.1 and 0.25 ppm and
found that increasing dietary Se markedly improved feed efficiency. Positive effects
of Sel-Plex on FCR were confirmed by Edens et al. (2001). In Serbia fodder
mixtures supplemented or not with organic selenium (Sel-Pex) or standard inorganic
selenium were fed to broiler chicks during the starter and finisher periods. It was
shown that organic selenium increased broiler chick performance, while selenium
deficient feeds decreased performance and increased chick mortality (Stolic et
al., 2002). Broiler diets containing 0.15 ppm feed-derived Se was supplemented
with 0.15 ppm Se in the form of sodium selenite or Sel-Plex. Chickens fed diets
containing organic selenium demonstrated the best production performance. In
contrast to selenite-supplemented birds, weight gain was improved by 4.2% and
feed efficiency by 9.8%. At the same time mortality was markedly decreased
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from 6.7% in selenite-supplemented group to 0.84% in Sel-Plex supplemented

birds (Vlahovic et al., 1998). Improved growth rate of broilers fed a Sel-Plex
supplemented diet could be also related to the increased concentration of the
active form of thyroid hormone in serum of chickens supplemented with organic
Se (Edens, 2001; Edens and Gowdy, 2004a; Jianhua et al., 2000).
Did you know that major advantages of Sel-Plex for broiler
production include increased growth, decreased mortality
and improved FCR?
A trial involved 2400 male Ross broiler chicks (1 to 42 days of age) was run in
Brazil (Arruda et al., 2004). The addition of 0.1 ppm as Sel-Plex in combination
with 0.2 ppm Se as sodium selenite resulted in an improvement of body weight
gain and FCR as compared to 0.3 ppm selenite. Furthermore, in another experiment,
the addition of 0.2 ppm Se, as Sel-Plex, in combination with 0.1 ppm Se, as
sodium selenite, improves growth performance of broilers (Anciuti et al., 2004).
It is interesting to note that in the experiment all doses of organic selenium (0.1,
0.2 or 0.3 ppm) improved FCR in comparison to birds fed only inorganic selenium.
In a study, conducted in Thailand, a total of 1,920 day-old, male chicks (Cobb
500) were allocated to 6 treatments, including sodium selenite or Sel-Plex at 0.2
ppm or no Se supplementation (Srimongkol et al., 2004). Chicks given Sel-Plex
had highest final body weight and uniformity. The results with several million
broilers showed that body weight and liveability were significantly improved when
selenized yeast (Sel-Plex) replaced selenite in the diet (Edens and Gowdy, 2004;
Table 7.12). Maximum body weight gain and best efficiency of food utilisation
were obtained in chicks fed diets containing 0.50 mg/kg Se and 300 IU/kg vitamin
E (Swain et al., 2000). Indeed an analysis of data obtained in various countries
indicate that replacement of sodium selenite by organic selenium in the form of
Sel-Plex in the broiler diets improved final body weight by 30-198 g and improved
FCR (Table 7.13).
Indeed, positive effects of organic Se on efficiency could be attributed to better
feathering of chickens fed on the diet supplemented with organic Se, particularly
during cold stress conditions (Edens, 1996; 1997; 2001). A group of 720 day-old
Cobb 500 chicks were allotted according to a 3x2x2 factorial design (3
environmental temperatures, 2 levels of Se and 2 sources of Se) and grown for 42
days (Dajhlke et al., 2004). Irrespective of the level of Se in the initial diet, organic
selenium (Sel-Plex) improved feathering during the critical phased of broiler
chicken thermal homeostasis, i.e. up to 21 days of age. Organic selenium induced
more rapid whole body feathering in the slow-feathering males as well as in the
normal-feathering females (Edens et al., 2001a). It is interesting to note that in the
study the influence of organic selenium in the form of Sel-Plex was evident from
21 through 42 d of age. Organic selenium in the form of Sel-Plex at 0.1 ppm
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improved feather score and decreased 24 h drip-loss and showed markedly higher
deposition rate in breast muscle and a lower excretion rate in the excreta compared
to the sodium selenite (Choct and Naylor, 2004).
Table 7.12 Results of field trials of Sel-Plex efficiency (Edens and Gowdy, 2003)
Se, ppm
Body weight, kg
0
Selenite, 0.2
Sel-Plex, 0.2
Selenite+Sel-Plex (0.1+0.1)
FCR
Mortality, %
North Carolina field trial (Arbor Acre male broiler for 42 days)
2.38
1.93
2.3
2.43
1.87
2.5
2.45
1.84
2.3
2.45
1.82
2.3
Selenite, 0.3
Sel-Plex, 0.3
Mexico field trial, Cobb broilers, 48 days

2.442
2.048
12.0
2.640
1.959
9.81
Selenite
Sel-Plex
India field trial, Avian 34 broilers, 35days

1393
1.805
1.84
1452
1.741
1.97
Selenite, 0.15
Sel-Plex, 0.15
Selenite 0.15 + Sel-Plex, 0.15
1.89
1.94
1.92
0.3 Selenite+ 0 Sel-Plex

0.2 Selenite + 0.1 Sel-Plex
0.1 Selenite + 0.2 Sel-Plex
0 Selenite + 0.3 Sel-Plex
Brazil field trial, male Cobb broilers, 42 days

2285
1.823
2.0
2380
1.729
2.2
2380
1.749
2.2
2392
1.730
2.3
Thailand field trial

2.11
1.99
1.73
5.63
6.72
6.94
Table 7.13 Field results of replacement of Selenite by Sel-Plex (Edens and Gowdy, 2004)
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Country
Body weight, g
FCR, feed/BW gain
USA
Brazil
Mexico
Thailand
India
+30
+126
+198
+50
+59
-0.03
-0.093
-0.089
-0.120
-0.061
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The form of dietary Se supplementation also appears to affect cut-up yields of

broilers, though data are limited. For example, inclusion of organic Se into the
diet improved eviscerated weight and breast yield of broilers (Naylor et al., 2000).
In another experiment (Edens, 2001), a percentage of carcass weight, yield of
viscera, feet, neck and leg yields were increased when organic Se was added to
the diet. However, in the same experiment pectoralis major yield decreased as a
result of organic Se supplementation (Edens et al., 2000). Again, a Se effect on
meat yield could be due to changes in thyroid hormone metabolism.
In commercial poultry production stress conditions are a common factor
decreasing productive and reproductive performance of birds. In this respect,
increased antioxidant supplementation is beneficial in preventing detrimental
effects of such stresses (Surai, 1999; 2000; Surai and Dvorska, 2001). Among
natural antioxidants in poultry feed, Se has a special place as a part of the first and
second levels of antioxidant defence. For example, in stress conditions (challenge
with E. coli) organic Se decreased production of heat stress proteins (Edens, 2001);
and as a result improved body weight (2291 vs. 2004 g) and feed conversion
ratio (1.84 vs. 1.98) and reduced mortality (5.7 vs 18.3%). Therefore, challenge
birds with pathogenic E. coli caused increased mortality and decreased body
weight. At the same time, replacement of sodium selenite by Sel-Plex substantially
decreased mortality and improved chicken growth (Table 7.14). Positive effects
of Se on stressed poultry were noted when immunostimulating properties of this
element were considered.
Did you know that organic selenium in the chicken diet could
help dealing with oxidative stress caused by various
environmental factors?
Table 7.14 Effect of Sel-Plex and E. coli on performance at 42 days of age of male broiler chickens
(Edens and Gowdy, 2004)
Body weight, g
No Selenium, no E. coli
Sel-Plex, no E. coli
No selenium + E. Coli
Sel-Plex + E. coli
2047
2283
1896
2098
FCR, g/g
1.95
1.82
2.09
1.93
Mortality, %
16.7
5.0
36.7
15.0
Recently, it has been shown that Se supplementation at 0.4 mg/kg for White
Leghorn type chickens reduced death or lesions from E. coli or sheep erythrocyte
antigen challenge from 86 to 21% and dietary additions of Se between 0.1 and
0.8 mg/kg resulted in a substantial (77%) antibody titre increase in chickens (Larsen
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et al., 1997). Similarly, inclusion of increased levels of Se (0.6 mg/kg) in the

chicken diet decreased morbidity and mortality from Mareks disease, increased
the ability to remove oxygen free radicals and lipid peroxide, and alleviated the
degree of tissue damage caused by oxygen-free radicals (Huang and Chen, 1996).
In another experiment, chicks were given basal diets containing 0.086, 0.3 or 0.6
mg/kg Se. They were infected with infectious bursal disease virus at 39 days of
age. Ten days later the mortality rates were 33.3, 12.4 and 10.6%, respectively;
and the infection-induced inhibition of T lymphocyte transformation was less in
the Se-supplemented birds (Bu et al., 1996). It seems likely that organic selenium
provided better protection in stress conditions than sodium selenite. Indeed, the
response of heat shock protein (hsp70) to heat stress was different in these two
groups: in the selenite-fed group it is increased, but in Sel-Plex fed group decreased
(Figure 7.13, Mahmoud and Edens, 2003). Furthermore, when chickens were
subjected to cold stress and fed aflatoxin-contaminated feed, a combination of
0.1 ppm organic Se with 500 mg/kg vitamin E decreased incidence of pulmonary
hypertension syndrome (PHS) and decreased mortality (Stanley et al., 1998).
Similarly, substitution of inorganic Se with organic Se significantly reduced
mortality associated with ascites in cold stressed broiler chickens while a
combination of organic Se (0.3 ppm) with vitamin E (250 ppm) in the diet (Roch
et al., 2000) gave the best protection from ascites.
5.5
Pre-stress
5.0
Post-stress
4.5
4.0
3.5
3.0
Basal (B)
B+0.2SN
B+0.2SP

Figure 7.13 Effect of dietary sodium selenite (SN) and Sel-Plex (SP) on hsp70 concentration in liver of 4
week old chickens, ng/mg TP (Adapted from Mahmoud and Edens, 2003)
In general, analysis of the published data indicates that selenium supplementation

at 0.2-0.3 ppm in the organic form (Sel-Plex) provides optimum Se status for
chicken growth and development. However, for immunomodulation doses of Se
could be higher and need further investigation.
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Se and meat quality

Selenium can affect meat quality by decreasing lipid peroxidation during meat
storage. It seems likely that there is species-specific difference in the Se metabolism
in muscles. For example, a comparison of GSH-Px activity in muscle of chicken,
turkey and duck fed similar Se levels in the diet indicated that GSH-Px activity in
duck muscles was several fold higher than that in chickens or turkey (Figure
7.14).
5.0
4.5
Breast
4.0
Thigh
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
Chicken
Turkey
Duck
Se content in the feed, ppm: chicken: 0.42; turkey: 0.49; duck: 0.44
Figure 7.14 GSH-Px activity in muscles, U/g (Adapted from Daun and Akesson, 2004)
It is interesting to note that duck muscles are characterised by increased Se level,

but the difference in Se concentration between duck and chicken muscles comprises
only 19-37% (Table 7.15). Indeed, when organic selenium was fed to the growing
broilers at 0.2 ppm, selenium concentration in the breast muscle increased more
than twice in comparison to the same dose of selenite (Krikova et al., 2003; Figure
7.15).
Table 7.15 Species-specific differences in Se content in muscles, ng/g (Adapted from Duan and
Akesson, 2004)
Species
Breast muscle
Thigh muscle
Chicken1
Turkey2
Duck3
109.1
67.4
148.5
116.6
109.5
139.1
1,2,3
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/selenium in the diet was 0.42, 0.49 and 0.44 ppm respectively
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18
Blood
16
Breast M
14
12
10
8
6
4
2
0
Basal (B)
B+0.2SN
B+0.2SP
B+0.7SP
Figure 7.15 Effect of dietary sodium selenite (SN) and Sel-Plex (SP) on selenium content in blood (mol/l)
and breast muscle of 7 week old chickens, mol/kg DM (Adapted from Kirikova et al., 2003)
Did you know that increased Se content in chicken muscles

could improve antioxidant defences?
In order to study effects of selenium form on meat quality we stored breast muscle
of breeders from the experiment described above for two years s at 20OC (Surai
and Dvorska, 2002; 2002a; Table 7.16). Dietary supplementation of organic Se at
0.4 ppm significantly increased vitamin E concentration in breast muscle.
Combinations of vitamin E and organic Se in the diet were more effective in
promoting vitamin E accumulation in breast muscle than vitamin E alone. The
semi-synthetic diet was comparatively low in Se; and this caused a significant
decrease in muscle Se-GSH-Px activity. On the other hand, inclusion of organic
Se in the commercial breeder diet increased Se-GSH-Px activity in muscle more
than two-fold. The difference in Se-GSH-Px activity between 0.2 and 0.4 ppm Se
supplementation was not dramatic, but higher Se supplementation was associated
with higher Se-GSH-Px activity. There was also a trend toward increased SeGSH-Px activity in muscle associated with vitamin E supplementation of the diet.
Combination of organic Se and vitamin E supplementation were associated with
the highest Se-GSH-Px activity in muscle.
As a result of significant differences in dietary antioxidant composition, the
initial level of MDA in the breast muscle after two years storage at 20 OC differed
significantly among the various groups. For example, the highest level of the
final product of lipid peroxidation was found in muscle from laying hens fed a
semi-synthetic diet and characterised by the lowest vitamin E and Se-GSH-Px
activity. The lowest initial level of MDA was found in muscle from birds fed diets
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supplemented with either 200 ppm vitamin E or 100 ppm vitamin E in combination
with 0.4 ppm organic Se.
Table 7.16 Effect of selenium and vitamin E on breast muscle quality during storage (n=5) (Adapted
from Surai and Dvorska, 2002)
Dietary Diet
group
1
2
3
4
5
6
7
8
9
Vit. E,
mg/kg
Semi- synthetic
Commercial (CD)
CD
CD
CD
40
CD
100
CD
200
CD
40
CD
100
Breast muscle composition
Se,
mg/kg
0.2
0.4
0.2
0.4
Vit.E,
g/g
1.34 a
1.66 a
1.88 a
2.14 b
2.55 bd
4.88 c
6.11 c
3.22 d
5.88 c
Se-GSHPx, %
63.5 a
100 b
226.4 c
257.2cd
109.3b
122.2b
119.4b
265.4cd
305.9ed
MDA, MDA, MDA, Feinitial

free stimulated
level, g/g peroxperoidation, xidation,
g/g
g/g
2.46 5a
1.55 b
0.82 3c
0.56 df
0.73 cd
0.51 ef
0.33 g
0.5 ef
0.31 g
3.96 a 10.22a
2.49 b
7.66b
c
1.44
3.28 c
df
0.81
2.99 c
ed
1.05
2.93 c
edf
0.72
2.06 ce
gf
0.66
1.01 d
gf
0.62
1.66 de
gf
0.51
0.96 fd
Values are means S.E.M; values in a column which do not have a common superscript are significantly
(P<0.05) different
a
The level of selenium in the semi-synthetic diet was 44 g/kg and in commercial diet 171 g/kg.
Selenium was supplemented in the form of Sel-Plex. Semi-synthetic and commercial diets contained 4.86
and 10.05 mg/kg -tocopherol. Both, commercial and semi-synthetic diets were balanced in other
nutrients.
Did you know that long-term meat storage at -20C is associated

with an accumulation of products of lipid peroxidation and
increased Se level in the meat could inhibit this detrimental
process?
Spontaneous and iron-stimulated lipid peroxidation in the muscle also reflected
dietary antioxidant composition and were lowest in muscle from birds fed diets
supplemented with both vitamin E and organic Se, or with high levels of either
vitamin E (100 or 200 ppm) or Se (0.4 mg/kg) alone. Therefore our results (Surai
and Dvorska, 2002; 2002a) showed that inclusion of organic Se in the breeder
diet can have beneficial effects not only on egg quality as shown previously
(Surai, 2000), but in addition breeders themselves benefit from better antioxidant
protection. In our case breast muscle storage stability was significantly improved
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as a result of dietary Se or Se+vitamin E supplementation. Considering consumer

demand for healthier meat, a combination of organic Se with increased vitamin
E supplementation could be an effective means of meeting these demands.
However, this approach requires further investigation with broiler chickens. Similar
selenium accumulation in the breast muscle as a result of dietary Sel-Plex
supplementation and its protective effects on prevention of MDA accumulation
during chicken breast muscle storage at 20C for 16 weeks was shown in Serbia
(Olivera Pesut, Personal communication).
Additionally, Avanzo et al. (2001) have shown that deficiencies of -tocopherol
and Se caused multiple alterations in the antioxidant system and adversely affected
the redox state of chicken superficial pectoralis muscle (Figure 7.16). It seems
likely that a stabilising effect of Se is also associated with maintaining muscle
membrane integrity. For example, Edens (1996) showed that drip loss was
decreased when organic Se was fed to broilers. Using a model system based on
red blood cell membrane stability, Edens (2001) confirmed membrane-stabilising
effect of organic Se. Edens (1997) also found an interaction between vitamin E
and organic Se in improving yield in growing chickens. In a trial during the spring
season organic Se improved feathering and reduced drip loss. More conclusive
results have been obtained recently in the University of New England in Australia
(Naylor et al., 2000). In this experiment, birds receiving dietary organic Se had
reduced drip loss. Further experiments by Edens (2001) revealed that organic Se
itself was not able to decrease drip loss, but sodium selenite in fact, increased it
due to its pro-oxidant properties. Therefore, replacement of selenite by Sel-Plex
organic Se was associated with decreased drip loss. Similarly, in pigs selenite had
a detrimental effect on loin quality, as reflected by higher drip loss and a paler
colour (Mahan et al., 1999). These data suggest that meat quality during storage
can be improved by inclusion of organic Se in the diet. It is also obvious that
increased Se supplementation (0.25 ppm) in the form of Sel-Plex had additional
positive effects (compared to 0.1 ppm) on prevention of drip loss.
Species-specific differences of the effects of Se and other antioxidants on meat
quality probably reflect differences in the levels of PUFA as well as some other
antioxidants (vitamin E, vitamin C, glutathione) and pro-oxidants (Fe, Cu etc.) in
both tissues and diets. The data indicate that Se is an important element in
maintaining meat quality during storage. It seems likely that optimal combination
of Se and vitamin E would be even more beneficial in preventing lipid peroxidation,
membrane deterioration, peroxide accumulation and maintaining meat freshness
and quality. Since lipid manipulation and enrichment of meat by n-3 fatty acids
are considered an important step in the improvement of human diet (Wood and
Enser, 1997), antioxidant supplementation in such cases could be of great
nutritional importance.
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1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
-E-Se
-E+Se
+E-Se
+E+Se
Figure 7.16 Effect of dietary selenium and vitamin E on lipid peroxidation in chicken muscle
mitochondria, nmol TBARS/mg protein (Adapted from Avanzo et al., 2001)
Se and egg quality

Egg freshness is one of the most important parameters determining consumer
perception and demand. During storage, egg freshness decreases. This process is
associated with biochemical changes in composition and structure of egg
membranes. In experiments conducted in Japan (Wakebe, 1999), inclusion of
Sel-Plex in the layer diet at 0.3 ppm Se/kg increased GSH-Px activity in the egg
yolk and white. The Haugh units were used as an indicator of egg freshness. The
value was high on day 1 in both treatment and control groups with no difference
due to Se supplementation. As time progressed, Haugh units of the control group
declined sharply while the declination was more moderate in the treatment group.
By day 7, it was evident that the Haugh unit is significantly higher in the treatment
group. Similar data were reported by Rutz et al. (2003). In Brazil, Sel-Plex (0.10.3 ppm) was added on top of a commercial premix, containing 0.15 ppm Se
from an inorganic source (Pan et al., 2004). A significant improvement in yolk
color and in Haugh units were observed as a result of organic selenium
supplementation. (Rutz et al., 2003). These results are in agreement with other
data from the same authors (Pan and Rutz, 2003). The data have commercial
significance indicating a possibility to improve egg quality maintenance during
storage.
There are also implications for incubated hatching eggs as well, since long egg
storage is associated with decreased hatchability. Indeed, organic selenium in the
form of Sel-Plex in the breeder diet improved Haugh units of eggs stored for 2
weeks (Pappas et al., 2004; 2004a) and there is an indication that organic selenium
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in the breeders diet could improve hatchability of stored eggs (Edens and Sefton,
2004; Sefton and Edens, 2004b). Furthermore, during storage at 20C lipid
peroxidation was significantly decreased in egg yolk enriched with Se, which
had increased GSH-Px activity (Yaroshenko et al., 2003). When yolk was incubated
at 37C, lipid peroxidation was enhanced and the protective effect of increased
dietary Se was significant (Dvorska et al., 2003). Therefore, inclusion of organic
Se in the diet of laying hens can be used to maintain quality during storage.
Furthermore, inclusion of organic selenium in the chicken diet can also increase
Se concentration in the perivitelline membrane. Indeed, as can be seen from the
Figure 7.17, inclusion of Sel-Plex in the commercial diet significantly increased
the Se level in the perivitelline membrane (Surai et al., 2004c). Therefore, this
could be an additional mechanism of a positive effect of Sel-Plex on egg freshness
during storage. The effect of Se on egg freshness probably depends on age of
hens, composition of diet and conditions of egg storage. For example, Paton and
Cantor (2000) were not able to show an effect of dietary Se form on the Haugh
units.
600
500
400
300
200
100
0
Semi-synthetic
Commercial (C)
C+0.2 Sel-Plex
C+0.4 Sel-Plex
Figure 7.17 Selenium in perivitelline membrane, ng/g (Adapted from Surai et al., 2004c)
Did you know that replacement of sodium selenite by organic

selenium could improve egg freshness during storage?
Organic selenium can also affect egg shell quality. For example, Paton and Cantor
(2000a) showed increased shell breaking strength due to feeding organic Se to
Babcock laying hens at 80 weeks of age. Furthermore, a partial or full replacement
of sodium selenite by organic selenium in the form of Sel-Plex was shown to
increase egg production, egg weight and weight of egg parts including shell, yolk
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and albumin (Rutz et al., 2003). Sel-Plex increased yolk and white weights relative
to control and trends toward improved egg production, weight and FCR were
noted with Sel-Plex addition (Pan and Rutz, 2003). Similarly, replacement of 50%
selenite (total supplemental Se was 0.4 ppm) in the laying hen diet by Sel-Plex
was associated with a significant increase in egg shell weight and thickness (Klecker
et al., 1997; 2001). Inclusion into the quail diet of high selenium (5.7 ppm) wheat
at the level of 60% was associated with increased Se content in tissues by 3-13
fold and eggshell thickness was significantly increased (Stoewsand et al., 1978).
Replacement of sodium selenite (0.2 ppm) in the diet by the same amount of
organic selenium as Sel-Plex significantly increased egg specific gravity after 9
weeks (Renema, 2004). In an experiment conducted in Brazil, ISA Brown laying
hens at age of 77 weeks were supplemented with Bio-PlexTM, a combination of
organic minerals, including Zn, Mn and Se (Xavier et al., 2004). Egg production
and feed conversion were improved. Furthermore, improvement in egg shell quality
(weight, thickness and specific gravity), Haugh units and increased yolk and
albumin weight were also observed.
Selenium was found in all parts of the egg, including shell and membranes
(Figure 7.18).
200
180
160
140
120
100
80
60
40
20
0
Albumin
Egg yolk
Perivitelline
membrane
Shell
membrane
Shell
Figure 18. Selenium distribution in egg, ng/g (Adapted from Surai et al., 2004)
In fact, the highest Se concentration was detected in the shell membrane and Se
concentration in the shell was comparable to that in the albumin. Selenium
concentration in quail shell ranged from 122.5 to 186.5 ng/g, which represented
about 12% of total egg Se (Surai et al., 2004). In comparison, shells of eggs of
roseate terns (Sterna dougallii) and herring gulls (Larus argentatus) in wild contained
about 1-5% of total Se of the egg (Burger, 1994). Inclusion of Sel-Plex in the quail
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diet (0.5 ppm) significantly increased Se concentration in the shell (up to 231.2458.3 ng/g, Figure 7.3). Therefore Se concentration in shells from control birds
comprised 152.6 11.4 ng/g and was almost doubled in the Sel-Plex group, comprising
307.640.5 ng/g. Furthermore, in Se-enriched eggs the shell contained about 9.2%
of the total egg selenium (Surai et al., 2004a; 2004b). This change could possibly
affect shell structure and could be potentially an additional source of Se for the
developing embryo. It has been recently appreciated that organic matrix of the egg
shell is responsible for crystal formation and ultimately determines shell quality.
However, selenium excess could be detrimental for egg production and shell quality.
For example, in Japanese quail hens 7 ppm of dietary Se depressed egg weights with
no effect on eggshell thickness (Stoewsand et al., 1978a).
Did you know that organic selenium could improve
shell quality?
Furthermore, it is possible that medullar bone in laying hens could accumulate Se
to some extent and use it for shell formation. It is interesting that both Se deficiency
and Se excess significantly decreased the biomechanical strength of rabbit bones
while the bones belonging to the Se-adequate control group always had the largest
modulus of elasticity (Turan et al., 1997). Molecular mechanisms of the Se effect on
shell/bone formation are not known at present. However, Se presence in bones has
been shown. For example in humans about 16% of total body Se was found in bones
(Zachara et al., 2001).
Recently a relationship between active vitamin D metabolites and the Se-dependent
enzyme thioredoxin reductase was established (Schutze et al., 1998; 1999). In fact
TrxR was identified as a 1,25(OH)2D3-responsive gene, providing a link between the
induction of a differentiation program by 1,25(OH)2D3 and the expression of the Seresponsive TrxR system in human osteoblasts. Similarly, the expression of TrxR and
its regulation by 1,25(OH)2D3 and selenite in monocytes might be important for
induction of differentiation and maintenance of function. An effect of TrxR activity
on 1,25(OH)2D3 would be expected, and this could be an important link between Se
and bone metabolism. On the other hand, antioxidant properties of various
selenoproteins could also be of importance to maintain antioxidant protection of the
oviduct during egg shell formation.
Practical approaches to improve selenium status of poultry

Poultry diets are supplemented with Se in order to provide a margin of safety against
deficiency, meet physiological requirement in selenium and to maintain high
productive and reproductive performance. In this respect, level and form of dietary
Se are of great importance. During evolution all animals, including birds, adapted to
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metabolising the organic form of Se (Surai and Dvorska, 2001). Indeed feed
ingredients contain Se only in organic form, mainly as SeMet. This means that the
digestive system is ill-prepared for inorganic Se (selenite or selenate). This fact is
underscored by the fundamental differences in absorption and metabolism of inorganic
and organic Se sources. Organic Se (SeMet) is actively absorbed in the intestine as an
amino acid employing similar processes as methionine. In contrast, inorganic Se is
passively absorbed. The chemical similarity between SeMet and methionine allows
the body to use them interchangeably in protein synthesis. This makes it possible to
build Se reserves in the body (mainly in muscle). Our data with chicken (Table 7.17)
and quail (Table 7.18) clearly indicate increased Se concentration in tissues of birds
fed organic selenium in the form of Sel-Plex. Since SeMet is not synthesised in animals
and only plants, bacteria and marine algae can synthesise this amino acid (Schrauzer,
2000), there is little if any Se reserve in the body when inorganic Se is used.
Table 7.17 Effect of various levels of organic selenium (0.1 vs 0.5 ppm) in the diet on the Se
concentration in tissues of laying hens, ng/g (Adapted from Pappas et al., 2004; 2004a; 2005a)
Tissue
0.1 Selenium
0.5 Selenium
Increased, fold
23 weeks
Liver
Kidney
Lung
Breast Muscle
Brain
152
250
120
94
135
Liver
Kidney
Lung
Breast Muscle
Brain
122
256
72
66
136
421
521
251
191
186
2.8
2.1
2.1
2.0
1.4
434
528
257
216
223
3.6
2.1
3.6
3.3
1.6
27 weeks
Table 7.18 Effect of various levels of selenium (0.3 vs 0.6 ppm) in the diet on Se concentration in tissues
of adult quail, ng/g (Adapted from Karadas et al., 2004b)
Tissue
0.3 Selenium*
Liver
Kidney
Leg Muscle
Breast Muscle
326
663
130
133
0.6 Selenium**
Increased, fold
821
1256
357.4
438.3
2.5
1.9
2.8
3.3
*/0.1 ppm Se from the diet + 0.2 Se from Selenite

**/0.1 ppm Se from the diet + 0.5 Se from Sel-Plex
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Did you know that major advantages of organic selenium in
comparison to sodium selenite are associated with Se reserves
built in tissues, which can be used in stress conditions when Se
requirement is increasing and feed consumption is decreasing?
This difference in tissue reserves of Se can explain why organic Se is more effective
than inorganic sources, especially in stress conditions. Consider two different
scenarios for Se supplementation of poultry. The first and most common scenario
is addition of inorganic Se to diets containing low levels of naturally occurring
organic Se (Figure 7.19). When overproduction of free radicals occurs as a result
of stress conditions, the body responds to by attempting to mobilise antioxidant
reserves; and more importantly, by synthesising additional selenoproteins. In this
scenario, the main limitation is absence of Se reserves and a restricted ability to
synthesise additional selenoproteins. Therefore in this scenario we would expect
a decrease in productive and reproductive performance of poultry as well as
compromised immunity defence.
Inorganic selenium scenario

Stress conditions
Absence of Se reserves
in the body
Inability to synthesise
additional
selenoproteins
Overproduction of free
radicals and lipid
peroxidation
Decreased productive and

reproductive performances,
increased susceptibility to
diseases
Figure 7.19 Selenium and stress conditions-1 (Adapted from Surai, 2002)
Selenium reserves are much higher in the scenario where organic Se sources
provide dietary Se (Figure 7.20). The major benefit would come from Se reserves
accumulated in the form of SeMet in muscles. Protein catabolism during stress
conditions releases SeMet to the free amino acid pool, thereby supplying Se needed
for the synthesis of additional selenoproteins to prevent damaging effects of free
radical overproduction. It must be emphasised that SeMet and SeCys are major
forms of Se in the body. However, cells do not contain free pools of these
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421
selenoaminoacids. They are present only as parts of various selenoproteins, or in

the case of SeMet in tissue proteins. When Se is supplemented in the form of
SeMet, this amino acid will be non-specifically incorporated into various proteins
in place of methionine, for example in muscle. This could be an advantage in
comparison with SeCys, since Se-Cys-containing proteins are antioxidants
themselves and more expensive to degrade, but SeMet-containing proteins are
not antioxidants and therefore would be less expensive for body antioxidant
defence. Thus in this scenario, during protein catabolism by proteasomes much
more Se will be released in the form of SeMet in comparison to SeCys.
Organic selenium scenario

Stress conditions
Prooxidants and free
radical production
Se reserves in
tissues
Selenoprotein synthesis and

prevention of lipid
peroxidation
Maintenance of high
productive and reproductive
performances, resistance to
diseases
Figure 7.20 Selenium and stress conditions-2 (Adapted from Surai, 2002)
Differences in Se status between birds that respond effectively to stress conditions

and those that do not are often not dramatic. For this reason, we recognise the
importance of marginal deficiencies in Se and other trace elements. These
differences are very difficult to evaluate, particularly when our practical means of
evaluating Se status tends to focus on only one of the functional selenoproteins.
For example, if hatchability is 82%, is there any room for improvement due to
improved Se status? The answer lies in the extent to which functional aspects of
Se are limiting fertility or chick development. Many aspects of reproduction and
embryonic and early chick mortality are sensitive to antioxidant/pro-oxidant
balance (Surai, 1999; 2002).
While improving Se status cannot counter all the ill effects of stresses such as
very high levels of mycotoxins or environmental temperature extremes, there is a
range of everyday stress conditions on commercial farms where the organic Se
supplementation model/scenario could be effective (Surai and Dvorska, 2001).
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Conclusions
It is quite clear that the roles of Se in avian nutrition and reproduction need new
consideration in light of better understanding of molecular mechanisms of Se action
at the cellular and sub-cellular levels. In particular, discovery and characterisation of
a range of new selenoproteins, better understanding of relationships between different
antioxidants as important parts of integrated antioxidant system with possibilities for
antioxidant recycling in vivo gave a new insight in this matter.
The most fascinating part of Se-related research is coming from understanding a
principal difference between various Se sources in the diet. The digestive system of
animals, including birds, adapted to metabolise organic Se from plant-based feedstuffs
during evolution. Therefore, inclusion of selenite or selenate in the diet is not the
natural situation and differences in assimilation, distribution and accumulation of
Se in tissues depend on source of Se. Furthermore, main Se compound of selenized
yeast, SeMet itself, possesses antioxidant properties, which could be beneficial during
digestion (Surai et al., 2003; 2004d). In contrast, selenite is pro-oxidant and in
combination with iron and zinc potentially could stimulate lipid peroxidation and
cause damages to enterocytes and as a result decrease absorption efficiency of various
nutrients, including antioxidants. In addition, the natural form of Se, selenomethionine,
contributes to Se tissue reserves thereby providing a better chance for animals to
adequately respond to stress conditions by synthesising additional selenoproteins.
However, most of Se-related research in food animals was until recently conducted
using inorganic Se. Therefore much of the data related to effects of Se on various
physiological processes and on the productive and reproductive performance of
animals need to be re-evaluated using natural sources of Se. The data presented
above and summarised in Table 7.19 indicate that replacement of sodium selenite by
organic selenium in form of Sel-Plex in the chicken diet improves antioxidant defences
and has a range of commercial advantages. Indeed, in breeders main advantages
include:
improved fertility
improved hatchability
increased early chick viability
In growing broilers Sel-Plex is associated with:
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better growth
improved FCR
decreased mortality
decreased drip loss
decreased lipid peroxidation during meat storage
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Table 7.19 Advances of organic selenium for poultry
Parameter
Effect of organic
vs inorganic
selenium
References
Chicken sperm morphology

Duration of fertility
Fertility
Hatchability
Egg production of breeders
Chicken early mortality
Se transfer to the egg
Chicken feathering
FCR in broilers
Improved
Improved
Improved
Improved
Increased
Decreased
Improved
Improved
Improved
Chicken growth
Improved
Eviscerated weight and

breast yield in broilers
Drip loss
Lipid peroxidation in
chicken meat
Chicken growth in stress
conditions
Negative effects of heat
stress for chicken
Ascites
Performance of laying hens
Egg freshness during storage
Egg shell quality
Improved
Edens, 2002; Edens and Sefton, 2003

Agate et al, 2001
Edens, 2002; Edens et al., 2003
Edens, 2002; Edens and Sefton, 2003
Renema, 2004
Lanning et al., 2000
Paton et al., 2002; Cantor et al., 1996
Edens, 1996; 1997; 2001; 2002
Naylor et al., 2000; Edens, 2001; Edens
and Gowdy, 2004
Edens, 2001; Vlahovic et al., 1998;
Stolic et al., 2002; Anciuti et al.,
2004; Srimongkol et al., 2004; Edens
and Gowdy, 2004
Naylor, 2000
Decreased
Decreased
Edens, 1996; Naylor et al., 2000

Surai and Dvorska, 2002; 2002a
Improved
Edens, 2001
Decreased
Mahmoud and Edens, 2003
Decreased
Improved
Improved
Improved
Chicks/hen housed
Increased
Se-enriched egg and chicken

production
Se-enriched turkey meat
production
Toxicity
Effective
Roche et al., 2000

Pan and Rutz, 2003
Wakebe, 1998; Pan and Rutz, 2003
Klecker et al., 1997, 2001; Paton and
Cantor, 2000; Rutz et al., 2003
Rutz et al., 2003; Edens and Sefton,
2003; Sefton and Edens, 2004c
Yaroshenko et al., 2003; 2004
Effective
Sims et al., 2003
Less toxic at
high doses
Gowdy et al., 2003
Selenium was detected in egg shell and Sel-Plex can increase Se concentration in
all parts of the egg, including egg shell, shell membrane and perivitelline
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membrane. Replacement of sodium selenite by Sel-Plex in the laying hen diet

was shown to:
improve FCR
improve shell quality
improve egg freshness during storage
decrease lipid peroxidation during storage
One more important advantage of Sel-Plex in poultry production is related to

possibilities of producing Se-enriched eggs, broilers and turkey (see chapter 12).
Applications of Sel-Plex for turkey and waterfowl production also are of great
importance. The data, accumulated for the last 5 years clearly indicate that the
time has come to reconsider Se supplementation of poultry and redefine Se
requirement. Indeed, full replacement of sodium selenite in poultry diets by organic
sources of selenium, such as Sel-Plex, is just the matter of time. Furthermore,
more research should be done with organic sources of Se in order to better
understand and exploit its physiological role, solve Se deficiencies and improve
general health of human and animals.
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Selenium in Pig Nutrition 445

8
SELENIUM IN PIG NUTRITION
Cheap meat never makes a good soup
Introduction
Among farm animals pigs have a special place providing tasty meat and many
different pork-related products. World pig meat production reached 95.8 million
metric tons in 2003 being higher than poultry meat (72.5 million tons) or beef
(61.9 million tons) production (Best, 2004). Indeed, pork is the most popular
type of meat in almost every country in the European Union. The only exception
is the United Kingdom, where poultry meat is slightly more popular than pork. In
2002, the US production of red meat was 47.3 billion pounds, including 19.7
billion pounds of pork and 27.2 billion pounds of beef and pork consumption per
capita was 51.5 pounds (American Meat Institute, 2004). Furthermore, in 2004
pork production in the US is expected to be 20.3 billion pounds, on a slaughter of
more than 102 million head of hogs. Recent achievements in pig breeding and
substantial increase in growth rate and percentage of lean meat is associated with
higher demand for balanced diet providing all necessary macro- and
micronutrients, including vitamins and minerals. In particular, role of natural
antioxidants in pig production is a topic of recent research all over the world.
Did you know that world pig meat production reached
95.8 million metric tons in 2003 being higher than poultry
meat (72.5 million tons) or beef (61.9 million tons) production
Indeed, oxidative stress, related to overproduction of free radicals and toxic
products of their metabolism, is responsible for decreased immunocompetence,
compromised reproduction, poor growth and development of farm animals
including pigs. During last few years deeper understanding of the role of maternal
diet in the health status and development of the progeny has been greatly
appreciated. Among different antioxidant compounds selenium has a special place
been responsible for regulation of antioxidant defences. Restricted transfer of
selenium and vitamin E via placenta makes newly born piglets vulnerable to
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environmental stresses. Therefore, improvement of Se status of neonate by

delivering selenium via colostrum and milk is a priority for pig nutritionists.
Furthermore, optimal Se supplementation of the diet in post-weaning period is
also of great importance for pig producers. The main advances in Se nutrition of
sows and pigs are summarised below.
Se-deficiency
Clinical Se deficiency is a rear event in commercial pig production; however,
sub-clinical Se deficiency could be responsible for decreased productive and
reproductive performance of pigs. Indeed, the incidence of selenium deficiency
is seen most frequently in the neonatal, postnatal and post-weaning periods (Close,
2003). It is well known that most feed ingredients in the USA contain much higher
Se concentrations than the same feedstuff growing in Europe. However, most
production of maize and soya in the USA is concentrated in the Midwest region
with poor Se level in the soils. Therefore, very often the corn and soybean meals
used in American pig diets are Se-deficient.
There have been several important changes in swine industry for the last
20-30 years that have contributed to Se-related problems (Mahan, 1991):
swine confinement has eliminated soil as an additional source of selenium

for pigs
swine confinement and progress in pig selection have resulted in increased
growth rate and increased Se requirement
increased crop yields depleted the soil of Se for incorporation into plants
and usage of synthetic fertilizer applications decreased Se availability for
plants
modern agricultural practises decreased soil pH and further decreased Se
availability from soil to plants
increased usage of simplified grain-soybean-based diets excluded some other
sources of Se from the swine diet
earlier weaning decreased Se reserved accumulated in the body of young
piglets by colostrum and milk consumption
increased sow productivity, early rebreeding, longer retention of sows in
the herd depleted Se maternal reserves
Furthermore, it is necessary to take into account a complexity of selenium-vitamin

E relationship in the body, which was addressed in Chapter 2. Indeed, in many
cases clinical signs of Se deficiency are complicated by simultaneous vitamin E
deficiency. It seems likely, that in many cases the compromised antioxidant system
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of the body (see Chapter 1) and oxidative stress with damages to PUFAs, proteins
and DNA are responsible for those clinical signs of Se deficiency.
There is great body of information showing detrimental consequences of Se
deficiency in swine. Indeed, the main consequences of selenium deficiency in
pigs are similar to those of vitamin E deficiency and include (Jenkins and
Hidiroglou, 1972; Jonsson, 1993; Nutrient Requirements of Swine, 1998; Reid,
2001):
Reduced pig performance (growth rate, feed intake, feed utilization and
the apparent digestion for dry matter, ether extract and nitrogen; Glinke and
Ewan, 1977). For example, selenium-deficient pigs (Se in the feed was 0.019
ppm) had a significantly lower daily food intake and daily weight gain than
control pigs (0.25 ppm Se), whereas, food conversion was identical within
both groups. Selenium concentrations in liver, muscle and serum, as well as
activity of GSH-Px in serum, was significantly lower in selenium-deficient
pigs (Kirchgessner et al., 1995).
Hepatic necrosis (hepatosis dietetica); an acute type with liver failure and
sudden death or a subacute type with ascites and jaundice with edema and
cardiomyopathy. The disease occurs most frequently in young pigs up to 3
months of age. Pigs often dying without clinical signs of deficiency (Jenkins
and Hidiroglou, 1972). Massive necrosis can be seen in the liver of affected
pigs and selenium concentration in the liver substantially decreased (Figure
8.1). Selenium inclusion into the feed or injection can prevent the disease.
The disease can occur in combination with nutritional muscular distrophy
and mulberry heart disease.
0.30
Healthy
NMD
HD
0.25
0.20
0.15
0.10
0.05
0.00
Liver
Heart
Figure 8.1 Se concentrations in tissues of normal pigs and pigs with Mulberry heart disease (MHD) and
Hepatosis diabetica (HD), mg/kg wet weight (Adapted from Pedersen and Simensen, 1977)
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Ulcers in the cutaneous mucous membrane of the stomach and esophagus,

cecal and colonic hemorrhages, possibly gut edema and post-weaning
diarrhea (Mahan, 1991) and bloody faeces.
Nutritional muscular distrophy (NMD, White muscle disease) is
characterised by bilateral paleness and distrophy of the skeletal muscles
and it is frequently observed in combination with hepatosis dietetica and/or
mulberry heart disease. Selenium concentration in major tissues is
dramatically decreased (Figure 8.2). Affected pigs display generelized
muscular weakness and walk with an unsteady gait. There is hyaline
degeneration with loss of striations, vacuolisation, and disruption of muscle
fiber groups. The longissimus, diaphragm and adductor muscles are most
commonly affected by NMD (Jenkins and Hidiroglou, 1972).
2.0
Healthy
1.8
NMD
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
Liver
Skeletal muscle
Heart
Pancreas
Figure 8.2 Se concentrations in tissues of normal pigs and pigs with nutritional muscular
distrophy (NMD), mg/kg DM (Adapted from Lindberg, 1968)
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Mullberry heart disease is dietetic microangiopathy (MAP) and often occurs

in rapidly growing pigs and may be combined with other Se-responsive
conditions such as subacute hepatosis dietetica. It is well known that the
mulberry heart disease of the pig represents a disease with major
manifestation at the heart, with vascular and myocytic lesions and
transudation to the serous cavities. The vascular lesions include fibrinoid
necrosis in intramyocardial small arteries and arterioles with fibrin
microtrombi in the myocardial capillaries (Jonsson, 1993). Affected animals
usually die suddenly from acute cardiac failure, without premonitory signs
(Jenkins and Hidiroglou, 1972). Clinical signs of the disease include dyspnea
and severe muscle weakness and the heart lesions vary considerably affecting
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atria and ventricles bilaterally. Selenium concentration in the liver and heart
is reduced (Figure 8.1).
Impaired reproduction. Total number and number of liveborn piglets and
number of piglets alive at weaning were significantly lower for sows in the
Se-deficient group in comparison to the sow group supplemented with 0.1
ppm Se. Average litter weights at birth and weaning were also lower in the
Se-deficient group (Mihailovic et al., 1989). Adding sodium selenite to Sedeficient diet during gestation (from 60 days before farrowing) and lactation
(28 days) improved weight gain by 7.5-12% and feed utilization by 1420% (Bonomi, 2001). Decreased semen quality (lower motility and increased
percentage of abnormal sperm, lower fertilizing rate of oocytes, MarinGuzman, 1997; decreased number of spermatozoal reserves and Sertoli cells,
Marin-Guzman et al., 2000) and reduced the conception rate of gilts to first
service (Edwards et al., 1977) were characteristic of Se deficiency.
Furthermore, decreased litter size (Mahan et al., 1974) and increased piglet
mortality (Nielsen et al., 1979) were observed as a result of Se deficiency.
Progeny from Se and vitamin E-deficient sows are weak at birth with a
reduced desire to nurse the dam (Mahan, 1991) and smaller litter size was
related to low Se-diet (Mahan and Peters, 2004).
Increased incidences of mastitis, metritis and agalactia and reduced milk
production. These are post-parturient diseases in sows with lactation failure
(Mahan, 1991a; 1994; 1999).
Impaired prolonged farrowing due to poor muscle tone with increased
piglet mortality, lethargic and weak piglets (Acda and Chae, 2002; Close,
2003) and reduced muscle tone and strength with an increased incidence of
stillborn piglets (Close, 1998)
Impaired immune response and increased susceptibility to infection (see
chapter 4)
Reduced tolerance to parenterally administered iron and iron toxicity in
baby pigs
Tissue damage (elevated activities of lactate dehydrogenase, glutamicoxaloacetic transaminase and glutamic-piruvic transaminase in serum)
It seems likely that Se deficiency can affect fatty acid profile of some tissues. For
example, Se-deficient pigs were characterised by increased levels of total and n6 PUFAs in liver neutral lipids and lower levels of total saturated fatty acids in
muscle total lipids than control pigs, whereas the fatty acid composition of serum,
erythrocytes and back fat was not affected by selenium deficiency. Increased
lipid unsaturation, simultaneously with compromised antioxidant defences in Se
deficient pigs could trigger a chain of events related to lipid peroxidation and
leading to various lesions described in literature.
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Did you know that the main consequences of selenium deficiency
in pigs are similar to those of vitamin E deficiency and include:
reduced pig performance; hepatic necrosis; tissue damage; ulcers
in the cutaneous mucous membrane of the stomach and
esophagus, cecal and colonic hemorrhages; nutritional
muscular distrophy; mullberry heart disease, impaired
reproduction and immune system; increased incidences of
mastitis, metritis and agalactia and reduced milk production;
impaired prolonged farrowing; reduced tolerance to parenterally
administered iron and iron toxicity in baby pigs?
There are several studies conducted to evaluate details of clinical, morphological,

microscopic and biochemical changes in Se or Se/Vitamin E deficiency in pigs.
For example, twenty two newborn pigs from 2 weeks of age to weaning were fed
a corn-torula yeast creep feed containing Se at the concentration of 0.03 ppm,
and from weaning to slaughter, a corn-soybean meal ration containing Se at the
concentration of 0.07 ppm and vitamin E at the concentration of 15.7 mg/kg (Van
Vleet et al., 1975). Subclinical Se-E deficiency was developed and characterized
by subtle muscular stiffness, significant increases in plasma activities of glutamic
oxalacetic transaminase (GOT) and creatine phosphokinase (CPK), and typical
residual lesions in heart and skeletal muscle, but not in liver, at slaughter at 165
days of age. The major alterations in injured fibers progressed from hyaline
degeneration, with subsequent macrophagic invasion and phagocytosis of disrupted
sarcoplasm, to muscle fiber regeneration by myoblastic proliferation, fusion, and
differentiation into fibers with mature myofibrils. When electron microscopic
techniques were used, myofibrillar lysis and disruption was observed. In particular,
disruption of mitochondria, sarcoplasmic reticulum, and plasma membranes was
evident in fibers with myofibrillar alterations (Van Vleet et al., 1976).
Selenium-vitamin E deficiency was produced in weanling swine by feeding a
semi-synthetic basal diet for 13 to 59 days. In hearts with vascular damage, gross
hemorrhages were observed in the myocardium and serosal surfaces (Van Vleet
et al., 1977a). The authors indicated that:
myocardial arteriolar damage was characterized by segmental fibrinoid

accumulation in vessel walls and by scattered fibrin thrombi;
extensive subendothelial and inner wall accumulations of dense granular
deposits of serum proteins and masses of fibrin in arterioles took place.
endothelial cells of affected arterioles were loosely attached to each other
and in arterioles with fibrin thrombi, the endothelium was disrupted.
The changes in arterioles depended on the severity of damage. From the one
hand, in mildly injured arterioles, increased endothelial permeability was associated
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with insudation of blood proteins into the vessel wall producing accumulation of
fibrinoid. On the other hand, in severely injured vessels, endothelial integrity was
completely destroyed, smooth muscle cells were necrotic and thrombosis had
developed. It seems likely, that lipid peroxidation and alteration in regulation of
antioxidant defences in endothelial cells are responsible for aforementioned
arteriolar lesions (Van Vleet et al., 1977a). It was shown that the lesions were also
scattered throughout the heart but were most severe in the atria. It is interesting
that the damaged fibers had many features of myofibrillar degeneration with
hypercontraction bands, myofibrillar lysis, and mitochondrial swelling, disruption,
and mineralization (Van Vleet et al., 1977; 1977b). Immune system was also
involved in the development of lesions. For example, numerous macrophages
passed through focal disruptions in the external lamina of the muscle cells and
engulfed sarcoplasmic and nuclear debris. Although vascular lesions were present
in the hearts of selenium and vitamin E deficient pigs, it was concluded that the
fiber alterations developed independently of the vascular changes. The
pathogenesis of this cardiomyopathy induced by selenium and vitamin E
deficiency is thought to be a result of the antioxidant system alteration (Van Vleet
et al., 1977). In fact, in pigs fed on diets depleted of vitamin E and Se, increases
in concentrations of markers of lipid peroxidation (4-hydroxynonenal and hexanal)
were observed (Nolan et al., 1995). However, skeletal myopathy and increased
lipid peroxidation in heart and supraspinatus muscle were only observed in extrastressed pigs, fed on diets depleted of vitamin E and Se and supplemented with
extra n-3 PUFAs in the form of vitamin E-stripped linseed oil.
An evaluation of the coagulation system has been conducted in vitamin E and/
or selenium deficient swine and confirmed that in selenium deficiency, the
prothrombin time was shortened (Fontaine et al., 1977). The platelet count and
platelet turnover were also greatly decreased by selenium deficiency and this was
associated with decreased half-life of fibrinogen labelled with 75 Seselenomethionine and increased turnover of fibrinogen. From these fibrinogen
kinetic findings, it was concluded that chronic low grade disseminated
intravascular coagulation could occur in selenium deficient animals and it is related
to the development of hepatosis dietetica or widespread microvascular damage.
Furthermore, some other factors, such as increased fibrinogenolysis also were
considered to be involved in the development of lesions of Se deficiency.
Furthermore, erythropoiesis appeared to be only slightly decreased in selenium
deficient pigs (Fontaine et al., 1977a). However, these alterations of erythropoesis
depend on the severity of Se/vitamin E deficiency. For example, sows of Swedish
Landrace x Yorkshire breed were fed a diet extremely deficient in vitamin E and
selenium during the last six weeks of pregnancy and compared to sows of the
same breed and age fed a normal commercial diet (Thoren-Tolling, 1984).
Selenium-vitamin E deficient piglets showed significantly lower hemoglobin values
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at birth than the normal piglets. Serum creatine phosphokinase activities were
found to be increased in association with selenium deficiency (Fontaine et al.,
1977b) and it reflects the occurrence of sub-clinical muscular dystrophy.
It is possible that disturbances in antioxidant defences are responsible for the
development of microangiopathy (MAP, mulberry heart disease) in pigs. However,
there is some controversy among data related to this disease and it is not clear at
present what role Se deficiency plays in the development of this disease. As
mentioned above, the mulberry heart disease of the pig represents a disease with
major manifestation at the heart, primarily affecting young animals with vascular
and myocytic lesions. The vascular lesions include fibrinoid necrosis in
intramyocardial amall arteries and arterioles with fibrin microtrombi in the
myocardial capillaries (Jonsson, 1993). However, it seems likely that apart from
the characteristic haemorrhagic appearance of the heart, the brain can also be
affected. In particular, histopathological changes include marked alteration of the
small cerebral blood vessels with fibrinoid degeneration, necrosis or infiltration
with pleomorphic cells (Stierstorfe et al., 2001). Furthermore, the vessel lumina
are frequently obliterated by thrombi, and as a consequence large areas of malacia
are found in brain parenchyma. It was reported that three 5-week-old pigs died of
the cardiac form MAP of selenium-vitamin E deficiency. It was observed that the
hearts had prominent hydropericardium and extensive serosal and myocardial
hemorrhage (Van Vleet and Ferrans, 1977). Histopathologic observations
demonstrated marked myocardial edema, congestion, and hemorrhage, with
numerous capillary hyaline microthrombi that stained red with the periodic-acid
Schiff rocedure. Ultrastructurally, these thrombi were composed of elongated,
dense masses of intertwined fibrin strands with occasional entrapped erythrocytes.
It was also shown that MAP persisted among young pigs in Denmark although
the diets of sows and pigs were supplemented with selenium and vitamin E (Nielsen
et al., 1989). Furthermore, the concentrations of selenium and vitamin E in the
liver and heart tissues of young pigs, which had died suddenly, and had the
characteristic lesions of mulberry heart disease post mortem, were not significantly
different from the concentrations found in pigs of the same age, which had died
suddenly for other reasons. Similarly, swine with dietetic MAP had lower heart
and liver -tocopherol concentrations than did control pigs. However, heart and
kidney Se concentrations and heart and liver PUFA concentrations were similar
in pigs of either group (Rice and Kennedy, 1989). Indeed, in spite of apparently
adequate amount of dietary -tocopherol, pigs with MAP had lower tissue tocopherol concentration than did control pigs. The authors concluded that
spontaneous MAP is associated with altered -tocopherol metabolism, but is
unrelated to alterations in tissue Se and PUFA concentrations and GSH-Px activity.
In another study, the significance of selenium deficiency was also investigated in
pigs that died suddenly of MAP. It was shown that hepatic selenium concentration
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in pigs with MAP (1.04 g/g dry weight) was only slightly lower than in healthy
pigs (1.23 g/g dry weight; Korpela et al., 1990). However, in 22.2% of MAP
pigs hepatic selenium concentration was below 0.5 g/g which reflects selenium
deficiency. Thus, there is a possibility that the low selenium status together with
vitamin E deficiency increases oxidative stress and thus contributes to the
development of oxidative damage. During a 2-year period from January 1, 1999,
to December 31, 2000, 77 diagnoses of mulberry heart disease were documented
at the Iowa State University Veterinary Diagnostic Laboratory (Pallares et al.,
2002). The level of vitamin E in the liver of pigs with the disease was significantly
decreased, however, liver selenium concentrations were adequate in all pigs. It
has been suggested that heredity could be involved in the development of such a
disease (Poulsen, 1993). Clearly, there is a need for further research to clarify
causes and molecular mechanisms of the development of mulberry heart disease
in pigs.
Se dietary supplementation is an effective way to deal with Se deficiency. In
some instances of severe Se deficiency an intramuscular administration of this
element is used (Van Vleet et al., 1973; Mahan et al., 1973). It is interesting that
even after 24 days post-injection, Se concentration in serum and tissues are still
elevated (Table 8.1). However, forms of selenium accumulated and its availability
for synthesis of functional selenoproteins have not been elucidated. As will be
shown below, a combined injection of iron and sodium selenite could have
detrimental effects on piglets. It seems likely, that usage of organic form of selenium
in the sows and pigs diets could help meeting Se requirements and there would
not be a need for Se injections.
Table 8.1 Effect of selenium injection on its concentration in pig tissues, mg/kg dry matter (Adapted from
Diehl et al., 1975)
Collection
period
Untitled-2
Se injection,
mg Se/kg BW
Kidney
Liver
Longissimus
muscle
Serum
Initial
Control
1.24
0.43
0.24
24 days
postinjection
Control
0.275
0.55
1.10
2.62
4.06
5.17
5.30
0.43
0.72
0.91
1.05
0.17
0.32
0.37
0.31
0.025
0.053
0.078
0.093
60 days
post-injection
Control
0.275
0.55
1.10
3.97
5.99
6.99
6.54
0.38
0.54
0.60
0.71
0.17
0.20
0.28
0.32
0.030
0.047
0.062
0.060
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454
From information provided above it is obvious that manifestations of Se deficiency

depend on a range of nutritional and environmental factors such as degree of
deficiency, presence of other antioxidants and pro-oxidants, activities of tissues
at time of deficiency and some others (Jonsson, 1993):
myopathy is more prevalent in young growing animals with active

myogenesis
liver necrosis is observed in pigs reared in confinement
fetal placenta, uterus and mammary glands are affected in reproductionlactating animals.
However, alterations in antioxidant defences and oxidative stress are responsible

for different metabolic and morphological changes on cellular and sub-cellular
levels leading to clinical manifestation of Se deficiency in pigs. Indeed sudden
heart failure caused by single Se deficiency or general antioxidant deficiency can
occur at various stages of pig development and related to negative balance between
antioxidants and pro-oxidants in stress conditions of modern pig production.
Indeed, movement of pigs from one pen to another, the stress of weaning, stress
of transportation or stress of farrowing (Close and Cole, 2000) could be important
triggers for various detrimental consequences in pigs including sudden death.
Se requirement
It has been suggested that most, if not all, aspects of swine production are affected
by feed levels of Se and vitamin E (Jenkins and Hidiroglou, 1972). After research
work showing that many regions in the USA are deficient in Se, great efforts have
been made to gain FDA approval for Se supplementation of animal diets (Ullrey,
1992). For example, results of a cooperative survey of the dietary selenium fed
growing swine in 13 states (Ku et al., 1972) showed that the highest Se
concentration was in South Dakota (0.493 ppm) and lowest in Virginia (0.027
ppm) being more than 15 fold lower (Table 8.2). In 1974 U.S. FDA approved
the addition of 0.1 ppm of selenium to all swine diets. The approval of selenite for
use in animal diets followed in other countries as well. For example, addition of
selenium to swine feed (max. 0.1 ppm) was legalized in Denmark in 1975
(Pedersen and Simesen, 1977). In 1991 the Denmark recommendation was
changed to 0.4 ppm for pre-starter diets and 0.22 ppm for both growing and
breeding pigs (Poulsen, 1993).
Did you know that in 1974 U.S. FDA approved the addition
of 0.1 ppm of selenium to all swine diets for the first time?
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Table 8.2 Selenium content in diets and in Longissimus muscle of pigs in the USA (Adapted from Ku et
al., 1972)
State
South Dakota
North Dakota
Nebraska
Iowa
Wisconsin
Wyoming
Arkansas
Idaho
Indiana
Michigan
New York
Illinois
Virginia
Dietary Selenium,
ppm
Se in longissimus muscle,
ppm (wet weight)
0.493
0.412
0.330
0.235
0.178
0.158
0.152
0.086
0.052
0.040
0.036
0.036
0.027
0.521
0.386
0.313
0.278
0.125
0.311
0.212
0.111
0.063
0.052
0.046
0.059
0.034
The current maximum accepted total dietary Se level in Denmark is 0.5 ppm.
Indeed, the inability to supplement selenium-deficient diets prior to FDA approval
has cost hundreds of millions of dollars. For example, annual losses to the beef
cattle, dairy cattle and sheep industry were estimated in 1975 at nearly $545
million (Ullrey, 1980). Later, in 1982, FDA reconsidered a level of Se
supplementation and allowed the addition of 0.3 ppm of selenium to diets for
pigs up to 20 kg (Nutrient Requirements of Swine, 1998). Ultimately, in 1987,
levels of supplemental Se in diets for chickens, turkeys, ducks, swine, sheep, and
cattle were approved at 0.3 ppm (Ullrey, 1992). Therefore, currently 0.3 ppm of
selenium in the diet is allowed for all pigs. This dietary Se allowance for pigs was
based on tissue Se concentrations, Se balance and the activity response of cellular
GSH-Px. In addition to dietary Se supplementation, other solutions of Se deficiency
are also available. For example, inclusion of selenite into fertilizers are shown to
be effective in increasing Se level in grains, animal diets and animal tissues (Table
8.3). However, this application found its commercial use only in Finland and
New Zealand. Indeed, environmental concerns of potential Se pollution from animal
production re-opened a question whether the Se supplementation was in excess of
dietary needs (Ullrey, 1992), but there was no sufficient evidence to change Se
supplementation and the level of 0.3 ppm has been maintained.
There is a great deal of difficulties to establish an optimal Se requirement of
pigs:
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456
Table 8.3 Selenium concentrations (mg/kg wet weight) in tissues of pigs and cattle in Finland (Adapted
from Venalainen et al., 1997).
Muscle
Liver
Year
pig
cattle
pig
cattle
1974
1981
1985
1986
1987
1988
1989
1990
1991
1992
1993
1994
1995
0.11
0.08
0.08
0.12
0.23
0.26
0.30
0.29
0.26
0.19
0.19
0.13
0.15
0.04
0.07
0.10
0.16
0.16
0.21
0.19
0.16
0.16
0.14
0.09
0.10
0.45
0.56
0.49
0.56
0.64
0.7
0.73
0.68
0.62
0.57
0.56
0.50
0.52
0.10
0.16
0.28
0.34
0.42
0.45
0.51
0.46
0.40
0.37
0.35
0.32
0.28
Se was introduced to all multi-nutrient fertilizers used in Finland after July 1984 at 16 mg/
kg fertilizers for cereal crops and 6 mg/kg for grassland crops. In 1990 the level of Se
inclusion to the all fertilizers was decreased to 6 mg/kg.
Untitled-2
Data obtained in the laboratory low-stress conditions cannot be the same in

commercial higher-stress conditions. For example, the size of groups in
which pigs are tested in experimental conditions is usually much smaller
than in commercial conditions. Health status of pigs and microbial
population in pig houses could also substantially differ between laboratory
and commercial conditions.
The level of other antioxidants and pro-oxidants in the diets are variable
and since Se is an essential part of antioxidant defences its requirement
could substantially vary depending on the diet used
The data obtained with average-productive animals cannot be transferred
directly to high productive stress-sensitive animals. Indeed pig genotype
determines their physiological features including sensitivity to stresses
The choice of the end points to assess requirement (see chapter 3). Indeed,
most of the data are related to Se level in plasma, whole blood, tissues or
GSH-Px activity. However, GSH-Px is only one from more than 25 various
selenoproteins identified in animal and human bodies. The usage GSH-Px
activity in plasma is not the best option to assess Se requirement and to
assess physiological consequences of Se deprivation or supplementation.
Technically, it is the easiest option, but its value is questionable. GSH-Px
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activity in the target tissues and a combination of various functional tests

would be more physiologically relevant tests.
Form of selenium used. In most of studies sodium selenite was used and it
is known that organic selenium is more efficiently assimilated from the diet
building Se reserves in tissues
Se requirement to maintain various physiological functions could vary
substantially. For example, to maintain high immunocompetence, Se
requirement is usually higher than for growth and development
In accordance with Nutrient Requirements of Swine (1998) pig requirement
in Se is defined at the level of 0.15-0.3 ppm (Table 8.4). However, it is
necessary to take into account that they are minimal Se requirements and in
commercial conditions Se requirements could be substantially higher.
Table 8.4 Selenium requirements of swine (Adapted from NRS, 1998)
Pigs, sows and boars

Pigs with bodyweight 3-5 kg
Gestating sows*
Lactating sows*
Sexually active boars*
mg/kg diet (90% dry matter)
mg/day
0.30
0.30
0.25
0.15
0.15
0.15
0.15
0.15
0.15
0.08
0.15
0.25
0.28
0.39
0.46
0.30
0.80
0.30
*/Recent recommendations by Close and Cole (2000) are 0.3 mg Se/kg diet
Selenium supplementation of the deficient diet could affect various parameters of

pig growth and development, however, the final results depend on many various
factors. For example, pigs from sows fed a diet deficient in Se and low in vitamin
E were fed a Torula yeast diet supplemented with 100 IU dl-alpha-tocopheryl
acetate/kg of diet and with Se at 0, 0.025, 0.050, 0.075 or 0.100 ppm (Adkins and
Ewan, 1984). Selenium supplement had no significant effect on average daily
gain, feed intake or gain to feed ratio for the 4-wk experiment. Serum Se increased
linearly with increasing supplemental Se and serum GSH-Px activity also increased
linearly with increasing supplemental Se. The correlation between serum Se
concentration and GSH-Px activity was 0.81 (Adkins and Ewan, 1984). Meyer et
al. (1981) showed that 0.3 mg Se/kg was required by weanling pigs based on
breakpoint regression analysis of liver GSH-Px1 and plasma GSH-Px3 activities.
It is necessary to take into account that relative activity distribution and age-related
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458
changes are different between GSH-Px1 and GSH-Px4 in pigs (Lei et al. 1997). In
particular, it was found that 0.1 mg Se/kg was insufficient and 0.3 mg Se/kg was
required for young pigs to express the full activity of GSH-Px1 in liver. However,
the authors did not test Se level at 0.2 mg Se/kg in their study. In an experiment
conducted by Lei et al. (1998) eight different tissues were collected from Seadequate male pigs aged 1, 28 and 180 days, and PH-GSH-Px, and GSH-Px
activities were assayed. Both mRNA and activity expression of PH-GSH-Px in
most assayed tissues was increased with age, but increases in PH-GSH-Px mRNA
levels between ages did not fully account for all changes in activity (Lei et al.,
1998). Expression of GPX activity was increased more than that of PH-GSH-Px
between day 1 and day 28. In this experiment, there was no difference in GSH-Px
activities between pigs fed 0.2 and 0.3 or 0.5 mg Se/kg. Therefore, it was
concluded that full activity expression of GSH-Px1 and GSH-Px4 in various tissues
of weanling pigs requires 0.20 mg Se/kg in the corn-soy-based diets (Lei et al.,
1998).
Did you know that current recommendations on Se requirement
of swine are 0.15-0.30 ppm, but in commercial conditions this
should be increased depending on level of stresses?
As mentioned above, pig Se requirement depends on the experimental conditions,
especially on amounts of stresses caused by environmental and technological
factors and in experimental laboratory conditions it seems to be lower than in
practical conditions of pig producing units. Indeed, in physiological conditions
Se requirements of the growing pigs are quite low. For example, using serum
GSH-Px activity as the measurement criterion, Mahan et al. (1999) showed that
the supplemental dietary Se requirement did not exceed 0.10 and 0.05 mg Se/kg
diet for the growing and finishing phases, respectively, when added to a basal
diet containing 0.06 mg Se/kg. Grower pigs fed sodium selenite had serum GSHPx activity that reached a plateau at 0.1 ppm Se and 0.3 ppm when the Se-enriched
yeast source was fed, but the interaction response was not significant. On the
other hand, during the finisher period, serum GSH-Px activity reached a plateau
at 0.1 ppm Se for both Se sources (Mahan and Parrett, 1996). In earlier work Se
requirement to maintain an optimal GSH-Px activity was shown to be higher. In a
trial with sows different dietary selenium levels (0.1; 0.3 and 0.5 mg/kg) were fed
and GSH-Px activity in plasma was determined at day 60, 90 and 110 of gestation,
day 5, 15 and 25 of lactation and day 7 post weaning. It was shown that GSH-Px
activities dropped within the treatments, independent of dietary selenium, from
day 60 of gestation to a minimum at day 5 and 15, respectively, of lactation and
increased again after weaning (Neumann et al, 1989). Highest GSH-Px activities
were observed at the 0.5 ppm Se level in the diet. However, reproductive
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performance ameliorated with increasing dietary selenium. For example, piglets
gain from birth to weaning and litter weight at weaning increased significantly.
The relation between selenium intake and plasma GSH-Px activity was investigated
in 178 German Landrace sows fed on barley-oat concentrate with Se 0.1, 0.5 or
0.9 mg/kg diet. It is concluded that the optimal Se supply for pregnant and nonpregnant sows in concentrate is 0.5 mg/kg. In particular, lowest percent of stillborn
piglets was seen in the group fed on the diet containing 0.5 mg/kg selenium
(Neumann and Bronsch, 1988; 1989). The authors concluded that the requirement
of the sow during the reproductive cycle is 0.5 mg selenium/kg diet.
Selenium applications in pig production

As mentioned above selenium is an integral part of a range of selenoproteins
playing important roles in regulation of various physiological processes. The most
important applications of selenium are in preventing damaging effects of free
radicals and toxic products of their metabolism in stress conditions. There are
several important periods of pig development where protective role of selenium
is difficult to overestimate. They include:
Gestation
Parturition and lactation
First few days after piglet birth
Weaning period
Maintenance of semen quality in boars
Maternal effect on the progeny: organic selenium vs selenite

Low-Se maternal diet is a risk factor for the sow and the developing pig embryo.
Indeed, concentrations of Se in maternal whole blood and liver decreased during
gestation in sows fed the low-Se diet compared to sows fed the Se-supplemented
diet (Hostetler and Kincaid, 2004). It is interesting that sodium selenite included
into the maternal diet was not effective in changing the concentration of Se in the
whole foetus. Indeed, fetal liver Se decreased in both treatments as gestation
progressed, however, the decrease was greater in liver of foetuses from sows fed
the low-Se diet. Similarly, a marked decrease in GSH-Px activity in fetal pig liver
at the time of birth in comparison to earlier stages of gestation was observed and
selenite supplementation of the maternal diet did not affect these changes (Hostetler
and Kincaid, 2004a). It is important to note that, maternal liver GSH-Px activity
was approx 12-fold greater than fetal liver GSH-Px activity regardless of dietary
treatment. Furthermore, lipid peroxidation indexes were greater in liver
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460
homogenates from gilts fed the low-Se diet, even though there was no difference
in GSH-Px activity. It could mean that other than GSH-Px selenoproteins were
affected and resistance of fetus to oxidative stress decreased.
Did you know that the most important periods of pig
development where Se is of great importance include:
gestation, parturition and lactation, first few days after birth,
weanling period, maintenance of semen quality in boars?
The activity of the selenium-dependent enzyme GSH-Px in the serum of piglets
was very low during the first week of piglet life despite the fact that the sows feed
had been supplemented with 0.35 mg inorganic selenium/kg (Pehrson et al., 2001).
The authors concluded that the selenium status of newborn piglets might be more
critical for their health than their vitamin E status. Similar conclusions were made
by Loudenslager et al. (1986) showing that plasma biological antioxidant state
(tocopherol content and GSH-Px activity) of pigs at birth was very low predisposing
piglets to oxidative stress. Indeed, mean concentrations of vitamin E (1.77 mg/
litre) and GSH-Px (682 U/litre) were low in the blood plasma of the piglets at
birth, but a large increase was observed on the next sampling occasion at the age
of 3 days (vitamin E: 5.31 mg/litre; GSH-Px: 996 U/litre) reflecting transfer of
these antioxidant compounds via colostrum (Hakansson et al, 2001). The authors
showed that the highest concentrations of vitamin E (17.9 mg/kg) and Se (200
g/litre) were found in colostrum, but with a large variation between sows. One
week postpartum the concentrations of vitamin E and Se in sow milk had
decreased, on average by 86 and 68%, respectively. Furthermore, sow milk
selenium and vitamin E declines with advancing parity (Mahan, 1991, 1994).
This means that adult sows need more vitamin E and Se to maintain their reserves
in the body and to transfer them to progeny. Furthermore, piglets born to older
sows could be at greater risk of oxidative stress at birth as well as during early
postnatal development.
In the experiment conducted by Mahan (2000) six dietary treatments were
used in a 2 x 2 factorial arrangement with two additional treatments. Inorganic
(sodium selenite) or organic (Sel-Plex) Se sources were added to the diet at 0.15
or 0.30 ppm Se. A non-Se-fortified corn-soybean meal basal diet served as a
negative control, and a sixth group was fed 0.15 ppm Se from both inorganic and
organic Se sources. A total of 43 sows were fed their treatment diets at 2.2 kg/d
from 6 d pre-partum to parturition and at full feed through a 14-d lactation period.
The major results can be summarised as follows:
Untitled-2
Firstly, it was concluded that Se dietary supplementation is an important

means to maintain antioxidant defences of sows. For example, when the
basal diet was fed, sow serum GSH-Px activity declined from 6 d prepartum
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and remained low throughout lactation (Mahan, 2000). Therefore, inclusion

of selenium into sows diet caused an increase in sow serum Se concentration
and serum GSH-Px activity at both 7 and 14 d postpartum.
Secondly, it was confirmed that colostrum is an important source of selenium
for newly born piglets. However, selenium transfer to the colostrum is
minimal if it is added to the sows diet in the form of sodium selenite. Indeed,
the short-term feeding of selenite at 0.15 or 0.30 ppm Se did not affect
colostrum Se content (Mahan, 2000). In contrast, inclusion of Sel-Plex into
sows diet significantly increased Se content of colostrum.
Thirdly, a positive effect of organic selenium was observed in relation to Se
concentration in the milk. For example, milk Se at 7 and 14 d postpartum
was 2.5 to 3 times higher when the organic Se source was provided.
Fourthly, low efficiency of selenite transfer to colostrum and milk was
confirmed by using a combination of inorganic and organic Se at 0.15 ppm
Se. Indeed, colostrum and milk Se contents were similar to those of sows
fed 0.15 ppm Se from the organic Se source (Mahan, 2000). It seems likely
that Se in the colostrum and milk is present in organic form and
selenomethionine represents a substantial proportion of those forms. Since
SeMet is not synthesised in animal body, only when organic selenium is
included into sows diet Se concentration in colostrum and milk substantially
increased.
Furthermore, organic selenium in the maternal diet was also effective in
increasing Se concentration in serum of piglets at 7 and 14 d of age.
Similarly, Se dietary supplementation of pigs for 105 days in the form of selenized
yeast was associated with increased Se concentration in whole blood and liver in
comparison to pigs supplemented with sodium selenite (Ortman and Perhson,
1998). Surprisely, there was no difference in Se concentration in the blood or
liver between pigs supplemented with 0.1 or 0.3 ppm of organic selenium.
Did you know that piglets are born with low antioxidant
defences and their postnatal development and health depends
on antioxidants transferred via colostrum and milk?
When diet for Crossbred barrows was supplemented with 0.1; 0.3 or 0.5 ppm
selenium in the form of selenite or Sel-Plex, selenium retention increased as dietary
Se levels increased, particularly when the Sel-Plex was provided (Mahan and
Parrett, 1996; Table 8.5). Similar results were reported when selenite or organic
selenium in the form of selenized yeast (Sel-Plex) was fed to grower and finisher
swine (Table 8.6; Mahan, 1999). Increasing selenite concentration in the diet from
0.05 to 0.3 ppm only slightly affected Se concentration in the loin and pancreas,
but substantially increased Se concentration in the liver. In contrast, organic Se in
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Table 8.5 Effect of dietary selenium on selenium level in tissues of grower and finisher swine, ppm
(Adapted from Mahan and Parrett, 1996)
Item
Basal
Serum
Serum
Loin
Liver
Pancreas
Kidney
Serum
Loin
Liver
Pancreas
Kidney
Inorganic selenium
0.1
0.3
0.5
Organic selenium
0.1
0.3
0.5
60 kg body weight (feeding from 22.2 kg BW)

0.056
0.142
0.162
0.162
0.115
0.114
0.167
0.170
0.189
0.216
0.229
0.615
0.682
0.756
0.533
0.421
0.526
0.519
0.494
0.532
1.248
1.603
2.043
1.824
1.708
0.159
0.300
0.679
0.645
1.870
0.170
0.362
0.820
0.604
1.756
105 kg body weight (feeding from 65.8 kg BW)

0.044
0.154
0.167
0.167
0.117
0.08
0.101
0.124
0.13
0.143
0.222
0.492
0.675
0.699
0.467
0.368
0.435
0.484
0.459
0.490
1.402
2.599
3.512
3.738
2.140
0.168
0.212
0.737
0.614
2.990
0.187
0.262
0.901
0.725
3.040
Table 8.6 Effect of dietary selenium on tissue selenium concentration, ppm (Adapted from Mahan et al.,
1999)
Item
Basal
Inorganic selenium
0.05
0.1
0.2
Loin
Liver
Pancreas
Kidney
0.087
0.195
0.314
1.305
0.091
0.425
0.327
1.716
0.103
0.464
0.360
1.771
55 kg body
0.113
0.526
0.400
1.76
Loin
Liver
Pancreas
Kidney
0.085
0.258
0.290
1.811
0.106
0.452
0.355
2.283
0.114
0.436
0.391
2.333
Organic selenium
0.3
0.05
0.1
0.2
0.3
weight
0.120
0.522
0.368
1.655
0.113
0.319
0.335
1.597
0.143
0.398
0.396
1.734
0.184
0.577
0.529
1.973
0.206
0.513
0.529
1.972
105 kg body weight

0.118
0.124
0.478
0.534
0.367
0.383
2.328
2.376
0.134
0.469
0.395
2.282
0.17
0.516
0.426
2.333
0.249
0.627
0.483
2.473
0.332
0.717
0.573
2.677
the swine diet was much more effective in transferring to loin and pancreas. It is
interesting to note, that as dietary Se levels increased, urinary Se excretion was
substantially increased when pigs were fed sodium selenite, whereas excess of
selenium in the form of Sel-Plex was mainly excreted via fecal route. Indeed, loin
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Se contents were higher in grower and finisher pigs as dietary Se levels increased
when the Se-enriched yeast was fed.
When sodium selenite or Sel-Plex at doses 0.1 and 0.3 ppm were fed to firstparity gilts starting approximately 60 days before breeding until weaning (21
days) it was shown that organic selenium has following advantages in comparison
to selenite (Mahan and Kim, 1996: Table 8.7):
Table 8.7. Effect of selenium fed to reproducing gilts on neonatal and weaning pig selenium
measurements (Adapted from Mahan and Kim, 1996)
Item
Inorganic Se, ppm

0.1
0.3
Loin Se, ppm

Liver Se, ppm
0.039
0.236
Neonate
0.055
0.250
0.068
0.283
0.085
0.310
Serum Se, ppm

Loin Se, ppm
Liver Se, ppm
0.062
0.101
0.352
Weaning, 21 d
0.075
0.121
0.388
0.082
0.129
0.535
0.102
0.244
0.509
0.1
Se-yeast, ppm
0.3
More effectively transferred via placenta resulting in higher Se content in

loin and liver in the neonate piglet
More efficiently transferred to the milk
More efficiently maintained Se status of the developing piglet until weaning
resulting in higher Se concentration in weaning pig loin
It is interesting that by increasing sodium selenite level in the sows feed by 9fold, it was possible to increase Se concentration in blood and colostrum only
moderately. For example, in sows, average whole blood Se concentrations near
parturition were 144.1 and 174.3 g/litre for sows given Se 0.1 and 0.9 mg/kg,
respectively. Average whole blood Se concentrations were 59.2 g/litre in piglets
from sows given Se 0.1 mg/kg diet and 79.9 g /litre in piglets from sows given
Se 0.9 mg/kg diet. Sows given Se 0.9 and 0.1 mg/kg had average concentrations
of Se in colostrum of 148.0 and 86.0 g/litre (Blodgett et al., 1989).
In general, it was shown that increased from 0.1 to 0.3 ppm Se supplementation
with both Se sources was related to increased Se concentration in neonatal loin,
milk and weaning pig loin with organic selenium to be more efficient. On the
other hand, gilt serum GSH-Px activity was generally similar at the 0.1 and 0.3
ppm Se level for either Se source, whereas serum Se was consistently higher
when the dietary Se level was 0.3 ppm. Furthermore weanling pig serum GSH-Px
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464
activity was similar regardless of the Se level or Se source fed to the dam, but
serum Se increased when the 0.3 ppm Se level and the Se-yeast was fed to the gilt
(Mahan and Kim, 1996). As parities progressed, sow milk Se concentration
decreased when the diet was supplemented with sodium selenite (Mahan and
Peters, 2004; Mahan et al., 1991; 1994) and the authors suggested that:
labile sow Se tissue reserves depleted with advancing parity

tissue reserves were not readily mobilized
mammary tissue of older females could not incorporate or transfer dietary
inorganic selenium or tissue Se reserves into milk as effective as young
sows.
Recently, maternal effect of selenium on piglets was characterized in great details

(Mahan and Peters, 2004; Tables 8.8-8.9). Indeed, Se from organic sources (SelPlex) was more effectively transferred to colostrum, milk and sow hair, and a
combination of organic and inorganic selenium was not effective in increasing Se
content of colostrum and milk. At 0.3 ppm dietary supplementation Se level in
the liver, loin and pancreas of the sows was substantially higher when organic
selenium was used in the diet. Similarly, in neonate pig liver and loin Se
concentration was twice higher than that in piglets from sows supplemented with
selenite. Furthermore, total Se contents in neonate piglets was doubled when
selenized yeast in the form of Sel-Plex was used in sows diet (Mahan and Peters,
2004). It is interesting to note that sodium selenite fed to sows had some detrimental
effects on piglets. In fact the percentage of piglet with splay legs (Figure 8.3) and
stillborn piglets (Figure 8.4) was increased by selenite supplementation of the
maternal diet. In contrast, in the same conditions, organic selenium had protective
effects (Mahan and Peters, 2004). It could well be that prooxidant properties of
selenite are responsible for those detrimental changes in sows progeny.
Table 8.8 Effect of dietary Se on sow and litter serum and milk Se profiles (Adapted from Mahan and
Peters, 2004).
Basal
Item
Sow serum Se, mg/l
70 d
110 d
Weaning
Colostrum Se, mg/l
Milk Se, mg/l
Sow hair, Se, mg/kg
Litter, weaning serum
Se, mg/l
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Organic Se
Inorganic Se
Combination
0.15
0.30
0.15
0.30
0.30
0.114
0.088
0.086
0.075
0.040
0.271
0.146
0.130
0.144
0.151
0.067
0.482
0.169
0.159
0.179
0.168
0.101
0.763
0.143
0.125
0.160
0.094
0.055
0.303
0.135
0.124
0.162
0.095
0.056
0.326
0.162
0.152
0.173
0.148
0.064
0.507
0.056
0.078
0.092
0.067
0.062
0.077
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Table 8.9 Effect of dietary Se on sow and pig tissue Se concentrations (Adapted from Mahan and Peters,
2004).
Basal
Organic Se
Inorganic Se
Combination
Item
0.15
0.30
0.15
0.30
0.30
Sow tissue Se, mg/kg

Liver
Kidney
Loin
Pancreas
0.298
2.23
0.071
0.300
0.583
2.40
0.203
0.487
0.781
2.50
0.329
0.510
0.450
2.68
0.088
0.334
0.571
2.94
0.092
0.369
0.565
2.91
0.217
0.467
Neonate pig Se, mg/kg

Liver
Loin
Total Se, mg/pig
0.197
0.037
0.075
0.348
0.066
0.123
0.424
0.107
0.200
0.238
0.046
0.079
0.279
0.046
0.100
0.344
0.074
0.116
35
30
25
20
15
10
Basal
0.15 Sel-Plex
0.30 Sel-Plex
0.30 Selenite
Figure 8.3 Effect of selenium in sows diet on splay leg (% live pigs) in progeny (Adapted from Mahan
and Peters, 2004)
Indeed, the progeny from sows between the 4th and 7th parity often are deficient
in selenium and their mortality rates can be increased. Furthermore, the higher
the level of productivity, the greater the loss of Se from the sows stores (Close,
2003). As mentioned above, if Se in the milk exists in the form of SeMet, which is
not synthesized in sows body, therefore, only when Se is supplemented in organic
form it can be effectively transferred to colostrum and milk. It could well be, that
SeMet reserves in sows body are depleted and this caused Se declining in milk
with advanced parity. Therefore, even high inclusion rate of selenite cannot
provide a form of selenium which is transferred to colostrum and milk and the
only solution for this problem is to use organic selenium, providing SeMet which
is effectively transferred to the colostrum and milk.
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0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
Basal
0.15 Sel-Plex 0.30 Sel-Plex 0.15 Selenite 0.30 Selenite
Figure 8.4 Effect of selenium in sows diet on number of stillborn piglets (No/litter) (Adapted from Mahan
and Peters, 2004).
Did you know that replacing sodium selenite by organic

selenium in the sows diet increases Se transfer via placenta,
increases Se concentration in the colostrum and milk?
Selenium yeast was added to the sows feed ration at the rate of 0.9 mg Se/head/
day, starting from the 3rd day prepartum to the 21st day of lactation (Kolacz et al.
2001). On the 7th day of lactation, mean selenium concentration in milk was 10
times higher when compared with the controls. On the 7th day of life, the mean
selenium concentration in the blood serum of piglets was nearly 5 times higher
than in the controls. On the 21st day, blood selenium concentration increased in
both experimental and control piglets by 51 and 211%, respectively. Despite the
sharp improvement in selenium concentration measured in control pigs, the
difference between supplemented and unsupplemented litters was still significant
(0.334 and 0.143 mg/litre, respectively). Pigs of the experimental group gained
weight faster and lower mortality was recorded, in comparison to control pigs.
Selenium yeast supplementation influenced fertilization rate as well as the size
and weight of the litter of the next parity (Kolacz et al. 2001). In the feeding
experiment with sows, the control group was fed with a diet containing 0.1 ppm
of Se and 37 IU vitamin E, and the treatment group was fed the same diet but
adding 0.2 ppm organic Se and 20 IU vitamin E, respectively. The number of
pigs born alive in the treatment group was 3.1% greater than in the control group.
The number of pigs weaned at the first to the second and to the ninth litters and
the survival rates of weanling pigs of the third to the fifth and sixth to the eighth
litters in the treatment group were significantly higher than those in the control
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group (Chen et al., 2000). Experimental sows were supplemented with selenized
yeast at the level of 0.3 ppm Se throughout the experiment. The experiment started
14 days prior to expected parturition and finished when piglets of the 2nd farrowing
had been weaned. It was proven that long-term organic Se supplementation
increased litter size by 0.8 piglets in the 2nd farrowing and the piglets of
experimental sows exhibited almost 400 g more live weight by the time of weaning
at 33 days of age (Rafai and Jakab, 1999). Similarly, it was shown that replacing
sodium selenite by organic selenium in the form of Sel-Plex to achieve dietary Se
level of 0.45 mg/kg was associated with decreased (10.54 vs 13.08%) pre-weanling
mortality and increased daily gain of piglets during pre-weaning period (221g vs
213; Janyk et al., 1998). Similarly, a combination of increased vitamin E
supplementation (400 IU/kg) and organic selenium (0.2 ppm) in the sows diet
was associated with increased percent of farrowing sows (87.5 vs 82.5%), total
number of piglet born and live born piglets, as well as decreased pre-weaning
mortality (Table 8.10, Janyk, 2001).
Table 8.10 Effect of selenium in combination with vitamin E (intramuscular injections at days 60 and 100
of gestation) on sows (Adapted from Janyk, 2001).
Number of sows served

Number of farrowing (total)
Percentage of farrowing
Litter data
Total born
Total born per litter
Live born
Live born/litter
Stillbirths
Piglet weight, g
Birth
Weaning (30 d)
Pre-weaning mortality, %
Basal diet + 0.25 mg/kg

Selenite + 40 IU/kg
vitamin E
Basal diet+ 0.2 mg/kg

Sel-Plex+ 400 IU
vitamin E
20
33
82.5
20
35
87.5
386
11.7
330
10
1.2
420
12
385
11
1.1
1480
7530
15.5
1530
7930
10.6
Recently, sows have been fed organic selenium instead of selenite at 0.15 or 0.3
ppm from the time when sows entered the breeding area and continued during
gestation and lactation period (Pineda et al., 2004). The advantages of organic
selenium can be summarised as follows:
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10.4% higher total piglet born

more piglet born alive
6% higher litter weight at weaning
Effect of selenium (Se) source on litter performance was evaluated in a side-byside comparison of Se-enriched yeast (Sel-Plex) with sodium selenite at 0.3 ppm
Se supplementation (Lampe et al., 2005). The trial encompassed 6 weeks of
farrowings within a 3,400 sow farrow to wean facility. Litter data were analyzed
from 380 sows per treatment. Females remained on their respective treatment
through weaning. Milk Se concentrations were significantly greater in Sel-Plex
group than in sodium selenite group (0.111 + .003 ppm vs 0.088 + .003 ppm).
Sows receiving Sel-Plex weaned more pigs (9.61 vs 9.26) and had a greater litter
weaning weight (53.15 vs 51.07 kg). Prewean mortality of Sel-Plex treatment
was 9.76% compared to 11.30% for sodium selenite treatment. In another
experiment, one thousand pigs were weaned, 500 from sows receiving Sel-Plex
and 500 from sows receiving sodium selenite (at 0.3 ppm each) as Se source
(Lampe et al., 2005a). The weaned pigs were placed in a commercial nursery and
penned in groups of 25 head. Average weight of pigs at entry to the nursery was
5.31 + 0.04 kg and was not different dependent on Se source of the sow. Pigs
were fed a 3-phase nutrition program during the 42 d nursery trial. The results of
the experiment indicated that fewer dead and culls (no value pigs) in the nursery
when pigs were from Sel-Plex fed sows. In fact, total mortality and culls in the
nursery was 3.2% for those pigs weaned from sows receiving Sel-Plex compared
to 5.4% for those pigs weaned from sows receiving sodium selenite. Therefore,
the authors concluded that in commercial production prewean piglet survivability
and piglet survivability in the nursery can be enhanced when Sel-Plex replaces
sodium selenite as a dietary selenium source of the sow.
It seems likely that feeding to sows a premix where all trace minerals (Cr, Cu,
Fe, Mn, Zn and Se) were replaced by organic minerals (BioplexesTM) can increased
number of pigs born and greater pigs gain during the nursing period (Peters and
Mahan, 2004). This could be the next step in the pig nutrition: to use only organic
minerals in the swine diets. The additional benefit from a mixture of organic
minerals can be related to prevention of pro-oxidant properties of inorganic iron
and copper in the digestive tract (Surai et al., 2003; 2004), prevention of some
mineral interactions and better assimilation efficiency of trace minerals.
Summarising data presented above it is necessary to underline that the selenium
status of the developing fetus and weanling pig are dependent on the Se status of
the mother. This is extremely important because about 90% of all pre-weaning
death occurs within the first 7 days (Varley, 1995). Indeed, organic selenium in
the maternal diet helps building Se reserves in the animal body. For a pregnant
sow it is important to have a sufficient amount of Se for body maintenance and
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for the transfer to the progeny. When organic selenium is used in the diet of
pregnant sows, more selenium is transferred via placenta, colostrum and milk.
This subsequently resulted in higher Se status (higher Se concentration in serum,
liver and loin) of the young pig at weaning (Mahan, 1999). As a result, postnatal
piglet would benefit in terms of better antioxidant defences, which ultimately
would lead to improved immune system development, would affect piglet growth
and development in general. Since the first week after birth is a fast growth period
for piglets when its body mass can be doubled, antioxidant protection at this
point of the development is of great importance.
Selenium nutrition of pigs at weaning period

Weaning represents a significant challenge to young piglets and the success of
the process determines future productive and reproductive characteristics of
animals. The transition from a liquid diet to one that is usually based on dry feed
ingredients is accompanied by major changes in digestive physiology, immune
status and social and physical environment needs. Indeed, successful weaning is
the key to effective growth, feed efficiency and the subsequent development of
the pig. This is stressful time for pigs and multiple stressors at weaning include
(Close and Cole, 2000):
loss of maternal relationship

separation from littermates
movement to a new environment including new housing system with limited
thermoregulation ability
the change of diet composition and transition from liquid feeding to dry
feed with major changes in digestive physiology
mixing with other pigs and need for social adjustment
possible crowding
This is a period of active pig growth and it is well accepted that growth rate of the
pig is determined by a number of factors and selenium is involved in most of
them (Huo et al., 2003):
Genotype
Genetic potential of pigs is constantly improving and therefore their ability
to utilise nutrients is also improved. However, the price for this improvement
is an increased sensitivity to various stresses and therefore antioxidant
defences are an integral part of the body to prevent damaging effects of
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various stressors. Indeed, genotypes with a higher potential for lean tissue
growth, sometimes are characterised by a lower feed intake, and therefore
need more nutritional attention than other genotypes.
Nutrition
To meet increased demands for nutrient of fast growing pigs is an important
challenge for the nutritionist. Since digestive system of the piglet at birth is
not mature and actively developing for the first few weeks, colostrum and
milk are extremely important sources of essential nutrients. Antioxidants
compounds such as vitamin E and selenium are transferred from mother to
progeny providing essentials for building antioxidant defences of fastgrowing piglet.
Environment
Temperature is the most important environmental factor affecting growth
and development of piglets. Indeed, thermoregulation of piglets is dependent
on thyroid hormone function. Selenium as part of iodothironine deiodinase
is involved in activation of thyroid hormone and in thermoregulation of the
piglets. Since the fat reserves in the piglet body at birth is limited (about 1%
of body mass), its efficient usage is a priority and aforementioned
selenoenzymes are involved in this process. Furthermore, environmental
pollutants such as ammonia, carbon monoxide, sulphated hydrogen cause
oxidative stress in piglets and need for additional antioxidant protection,
provided by selenoproteins is increased.
Health status
Immune system of the newly born piglets is very weak and therefore they
are extremely sensitive to various pathogens. Indeed colostrum and milk
provide immunological protection, but a building of immune system of
piglets usually takes several weeks. During this period of time antioxidants
plays an important immmunoregulating role (see Chapter 4).
Management
The decisions about time of weaning, choice of diets and supplements and
some other management factors affect growth and development of piglets.
In particular, a choice of feed supplements could have a positive or
detrimental effect on growth performance of pigs. Indeed, the daily and
long-term management decisions made by swine personnel will greatly affect
the efficiency of pig production.
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Young pigs enter the postweaning period with a variable Se reserves with following
declining Se concentrations in pig serum and tissues. This period of postnatal
development of pigs is critical in terms of Se deficiency development and still 12% death rate occurs in the nursery and the occurrence of the Se deficiency is still
prevalent on most swine farms (Mahan, 1996). In general, Se deficiency can
affect growth and development of growing pigs with the largest, fastest growing
weanling pigs being more sensitive to sudden death. It has been shown that weaning
stress can exacerbate Se deficiency if Se reserves in the body are not sufficient. In
particular, Mahan (1991) suggested that the 21-day-old weaning pig fed a practical
diet requires 0.5 ppm supplemental Se for about 2 weeks and it could be reduced
to 0.35 ppm by 5 weeks postweaning.
Did you know that postnatal growth rate of the pig is
determined by such factors as genotype, nutrition,
environment, health status and management and selenium
is involved in most of them?
In an experiment conducted in China effect of organic selenium in the form of
Sel-Plex on growth performance and diarrhea incidence of weaning piglets from
days 21 through 42 was investigated (Huang et al., 2004). A total of 108 piglets
weaned at 21 days were randomly allotted to three dietary treatments, with three
pens and 12 piglets in each pen. When dietary sodium selenite (0.2 ppm) was
replaced by the same amount of organic selenium in the form of Sel-Plex the
following advantages were seen:
average daily gain significantly increased (290.9 vs 274.1 g/day)

decreased incidence of diarrhea (1.32% vs 1.72%)
decreased the feed cost/kg weight gain by 11%
Se metabolism in growing pigs was extensively studied. Four-week-old weaned

pigs were fed a corn-soybean meal diet supplemented with 0, 0.3, 0.5 or 1.0 ppm
Se from sodium selenite for 5 weeks (Mahan, 1985). It was shown that fecal Se
excretion increased with increasing dietary Se level. In contrast, urinary Se
decreased during the postweaning periods for pigs fed the basal diet, but increased
linearly as dietary Se increased. It was reported that unsupplemented diet was
deficient in Se, since there was a net loss of Se from the body during the initial 2
weeks postweaning, whereupon, it became positive. It was shown that Se retention
in the postweaning pig increased in direct proportion to the amount consumed
when diets contained up to 1.0 ppm Se. In general, apparent digestibility of Se
was approximately 70%, whereas it ranged between 30 to 40% for pigs fed the
basal diet during 5 weeks (Mahan, 1985). Balance experiments with Se in pigs
showed the following:
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Low selenium supplementation (0.1 ppm) was associated with increased

percent of Se retention in the body (from 45.5 to 61.7%, Groce, 1971; Table
8.11);
High selenium dietary supplementation (0.5 ppm) substantially decreased
Se retention in the body (15.2%; Groce, 1971).
Inclusion in the grower swine diet of organic selenium in the form of
selenized yeast (Sel-Plex) was associated with a substantial increase in
percent of retention of Se in the swine body (Table 8.12, Mahan and Parret,
1996)
Table 8.11 Daily Se balance in pigs supplemented with sodium selenite (Adapted from Groce et al.,
1971)
Item
Basal
Ppm Se (as fed)

Se intake, g
Total Se excretion, g
Fecal Se, g
Urinary Se, g
Se retention, g
Percent retention
Basal+0.1 ppm Se
0.042
18.9
10.3
8.4
1.9
8.6
45.5
Basal+0.5 Se
0.147
66.2
25.3
18.4
6.9
40.9
61.7
0.543
244.4
207.3
57.7
149.6
37.1
15.2
Table 8.12 Effect of supplemental Se on balance measurements in grower swine (Mahan and Parret,
1996)
Selenite, ppm Se
Se balance, mg/d
Intake
Urine
Feces
Retention
Apparent absorption, %
Retention, % of intake
Retention, % of absorbed
Se-yeast, ppm Se
0.1
0.3
0.5
0.1
0.3
0.5
0.271
0.076
0.070
0.125
0.576
0.233
0.146
0.198
0.941
0.442
0.185
0.314
0.299
0.036
0.087
0.176
0.670
0.139
0.166
0.365
1.006
0.238
0.270
0.499
73.9
46.1
62.2
75.1
34.4
46.0
80.5
33.4
41.5
70.8
58.9
87.1
75.2
54.4
72.4
73.3
49.6
67.8
Selenium concentration in the pancreas increased linearly and quadratically in

response to increasing level of Se supplementation (Adkins and Ewan, 1984a ).
The correlation between pancreas Se level and GSH-Px activity was 0.36.
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Supplementation of Se had no effect on pancreas weight, protein content or the
activity of pancreatic trypsin, chymotrypsin, alpha-amylase or lipase. Apparent
digestibility increased linearly for dry matter and N as Se supplementation
increased, however, there was no significant effect on ether extract digestibility
(Adkins and Ewan, 1984a ).
Selenium in grower-finisher swine and meat quality

Effect of antioxidants on pork quality is extensively studied and it seems likely
that a combination of increased doses of vitamin E with organic selenium is an
optimal solution for pig industry. For example, finishing pigs before slaughtering
were supplemented with high vitamin E levels (200 ppm) or organic selenium
(0.3 ppm) or both vitamin E and organic Se for the last 60 days (Krska et al.,
2001). A significant difference in colour stability (reflectance) at 7 day post mortem
was observed in the group supplemented with a combination of vitamin E and
selenium. Indeed, positive effects of vitamin E and Se on colour in psoas major
muscle refrigerated for 7 days were related to significantly lower levels of lipid
peroxidation in pig tissues.
Did you know that feeding sodium selenite to growing pigs can
increase drip loss in meat and by replacing sodium selenite by
organic selenium this problem can be solved?
In a trial at INZOs research farm (Montfaucon, France) growing pigs were
supplemented with 0.3 ppm selenium as sodium selenite or Sel-Plex. Selenium
analyses of pig meat indicated that replacement of sodium selenite with Sel-Plex
was associated with 2-fold increase in Se content of raw muscle (from 0.72 to
1.42 g/g DM ) and 2.5-fold increase in Se content in cooked ham (from 0.66 to
1.73 g/g DM) (INZO, Personal communication). These data clearly indicate an
opportunity to produce Se-enriched pork by using organic selenium in the form
of Sel-Plex (see chapter 12).
Three hundred and fifty one crossbred pigs at an average body weight of 20.4
kg were fed diets containing Sel-Plex (organic) or sodium selenite (inorganic),
each at 0.05, 0.10, 0.20, or 0.30 mg Se/kg diet until 105 kg body weight. A nonSe-fortified basal diet containing 0.06 ppm Se was a ninth treatment group (Mahan
et al., 1999). Tissue Se contents increased linearly as the dietary Se level increased,
but the increase was markedly higher when organic Se was fed. Organic selenium
did not affect loin quality in terms of drip loss, pH and lightness. In contrast, there
was a trend for a higher drip loss and a linear increase in loin paleness when the
inorganic Se level increased (Table 8.13). The pro-oxidant properties of selenite
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could be responsible for these detrimental effects on meat quality. Indeed, selenite
can produce free radicals when reacting with glutathione or some other substances
in the gastro-intestinal tract (Surai et al., 2003) and this could ultimately lead to
the compromised antioxidant system in the digestive tract and in tissues.
Table 8.13 Effect of dietary selenium on carcass characteristics of pigs (Adapted from Mahan et al.,
1999)
Item
Basal
0
24- 72 h, 3C 3.35
72-120 h, 2C 2.33
72-120 h, -10C 2.53
24-120 h, 3C 5.68
24-120 h,
3C/-10C
5.88
24-120 h,
average
5.78
Hunter L
value
46.48
Inorganic selenium
0.05
3.95
2.52
2.67
6.47
0.1
0.2
Organic selenium
0.3
Drip loss (loin) % moisture

3.38
3.68
4.09
2.27
2.25
2.44
4.28
2.41
2.46
5.65
5.93
6.53
0.05
0.1
0.2
0.3
3.19
2.11
2.25
5.30
3.51
2.46
3.00
5.97
2.96
2.56
3.04
5.52
2.95
2.40
2.47
5.35
6.62
7.66
6.09
6.55
5.44
5.46
6.00
5.42
6.55
6.66
6.01
6.54
5.37
5.72
5.76
5.39
46.93
47.68
48.55
49.82
46.57
48.64
46.48
47.36
Selenium-vitamin E combinations
It is well known that Se and vitamin E are working together in concert providing
an effective antioxidant defence (see chapter 2). There is a great body of evidence
to show that a combination of these two antioxidants is more effective in
comparison to their individual use (Surai, 2003). For example, four groups of 12
pregnant sows of the same genetic background, with similar litter size, living
under the same housing conditions and having the same hygienic and nutritional
standards, were used in an experiment conducted by Mavromatis et al. (1999).
In control animals, a basic feed was provided, which contained 20 mg/kg tocopherol and 0.45 mg/kg selenium. Sows in the second group received the
same basic feed but additionally supplemented with 30 mg -tocopherol per kg.
The third group was fed on basic feed but additionally they were injected with 30
mg Se (sodium selenite) on days 30, 60 and 90 of pregnancy. The sows in the
fourth group was fed vitamin E-supplemented feed as in the group 2 and injected
with selenite as in group 3. The experiment started on day 30 of pregnancy and
lasted until weaning day (28 days post-farrowing). It was shown that piglets born
from sows supplemented with vitamin E and injected with selenium (group 4)
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had the highest immunoglobulin concentration level up to weaning day.
Furthermore, number of piglets born/litter, number of weaned piglets/litter, and
piglets average body weight at birth and on weaning day were greater in this
group in comparison with the animals of the other groups. In addition, those
piglets had lowest diarrhoea problems in comparison to other groups (Mavromatis
et al., 1999).
Selenium and iron injection

Iron deficiency is a challenging problem for fast growing piglets resulted from
low efficiency of Fe transfer via placenta and low Fe concentration in sows
colostrum and milk (about 1 mg/l). Indeed, newborn piglets have insufficient
iron reserves in their body (about 50 mg) to maintain physiological blood levels
of haemoglobin until weaning, and as mentined above sows milk provides only
small amounts of iron. Even high levels of iron fed to sows during late gestation
or parenteral administration of iron dextran to sows in gestation were not able
substantially increase placental transfer of iron to fetus (Nutrient Requirements of
Swine, 1998). Furthermore, modern fast growing breeds of pigs require more
iron to maintain the same level of blood haemoglobin than slower growers and
suckling piglets need iron supplementation. Therefore, iron supplementation or
injection during first days (the injection is usually used before piglets are 72 hours
old) after piglet birth is a common commercial practise. Otherwise, piglet anaemia
causing ill thrift, develops if there is no iron supplement. Anaemic piglets are
pale, and accumulate fluid around the throat, brisket and internal body spaces.
They are likely to scour and are susceptible to various other conditions and disease.
Anaemia is responsible for about 10% of pre-weaning deaths (Webster et al.,
2001).
Did you know that iron injection to Se-deficient pigs
could cause mortality?
It seems likely that iron injections, and oral dosing with organic iron preparations
within 18 hours of birth, are the most efficient methods of supplementation (Webster
et al., 2001). As the authors stated, staining, lameness and infection may follow
iron injections and regurgitation and reduced gut absorption in scouring pigs are
disadvantages of oral iron administration. When considering iron it is necessary
to take into account that inorganic iron is a prooxidant. Indeed, iron can induce
myodegeneration, resulting in peroxidative damage to muscle cells (Mahan, 1991).
Combined preparations of iron with antioxidants are used for injections for pigs.
In particular, it was shown that plasma tocopherol concentrations of Fe-injected
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pigs was decreased (Loudenslager et al., 1986). It has been shown that injectable
iron is toxic to pigs from Se and vitamin E-deficient dams (Nutrient Requirements
of Swine, 1998; Mahan, 1991). Consequently, vitamin E was found to be an
effective means in preventing toxic detrimental effects of iron consumption or
injection (Tollerz, 1973; Hill et al., 1999). However, injection of iron in combination
with sodium selenite was associated with depressed hemoglobin concentration
and decreased growth rate of piglets (Hill et al., 1999). Indeed, pro-oxidant
properties of selenite could be responsible for these detrimental effects of a
combination of iron and sodium selenite. It is interesting to note that recently in
the most intensive swine producing region of Columbia, a sudden 80% mortality
outbreak has occurred on one day old piglets from a two site farrow-to-finish
production system of 270 sows as a result of iron toxicity (Velasquez and Aranzazu,
2004). This happened due to attempts of cutting down the feed costs by lowering
vitamin E supplementation and changing from an organic to a cheaper inorganic
form of selenium supplementation. As the authors stated, several months after
such changes newborn piglets were affected by a deadly hemorrhagic clinical
entity developed after a normal iron dextran injection. An outbreak of acute
selenium poisoning among suckling pigs was reported by Sivertsen et al. (2003)
when 92 piglets were found dead or moribund without preceding symptoms.
Necropsy revealed acute congestion of liver and small intestine. It is interesting
that the source of toxicity was a powdered iron supplement contaminated by
sodium selenite. It is likely that pro-oxidant properties of selenite, especially in
combination with iron, are responsible for free radical production consequently
causing toxicity.
Conclusions
From the data presented above it is clear that Se plays an important role in swine
nutrition. The requirement of swine in selenium varies depending on many
environmental and other conditions and in general is considered to be 0.15-0.30 mg/
kg feed. It seems likely that reproducing sows and boars are especially sensitive to Se
deficiency (Figure 8.5) and to meet their requirement in this important element is a
great challenge for pig nutritionists. Indeed, in many countries in the world there are
legal limits of how much Se can be included into the diet (Table 8.14) and this restricts
flexibility of a nutritionist in terms of addressing Se needs of the developing and
reproducing swine. For example, immunomodulating properties of Se are usually
seen at doses substantially higher than those needed to maintain pig growth and
development. Indeed, organic selenium in the form of selenized yeast is proven to be
an important natural solution for pig industry. The advantages of organic selenium in
comparison to selenite (Table 8.15) are obvious and include better Se transfer from
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Oestrus and ovulation rate
Semen
Fertilization rate
Se
Embryo survival
Uterine capacity
Se
Newborn piglets
Se
Sow fertility
Se
Weaned piglets
Figure 8.5 Potential role of selenium in sow reproduction (Adapted from Close, 1999; Close and Cole,
2000).
Table 8.14 Approved levels of selenium supplementation in feed (Adapted from Mahan and Kim, 2001)
Countrya
Canada
Denmark
Finland
Norway
Sweden
United Kingdom
United States
Complete feed
Concentrate
supplement
Salt/mineral
mixes
0.1
0.2
0.3
0.2b
0.3
0.5
0.3
0.7
-c
25
20
30
90 d
/In New Zealand selenium is largely applied in fertilizers

/Extended to 0.3 ppm in broiler diets
c
/Allowable in amounts not to exceed daily intake of 0.7 mg Se for sheep or 3.0 mg for cattle
d
/Extended to 120 ppm in salt/mineral mixtures for cattle
b
mother to the progeny via placenta, colostrum and milk, which could be translated
into better immune system development in piglets resulting in improved piglet
viability and decreased pre-weaning mortality, improved their growth and
development and improved meat quality. Indeed, in stress conditions, when
requirement for antioxidants substantially increased, but feed consumption is
usually decreased Se reserves, accumulated in tissues, could
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478
Table 8.15 Advances of organic selenium for pigs
Parameter
Effect of organic vs
inorganic selenium
References
Serum, liver, colostrum and

milk selenium
Toxicity
Tissue Se concentration
Drip loss
Meat colour
Liver Se
Blood Se
Weanling pig loin Se
Placental Se transfer
Se transfer to the fetus and
status at birth
Total Se in neonate
Gilt tissue Se level
Muscle Se level
Se excretion
Backfat depths
Loin-eye area
Increased
Kim and Mahan, 2001, Mahan,

2000, Mahan and Peters, 2004
Kim and Mahan, 2001a; 2001b
Mahan et al., 1999
Mahan et al., 1999
Mahan et al., 1999
Ortman and Pehrson, 1998
Mahan and Kim, 1996
Mahan and Kim, 1996, p.2711
Mahan and Jacques, 1998
Mahan and Peters, 2004
Mahan and Parrett, 1996
Mahan and Parrett, 1996
Wolter et al., 1999
Wolter et al., 1999; Miller et al.,
1997
Mahan, 1996
Se bioavailability in sow milk

to the nursing pig
Piglet weight at birth and
weaning and daily gain
pre-weaning
Total piglet born and piglet
born alive
Pre-weaning mortality
Less toxic
Increased
Decreased
Improved
Increased
Increased
Increased
Increased
Improved
Increased
Increased
Increased
Decreased
Decreased
Increased
Increased
Increased
Janyk et al., 1998; Janyk, 2001;

Pineda et al., 2004
Increased
Pineda et al., 2004
Reduced
Janyk et al., 1998, Janyk, 2001;

Close, 2003; Lampe et al., 2005
Lampe et al., 2005a
Janyk et al., 1998, Janyk, 2001;
Bobcek et al., 2004
Bobcek et al., 2004
Piglet survivability in the nursery

Number of stillbirths
Splay legs, % live pigs
Growth rate
Increased
Decreased
Decreased
Increased
FCR
Improved
be a key for the resistance of swine to detrimental effects of free radicals and
toxic products of their metabolism. Environmental concerns about selenium as a
potential pollutants lead to legal restrictions of levels of Se, which can be included
into animal diets. Therefore, better assimilation of selenium from organic sources
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could potentially help pig nutritionists to overcome consequences of such a
limitation.
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Selenium in Ruminant Nutrition 487

9
SELENIUM IN RUMINANT NUTRITION
Better a good cow than a cow of good kind
Introduction
Despite substantial achievements in dairy and beef cattle industries for the last 20
years there are several problems which need to be solved. It seems likely that
three weakest links include:
immunity
high somatic cell counts
mastitis
reproduction
low conception rate
retained placenta
metritis
cystic ovaries
viability in early postnatal life (calf death loss at birth or between birth and
weaning is a significant source of reduced reproductive rate
Among many different approaches used to address above problems diet

optimization is considered to be the most important one. Taking into account
various stresses of commercial dairy, beef and sheep production it is necessary to
pay more attention to antioxidant-prooxidant balance in the feed, digestive tract
and tissues (Surai, 2002; Surai et al., 2003; 2004). As mentioned above, Se as an
integral part of a range of selenoproteins plays an important role in regulation of
various physiological functions including immunity, reproduction and early
postnatal viability. It is important to note that Se deficiency in dairy cows persists
globally independently on current practise of selenium dietary supplementation.
Therefore it seems reasonable to suggest that by addressing Se requirement it
would be possible to deal with those weakest links in dairy, beef and sheep
production.
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Selenium deficiency in ruminants

Selenium deficiency alone or in combination with vitamin E are related to the
development of various diseases in ruminants (Table 9.1; Pehrson, 1993; Kolb
and Seehawer, 2001) including:
nutritional muscular dystrophy (NMD). NMD or white muscle disease

(WMD) is the most well documented disease related to Se/vitamin E
deficiency in ruminants. Indeed, relationship between Se status and NMD
was first described in 1958 (Hogue, 1958; Muth et al., 1958). A model for
nutritional degenerative myopathy in ruminant cattle was developed using
vitamin E- and selenium-deficient diet with additional supplementation of
PUFAs (Kennedy et al., 1987). Necropsy revealed widespread severe skeletal
myodegeneration and myocardial lesions with preferential involvement of
the left ventricular myocardium. Clinical signs and pathological changes
were similar to those reported in field outbreaks of nutritional degenerative
myopathy in ruminant cattle.
Table 9.1 Selenium responsive diseases in ruminants (Adapted from Gates and Johnson, 1995; Pehrson,
1993; NRC, 1996)
Disease
Major clinical signs
NMD
Acute onset: stiffness, skeletal and/or cardiac muscles affected.

Signs vary from sudden death to chronic lameness
Unthriftiness
Decreased feed efficiency and weight gains, anthrifty appearance
Retained placenta
Retained placenta
Abortions, stillbirths
Late pregnancy abortions and stillbirths
Neonatal weakness
Calves and lambs born weak
Diarrhea
Diarrhea, usually profuse, and weight loss in young and adult cattle
Compromised immune Suppressed immune response to vaccines and environmental microsystem
organisms
Infertility
Decreased conception rate, irregular estrus cycles
Mastitis
Udder inflammation
Metritis
Sub-optimal
Decreased milk production. growth and development
productivity
Peridontal disease
Sheep
Anemia with presence Anemia
of Heinz bodies
Increased mortality and Decreased animal viability
reduced calf weaning
weight
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It is possible to distinguish two forms of NMD (Sobiech and Kuleta, 2002):
subclinical, which is manifested by an increased serum activity of various

enzymes
clinical, in which dyspraxia accompanies muscular atrophy and anatomopathological changes in muscular tissues
Did you know that nutritional muscular dystrophy (NMD)
or white muscle disease (WMD) is the most well documented
disease related to Se/vitamin E deficiency in ruminants
The disease mainly affects both the skeletal and cardiac muscles. However, rib
muscles and diaphragm may also be affected (McDowell et al., 2002). When the
skeletal muscles are affected, symptoms vary from mild stiffness to obvious pain
upon walking, to an inability to stand. Muscular tremor may take place and the
affected muscles tend to be swollen and firmer than normal. The diseased animals
have a stiff gait, arched backs and are unwilling to move. Lambs/kids may tremble
in pain when held in a standing position. Indeed, affected young animals are
characterised by difficulty walking and may be unable to rise and nurse. They
gradually loose strength and become weak with decreased foraging with a possible
death from starvation. Therefore the main clinical symptoms of calves with WMD
were motor disturbances including recumbency and stiffness and weakness
(Hoshino et al., 1989). In addition, heart failure, paralysis of the hind legs and a
dystrophic tongue may be evident. Mortality of diseased animals can be as high
as 30% (Telford and Einarson, 1971). Because of weakening immune system
secondary infections can also be found in affected animals. In fact, some animals
show signs of respiratory distress and may be treated for pneumonia by antibiotics
without success and they can die as a result of fluid accumulation in the lung.
This disorder has been observed in all farm animal species, especially in rapidly
growing lambs and calves in areas where the feeds consumed contain between
20 and 30 ppb selenium in dry matter. Two etiologic forms of the disease have
been described, congenital and juvenile. The congenital form usually results in
abortion, stillbirths, or death shortly after birth with degenerative changes of the
myocardium (Andrews et al., 1968; Hartley and Grant, 1961; Hidiroglow, 1980).
For example, a 13-hour-old calf was involuntary recumbent since birth. Congenital
nutritional muscular dystrophy was diagnosed based on clinical findings, increased
serum creatine kinase and decreased serum vitamin E and selenium levels
(Abutarbush and Radostis, 2003). Recovery followed after supportive therapy
and parenteral vitamin E and selenium (Table 9.2). Similar case was described
with 18-hour-old recumbent calf developing dyspnea (Gawley and Bradley, 1979).
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Table 9.2 Effect of Se and vitamin E treatment on a beef calf with congenital muscular distrophy
(Adapted from Abutarbush and Radostitsm 2003)
Reference range
Day 2 (before treatment)
Day 3
Day 4
Day 5
Day 6
Day 7
Creatine kinase, U/l
Aspartate aminotransferase, U/l
35-280
29280
6200
966
267
119
166
78-132
299
198
145
97
78
Therefore, cows receiving a Se-deficient diet during gestation may give birth to
offspring suffering from NMD. Furthermore, the calves may be born dead or
extremely weak and die during the first few days of life (Hansen et al., 1993).
An outbreak of nutritional muscular dystrophy which caused the perinatal death
of 11 calves in a herd in the Las Colonias region of Santa Fe Province was
described (Rodriguez et al., 1998). Post-mortem examination of the calves showed
skeletal muscle degeneration as well as heart, liver, kidney and spleen lesions.
Furthermore, CPK and AST levels were higher than normal in the sick animals.
After parenteral treatment of calves and pregnant cows with Se and vitamin E,
blood chemical values returned to normal. Indeed, selenium deficiency in bovine
fetuses may be responsible for myocardial necrosis and heart failure (Orr and
Blakley, 1997). In general, the juvenile or delayed form of the disease most
commonly affect calves 3 to 8 weeks old with predominant sign in muscular
weakness (Campbell et al., 1990).
Muscular dystrophy in adult selenium-deficient dairy cows due to increased
locomotor activity and stress associated with the change in housing conditions
was described (Pavlata et al., 2001). In particular, the investigations were conducted
in a herd of Bohemian Red Pied cattle at the time of a transfer from a stanchion
barn into loose boxes. Clinical muscular dystrophy, manifested by general
weakness, subcutaneous oedema and downer syndrome, accompanied by marked
increases in creatine kinase and lactate dehydrogenase activities was observed in
dairy cows of the transferred group.
Similar to calves, there are two major critical ages for NMD in lambs. Firstly,
during first days of life, lambs born to Se-deficient sheeps were weak and died
within a few hours after various stresses, such as physical exercise. Secondly, Sedeficient lambs between three and six weeks were also very sensitive to NMD
(Pehrson, 1993). Furthermore, lambs and calves three months old are also affected
(Shamberger, 1983). In general, lambs are more affected by NMD than calves.
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Nutritional myodegeneration was also described in goats. In particular it was
established that pathological changes affect skeletal and cardiac muscles with the
muscular form of the disease being more frequent than the cardiac one (Ivandija,
1987).
Experimental vitamin E-Se deficiency in cattle produced preferential degeneration
and necrosis of Purkinje cardiocytes (Kennedy and Rice, 1988). Alterations in necrotic
cells included mitochondrial mineralization, sarcoplasmic condensation, and
plasmalemmal fragmentation. Necrosis of Purkinje cells was followed by macrophagic
penetration of the external lamina, phagocytosis of necrotic sarcoplasm, and repair
by fibrosis (Kennedy and Rice, 1988).
Microscopic lesions, comprising multifocal or diffuse cardiocyte degeneration and
necrosis, were seen in atrial and ventricular myocardium of affected calves (Kennedy
and Rice, 1992). Histopathological examination revealed a mild focal to an extensive
diffuse greyish white discoloration of the subendocardial myocardium. In affected
muscles (diaphragm, intercostals, myocardium, pelvic and hindleg muscles)
degenerative changes with the pale areas were observed. Ultrastructurally, sublethally
damaged cardiocytes had lysed contractile material; vacuolated sarcoplasm; altered
mitochondria, sarcoplasmic myelin figures, and lipofuscin granules; and multiple
nuclei and plasmalemmal disruption; the external lamina remained largely intact.
Necrosis was followed by macrophage invasion and phagocytosis of necrotic debris.
Repair of the lesions was by deposition of collagen and elastin fibres (Kennedy and
Rice, 1992). Furthermore, the lesions range from hyaline degeneration to a coagulation
necrosis of the muscle fibers with the appearance of macrophages and lymphocytes
and areas of calcification (Shamberger, 1983).
Pathologically, the left ventricle and the interventricular septum showed hyaline
myocardial dystrophy. Calves died peracutely and nonpredictably at an average
age of 17.3 days. The myocardial form of nutritional muscular dystrophy occurred
in 3% young calves from 4 counties of Southeast Bohemia in 1985. The same
disease was diagnosed in 11% and 19% calves in 1986 and 1987 respectively
(Sterba and Vavra, 1987). Indeed, damages to cardiac muscle could result in
sudden death (Hansen et al., 1993; Pehrson, 1993). For example, outbreaks of
sudden death of cattle, with incidence of up to 20%, caused by selenium deficiency,
were described in several counties in the Liupan Mountain areas of China (Guo
DeBao et al., 1997). Similarly, the Se-vitamin E deficiency was responsible for
deaths of dairy cows occurred in spring for 5 successive years in Yanbei prefecture,
Shanxi, China in 1981-1985. The sick animals were mainly calves and cows just
before or after calving, and the disease was characterized by exudative lesions
and functional disturbance of the alimentary, locomotory and reproductive systems
with mortality reaching 22% (Zhang Kong et al., 1996). Injection with sodium
selenite and vitamin E resulted in a 98% cure. Therefore, selenium dietary
supplementation or injections were able to prevent the disease.
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Biochemical examination showed severe selenium deficiency with whole blood

selenium values being 5-30 g/L and very low levels (<0.1 ppm) in the liver of
cattle or sheep affected by NMD (Pehrson, 1993). Indeed, affected animals were
characterised by extremely low Se status. For example, in Germany average Se
values were 19 g/L (Grunder, and Auer, 1995) while in Japan affected animals
had Se values less than 35 g/L (Hoshino et al., 1989). Increased activities of
enzymes showing damages to tissues are also characteristic for NMD (Table 9.3).
Indeed, the activities of lactate dehydrogenase, creatine phosphokinase, 5 1nucleotidse, and glutamic, oxalic, and pyruvic transaminases in the serum or plasma
are increased (Selenium in Nutrition, 1983).
Table 9.3 Serum muscle-specific enzymes (IU/l) in lambs (Adapted from El-Neweehy et al., 2001)
Group
No of lambs
ALT
AST
LDH
CPK
20
20
18
4.9
17.2
6.8
26.8
238.8
42.7
684.8
1815.7
642.5
43.4
1168.9
153.3
Control
NMD
NMD after treatment
It seems likely that lipid peroxidation in muscle membranes is involved in the

development of NMD. Damages to membranes are associated with accumulation
of Ca with following injuries to mitochondria. In fact, calcium salts may be
deposited among the muscle fibers in certain parts of the bodies of affected animals.
These salts give the areas involved a whitish appearance (Hansen et al., 1993).
Since mitochondria are considered to be major source of free radicals in biological
system, those changes in mitochondria homeostasis further increase free radical
production with following cell death and segmental necrosis and probably
apoptosis. Indeed, selenium deficiency is a major factor in the development of
NMD. However, such stress factors as vitamin E deficiency, high dietary levels of
polyunsaturated fatty acids, sudden muscular activity can also be involved in the
development of NMD. Calves deficient in both vitamin E and Se or deficient in
vitamin E alone showed elevated muscle concentrations of thiobarbituric acidreactive substances, ascorbate-induced TBARS, ascorbate-induced hexanal and
iron-induced 4-hydroxynonenal (Walsh et al., 1993). However, muscle tissues
of calves depleted of Se alone showed no increases in these indices of lipid
peroxidation.
Diagnosis is usually based on clinical findings:
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elevated levels of muscle enzymes (CK, AST, LDH);

low levels of vitamin E and selenium in the diet, tissue, and serum (whole
blood);
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low GSH-Px activity in plasma/serum, whole blood or tissues;

muscle degeneration with clinical and histopathological changes
positive responses to Se/vitamin E treatment
Protective effect of selenium and vitamin E was shown in sheep and in cattle
(Shamberger, 1983, Pehrson, 1993). Indeed, early cases of NMD can be treated
with parenteral injection of vitamin E and selenium. As can be seen from Table
9.2, elevated activities of muscle-related enzymes in plasma are normalised in 56 day period. The major preventive measure is optimal dietary supplementation
of selenium (preferentially in organic form) and vitamin E. It has been shown that
maternal nutrition is the most important factor in preventing their progeny from
early development NMD (Hidiroglou et al., 1972; Jenkins and Hidiroglou, 1972).
However, in spite of sodium selenite dietary supplementation of the cows, many
practitioners report that NMD still is a common disease among fast-growing calves,
particularly during their first month of life. It seems likely that this is a result of
poor Se transfer to the colostrum and milk from sodium selenite associated with
inadequate Se status of calves. Therefore NMD in nursing calves can be prevented
if the diet of the cows are supplemented with organic selenium (Pehrson, 2004).
Did you know that the major preventive measure against NMD
is optimal dietary supplementation of selenium
(preferentially in organic form) and vitamin E?
As a result of degenerative myopathy from Se deficiency a disease condition
called shoulder lameness or flying scapular was described (Buergelt et al.,
1996). It is characterised by bilateral dorsal scapular displacement which occurs
suddenly and leads to stiff gait and reluctance to move (Gunning and Walters,
1994).
Retained placenta (retained foetal membranes) and metritis

Three basic physiological functions are considered to be most important in disease
prevention during periparturient period (Goff and Horst, 1997):
adaptation of the rumen to lactation diets that are high in energy density
maintenance of normocalcemia
maintenance of a strong immune system
It is well accepted that, the incidence of both metabolic and infectious diseases is
greatly increased when one or more of these physiological functions are impaired
First two weeks of lactation are considered to be an important stress-period for
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cows. This is a most sensitive part of transition period, which lasts from 3 weeks
before parturition to 2-3 weeks after parturition (Drackley, 1999). This is a
transition from non-lactating pregnant status to non-pregnant lactation status, a
period critically important to health, production and profitability of dairy cows.
Indeed, most infectious diseases and metabolic disorders including retained
placenta, metritis and mastitis take place during this time. It is well established
that a significant suppression of immune function takes place in the peripartum
period. The physical and metabolic stresses of pregnancy, calving, and lactation
may contribute to this decrease in host resistance and the subsequent increase in
disease incidence (Goff and Horst, 1997; Mallard et al., 1998). Indeed, substantial
evidence indicates that innate and acquired defense mechanisms are lowest from
3 wk precalving to 3 wk postcalving. This leads to the increased incidence of
peripartum disease.
Did you know that retained placenta has remained a
problem for dairy industry affecting about 9% of all
calvings in US ?
Retained placenta (RP) is a reproductive abnormality unique to the cow and water
bufallo among domestic ruminants with the incidence varying from 3 to 39% of
parturition (Kimura et al., 2002). Indeed RP has remained a problem for dairy
industry affecting about 9% of all calvings in US (Kellogg et al., 2001). In fact,
RP is responsible for decreased milk production (Lucey et al., 1986). In
accordance with various estimations total cost associated with RP ranges from
about $100 to $280/case (Joosten et al., 1988; Kelton et al., 1998; Weiss, 2003).
In particular, substantial economic losses in dairy industry were related to RP
with the cost estimated per case to be $285 (Guard, 1999).
It has been shown that, major reproductive disorders (dystocia, retained placenta
and metritis) are interrelated and can affect the length of calving interval, the
number of days open, and the reproductive efficiency in general (Rajala and
Grohn, 1998). In general, cows with RP are those which are at high risk of
development of metritis and low fertility (Coleman et al., 1985). Furthermore,
dystocia and stillbirth increased the risk of retained placenta, while dystocia and
retained placenta increased the risk of metritis (Emanuelson et al., 1993; Table
9.4). On the one hand, a cow with a retained placenta was 4.4 times more likely to
develop early metritis and 2.5 times more likely to develop late metritis than one
without. On the other hand, a cow with endometritis was 1.5 times more likely to
develop cystic ovaries and silent heat. The involution of the uterus is prolonged,
the resumption of ovarian function is delayed, fertility is impaired and the period
between two calvings extended (Kudlac, 1991). Cows with retained placenta had
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a 14%, those with metritis a 15%, and those with ovarian cysts a 21% lower
conception rate than cows free of these disorders (Table 9.4; Grohn and RajalaSchultz, 2000). Cystic ovaries, cystic ovary degeneration, ovarian follicular cysts
and ovarian cysts are all terms used synonymously to describe an ovulatory
condition when a follicular structure grows to and surpasses ovulatory size but
fails to ovulate. Cystic ovaries comprise an economic problem for dairy industry
since cows are infertile as the condition persists. Affected cows have extended
calving intervals of approximately 50 days over healthy cows. This disorder
occurs in 10 13% of the US dairy herd population annually (Garverick, 1997).
Table 9.4 Risk factors for cow diseases (Adapted from Grohn and Rajala-Schultz, 2000).
Risk factors
Dystocia Retained
placenta
Dystocia
Abortion
Reteined placenta
Early metritis
Late metritis
Silent heat
Ovarian cysts
Other infertility
Ovarian
cysts
4.1
19.8
Early
metritis
3.2
3.7
4.4
1.5
2.9
1.5
5.4
2.8
Late
metritis
Silent
heat
Other
infertility
3.5
2.5
1.6
2.1
1.3
1.5
3.0
1.7
2.4
2.2
3.6
1.6
2.6
Did you know that in accordance with various estimations total

cost associated with RP ranges from about $100 to $280/case?
It was suggested that inadequate dietary antioxidants may increase oxidative stress
and incidence of RP in dairy cows (Brzezinska-Slobodzinska et al., 1994).
Therefore, it seems likely that selenium supplementation is an effective measure
to decrease incidence of RP (Table 9.5; Figure 9.1; Harrison and Hancock, 1999).
The first report on relationship between retained placenta and selenium/vitamin E
status of cows was published by Trinder et al. (1969). Since then, this subject has
received substantial attention. For example, of the 102 cows with selenium
concentrations of 30 ng/ml or less, 21.5% had RP (retention for more than 12
hours after calving) compared with 5.6% of those cows with concentrations of
over 30 ng/ml (Jaskowski, 1987). Blood selenium concentrations 2-4 weeks before
and/or 2-3 months after parturition were lower in cows with placental retention,
stillbirths and infertility (Jaskowski, 1991). High incidence of RP (40%) was
observed in cows with low Se level in blood (30 ng/ml) in a 110-cow herd in the
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Table 9.5 Reported positive effects of vitamin E and Se on the incidence of retained placenta (Adapted
from Allison and Laven, 2000).
Percentage
incidence
Control
Treatment
28.3
8.9
16
37.7
20.2
20.6
25.4
51.2
16.3
14.9
8.8
37
Treatment
Year
Injection 20 days prepartum 70 IU vitamin E and

2.3 mg Se
1000 IU vitamin E per animal per day plus
injection 21 days prepartum of 1 mg Se/kg
bodyweight
injection 15 to 40 days prepartum of 680 IU
vitamin E and 50 mg Se
injection 20 days prepartum of 680 IU vitamin E
and 50 mg Se
injection 40 and 20 days prepartum of 680 IU
vitamin E and 50 mg Se
injection one month prepartum of 680 IU vitamin
E and 15 mg Se
1985
1984
1980
1981
1976
1969
35
30
25
20
15
10
5
0
Control
Vitamin E
Selenium
Se + vit. E
Figure 9.1 Effect of Se and vitamin E on incidence (%) of retained placenta (13 studies; Adapted from
Harrison and Hancock, 1999)
Netherlands. Dietary supplementation with Se restored normal Se values and

placental retention ceased to occur (Wentink et al., 1988). Similarly in Korea
prevalence of placental retention was 34.5% in Se-vitamin E deficient cattle and
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decreased to 9.7% in animals injected i/m with sodium selenite and vitamin E 21
days before parturition (Shin and Jo, 1987).
A series of experiments was conducted over 3 years with pregnant dairy cows
in a high-producing herd (9000 kg milk/yr) with a history of retained placenta in
17% of the primiparous and 28% of the multiparous animals (Eger et al., 1985). It
was indicated that Se ranged from 0.035 to 0.109 ppm in the prepartum diet and
from 0.16 to 0.2 ppm in the postpartum diet. Doses of injected selenium
(intramuscularly 3 weeks prepartum) ranging from 2.3 to 23.0 mg reduced almost
twice the incidence of retained placenta in 186 primiparous and in 428 multiparous
cows to 7 and 15% respectively. Selenium alone was at least as effective as a
combination of selenium and vitamin E. In another study, a combination of Se
injection and oral vitamin E decreased incidence of RP from 17.5% to 0% (Harrison
et al., 1984). By increasing the mean daily intake of selenium at least 3 wk
prepartum from 0.23 mg to 0.92 mg daily, overall incidence of retained placenta
was reduced from 38% to 0% (Julien et al., 1976). A positive prophylactic effect
was achieved regardless of whether -tocopherol was supplemented as well.
Therefore, when dairy cows were deficient in selenium, the supplementation of
this element reduced the incidence of retained placenta. In a series of field
experiments in Ohio involving 193 parturient cows of the Holstein and Guernsey
breeds, the prophylactic efficacy of selenium and vitamin E was tested under
field conditions. For this purpose herds initially were chosen because of a chronic
problem with RP which could not be related to a known etiological factor. It was
shown that a single 20 day prepartum injection of 50 mg of sodium selenite and
680 IU of -tocopherol acetate was able to reduce incidence of retained placenta
from a mean of 51.2% in control cows (without injections) to 8.8% (Julien et al.,
1976a).
Did you know that an optimal dietary Se supplementation is
an effective means of RP prevention in cows?
In Poland, 288 cows on 6 farms were injected i.m. with one of 12 combinations
of vitamin E (60, 300 or 600 mg) and selenium (0, 5, 25 or 50 mg) about 10 days
before calving. The results showed protective effect of Se against various disorders.
In fact, the higher the dose of Se used, the lower the incidence of placental retention
(23.6% without Se, 6.9% at 50 mg Se) was registered (Jaskowski, 1993). It is
interesting to note that selenium effect was seen regardless of vitamin E content.
There were also reductions in endometritis, ovarian dysfunction and ovarian cysts.
In an experiment with 18 cows, the 12 injected i.m. with 50 mg selenium either
21 or 10 days before expected parturition showed better uterine involution and a
lower frequency of endometritis and ovarian disorders than the controls with the
best results being in those given selenium 10 days before parturition (Jaskowski,
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1991). Peripartum intramammary infection (IMI) and retained placenta were

investigated with 50 multiparous Holstein cows supplemented with vitamin E
and/or selenium. Treatments given orally by capsule from 6 wk before reported
calving until parturition included: control, 3 mg Se/head per day as sodium selenite;
1000 IU vitamin E/head per day as D,L- alpha-tocopheryl acetate; and 3 mg Se/
head per day plus 1000 IU vitamin E.. The incidence of IMI during early lactation
was lower in the Se-vitamin E group than in the other 3 treatments evaluated
(Finkelstein et al., 1992). Furthermore, the incidence of RP was numerically lower
in the Se-vitamin E group than in the other 3 treatments. At the beginning of the
dry period and 30 days later, cows were injected with 50 mg sodium selenite +
600 g -tocopherol. Treatment reduced the incidence of retained placenta by
7% and involution time of the uterus by 23%, and reduced the service period by
16.3 days compared with these traits in untreated cows (Kolchina et al., 1991). In
an experiment conducted in Yugoslavia vitamin E added to the diet at 1 g/cow
daily from 60 or 21 days to the expected calving date and selenium, 0.1 mg/kg
body weight, given once on day 21 before calving reduced the incidence of retained
placenta from 51.2% to 8.8% or from 17.5 to 0%, respectively (Ivandija, 1987a).
The same treatment reduced the incidence of metritis and cystic ovaries. In
particular, in cows with metritis, Se injection reduced the number of days required
for involution of the uterus. Vitamin E and Se also reduced the incidence of mastitis.
Furthermore, the offspring from the cows given Se had higher weaning weights
than those from control cows. It is interesting that Se-vitamin E combination not
only significantly reduced the incidence of retained placenta but also reduced the
days required for calving the first service (Kim et al., 1997). A farm was described,
on which the placenta was retained in approximately forty per cent of the cows
and on which selenium deficiency (30 ng/ml) and reduced activity of GSH-Px in
the erythrocytes (65.2 U/g Hb) were observed. When 0.5 mg of Se per kg of dry
matter of a mixture of concentrated feeds were added, the concentrations of Se
and GSH-Px were restored to normal and the symptom consisting in retained
placenta disappeared (Wentink et al. 1988). Sixty-four multiparous dairy cows
were used in an experiment conducted by Brzezinska-Slebodzinska et al. (1994).
Treatments given daily be gelatine capsule during the last 6 weeks prepartum,
were 1000 IU of vitamin E par cow; 3 mg of Se per cow or a mixture of vitamin
E and selenium. Incidence of RP, compared to the unsupplemented control was
reduced by 33% by vitamin E, 47% by Se and 50% by vitamin E and Se combined.
It is unclear why some cows fail to expel the placenta following calving. Both
the mechanism of placental expulsion and the cause of RP remain unclear. Various
factors including age, species, heredity, environment, hormones and nutrition have
been considered to be involved in the development of RP. It was suggested that
immunosuppresion at calving is related to problems of expulsion of fetal
membranes. It has been shown that leukocytes of cows that would expel the
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placenta normally had a strong chemotactic response to cotyledon material. In
great contrast, cows that failed to expel the placenta normally had peripheral
blood leukocytes that exhibited little to no chemoattraction to the cotyledon
suspension (Gunnink, 1984; 1984a; Goff and Horst, 1997). The authors suggested
that at the time of parturition, placental tissue becomes a dead foreign body that
the cows body must recognize and reject in order to complete seperation of the
fetal membrane. Therefore, RP is related to failure of the immune system to break
down the placentome promptly after delivery of fetus (Kimura et al., 2002). It
seems likely that impaired neutrophil function causes retained placenta. For
example, neutrophils isolated from blood of cows with RP had significantly lower
neutrophil function before calving, and this impaired function lasted for 1 to 2 wk
after parturition (Kimura et al., 2002). A loss in neutrophil chemoattraction for
fetal membrne tissue after parturition and decreased superoxide radical production
by neutrophils were observed in those cows that would develop metritis and
characterised by RP (Cai et al., 1994). It is well accepted that neutrophils are the
primary mechanisms of uterine (Bondurant, 1999) and mammary (Mallard et al.,
1998) immune defence. Aberrant immune function before and after calving seems
to predispose cows to severe uterine infections. Few cows die from uterine
infections, but cows with uterine infections are more likely to be culled for poor
reproductive performance. Also, uterine infections can reduce milk production,
and some treatments contaminate milk (Lewis, 1997).
Did you know that RP is related to failure of the immune system
to break down the placentome promptly after delivery of fetus?
It seems likely that effect of supplementary selenium on retained placenta depends
on the Se status of cows. Indeed, when Se status is adequate additional Se
supplementation will not affect rate of retained placenta and other reproductive
problems which might not be related to Se deficiency. A 2-year study was conducted
to evaluate effect of vitamin E and Se on cows consuming diets that contained ample
amounts of selenium (0.1 to 2.0 ppm selenium). When cows were injected with 68
IU vitamin E and 5 mg selenium per 45.4 kg body weight approximately 21 days
prepartum there was no effect on incidences of retained placenta and calving difficulty
in comparison to control animals without injections (Schingoethe et al., 1982)
The terms metritis and endometritis are used interchangeably to describe an
inflammation of the endometrial lining of the uterus without systemic signs, which is
associated with chronic postpartum infection of the uterus with pathogenic bacteria,
primarily Arcanobacterium pyogenes (LeBlanc, 2002). Uterine disorders, primarily
nonspesific uterine infections, reduce the reproductive efficiency of dairy cows. In
some herds. 40% of the portpartum cows may be diagnosed with uterine infections
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(Lewis, 1997) and the cost to producers for each lactating dairy cow with a uterine
infection (i.e. metritis) was $106. Therefore, metritis has a major impact on the
profitability of dairy operation.
It was shown (LeBlanc, 2002) that of 1865 cows examined, 316 cows (16.9%)
were classified as having clinical endometritis. Furthermore, cows with endometritis
had a longer time to pregnancy and therefore affected cows had a 30% relative reduction
in the probability of pregnancy at first insemination. Furthermore, cows with clinical
endometritis were also less likely to become pregnant at all, and correspondingly
70% more likely to be culled for reproductive failure. It took approximately 32 d
longer for half the cows with endometritis to become pregnant than it did for unaffected
cows, adjusted for herd, parity, ovarian structures, and treatment (LeBlanc, 2002).
It seems likely that Se-deficiency is a factor related to metritis development and Se
supplementation could decrease incidence of this disorder. For example, a 104-cow
herd was divided into 2 groups, each given daily 150 g of a mineral supplement
containing either 0.5 or 19 mg of Se per kg of supplement for 15 months. For the first
3 months there was little difference between the groups, but subsequently the Sedeficient cows had a higher incidence of mastitis (22 versus 12 cases), endometritis
(17 vs. 8 cases) and embryonic mortality (5 vs. 0 cases) (Klawonn et al., 1996).
Decreased incidence of metritis in Se-treated dairy cows provides an example of an
association between Se deficiency and decreased disease resistance (Suttle and Jones,
1989). Injection of sodium selenite (50 mg) at 21 days prepartum was associated
with a significant reduction of metritis insidence (from 83% to 57%) in cows (Harrison
et al., 1984). In the same study the incidence of cystic ovaries decreased from 47 to
19% (Figure 9.2). Furthermore Se supplementation of cows with metritis resulted in
more rapid uterine involution (Harrison et al., 1986).
60
50
ab
Means differ, P<0.05
Se (ppm)
a
40
30
b
20
10
Control
Selenium
Vitamin E
Se + E
Figure 9.2 Incidence (%) of cystic ovary depending on Se and vitamin E status of cows (IM Se of 50 mg
21 days prepartum, 740 m g Vit E/day orally; Adapted from Harrison et al., 1984)
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When pregnant multiparous crossbred cows were injected with 500 mg vitamin E
and 15 mg selenium at 4 weeks before expected calving the placental retention
24 h after parturition decreased from 25% in control to 15% in the treated group
(Kumar and Mahmood, 2001). Endometritis occurred in 100% of the animals
following placental retention as compared to 10.2% of those with normal expulsion
of the fetal membranes. In experiment conducted in Yugoslavia vitamin E added
to the diet at 1 g/cow daily from 60 or 21 days to the expected calving date and
selenium, 0.1 mg/kg body weight, given once on day 21 before calving reduced
the incidence of retained placenta from 51.2% to 8.8% or from 17.5 to 0%,
respectively (Ivandija, 1987a). The same treatment reduced the incidence of cystic
ovaries. In particular, in cows with metritis, Se injection reduced the number of
days required for involution of the uterus. Furthermore, the offspring from the
cows given Se had higher weaning weights than those from control cows.
Increased SSC and mastitis

Mammary gland health is an important issue in dairy industry and intramammary
infections (IMI) are responsible for decreased milk production and compromised
milk quality. Milk somatic cells include several cell types, including neutrophils,
macrophages, lymphocytes and smaller percentage of epithelial cells of the
mammary gland. In the healthy lactating mammary gland, total somatic cell count
(SCC) is often <100,000 per ml of milk (Sordillo et al., 1997). It has been shown
that during bacterial IMI, total SCC can increase to 1000,000 per ml of milk
within a short period of time with neutrophils representing >90% (Harmon et al.,
1994). For example, under New Zealand Livestock Improvement Corporation
criteria, 2-year-old and older cows having SCC greater than 120,000 and 150,000
cells/ml respectively are considered likely to have infected udders (Grace et al.,
1997). In fact the severity and duration of mastitis is related to the promptness of
the leukocyte migratory response and the bacterial activity of SCC at the site of
infection. The duration and severity of the inflammatory response have a major
impact on the quantity and quality of the milk produced. In particular SSC is
considered to be an important characteristic of milk quality, which is strictly
regulated in various countries. For example, milk quality regulations in Germany
stated that milk must contain <400000 cells/ml averaged over 3 months. When
these limits are exceeded, milk price is reduced by DM 0.02/litre (Tran, 1995).
Indeed, decreasing SSC in the milk builds consumer confidence and safety.
Did you know that milk somatic cells include several cell types,
including neutrophils, macrophages, lymphocytes and smaller
percentage of epithelial cells of the mammary gland?
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Analysis conducted by Baltay and Bedo (2000) showed an importance of

physiological, environmental and production factors affecting SCC:
Physiological factors:
stage of lactation
number of lactations
milk yield
Environmental factors:
climate
season
time of day
Production factors:
technological environment
feeding
milking
It was shown that SCC increases immediately after calving and the elevated count
is seen for 5 days to 2 weeks. Subsequently, SCC drops to the normal value,
though it tends to increase during late lactation. SCC also increases with lactation
number or age (Baltay and Bedo, 2000). A significant negative correlation between
milk yield and SCC during lactation has been also described (Kehrli and Shuster,
1994). Indeed, increased SCC is associated with milk loss (Table 9.6). It is
necessary to take into account that high-yielding cows are especially susceptible
to stress, therefore they need additional attention to maintain low SCC.
Table 9.6 Estimated differences in lactation milk production associated with an increase in SCC score
(adapted from Harmon, 1994; Raubertas and Shook, 1982).
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Mean SCC score
Mean SCC x 103/ml
0
1
2
3
4
5
6
7
12.5
25
50
100
200
400
800
1600
502
Difference in milk production, kg/305 days

Lactation 1
Lactation 2
-91
-181
-272
-363
-454
-181
-363
-544
-726
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Strategies for lowering somatic cell count include:
breeding
husbandry
hygiene
milking technique
nutrition
It seems likely that optimal nutrition is of major importance to maintain general

health and low SCC. Therefore in addition to correct feeding dietary
supplementation with selenium and vitamin E is recommended to reduce the
incidence of mastitis and thus somatic cell counts (Baltay and Bedo, 2000). These
suggestions are based on the scientific evidence indicating a strict relationship
between antioxidants and SCC. Indeed, a range of various studies have linked
low Se and/or vitamin E status to increased susceptibility of dairy cows to
intramammary infections (Spears, 2000; see Chapter 4). This was clearly showed
by Maddox et al. (1991); Smith et al., (1984; 1997) and Smith and Conrad (1987).
Furthermore, individual serum Se concentrations were negatively related to logtransformed somatic cell counts at the completion of the trial (Fisher, 1995; Fisher
et al., 1995). Indeed, bulk tank SCC decreased significantly as Se concentration
in plasma increased and plasma glutathione peroxidase was correlated positively
to Se intake but negatively to SCC (Weiss et al., 1990).
Did you know that bulk tank SCC decreased significantly as Se
concentration in plasma increased and plasma GSH-Px was
correlated positively to Se intake but negatively to SCC?
Regression of bulk tank SCC on average herd serum Se concentration gave the
following equations:
SCC= 6.06 7.64 (Se, g/ml), r2= 0.70
SCC= 5.69 0.99 (GSH-Px, IU/ml), r2= 0.46
SCC= 6.18 7.59 (Se, g/ml) 0.56 (GSH-Px, IU/ml), r2=0.82
Therefore the highest negative correlation was seen when both Se concentration
and GSH-Px activity were included into the equation. In dairy ewes, a significant
correlation was observed between GSH-Px activity and log 10 SCC (r = -0.22,
P<0.05; Morgante et al., 1999). The effect of parenteral administration of two
subcutaneous injections of selenium and vitamin E during the dry period on the
mammary health and milk somatic cell counts of 25 dairy ewes was studied (Table
9.7). As can be seen from the table supplementation reduced somatic cell counts,
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during the subsequent lactation, but the effect on mastitis and intramammary
infections was not significant (Morgante et al., 1999). This was associated with a
redistribution of various cell counts in the milk. Similar results were reported in
goats (Atroshi et al., 1985). Indeed, Se supplementation was associated with a
decrease in the output of milk somatic cells in cows (Malbe et al., 1995). This was
confirmed by Hemingway (1999) using Se boluses and showing 2-fold decrease
in SCC. A significant negative correlation between the activity of GSH-Px in whole
blood and the bulk milk somatic cell counts in the herds with a low incidence of
mastitis was shown (Ndiweni et al., 1991). The authors suggested an existence of
an association between the incidence of subclinical mastitis or inflammation and
the selenium status of these herds. The herds with the low SCC (less than or equal
to 150000 cells/ml) had higher mean blood GSH-Px activity (35.6 2.95 mU/mg
of haemoglobin) than did the herds with the high SCC (20.2 2.38 mU/mg of
Hb). Whole blood concentrations of Se were higher in herds with low SCC (0.133
0.01 mg/l of blood) than in herds with high SCC (0.074 0.007 mg/l of blood).
Furthermore significant negative correlations were found between the prevalence
of intramammary infection with major pathogens and mean herd activity of GSHPx and mean herd concentrations of Se (Erskine et al., 1987).
Table 9.7 Effect of selenium and vitamin E on ewes (Adapted from Morgante et al., 1999).
Control
Experimental*
Blood
Lysozyme, g/ml
GSH-Px, U/ml of packed cell volume
White blood cells, x 106/ml
Lymphocytes, x 106/ml
4.3
86.3
10.8
6.3
4.8
139.5
9.0
4.9
Milk
SCC, x 103/ml
SCC, log10
2733
6.0
1272
5.4
Differential cell count

Macrophages, %
Polymorphonuclear neutrophils, %
Lymphocytes, %
Epithelial cells, %
Eosinophils, %
38.4
50.7
7.6
0.8
1.4
48.8
40.1
8.4
1.0
0.7
*Two subcutaneous injections of Se (0.1 mg/kg of BW) and vitamin E (5 mg/kg BW) at 2
weeks intervals from about 1 month before lambing
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Mastitis, or inflammation of the mammary gland, represents another expensive
problem for dairy farmers and is recognised as the most costly disease facing the
worlds dairy industry (Dingwell et al., 2003; Spain, 2005). The milk production
loss from clinical mastitis was estimated as 1181 kg (Wilson et al., 2004). Even on
well-managed farms, approximately 50 cases of clinical mastitis can be expected
per 100 cow-years (Weiss, 2003). Again, the total costs associated with clinical
mastitis is in a range of $100 - $140 per case (Hoblet et al., 1991) or $150-200/
cow/year (Smith et al., 1998) or about $17,500 annually for an average 100-cow
herd (Smith et al., 1998). Much higher cost of mastitis was calculated u in the
UK. In the case of a mild case, the direct cost of mastitis add up to 77.03 and the
total cost is 137.94, while in the case of severe mastitis the total cost of mastitis
is 400.83 per cow (Esslemont and Kossaibati, 2002). The authors calculated that
90% of mastitis cases are mild, 9.8% are severe and 0.2% fatal with everage cost
per affected cow to be 166,31. The annual cost of mastitis to US dairy farmers in
terms of reduced milk production, discarded milk, veterinary and drug costs and
premature culling is estimated to be $2 billion (Smith, 1988). It is interesting to
note that despite of great efforts from dairy scientists to prevent the disease, mastitis
infections in dairy cows have not decreased (Spain, 2005) or even increased in
many countries (Pyorala, 2002).
Did you know that the total costs associated with clinical mastitis
is in a range of $100 - 200/cow/year or about $17,500
annually for an average 100-cow herd?
Factors predisposing cattle to mastitis are categorised as animal factors and
environmental factors (Spain, 2005). Animal factors are related to anatomical
structure and physiological functions of mammary gland and teats. In relation to
the metabolic factors it was admitted that the incidence of mastitis occurred in
two distinctive periods of the dry period. These are the first three weeks after dry
off and last two weeks of gestation and early lactation. This probably relates to
the decreased efficiency of immune system at these periods of ontogenesis. For
example, the proportion of CD62L+ neutrophils decreased significantly at calving
(Meglia et al., 2001). In fact, these changes at calving may result in less migration
of blood neutrophils into the tissues, and might contribute to the increased
susceptibility to infections at this time. It is interesting to note that higher producing
cows are at greater risk of mastitis (Spain, 2005). Indeed, cows with higher milk
yield in the previous lactation are more likely to have retained placenta, early
metritis, silent heat, ovarian cysts, and other infertility than are cows with lower
milk yield (Grohn and Rajala-Schultz, 2000).
The following areas of management strategies to control coliform mastitis are
considered to be of great importance (Smith et al., 1993):
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Reducing teat end exposure to coliform mastitis

housing
bedding and milking time hygiene. For example, use of inorganic bedding
materials can significantly reduce clinical coliform mastitis, while predipping can reduce exposure to the coliform bacteria during the milking
process
Optimising mammary resistance to infection
preventing teat injuries
diet optimisation; cows resistance to infection can be optimised by
reducing stress and feeding well-balanced diets containing adequate
amounts of vitamin E and selenium
immunization
usage of antibiotics
Roles of selenium and vitamin E deficiency in the mastitis development was

addressed in a series of publications during last 20 years and summarised in the
Chapter 4. Improvement of cow immunity as a result of Se supplementation is
responsible for reduction of mastitis cases at dairy farms. For example, for 14
months, 2 groups of cows, matched for breed, age, lactation number and lactation
stage were at pasture during summer, and were given mixed diets of grass silage,
maize silage and sugar beet pulp in winter. The cows were supplemented with Se
at 0 and 19 mg/kg in the mineral supplement. It was shown that Se-supplemented
cows had significantly fewer udder inflammations and less mastitis treatment
(Salewski and Seegers, 1994). Supplementing the feed of selenium-deficient dairy
cows (25 cows per group) with Se at a level of 0.2 ppm induced self-cure of
subclinical mastitis. In fact, the prevalence of quarters harbouring subclinical
mastitis decreased 2-fold during the 8-week supplementation period. Furthermore,
the recruitment of phagocytes to the infected milk compartment of the udder was
improved due to Se-supplementation. Selenium supplementation also induced an
unspecified antibacterial activity in milk lactoserum (whey), inhibiting in vitro
growth of the major mastitis pathogens: Escherichia coli, Staphylococcus aureus,
Streptococcus agalactiae and Streptococcus uberis (Ali-Vehmas et al., 1997).
The effects of selenium was examined in dairy cows having oral supplementation
with 5 mg selenium or oral supplementation with 5 mg selenium plus 500 IU
vitamin E. Selenium supplementation increased serum selenium levels and reduced
the incidence of sub-clinical mastitis (Zanetti et al., 1998). For example, calves
from supplemented cows had serum levels of selenium 66% greater than controls.
Se supplementation had a positive effect on udder health. The percentage of
quarters harbouring mastitis pathogens dropped from 22.9 to 13.0 in Se-yeastsupplemented and from 18.4 to 7.4 in the selenite-supplemented group (Malbe et
al., 1995).
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The effects of selenium supplementation on mastitis parameters in milk and on
GSH-Px activity in blood were evaluated. Fifty-five Estonian dairy cows were
allocated to selenium-supplemented (n=39) and non-supplemented (n=16) groups.
The supplemented group received 0.2 ppm organic selenium in the form of
selenium yeast in their diet daily for 8 weeks. The non-supplemented cows received
their standard diet with no selenium supplementation. Mastitis parameters (i.e.,
bacteriologic findings and somatic cell count, N-acetyl-beta-D-glucosaminidase,
and bovine serum albumin concentration) and GSH-Px activity were monitored
(Malbe et al., 2003). The increase in the activity of GSH-Px was significantly
(P<0.001) greater in selenium-supplemented cows than in non-supplemented ones.
Milk samples from each quarter were examined before and 8 weeks after initiation
of the study. The proportion of quarters still pathogen-free after 8 weeks was
significantly (P<0.001) higher in selenium-supplemented cows than in nonsupplemented cows. Differential positive rate (Youdens index) revealed that
individual quarters were more prone to be infected by pathogens when the blood
GSH-Px activities in cows were below the cut-off value of 3.3 microkat/g
hemoglobin than when GSH-Px activity was above this value. It was concluded
that selenium supplementation in cows with low GSH-Px activity improves udder
defense mechanisms that are responsible for reduction of the incidence of new
mastitis cases.
Did you know that an optimal dietary Se supplementation is
an effective means of mastitis prevention and decreasing
SSC in cows?
When Se and vitamin E deficient cows in Argentina were supplemented with
these antioxidants there was a reduction of udder infections by 42% and of clinical
mastitis by 32% (Forte et al., 1987). Dairy cows receiving dietary supplements of
selenium and vitamin E during the dry period and continuing into lactation have
a significantly reduced incidence of mastitis compared with controls (Smith, 1988).
Peripartum intramammary infection (IMI) and retained placenta were investigated
with 50 multiparous Holstein cows supplemented with vitamin E and/or selenium.
Treatments given orally by capsule from 6 wk before reported calving until
parturition included: control, 3 mg Se/head per day as sodium selenite, 1000 IU
vitamin E/head per day as D,L- -tocopheryl acetate and 3 mg Se/head per day
plus 1000 IU vitamin E. The incidence of IMI during early lactation was lower in
the Se-vitamin E group than in the other 3 treatments evaluated (Finkelstein et al.,
1992). Furthermore, the incidence of retained placenta was numerically lower in
the Se-vitamin E group than in the other 3 treatments. The preparation containing,
apart from other minerals, 0.30-0.35 mg selenium and 6.5-7.0 mg iodine per 1 ml
was injected (15-20 ml) to cows with clinical mastitis and decreased percentage
of animals with mastitis from 70% to 20% (Bogush et al., 2000).
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As mentioned above, positive responses of cows to Se supplementation in

terms of mastitis prevention were related to improved immunity (see Chapter 4).
For example, dietary Se supplementation of cattle results in a more rapid neutrophil
influx into milk following intra-mammary bacterial challenge and increased
intracellular killing of ingested bacteria by neutrophils (Hogan et al., 1992). It is
generally accepted that one of physiological consequences of -tocopherol or Se
deficiency is reduced neutrophil activity. Se-dependent GSH-Px and vitamin E
are antioxidants that protect neutrophils from the destructive action of toxic oxygen
molecules necessary for intracellular kill of ingested pathogens (Hogan et al.,
1993). It was shown that feeding dams Se-enriched yeast enhances the neutrophil
and lymphocyte functions of suckling calves, to which Se is transferred through
milk. Twelve postpartum, nursing Angus and Hereford beef dams were divided
into 2 groups. The experimental group was supplemented with Se-yeast at 0.525
mg Se/100 kg of body weight daily for 11 weeks and the control group was not
supplemented with selenium. After 3 weeks, serum Se was significantly higher in
calves of dams receiving Se supplements than in those of control dams. Various
assays showed that, at 5 weeks, macrophage ability to generate free radicals was
improved in comparison to control calves. Furthermore, after 5 weeks, lymphocyte
function (response to concanavalin A) was significantly higher in experimental
animals than that in control animals (Matsui et al., 2000a).
As mentioned in the Chapter 4, a detailed meta-analyses of publications related
to the effect of selenium and/or vitamin E on mastitis in cows was published in
1999. In particular, it was shown that from 19 studies conducted all over the
world including Mexico, Italy, UK, Israel, Canada, Portugal and Korea and
published in peer-review journals in 13 studies (68%) positive effect of selenium/
vitamin E supplementation was reported. On average, incidence of mastitis was
decreased 3-fold, in control animals mastitis was registered in 32% and in Sesupplemented cows mastitis was evident only in 10% (Harrison and Hancock,
1999).
Acceptable target levels to influence resistance of cows to mastitis are for
selenium in blood 0.2-1.0 g/ml and for vitamin E >4 g/ml. It is recommended
that intake of Se to be 3 mg daily for dry cows and 6 mg daily for lactating cows
and intake of vitamin E to be 1000 IU daily during both the dry period and lactation
(Smith et al., 1989). Effect of Se and vitamin E supplementation on cows was
reviews by Harmon (2002) showing:
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42% reduction in prevalence of infection at calving

57% reduction in clinical mastitis in early lactation and 32% reduction
throughout lactation
40-50% reduction in duration of infections
decreased somatic cell counts for the lactation
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Unthriftiness and reduced growth rate

Se-responsive unthriftiness (SeRU), varying from subclinical growth depression to
the progressive loss of condition usually associated with diarrhea, can occur in cattle
and sheep of all ages (Andrews et al., 1968). For example, SeRU and high mortalities
in lambs, and low lambing percentage from ewes, have been considered as important
consequences of Se deficiency in New Zealand livestock (Sheppard et al., 1984;
Table 9.8). A trial was carried out to investigate some features of SeRU in the Strathbogie
ranges of Central Victoria where unthriftiness of young sheep has been a problem for
10 to 20 years. Eighty Merino ewes and lambs were allotted to one of 4 groups in a 2
x 2 factorial designed trial in which sodium selenite (0.1 mg/kg) was given orally to
ewes and/or lambs at marking time and to treated lambs at 3 monthly intervals
thereafter. Selenium treatment of the lambs produced significant responses: mortality
in treated groups was 0% compared with 17.5% in untreated groups; body weight
gains were 1.9 kg higher at both weaning and one year of age in treated than in
untreated lambs; mean fleece weight was 14.4% higher in treated lambs and they
produced 39% more wool than the surviving untreated lambs (McDonald, 1975).
Table 9.8 Effect of Se deficiency in breeding ewes (Adapted from Sheppard et al., 1984)
Trial A
Sedeficient
Ewes to ram
Se in blood of
At joining flock to rams
ewes, ng/ml
A month before start of lambing
No. of barren ewes
No. of lambs born
No of ewes with twins
Wt. gain, kg
One month before joining to
one month before lambing
Ewe mortalities
Between joining and lamb
docking
During late pregnancy
Wt. of single
lambs at birth, kg
Trial B
Sedosed
Sedeficient
Sedosed
108
9.1
9.8
45
46
4
-5.6
108
48.0
23.0
15
96
15
-0.4
200
8.0
8.6
79
139
28
200
55.0
25.0
14
249
68
22
12
10
4.1
2
4.6
Diagnosis of SeRU in lambs is usually based on measurements of lamb growth

response to Se supplementation (McLean et al., 1959). The authors showed that
SeRU in lambs developed when the Se blood concentration decreased below 10 ng/
ml. Furthermore, lamb death was observed when Se level in blood decreased below
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5 ng/ml. For Se-deficient lambs, the potential for a growth response to Se

supplementation was strongly related to blood Se concentration. In fact, economically
significant decrease in liveweight gains of >10 g/day were observed when initial
blood Se concentrations were <130 nmol/L (Grace and Knowles, 2002). A similar
conclusion was drown earlier by Sheppard et al. (1984).
The effects of selenium deficiency on growth was studied in 3 groups of 20
heifers between 6 and 18 months of age. Heifers that received recommended
amount of selenium in the diet had higher daily weight gain and achieved higher
body weight both at 12 and 18 months of age (Prytkov, 1999). Se supplementation
can also increase weight gain in selenium deficient grazing dairy heifers. For
example, calve growth was reduced when dietary Se was <100 ng/g DM (Angelov
and Yanchev, 1991). An experiment was conducted with selenium deficient
grazing dairy heifers from two dairy farms from the south of Chile (Oblitas et al.,
2000). The experimental animals were administered with sodium selenite (i.m.),
in a single dose of 5 mg of Se/100 kg body weight. The liveweight gain up to the
third month was greater in the treated group than in the control with a mean of
234 and 220 g/day in the growing and mating treated heifers, respectively in
comparison with 169 and 148 g/day in the control groups. An experiment was
carried out with 81 calves in the first 8 weeks after birth having plasma Se less
than or equal to 44 ng/ml. The calves in experimental group were treated with
subcutaneous injections of Se 0.2 + vitamin E 60 mg/kg body weight on day 1
and 3 post-partum and a further treatment was used with half these amounts at 4
weeks old. The animals in control group were injected with saline. Weight gains
were higher in Se injected calves at 4 weeks old and incidence of infections was
lower and duration of infections shorter, especially pneumonias (Jachens, 1993).
For 60 days 3 groups of 8 Black Pied calves, initial body weight 142 kg, in an
area deficient in selenium (Se in forage 0.072 mg/kg) were given diets of maize
silage and concentrates without or with supplementary Se 0.06 or 0.12 mg/kg
body weight daily. Mean gain was 24.1 9.6, 32.8 8.4 and 35.8 12.1 kg in the
3 groups, respectively. Activity of GSH-Px was greater and aspartate
aminotransferase activity was lower in experimental groups (Wang and Hong,
1993).
Did you know that selenium treatment of the Se-deficient lambs
or calves reduced significantly their mortality and
increased body weight gains?
In 2 herds, yearling dairy heifers were given 2 or 4 intraruminal pellets containing
elemental selenium (3 g) or were not treated. During 7 months after treatment
mean whole blood GSH-Px activity was higher in treated than in untreated heifers.
In one herd, which had the lowest initial values liveweight gain was 10% higher
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than in the untreated heifers (Wichtel et al., 1992). Eighty beef cows 3 to 10 years
old in mid-pregnancy were allotted by breed and age to 4 treatment groups. Control
group cows received no supplementary selenium. Cows from the experimental
groups on day 0 of feeding received by injection Se (0.1 mg/kg body weight) and
were given the salt-mineral mixture containing Se (120 mg/kg). Calves from
experimental groups were also given Se by injection (at 0.1 mg/kg body weight)
about 24 h old. Supplementary Se increased whole-blood Se concentrations in
the cows. It is important to mention that whole-blood Se concentrations of the
newborn calves correlated positively with those of their dams at parturition, but
were significantly higher (Swecker, et al., 1991). Calf birthweight was not affected
by Se treatment of cows but calf average daily gain for 60 days was 790 g in
control group and 830 - 930 g in experimental groups.
It seems likely that the effects of Se deficiency in grazing calves may be
mediated by alterations in thyroid hormone metabolism. It has been shown that
type I iodothyronine 5'-deiodinase activity occurs in the thyroids of rats, mice,
guinea pigs and man. Therefore, all those species have a potential mechanism for
maintaining plasma T3 concentrations in Se deficiency and in hypothyroid stress.
In great contrast, farm animal species such as cattle, sheep, pigs, lamas, goats and
rabbits have no significant activity in the gland and they may be more susceptible
to thyroid-related effects of Se deficiency (Arthur et al., 1993). Selenium deficient
calves when compared to Se supplemented calves had increased plasma thyroxine
concentrations and decreased plasma tri-iodothyronine concentrations. These
changes in the selenium deficient calves were accompanied by significant increases
in plasma urea and creatinine concentrations and decreased plasma alkaline
phosphatase activity (Arthur et al., 1988). The effects of selenium supplementation
on growth rate and on thyroid were examined in heifer calves from 2 herds at
pasture. Supplementation of calves with intraruminal Se pellets (designed to release
Se about 3 mg/day) increased the basal plasma concentration of 3,5,3'triiodothyronine and reduced the basal plasma concentration of thyroxine for
both herds (Wichtel et al., 1996). Furthermore, for one herd, supplementation
with Se increased the triiodothyronine response to challenge with thyrotropinreleasing hormone, increased body weight gain, and tended to increase the plasma
concentration of insulin-like growth factor I. Serum growth hormone (total, free
and binding forms), prolactin (total and binding forms) and thyrotrophic hormone
of day old calves were significantly increased after dam supplementation. It is
interesting to note, that selenium exerted its positive effect on growth hormone
and thyrotrophic hormone up to 40 days post-natal. Calve growth was reduced
when dietary Se was <100 g/kg DM (Angelov and Yanchev, 1991). Ten Friesian
cows in their late stage of gestation were intramuscularly injected with 100 mg of
selenium as sodium selenite at a rate of two doses with one-week interval, while
seven pregnant animals were left without treatment as controls. Selenium
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512
supplementation improved the live body weight of calves. The overall mean of
weight gain and relative growth rate of calves per week were significantly increased
(4.24 vs. 3.64 kg and 8.42 vs. 7.84%, respectively; Youssef et al., 2000).
In experiment conducted in Italy selenium was added to the calve diets during
weaning to increase their selenium content from 0.06 to 0.1, 0.15 and 0.2 ppm
for 120 days (Bonomi et al., 2001). There was an improvement in calves weight
gain by 7.0, 12.5 and 17.0% respectively. Selenium supplementation improved
the feed utilisation efficiency of the calves by 5, 9 and 12% respectively.
Furthermore, selenium supplementation, though limited to doses 0.15 and 0.2
ppm, improved the reproductive efficiency of the heifers: the number of services
per conception was 1.8 in the control group and 1.4 and 1.2 in experimental
groups respectively (Bonomi et al., 2001a). In another experiment selenium was
added to the milk replacer at a dose of 0.3 or 0.4 ppm and this improved the
weight gain of the calves (by 9 and 12%, respectively) and feed efficiency (by 9
and 14%; Bonomi et al, 2001b). Furthermore, it improved the carcass yield (by
4.5 and 8.0%), meat digestibility and meat tenderness. Selenium supplementation
also positively affected the health status of the calves, with a strengthening of the
organic resistance of the animals to respiratory and gastro-intestinal diseases.
Similar positive results were reported for young bulls fed from 300-650 kg
liveweight a selenium deficient diet (0.06 ppm Se) or the same diet supplemented
with selenium to give Se level 0.3-0.4 ppm (Bonomi, 2001).
There are also other detrimental consequences of Se deficiency in cattle.
For example, swelling of the hocks with lameness developed in 80 heifers of a
herd, commencing just after calving in Germany. It responded to sodium selenite
injection and a daily supplement of 5 mg Se for cows, or 2-3 mg Se for calves
(Eicken et al., 1992).
Reduced reproduction rates

Selenium plays an important role in animal reproduction (for details see Chapter
5). Significant correlation coefficients were found for both services per conception
(r=0.45) and days open (r=0.49) with the serum selenium concentration 14-21
days post-partum (Larson et al., 1980; Hemingway, 2003). Fertility problems in
sheep and dairy cattle in many cases can be related to Se deficiency. Indeed
infertility has increased in ewes grazing pastures low in selenium. It was shown
that Se infertility in ewes is more prevalent in some areas and in some seasons,
but the actual cause of this malady and the continuing role of additional factors
are unknown (Hidiroglou, 1979). Furthermore reproductive performance of ewes
may be improved by Se or Se+vitamin E supplementation/injection. For example,
recent data (Table 9.9) indicate that Se injection (4 weeks before the mating season
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and 4 weeks before lambing) was associated with higher percentage of ewes with
oestrus and lambing, more lambs born per ewe, and higher lamb daily gain
(Gabryszuk and Klewiec, 2002). It is interesting to note, that positive effect was
significant only in 3 year-old ewes, but not in 2-year-old animals. Similarly survival
of lambs, live weights at birth and at weaning were increased by Se
supplementation (Langlands et al., 1991a, b). Furthermore, positive effects of Se
and Se+vitamin E were also observed by other authors (Scales, 1974; Segerson
and Ganapathy, 1980). It seems likely that this effect of Se depends on the severity
of Se deficiency. For example, there was no effect of Se on fertility in experiments
conducted by Davis (1966) and Segerson et al. (1986).
Table 9.9. Effect of i.m. Se (2.09 mg) and vitamin E (250 mg) injections to 3-year old ewes on
reproduction and rearing of lambs (Adapted from Gabryszuk and Klewiec, 2002).
Parameter
Control
Se-injection
Se + Vitamin E
injection
Ewes in oestrus, %
76
100
Ewes lambing, %
68
100
Number of lambs born of ewes lambing
100
112
Lamb live weight at birth, kg
4.2
4.4
No. lambs reared to 28 days
15
25
Lamb live weight at day 28, kg
9.4
10.9
Daily live weight gain for 28 days, g
197.7
241.8
.
* Four weeks before the mating season and 4 weeks before lambing
92
76
105.3
4.3
19
10.0
216.1
Did you know that selenium deficiency is associated with

decreased reproduction from the male (fertility) and female
(conception rate) sides?
In two experiments selenium-deficient Friesian/Holstein heifers were given a single
subcutaneous injection of barium selenate just before mating. This treatment
effectively restored normal Se status and the number of days from first insemination
to calving was significantly reduced for the Se-treated heifers. This was due to a
significant improvement in combined first- and second-service conception rates
(MacPherson et al., 1987). When two Se deficient herds were supplemented with
0.1 ppm Se number services per conception, days open and calving interval were
reduced (Table 9.10, Cortese, 1988). In studies on 117 cows in three groups,
(each of 39 cows), experimental animals were injected i.m. with 50 mg Se as
sodium selenite 21 and 10 days before parturition, respectively, while control
group was not treated. The main result are shown in Table 9.11 (Jaskowski, 1990).
Improved Se status of cows was associated with decreased RP, stillborn calves,
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514
endometrites and ovarian disorders. Furthermore, days to first service and service
period were reduced. Cows (296) from a Se-deficient herd, with blood serum Se
concentration <25 ng/ml, were divided randomly into two groups and injected
i.m. with 0 or 20 mg Se as sodium selenite at about 3 weeks after parturition. The
frequency of ovarian cysts, conception rate and service period in treated cows in
one herd were 20.8%, 55.8% and 97.6 days, compared with 39.6%, 40.2% and
121.4 days in controls (Jaskowski and Rogoziewicz, 1990). At the beginning of
the dry period and 30 days later, cows were injected with 50 mg sodium selenite
+ 600 g alpha-tocopherol. Treatment reduced the incidence of retained placenta
by 7% and involution time of the uterus by 23%, and reduced the service period
by 16.3 days compared with these traits in untreated cows (Kolchina et al., 1991).
Three selenium supplementation trials, conducted from 1986 to 1991, at the
University of Arkansas Southwest Research and Extension Center. The results
showed that Se supplementation increased fertility of cattle from 55 to 80%, and
increased weaning weight by 41 lb (Phillips et al., 1992). The authors admitted
that when cattle are deficient or low in Se, it takes 60 to 90 days for body stores of
Se to accumulate for production levels to exceed maintenance requirements.
Table 9.10 Effect of Se-supplementation (0.1 ppm) of Se-deficient cows in two herds (Adapted from
Cortese, 1988).
1st herd
Control
Number of services per conception
Days open
Calving interval, days
Blood selenium concentration
immediately after calving, mg/L
Blood selenium concentration at peak
of lactation, mg/L
2nd herd
SeControl
Sesupplemented
supplemented
2.34
136.5
421.5
0.038
2.10
116.1
401.1
0.081
2.82
167.6
452.6
0.021
1.81
98.0
383.0
0.073
0.053
0.088
0.047
0.076
Table 9.11 Effect of Se injection on cow reproduction (Adapted from Jaskowski, 1990)
Retained placenta,%
Stillborn calves, %
Endometritis, %
Ovarian disorders
Days to first service
Service period, days
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Control
50 mg Se, 21 d
before parturition
17.9
5.1
30.8
35.9
79.2
128
10.2
0
33.4
25.6
57.4
86.9
50 mg Se, 10 d
before parturition
10.2
2.6
18.0
23.2
59.9
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Total of 198 cows were randomly assigned to treatment with a single intramuscular
injection of 10 ml of a preparation containing vitamin E and selenium or placebo
3 wk before expected parturition. Incidence of retained fetal membranes was
3.0% for the treated group and 10.1% for the control group (P=0.06). Administration
of MU-SE also increased the percentage of cows pregnant to the first service
(41.2 vs 25.3%; P=0.02), reduced the number of services per conception (2.3 vs
2.8; P<0.05), and reduced the interval from calving to conception (121 vs 141
days; P=0.06). Therefore, prepartum supplementation with vitamin E and selenium
can decrease the incidence of retained fetal membranes, increase pregnancy rates
and, thereby, reduce the interval from calving to conception in lactating dairy
cows (Arechiga et al., 1994). Injection of vitamin E and selenium can increase
fertility in cattle that did not become pregnant at first service. For example, in a
trial, cows in a temperate climate received intramuscular injections of vitamin E
(500 mg) and selenium (50 mg) at 30 d postpartum (n = 97) or were untreated
controls (n = 89). The treatment resulted in increased pregnancy rate at second
service (69.8 vs. 52.1%), reduced the number of services per conception (1.7 vs.
2.0; P<0.05) and the interval from calving to conception (84.6 vs. 98.1 days,
P<0.05) (Table 9.12, Arechiga et al., 1998). Selenium and vitamin E (5 ml
containing 1 mg of Se and 68 IU of vitamin E/ml) were injected into beef cows 5
days before calving (Cohen et al., 1989). The treatment was related to reduced
placenta expulsion time, cows were in better body conditions and lost less weight
during first 4 weeks of the breeding season and there was a positive trends for
higher pregnancy rates (85.5% vs 78.4%) and for greater weaning weight and
there were also fewer calf death losses in the experimental calves.
Table 9.12. Effect of postpartum administration* of selenium and vitamin E on reproductive function of
lactating dairy cows (adapted from Arechiga et al., 1998)
Interval, calving to first service, days

Pregnancy rate at first service, %
Pregnancy rate at second service, %
Services per conception
Interval, calving to conception, days
Control
Se/vitamin E
63.3
46.1
52.1
2.0
98.1
61.7
45.4
69.8
1.7
84.6
*A single intramuscular injection of selenium (50 mg) and vitamin E (500mg) at day 303
postpartum
Recent analysis of the effect of Se/vitamin E supplementation of the Se-deficient

sheeps conducted by Hemingway (2003) indicated positive responses including
increased the lambing percentage of ewes, reduction in the proportion of barren
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ewes, increased proportion of ewes with multiple births and some others. Selenium
prepartum injections also resulted in higher colostrum and milk concentrations of
Se (Cuesta et al., 1995).
It seems likely that ROS may function as intracellular regulators of
steroidogenesis in the corpus luteum (Carlson et al., 1993). Therefore, luteal cells
can employ reactive oxygen species at specific sites in controlling the production
of progesterone over the course of the reproductive cycle and in inhibiting its
synthesis during regression at the end of the cycle. Se supplementation of cows
(0.5 ppm) did not affect the length of the oestrous cycle, but it did significantly
increase the concentration of plasma progesterone during the oestrous cycle
(Kamada and Hodate, 1998).
Immunosuppression and increased susceptibility to various diseases

As clearly shown in the Chapter 4, immunosuppressive effect of Se deficiency
can be overcome by Se supplementation with an improvement in both natural
and adaptive immunity. As a result, animal resistance to various pathogens and
eventually to diseases can be improved. For example a study was carried out in
order to examine the efficiency of selenium/vitamin E on the health status in the
first eight weeks of life in Se-deficient neonate calves.
Did you know that immunomodulating properties of selenium
are associated with immunocompetence maintenance of
cows in stress-conditions of commercial dairy production?
Animals with low selenium level (< 40 ng/ml) were randomly divided into two
groups: to be treated or to serve as control. The calves of experimental group
were treated with 0.2 mg selenium and 60 mg vitamin E per kg body weight
subcutaneously on the first and third day of postnatal development; they got
another treatment with half of the dosage in the fourth week of life. Control group
received a placebo (0.9% sterile NaCl-solution). The experimental group showed
a lower infection rate and the duration of infection (especially in the case of
pneumonia) was shorter than in the controls (Feldmann et al., 1998).
Sub-optimal productivity and early mortality

While failure to get beef females pregnant is the major cause of reproductive loss
in beef herds, calf death loss at birth or between birth and weaning is also a
significant source of reduced reproductive rate. In a summary of three research
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studies by Bellows and Short (1994), the average percentage of calves born dead
was 7.0% of those calving while subsequent losses to weaning averaged another
6.7% of those born alive. A survey of Kansas ranches published by Simms et al.
(1986) indicated that 5.8% of calves born didnt survive until weaning. A summary
of over 4,000 calvings over a 21-year period in Colorado (Holland et al., 1992)
showed a loss of 4.7% of the calves in the first 21 days of life and another 3.4 %
loss from 21 days to weaning. Thus, numerous studies have shown that calf
death loss is a significant factor in net reproductive rate and has a very significant
impact on overall profitability in the beef cattle industry.
Stillbirths are defined as a calf that dies just prior to, during, or within 24 to 48
h of parturition (Meyer et al., 2000). It has been calculated that about 7% of the
Holstein calves born in the United States die within 48 h of birth. Therefore, the
cost of stillbirths to the dairy industry is very high and, for example in the US in
1980th, it was about $132 million per year (Thompson et al., 1981). Factors
influencing stillbirths are diverse, including genetic, environmental and
management factors. However, dystocia, difficulty of a birth, has been implicated
as the major cause of stillbirths (Meyer et al., 2000). Furthermore, pneumonia,
diarrhea and cold exposure are among other important causes of stillbirths
(Kvasnicka et al., 1999). In Denmark, a total of 240 calves and 66 abortions were
submitted corresponding to a calf mortality rate of 7%. About 43% of the calves
died at day 0, while 22% were aborted, 15% died during the first week of life, 9%
died from 1 to 4 weeks of age, and 11% died at the age of 1 to 6 months (Agerholm
et al., 1993). The most common cause was neonatal stillbirth followed by foetal
infections, pneumonia, and septicaemia. The high incidence of stillbirth in Swedish
Holstein heifers has increased continuously during the last 15 years to an average
of about 11% today (Kornmatitsuk et al., 2004). For example, comparatively
high levels of stillbirths (10.3%) were observed in first-calving Swedish Holstein
cows in 2002 (Berglund et al., 2003). It is interesting to note that as much as one
third of the dead calves seemed clinically normal with no obvious reason for
death and the pathological reasons behind the increased incidence of stillbirth are
unknown. Neonatal lamb mortality is also a great problem in sheep operations.
For example, Hatfield et al. (1999) indicated that lamb mortality rates of 10-35%
are considered acceptable in various operations in the USA, Australia and other
countries. They also mentioned that the majority of lamb mortality occurs in the
first few days of life. It has been shown that nutrition and management of the cow
during the dry period may have a profound effect on the survival, health and
growth of newborn calves (Quigley and Drewry, 1998).
Did you know that calf death loss is a significant factor in net
reproductive rate and has a very significant impact on overall
profitability in the beef cattle industry?
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Selenium in combination with vitamin E is shown to affect early animal mortality.

Medium wool ewes were injected with vitamin E and (or) Se over a 2-yr period to
evaluate the influence of these treatments on reproduction. Ewes were divided
randomly into four groups, consisting of a control, plus groups receiving monthly
s.c. injections of either 272 IU vitamin E, 4 mg Se or 272 IU vitamin E plus 4 mg
Se during pregnancy. Selenium administration increased (P<0.05) ewe blood Se
concentrations. Pre-weaning survival of lambs was increased (P<0.05) by ewe
treatments with either Se or vitamin E and thus, treated ewes weaned approximately
20% more lambs/ewe mated than did control ewes. (Kott et al., 1983). SeleniumE injections significantly (P<0.05) reduced calf death losses (from 15.3% to 4.2%)
and slightly increased adjusted calf weaning weights (Spears et al., 1986).
Selenium deficiency in grazing animals can be prevented by the parenteral
administration of 50 mg selenium and 3,000 mg vitamin E on the 21st and 5th
day before calving (Kolb and Seehawer, 2001). This has a favourable effect on
the vitality of the newly born calf. It could well be that Se transfer via placenta,
colostrum and milk could be an important route for improvement of antioxidant
defences of newly born calves or lambs. Indeed, selenium prepartum injections
resulted in higher colostrum and milk concentrations of Se (Cuesta et al., 1995).
Furthermore, by using organic selenium in the maternal diet it is possible to achieve
significantly higher levels of Se in colostrum and milk in comparison to animals
fed sodium selenite.
Selenium deficiency in dairy and beef industries

As mentioned above, deficiencies of selenium in cattle and sheep have been
confirmed under natural grazing conditions in many countries all over the world.
In this regard Grant and Sheppard (1983) have shown that Se deficiency occurs
in flocks grazing pastures that have Se concentrations <0.030 mg Se/kg dry matter.
Overt signs of inadequacy such as white muscle disease (nutritional muscular
dystrophy) occur primarily in young calves or lambs born to selenium deficient
dams. In general, clinical signs of deficiency have not occurred in older animals
such as finishing beef cattle and lactating dairy cows. However, sub-clinical
deficiencies of selenium are not determined easily, and thus an inadequacy of the
element may be limiting maximum animal performance under certain
circumstances of animal feeding (Ammerman and Miller, 1975).
In the US survey, 36% of herds from the southeast were classified as deficient
or severely deficient compared to 13.5% deficient animals nationally (Table 9.13,
Dargatz and Ross, 1996). Se-deficiency diseases were diagnosed in 46 states and
were reported to be an important livestock problem in regions of 37 states;
deficiencies were also diagnosed in wildlife in 10 states (Edmondson et al., 1993).
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Analysis of serum samples (n=204) submitted to the Iowa State University
Veterinary Diagnostic Laboratory between January 2000 and July 2001, showed
a mean serum selenium value of 68.417 ppb for adult Holstein cows in the Iowa
and Wisconsin areas (Villar et al., 2002). The authors suggested that the Se levels
found in the survey could be considered adequate for reproductive performance,
but marginal for optimal resistance to mastitis pathogens or for adequate transfer
of selenium to the suckling calf. The data concerning the role of trace elements in
the perinatal diet of dairy cattle is quickly accumulated. The most important for
stimulation of immune response are selenium, zinc and copper, which are more
effective when administered in organic, rather than inorganic form (Pinotti et al.,
2001).
Table 9.13 Percentage of cattle with various blood Se contents in the US (Adapted from Dargatz and
Ross, 1996)
Supplementation
No supplementation
Individual cattle status
West
Central
South
east
West
Central
South
east
Severely deficient, <50 ppb

Marginally deficient, 51-80 ppb
Adequate, 81-160 ppb
High adequate, >161 ppb
4.4
8.0
20.3
67.3
0
3.6
30.9
65.5
16.7
23.3
40.9
19.1
4.9
4.4
13.1
77.6
7.9
9.2
36.4
45.6
29.1
32.0
30.8
8.1
Did you know that Se-deficiency is a common problem in dairy

industry world-wide irrespectively of sodium selenite
supplementation?
Selenium deficiency in Europe is even more pronounced than in the US. For
example, Se status was assessed directly by the determination of selenium
concentration, or indirectly by the measurement of GSH-Px activity in whole
blood samples collected from 879 cattle (733 cows, 63 calves, 42 heifers and 41
finishing bulls) reared on 93 farms in 12 of the 14 regions of the Czech Republic
(Pavlata et al., 2002). The blood samples were collected from 1999 to 2001.
Selenium deficiency or marginal values were found in 50% of the tested animals
and on 54% of the farms. In terms of animal categories, deficient or marginal
selenium status was found in 42% of cows, 80% of calves, 100% of heifers, and
90% of bulls. Selenium-deficient herds were detected in almost all regions of the
Czech Republic. The same authors collected samples from 44 cattle at slaughter.
Poor selenium status, as assessed from blood, muscle and liver selenium
concentrations, was found in 80%, 70% and 73% of the tested animals, respectively
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(Palvata et al. 2001a). In Poland, a total of 262 animals were evaluated: 151
horses, 60 cattle and 51 sheep. In 70.1% of the animals, Se levels were either low
or very low (<10 ng/ml, Ramisz et al., 2001). When 11 dairy herds from the
central regions of Estonia were studied, it was concluded that the selenium status
of cows was deficient, similarly to that in Sweden, Norway and Finland (Pehrson
et al., 1997). When 18 selenium unsupplemented or irregularly supplemented
herds in Slovenia were evaluated based on selenium concentration in the liver,
34.3% of animals were classified as adequate, 37.2% as marginal and 28.5% as
Se-deficient (Zust and Hrovatin, 1994). Similarly, in late lactation and the dry
period when Se was given as a mineral mixture, about 60% of the cows had
marginal blood Se values (Zust et al., 1995). Blood selenium was estimated in a
total of 172 calves, 163 heifers and 399 cows examined in a cattle clinic in Giessen,
Germany, from May 1990 to May 1991. Average values were Se 0.046 mg/litre,
compared with literature values of <0.04, 0.04-0.07 and >0.07 mg/litre, identified
as Se-deficient, marginal supply and adequate supply, respectively. Indeed, 50%
of individual cattle were deficient, 25% marginal and only 25% were sufficient in
Se (Grunder and Auer, 1995). In Ireland in 1993 soil Se total content ranged from
0.2 to 6.0 mg/kg DM with over 90% of herbage samples being deficient in Se.
Furthermore, low or very low blood Se status was found in 17% of herds (Mee et
al., 1996). Selenium status of 50 dairy herds in the Irish Republic were studied
during the spring and autumn of 1991. Only dry cows were sampled in spring
and only lactating cows were sampled in the autumn. Herds were classified as
very low, low, marginal, normal, high or very high Se status. In the spring 18
herds were very low in Se with a further 9 herds having low Se status, while in the
autumn, 20 herds were low or very low in Se (Mee et al., 1993). Similarly, out of
the 205 cattle slaughtered in the area of Thessaloniki, Greece, 78% presented
deficient concentration of Se in liver (0,110-0,600 g/g DM), 17% marginally
deficient concentration (0,601-0,900 g/g DM) and only 5% normal concentration
(0,901-1,512 g/g DM; Christodoulopoulos et al., 2000a). Plasma selenium was
studied in dairy cows within the region of Thessalonica, Greece. The survey
included 65 dairy farms from which samples of blood were collected from 10
cows from each farm. Out of the total 650 cows examined, 40% presented deficient
concentration of Se in blood (<0.08 mg/L), 29% presented marginally deficient
concentration (0.08-0.12 mg/L) and only 31% had a normal concentration (>0.12
mg/L; Christodoulopoulos et al., 2000). Regional and seasonal selenium
nutritional status was studied in cattle and sheep, in 5 regions of Turkey, using
neutron activation analysis of blood. Blood Se was 51.0 to 75.0 ng/ml. In particular,
in the Bursa region cattle on a state farm were severely Se deficient (33.0 ng/ml,
Oncuer et al., 1996).
Many Latin America countries are also Se deficient. For example, the content
of Se in forage samples and the blood activity of GSH-Px of lactating cows and
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heifers were evaluated between autumn and spring of 1999 in 12 dairy herds in
Chile (Wittwer et al., 2002). There was a significant positive correlation between
the content of Se in forage and the blood activity of GSH-Px in heifers (r=0.74).
The content of Se in most of the forage produced in the region was below the
nutritional requirements for grazing dairy cattle and heifers are mostly affected
by Se deficiency. Selenium deficiencies in cattle in Valdivia (Chile) were observed.
In particular, older calves (36.4%) and heifers (23.6%) showed the highest
percentages of animals with GSH-Px activities below physiological limits (Ceballos
et al., 1998). Trace element concentrations in the soil, plants and cattle in 6
important cattle-producing regions of Nicaragua were examined. Fourteen farms
within 6 regions during the wet season and 8 farms within 2 regions during the
dry season were evaluated. Percentage of serum samples deficient in Se (<40 ng/
ml) was in the range 13-93% in the wet season (Velasquez-Pereira et al., 1997).
Asian countries are not exception from global Se deficiency. Indeed, a study
was conducted to determine the selenium status in soil, forage and serum of
prepuberal Jersey heifers in different districts of Assam (India). It was shown that
practically 100% serum samples analysed were deficient in selenium (Anubha
Baruah and Baruah, 2000). The selenium status of beef cattle in the southern part
of Thailand was investigated and it was shown that beef cattle grown in these
areas suffered from chronic selenium deficiency (Kamada et al., 2000). The Se
status of livestock on 12 Wendon Valley farms in New Zealand was determined
and marginal Se deficiencies in lambs were found on four farms (Grace et al.,
2000).
Therefore Se deficiency in ruminants is a global problem in European, Asian
and American countries and this results in decreased immunocompetence,
productive and reproductive performance of animals. Data are actively
accumulating to prove that Se supplementation can improve Se status of animals
(Table 9.14).
Important features of Se metabolism in ruminants

Se is absorbed in the lower parts of the small intestine. In fact, 75Se as selenite or
selenate was unabsorbed from the rumen, slightly absorbed from the abomasums,
and secreted into duodenum and jejunum with net absorption in the lower portion
of the small intestine (Wright and Bell, 1966). Therefore, the duodenum is the
primary site of selenium absorption, with no absorption from the rumen or
abomasums. It is necessary to underline that there are several factors affecting
efficiency of Se absorption including:
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Table 9.14 Effect of Se supplementation on Se status of cows (Adapted from Hemigway, 1999)
Treatment
Results
References
1 mg Se/day (selenite) to cows

during pregnancy
Over the first 28 weeks whole

blood Se from 0.06 to
0.16 mg/l in unsupplemented
cows: 0.02-0.03 mg/l
Lein et al., 1980
Basal diet (2.3 mg Se/day +5 mg Whole blood Se from 0.12 to

Se/day for 10 days
0.19 mg/l; plasma Se from
0.05 to 0.11 mg/l
Basal diet (0.5 mg Se/day) at
day 21 from calving injection
Se (65 mg)
Harrison and Conrad,

1984
Plasma Se from 0.035 to

0.092mg/l on the day following
injection, to 0.04 mg/l by the
day of calving; without injection:
0.03-0.035 mg/l
1 or 2 mg supplementary Se/day Without supplementation serum de Toled and Perry,

from 60 days before to 20 days Se from 0.02 to 0.019 mg/l at
1985
after calving
calving and to 0.015 mg/l after
20 days lactation. 1mg/day: 0.026;
0.025 and 0.019 mg/l respectively;
2 mg/l:0.024; 0.037 and 0.026 mg/l
respectively
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Heifers were on low Se diet

(0.6 mg Se/day) from 90 days
before to 35 days after calving
+ 2 mg Se/day (selenite)
Whole blood Se from 0.037 to

0.128 mg/l
Grasso et al., 1990
Beef cows given two Secontaining sustained-released

ruminal boluses
Se in the milk from 0.044 to

0.066 mg/l over a 7-month
period
Hidiroglow et al.,
1987
Se in the cows diet was

increased from 0.05 to 0.75 mg
Se/day by selenite or Se-yeast
Plasma Se over 20-40 days

from 0.03 to 0.11 mg/l Milk Se
from 0.01 to 0.04 mg/l
Conrad and Moxon,

1979
From mid-lactation cows were

given daily 4 doses of selenite
Increasing Se intake from 2 to

Maus et al., 1980
6 mg Se/day plasma Se from
0.06 to 0.11 mg/l and milk Se
from 0.01 to 0.05 mg/l; increasing
Se intake from 6 to 14 mg/day did
not affect Se concentration
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Table 9.14 (Contd.)
Treatment
Results
Lactating cows received 0, 2.5,

5.0 or 10 mg Se/day (selenite)
for 8 weeks
Plasma Se in untreated cows was Stove et al., 1988

0.023 mg/l; 2.5 mg Se/day plasma
Se to 0.038 after 1 week and
remained 0.052-0.056 mg/l
thereafter; 5 mg Se/day 0.054 mg/l
after 1 w and 0.070 mg/l thereafter;
10 mg Se/day: 0.07 mg/l over the
whole period
Se (selenite) at 2 mg/day was

given to cows over two
lactations
Serum Se from 0.033 to

Stove et al., 1988
0.054 mg/l; during pregnancy
serum Se from 0.049 to 0.062 mg/l;
at parturition; 0.028 and 0.041 mg/l
and by day 7 of lactation 0.027 and
0.044 mg/l
Cows wee on a basal diet

from 60 days before calving
(0.1 mg Se/day) or + 0.2 mg
Se/kg DM
At calving plasma Se from 0.085 Weiss et al., 1990

to 0.090
Cows at mid-lactation were

given sodium selenite or
Se-enriched yeast, 2.6 mg
Se/day
Over an 8-week period, whole

Malbe et al., 1995
blood Se concentration increased
from 0.005 to 0.007 mg/l in
unsupplemented cows, from
0.007to 0.091 mg/l in selenite
group and from 0.005 to
0.107 mg/l in Se-yeast group
Lactating cows with Se in

blood 0.025 mg/l were given
2 or 4 mg Se/day as a selenite
of Se-Yeast
Se in blood increased over 133

day period to 0.08 and
0.144 mg/l with selenite
supplementation and to 0.144
and 0.184 mg/l with se-yeast
supplementation
References
Knowles et al., 1999
the amount that was ingested

other dietary factors: Ca, arsenic, cobalt and sulfur may decrease Se
absorption by 50% or more
Selenium absorption measured by the balance technique ranged from 17 to 50%

of Se intake in nonlactating dairy cows fed a variety of diets with or without Se
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supplementation (Harrison and Conrad, 1984). Absorption of inorganic 75Se in

steers was estimated to be only 13% (Costa et al., 1985). Similar true Se absorption
(11%) was reported in nonlactating cows fed hay supplemented with inorganic
selenium (Koenig et al., 1991). In another experiment it varied at 10-16% of daily Se
intake (Koenig et al., 1991a).
Selenium is transported to the liver bound to carrier proteins or in a free form. Se
enters the blood stream bound to alpha- and gamma-globulins, delivered to the liver
and after metabolic changes forms selenoproteins, such as glutathione peroxidase.
The incorporation of Se into proteins is genetically determined (Ceballos, and Wittwer,
1996). After absorption, Se metabolism in ruminants is similar to non-ruminants.
Factors that affect Se metabolism, include:
chemical forms of Se
level of Se in blood and tissues before supplementation
presence of various minerals and amino acids in the diet
concentration of Se in the diet
Did you know that inorganic Se assimilation in ruminants is quite
low: rumen bacteria reduce it to unavailable metallic Se and Se
incorporated into bacteria proteins is characterised by low
availability?
In a Se-balance trial with cows in the late pregnancy and lactation period, feeding of
diets containing Se 0.106 mg/kg resulted in daily Se intake of 973.3 g and daily Se
accumulation of 136.9 g (Kamada et al., 1998). Therefore only 14% Se consumed
was accumulated in the body. Estimates of total body selenium in cows are extremely
limited; in dry dairy cows fed diets with 0.03 mg Se/kg DM estimated total body
selenium was 44 mg (Koenig et al., 1991a).
The metabolism of intravenously dosed 75Se was studied in 10 Holstein bull calves
fed ad libitum on a commercial diet. The total endogenous 75Se in the faeces over the
4-day collection period was about 4.14% of the dose. About 97% of the 75Se dose
disappeared from the calfs blood within 6 h after dosing (Neathery et al., 1990). The
principal route of Se excretion in ruminants is the faeces (Wichetel, 1998; Podoll et
al., 1992), but selenium is also expelled from the body via the lung, especially in the
case of high Se consumption. When Se is administered by intravenous or subcutaneous
injection to ruminants, urine is the major route of excretion (Wright and Bell, 1966).
Urinary Se excretion in sheep was shown to account for 23-33% with the remainder
voided via feces (Koenig et al., 1997).
Urinary excretion of Se in lambs and goats was higher when sodium selenite was
used as a dietary supplement in comparison to SeMet (Ehlig et al, 1967; Aspila,
1991). A significantly higher faecal excretion, lower apparent absorption and lower
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retention of selenium were observed in sheep fed a low-Se diet (0.01 ppm) and
receiving continuous intraruminal infusion of selenite, compared with SeMet (Table
9.15; Peter et al., 1982). It is widely believed that urinary Se excretion is a primary
mechanism to decrease selenium retention and maintain Se homeostasis. In cows,
urine accounted for approximately 30% of total Se excretion by cows fed 0.3 mg/kg
of Se compared with 18% for cows fed 0.1 mg/kg of Se (Ivancic and Weiss, 2001).
The authors also showed that when cows Se intake was 2.5 mg/day, Se apparent
digestibility was 38.6% and true digestibility 54.7 %. At the same time fecal, urinary
and milk Se output comprised 1.46, 0.52 and 0.48 mg/day (Table 9.16).
Table 9.15 Selenium absorption and retention in sheep (Adapter from Peter et al., 1982).
Period I
Period II
Selenite
SeMet
Selenite
SeMet
Se intake, ug/day
60.4
55.5
56.0
55.8
Faeces
Se output, ug/day
34.0
43.9
22.8
59.0
31.1
44.4
24.2
56.8
Urine
Se output, ug/day
Output/Intake,%
Se retention, % intake
3.6
6.0
38.3
2.5
4.4
54.5
5.4
9.7
34.7
5.2
9.4
47.4
Table 9.16 Selenium balance in lactating cows (Adapted from Ivancic and Weiss, 2001)
Parameter
Dietary Se supplementation
0.1 ppm Se
0.3 ppm Se
Se intake, mg/day
Se apparent digestibility, %
Se output, mg/day
2.50
38.6
4.82
46.9
Fecal
Urinary
Milk
Total
Absorbed Se, mg/day
Se balance, mg/day
1.46
0.52
0.48
2.46
1.04
0.04
2.58
1.40
0.39
4.36
2.24
0.46
When dose of Se was increased to 4.82 mg/day fecal, urinary and milk Se output
was 2.58, 1.4 and 0.39 mg/day. Therefore, increasing selenite dose was associated
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with lower transfer Se to the milk. The authors showed that milk Se concentrations
were related with plasma Se:
Milk Se (g/ml) = 037 x plasma Se (g/ml) 4.2
The amount of Se excreted per day via urine was also related with plasma Se
concentrations:
Urinary Se (g/day)= 0.031 x plasma Se (g/ml) 0.91
It is well accepted that absorption of selenium (sodium selenite) in ruminants is
much lower than in monogastric animals, while the bioavailability of selenium
from selenite and selenate is similar in ruminants (Table 9.17; Podoll et al., 1992;
Ortman and Pehrson, 1999). Indeed, absorption of orally administered 75Se was
85% in swine and only 34 % in sheep (Wright and Bell, 1966). Similarly, in the
rat, about 86% of labelled selenium ingested orally either in the form of selenite
or selenate was absorbed (Mason and Weaver, 1986).
Table 9.17 Effect of selenium supplementation (0.3 ppm) on farm animals (Adapted from Podoll et al.,
1992)
Species
Dairy cattle
Sheep
Horses
Dairy cattle
Sheep
Horses
Se form
0
Days of supplementation
14
42
Selenate
Selenite
Selenate
Selenite
Selenate
Selenite
Se, mg/l
0.047
0.050
0.084
0.076
0.093
0.101
0.079
0.075
0.114
0.122
0.157
0.165
0.087
0.078
0.131
0.132
0.190
0.199
Selenate
Selenite
Selenate
Selenite
Selenate
Selenite
GSH-Px, U/ml
0.135
0.165
0.089
0.141
0.677
0.674
0.113
0.115
0.123
0.129
0.505
0.606
0.087
0.112
0.152
0.185
0.559
0.610
It seems likely that rumen-reducing conditions and micro-organisms there affect

Se metabolism and sodium selenite could be converted into insoluble forms such
as elemental selenium or selenides (Peterson and Spedding, 1963; Cousins and
Cairney, 1961; Spears, 2003), which cannot be absorbed. Indeed, studies
conducted at Division of Animal Production of CSIRO (Australia) demonstrated
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that significantly more Se from selenite than SeMet was converted to insoluble,
inorganic form by rumen microbes (Peter et al., 1982). Furthermore, rumen
bacteria can use Se for synthesis of various compounds and it seems likely that
this process depends on many factors and regulated metabolically. For example,
uptake of 75Se by rumen microorganisms in vitro was related inversely to selenium
and vitamin E status of sheep (Hidiroglou et al., 1968; Whanger et al., 1968).
Selenium in the liquid phase of digesta was largely protein-bound and following
hydrolysis of the microbial cell protein, the element was absorbed as Se-containing
free amino acids. Furthermore, 6 h post dosing 50% of the 75Se activity was in the
rumen bacterial protein (Hidiroglou and Jenkins, 1974).
It is interesting to note that the Se concentration of bacteria isolated from sheep
rumen was 2.3 and 3.9 times the original dietary Se concentration of the forage
and concentrate diets, respectively (Koenig et al., 1997). It seems likely that the
metabolism of Se by rumen bacteria is dependent on the particular species of
bacteria. Indeed, pure cultures of Selenomonas ruminantium and Butyrivibrio
fibrisolvens incorporated 75Se into selenoaminoacids, while Bacteroides ruminicola
produced only elemental Se (Hudmann and Glenn,1984; 1985). In particular, it
was shown that Selenomonas ruminantium incorporated 75 [Se]-Selenite into
selenocystine, selenoethionine, selenohomocysteine, and selenomethionine and
was also reduced to red elemental selenium. 75[Se]Selenite was converted to
selenocystine, selenohomocystine and selenomethionine by B. fibrisolvens, while
Bacteroides ruminicola did not incorporate [75Se]selenite into organic compounds,
but did reduce it to red elemental selenium.
However, Se bioavailability from microbe mass seems to be low. For example,
a tissue uptake experiment was conducted to determine the bioavailability of rumen
bacterial Se in mice. The donor animal was wether fed a diet containing 0.2 mg
Se/kg dietary dry matter (DM). Ruminal fluid was collected 2 h postprandially
and bacterial-rich precipitate was obtained by differential centrifugation of the
ruminal fluids. This was later freeze-dried and mixed in the diet and used in feeding
experiment with the mice. Diet 1 and Diet 2 were supplemented with selenite to
give an Se content of 0.025 and 0.1 mg/kg dietary DM, respectively. In Diet 3,
rumen bacterial matter was 20% of the diet, which gave an equivalent of 0.1 mg
Se/kg dietary DM. The obtained results showed that those mice fed a selenitesupplemented diet (Diet 2) had significantly higher (P < 0.05) tissue Se
concentrations than those mice fed the other two diets. No statistical differences
were observed on various tissue Se concentrations between Diet 1 and Diet 3,
indicating that Se availability from rumen bacteria to be about 25% of that in
selenite (Serra et al., 1997). Therefore, bacterial Se collected from the rumen of
wether was not fully available for absorption in the intestine of the mice.
It seems likely that dietary Se can affect rumen conditions and metabolism.
For example, positive effects of Se on rumen gas production were detected at 0.2
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and 0.4 mg Se/kg DM. In particular, the production of short chain fatty acids was
optimal at 0.2 and 0.4 mg Se/kg DM but decreased significantly for concentrations
greater than 12.8 mg Se/kg DM (von Brehm, 2001). These data are consistent
with previous observations indicating that Se supplementation can influence rumen
microbial fermentation and that Se compounds differ in this regard (Kim et al.,
1997a)
Meeting selenium requirements

Recent NRC (2001) set the Se requirement at 0.3 ppm for lactating and dry cows.
It has been suggested that there is no need for additional selenium above 0.3 mg/
kg of diet when dietary antagonists are not present (Weiss, 2003). On the other
hand, the beef cattle requirement for all ages was set at 0.1 ppm and maximal
tolerable concentration to be 2 ppm (NRC, 2000). Sheep Se requirement was set
at 0.1-0.2 ppm with 2 ppm to be maximal tolerable level (NRC, 1985).
The current US FDA regulation allows ruminant diets to be supplemented with
0.3 ppm selenium from either sodium selenite or selenate. Recently (2003) organic
source of supplemented selenium (Sel-PlexTM) was cleared by FDA for dairy and
beef cattle and is considered to be an effective source of Se for ruminants.
An animals requirement for selenium depends on many factors including
age
physiological stage
rate of productivity
health status
amount of stresses
balance of nutrients (sulphur, lipids, vitamin E, proteins, amino acids and
several microelements)
species
In particular, increases in the growth rate, milk yield, and reproductive performance
are associated with increase in the Se requirement. It has been shown that increasing
dietary sulphur linearly reduced apparent and estimated true Se digestibility
(Ivancic and Weiss, 2001). Therefore, dietary S from sulfate can reduce Se balance
especially when cows were fed diets with less than 0.3 mg of Se/kg of diet dry
matter. Similarly nitrates in water may tie up selenium and prevent absorption and
high dietary calcium may decrease intestinal absorption of selenium (Harrison
and Conrad, 1984b; Valle, 2001). Excessive consumption of copper, zinc or iron
also reduced Se availability (Smith et al., 1998).
It is important to mention that Se requirements of growing cattle, growing pigs
and the recommendations for children (on the basis of daily intake per kg body
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weight) are in the same order of magnitude (Gurtler and Anke, 1993). The major
benefits, observed experimentally, of maintaining optimal Se intakes include
minimizing the incidence of mastitis and preventing calf losses associated with
myopathy and (or) respiratory disease. Indeed, immunomodulating properties of
this mineral are of great importance for dairy industry. For example, in Finland,
511 dairy cows from commercial herds were studied for 1 year (1989-1990).
The mean selenium content of whole blood was 191 g/litre. An increase in Se
concentration was associated with a decrease in all infections, including infections
by Staphylococcus aureus, Actinomyces pyogenes and Corynebacterium spp.
(-17.7, -31.7 and -70.6%, respectively, Table 9.18).
Table 9.18 Prevalence of infection in groups of cows with low and high Se (adapted from Jukola et al.,
1996)
All pathogens
Cows infected
CNS
Cows infected
SA
Cows infected
APC
Cows infected
no.
no.
no.
no.
Se<150 ng/ml
S>150 ng/ml
108
700
53
345
Se <150 ng/ml
Se>200 ng/ml
108
289
Se <180 ng/ml
Se >200 ng/ml
310
289
28.3
20.8
-27%
28.3
19.7
-31%
28.3
19.3
-32%
24.6
19.3
-21%
11
29
108
498
38.1
29.1
-24%
38.1
30
-21%
38.1
29.5
-23%
30.2
29.5
-2%
34
221
Se<150 ng/ml
Se>180 ng/ml
55.7
45.7
-18%
55.7
45.9
-18%
55.7
45.8
-18%
47.7
45.8
-4%
11.3
3.3
-71%
11.3
3.5
-70%
11.3
3.9
-65%
5.3
3.9
-26%
53
251
53
143
147
143
34
731
34
82
111
82
11
21
11
14
19
14
CNS= Staphylococcus auerus; SA= Staphylococcus auerus; APC=Actinomyces pyogenes or

Corynebacterium sp.
Therefore the Se concentration of 200 g/litre was accepted as a target value to

optimise udder health (Jukola et al., 1996). Indeed, significant negative correlations
were found between the prevalence of intramammary infection with major
pathogens and mean herd activity of GSH-Px (r = -0.62) and mean herd
concentrations of Se (r = -0.66; Erskine et al., 1987). Furthermore, selenium
supplementation of Se-deficient diets in dairy and beef cattle have been associated
with the following clinical responses (Weiss, 2003):
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reduced prevalence of retained fetal membranes (estimated cost of $100120/case; Harrison et al., 1984; Smith et al., 1998),
reduced severity and prevalence of clinical mastitis (estimated cost $120/
case; Smith et al., 1997; 1998),
reduced milk somatic cell counts (potential quality bonuses for milk; Weiss
et al., 1990; Smith et al., 1998),
reduced calf mortality (Spears et al., 1986).
Did you know that the Se requirement of dairy cows is set by

NRC (2001) at 0.3 ppm, and beef cattle (NRC, 2000) at
0.1 ppm and sheep (NRC, 1985) at 0.1-0.2 ppm?
Mineral supplementation is a way to guarantee the optimal Se status and animal
health. There are several important periods of the animal ontogenesis where
antioxidant defences and Se status are critical. For example, in cattle, low selenium
status becomes critical near calving when cows are more susceptible to
intramammary infections, retained placenta, or infections in the neonate (Villar et
al., 2002). Furthermore, requirements for selenium are increased in dairy cattle,
during the ninth month of pregnancy and the first month after parturition
(Christodoulopoulos et al., 2001). It has been suggested that diets fed to animals
at all stages of life (calves, heifers and lactating and dry cows) should be
supplemented with 0.3 ppm selenium (Smith et al., 1998). Supplementation of
dairy cattle diets with 0.3 mg of Se/kg of DM usually maintains plasma
concentrations of Se within normal ranges (Maus et al., 1980) while at 0.1 mg of
Se/kg of DM, the amount of Se was insufficient to maintain Se in blood of cows in
late gestation. and the transfer of Se to the calf via the placenta and colostrum was
reduced (Abdelrahman and Kincaid, 1995).
When considering Se requirement of ruminants it is important to understand a
body balance of selenium. For example, it was shown that Se absorption and
retention are linear when dietary Se intakes ranged from 400 to 3100 g/day,
while zero retention of Se was seen with Se intake less than 332 g/day (Harrison
and Conrad, 1984a). This means that cows consuming <10 kg dry matter/day,
with Se content of <0.3 g/g dry matter, could be in negative Se balance (Ville,
2001). Indeed, Se concentration increased linearly from about 80 to 120 ng/ml
of plasma and about 30 to 55 ng/ml of milk as intake of selenium increased from
about 2 to 6 mg/day. However, increase in selenium intake from 6 to 12 mg/day
resulted in little change in plasma and milk selenium. It has been proposed that
Se intakes of pregnant, lactating dairy cows should be 5 to 7 mg/day to sustain
adequate Se concentration in serum (Stowe et al., 1988). Furthermore, it was
shown that Se concentration in whole blood at 0.16-0.25 ppm can provide an
adequate Se transfer to the fetus (Koller et al., 1984). Those suggestions are in
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agreement with data indicating that 2.5 mg of Se/day were inadequate to maintain
Se concentrations in plasma or in whole blood during the dry period of Holstein
cows (Weiss et al., 1990). For lactating cows, intakes of >6 mg of Se/day are
needed to maintain adequate Se concentrations in serum (Gerloff, 1992). However,
5 mg of Se/day for dry cow were sufficient to maintain their adequate Se status
(Weiss et al., 1984). It seems likely that more than 4 months are required to fully
deplete or replete blood Se (Nicholson et al., 1991a).
Methods for Se status assessment

Main methods of Se status assessment include determination of (Kincaid, 1999):
Se concentration in serum
Se concentration in whole blood
Se concentration in liver
GSH-Px activity in erythrocytes
GSH-Px activity in liver
mRNA levels for GSH-Px
PH-GSH-Px activity in tissues
Furthermore, additional tests of Se status evaluation could be:
Se concentration in muscles
Se concentration in milk and colostrum
Selenoprotein P concentration and expression in various tissues
Deiodinase activity in tissues
TR activity in various tissues
It is necessary to mention that each method mentioned above has its advantages
and disadvantages and data obtained with various methods are interrelated but
are not the same. For example, the concentration of Se in blood plasma was
positively correlated to concentration of Se in the diet when the data set contained
just dry cows (Table 9.19; Weiss et al., 1990):
Plasma Se = 0.059 + 0.044 Feed Se (r2=0.39)
However, no relationship existed between dietary Se and plasma Se in lactating cows.
It is necessary to mention that Se concentration in whole blood is approximately 3fold higher than that in serum (Scholz and Hutchinson, 1979; Smith et al., 1998).
The ratio of whole blood Se to serum Se is approximately 4 to 1 in sheep with low Se
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Table 9.19 Regression equations for predicting blood values from dietary data (Adapted from Weiss et
al., 1990; Knowles et al., 1999)
Intercept
Slope
r2
Se intake, mg/d
Se intake, mg/d
Se intake, mg/d
0.033
0.144
45.67
0.014
0.019
4.49
0.81
0.53
0.40
Diet Se, ppm

Diet Se, ppm
Diet Se, ppm
0.031
0.142
45.46
0.215
0.295
66.25
0.84
0.54
0.39
0.13
1.33
6.7 x 10-5
6.1
0.24
0.63
0.69
0.73
Dependent
Independent
Plasma Se, g/ml

Whole blood Se, g/ml
Whole blood GSH-Px,
mEU/ mg Hb
Plasma Se, g/ml
Whole blood Se, g/ml
Whole blood GSH-Px,
mEU/ mg Hb
Milk Se, nmol/l
Liver Se, nmol/kg FW
Liver GSH-Px, U/mg protein
Liver GSH-Px, U/mg protein
Blood Se, nmol/l

270
Blood Se, nmol/l
Blood Se, nmol/l
Liver Se, nmol/kg FW -
intake (Stowe and Herdt, 1992). Indeed, Se concentration in whole blood is a

preferential test since haemolysis can increase Se concentration in serum giving
false values. In fact, it was shown that the accuracy of estimating blood Se from a
known serum Se was not useful for diagnostic purposes (Maas et al., 1992). The
authors concluded that the use of serum Se concentration to assess nutritional
status of cattle with respect to Se was not appropriate. However, other authors
(Smith et al., 1998) argued that Se concentration in plasma is a useful test to
assess short-term Se status. In general, relationship between serum selenium and
whole blood selenium can be described as follows (Thompson et al., 1998):
Serum Se (nmol/L) = 0.81 x whole blood Se + 0.48 (R2= 0.85)
The summarised data on Se status assessment are shown in Table 9.20. For
ruminant plasma, selenium concentrations exceeding 70 ng/ml are considered to
reflect adequate Se status (Kincaid, 1999). Furthermore, Dargatz and Ross (1996)
classified cattle as Se deficient, marginally deficient, adequate and high adequate
with Se concentration in total blood (ng/ml): <50; 51-80; 81-160 and >161
respectively. In accordance with Smith et al. (1998) dairy cows are classified as
adequate, marginal and deficient when Se level in plasma/serum were >75; 50-75
and <50 ng/ml or in whole blood >200; 140-200 and <140 ng/ml respectively.
Similarly, according to Puls (1994) Se status of cattle can be expressed as deficient,
marginal, adequate and high when Se concentrations in whole blood are <50; 5080; 80-1200 and >3000 ng/ml or in plasma/serum: <30; 30-60; 60-400 and >2500
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ng/ml. Thompson et al. (1998) suggested Se reference ranges for whole blood in
cattle as follows: responsive, marginal and adequate to be <130; 130-250 and
250-2000 nmol/l. The same classification based on Se concentration in serum is
<85; 85-140 and >140 nmol/l while in the liver: <600; 600-850 and >850 nmol/
kg fresh tissue respectively. Based on Se concentration in the liver Caple and
McDonald (1983) classified sheep as Se deficient, marginal. adequate or high
when they contained <200; 200-500; 500-3000 and >6000 ng/g DM.
Table 9.20 Criteria for classification of cattle on selenium in blood and liver (Adapted from Kincaid,
1999; Zarski et al., 1998)
Status
Se in whole
blood, ng/ml
Severely deficient
<60
Marginally deficient 60-200
Adequate
210-1200
High adequate
>1200
Se in liver
of adults,
ng/mg DM
Se in livers
of newborns,
ng/mg DM
Se in milk,
ng/ml
0.1-0.5
0.6-1.25
1.25-2.5
>2.5
<1.1
1.1-2.2
2.3-8.0
-
<5.5
5.5-10.3*
>10.3
-
*5.5-7.9 moderate deficiency; 7.9-10.3- slight deficiency
Did you know that the Se status of cattle could be assessed

be various methods, but there is no single method which could
be universal?
In general Se concentrations below 30 ng/ml whole blood indicate a risk of NMD
and concentrations below 50 ng/ml indicate a Se-deficient state (Ortman, 1999).
As mentioned in the review conducted by Ortman (1999) the borderlines between
suboptimal and optimal Se status varied in different publications and Se
concentrations from 50-75 ng/ml up to 100 ng/ml are considered to be marginal.
Furthermore, when preventive effects of Se against various diseases were
considered, optimal Se concentration to be 200 ng/ml.
Therefore, for cows, a reference range of 70 to 100 ng of Se/ml of bovine
serum is an acceptable target concentration. This range can be achieved most
often by providing more than 6 mg of supplemental Se/animal/day, but several
factors affect serum Se responses of different cows to specific Se intakes (Gerloff,
1992). These factors may include:
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forage type and source

rumen environment
supplemental fat
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534
dietary calcium
dietary sulphur
trace elements
genetics
It is interesting that Se level in blood also depends on species of animals. For

example, blood Se concentrations in Dorper sheep, Boer goats and Simmental
cattle under extensive grazing conditions were compared in South Africa. It was
shown that blood Se concentrations in sheep was 200-350 ng/ml, while in goats it
was lower: 140-250 ng/ml and lowest Se concentration was found in cattle: 55150 ng/ml (Niekerk et al., 1990).
One of the most effective ways of determining Se deficiency is a liver analysis.
Liver levels of 0.25 to 0.5 ppm on wet basis (0.8-1 ppm dry base) are considered
to be adequate, and levels below 0.2 ppm considered being deficient (Corah,
1996). Based on published values of adequate adult liver selenium concentrations
and maternal-fetal relationships, Van Saun et al (1989) suggested an adequate
liver selenium concentration in the bovine fetus to be greater than 2.2 g/g liver
dry wt, and in whole blood, greater than 120 ng/ml. Selenium reference ranges
for liver and serum to diagnose Se deficiency in cattle are shown in Table 9.21.
Table 9.21 Selenium reference ranges for liver and serum to diagnose Se deficiency in cattle (Adapted
from Thompson et al., 1998)
Whole blood Se,

nmol/l
Liver Se,
nmol/kg fresh tissue
Serum Se, nmol/l
<130
130-250
250-2000
<600
600-850
>850
<85
85-140
>140
Responsive
Marginal
Adequate
In young sheep liver Se concentrations <250 nmol/kg fresh tissue are considered
deficient, 250-450 nmol/kg fresh tissue, marginal and >450 nmol/kg fresh tissue
are considered adequate (Andrews et al., 1976). Indeed, blood and liver Se
concentrations are highly correlated in sheep (R2=0.97; Sheppard et al., 1984).
Similarly concentrations of Se in whole blood of the lambs were positively
correlated to the Se concentrations in liver (r=0.77; Rock et al., 2001).
Selenium concentration in milk can also be used for Se status assessment.
Indeed, Givens et al. (2004) calculated linear regressions for the relationships
between milk and dietary selenium concentrations:
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when selenite was the main source of Se in the diet:
milk Se (ng/ml) = 8.03 + 12.04 diet Se (g/kg DM), r2= 0.96
when organic selenium (Sel-PlexTM) was the main Se source in the diet:
milk Se (ng/ml) = -8.83 + 96.32 diet Se (g/kg DM), r2= 0.99
It is generally accepted that Se concentration in serum or plasma reflects shortterm Se nutritional status, while whole blood (erythrocyte) Se and GSH-Px activity
are more indicative of long-term Se status (Thompson et al., 1980). Indeed, Se is
incorporated into red cells at time of their formation, therefore, taking into account
life span of the red blood cell (90-120 days) Se content of the erythrocytes reflects
Se intake 1-3 months previously (Smith et al., 1998).
Activity of GSH-Px in plasma was correlated weakly with concentration of Se
in the diets of dry and lactation cows as follows (Weiss et al., 1990):
GSH-Px (EU/ml)= 0.18 + 0.18 Feed Se (ppm), r2=0.21 or
GSH-Px (EU/ml)= 0.16 + 0.011 Feed Se (mg/day), r2=0.29
It was shown that serum Se concentrations (up to 789 ng/ml) correlated with
GSH-Px activity. Therefore, the relation between plasma Se (g/ml) and the RBC
GSH-Px activity (nmoles of NADPH oxidised per minute per mg of Hb) for cattle
was presented as follows (Stevens et al., 1985):
RBC GSH-Px= 216 Plasma Se + 35.6; r2=0.9; P<0.001)
In particular GSH-Px activities in the range of 45 to 85 U/g of Hb was considered
to be normal values for cows (Erskine, 1993). As suggested by Knowles et al.
(1999) GSH-Px as a diagnostic criterion of Se status may be appropriate only in
cows that do not receive Se supplements or in cases with low Se supplementation.
Did you know that the Se status assessment based on the activity
of GSH-Px should be reconsidered since GSH-Px is one from
the 25 known selenoproteins?
It is proven that Se concentration and GSH-Px activity in bovine plasma, serum
or whole blood depend on Se dietary concentration (Perry et al., 1978), duration
of supplementation (Echevarria et al., 1988) and source of Se (Henry et al., 1988;
Pehrson et al., 1989). Injecting Se generally elevates plasma or serum selenium
concentrations for up to 60 days postinjection compared with concentrations in
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536
un-injected cattle (Harrison et al., 1984; Thompson et al., 1980). Dietary Se is

highly correlated to GSH-Px when animals are deficient in Se, but the relationship
weakens as animals become adequate in Se (Combs and Combs, 1984). For
example, it has been shown that whole-blood GSH-Px activity was poorly correlated
with whole-blood Se concentration in grazing beef cows and ewes consuming
herbage with 0.09-0.24 mg of Se/kg DM (Stadmore et al., 1982). In contrast,
concentration of Se and activity of GSH-Px were highly correlated in blood of
cows and sheep (Kincaid, 1999; Rock et al., 2001). However, this correlation
depends on Se status. After reaching a certain concentration of Se in the blood,
which is necessary for maximum expression of GSH-Px, there is no further increase
in the enzymatic activity in response to increasing Se concentration. It was
suggested that serum Se in excess of 85 ng/ml is adequate for newborn cria and
dams with serum Se in excess of 160 ng/ml can be predicted to give birth to cria
with adequate Se status (Herdt, 1995).
In general, a correlation between Se and GSH-Px in whole blood in beef calves
was found to be high (r=0.82; Harapin et al, 2000) and the correlation between
blood selenium and blood GSH-Px activity was even higher (r=0.97; Ceballos et
al., 1999). Selenium levels in the various tissues and activity of GSH-Px are
interrelated. For example samples were collected from 44 cattle at slaughter and
analysed (Pavlata et al., 2001; 2001a). The relations were expressed by the
following equations and correlation coefficients:
blood Se vs. liver tissue Se y=1.20x+31.58, r=0.78;
blood Se vs. muscular tissue Se y=0.53x+11.97, r=0.83;
blood Se vs. GSH-Px y=8.29x-68.77, r=0.93.
Similarly, relationship between Se concentration in the cattle liver and whole blood
can be described as follows (Thompson et al., 1998):
Liver Se (nmol/kg FW) = 0.56 x whole blood Se + 3.69 (R2=0.56)
Highly significant correlations were also found between selenium concentrations
in the liver and striated muscles (r=0.78), the liver and myocardium (r=0.85), and
in striated muscles and the myocardium (r=0.94). Furthermore, there were positive
correlations between the concentration of Se in cow milk and the concentration
of Se in whole blood (r=0.64) and plasma Se (r=0.68) and the activity of GSH-Px
in the erythrocytes of calves (r=0.59; Table 9.22; Ortman, 1999).
The main problem in using GSH-Px activity as a test for Se status assessment is
related to variability of results between different laboratories. This is associated
with differences in methodologies used for GSH-Px analysis and problems in
sample storage and transportation, since the enzyme can be easily inactivated if
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conditions are not appropriate. Furthermore, the activity of GSH-Px in bovine
plasma/serum is very low comprising only 0.5-2.0% of the total activity in bovine
blood (Ortman, 1999). Therefore even traces of erythrocyte haemolysis would
dramatically change GSH-Px activity in plasma or serum substantially decreasing
validity of the test.
Table 9.22 Correlation matrix for milk, blood and serum selenium concentrations and activities of GSHPx in erythrocytes in 62 cows with milk Se concentrations ranging between 7 and 33 ng/ml (Adapted
from Ortman, 1999).
Milk Se
Whole blood Se
Erythrocyte GSH-Px
Serum Se
Milk Se
Whole blood Se
Erythrocyte GSH-Px
Serum Se
1.00
0.78
0.70
0.71
0.78
1.00
0.96
0.85
0.70
0.96
1.00
0.76
0.71
0.85
0.76
1.00
Routes of selenium supplementation

Similarly to other classes of livestock, including poultry and swine, mineral
supplements for feedlot cattle and dairy cows are incorporated into concentrate
diets. However for graizing cattle which received little concentrates and depend
on forages other routs of supplementation have been developed including
(McDowell, 1996; Wichtel, 1998; Hemingway, 2003):
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licks (a free-choice selenium mineral mix; potential toxicity of supplements,

sporadic intake, individual animal variability restrict their usage)
drenching with Se compounds such as selenite/selenate or selenized yeast
(difficult to use when dairy and beef cattle are exclusively on pasture)
intraruminal boluses (oral administration of intraruminal pellets containing
Se; compressed metallic selenium powder with iron powder. There are
uncertainties regarding possible surface coating with an impervious layer
of calcium phosphate; the exact mechanism of bioavailable Se release from
the pellets is uncertain. There are also soluble glass pellets and main
disadvantage of these pellets is that they remain in the rumen and may
affect the quality of slaughterhouse by-products used for animal feed
(McDowell, 1996)
selenite or selenate injections (provide only a short-term response in the
plasma). Injected selenite did not alter Se status of deficient/marginal Holstein
calves or tissue Se level (Pavlata et al., 2001), while organic selenium given
via started feed can solve this problem.
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538
depot injections (injections of long-lasting selenium compounds such as

barium selenate sub-cutaneously and intra-muscularly; they have potential
tissue residue problems. Injecting cows pre-partum with selenite or selanate
does not result in substantial transfer of Se to the calf via colostrum and
milk (McDowell et al., 2002).
adding sodium selenite to the drinking water (difficult to control in
commercial conditions)
various methods of pasture and soil application (selenium fertilization;
this method is shown to be effective in Finland and New Zealand, but has a
limited use in other countries because of environmental issues)
Irrespective of methods of Se supplementation used, Se inadequacy in ruminants

is still a global problem. This is partly due to usage of inorganic forms of selenium
with low Se availability. Therefore, the solution for Se deficiency eventually moved
from nutritionists to veterinarians, who trying to correct deficiency by Se injections
in critical periods of ontogenesis. However, for the last few years information has
been actively accumulated indicating that introduction of organic selenium in the
form of selenized yeast could substantially improve Se status of dairy and sheep
and there will not be a need for veterinarian intervention to correct Se deficiencies.
Supplemental sodium selenite and sodium selenate by either oral administration
or parenteral injection have prevented clinical signs of selenium deficiency and
animal losses in both ruminant and non-ruminant animals. In general, organic
forms of selenium are absorbed more readily by animals than are inorganic
compounds (Ammerman and Miller, 1975). In 1993-1994 in the USA only 49%
of the 251 operations studied used Se supplementation of cattle. Most of them
(97.6%) used a mineral supplement with additional Se, while 4.1% added Se to
the cattle diet and 4.1% used Se injections (Dargatz and Ross, 1996). Furthermore,
some operations used more than one method of Se supplementation. As showed
by Ortman (1999) advantages of using a Se-supplemented mineral feed compared
with injecting or drenching the animals with selenium include:
much simpler in practice

decreased stress put on the animals
maintenance of steady Se status during supplementation, while a drench
will increase the Se status of the calves only for a period of 2-3 months
Practical applications of Se nutrition of ruminants

The importance of a successful transition from late pregnancy to early lactation is
well appreciated by dairy farmers. Indeed, health problems during the transition
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period can be extremely costly. However, the incidence of health problems on
dairy farms in various parts of the world today remains very high. In fact, only
50% of all cows calving without health problems (Ferguson, 2001). In many
cases health problems are related to various stresses. Indeed, stress in cattle can
be caused by infections, operations, long lasting heat or cold, transport or some
other nutritional or environmental factors.
During the transition period various stressors could impact dairy cows in several
ways resulting in the following (Drackley, 2001):
decreased dry matter intake

redistribution and re-direction of nutrients in the body away from the critical
functions of fetal growth, lactogenesis, and preparation of support functions
for lactation to support the stress response
activation of the sympathetic nervous system and release of the stress
hormones, such as glucocorticoids and epinephrine, which generally are
antagonistic to milk production.
changes in hormonal balance in the body: hormones and cytokines
associated with the stress response may decrease secretion of hormones
important for lactogenesis, such as growth hormone.
activation of lipolysis and fat infiltration in the liver.
suppression of immune function, leading to increased susceptibility to
infectious disorders.
disruption of the normal metabolic adaptations to lactation and wasting of
muscle tissue, increased fat mobilization, and increased fat deposition in
the liver
Depending on the level of stress the cow can adapt without detrimental
consequences for productive and reproductive traits or, if stress exceeds adaptive
ability of the animal, the cow can loose its productive potential. In general oxidative
stress was observed in cows during transition period (Miller et al., 1993;
Formingtoni et al., 1997; Ronchi et al., 2000; Bernabucci et al., 2002), which
could be an important contributing factors to various disorders and metabolic
diseases. In particular, a hot environment is responsible for oxidative stress which
worsens already weak antioxidant defences in transition cows (Bernabucci et al.,
2002).
Did you know that depending on the level of stress the cow can
adapt without detrimental consequences for productive and
reproductive traits or, if stress exceeds adaptive ability of the
animal, the cow can loose its productive potential?
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It seems likely that selenium is an important factor in cow adaptability to different

stresses. At parturition, tissue nutrient reserves are often at their lowest point.
Indeed, transfer of nutrients to the developing fetus, then into colostrum and milk
represents a tremendous drain on cow Se reserves. In particular, some of the
mentioned above problems in dairy and beef industries related to calf viability
could be associated with Se deficiency. For example, data published in 1997
indicated that in the USA 4.3% of newborn calves died and that 37% of the calf
losses were related to either weather (21%) or respiratory (16%) causes (NAHMS,
1997). Therefore, those loses could be related to inadequate thermoregulation
and low immunity. It seems likely, that antioxidant system of the newly born calf
and lamb is quite weak, since only limited amount of natural antioxidants is
transferred via placenta. However, high correlation (r=0.74) was found between
maternal Se blood concentration and its Se status of the newborn calves (Kincaid
and Hodgson, 1989) indicating that Se is transferred via placenta. Relationship of
Se content of components of the conceptus to day of gestation during late pregnancy
in Holstein cows is shown in Table 9.23. The equations describe changes in Se
content during the third trimester of pregnancy. As indicated by negative values
for the zero time intercepts, the functions should not be used to predict Se
composition prior to 190 d of gestations. Evidence exists linking Se deficiency to
weak calves at calving time (Smart et al., 1981) and increased incidence of early
embryonic death (Corah and Ives, 1991). It was shown that if cows have
concentrations of Se <120 ng/ml in whole blood, calves will have insufficient
tissue Se to prevent reduction in blood Se (Weiss et al., 1983).
Table 9.23 Relationship of Se content of components of the conceptus to day of gestation during late
pregnancy in Holstein cows (Adapted from House and Bell, 1994)
Component
Uterine tissues
Caruncles
Cotyledons
Fetal membranes
Fetus
Entire conceptus
Equation
Linear functions
y= -0.286 + 0.0039t
y= - 0.409 + 0.0038t
y= - 0.152 + 0.0015t
y= - 0.072 + 0.0006t
y= - 7.278 + 0.0414t
y= - 8.839 + 0.0552t
0,75
0,75
0.59
0.75
0.97
0.98
y milligrams of Se in component; t- day of gestation
There was a positive correlation between Se concentration in the whole blood of

cows and the whole blood of the calves (r=0.78; Ortman, 1999). A trial was
conducted to determine the effect of maternal supplementation of Se on transfer
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of Se to the fetus during late gestation. Selenium supplementation (3 mg/d
supplemental Se) significantly increased concentrations of Se in blood of dams at
parturition and their calves had higher Se in blood and liver. Therefore maternal
supplementation of Se increased Se reserves in the liver of the newborn and in
colostrum and carryover effects of Se supplementation of dams were evident in
calves at 42 d of age (Abdelrahman and Kincaid, 1995). In fact, a significant
correlation existed between Se in blood of calves at birth and in liver at 42 d of
age (r=0.55). The authors recommended using 3-5 mg Se/day to ensure adequate
Se reserves in tissues of newborns. Information is actively accumulating to indicate
that efficiency of Se transfer to newborns depends on the form of Se in the maternal
diet. Indeed, more Se transferred from the dam to the fetus and into milk when
organic selenium in the form of selenized yeast (Sel-PlexTM) was used in the diet
(Knowles et al., 1999; Gunter et al., 2003: Pehrson, 2005). Swecker et al. (1995)
showed that dietary Se (120 mg of Se/kg of salt mineral mix) increased colostral
IgG and the absorption of colostral IgG of beef cattle.
In addition to placental transfer, if selenium and vitamin E were delivered via
colostrum and milk, the calf would be able to build effective antioxidant defences
to deal with aforementioned stresses. First of all, improved immunity would
increase abilities of animals to withstand stresses and pathogen challenges (see
Chapter 4). Secondly, an effective thermoregulation of the calf at time of birth
could improve animal chances to survive cold stress at parturition, especially if
this happens in cold climate conditions. Indeed, production of heat to maintain
homeothermy in the neonate depends on shivering thermogenesis in the muscle
and nonshivering thermogenesis in brown adipose tissue (BAT). It is well known
that BAT is a specialised type of tissue with the ability to release heat in response
to cold stress. Therefore, newborn lambs or calves depend on BAT as the primary
source of nonshivering heat production and this process is under the thyroid
hormone control. At the same time, as mentioned in Chapter 2, Se-dependent
enzyme iodothyronine deiodinase is responsible for activation of the thyroid
hormone. For example, Arthur et al. (1991) was the first to show that in Se deficient
rats cold-induced increase in 5 1-deiodinase activity in BAT was dramatically
attenuated. The authors concluded that prenatal Se deficiency might impair the
thermogenic capacity of BAT in cold-exposed newborn ruminants. Therefore,
when Se status is low, activities of the deiodinases will be compromised and this
would lead to compromised thermoregulation of animals and increased mortality
during first days after birth. Indeed, Se-deficient steers and heifers were
characterised by reduced T3 and elevated T4 concentrations in serum in
comparison to Se-supplemented controls (Arthur et al., 1988; Wichtel et al., 1996).
The confirmation of the dependence of thyroid metabolism in ruminants from the
Se status came from studies demonstrating that prenatal Se supplementation
increased plasma T3 and decreased plasma T4 concentrations in newborn lambs
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542
and calves (Table 9.24; Donald et al., 1994; Awadeh et al., 1998b). Similarly,
pregnant ewes, which were fed additional Se had higher concentrations of triiodothyronine (T3) and thyroxine (T4), however, differences in concentrations of
these hormones in progeny (in lambs at 12 h of age) were not significant and only
trend (P<0.1) for higher T3 level was admitted (Rock et al., 2001). It seems likely
that effect of Se supplementation on the thyroid metabolism is dependent on the
Se status of the animals and when the status is not deficient there is no additional
benefit of selenium. Furthermore, activation of BAT causes large increases in
oxygen consumption and consequently causes increases in free radical production
(Hatfield et al., 1999) and such selenoproteins as GSH-Px, TR and MSRB could
be involved in maintenance of antioxidant defences. As mentioned above, when
Se and vitamin E were injected into cows and calves which were fed diets
marginally deficient in Se calf death loses compared to controls from birth to
weaning reduced from 15.3% to 4.2% (Spears et al., 1986). Indeed a proper Se
supplementation of the dry cow prior to calving and ingestion of colostrum by
calves is critical to providing sufficient Se to neonatal calves and maintain their
antioxidant defences.
Table 9.24 Effect of maternal intakes of selenium on selenium status and thyroid hormones in day old
calves (Adapted from Awadeh et al., 1998)
Concentration and chemical form of Se in salt mixes

20 ppm Se
60 ppm Se as
120 ppm as
60 ppm as
as selenite
selenite
selenite
Sel-Plex
Year 1
Se in blood, g/ml
GSH-Px in blood, EU*
Se in liver, g/g
0.12
0.6
3.1
0.14
0.8
3.9
0.14
0.8
3.1
Year 2
Se in blood, g/ml
GSH-Px in blood, EU
T3, ng/ml
T4, ng/ml
Ratio T3:T4
0.12
0.8
3.4
85.3
0.032
0.14
0.8
3.0
99.2
0.033
0.15
0.7
4.3
70.1
0.053
0.17
0.9
3.4
0.16
0.7
5.3
116.4
0.04
It has been shown that there are some species-specific differences in Se transfer
from the dam to the fetus. For example, in ewes maternal tissues had higher
concentration of labelled Se than the corresponding fetal tissues (Hidiroglou et
al., 1969). Furthermore in sheep the concentrations of labelled selenium in the
maternal plasma was 11.7-fold higher than that in fetal plasma (Wright and
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Bell,1964). Therefore, only limited amount of Se was transferred via the placental
membrane. In great contrast, in beef cattle Se more readily crossed the placenta
and the fetus can have Se concentration higher than that of the mother (Koller et
al., 1984; Campbell et al., 1990; Table 9.25). Therefore there is a high correlation
(r=0.872) between Se concentration in the cow and calf and blood Se concentration
in calf can be predicted from the following equation:
y= 0.02 + 1.03x
where x is cow blood Se concentration (Campbell et al., 1990).
Table 9.25 Effect of various levels of Se in the diet on cows and their progeny (Adapted from Koller et
al., 1984)
Alfalfa hay+
soybean+ Se in
mineral mix
Cows
Se in whole blood at time of parturition,
mg Se/kg of blood
GSH-Px in whole blood at time of parturition,
mU/mg of hemoglobin
Alfalfa hay+
soybean
Alfalfa hay
0.250
0.162
0.052
144
80
30
0.175
113
0.081
50
Calves
Se in whole blood, mg Se/kg of blood
0.242
GSH-Px in whole blood, mU/mg of hemoglobin 154
It has been shown that plasma concentrations of Se of calves at birth were correlated
positively with that of their dams at calving (Hidiroglou et al., 1994). Similarly
the RBC GSH-Px activities of 15-day-old calves (U/g of Hb) were significantly
correlated with values of their dams (Enjalbert et al., 1999):
Calf RBC GSH-Px = 59.8 + 1.26 Maternal RBC GSH-Px (r2=0.52; P<0.001)
It is interesting to note that in the aforementioned study using high Se
supplementation (13-45.5 mg of Se/day for 15 days) the RBC GSH-Px activity in
cows and their calves were also significantly correlated 77 to 115 days after calving
(r2=0.44, P<0.001). A significant correlation of Se concentration of the calf at
birth with that of the dam at 2 days after calving (r=0.5) was also reported by
Hidiroglou et al. (1994).
Campbell et al. (1990) suggested several possible mechanisms of the active
accumulation of Se in the fetus:
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the transfer of Se may be a one-way process across the placental membrane

from cow to fetus;
the metabolism of Se in the fetus may be minimal prior to birth resulting in
Se reserves;
there may be a specific period during gestation in which Se can be actively
transferred across the placenta and after that, it is trapped in the fetus
There are no data available to show in which form Se is transferred to the fetus.
Indeed, from the one hand if SeMet is present in the diet as an integral part of feed
ingredients one could expect SeMet to be transferred to the fetus and nonspecifically incorporated into the body proteins building Se reserves which can
be used in stress conditions when Se requirement increases. On the other hand,
inorganic Se in the form of sodium selenite or sodium selenate will be transferred
to the fetus most likely as SeCys and will be specifically incorporated into
selenoproteins. However, the rate of synthesis of selenoprotein during fetus
development is not known. Furthermore, the role of rumen bacteria in synthesis
of various selenocompounds with their possible transfer via placenta is not
investigated yet.
When fetuses were collected from pregnant primaparous beef cows it was shown
that in fetal liver Se concentration decreased significantly during the final 60 days
of gestation (Abdelrahman and Kincaid, 1993). Therefore Se dietary
supplementation during late gestation is of great value for the developing fetuss.
Furthermore, supplementation with Se during the dry period increased
concentrations of Se in the liver of calves at birth and at 42 days of age
(Abdelrahman and Kincaid, 1995). The authors showed that supplementation of
dams with >3 mg of Se/day was needed for calves to be born with Se concentration
in plasma of 50 ng/ml.
Did you know that there is a limited ability of the cow to
transfer Se into the colostrum and milk when sodium selenite
is used as a supplement?
The transfer of Se to the colostrum and milk is of great importance to the newly
born calf. Selenium concentration in the milk depends on the Se level and form in
the maternal diet. Indeed, a small proportion of added (as sodium selenite) dietary
Se (4.8%) was transferred to the milk with a deficient diet but only 0.9% of the
amount of added selenium was in milk of cows consuming diets adequate in
selenium. In contrast, 19% of the organic selenium furnished in brewers grains
appeared in the milk when the ration was deficient in selenium (Conrad and Moxon,
1979). In more recent study by Givens et al. (2004) for cows fed the Se-yeast the
efficiency of Se transfer to the milk ranged from 9.9 to 12.5%, compared with
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2.4-4.1% for cows fed sodium selenite. Similarly only 1.5% of radioactive sodium
selenite was found in the milk after oral dosage (Waite et al., 1975). Increasing Se
intake from 2 to 6 mg/day was associated with a 2-fold increase milk Se (Maus et
al. 1978). The Se bolus or Se pellets were also able to increase Se level in the
colostrum from 17 ng/ml (Se-deficient cows) to 52-72 ng/ml (Campbell et al.,
1990).
A study was conducted to determine the effect of selenium sources (salt free
choice mineral mixes (yeast form) or injectable form, Deposel (barium selenate,
long lasting) and Mu-Se (sodium selenite, short lasting) on the blood, liver, milk
and colostrum selenium level of cows. A total of 75 pregnant Angus cows were
subjected to a two-year study (December 1996-December 1998). It was shown
that Deposel provided long lasting reliable protection from selenium deficiency
while the yeast form of selenium provided continuous and highest levels of
selenium in blood, liver and milk samples (Table. 9.26; McDowell, 2002; Valle et
al., 2003).
Table 9.26 Selenium concentrations (g/ml) in colostrum and milk from Angus cows (Adapted from
McDowell et al., 2002)
Colostrum
st
Control
1
Mu-Se (selenite)
2
Deposel (selenate)
3
Sel-Plex-1
3
Sel-Plex-2
nd
Milk*
st
Plasma*
nd
st
1 year
2 year
1 year
2 year
1 year
2nd year
0.032
0.056
0.071
0.092
0.092
0.024
0.035
0.049
0.039
0.065
0.01
0.02
0.02
0.03
0.03
0.02
0.01
0.02
0.02
0.03
0.02
0.02
0.02
0.07
0.07
0.02
0.02
0.02
0.06
0.07
*/180 days postpartum; plasma of calves from cows supplemented with selenium
1
/Subcutaneous injection of 5 ml of Mu-Se (5 mg Se per ml as sodium selenite)
2
/Subcutaneous injection of 9 ml of Deposel ( 50 mg Se per ml as barium selenate)
3
/ The free choice mineral mixture containing 54.5 g/kg Sel-Plex resulting in a Se
concentration of 30 mg/kg in the mineral mixture with average daily Se intake to be 2.1 mg
per cow
Selenium distribution in cow milk was studied. Skim milk contained 93% of the
total milk selenium. Irrespective of the separation technique used, skim milk
selenium was mainly found to be associated with the casein fraction (>55%). In
fact selenium was mainly protein bound. Of all the milk proteins, beta-casein
contributed most to the total skim milk selenium (33% of total Se), while 17% of
the total skim milk selenium was associated with whey proteins (Van Dael et al.,
1991). In goat milk also about 80% of Se was associated with casein fraction
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(Allen and Miller, 1981). Technological processes such as milk heating (UHT,
pasteurisation) and the preparation of milk powder do not provoke substantial
losses of Se. During the production of cheese the Se concentration in dry matter
increases by a factor of 1.4 in comparison to the starting milk (Haldimann et al.,
1999). Recently, the selenium distributions in the different milk phases have been
studied in 14 whole milk samples, and the highest selenium levels were found in
milk whey (47.2-73.6%), while the lowest level was found for the fat phase (4.816.2%). A strong correlation was found between the selenium levels in whole
milk and the selenium levels in the milk components (Muniz-Naveiro et al., 2005).
Polyunsaturated fatty acids of milk can be oxidized causing problems with
milk rancidity. Predisposing factors associated with the development of rancid
flavours in milk include (Duval, 1995):
the presence of mineral deposits on dairy equipment

the presence of high levels of iron and copper in feeds
feeding with forage or silage which has been stored for >1 month or in
poor conditions
high levels of unsaturated fats in feeds
The use of supplementation of feeds with antioxidants such as vitamin E and

selenium to prevent oxidation of milk and the development of a rancid flavour is
considered to be effective. It is recommended that daily rations should contain 56 mg selenium and 650-800 IU vitamin E per cow. To correct an occurrence of
rancidity, vitamin E supplementation of rations to a level of 7000 IU/cow per day
may be carried out, with levels of vitamin E supplementation reduced to 3000 IU/
cow per day after 4 weeks (Duval, 1995). It seems likely, that Se-enriched milk
could be characterised by increased stability against oxidation. Indeed, such milk
had lower formation of dityrosine, a specific protein oxidation product, following
oxidative challenge with hydrogen peroxide (Stagsted and Nielsen, 2004).
Selenized yeast vs selenite

In a recent review of the literature related to Se metabolism in ruminants Weiss
(2003) indicated that in dairy cows and heifers using diets with typical selenium
concentrations there is no significant difference between sodium selenite or
selenate. On the other hand, organic Se is more efficiently absorbed and assimilated
than inorganic Se in lactating dairy cattle. In the review Weiss (2003) analysed
eight studies with cows, heifers, or steers where direct statistical comparisons on
the effects of selenium from selenite or selenium yeast were made. From them 5
studies reported various benefits of Se-yeast, while 3 studies reported no overall
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difference in whole blood selenium when two Se sources were compared. However,
it seems likely that other parameters such as Se in milk and colostrum as well as
Se transfer via placenta would be more appropriate tests for Se efficiency
comparison between various Se sources. Indeed, comparing Se in cows milk
Weiss (2003) indicated that 7 studies (from 8 analysed) reported increases in milk
selenium concentrations due to Se-yeast consumption in comparison to sodium
selenite. Five studies showed a significant increase, one study with beef cows
showed nonsignificant increase and one more study showed a high numerical
increase without statistical comparison.
Indeed, cattle supplemented with Se yeast had higher serum Se concentrations
at the end of the trial. In the study, individual serum- and milk Se concentrations
tended to be uniformly higher in animals given the Se yeast (Fisher, 1995; Fisher
et al., 1995). The effects of organic Se (selenized yeast) and sodium selenite
were compared in a feeding experiment with 100 Se-deficient dairy cows.
Supplementing the feed with organic Se 0.2 mg or sodium selenite for 8 weeks
increased blood Se within this period from the background level of 5.6 g/litre to
167 (Se-yeast) and 91 g/litre (selenite). Corresponding values for GSH-Px were
0.22, 3.0 and 2.3 Kat/g Hb (Malbe et al., 1995). In general, the bioavailability of
yeast Se was superior to selenite. Relative bioavailability (selenite = 1) of yeast Se
was 1.4 if blood GSH-Px, 1.9 if blood Se, and 2.7 if milk Se was used as the
response criterion (Malbe et al., 1995). The bioavailability of selenium from
sodium and cobalt selenite and Se from Se-containing yeast was evaluated by
glutathione peroxidase activity in the erythrocytes of 28 Se-deficient heifers. Se
supplement was added to the feed as a premix for 12 weeks. There was no
difference between the availability of Se from Na or Co selenite, but the
bioavailability of Se from the Se-containing yeast product was about twice that
from the inorganic sources (Pehrson et al., 1989).
Did you know that a common practice of Se injections in dairy
and beef industries is a result of low efficiency of sodium
selenite assimilation by cows? This practice could be
changed when organic selenium is used as a feed supplement
Similar results were reported by other authors. For example, Se (0.75 mg daily)
from the Se-yeast maintained Se concentrations in whole blood and milk at the
same levels as Se 3.0 mg in the form of sodium selenite. Furthermore, Se (3.0 mg)
from the yeast product increased blood Se by 40% and that in milk by 100%
(Ortman and Pehrson, 1997). In an experiment conducted in Italy, ten lactating
Holstein cows were randomly assigned to diets containing Se from either Na
selenite or Sel-Plex for 6 weeks. Cows given Sel-Plex diet had 67% higher milk
Se content within 2 weeks of beginning of supplementation. Furthermore, SSC
were significantly reduced as early as 2 weeks after inclusion of Sel-Plex (30%)
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548
and continued this trend throughout the test period (Duarte et al., 2004). Similarly,
inclusion of selenium (0.2 ppm) in the form of Sel-Plex (0.2 ppm) into the cows
diet providing 2.2 mg Se/day in the form of sodium selenite for 8 weeks was
associated with an increased Se level in the milk (0.138 mg/l vs 0.048 mg/l) and
decreased SSC (174,500 vs 229,300 cells/ml; Foltys et al., 2004). Field trial results
with Sel-Plex (Alltech, Inc.) confirm earlier university work indicating that a 20
to 30% increase in whole blood Se and an 80 to 100% increase in milk Se levels
can be associated with selenium yeast supplementation (Eliott et al., 2005).
In a 2 x 2 factorial design experiment, 20 early-lactation Holstein-Friesian
cows, that had been receiving a diet containing 0.33 ppm supplemental Se as
sodium selenite (SS), were fed Se supplement in the form of selenium-enriched
yeast (SY) or SS, each at levels that provided 0.15 and 0.33 ppm supplemental
Se, for a period of 10 weeks following a 2-week period with no supplemental Se
(Hemken et al., 1998). Blood Se levels were significantly higher in cows fed SY
than SS, indicating that the bioavailability of Se in SY is about twice that in SS.
The possibility of improving Se status of pregnant dairy cows and their calves by
supplementing dairy cows with inorganic or organic forms of Se was studied. A
Se-poor diet (0.03 ppm) for 30 dry cows was supplemented with Se. Two months
before calving, cows were divided into 3 groups and supplemented with inorganic
(0.23 ppm) or organic (0.105 ppm) selenium until calving. Se source and
supplementation influenced GSH-Px activity in whole blood of dams and their
calves. Indeed, usage of Se from yeast product for GSH-Px synthesis by cows
was 3.5-fold higher than that of Se from the mineral mixture (Ling and Ploom,
1999). In an experiment conducted at Agricultural Grasslands Research Centre
(New Zealand) Se-enriched yeast or sodium selenate was administered per os
(equivalent of 2 or 4 mg Se/day) three times a week for 133 days to previously
unsupplemented cows that were grazing low-Se pastures. Control cows were not
supplemented with selenium (Knowles et al., 1999). Selenized yeast was 2-3times more effective than was sodium selenite in increasing Se level in blood. In
the first 100 days of lactation cows fed selenium-supplemented rations had
significantly elevated serum selenium concentrations: 21.4 g/l (no
supplementation), 45.3 g/l (sodium selenite, 4 mg/cow/day), 65.4 g/l (selenized
yeast, 4 mg/cow/day) and 57.3 g/l (selenized yeast, 2 mg/cow/day; Falkowska
et al., 2000). Indeed, selenium from yeast was utilized better than sodium selenite.
Supplemental organic Se gave higher blood Se and GSH-Px activity than inorganic
Se. After 4 months of Se supplementation (1 mg/day) both Se concentration in
whole blood of beef calf and GSH-Px activity were higher for calves given Seenriched yeast those given inorganic Se (Nicholson et al., 1991a,b). Se
supplementation with Se-enriched yeast 5 mg/day increased Se in milk from 13.2
to 34.7 g/litre (Charmley et al., 1993). It is interesting that efficiency of Se transfer
from feed to the milk was highest (up to 24.6%) when organic selenium in the
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form of Sel-Plex was used, while pure selenomethionine (17.1%) or sodium
selenite (11.7%) were less effectively transferred to the milk (Manner and Lablin,
2000). For example, milk from cows consuming 3.4 mg Se per day (as feedderived Se plus Sel-Plex) contained 71 g/l Se, while milk from cows consuming
similar amount of Se (3.5 mg per day, as feed-derived Se plus selenomethionine)
contained only 37 g/l selenium. It is also interesting to note that adding sodium
selenite into the water was not effective in increasing Se level in the milk.
Supplementation of the suckler cows diet with organic Se in the form of Se
yeast rather than sodium selenite can improve the Se status of their calves. A trial
was conducted in Sweden to determine effect of maternal selenium on the selenium
status of suckling calves. Hereford cows were divided into two groups and had
free access to a mineral supplement that contained Se 30 mg/kg as Se-enriched
yeast or sodium selenite. The basal feed was quite low in Se and contained Se
0.02 mg/kg DM and mean daily consumption of the mineral supplement was
approximately 110 g/cow. The results can be summarised as follows (Pehrson et
al., 1999):
Se concentration in whole blood and the GSH-Px activity in the erythrocytes

of the cows and calves in the yeast group were higher than in comparison
to the animals in the selenite group.
Se concentrations in whole blood from calves in the yeast and selenite
groups were 130 and 84 g/litre, respectively, and plasma concentrations
were 48 and 34 g/litre, respectively.
Se concentration in the milk from the yeast group (17.3 g/litre) was higher
than that in milk from the selenite group (12.7 g/litre).
There were significant correlations (r = 0.59 to 0.68) between the
concentrations of Se in the cows milk or cows whole blood compared
with Se concentrations in the calfs whole blood and plasma or with the
erythrocyte GSH-Px activity of the calves.
In another experiment conducted in the Swedish University of Agricultural Sciences

dairy cows (n=42) were fed a basal diet containing 0.10-0.12 mg Se/kg DM for 5
months and were then divided into four groups. During the next 84 days, the
cows in three of the experimental groups were supplemented with 3 mg of Se
daily (as sodium selenite, sodium selenate, or Se-yeast). The results could be
summarised as follows (Ortman and Pehrson, 1999):
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At the end of the trial, the mean whole blood Se concentrations increased
from 104 g/l (control group) up to 138, 141, 165 g/l in the selenite,
selenate, and yeast groups, respectively.
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550
Plasma Se reached a plateau level within 4 weeks, at 75 g/l in the selenite

group, 80 g/l in the selenate group, and 90 g/l in the yeast group, while in
the control group it remained at 50 g/l.
The milk Se concentration reached a plateau within 1 week after the start of
Se supplementation. being 14.0, 16.4, 16.4, and 31.2 g/l in the control,
selenite, selenate, and yeast groups, respectively.
These data were confirmed by McIntosh and Royle (2002) showing that inclusion
of Sel-Plex in the cows diet to provide 2 or 6 mg Se/day was associated with
increased Se concentration in the milk from 6.9 up to 15.0 and 25.2 ng/ml
respectively (Figure 9.3). Similarly, in commercial conditions of Florida dairy a
replacement of sodium selenite with Sel-Plex was associated with a significant
increase in Se concentration in blood and colostrum (Figure 9.4). Furthermore,
increase Se supplementation in the form of Sel-Plex from 2.67 to 6 mg/day was
associated with an increase in Se concentration in the colostrum for the first two
milkings by 42% and for the first 8 milkings by 35% (Lewis, 2004).
30
25
Se (ng/ml)
20
15
10
5
0
0mg Se/day
2mg Se/day
6mg Se/day
Figure 9.3 Effect of Se-Yeast on milk Se status (Adapted from McIntosh and Royle, 2002)
Did you know that important advantages of organic selenium in

the ruminant diet include higher Se concentration in colostrum
and milk, which could help to build antioxidant system of the
newly born calves or lambs?
In an experiment, conducted by Gunter et al. (2003) 120 pregnant cows were
used to determine the effects of supplemental Se and its source on performance
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Blood Se
Colostrum Se
abc
Se (ppb)
170
Means with different superscripts

differ (P<0.05)
b
120
b
70
a
20
Pre-Sel-Plex
1 month on
2 months on
Date sampled
Figure 9.4 Blood and colostrum Se at calving at Florida dairy (Adapted from Eliott et al., 2005)
and blood measurements. Cows had ad libitum access to one of three free-choice
minerals: 1st: no supplemental Se, 2nd: 26 mg of supplemental Se from sodium
selenite/kg, and 3d : 26 mg of supplemental Se from seleno-yeast/kg (designed
intake = 113 g/cow daily). At the beginning of the calving and breeding seasons,
cows supplemented with Se were characterised by greater whole blood Se
concentrations and GSH-Px activities in comparison to cows receiving no
supplemental Se; cows fed selenoyeast had greater whole blood Se concentrations
than cows fed sodium selenite, but GSH-Px did not differ between the two sources.
At birth and near peak lactation, calves from cows supplemented with Se had
greater whole blood Se concentrations than calves from cows fed no Se and calves
from cows fed seleno-yeast had greater whole blood Se concentrations and GSHPx activities than calves from cows fed sodium selenite (Tables 9.27-9.28, Gunter
et al., 2003). Furthermore immunity of weaned beef cattle was improved. Indeed,
lymphocyte proliferation (non-stimulated) and macrophage phagocytosis was
significantly improved by Se-Yeast in comparison to sodium selenite or nonsupplemented animals (Table 9.29).
A field trial was conducted at a large Florida dairy to evaluate the response in
whole blood and colostrum selenium to organic selenium supplementation (SelPlexTM). In the trial Sel-Plex replaced 100% of the sodium selenite in the dry cow
supplement and provided 0.3 ppm Se. Whole blood samples were taken from 16
cows as they entered the dry lot (pre-Sel-Plex). Samples were also taken 1 mo
and 2 mo on Sel-Plex from the same cows. An additional set of blood and colostrum
samples were collected from 4 randomly selected cows after calving (Harrison et
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Table 9.27 Whole blood Se concentration in beef cows depending on selenium supplementation
(Adapted from Gunter et al., 2003)
Sel-PlexTM**
Whole blood Se, ng/ml
No Se
Sodium selenite*
Cows
Beginning of mineral feeding
Start of calving season
Initiation of breeding season
101
108
93
111
142
150
122
174
187
Calves
Birth
Peak of lactation
105
51
134
66
203
122
*/26 mg of supplemental Se/kg of free choice mineral from sodium selenite

**/26 mg of supplemental Se/kg of free choice mineral from Sel-Plex
Table 9.28 Whole blood GSH-Px activity in beef cows depending on selenium supplementation
(Adapted from Gunter et al., 2003)
Whole blood GSH-Px, EU/g Hb
No Se
Sel-PlexTM**
Sodium selenite*
Cows
Beginning of mineral feeding
Start of calving season
Initiation of breeding season
57
74
58
46
101
150
52
106
187
Calves
Birth
Peak of lactation
56
96
62
121
115
190
*/26 mg of supplemental Se/kg of free choice mineral from sodium selenite

**/26 mg of supplemental Se/kg of free choice mineral from Sel-Plex
Table 9.29 Effect of Se supplementation on immunity of weaned beef calves* (Adapted from Gunter et
al., 2003)
Item
No Se
Whole blood Se, ppm

GSH-Px, EU/g Hb
Ubstimulated
ConA
PHA
PWM
Macrophage phagocytosis, %
Sodium selenite**
Sel-PlexTM***
54.7
40.2
105.0
119.1
172.0
143.9
1,426
65,586
12,286
13,441
5.0
867
77,658
18,414
10,077
4.2
1,408
92,261
24,066
16,238
9.2
*22 days post-weaning; the weaning supplement was calculated to supply 0.24 ppm Se (DM
basis).
**/26 mg of supplemental Se/kg of free choice mineral from sodium selenite for cows
***/26 mg of supplemental Se/kg of free choice mineral from Sel-Plex for cows
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al., 2005). It was shown that blood Se from the set of 16 cows averaged 99, 113,
and 129 ppb for the pre-Sel-Plex, 1 mo and 2 mo samples, respectively. Therefore,
blood Se increased linearly (P=0.0012) and was different between pre-Sel-Plex
and 2 month on Sel-Plex samples (P<0.05). Furthermore, colostrum Se averaged
43, 90, and 95 ppb for the pre, 1 mo and 2 mo samples, respectively, and was
higher after 1 or 2 months on Sel-Plex than the pre Sel-Plex values (P<0.05).
Indeed, colostrum Se was 112% higher after 1 month and 124% higher after 2
months compared to pre Sel-Plex values.
A field trial was conducted at a New York dairy to evaluate the response in
whole blood and milk selenium to organic selenium supplementation (Sel-PlexTM).
Sel-Plex replaced 100% of the sodium selenite in the lactating cow supplement
and provided 0.3 ppm Se. A group of 15 cows were selected and whole blood
samples were pulled on February 6 (pre-Sel-Plex) and on April 26 (post-Sel-Plex;
Harrison et al., 2005a). Blood Se increased from 186 to 225 ppb (+21.2%) after
the inclusion of Sel-Plex in the diets (P<0.0001). Milk Se increased from 22 to 39
ppb (+78%) after the addition of Sel-Plex when the pre- and post-Sel-Plex samples
were compared (P<0.0001). Therefore, replacing inorganic Se with Sel-Plex
resulted in improved Se status of cows.
In an experiment the cows were randomly allocated into five groups. Control
group was not supplemented with selenium, 1 st experimental group had
subcutaneous injection of sodium selenite (SS), 2 nd experimental group had
subcutaneous injection of barium selenate (BS) and two other groups had
supplementation of mineral mix with Se-yeast. The results indicated that two freechoice salt mineral mixture treatments provided adequate selenium levels in liver
at 6, 12 and 24 months after treatment began (Valle et al., 2003). During the
second year, selenium concentrations for SS treatment declined, while the
concentrations were maintained in the BS and both free-choice mineral mixture
treatments. Indeed, the yeast form of selenium provided continuous and highest
levels of selenium in blood, liver and milk samples.
Did you know that by using organic selenium in the cows diet it
is possible to build Se reserves in the body which can be
effectively used in stress conditions when Se requirement
increased while feed consumption decreased?
Angus cows at 125-150 days of gestation received either no Se supplementation
(control), 5 ml sodium selenite via subcutaneous injection (Mu-Se) every four
months, 9 ml barium selenite via injection (Deposel), sodium selenite or selenized
yeast (Sel-Plex) via free-choice salt-based mineral mixtures (26 ppm Se) for one
year (Davis et al., 2004). Calf whole blood Se concentrations were determined at
birth, 30, 90 and 180 days of age. Neither injectable source was effective for
maintaining blood Se concentration during this experiment resulting in Se
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deficiency at 180 days of age. Sel-Plex was able to maintain physiological

concentration of Se in calf blood during the same period while calves fed on
mineral mixture containing sodium selenite were marginally deficient at 180 days
of age. Indeed, at this age Se concentrations in the calves blood was 0.037; 0.034;
0.036; 0.068 and 0.19 ppm in control, Mu-Se, Deposel, selenite mineral mixture
and Sel-Plex mineral mixture respectively. Results of the experiment conducted
by Richards et al. (2004) indicate that supplementing 0.27 mg Se/kg diet (DM)
from Se yeast in a feedlot diet containing adequate Se did not increase animal
performance, but resulted in increased loin Se concentrations (0.258 vs 0.135
ppm) and liver Se levels (0.831 vs 0.426 ppm).
In an experiment conducted in Finland, calves were fed, from birth to 13 to 14
months old, on a basal diet alone containing 0.03 mg Se/kg DM. An experimental
group of calves was supplemented with inorganic Se, as sodium selenite, to contain
0.25 mg/kg DM. The source of Se for another experimental group of calves was
plant Se, intrinsic in silage and barley, plus high-Se yeast mixed into a liquid milk
formula to provide diets with Se 0.25 and 0.40 mg/kg (Ekholm et al., 1991). At
slaughter, samples of 4 muscles and 10 other tissues and organs were taken for Se
analysis. The added Se significantly increased the Se concentration of all the
tissues studied. The greatest response was with plant Se added at 0.40 mg/kg. The
highest response to organic selenium (10-12-fold increase) was observed in
muscles. In other tissues, the increment was 1.5-7.5-fold, depending on tissue
type. Similar responses to organic selenium reported by Ortman and Pehrson
(1997) are presented in Figure 9.5.
170
150
130
110
90
70
50
3mg
selenite
3mg
selenite
0.75mg 0.75mg
Se yeast Se yeast
3mg Se
yeast
3mg Se
yeast
Figure 9.5 Selenium concentration (ng/g wet weight) in skeletal muscle of cows supplemented with
selenium (Adapted from Ortman and Pehrson, 1997)
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In a study, 12 mature cows were given one of four experimental salt mixes
containing Se 20, 60 or 120 mg sodium selenite/kg of salt mix; the fourth treatment
was Se 60 mg selenized yeast/kg of salt mix. Within serum, the highest
concentration of Se was in the selenoprotein P fraction (31.6 ng/ml), the smallest
concentration was in the glutathione peroxidase fraction (4.7 ng/ml) and an
intermediate amount of Se was obtained from the albumin fraction (8.5 ng/ml;
Table 9.30; Awadeh et al., 1998). Furthermore, SeP selenium comprised 54-75%
of total plasma selenium, while GSH-Px was responsible for 6-17% selenium in
plasma. It has been shown that Se-yeast consumption only slightly changed Se
distribution between various protein fractions of serum.
Table 9.30 Distribution of Se among different fractions in serum of cows given free access to salt
containing selenium (Adapted from Awadeh et al., 1998).
Serum fraction
20 mg/kg
Albumin
GSH-Px
Selenoprotein P
29.3
14.6
53.6
60 mg/kg
120 mg/kg
% of Se in protein fraction
20.1
17.1
73.0
60 mg/kg (SeY)
11.8
6.0
73.9
15.6
9.3
75.2
Evidence is actively accumulating that organic selenium is more efficient than

sodium selenite for growing calves. For example, Angus calves were supplemented
with organic and inorganic selenium at two levels (0.1 and 0.3 ppm). The group
fed 0.1 ppm organic selenium was characterised by the highest liveweight gain
(161 kg). Group fed 0.3 mg/kg Se-yeast had the highest plasma and liver selenium
concentration (Valle et al., 2001a). The lowest plasma selenium level was observed
in the unsupplemented control calves. Plasma selenium concentrations of control
animals tended to decrease with time. The selenium supplementation at 0.3 mg/
kg in the form of Se-yeast substantially slowed down the Se decline with time. It
is interesting to note, that in terms of increasing Se status, dietary supplementation
with organic selenium is more efficient than selenium injections. For example,
selenium concentrations were investigated in calves treated parenterally with
inorganic selenium (40 mg in two injections, Group II) or treated orally for two
months with organic selenium (0.3 mg/kg feed, Group III), in comparison to
untreated control calves (Group I). Both treated groups showed highly significant
increases in whole blood selenium concentrations (Pavlata et al., 2001).
Comparison of tissue concentrations revealed highly significant differences in
the liver (213.3 56.8, 206.5 36.2 and 424.7 88.4 g/kg wet tissue), striated
muscles (92.4 29.2, 81.412.1, and 263.4 47.4 g/kg) and myocardium (121.5
31.8, 108.3 9.6, and 251.4 51.0 g/kg for Groups I, II and III, respectively).
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Growing beef and dairy cattle were supplemented with sodium selenite or selenized
yeast to supply 1 mg Se/kg supplement and a comparison was made with
unsupplemented cows (Nicholson et al., 1991a). Blood Se concentration was
significantly (P<0.01) higher for the organic (141 ng/ml) than for the inorganic (102
ng/ml) or control (45 ng/ml) groups. Similarly, the cows receiving Se from fertilized
alfalfa silage were characterised by increased Se concentration in the milk (58 ng/ml)
or plasma (111 ng/ml) in comparison to sodium supplemented or unsupplemented
animals (Nicholson et al., 1991b). It is interesting, that increased Se concentration in
beef as a result of Se-yeast dietary supplementation is associated with improvement
of beef quality, in particular with decreased drip loss (Table 9.31).
Table 9.31 Effect of organic selenium (Sel-Plex, per os 4 mg/heard/day) on beef quality (Adapted from
Simek et al., 2002)
Group and treatment
Se in Longissimus
dorsi muscle, mg/kg
Control
7 day supplementation
30 day supplementation
0.107
0.168
0.223
Drip loss, %
Vacuum-stored for
four days, %
0.92
1.17
0.50
0.29
0.45
0.03
Therefore, it has been proven that organic selenium in the form of Selenized yeast
(Sel-PlexTM) is more effectively increases Se concentration in blood and milk in
comparison to selenite (Ortman and Pehrson, 1999; Knowles et al., 1999). In lambs
organic selenium is also more effectively accumulated in skeletal muscles and other
tissues (Ehlig et al., 1967). Furthermore, 90 ppm Se as selenized yeast (Sel-PlexTM) or
sodium selenite in the trace mineralised salts was fed to sheep from Day 56 of the
experiment until lambing and providing Se intake equivalent to about 0.2 mg Se/kg
of diet DM (Rock et al., 2001). The results of the experiment are summarised in Table
9.32 and indicate increased (in comparison to selenite) Se concentrations in the whole
blood and liver as well as in colostrum as a result of organic selenium supplementation.
Table 9.32 Effect of supplemental Se during gestation on concentration of Se in colostrum and liver and
blood and activity of GSH-Px in newborn lambs (Adapted from Rock et al., 2001).
Whole blood Selenium, g/ml

Whole blood GSH-Px, EU/ml
Liver selenium, g/g
Colostrum selenium, g/g
Control
Selenitea
SeYb
0.101
145
0.63
0.012
0.234
414
1.34
0.132
0.434
640
1.80
0.226
/90 ppm Se as sodium selenite in the trace mineralised salts fed from Day 56 of the experiment
until lambing and providing Se intake equivalent to about 0.2 mg Se/kg of diet DM
b
/90 ppm Se as selenized yeast (Sel-PlexTM) in the trace mineralised salts fed from Day 56 of
the experiment until lambing and providing Se intake equivalent to about 0.2 mg Se/kg of
diet DM
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Advantages of organic selenium were summarised by Ortman (1999) and Valle
(2001) in their thesis. It was shown that
Se-yeast supplement caused a higher concentration of selenium in the blood

and tissues of cattle and in cows milk than inorganic selenium supplement.
Long-term supplementation of dairy cows with Se-yeast did not result in
toxic accumulations of Se in their tissues.
Suckler beef calves whose dams were supplemented with Se-yeast had a
higher Se status than calves whose dams were supplemented with selenite.
Se-yeast proved to be more effective in stimulating weight gains and liver
Se concentrations than sodium selenite for Angus steers and heifers
Se-yeast was more effective than sodium selenite in raising and maintaining
adequate Se concentrations in tissues of beef cattle
Improved Se status of cows and their calves is translated in improved immunity

and health. For example, in experiment conducted in China, thirty healthy HolsteinFriesian pregnant dairy cows were assigned to 5 groups, 4 test groups (T1, T2, T3
and T4) and a control group. Tested Se sources included Se-yeast (Sel-Plex 50), a
mixture of Se compounds (70% Se-Plex 50 + 30% Na selenite), a Se amino acid
complex (mainly selenomethionine) and Na nitrite and were supplemented to
cows in the test groups for 168 days (Huang Zhi Jian et al., 2002). Blood GSH-Px
and serum Se concentrations of cows in groups T1, T2 and T3 were significantly
higher than levels found in control cows. The highest significant responses in
terms of lymphocyte transformation rate and T lymphocyte count measured on
day 70 of Se supplementation were observed in cows supplemented with SeYeast compared to those in other groups. Supplemental organic Se significantly
improved the cows reproductive performances, reduced services per conception
and decreased the incidence of retained placenta and endometritis.
Did you know that by using organic Se in the cow or sheep diets
it is possible to maintain the high Se status during all periods
of ontogenesis and to maintain high productive and
reproductive performances in stress conditions of
commercial meat and milk production?
The effect of dietary supplementation of organic Se in the form of Sel-Plex on
various aspects of Se status of dairy goats was investigated using 12 healthy milk
goats in a 10-week trial. The goats were allocated to two groups of 6 animals and
the control group was fed a basal diet containing 0.045 ppm Se. An experimental
goats received 0.15 ppm Se in the form of Sel-Plex (Hhaled and Illek, 1999). The
concentration of Se in the whole blood, plasma and milk and GSH-Px activity in
erythrocytes were significantly increased in the experimental group. There was
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558
also a significant effect of organic selenium on concentration of thyroid hormones

in goats. Furthermore, organic selenium in the form of Sel-Plex was shown to
decrease SCC in experimental (Figure 9.6) and commercial (Figure 9.7) conditions.
Indeed, field data from 16 herds across US showed a significant reduction in SCC
of about 50,000 cells/ml in first test after Sel-Plex introduction to the diet (Eliott et
al., 2005). Similarly, SCC expressed on the linear score scale was also lower after
Sel-Plex supplementation in 16 commercial dairy herds in the USA (4.50 vs.
4.18; P<0.05; Harrison et al., 2005b).
170
150
SCC x 103
130
110
90
70
50
0mg Se/day
2mg Se/day
6mg Se/day
Figure 9.6 Effect of Se-Yeast on SCC (Adapted from McIntosh and Royle, 2002)
350
Pre
Post
SSC x 103
300
250
200
150
100
1.0
2.0
3+
All cows
Lactation number
Figure 9.7 Effect of Sel-Plex on SCC in the USA (16 herds, 1607 cows; Adapted from Eliott et al., 2005)
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Furthermore, cows supplemented with organic selenium in the form of Sel-Plex
were characterised by improved health, productive and reproductive parameters,
including decrease mastitis, retained placenta and metritis and increased conception
rate (Elliot, personal communication).
Conclusions
Selenium nutrition of ruminants has some specific features, which create specific
problem in dairy and beef industries. In particular, in many places in the world Se
level in feed ingredients are not adequate to meet high Se demand of growing,
reproducing and lactating animals. A common practise of dietary Se
supplementation in inorganic form proved to be of low efficiency. That is why in
many cases veterinarians are trying to correct problems of inadequate nutrition
and Se injections are still a common practise in dairy industry. Indeed, part of
selenite consumed is reduced to metallic Se or selenide by rumen bacteria and
both are not available for further metabolism. The second part of selenite is
incorporated into proteins synthesised by rumen bacteria and it seems likely that
Se is also of low availability for animals. The replacement of sodium selenite by
organic Se sources, in particular, by selenized yeast in the form of Sel-Plex, is
proven to be effective means in solving Se problems in dairy, beef and sheep
industries. The data accumulated for the last 10 years clearly indicate advantages
of such replacement (Table 9.33). This includes increased Se concentration in
blood and GSH-Px activity, approximately doubled Se concentration in colostrum
and milk, higher Se transfer via placenta. As a result, cows health is improved
with lower somatic cell counts, decreased mastitis and retained placenta and
improved conception rates. The benefit to the newly born calves is coming from
improvement of their antioxidant defences and thermoregulation leading to better
immunity, viability and lower mortality during first months of the postnatal
development. Similarly to monogastric animals, when organic selenium is used
ruminants can also build Se reserves in their tissues, in particular in muscles, and
those reserves can be effectively used by animals in stress conditions when Se
requirement is increasing while feed consumption is declining.
Indeed, there are still many questions related to Se metabolism in ruminants
which need further clarification and this warrants new studies in this scientific
field.
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560
Table 9.33 Advances of organic selenium for ruminants
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Parameter
Effect of organic
vs inorganic
selenium
References
Drip loss of beef

Se in cow plasma
Decreased
Increased
Se in cow milk
Increased
Se in cow colostrum
Se in whole blood of calves
Se in plasma of calves
Se in whole blood of calves
GSH-Px in erythrocytes of calves
Se in cow whole blood
Increased
Increased
Increased
Increased
Increased
Increased
Se in whole blood of calves at birth

Se in cow blood, liver and milk
Increased
Increased
Se in cow serum
Se in calve liver
GSH-Px in erythrocytes of yearling
heifers and cows
GSH-Px in whole blood of cows
GSH-Px in erythrocytes of calves
at birth
Se in goat milk, plasma and whole
blood
GSH-Px in whole blood of goats
Casein selenium
Triiodothyronine (T3) in plasma of
calves at birth
IgM in cow serum
Proportion of Se in serun albumin
fraction
Se in skeletal muscles of lambs
Se in skeletal muscles of cows and
calves
Increased
Increased
Increased
Simek et al., 2002

Pehrson et al., 1999; Hemken et
al., 1998
Pehrson et al., 1999; Conrad and
Moxon, 1979; Ortman and
Pehrson, 1999; Malbe et al.,
1995; Knowles et al., 1999;
Pehrson et al., 1999
Gunter et al., 2003
Fisher et al., 1995; Hemken et
al., 1998; Gunter et al., 2003;
Malbe et al., 1995; Knowles et
al., 1999; Awadeh et al., 1998a
Gunter et al., 2003
Valle et al., 2003; Harrison et
al., 2005a
Fisher et al., 1995
Valle, 2001
Pehrson et al., 1989;
Increased
Increased
Malbe et al., 1995

Gunter et al., 2003
Increased
Khaled and Illek, 1999
Increased
Increased
Increased
Khaled and Illek, 1999

Knowles et al., 1999
Awadeh et al., 1998
Increased
Decreased
Awadeh et al., 1998

Awadeh et al., 1998a
Increased
Increased
Ehlig et al., 1967

Ortman and Pehrson, 1997;
Pavlata et al., 2001
560
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Table 9.33 Contd.
Parameter
Effect of organic
vs inorganic
selenium
References
Urinary Se excretion in lambs

Urinary Se excretion in goats
Se in bull muscles
Daily gains in calves
Somatic cell counts
Decreased
Decreased
Increased
Increased
Decreased
Retained placenta
Reduced
Postpartum endometritis morbidity
Decreased
Services per conception
Decreased
Nutritional muscular degeneration

in nursing calves
Decreased
Ehlig et al., 1967

Aspila, 1991
Ekholm et al., 1991
Valle, 2001
Diaz et al., 2004; Foltys et al.,
2004; McIntosh and Royle,
2002; Eliott et al., 2005;
Harrison et al., 2005b
Erokhin and Nikonov, 2001;
Huang Zhi Jian et al., 2002:
Eliott et al., 2005
Eliott et al., 2005
Huang Zhi Jian et al., 2002;
Eliott et al., 2005
Pehrson, 2004
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10
SELENIUM IN NUTRITION OF OTHER ANIMALS:
HORSES, DOGS, CATS AND FISH
You can take a horse to the water,
but you cant make him drink
SELENIUM IN HORSE NUTRITION
Introduction
Antioxidant-prooxidant balance in horses plays an important role in maintenance
of their health. Indeed, it is generally accepted that mitochondria are the main
source of free radicals in biological systems (see chapter 1). It has been shown
that increased exercise is related to higher oxygen consumption and in many
cases to overproduction of free radicals. In fact, exercise-induced oxidative stress
can contribute to accelerated muscle fatigue and muscle fibre damage, leading to
exercise intolerance and poor performance (Sen and Packer, 2000). It seems likely
that horses can adapt to regular exercise and racing by increasing activities of the
antioxidant enzymes. However, a dietary provision of minerals, which are essential
part of GSH-Px (Se) and SOD (Zn, Cu and Mn) in optimal amounts is a challenging
task for horse nutritionist.
Se deficiency
Selenium deficiency in horses is related to muscle degeneration. Indeed, nutritional
myopathy (white muscle disease, WMD) involves skeletal and cardiac muscles
and results in (NRC, 1989):
weakness
impaired locomotion
difficulty in suckling and swallowing
respiratory distress
impaired cardiac function
WMD of foals is caused by a dietary deficiency of selenium and vitamin E, usually

in association with predisposing factors such as a high intake of dietary unsaturated
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fats or unaccustomed exercise. WMD has been observed in foals from birth to 1
year of age, particularly those foals born to dams fed selenium-deficient diets,
during gestation (Lofstedt, 1997).
Did you know that white muscle disease of foals is caused by a
dietary deficiency of selenium and vitamin E, usually in
association with predisposing factors such as a high intake of
dietary unsaturated fats or unaccustomed exercise?
There are two major forms of the disease in foals: an acute, fulminant syndrome,
which is rapidly fatal, or a subacute syndrome characterized by profound muscular
weakness. The disease frequently has complications in the form of aspiration
pneumonia and stunting. On the biochemical level, markedly increased muscle
enzyme and low GSH-Px activities are common findings in affected foals. If
affected foals with the subacute form of the disease are supplemented with Se at
early stages of the disease they may survive; however, mortality rates remain
high (30%-45%; Lofstedt, 1997).
In foal affected by WMD serum selenium levels were below 65 ppb, showing
deficient levels. The mares of affected foals had significantly lower Se levels than
other mares (Higuchi et al., 1989). There was a good correlation between serum
Se concentration and blood GSH-Px activity (r = 0.81). Selenium levels in the
liver of affected foals were lower than in the foals, which succumbed with other
diseases. In acute disease vitamin E and Se should be given parenterally (5 mg
Se/horse) or by oral application of sodium selenite (46% selenium, 20 mg/horse),
in addition to symptomatical treatment (Zentek, 1991).
In Germany, post mortem studies on 143 foals over three years showed a total
of 46 (32.2%) cases of muscular dystrophy (Sandersleben and von Schlotke, 1977).
Histological examination of tissue from foals with subacute muscular dystrophy
showed hyaline skeletal-muscle fibre disintegration with calcification. Grossly,
the muscular lesions were not apparent even in the advanced stages. Most cases
were diagnosed within one week of birth. Vitamin E/selenium deficiency has been
implicated in the aetiology of the disease, and administration of this combination
to the mare during the lactation period is recommended (Sandersleben and von
Schlotke, 1977).
Se excess
Similar to other animals, Se access for horses can be toxic. It is interesting that,
explorer Marco Polo in the 13 th century was the first to describe selenosis in
horses which caused drop off of the hoofs of the animals as a result of consumption
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of specific plants known as selenium accumulators (see Chapter 3). Acute selenium
toxicity (blind staggers) in horses is characterized by (Rosenfeld and Beath, 1964,
NRC, 1989):
blindness
head pressing
perspiration
abdominal pain
colic
diarrhea
increased heart and respiration rates
and lethargy
Chronic selenium toxicity (alkali disease) is characterized by (Rosenfeld and

Beath, 1964; Traub-Dargatz et al., 1986):
alopecia, especially about the mane and tail

cracking of the hooves around the coronary band
In August 1991, 4 cases of naturally occurring selenosis in horses have been

detected in Wyoming State Veterinary Laboratory. Clinical signs were most often
referable to epithelial damage, e.g., hoof lesions and loss of mane and tail
(Raisbeck et al., 1993). A 9-year-old gelding showed a swollen prepuce, and
vesicles on the mucosa of the nostrils and mouth. After 6 days lameness occurred
and laminitis was diagnosed by radiography. Exudation at the coronary band was
suspicious for selenium toxicosis (Coenen et al., 1998). The expected selenium
intake was 2.1 mg/day, but in this case it reached 153.4 mg/day. Plasma selenium
reflected the selenium over-dosage (307 ng/ml). In mixed hair samples (coat and
mane) selenium concentrations were 0.3-0.64 mg/kg dry matter (DM) while in
mane hair near the skin (representing the most recent ground of hair) 2.63 mg Se/
kg DM were found. The rapid onset of clinical signs of severe chronic selenosis
(hair loss, cracked hooves, depression, and lameness) was observed in a fouryear-old mare grazing seleniferous pastures in County Meath. Concentration of
selenium in blood of the 3 animals ranged from 635 to 6350 ng/ml (8-80 mol/l)
while the concentration up to 200 ng/ml (2.5 mol/l) was considered normal
(McLaughlin and Cullen, 1986).
Did you know that the maximal tolerable level of selenium in
horses is estimated at 2 mg/kg of diet and the LD50 for orally
administered selenium is considered to be approximately
3.3 mg of selenium?
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The maximal tolerable level of selenium in horses is estimated at 2 mg/kg of diet

and the LD50 for orally administered selenium is considered to be approximately
3.3 mg of selenium (as sodium selenite)/kg of body weight (NRC, 1989). After
oral administration of sodium selenite lethal dose was 3.3 mg/kg bodyweight
(Zentek, 1991). Indeed, in feedstuffs selenium concentration must be below 5
mg/kg dry matter (Traub-Dargatz et al, 1986).
Se requirement
In accordance with NRC (1989) physiological requirement of horses is 0.1 mg/
kg of diet, however, it seems likely that horses at high work intensities may have
higher Se requirement (Shelle et al. 1985). Furthermore, foals, pregnant or lactating
mares, and racing horses have increased Se requirements (Zentek, 1991). In fact
recently it has been suggested that the appropriate Se concentration in the total
horse diet to be 0.3 ppm (Stowe, 1998) and calculation conducted by Pagan et al.
(1999) indicated that the grain mix for horses should contain about 0.6 ppm Se.
Did you know that recently it has been suggested
that the appropriate Se concentration in the
total horse diet to be 0.3 ppm?
In general, when sodium selenite was added to the diet, plasma Se concentration
of mature horses reached a plateau at about 140 ng/ml in horses fed 0.14 mg Se/
kg of diet (Shellow et al., 1985). It has been shown that in Bavaria 52% of horses
received Se at 1.25 g/kg BW, which is only half of the recommended daily Se
intake for horses (Wichert et al., 2002). At the same time Se level in plasma (g/
l) was <50 in 25% horses, >50-75 in 27%, >75-100 in 19%, >100-150 in 27%.
Taking the reference values for horses at 100-250 (g/l) it is clear that more than
70% of horses were Se deficient.
Selenium and GSH-Px

Similar to other species, with the moderate Se level in the diet, in horses there is a
good correlation between Se status and GSH-Px activity in blood and tissues. For
example, the activity of GSH-Px was measured in the blood of horses and it was
positively related to the blood selenium concentration (r=0.98) over the range of
enzyme activities of 8.2 to 140 units (mmoles NADP-oxidised/min/g Hb) and
selenium concentrations of 0.24 to 2.74 mol/l (Caple et al., 1978). A quadratic
relationship was also demonstrated between erythrocyte GSH-Px and whole blood
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or serum selenium concentration (Blackmore et al., 1982). Similarly, when blood

Se concentrations and GSH-Px activity was measured in 30 standardbred horses
a significant correlation was found (r =0.97). However, there was no difference in
GSH-Px activity between the healthy foals and the foals affected by muscular
dystrophy or between the corresponding mares (Roneus, 1982). It seems likely
that the blood GSH-Px in foals reflects the amount of selenium given to the mare
during pregnancy (Roneus and Lindholm, 1983). A survey of selenium and vitamin
E concentrations in horses was conducted at four breeding farms in New York.
The mean blood selenium concentration in this survey for horses fed local feed
was 77 g/l. Horses fed commercial feed had a mean blood selenium concentration
of 156 g/l (Maylin et al., 1980). Furthermore, a 0.94 correlation coefficient was
found between blood GSH-Px activity and blood selenium concentrations in horses.
In Germany Se concentration in plasma and the activity of GSH-Px in blood
were measured in 304 horses, aged four months to 29 years. GSH-Px activity
varied from 2 to 190 U/g Hb and plasma Se concentration ranged from 16 to 291
ng/ml (Vervuert et al., 2000). The relationship between GSH-Px activity in blood
and plasma selenium concentration was shown to be described by the linear
regression equation y = 0.78 x + 43.1. There was a high variability within the
herd levels even if the horses were maintained under the same feeding and
management conditions. A survey of blood Se concentrations and GSH-Px
activities in breeding mares was conducted on two horse farms located in different
regions of Serbia (Mihailovic et al., 1996). There were no significant differences
in mean blood plasma Se concentrations in the horses on the farms (73,3 and
72.1 ng/ml, respectively), although they were fed diets containing different Se
concentrations. High correlation coefficient (0.84) was found between blood GSHPx activities and plasma Se concentrations in horses at both farms. A survey was
conducted in 7 counties in Virginia and 4 counties in Maryland to determine
relationships among blood selenium concentrations in 346 horses and management
and dietary factors which significantly affect blood selenium concentrations
(Crisman et al., 1994). Blood Se concentrations for the population surveyed ranged
from 27 ng/ml to 266 ng/ml with a mean of 125 ng/ml. Factors significantly
affecting blood selenium concentration included feeding supplements, origin of
hay and amount of time spent on pasture. There was also evidence that level of
exercise and gender of the horse may contribute to differences in blood selenium
concentrations.
Se metabolism and effective sources

Selenium metabolism in horses is similar to that described in previous chapters
for other animal species. Indeed, the main form of Se, which horses get from feed
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ingredients, is SeMet. Since Se level in feed ingredients is highly variable and

very often low, Se supplements have become an essential part of mineral premixes
for animals, including horses. It has been shown that horses supplemented with
sodium selenite excreted more fecal selenium than when supplemented with
organic selenium in form of Sel-Plex (Pagan et al., 1999; Table 10.1).
Table 10.1 Effect of selenium source on balance measurements in exercised horses (Adapted from
Pagan et al., 1999).
Selenium balance, mg/day

Intake
Urine
Feces
Retention
Retention, % of intake
Retention, % of absorbed
Whole blood Se, ng/ml
Pre
Post
4h
24 h
Sodium selenite
Sel-Plex
3.76
1.16
1.85
0.75
51.1
20.4
39.3
3.72
1.10
1.58
1.04
57.3
27.8
48.6
205.1
216.8
209.2
202.8
201.0
224.5
222.1
198.8
Did you know that organic Se is more effective in

horse nutrition than sodium selenite?
The apparent absorption of Se from selenite and Sel-Plex was shown to be 51.1%
and 57.3% respectively. Selenium retention in the body was also increased in
organic selenium-supplemented horses (48.6%) in comparison to selenite-fed
animals (39.3%). The authors showed that both plasma and whole blood Se
increased post-exercise, however, 24 hours post-exercise plasma Se retuned to
initial level in selenite-fed horses, but remained elevated in Sel-Plex fed horses.
This could be an indication of better adaptability of organic Se-fed horses to
stress conditions of exercising. Furthermore, at 0.3 ppm supplementation of horses,
there was no difference between sodium selenite or selenate in terms of maintaining
serum Se concentration or GSH-Px activity (Podoll et al., 1992). The injection of
barium selenate at a deep intramuscular site was efficacious in correcting the
selenium status of mares grazing pasture with a low (0.01-0.07 mg/kg DM) Se
concentration. However, some swelling and fibrosis at the injection site was
apparent at all dose rates used (Wichtel et al., 1998). Therefore, it seems likely
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that using organic selenium supplements in the horse diet would be a practical
way to maintain their optimal Se status.
Antioxidant defences and exercise

It is well known that physical exercise increases both tissue needs for oxygen and
cellular respiration and causes an overproduction of free radicals. When free
radical generation exceeds the cells antioxidant capacity tissue-damage develops
due to oxidative stress. Therefore, the optimal nutrition of training horses is a key
element of their management. In the equine species, an exercise-induced oxidative
stress has been described (Mills et al., 1997; Art et al., 1999; Balogh et al., 2001;
White et al., 2001; Hargreaves et al., 2002; Marlin et al., 2002).
However, antioxidant defence system in horses is poorly characterised yet.
For example, selected antioxidants including SOD, GSH-Px, total antioxidant status
(TAS), ceruloplasmin (CP), bilirubin, uric acid, zinc, copper and selenium were
determined in blood of 80 clinically healthy horses. Antioxidant parameters
significantly varied between horses from different environments or different breed
and sex. Nevertheless, age of horses had no significant effect on antioxidant
parameters (Gorecka et al., 2002). Significant correlation coefficients were
observed between enzymatic and non-enzymatic antioxidants. The authors have
shown that the nature of the relationship between the antioxidant system in horses
with respect to environmental factors was rather complex and to date only a part
of system is understood. To evaluate effects of exercise on antioxidant system of
horses activities of 3 major antioxidant enzymes in equine erythrocytes (SOD,
GSH-Px and catalase) were investigated. Blood samples were obtained from 11
thoroughbred horses before and immediately after vigorous exercise (Ono et al.,
1990). Firstly, it was shown that used level of exercise induced the increase of
plasma lipid peroxide concentration. Secondly, following the exercise, the GSHPx activity in erythrocytes was significantly reduced, whereas SOD and catalase
activities were not affected. Furthermore, to address an ability of racing
Standardbred horses to adapt to stress caused by exercising, samples were collected
immediately before and after a training jog at a commercial race track (Gallagher
and Stowe, 1980). It was shown that hemoglobin, hematocrit, and serum selenium
increased significantly, whereas RBC-reduced glutathione decreased significantly
immediately after exercise. GSH-Px and glutathione reductase were not altered
by exercise.
Did you know that exercise increases free radical production
and Se supplementation in an optimal organic form can be
protective helping to maintain optimal health of horses?
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Field studies have shown that horses during training undergo significant changes
of several blood antioxidant markers and that oral antioxidant supplementation
might help maintaining antioxidant defences. In fact, an antioxidant imbalance
was observed after three months under field conditions in the trained thoroughbred
horses, reflected by a significant decrease in GSH, SOD, GSH-Px, Se and a
significant increase in GSSG (Table 10.2; de Moffarts et al., 2005). The antioxidant
supplement prevented GSH-Px and Se decrease and significantly increased plasma
antioxidant capacity, -tocopherol and -carotene. Similarly, effects of an
antioxidative mixture, containing selenium and vitamin E (Se-E), on the response
of antioxidant enzyme activities following the exercise were examined. For this
purpose seven horses were injected intramuscularly with Se-E (Se: 25 mg, vitamin
E: 54.8 mg) and received the same vigorous exercise. It was shown that the Se-E
treatment decreased plasma lipid peroxide levels before the exercise. However,
SOD, GSH-Px, and catalase activities were not affected. Furthermore, the Se-E
treatment only slightly prevented the decrease in GSH-Px activity and the increase
in plasma peroxide level after the exercise, having no effect on SOD and catalase
activities (Ono et al., 1990).
Table 10.2 Blood antioxidant concentration of healthy trained thoroughbred horses before (T0), after 6
(T6) or 12 (T12) weeks of oral placebo (C) or antioxidant (E) supplement* (Adapted from de Moffarts et
al., 2005)
Marker
Group
TO
T6
T12
C
E
C
E
C
E
C
E
C
E
18.6
19.6
1654
1584
17.8
25.7
3.03
2.64
108
101
37.6
49.9
1512
1485
40.4
39.0
3.30
7.10
82
156
21.1
21.4
1400
1378
41.3
1378
3.30
7.40
73
153
Ascorbic acid, M/l

GSH, M/l
GSSGH, M/l
Vitamin E, M/l
Se, g/l
*The supplement provided the following daily quantities of vitamins and trace elements:
ascorbic acid: 11.5 g, -tocopherol acetate: 7 g, -carotene: 0.5 g, copper: 187 mg, zinc:
769 mg and Se: 7 mg
In addition, it has been shown that the controlled training and diet supplements
were able to significantly increase horse antioxidant defences in both the
extracellular fluids and blood cells of horses, thus decreasing peroxidative
phenomena following physical exercise (Avellini et al., 1999). In an experiment
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3-year-old racehorses underwent a series of different physical exercise trials before

and after 70 days of daily training and dietary supplements (vitamin E and
selenium). The above treatments were able to increase both red blood cell resistance
to the peroxidative stress induced in vitro and the GSH-Px activity in lymphocytes.
Moreover, they were also able to decrease indices of lipid peroxidation in the
plasma and the mobilisation of low molecular weight antioxidants (total peroxylradical trapping activity) following the physical exercise trials.
An oxidant/antioxidant imbalance in favour of oxidants has been identified as
playing a decisive role in the pathogenesis of chronic inflammatory airway diseases.
Therefore nutritional antioxidant supplementation might reduce oxidative damage
by enhancing the antioxidant defence, thereby modulating inflammatory processes
and airway inflammation of heaves-affected horses. In fact, it was shown that the
antioxidant treatment (consisting of a mixture of natural antioxidants including
vitamin E and selenium) significantly improved exercise tolerance of horses and
significantly reduced endoscopic inflammatory score (Kirschvink et al., 2002).
Plasma uric acid concentrations were also significantly reduced, suggesting
downregulation of the xanthine-dehydrogenase and xanthine-oxydase pathway.
When considering antioxidant supplementation of horses it is necessary to take
into account the fact, that only optimal antioxidant supplementation is beneficial
in terms of health maintenance. Therefore antioxidant overdose would not give
any additional benefits. For example, in healthy horses on a diet containing adequate
levels of antioxidants, additional antioxidant supplementation (an antioxidant
mixture containing selenium for 4 weeks) had no apparent beneficial or detrimental
effect on pulmonary function during moderate intensity exercise (Deaton et al.,
2002). Therefore, the importance of antioxidant supplementation become
especially apparent if the diet is deficient in antioxidants or if exercise intensity is
high, prolonged, or if disease or additional stresses are present.
Se and maternal effect

It has been suggested that low intake of selenium damages the development of
horse embryo and foetus, but also the viability of the newborn foals causing
muscle disease (Meyer and Klug, 2001). Similar to data presented in previous
chapters in relation to maternal effect of Se in pigs and ruminants, in horses
antioxidant transfer via placenta is restricted and increased Se delivery via
colostrum and milk would have major nutritional and physiological importance.
In fact, parenteral supplementation trials with mares at late gestation indicate that
only limited amounts of selenium cross the placental barrier, when sodium selenite
was used (Maylin et al., 1980). Therefore parenteral supplementation of mares
during gestation and lactation as well as supplementation of foals beginning at
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birth are essential procedures to increase blood selenium levels in foals to optimal
levels. The serum selenium of foals from selenium-adequate mares is typically
much lower than their dams and ranges from 70 to 80 ng of selenium per ml of
serum (Stowe, 1967). According to Blackmore et al. (1982), serum selenium values
below 65 ng/ml are indicative of deficiency.
Did you know that antioxidants are poorly transferred via
placenta and by using organic Se in the maternal diet it
is possible to improve Se status of foals?
Se status of 123 mares and 87 foals during pregnancy, lactation and rearing was
investigated at four thoroughbred breeding farms in different regions in the
southwest of Germany (Schonthaler, 1998). Mean plasma Se concentration in the
different feedlots related to Se intake, however, within the herds with equal Se
intake, plasma Se concentration showed great individual differences. In particular,
it was shown that plasma Se concentration of unweaned foals up to 4 months of
age was significantly lower than that in the mares. The ratio between plasma Se
concentration of mare and foal was roughly 2:1. A decrease of plasma Se
concentration in mares that were in an advanced state of pregnancy (week 40-48)
was also shown. After weaning, plasma Se concentration increased significantly.
The Se concentration in plasma and whole blood correlated closely, the ratio of
the two being roughly 1.4:1 (Schonthaler, 1998). The relationship between blood
Se and GSH-Px activity was higher in mares than in foals at parturition. The Se
status of foals was also significantly correlated with that of the respective mares at
parturition under practical management conditions where wide variations in dietary
Se during gestation were present (Lee et al., 1995). Concentrations of selenium in
the blood plasma of 12 mares and their foals and in maternal milk were measured
over several days. At parturition, the selenium concentrations in plasma samples
from mares were almost twice as high as those in plasma samples from foals
(Hospes et al., 1996). Within a few days, the plasma selenium levels in mares
decreased and the plasma concentrations in foals increased steadily. In milk, the
selenium concentrations decreased to below the detection limit within 12 h. In
fact, selenium concentration of milk was less than 25% of that in colostrum within
one day after foaling and was considered a minor source of Se for the foals
regardless of Se status of the mares during gestation (Lee et al., 1995).
Fetal development, parturition and lactation strain maternal trace mineral
reserves critical; to postpartum health of both mare and foal. It has been shown
that organic selenium in the form of Sel-Plex in the gestation diet improves mare
post-partum selenium status as well as that of her foal. In an experiment mares
received 1 or 3 mg Se/day as selenite or 3 mg Se/day as Sel-Plex beginning 55
days pre-foaling to 56 days post-foaling. It was shown that (Janicki et al., 2001):
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prior to foaling mare serum Se did not differ

serum Se was greater in mares receiving Sel-Plex immediately post-foaling
and at 4 and 8 weeks despite increased milk Se output, indicating improved
ability to maintain mare reserves
higher milk and colostrum Se was reflected in continued higher foal serum Se
status: foal serum Se was consistently higher when mares were fed Sel-Plex.
colostrum Se was highest when mares received Sel-Plex (+45%)
milk Se was 29% higher when mares received Sel-Plex in comparison to the
same dose of selenite
placental expulsion in mares given Sel-Plex required half the time in comparison
to mares fed selenite
Se and immunity
Immunomodulating properties of Se are characterised in previous chapters and
horses are not exception from the rule. For example, fifteen Shetland ponies were
used in a 7-wk trial to study the effect of supplemental Se on humoral antibody
production. Ponies were depleted of Se before being assigned randomly to either
a low Se (0.02 ppm) or higher Se (0.22 ppm) diet (Knight and Tyznik. 1990). It
was shown that Se supplementation was associated with a significant increase in
GSH-Px activity and blood Se concentrations during the later weeks of the
experiment. An enhanced primary response was also observed in Se-supplemented
ponies as evidenced by increased hemagglutination titers. Furthermore,
significantly higher IgG concentrations were observed in the Se-supplemented
group. In another experiment, fifteen horses used for serum production were
maintained on low vitamin E and selenium diets.
Did you know that dietary Se supplementation can improve
immunocompetence of horses?
They were divided into four groups receiving no supplements, vitamin E, selenium
or a combination of vitamin E and selenium. The humoral immune response to
novel antigens, such as tetanus toxoid and equine influenza virus, was increased
in groups receiving either vitamin E or selenium/vitamin E (Baalsrud and Overnes,
1986). However, no effects were recorded on the titres against Escherichia coli or
the levels of immunoglobulin G.
Conclusions
From the data presented above it is clear that antioxidant-prooxidant balance in
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horses plays an important role in their health maintenance. A specific emphasis

should be given to optimal levels of Se in horse diets. This will give an opportunity
to increase horse adaptability to high intensity exercises. In fact, in long term
optimal Se and vitamin E supplementation could improve performance of racing
horses as well as helping keep them healthy during exercising and at periods of
their recovery and rest. Similar to other animal species organic Se supplementation
of the horse diets is considered to be an effective means to maintain their optimal
Se status.
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SELENIUM FOR COMPANION ANIMALS

Every dog has its day
Introduction
Domestic cats and dogs both belong to the order Carniivora and have some
specific features of general metabolism and antioxidant defences. In particular,
cats cannot convert beta-carotene into vitamin A and require vitamin A to be
supplied with the diet; they have high protein requirement but poorly metabolised
carbohydrates and for cats taurine and arginine are essential amino acids. Dogs
cannot synthesise vitamin D and are characterised by low synthesis of arginine.
Furthermore, cats require preformed long chain polyunsaturated fatty acids such
as arachidonic acid and docosahexaenoic acid to be delivered with the diet. Since
life expectancy of cats and dogs has increased substantially for the last decades,
a special emphasis should be given to health maintenance of elderly animals
similar to human. In fact, in many cases cats and dogs suffer the same metabolic
disorders and diseases as human does.
Did you know that in many cases cats and dogs suffer the same
metabolic disorders and diseases as human does?
Antioxidants and animal health

Dietary antioxidants play a crucial role in the health of companion animals being
a major protection for animals exposed to elevated levels of toxins and free radicals.
The results of recent in vivo assessments, clinical trials, and research studies showed
that oral supplementation with vitamin E, selenium, glutathione, and taurine were
beneficial for both maintaining natural antioxidant-prooxidant balance in the body
and protecting against a number of degenerative diseases associated with free
radical damage and toxin exposure (Scanlan, 2001). Therefore optimal levels of
certain dietary nutrients have been shown to increase life span, improve life quality,
reduce symptoms and physical evidence of disease, and decrease mortality rates
in these animals. There are various stress-related disorders affecting companion
animals, including feline idiopathic cystitis, irritable bowel disease, gastric
dilatation-volvulus syndrome, eating disorders, fears, anxiety and phobias and
separation anxiety syndrome (Aldrich et al., 2005). Indeed, natural antioxidants
are important nutritional modulators decreasing detrimental effects of stresses.
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Food quality
Petfood quality control is very strict and ensure only high quality ingredients to
be included into the diet. However, since environmental pollutants such as heavy
metals or mycotoxins are very widely distributed it is almost impossible to
completely avoid their presence in food ingredients. For example, an analytical
survey of 42 elements in 31 commercial canned pet foods was conducted (Furr et
al., 1976). Arsenic, bromine, cadmium, chromium and mercury were highest in
fish-containing cat foods. Lead was most consistently high in those products
containing chicken. Barium, nickel and tin also appeared high in certain of the
samples. An analytical survey of mutagens, nitrosamines, polychlorinated
biphenyls, toxic elements as well as the toxicologically protective constituents
zinc, selenium, and vitamin C, in 48 pet foods was conducted (Mumma et al.,
1986). High concentrations of fluoride and iodide in some samples and increased
concentrations of mercury and selenium in certain cat foods containing fish were
detected. Polychlorinated biphenyls were only detected in one cat food. The
occurrence of ochratoxin A (OTA) in canned (26 samples) as well as dry pet
foods (17 samples) for cats and dogs was investigated (Razzazi et al., 2001). OTA
was detected in 47% of the pet food samples at the levels of 0.1-0.8 ng/g food.
Only two pet food samples contained higher (3.2 and 13.1 ng/g food) OTA levels.
OTA was also detected in 62% cat kidneys (0.35-1.5 ng/g tissue). When one
hundred samples of pet foods including 35 samples of cats food were analysed,
low levels of aflatoxin B1, OA and fumonisins were detected in some of them
(Scudamore et al., 1997).
Did you know that petfood can be contaminated by variety of
pro-oxidants and therefore Se dietary supplementation is an
important factor in preventing oxidative stress
in companion animals?
Aflatoxin B1 in cat food was higher in comparison to the dog food (Sharma and
Marquez, 2001). Recently mycoflora of 21 dry pet foods (12 belonging to dogs
and 9 to cats) that corresponded to 8 commercial brands made in Argentina and
imported has been analysed (Bueno et al., 2001). Ten genera and fungi classified
as Mycelia sterilia were identified. The predominant genera were Aspergillus
(62%), Rhizopus (48%), and Mucor (38%). The most prevalent among Aspergillus
was Aspergillus flavus followed by Aspergillus niger and Aspergillus terreus which
are potential sources of mycotoxins. There are also other contaminants in cats
food. For example, the concentration of bisphenol A ranged from 13 to 136 ng/g
in canned cat food (Kang and Kondo, 2002).
Lipid peroxides are the major petfood nutritionist concern. Since vitamin E in
the food is usually used in esterified form it does not possess antioxidant activity
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per se and only become active after de-esterification in the intestine. Therefore
various synthetic antioxidants are a choice for prevention of lipid peroxidation
during petfood storage. The effects of three different preservative treatments
(ethoxyquin and butylated hydroxyanisole, EX/BHA; mixed tocopherols, TC/TC
and ascorbyl palmitate and mixed tocopherols ATL/TC) applied to extruded dog
food were studied (Gross et al., 1994). After processing the dog foods were
placed in bags and stored for 16 wk at 48.8 degrees C or for 12 months at 22.2
degrees C. The best results were observed with EX/BHA. It is interesting that in
both the high and ambient temperature tests the dogs consumed more of the foods
with the lowest peroxide value when given a two-bowl choice. It is interesting
that dietary oxidised lipids compromised antioxidant defences and altered some
neutrophil and monocyte functions in dogs (Turek et al. , 2003).
Anorexia in companion animals and its connection to antioxidants

One of the most frequent motivations for seeking veterinary attention for a cats
and dogs is when the owner recognises a loss of normal appetite (anorexia) in
his/her pet (Michel, 2001). The mechanisms underlying decreased food intake
are complex and not completely understood. It is necessary to take into account
that the regulation of appetite involves interaction of external stimuli with signals
from the gastrointestinal tract and central nervous system (Michel, 2001). It is
likely that lipid peroxidation in the intestine could be a triggering factor for anorexia
development. If animals have a choice they can choose a food with lower level of
peroxides (Gross et al., 1994). The deficient concentration of erythrocyte
tocopherol together with the altered antioxidant enzyme activities suggest a certain
degree of oxidative damage in anorexia nervosa owing to both factors deficient
micronutrient intake and oxidative stress (Moyano et al., 1999). It seems likely
that cytokines play a specific role in anorexia development. In fact the proinflammatory cytokines and oxidant molecules produced during the inflammatory
response, may be beneficial, or detrimental to the patient, depending on the amounts
and contexts in which they are produced (Grimble, 1998). In particular, aberrant
or excessive production of those molecules has been implicated in inflammatory
disease, and sepsis. Complex systems exist for the control of cytokine production
and oxidant actions including the hormones of the hypothalamo-pituitary-adrenal
axis, acute phase proteins, and endogenous inhibitors of interleukin-1 and tumor
necrosis factor as well as endogenously synthesized antioxidants, such as
glutathione and dietary antioxidants, such as tocopherols, ascorbate and cachectins
(Grimble, 1998). At the same time, nutrients change cytokine production and
potency by influencing tissue concentrations of many of the molecules involved
in cytokine biology. It is well recognised, for example, that low antioxidant intake
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results in enhanced cytokine production and effects. The anorexia that in many cases
follows infection and injury, may be purposeful to permit release of substrate from
endogenous sources to support and control the inflammatory process (Grimble, 1998).
Therefore, prior as well as concurrent antioxidant intake are of great importance in
determining the outcome of the inflammatory response. It is interesting that anorexia
was one of the major clinical signs of toxicosis in aflatoxin B1-treated calves (Brucato
et al., 1986) and Se was demonstrated to improve feed intake in mycotoxin-treated
animals. On the other hand, vitamin E-selenium deficiency in wild ducks was also
associated with the development of anorexia and diarrhea (Dhillon and Winterfield,
1983). Similarly, silver toxicity in weanling swine was associated with lesions typical
of selenium-vitamin E deficiency including anorexia and diarrhea (Van Vleet et al.,
1976). It seems likely that Se deficiency can cause decrease in food consumption
and administration of SeMet increased voluntary feed consumption of seleniumdeficient chickens within 2-3 hours (Bunk and Combs, 1980). Therefore dietary
antioxidants could be supplements of the choice when dealing with anorexia in cats.
Large intestinal disease, especially colitis, is also a common problem in cats and no
single etiologic agent is responsible for this (Simpson et al., 1998). Furthermore,
renal disease, arthritis and compromised immunity are of great importance for cats as
well (Swanson et al., 2003). It seems likely that natural antioxidants, such as selenium,
could be of great value in prevention and treatment of the diseases.
Antioxidant deficiencies in companion animals

Signs of antioxidant deficiency in companion animals are usually very similar to
those described for laboratory and farm animals. For example, a case of nutritional
myopathy in a cat primarily caused by vitamin E deficiency was described (Dennis
and Alexander, 1982). The animal which had been fed a diet consisting almost entirely
of boiled Norwegian coley, was shown to have swollen muscles in both the hind and
fore legs. Analyses of biopsy material revealed chronic, severe myositis, with normal
muscle tissue undergoing a series of degenerative changes. It is interesting that
multivitamin and mineral supplementation, led to a complete clinical recovery, the
cat regaining full use of its legs within 14 days.
Experimentally induced vitamin E-selenium deficiency in the growing dog was
also characterised (Van Vleet, 1975). Beagle pups (5-8 week old) were fed a
semisynthetic basal diet (BD) deficient in vitamin E and selenium (Se) for 55 to 70
days. Clinical signs of vitamin E-Se deficiency developed after 40 to 60 days and
were characterised by:
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subcutaneous edema
anorexia
depression
dyspnea
eventual coma
Necropsy examination showed following gross lesions:
ventral subcutaneous edema

generalized skeletal muscular pallor and edema with scattered white
longitudinal streaking
prominent brownish yellow discoloration of the intestinal musculature
a layer of white chalky material at the renal corticomedullary junction
Microscopic examination revealed:

1.
2.
3.
4.
extensive skeletal muscular degeneration and regeneration

focal subendocardial necrosis in the ventricular myocardium
intestinal lipofuscinosis
renal mineralization
Did you know that clinical signs of vitamin E-Se deficiency
developed after 40 to 60 days and were characterised by:
muscular weakness, subcutaneous edema, anorexia,
depression, dyspnea and eventual coma?
Organic selenium vs sodium selenite

As mentioned above, animal can synthesise antioxidant enzymes but dietary
availability of Se, Mn, Zn and Cu are among major restrictions for this synthesis.
For example, recently it has been shown that availability and efficiency of selenium
in animal nutrition greatly depends on the dietary sources of this element. It seems
likely, that in cats and dogs, similar to other animals, organic selenium is more
efficiently assimilated from the diet. Indeed, the absorption of amino-organic
selenium is accelerated by the specific amino acid active transport mechanisms in
the gut mucosa. Sodium selenite is absorbed more slowly, possibly by simple
diffusion through the intestinal mucosa, than the amino acid-bound selenium
compounds. Indeed, the perfusion method was used to measure the rate of Se
absorption from the jejenum of healthy dogs (Reasbeck et al., 1985). It was shown
that Se absorption from the test segment (expressed as percent administered dose
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per centimetre) was 1.97 from D,L-selenomethionine, 1.15 from D,L-selenocystine,

and 0.51 from sodium selenite. It is interesting that Se concentration in serum of
cats and dogs was substantially higher than that in human or farm animals (Table
10.3). Hematologic samples (heparinized whole blood, plasma, and RBC fractions)
were obtained from 43 healthy euthyroid cats and 7 hyperthyroid cats. Plasma
concentration of Se and GSH-Px activity in whole blood or plasma did not differ
significantly among cats from the 4 regions. However, cats had a plasma
concentration of Se that was approximately 5 times the concentration reported in
rats and humans (Foster et al., 2001). The GSH-Px activity in whole blood or
plasma in cats generally was higher than values reported in rats or humans. In a
recent study with cats Se levels in the four diets ranged from 0.95 to 1.03 mg/kg
(Hendriks et al., 2002). The total selenium concentrations in petfoods (n=89)
commercially available in New Zealand was determined (Simcock et al., 2005).
Mean total selenium concentrations were similar (0.61-0.80 mg/kg DM) in petfoods
produced in Australia, New Zealand and the USA, but higher (mean 3.77 mg/kg
DM; p<0.05) in petfoods produced in Thailand. Seafood-based flavours had the
highest selenium concentrations in both cat and dog foods however, it is necessary
to underline that Se availability from various pet foods is quite low (Wendekind et
al., 1997; 1998). It seems likely that similar to other species Se metabolism in cats
and dogs is controlled by urinary excretion (Todd and Hendrix, 2005).
Table 10.3 Reference values of serum selenium concentration in Switzerland (Adapted from Forrer et al.,
1991).
Species
Cat
Dog
Pig
Chicken
Humans, 20-60 years
Humans, 60-100 years
Horse
Goat
Calves, 3-9 month old
Cattle, >9 months old
Sheep
Range, mmol/l
Range, gl/l
3.60-10.09
1.90-4.31
1.97-3.32
1.68-4.28
0.78-1.48
0.61-1.73
0.36-1.68
0.14-1.42
0.19-0.65
0.10-0.82
0.09-0.54
285.7- 800.8
250.8- 342.1
156.4- 263.5
133.3- 339.7
61.9- 117.5
48.4- 137.3
28.6 133.3
11.1-112.7
0.19-0.65
0.10-0.82
0.09-0.54
Did you know that cats are characterised by a plasma

concentration of Se that is approximately 5 times the
concentration reported in rats and humans?
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On the other hand, kitten given the low-Se diet had significantly reduced plasma
Se concentration and GSH-Px activity and compromised thyroid hormone
metabolism. In fact plasma total T4 increased and T3 decreased significantly (Yu
et al., 2002). This could affect kitten thermoregulation, growth and development
and ultimately lead to increased mortality.
Kitten requirement in Se has been recently estimated to be 0.15 mg Se/kg diet
(Table 10.4; Wedeking et al., 2003). As pet foods for cats contain a high proportion
of animal protein with a Se bioavailability of 30%, it is recommended that
commercial diets for cats contain 0.5 mg Se/kg DM. Recently Se requirement of
puppies was re-evaluated. Thirty beagle puppies (average = 8.8 weeks old) were
used in a randomized complete block design. Puppies were fed a low Se (0.04
mg Se/kg diet) torula yeast-based diet for 14 days (pre-test period) after which
this same diet was supplemented with five levels of sodium selenite for 21 days
(experimental period) to construct a response curve (0, 0.13, 0.26, 0.39 or 0.52
mg Se/kg diet; Wedekind et al., 2004). Based on the result of the study the
authors suggested Se requirement to be 0.21 mg Se/kg diet.
Table 10.4 Response of kittens fed graded levels of sodium selenite (Adapted from Wedekind et al.,
2003)
Treatment Dietary
Se,mg/kg
1
2
3
4
5
6
GSH-Px,
RBC, GSH-Px,
Plasma, U/ml U/106 cells
0.027
0.073
0.100
0.122
0.210
0.314
0.31
1.03
1.38
1.72
2.32
2.60
1.90
3.85
5.23
5.92
5.65
5.81
Plasma Se,
mM/l
T3,
nmol/l
0.22
0.99
1.56
2.06
4.12
4.61
0.73
0.86
0.92
0.91
0.91
1.27
Did you know that kitten requirement in Se has been recently

estimated to be 0.15 mg Se/kg diet and puppy Se requirement is
0.21 mg Se/kg diet?
It is interesting to note that, concentration T3 increased linearly with increasing
Se intake, whereas T4 was unchanged as a result the ratio of T4:T3 decreased
linearly in response to supplemental Se. In general, Se metabolism in cats has
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some specific features and when dogs and cats fed on the same diet were compared,
Se concentration in cats serum was 50-70% higher than that in dogs (Wedeking
et al., 2003; Todd and Hendrix, 2005). The authors indicated comparatively high
tolerance of cats and dogs to dietary selenium.
Taurine for cats: antioxidant properties and beyond

Taurine, a sulphur containing amino acid (2-aminoethane sulfonic acid) is
synthesised from methionine and cysteine in the presence of vitamin B6 and
found in almost all tissues in mammals. It is the most abundant intracellular amino
acid in humans which is not incorporated into proteins. However, cats have limited
ability to synthesise taurine and unable to conserve it in bile salt production and,
therefore, this amino acid becomes essential for feline. For example, feline central
retinal degeneration was shown to be a result of taurine deficiency (Hayes et al.,
1975). Compromised reproduction in cats is another consequence of taurine
deficiency (Sturman et al., 1985; 1986). Furthermore, taurine concentration in
cats decreased in various clinical conditions. For example, in cats with dilated
cardiomyopathy, mean plasma taurine concentration was the lowest of that in
cats of any group, being only 38% of the value in healthy cats (Fox et al., 1993).
In healthy individuals the diet, especially seafood and meat, is the usual source of
taurine, however, taurine is absent from plant food. It is not toxic and implicated
in numerous biological and physiological functions (Bidri and Choay, 2003;
Schuller-Levis and Park, 2003; Lourenco and Camilo, 2002; Degim et al., 2002):
antioxidant effects
bile acid conjugation and cholestasis
xenobiotic detoxification
modulation of intracellular calcium levels
participation in retinal development and function
endocrine/metabolic effects
osmoregulation, neuromodulation and stabilization of the membranes
modulation of cell proliferation, inflammation and collagenogenesis
reproduction: preservation of the motility of the spermatozoa, support of
their capacitation, improvement of the chances of success of fertilization
and the early embryonic development
immunomodulation
Early work on antioxidant properties of taurine were somewhat disappointing.

For example, taurine did not react rapidly with O2*-, H2O2 or *OH suggesting that
taurine does not function as an antioxidant in vivo (Aruoma et al., 1988). In
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another experiment, taurine suppressed erythrocyte hemolysis but did not affect
peroxidation (Nakamura et al., 1993). Taurine was also shown to be a weak
scavenger of peroxynitrite and did not attenuate sodium nitroprusside toxicity to
cells in culture (Mehta and Dawson, 2001).
Did you know that taurine is an essential amino acids for cats?
However, later antioxidant properties of taurine were shown in various model
systems in vivo and in vitro. For example, in Chinese hamster ovary cells cultivated
in vitro, taurine scavenged oxygen species (Cozzi et al., 1995). In non-diabetic
rats high glucose caused a significant increase in levels of MDA and 4hydroxynonenal, which were reduced by taurine (Haber et al., 2003). Wistar male
rats were fed on taurine in addition to high fat diet (11% coconut oil w/w) for 6
months. Taurine significantly reduced aortic cholesterol and TBARS and increased
GSH concentration (Sethupathy et al., 2002). Similarly, taurine supplementation
of rats restored kidney GSH content and GSH-Px activity and reduced MDA
production levels in the kidney tissue following cisplatin treatment (Saad and AlRikabi, 2002). When fuctose-fed rats received taurine in drinking water,
peroxidative damage was minimal in both plasma and liver (Nandhini et al.,
2002). Taurine also decreased TBARS (by 26%) in apoE-deficient mice (Kondo
et al., 2000).
In general, taurine can show antioxidant-related protective effects similar to
other antioxidants. For example, it was shown that oxygen persufflation
significantly enhanced the viability of livers during cold preservation only in
combination with SOD or taurine, which were both equally effective in reducing
lipid peroxidation, enzyme release, and vascular resistance upon post-ischemic
reperfusion (Lauschke et al., 2003). The isolated guinea pig lungs previously
being perfused by oxygenated Krebs-Henseleit solution were put in normothermic
ischemic conditions. Decreased pulmonary artery pressure, tissue perfusate MDA
levels and increased perfusate GSH levels were observed in taurine added group
(Oz et al., 2002). It was shown that 3 intraperitoneal injections of 200 mg/kg of
taurine prevented hypoxia-induced lactate accumulation and lipid peroxidation
(LP) in brain, liver, and heart tissues (Mankovskaya et al., 2000). The authors
suggested that the effect of taurine on LP could be due to its membrane stabilising
activity. Exposure of isolated frog rod outer segments to light (5000 lux) induces
membrane disorganization and swelling. An increase of about 50% in lipid
peroxidation accompanied the light-induced damage. Taurine and hypotaurine
(25 mM) prevented the increase in lipid peroxidation, and provided an entire
protection of rod outer segment structure (Pasantes-Morales and Cruz, 1985).
Antioxidant effect of taurine is not restricted to PUFAs. For example, Ogasawara
et al. (1993; 1994) showed an inhibiting effect of taurine against the modification
of protein, as well as an antioxidative effect through the reactions of taurine with
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aldehydes in vivo. Taurine ameliorated oxidative stress and cholesterol

accumulation in the aorta of rabbits fed on the high-cholesterol diet (Balkan et
al., 2002) and the results suggested that the anti-oxidative effects of taurine are
related to its anti-atherosclerotic actions (Kondo et al., 2001). In streptozotocindiabetic rats, dietary taurine supplementation ameliorates MDA levels, GSSG/
GSH, and NAD+/NADH (Obrosova and Stevens, 1999). Taurine reduced
significantly a decrease of glutathione antioxidant system activity protecting tissues
against GSH pool depletion and preventing a decrease of glutathione reductase
and glutathione peroxidase activities in acute severe hypoxia (Mankovska et
al., 1998).
It seems likely that in many cases in biological systems taurine could interact
with other antioxidants being an important part of an integrated antioxidant system.
For example, taurine counteracts oxidative stress and the nerve growth factor
deficit in early experimental diabetic neuropathy in rats. Antioxidant effects of
taurine in peripheral nerve were suggested to be mediated through the ascorbate
system of antioxidative defence (Obrosova et al., 2001). Taurine and GSH, were
investigated individually and in combination with retinol in a model system based
on phospholipid liposomes. In that system GSH protected PUFAs, but taurine
alone showed little antioxidant activity. However, in combination with retinol it
protected lipids twice as much as retinol alone (Keys and Zimmerman, 1999). In
another study, presence of taurine and zinc protected cells from retinol-induced
injury. For example, taurine (20 mM) and zinc (100 M) added simultaneously
abolished cell swelling and increased cell viability from 7 to 55%. Tocopherol
(200 M) was also effective in protecting these cells from retinol. The three
compounds together afforded complete protection. The effects of retinol and of
taurine, zinc or tocopherol seem to be unrelated to lipid peroxidation and a
membrane stabiliser action is proposed as the mechanism underlying the protective
effect of taurine and zinc or of tocopherol (Pasantes-Morales et al., 1984).
Similarly, effects of taurine on lipid peroxidation was considered to be due to its
membrane stabilising activity (Mankovskaya et al., 2000). In fact, taurine can
maintain membrane structure in stress conditions. For example, taurine (10 mM),
markedly attenuated the cell volume-increasing effect of ammonia (Zielinska et
al., 2003). Perchloroethylene induced membrane damage may be associated with
hemolysis, which can be effectively prevented by taurine (Ebrahim et al., 2001).
Ultrastructural observation revealed a highly protective effect on tissue damages
due to hyperoxia in taurine-treated rats (Pitari et al., 2000).
Antioxidant and membrane-stabilising effects of taurine were most pronounced
in conditions of oxidative stress caused by various toxic agents. For example, rats
were given water containing 20% ethanol (v/v) as drinking water for 3 months.
Taurine treatment starting after ethanol cessation caused a significant decreases
in serum transaminase activities and hepatic total lipid, triglyceride, MDA, and
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diene conjugate levels and significant increases in hepatic GSH glutathione, vitamin
E, and vitamin C levels (Balkan et al., 2002). Taurine seems to be capable of
fortifying cells against lead-induced oxidative attack without decreasing lead levels
(Gurer et al., 2001). Similarly, taurine treatment of rats, together with thioacetamide
administration, diminished the severity of the liver injury by decreasing oxidative
stress (Dogru-Abbasoglu et al., 2001). Taurine treatment of rats after 3nitropropionic acid administration protects the striatum from damage along with
increased survival rates (Rivas-Arancibia et al., 2001). Similarly, taurine treatment
of rats reduced gentamicin-induced increases in serum creatinine, tissue lactate
and TBARS levels (Erdem et al., 2000). The protective effect of taurine against
indomethacin-induced gastric mucosal injury was also due to inhibition of lipid
peroxidation and neutrophil activation (Son et al., 1996). It is interesting that
taurine enhanced the hepatic drug-metabolizing systems, leading to the stimulation
of the ascorbic acid metabolism in rats fed diets containing polychlorinated
biphenyls (Mochizuki et al., 2000). Taurine showed a protective role to reduce
cellular damage associated with both NO and metal-stimulated catecholamine
oxidation (Biasetti and Dawson, 2002) as well as ameliorating chronic streptozocininduced diabetic nephropathy in rats (Trachtman et al., 1995). The beneficial
effect of taurine was related to reduced renal oxidant injury with decreased lipid
peroxidation and less accumulation of advanced glycosylation end products within
the kidney. Taurine prevented galactose-induced cataracts in New Zealand white
rabbits (Malone et al., 1993) and its inverse relationship with lens MDA suggests
this is an antioxidant effect.
Did you know that taurine is an important part
of antioxidant defences?
Protective effects of taurine could be related to other functions. For example, the
protective effects of taurine supplementation against renal damage induced by
salt loading in Dahl salt-sensitive rats was suggested to be attributed to the
suppression of LOX-1, probably through its antioxidant effects (Chiba et al., 2002).
Similarly, it was concluded (el Tahir et al., 1987) that endogenous taurine may
participate in regulation of prostaglandin synthesis and that prostanoids may
contribute to its known actions. It was also suggested that taurine promotes the
bioavailability of the lipid soluble vitamins A, D, E, K, and F, probably by forming
different types of water soluble, easily hydrolyzable complexes (Petrosian and
Haroutounian, 2000).
During pregnancy, taurine accumulates in the maternal tissues and later released
to the fetus via the placenta and to the newborn via the maternal milk and taurine
deficiency in the mother leads to growth retardation of the offspring, and to
impaired perinatal development of the central nervous system and of the endocrine
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pancreas (Aerts et al., 2002). The adult offspring of taurine-deficient mothers

display signs of impaired neurological function, impaired glucose tolerance and
vascular dysfunction and developed gestational diabetes and transmit the effects
to the next generation (Aerts et al., 2002). In order to maintain optimal taurine
status of cats fed on a commercial canned diet it is recommended to make sure
the diet contains at least 2,000-2,500 mg taurine/kg DM (Earle and Smith,
1991;1994). Significant amounts of taurine and hypotaurine were found in
spermatozoa, seminal plasma and epididymal flushing fluid indicating that they
may be synthesised in cat testes or epididymides (Buff et al., 2001).
Molecular mechanisms of regulation of taurine synthesis in tissues are not well
understood but it seems likely that redox status of the cell could be involved in
regulation of this process. For example, an increase in taurine transporter gene
expression in human cells cultured was observed under conditions of elevated
levels of NO (Bridges et al., 2001). Similarly, taurine attenuates hepatocyte
apoptosis and necrosis through inhibition of both NO and reactive oxygen
intermediate. While taurine acts directly as an antioxidant, its effects on NO may
occur at the messenger RNA level (Redmond et al., 1996).
Antioxidants and immune system

One of the most important applications of antioxidants in companion animal
nutrition is modulation of immune system. Among many different nutrients
involved in immune system regulation natural antioxidants have a special place
being effective immunomodulating agents (for review see Surai, 2002). It is proven
that such antioxidant compounds as vitamin E, selenium and carotenoids are
involved in immunomodulation. However, the levels of the dietary antioxidants
showing those effects are usually several times higher than that necessary for
animal growth and development. Furthermore, other trace minerals, including
Zn, Cu and Iron are also involved in immune system regulation as parts of
antioxidant enzymes as well as many enzymatic systems. Unfortunately, available
data on effect of antioxidants on companion animal immunity are restricted to
several recent publications.
For example, the immuno-modulatory action of lutein was shown in domestic
cats (Kim et al., 2000a). Female Tabby cats (10-month old) were supplemented
daily for 12 weeks with 0, 1, 5 or 10 mg lutein. The DTH response to vaccine
increased in a dose-dependent manner on Week 6. Compared to control, cats fed
lutein also showed enhanced concanavalin A and pokeweed mitogen-stimulated
peripheral blood mononuclear cells proliferation. Dietary lutein also increased
the percentages of CD4+ and CD21+ lymphocytes on Week 12. Plasma IgG was
higher in cats fed 10 mg lutein on Weeks 8 and 12 (Kim et al., 2000a). Similarly,
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dietary -carotene increased cell-mediated and humoral immune responses in

Female Beagle dogs (Chew et al., 2000). In particular, compared with
unsupplemented dogs, those fed 20 or 50 mg of -carotene had higher CD4+ cell
numbers, CD4:CD8 ratio, plasma IgG concentration. Furthermore, the delayedtype hypersensitivity response to phytohemagglutinin (PHA) and vaccine was
heightened in -carotene-supplemented dogs. On the other hand, immune response
was impaired in dogs classified as low -carotene absorbers. Dietary lutein
stimulated immune responses in the domestic canine in a similar fashion (Kim et
al., 2000). Female Beagle dogs (17-18-month old) were supplemented daily with
0, 5, 10 or 20 mg lutein for 12 weeks. Delayed-type hypersensitivity (DTH)
response to saline, phytohemagglutinin (PHA) and a polyvalent vaccine was
assessed on Weeks 0, 6 and 12. The results of the study showed that luteinsupplemented dogs had stimulated DTH response to PHA and vaccine by week
6. Furthermore dietary lutein also increased lymphocyte proliferative response to
mitogens and increased the percentages of cells expressing CD5, CD4, CD8 and
major histocompatibility complex class II molecules. The production of IgG also
increased in lutein-fed dogs after the second antigenic challenge. (Kim et al.,
2000).
The subcellular location of taurine, and its precursor, hypotaurine, within human
neutrophils has been examined and it was shown that they both resided within the
cytosolic compartment of the cell. The ratio of taurine to hypotaurine is
approximately 50:1 (Green et al., 1991). In fact, taurine is considered to be the
most abundant free amino acid in leucocytes reaching up to 50mM concentration
(Schuller-Levis and Park, 2003) and comprising greater than 50% of the free
amino acid pool of lymphocytes (Redmond et al., 1998). The antioxidant function
of taurine in macrophages is of great importance (Pasentes-Morales and Cruz,
1985). Hypochlorous acid (HOCl), the major strong oxidant produced by the
phagocyte enzyme myeloperoxidase, reacts readily with free amino groups to
form N-chloramines. In biological fluids N-chloramines react more readily with
protein thiols than with methionine residues or ascorbate, and thus may cause
biologically relevant, selective loss of thiol groups (Carr et al., 2001). Taurine is
an efficient scavenger of HOCl and can prevent neuronal damage caused by
HOCl (Kearns and Dawson, 2000). These data suggest an important role of oxygendependent mechanisms in the regulation of eosinophil survival and apoptosis, in
fact, eosinophil apoptosis may be related to the ability of the cell to maintain an
appropriate oxidant-antioxidant balance (Wedi et al., 1999). Taurine depletion is
potentially deleterious to alveolar macrophages and pulmonary tissue (Zhang and
Lombardini, 1998). Administration of taurine reduce the inflammatory parameters
in the inflammatory bowel disease rat model by increasing defending capacity
against oxidative damage (Son et al., 1998; 1998a) and it protects the guinea pig
heart from neutrophil-induced reperfusion injury and oxidative stress (Raschke
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et al., 1995). Because respiratory burst activity of PMN was also significantly
reduced in the presence of taurine, the beneficial effect appears to be mediated by
antioxidative properties of taurine. In rats the host inflammatory response induced
by the xenotransplantation of neurons derived from the human teratocarcinoma
cell line was studied and taurine was able to facilitate graft survival and attenuate
the immune response generated by the xenograft (Rivas-Arancibia et al., 2000).
Taurine ameliorates endotoxin-induced leukocyte-endothelial cell interactions
associated with sepsis, thereby suggesting that taurine may have a therapeutic
role in the prevention of endothelial damage in sepsis (Egan et al., 2001).
Taurine at concentrations normally found in cells can inhibit oxidative damage
to DNA (Messina and Dawson, 2000) and anti-apoptotic action of taurine is the
most important part of its immunomodulating properties. For example, neutrophils
were stimulated with Fas monoclonal antibody in the presence or absence of
taurine. Fas receptor ligation resulted in significant neutrophil apoptosis at 18 h.
Apoptosis was inhibited and intracellular calcium levels were maintained in the
presence of taurine (Condron et al., 2003). Taurine attenuates hyperglycemiainduced apoptosis (by 78%) in human tubular cells via an inhibition of oxidative
stress (Verzola et al., 2002) and decreased hyperglycemia-induced human
umbilical vein endothelial cell apoptosis through ROS inhibition and [Ca 2+)]
stabilization (Wu et al., 1999). Taurine was significantly protective against arseniteinduced apoptosis of human polymorphonuclear leukocytes (Watson et al., 1996).
Age-related decline in cats immune system (Harper et al., 2001) are of great
importance and mechanisms of a decrease in total plasma antioxidant capacity of
older cats (Harper and Frith, 1999) await further clarification. It is necessary to
mention that after experimental infection with feline immunodeficiency virus,
aged cats developed more severe disease than young adult cats (George et al.,
1993). It is interesting that vitamin E alone in doses up to 4.3 IU/g was not able to
improve immune status of cats (Hendriks et al., 2002). However, a mixture of
antioxidants including lycopene, -carotene, lutein, vitamin E, taurine and
ascorbate were shown to improve antibody response to vaccination in cats (Harper
et al., 2001). Dogs consuming Se-vitamin E supplement had a significantly greater
serum Se and alpha-tocopherol than un-supplemented animals (Kandil et al.,
2005).
Did you know that Se and vitamin E supplementation
can improve immune response in dogs?
The supplement improved immune response against T. hydatigena as evidenced
by increase production of antibody titer and IgG concentration in comparison
with either vaccinated un-supplemented or control unvaccinated animals.
Moreover, the highest protection level against T. hydatigena infection was observed
in the same Se-vitamin E supplemented dogs. It seems likely that antioxidant
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compounds, including organic selenium, vitamin E, carotenoids and taurine in a

combination with other immunomodulators such as omega-3 fatty acids, could
be beneficial for the improvement of immune system of cats.
Conclusions
From results presented above it is clear that in many cases effects of antioxidants
on companion animals are very similar to those described for humans, since they
share common environments, stresses and in some cases diseases. Interactions
between antioxidants and prooxidants starts at the level of stomach and small
intestine as described for humans (Surai, 2002; Surai et al, 2003; 2004). Therefore
dietary supplementation with various antioxidants is an effective strategy to prevent
damaging effects of free radicals and toxic products of their metabolism on the
companion animals. In particular a specific attention should be paid to the form
of antioxidants used. For example, organic selenium (Sel-Plex) can improve
antioxidant system of the digesta and selenite in the same conditions could promote
peroxidation. It should also be taken into account that with the same amount of
organic selenium in the diet more selenium would be accumulating in tissues in
comparison to selenite building selenium reserves which could be useful in stress
conditions. Organic selenium is also effectively transferred to the colostrum and
milk providing important means of antioxidant system maintenance of the newly
born kitten. Furthermore, inorganic copper and iron are the major stimulators of
lipid peroxidation in digestive tract and use of organic forms of those elements
could avoid their possible detrimental actions. Use of various plant-derived
antioxidants, such as flavonoids, could also help to prevent oxidative damage in
the gastro intestinal tract (GIT) due to their antioxidant properties and chelating
ability in relation to iron and copper.
Of particular interest for companion animal nutritionist are data showing
protective effects of natural antioxidants in animal and human exercise as well as
their immunomodulating properties. It is well known that vitamin E and carotenoids
are not toxic for animals even at very high level of supplementation. However,
more research is needed to establish optimal antioxidant supplementation of the
companion animals. For example, significant systemic toxicity has been observed
in kittens treated daily with 50-1,000 mg/kg/day parenteral vitamin E for 3 weeks
(Phelps, 1981). The toxicity was dose-related and resulted in significant mortality
at doses over 100-200 mg/kg daily. Functional food business is quickly developing
all over the world. Indeed functional food enriched with various antioxidants in
particular selenium, vitamin E, carotenoids and flavonoids for cats already found
their way to the supermarket shelves (Swanson et al., 2003).
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SELENIUM IN FISH NUTRITION

Do not teach fish to swim
Introduction
Aquaculture accounted for 29% of global fisheries production in 2000 and of the
38 million tonnes from aquaculture around 5-10% was farmed intensively using
nutritionally complete feeds as the sole source of nutrition (Carter, 2003). Intensive
farming represents the majority of aquaculture in developed countries.
Considerable advances have been made in understanding the nutrient requirements
of intensively farmed finfish and intensive production offers opportunity for
increasing growth efficiency and controlling product quality through optimal
nutrient supply. Fish nutrition is associated with high levels of fat and in particular
polyunsaturated fatty acids in the diet. Therefore, an oxidative stress and
antioxidant protection are among major issues for fish nutritionist. Taking into
account recent data on fish nutrition it should be mentioned that Se is of great
importance for maintenance of fish health, in particular fish immunity, as well as
for fish growth, development and flesh quality.
Did you know that aquaculture accounted for 29% of global
fisheries production in 2000 and of the 38 million tonnes from
aquaculture around 5-10% was farmed intensively using
nutritionally complete feeds as the sole source of nutrition?
Se deficiency
Se deficiency in fish is associated with oxidative stress and causes similar clinical
signs as in various birds and mammals. In particular increased mortality and
compromised immunity are indicative for this deficiency. For example,
unacceptably high mortalities in rainbow trout fry (Oncorhynchus mykiss) six to
10 weeks after they started to feed were recorded in two spring water trout
hatcheries in Northern Ireland in May 1989 (McLoughlin et al., 1992). Muscle
degeneration and necrosis were consistent with histopathological findings in both
outbreaks, and this myopathy was similar to that previously described in salmonids
and other species associated with vitamin E and selenium deficiency.
Experimentally induced Se deficiency was related to similar changes. The trout
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given the diets without added Se or alpha-tocopherol for about 10 weeks (Oberbach
and Hartfiel, 1987) were characterised by:
lost appetite
pale, swollen gills with shortened gill lids
brown-yellow-coloured livers
anaemia
All those given Se or alpha-tocopherol developed normally. Selenium deficiency

causes growth depression in rainbow trout (Hilton et al., 1980) and catfish (Gatlin
and Wilson, 1984), but selenium deprivation alone does not produce pathological
signs in these fish. Supplements of both vitamin E and selenium were necessary
to prevent muscular dystrophy in Atlantic salmon (Poston et al., 1976) and
exudative diathesis in rainbow trout (Bell et al., 1985).
Did you know that Se and vitamin E deficiency in trout is
characterised by lost appetite, pale, swollen gills with shortened
gill lids, brown-yellow coloured livers and anaemia?
Two duplicate groups of rainbow trout (Salmo gairdneri; mean weight 27 g) were
given for 30 weeks diets containing selenium 0.025 or 1.022 mg/kg. There were
no significant differences between treatments in weight gain but packed cell
volume, liver vitamin E and liver and plasma Se concentrations were all
significantly lower in the Se-deficient trout (Bell et al., 1986). Ataxia occurred in
about 10% of the Se-deficient trout and histopathologies were evident in nerve
cord (damage to axon sheath) and liver (loss of integrity in endoplasmic reticulum
and mitochondria with appearance of increased vesiculation). GSH-Px activity
was significantly reduced in liver and plasma of Se-deficient fish but there was no
indication, from differential analysis, of any non-Se-dependent glutathione
peroxidase activity. However, glutathione transferase activity was significantly
increased in Se-deficient trout. Either simultaneous or separate dietary deficiencies
of vitamin E and Se in Atlantic salmon (Salmo salar) during the first 4 weeks of
feeding caused twice the mortality shown in fish given both additional vitamin E
and Se. Subsequent dietary repletion with both vitamin E and Se significantly
reduced mortality during the following 2 weeks (Poston et al., 1976). Vitamin E
deficiency with or without Se caused extreme anaemia, pale gills, anisocytosis,
poikilocytosis, increased plasma protein, exudative diathesis, dermal
depigmentation, yellow-orange liver colour, yellow-brown intestinal contents,
enlarged gall bladder distended with dark green bile, muscular dystrophy, increased
carcass fat and water and a response to handling characterized by a transitory
fainting with interruption in swimming. In particular, addition of both vitamin E
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and Se was necessary to prevent muscular dystrophy. Fingerling channel catfish

were fed purified diets either unsupplemented and deficient in both selenium and
vitamin E, deficient in either selenium or vitamin E, adequate in both selenium
(0.2 mg/kg) and vitamin E (60 mg/kg), or excessive in both nutrients (four times
the recommended levels; Wise et al., 1993). Hepatic, selenium-dependent
glutathione peroxidase activity was suppressed in fish fed diets deficient in
selenium when compared with this enzyme activity in fish fed diets containing
recommended or higher levels of selenium.
Se toxicity
Selenium toxicity in fish is well studied as a result of environmental concern
related to Se contamination of various ponds, lakes and rivers. For example, Belews
Lake, North Carolina (USA) was contaminated by selenium in wastewater from a
coal-fired power plant during the mid-1970s, and toxic impacts to the resident
fish community (20 species) were studied for over two decades. Symptoms of
chronic selenium poisoning in Belews Lake fish included (Lemly, 2002):
telangiectasia (swelling) of gill lamellae

elevated lymphocytes
reduced hematocrit and hemoglobin (anemia)
corneal cataracts
exopthalmus (popeye)
pathological alterations in liver, kidney, heart, and ovary reproductive failure
teratogenic deformities of the spine, head, mouth, and fins.
Experimentally it was proven that toxic Se doses are much higher than those used
for diet supplementation and they are of the same magnitude as in various farm
animals. In fact, selenium toxicity occurred in rainbow trout and catfish when
dietary selenium exceeded 13 and 15 mg/kg dry feed, respectively (Hilton et al.,
1980; Gatlin and Wilson, 1984; Hilton and Hodson, 1983). The major effects of
Se excess include:
reduced growth
poor feed efficiency
high mortality
renal calcinosis
Indeed, fish food was spiked with SeMe to contain 4.6, 12, and 18 mg/kg (dry
weight) of Se. Fish exposed to SeMe for 90 days exhibited a significant decrease
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in body weight and fork length in the 4.6 and 12 mg/g Se treatments compared
with controls (Vidal et al., 2005). Based on decreased growth after 90 days, a
dietary Se lowest observed-effect concentration (LOEC) value of 4.6 mg/kg and
a Se body burden LOEC of 1.20 g/g (wet weight) were estimated. The chronic
effects of dietary Se exposure in juvenile Sacramento splittail (Pogonichthys
macrolepidotus) were investigated in the laboratory. Purified casein-based diets
were supplemented with selenized yeast for 9 months giving 0.4 (control), 0.7,
1.4, 2.7, 6.6, 12.6, 26.0, and 57.6 mg of Se/kg dry weight. Mortalities occurred
only in the two highest Se treatments and were accounted for 8.3 and 18.3% at 5month and 10.0 and 34.3% at 9-month, respectively (Teh et al. 2004). However,
Se-induced deformities were observed in fish fed > or =2.7 mg of Se/kg diets at 5month and in fish fed > or =0.7 mg of Se/kg diets at 9-month. The authors showed
that chronic exposure to 6.6 mg of Se/kg diet induced deleterious health effects
that can potentially impact survival of juvenile splittail. The toxicity of 2
organoselenium diets (high Se fish meal based diet (FM) or SeMet-supplemented
diet) was evaluated in 90- to 120-day partial life cycle tests with 2 life stages of
chinook salmon (Oncorhynchus tshawytscha). After 90 days of exposure in
freshwater, survival was reduced in fish fed Se more than or equal to 9.6 g/g of
either diet, and growth was reduced in fish fed Se more than or equal to 5.3 g/g
of FM diet or Se more than or equal to 18.2 g/g of the SeMet diet (Hamilton et
al., 1990). After 120 days in brackish-water, survival was unaffected but growth
was reduced in fish fed Se more than or equal to 18.2 g/g of FM diet or Se 35.4
g/g of the SeMet diet. After the 120-day feeding experiment, survival during a
10-day seawater challenge test was reduced in fish fed Se 35.4 g/g of either diet.
Casein-gelatin diets containing graded levels of supplementary selenium (as sodium
selenite) 0 to 15 mg/kg were given to fingerling channel catfish (Ictalurus
punctatus) for 15 weeks. Weight gain values generally indicated that the basal
diet containing Se 0.06 mg/kg diet caused growth depression, and a supplementary
Se level of 15 mg/kg also reduced growth response, which indicated Se toxicity
(Gatlin et al., 1984). Chronic dietary Se toxicity occurred at dietary Se 13 mg/kg
dry feed. Major effects of Se toxicity were reduced growth rate, poor feed efficiency
and a high number of mortalities (Hilton et al., 1980). No histopathological lesions
or significant deviation in blood values or liver somatic index were detected in
trout raised on diets with Se 13 mg/g dry feed. Tissue Se analysis indicated that
trout can maintain homeostasis with dietary Se levels up to 1.25 mg/kg dry feed.
To determine if elevated concentrations of waterborne selenium, caused by
coal mining, in the Elk River in southeastern British Columbia, may be causing
reproductive or teratogenic effects in wild cutthroat trout (Oncorhynchus clarki
lewisi), fertilized eggs from exposed and reference fish were raised in the laboratory
(Kennedy et al., 2000). Selenium concentrations in the eggs was 21.0 g/g (range:
8.7 to 81.3). Despite these elevated egg Se concentrations, there was no significant
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effect on fertilization; time to hatch; percent hatch; or egg, larvae, and fry
deformities or mortalities. The lack of any toxic response in this study the authors
explained as a result of an evolved tolerance to higher tissue Se concentrations in
a population of fish living in a seleniferous river system.
Se requirement
To determine Se requirement of fish is a complex task. For example, there are a
large number of species from different phyla, each species may have several
different life-history stages, time to harvest may be as long as five years, a single
species may be exposed to a wide range of environmental conditions, and
production cohorts are exposed to different patterns of day to day variation in
key environmental parameters (Carter, 2003). Similar to other animal species, the
selenium requirement of fish varies with the form of selenium ingested,
polyunsaturated fatty acid and vitamin E content of the diet, and concentration of
waterborne selenium (NRC, 1993). It seems likely that Se requirement in fish is
similar to those established for various farm animals. In rainbow trout (Salmo
gairdneri) peak plasma GSH-Px activity was obtained at dietary Se value between
0.15 and 0.38 mg/kg dry feed (Hilton et al., 1980).
Did you know that Se requirement of rainbow trout is 0.15-0.38
ppm, and fingerling channel catfish 0.1-0.5 ppm and it is
recommended to provide with a mineral premix for Channel
catfish 0.25-0.3 ppm selenium and for rainbow trout 0.3 ppm?
Liver and plasma Se-GSH-Px activities indicated the Se requirement of fingerling
channel catfish was between 0.1 and 0.5 mg/kg diet. Growth results and liver and
plasma Se-GSH-Px activities indicated that the minimum Se requirement of
fingerling channel catfish fed on adequate vitamin E diet was 0.25 mg/kg dry diet
(Gatlin and Wilson, 1984) and it is recommended to provide with a mineral premix
for Channel catfish 0.25-0.3 ppm selenium and for rainbow trout 0.3 ppm (NRC,
1993). GSH-Px activity in plasma and liver is a useful index of selenium status in
fish (NRC, 1993).
When considering Se requirement and physiological concentrations of Se in
fish tissues it is important to have a comparison with fish in wild. It seems likely
that in most of cases Se concentration in wild fish is several-fold higher than that
in farmed fish. For example, selenium was estimated in whole livers from adult
salmon caught at the entrance to the Strait of San Juan de Fuca, caught in Puget
Sound or its estuaries, fish returning to a hatchery and fish reared in saltwater net
pens. In each case, Se concentrations in wild coho salmon (Oncorhynchus kisutch)
were about twice as high as those in hatchery-reared fish (Felton et al., 1990).
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Tissue levels of zinc, iron, copper and selenium in liver, kidney, gills and gonads
of Atlantic salmon were analysed in fish caught near a spawning ground, in the
open sea or from farmed fish. There were significant differences in liver
concentrations of the 4 trace elements between wild and farmed salmon. In fact,
Se varied 10-fold, Cu 5-fold, Fe 3-fold and Zn 2-fold (Maage et al., 1991).
Concentrations of selenium in liver and serum were higher in wild salmon than in
most domesticated animals (Poppe et al., 1985). Liver samples from 37 wild
salmon, 233 healthy farmed salmon were analysed and it was shown that Se
concentrations were 6 to 7 times higher in wild salmon than in farmed salmon
(Poppe et al., 1986).
It is interesting to note that it is difficult to achieve Se concentration in fish
similar to that in wild when sodium selenite is used. Hatchery-reared coho salmon
(Oncorhynchus kisutch) were fed on increased amounts of sodium selenite to
raise eviscerated body concentrations to those seen in wild counterparts.
Eviscerated body Se concentration, GSH-Px and SOD levels were measured during
and at the end of the 9-month freshwater (FW) feeding trial. Se supplements were
added to a commercial ration to give final concentrations of 1.1, 8.6, 11.1 and
13.6 mg/kg (Felton et al., 1996). The results indicated that a dietary Se concentration
as high as 8.6 mg/kg was necessary for inducing eviscerated body loads similar
to those found in wild fish producing healthy coho. This Se supplemental level is
very close to toxic one. Therefore, by using organic selenium in the fish diet it is
much easier to achieve the same Se status as in wild fish.
GSH-Px activity and Se concentrations in blood and tissues are used for
assessment of Se status of fish. For example, Tilapia [Oreochromis] fingerlings
were assigned to 4 groups and fed for 30 days on basic diets low in Se (0.03 mg/
kg) without vitamin E supplementation, supplemented with Se 0.1 mg/kg feed,
vitamin E (500 mg/kg) or a combination of both. Supplementation with Se and
vitamin E separately or together, increased blood Se from 90 to 199 ng/ml
(Dimanov et al., 1998). GSH-Px activity in the blood of experimental fish increased
from 5 to 14 mU/mg Hb.
Health and immunity

Research on several fishes, including some marine and diadromous species such
as salmonids, has established that immunocompetence and disease resistance can
be compromised by deficiencies of various nutrients, especially certain vitamins
and minerals (Sealey et al., 1999). Thus, adequate levels of these micronutrients
must be supplied in prepared diets to support optimal growth and production
efficiency of fish in aquaculture. In addition, dietary supplementation of some of
these micronutrients in excess of minimum requirement levels has been shown to
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significantly enhance immune responses and disease resistance of various animals.

Increased fortification of such nutrients as vitamin C, vitamin E, and selenium
have shown positive influences on immunity and disease resistance in some studies
but no effects in other studies (Sohn et al., 2000). Indeed, intracellular superoxide
anion production of macrophages was higher in fish fed the diet fortified with
four times the recommended levels of those nutrients than in fish fed the other
diets (Wise et al., 1993). The results of this study indicate that higher than
recommended levels of one or more nutrients may enhance macrophage function.
Groups of juvenile spring chinook salmon naturally infected with Renibacterium
salmoninarum, the causative agent of bacterial kidney disease, were maintained
for 214 days in fresh water and 110 days in sea water and given vitamin E 53 and
299 mg/kg dry diet combined with sodium selenite to give selenium concentrations
of 0.038 or 2.49 mg/kg dry diet (Thorarinsson et al., 1994). No mortality occurred
in the group given high Se + high vitamin E diet, whereas mortality was 3% in
groups given low Se + high vitamin E diet or high Se and low vitamin E diets, and
31% in the group given low Se and low vitamin E diet. GSH-Px activity in blood
plasma was significantly higher with in high Se fishes in comparison to low Se
ones.
Selenium and fish quality

It is well known that salmon coloration is an important quality parameter
determining its price and acceptability among customers. Indeed, wild salmon
can have an access to various carotenoid sources which provide deep coloration
of its flesh. Astaxanthin is the major carotenoid in wild salmonoids. Therefore,
the addition of pigmenting carotenoids is an essential process in order to ensure
that the characteristic pink/red flesh coloration is maintained during the production
of farm-raised trout and salmon in many countries. Indeed, the total market for
astaxanthin in the salmon industry in 2001 was approximately US$250 million
(Davis, 2005). For Atlantic salmon key changes in product quality are demonstrated
by muscle n-3 and n-6 fatty acid content in relation to source of fish (wild or
farmed) and the oil source (Carter, 2003). Research into aquaculture nutrition has
increased dramatically over recent years. Major themes are on unifying the
approach for nutrient requirement determination, the replacement of marine
ingredients and in controlling product quality through nutrition. However, inclusion
of astaxanthin into the diet (up to 80 ppm) increases substantially final feed cost
and there were a variety of various attempts do decrease astaxanthin dose with
the same colouring effect. It has been shown that carotenoid deposition in tissues
depends on many different factors including diet composition, level of fat, fatty
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acid composition of the diet, presence and concentration of various antioxidants,

etc. It is well known that carotenoids, including astaxanthin are easily oxidized
and need stabilization. For example, the results of the recent study of Bell et al
(2000) suggest that both vitamin E and astaxanthin have antioxidant functions in
Atlantic salmon. It is necessary to underline that salmon flesh colour depends on
various factors with carotenoid composition, concentration and flesh texture being
the major ones. Therefore usage of natural antioxidants to improve flesh coloration
is of great importance in recent research and practical application. In fact, fish
nutritionists consider vitamin E as a primary antioxidant for this purpose. However,
information presented in previous chapters clearly shows that a combination of
organic selenium with vitamin E would be an optimal choice for this purpose.
Indeed, better Se accumulation and retention from Se-yeast would improve flesh
quality as well as decrease astaxanthin oxidation during fish storage and display.
Did you know that organic Se dietary supplementation could
improve salmon flesh texture and coloration?
In an experiment conducted in Chile one hundred forty Atlantic salmon initially
weighing 2.6 kg were allocated to 2 sea pens for the 30 day feeding trial. The diet
fed the control pen contained fish meal as the sole source of selenium while the
diet given the test group contained organic selenium in the form of Sel-Plex at a
rate to supply 0.25 ppm selenium (De Lyons, 1998). The results of the feeding
trial are presented in Table 10.5. Supplementation of the salmon diet with SelPlex had no effect on weight gain but improved flesh quality measurements. In
particular, flesh texture improved by almost 10% at the same time flesh oil content
decreased by 7% and GSH-Px activity practically doubled. These changes together
with the 10.5% increase in pigment deposition clearly indicated beneficial effect
of organic selenium of salmon quality. Indeed, a combination of organic selenium
with increased vitamin E supplementation could provide further benefit including
pigment stability during salmon storage.
Table 10.5 Effect of organic selenium on flesh quality of Atlantic salmon (Adapted from de Lyons, 1998)
Weight gain, g
Colour, Roche score
Pigment, mg/kg
Texture, kg/cm2
Flesh oil, %
GSH-Px, U/g Hb
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Control
Sel-Plex
1.403
14.9
6.7
11.4
10.1
53.3
1.408
15.2
7.4
12.5
9.4
107.3
Change, % of control
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+0.36
+2.01
+10.45
+9.65
-6.93
+101.3
624
Effective forms of Se in fish diets

Information is actively accumulated indicating that organic selenium has the same
advantages in fish as it was shown for other animal species. In particular, it has been
shown that organic selenium in the form of selenium-yeast acted more efficiently on
antioxidant system of the carp fingerlings than inorganic selenium salts. For example,
in the group supplemented with sodium selenate, no significant changes in the activity
of the antioxidant enzymes were recorded in both the erythrocytes and in the liver,
with the exception of GST activity that was significantly reduced in the plasma
compared with the controls (Jovanovic et al., 1997). In the group supplemented with
Se-Yeast, the activities of GSH-Px, CAT, and SOD were significantly increased in
erythrocytes, whereas GST activity and plasma content of GSH + GSSG were reduced
compared with the controls. At the same time, the activities of hepatic SOD and GST
were increased compared with the controls.
Did you know that in fish organic Se is more efficiently
assimilated than sodium selenite and could be a supplement
of the choice to maintain optimal Se status?
It has been shown that channel catfish challenged with E. ictaluri are responsive to
dietary selenium and that organic selenium sources have greater potency than inorganic
sources For example, Channel catfish (Ictalurus punctatus) fingerlings (average initial
weight, 1.70 g) were fed a casein-based purified diet supplemented with 0, 0.02,
0.06, 0.20, or 0.40 mg of selenium/kg from sodium selenite, Se-Met or Se-Yeast for
9 weeks (Wang et al., 1997). Subsequently, fish were challenged by immersion with
a virulent strain of Edwardsiella ictaluri (105.9 cells/ml), and mortalities were recorded
during a 14-day period. Dietary selenium concentrations for maximum weight gain
were 0.20 mg/kg from Se-Met and Se-Yeast and 0.40 mg/kg from sodium selenite.
Dietary selenium concentration for maximum survival from E. ictaluri challenge was
0.20 mg/kg for fish fed Se-Met and 0.40 mg/kg for fish fed Se-Yeast and sodium
selenite. Antibody production generally increased as dietary concentration of selenium
increased. It is interesting that antibody titre was highest for fish fed Se-Yeast,
intermediate for fish fed Se-Met, and lowest for fish fed sodium selenite. In another
experiment conducted in the same department, a total of 1170 channel catfish
fingerlings, initial weight 1.70 g, were stocked into 39 aquaria, and fed on caseinbased purified diets with the same experimental design (Wang and Lovell, 1997).
Fish fed on the basal diet showed low live weight gain, poor feed conversion efficiency,
low plasma and liver GSH-Px activities, and low Se content in the liver and muscles,
compared with those fed on Se supplements. Weight gain and apparent feed conversion
efficiency responded quadratically to the graded dietary Se levels from all 3 sources.
Regression of weight gain on dietary Se supplementation was highest for Se-Met,
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followed by Se-Yeast and sodium selenite. Broken-line analysis showed that minimum
supplemental dietary Se requirement as sodium selenite, Se-Met and Se-Yeast was
0.28, 0.09 and 0.11 mg/kg diet for live weight gain, and 0.17, 0.12 and 0.12 mg/kg
diet, for liver GSH-Px activity, respectively. Relative bioavailability values of Se-Met
and Se-Yeast compared with sodium selenite were 336 and 269% for growth, and
147 and 149% for GSH-Px activity, respectively. Se from Se-Met and Se-Yeast showed
significantly higher rates of accumulation in liver and muscle than Se from sodium
selenite (Table 10.6 and 10.7; Lovell and Wang, 1997).
Table 10.6 Selenium requirement and relative bioavailability in channel catfish (Adapted from Lovell and
Wang, 1997)
Criterion
Se source
Weight gain
Selenite
SeMet
Sel-Plex
Plasma GSH-Px Selenite
SeMet
Sel-Plex
Liver GSH-Px Selenite
SeMet
Sel-Plex
Liver Se
Selenite
SeMet
Sel-Plex
Muscle Se
Selenite
SeMet
Sel-Plex
Equation
Se requirement*
Relative
bioavailability, %**
y=10.8+ 34.9x
y=10.6 + 117x
y=10.2 + 93.8x
y=2.0 + 4.4x
y= 2.3 + 5.1x
y=2.0 + 5.0 x
y=22.5 + 600.7x
y=22.5+ 883.7x
y=21.4+ 891.8x
y=0.06 + 10.3x
y= 0.77 + 20.3x
y=0.74 + 19.0x
y= 0.16 + 0.7x
y=0.16 +3.3x
y=0.17 + 3.1x
0.28 0.031
0.09 0/007
0.11 0.012
0.17 0.024
0,12 0.021
0.12 0.020
0.18 0.045
0.09 0.015
0.11 0.025
0.10 0.022
0.10 0.024
100
336
269
100
116
116
100
147
149
100
197
184
100
478
453
*/ Breakpoint in the regression line;

**/The ration of the slope of SeMet or Sel-Plex regression line to the slope of selenite
regression line
Table 10.7 Antibody titer of channel catfish depending on Se in the diet (Adapted from Lovell and Wang,
1997)
Dietary Se, ppm

0
0.020
0.06
0.2
0.4
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Selenite
SeMet
Sel-Plex
64
80
149
192
365
64
70
213
352
512
64
75
320
554
717
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Effects of supplementing a fish meal-based diet with selenium as selenite or SeMet

were compared using 5 duplicate groups of Atlantic salmon parr (mean weight
4.5 g) given a basal diet unsupplemented or supplemented with Se 1 and 2 mg/
kg as selenite or SeMet respectively, for 8 weeks. Weight gains were similar in all
groups. Se concentrations in the liver were highest in salmon given selenite while
muscle and whole body Se were highest in the groups given SeMet (Lorentzen et
al., 1994). Minor increases in muscle Se were observed when selenite was
supplemented. There were no significant differences in activity of the Se-GSHPx, between dietary groups, indicating a sufficient Se status in all groups. It was
shown that net absorption of SeMet in channel catfish was higher than that of
selenite in a purified egg white-based diet (90.8 vs 62.8%) or in a soybean mealbased diet (88.9 vs 69.7%; Lovell and Wang, 1997). It is well known that fish
meal is a rich source of selenium. However, as indicated in previous chapters Se
availability from this source is comparatively low. The same is true for fish feed.
For example, groups of Atlantic salmon (Salmo salar), mean weight 68 g, were
given diets for 4 weeks, in which selenium was supplied from fish meal, sodium
selenite, DL-selenomethionine or DL-selenocystine. SeMet was the most digestible
(92%) and fish meal (47%) the least digestible source of Se (Bell and Cowey,
1989). This was reflected in plasma Se concentration which was highest in fish
given SeMet, although the activity of GSH-Px was not increased.
Se-fish as human food

There is evidence indicating that many kinds of fish are characterised by
comparatively high Se content. For example, in Spain, total selenium concentration
was high in sardine, swordfish, and tuna (0.43; 0.47 and 0.92 mg/g, respectively),
but low in mackerel shad and octopus (0.26 and 0.13 g/g, respectively; Cabanero
et al., 2005). Selenium speciation in fish is a very difficult task and many questions
remain without answer. For example, SeMet was the only selenium compound
identified in the fish samples with higher selenium content (Cabanero et al., 2005).
In contrast to fish meal Se availability from fish is shown to be comparatively
high. For example, subjects consumed the labelled foods in four meals over two
consecutive days and absorption was measured by the luminal disappearance
method over 10 days (Fox et al., 2004). Apparent absorption of selenium from
fish was similar to selenate and there was no difference between the two processing
methods used. However, retention of fish selenium was significantly higher than
selenate.
Similar results were obtained in animal model studies. For example, selenium
deficient male Wistar-Moll rats about 45 g were fed on diets containing cod fillet
(Fish-Se), selenomethionine (seleno-L-methionine Se 0.2 mg/kg diet) or selenite
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(sodium selenite 0.2 mg/kg diet). GSH-Px activities in serum and liver and hepatic
Se concentrations were similar in all rats after 12 days. Serum Se concentration
was highest in the group fed on cod fillets (Knudsen et al., 1992). Rats fed on cod
fillets and selenomethionine had significantly higher femur Se concentration than
rats fed on selenite. It was concluded that Se in cod fillets was as biologically
available to rats as SeMet and selenite. The experimental results with Se deficient
rats showed that enrichment of fish fillets with SeMet yields fillets with high Se
bioavailability. In fact, Se bioavailability decreased in the order raw salmon >
cured salmon > selenite (Ornsrud and Lorentzen, 2002). This was supported by
the observations for Se accumulation in femur and muscle and induction of GSHPx activity. However, curing salmon altered the utilisation of Se. In a line with
these results are other experimental data. After 9 weeks of dietary Se repletion,
relative activity of liver GSH-Px from the different dietary groups compared with
control rats (100%) was: flounder 106%, tuna 101%, pork 86%, sodium selenite
81%, SeMet 80%, beef 80%, chicken 77%, veal 77%, and lamb 58% (Wen et al.,
1997). From the data presented above it is clear that fish is a good source of Se
and there is a good opportunity to produce Se-enriched fish by using organic
selenium supplements in the fish diet. Furthermore, comparatively high Se
availability from fish makes the issue of producing Se-fish even more attractive.
Did you know that by inclusion of organic selenium
in fish diet it is possible to produce Se-enriched fish
which could be a good source of Se for human consumption?
Conclusions
Fish nutrition is quickly developing in different countries and in many cases fish
nutritionists can learn from other industries, such as poultry. In fact, fish nutritionists
face the same problems as poultry or pig nutritionists. Indeed, antioxidant
misbalance and oxidative stress are major problems in fish nutrition. In fact,
immunocompetence can be compromised and fish viability substantially decreased
as a result of oxidative stress. Importance of Se in fish nutrition is well recognised.
Similar to other food industries, organic selenium is shown to be more effective
in fish nutrition than sodium selenite. Limited results from wild fish studies indicate
that Se level in wild fishes usually several-fold higher than those in farmed fishes.
Possible stabilizing effect of Se on astaxanthin absorption and metabolism resulting
in better flesh coloration needs further investigation. Production of Se-enriched
fish can help solving global Se deficiency. Using organic selenium in the fish diet
it is possible to achieve desirable Se concentration in the fish flesh.
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11
SELENIUM AND HUMAN HEALTH
Diet cures more than lancet
Introduction
From information on effects of natural antioxidants on animals including birds
presented in previous ten chapters of this book it has become clear that natural
antioxidants have important roles in maintaining not only animal health but could
also be beneficial for human. In fact, many physiological processes in human
body are dependent on antioxidant/prooxidant balance in every cell and in whole
body. In this regard diet provides a wide range of antioxidant compounds and is
able to affect our health.
Nutrition and health

Hippocrates observations on the relationship between health and food choices began
discussions about the factors that determine our health many centuries ago. However,
during the last decade it has become obvious that while our lifestyle, including diet,
stress, smoking, medical attention, exercise and genetics, is a major determinant of
our health status it is diet that has the pivotal role. The effect of nutrition on human
health has received tremendous attention and traditional medical teaching that diet
and nutrients play only limited roles in human health is being revised. In most developed
countries nutritional practice has changed the focus from combating nutrient
deficiencies to addressing nutrient requirements for maintaining good health
throughout the life. Indeed, collectively, cardiovascular disease (including stroke),
cancer, and diabetes account for approximately two thirds of all deaths in the United
States and about $700 billion in direct and indirect economic costs each year (Eyre
and Kahn, 2004). They account for nearly 2 of every 3 deaths in the United States close to 1.5 million people in 2001 (Anderson and Smith, 2003). The economic costs
of cardiovascular disease, cancer, and diabetes in the United States in 2003 were
estimated to be $351.8 billion, $189.5 billion, and $132.0 billion, respectively (National
Institutes of Health, 2004; Hogan et al., 2003).
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Did you know that Hippocrates suggested the relationship
between health and food choices many centuries ago?
Three major areas of concern are improvement of the diet, increase physical
activity and reduction of prevalence of tobacco use the major risk factors for
these diseases. In this chapter relationship of the balanced diet to human health
will be considered with a special emphasis to selenium.
Are there ideal diets?

Arguably one of the most fascinating ideas in human nutrition is that selected
foods and their components can improve physical or mental performance or
decrease disease risk (Milner and Craig, 2000; Milner, 2000). Thus, beyond
meeting nutrition needs, diet may modulate various body functions and may play
detrimental or beneficial roles in the development of some diseases (Roberford,
2000). For example, dietary factors are considered to be major contributors to
the leading causes of death of Americans, namely coronary heart disease and
certain types of cancer (Milner, 2000). Indeed, the last 50 years have been
characterised by the understanding of the impact of nutrition and dietary patterns
on health. In particular, oxidation of lipids, nucleic acids or proteins has been
suggested to be involved in the aetiology of several chronic diseases including
cancer, cardiovascular disease, cataract, age-related macular degeneration and
aging in general (Berger, 2005). Epidemiological findings, supported by animal
studies, have led to recommendations that people should consume at least two
servings of fruit and three servings of vegetable daily (Diplock et al., 1998) in
addition to at least two servings of fish weekly (Krauss et al., 2001). While
findings and reports such as these have had an impact on the type and quantity of
the food that many of us eat (Margetts et al., 1998) the majority of adults in
developed countries fall well short of meeting healthy eating guidelines.
In scientific literature three major so-called ideal diets have received
substantial attention for the last few years. They are Mediterranean, Japanese and
Hunter-gatherer diets (Table 11.1). The Mediterranean diet is associated with
decreased rates of various diseases. For example, this diet, based on green and
yellow vegetables, fruits, grains and olive oil, has been observed to have a
protective effect against development of atherosclerosis. The Japanese diet reflects
the highest life expectancy (Table 11.2) and the Hunter-gatherer diet is based on
a nutrient consumption similar to our ancestors during human evolution. These
three diets are different in composition but they have some similarities in terms of
low saturated fat and high levels of naturally occurring antioxidants.
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Table 11.1 Some characteristics of ideal diets
Mediterranean
Japanese
Hunter-gatherer
High consumption of legumes

and cereals, fruits and
vegetables
High consumption of
soya, seaweeds, raw fish
and rice
High consumption of many

different parts of plants and
animals, high in fibre
Moderate consumption of
milk and dairy products and
alcohol
Moderate consumption
of salt
Arachidonic acid and DHA

are major PUFAs
Low consumption of meat

and meat products
Low consumption of fat

and sugar
Low in saturated feed, low n6/n-3 ratio
Variable amount of olive oil,

some fish, cheese and nuts,
little sugar
It does not include cereals,

milk, refined sugar, salt or
alcohol
(Adapted from Truswell, 1998; from Trichopoulou et al., 1998)

Table 11.2 Countries with the highest life expectances
Country
Life expectancy
Japan
Greece, Hong Kong, Spain, Sweden, Switzerland
Australia, Canada, France, Israel, Italy, Netherlands, Norway
Austria, Belgium, Denmark, Finland, Germany, New Zealand, UK, USA
Ireland, Portugal, Singapore
79
78
77
76
75
(Adapted from Truswell, 1998).
Hunter-gatherer diet. The evolution of man is connected with a life-style of

hunting and gathering. The food sources (animal and plant) remained the same
during the evolution, but the proportions of foods, preferences, preparations and
the attainability changed (Haenel, 1989). It has been suggested that the nutritional
patterns of Paleolithic humans influenced genetic evolution during the time period
within which defining characteristics of contemporary humans were selected
(Eaton and Eaton, 2000). Because human genome has not changed much since
the beginnings of agriculture, genetically, humans remain adapted for a Paleolithic
dietary regimen. Such diets were based mainly on:
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fish;
uncultivated plant foods.
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Did you know that Mediterranean, Japanese and

Hunter-gatherer diets are considered to be ideal for human?
In comparison to Paleolithic man, today our diet is characterised by substantial
increase of some substances and decrease of others (Table 11.3). For example, in
comparison to the Paleolithic period, current U.S. intake of vitamin E decreased
by 4 times (7-10 vs 32.8 mg/day), carotene by 2 times (2.05-2.57 vs 5.56 mg/
day) and ascorbate by 6 times (77-109 vs 604 mg/day) (Simopoulos, 1998). In
the history of mankind, anthropology shows that humans were gatherers of fruits
and vegetables for their daily nutritional needs. This tradition has changed
dramatically with the development of agricultural industries (Weisburger, 1997).
Furthermore, the relocation of people from rural areas to cities also decreased
fruit and vegetable consumption. Therefore, in contrast to our ancestors who used
to eat a variety of various wild plants in large amounts, todays diet includes a
limited number of plants, consumed in limited amounts, and as a result is
comparatively poor in antioxidant compounds (Simopoulos, 1998). In general it
seems likely that Paleolithic diet contained quite high concentrations of various
antioxidants, including selenium.
Table 11.3 Comparison of modern and Paleolithic diets
Modern diet in comparison to Paleolithic one is characterised by

Increased
Decreased
Energy content
Saturated fat
Omega-6 PUFA
Trans fatty acids
Cereal grains
Sodium
Saccharose
Lactose
Alcohol
Dairy products
Energy expenditure
Omega-3 PUFA
Complex carbohydrate
Fiber
Fruits and vegetables
Animal protein
Antioxidant
Phytochemicals
Potassium and calcium
Vitamins
(Adapted from Simopoulos, 1998; Eaton and Eaton, 2000 and Haenel, 1989)
The main advantage of the described diet is an adaptation of the human digestive
system to most of those nutrients. This means that high efficiency of assimilation
from the diet and distribution in the body could be a driving force in health
promoting properties of various compounds, including antioxidants and
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phytochemicals. Possible implications of this kind of diet on the digestive tract

need further investigations.
Mediterranean diet. In the early 1950s in Naples very low incidences of coronary
heart disease was associated with a specific diet, which later was called the good
Mediterranean diet (Keys, 1995). That diet was mainly vegetarian, and differed
from American and northern European diets in that it was much lower in meat
and dairy products and uses fruit for dessert. That observation led to subsequent
research in the Seven Countries Study. At the end of the 1950s that study was
designed to investigate the relations between diet and cardiovascular diseases.
The comparison was made between Finland, Greece, Italy, Japan, The Netherlands,
United States, and Yugoslavia. The study provided important information on
national differences in diet preferences. For example, in Finland the intake of
milk, potatoes, edible fats, and sugar products was very high. A similar but lower
intake pattern was observed in The Netherlands. Fruit, meat, and pastry
consumption was high in the United States; cereal and alcoholic drink consumption
was high in Italy; and bread consumption was high in Yugoslavians except for
those in Belgrade. In Greece the intake of olive oil and fruit was high and the
Japanese cohorts were characterised by a high consumption of fish, rice, and soy
products (Kromhout et al., 1989).
Therefore interest in Mediterranean diet began more than 30 years ago, when
Ancel Keys published the results of the famous Seven Countries Study. The term
Mediterranean diet is associated with dietary patterns found in olive-growing areas
of the Mediterranean region and described in the 1960s. It was argued that there
is no single ideal Mediterranean diet, since there are important differences in the
food habits of the Mediterranean countries (Noah and Truswell, 2001). Indeed,
the countries around the Mediterranean basin have different diets, religions and
cultures. As a result their diets differ in many nutrients and the rates of coronary
heart disease and cancer, with the lower death rates and longer life expectancy
occurring in Greece (Simopoulos, 2001). The Greek version of the Mediterranean
diet is dominated by the consumption of olive oil and by high consumption of
vegetables and fruits (Trichopoulou and Vasilopoulou, 2000). In general, there
are several variants of the Mediterranean diet, but some common components
can be identified (Trichopoulou and Lagiou, 1997; Table 11.1).
Analyses of the dietary pattern of the diet of Crete shows a number of protective
substances (Simopoulos, 2001), such as:
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selenium;
glutathione;
a balanced ratio of (n-6):(n-3) PUFAs;
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high amounts of fiber;

rich in antioxidants (resveratrol from wine and polyphenols from olive oil),
vitamins E and C.
Growing evidence demonstrates that the Mediterranean diet is beneficial to health

with the stronger evidence for coronary heart disease, but with possible application
to some forms of cancer (Trichopoulou and Lagiou, 1997). In fact, it has been
shown that the Mediterranean diet substantially reduces the rate of recurrence
after a first myocardial infarction. The Mediterranean diet has strong secondary
preventive effects after myocardial infarction resulting in a decrease of total
mortality, cardiac death and non-fatal reinfarction (Ballmer, 2000). Albanian
paradox of high adult life expectancy in very low income country could be
another example of protective effect of Mediterranean lifestyle (Gjonca and Bobak,
1997). In particular it was shown that in Albania adult mortality, including mortality
from cardiovascular diseases, was similar to that in other Mediterranean countries
being less than half the rate in the UK but similar to that in Italy. Analysis of the
geographical distribution of mortality within Albania showed that mortality was
lowest in the southwest where most of the olive oil, fruits, and vegetables are
produced and consumed.
Probably the high proportion of monounsaturated fatty acids in olive oil can
cause a decrease in LDL cholesterol and an increase in HDL cholesterol.
Furthermore, such antioxidants as vitamin E, C and carotenoids and phenolic
substances possessing antioxidant activity (quercetin and resveratrol) could
decrease the oxidation of LDL and thus reduce atherogenicity. To explain the
beneficial effects of the diet the antioxidant properties of several plant foods in
the Mediterranean diet were postulated to be critical mediators of the beneficial
effects of this diet (Trichopoulou et al., 1999). Antioxidants represent a common
element in the components of the Mediterranean. In particular wild edible greens
frequently eaten in rural Greece in the form of salads and pies contain very high
quantities of flavonoids (Trichopoulou and Vasilopoulou, 2000). While there is
no direct evidence that these antioxidants are the main player to the benefits of
the Mediterranean Diet, indirect evidence from epidemiological data and the
increasing understanding of their mechanisms of action suggest that antioxidants
may play a major role. Dietary antioxidants may prevent oxidation of the
atherogenic cholesterol rich LDL lipoproteins. Observational research has shown
that flavonols, polyphenols with strong antioxidant properties present in plant
foods, may protect against coronary heart disease (Kromhout, 2001).
Therefore from data presented above an important conclusion can be drown
about beneficial role of natural antioxidant in healthy image of the Mediterranean
Diet. Unfortunately today the healthy Mediterranean diet is changing under the
strong influence of the Western culture.
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Japanese diet. The traditional Japanese diet has attracted the attention of many
researchers since it is beneficial for preventing ischemic heart disease and certain
type of cancers. It has been shown that this diet, containing little fat but enriched
in complex carbohydrates and n-3 fatty acids of marine origin, may be related to
the low atherogenic index in rural areas in Okayama Prefecture in Japan (Okita et
al., 1995). Main characteristic features of this diet is low level of fat and low
calorie which remained constant at the level of developing countries for the last
40 years (Hoshi, 2000). A wide verity of marine products including seaweeds
and root-vegetables make this diet different from others. Thus, the dietary habits
of the Japanese have made possible a high n-3 PUFA intake within a low-fat
regimen. They are currently consuming, on average, approximately 26% of energy
as fats with ratios of polyunsaturated to saturated fats and n-6 to n-3 fatty acids of
approximately 1.2:1 and 4:1, respectively (Sugano and Hirahara, 2000). For
comparison, in the United States the ratio of n-6 to n-3 fatty acids is approximately
9.8:1 (Kris-Etherton et al., 2000).
Among Japanese, flavonoids and isoflavones originated from soya-related
products are the main components among nonnutrient phytochemicals with
antioxidant potential in the diet and their high consumption may contribute to
low incidence of coronary heart diseases in Japanese women (Arai et al., 2000a).
Isoflavones have a limited distribution in nature, and, for practical purposes,
soyfoods are the only nutritionally relevant dietary source of these phytochemicals
(Messina and Bennink, 1998). Seaweeds in the Japanese diet provide substantial
amounts of various antioxidant compounds. Plants consumed by humans contain
thousands of phenolic compounds. Among them the effects of dietary polyphenols
are of great current interest due to their antioxidative and possible anticarcinogenic
activities. For example, soy isoflavones are capable of inhibiting lipoprotein
oxidation in vitro and suppressing formation of plasma lipid oxidation products
in vivo (Patel et al., 2001). It has been also shown that soy isoflavone
supplementation decreases levels of oxidative DNA damage in humans (Djuric et
al., 2001) and this may be a mechanism behind the cancer-preventive effects of
soy isoflavones. The study of Davis et al. (2001) demonstrated that soy isoflavone
supplementation protect cells from oxidative stress-inducing agents by inhibiting
NF-kappaB activation and decreasing DNA adduct levels. In the study of Exner
et al. (2001) genistein, a flavonoid derived from soy has been shown to prevent
atherogenic modification of LDL by glucose autoxidation radical products. As
the protective effect of genistein on LDL atherogenic modification was found at
glucose/genistein molar ratios which may occur in vivo, the authors suggested a
beneficial action of a soy diet in preventing chronic vascular diseases and early
atherogenic events. Similarly, when soy breakfast cereals (providing 168 mg/d
isoflavones) were used for 3 weeks, the level of oxidized LDL in human was
significantly reduced (Jenkins et al., 2000).
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Isoflavones are weak oestrogens but possess other potentially important

biological attributes independent of their ability to bind to the oestrogen receptor.
It is well known that Japanese diet is rich in isoflavones (daidzein and genistein).
The median intake of daidzein was 9.5 and 12.1 mg/day among two groups of
people who completed dietary records and the corresponding values for genistein
were 14.9 and 19.6 mg/day. The top four foods (tofu, miso, natto, and fried tofu)
covered about 90% of the population intake of these isoflavones (Wakai et al.,
1999). In other studies the dietary intake of daidzein and genistein among Japanese
women revealed by questionnaire was 16.4 and 30.1 mg/day/capita (Arai et al.,
2000) and using food analyses these figures were estimated at 12.02 and 13.48
mg/day (Nakamura et al., 2000). For comparison, in US adults mean intake of
daidzein and genistein was 4 6 and 7 10 mg/day, respectively and fifteen soy
foods contributed 95% of the total genistein and daidzein intake (Kirk et al.,
1999). It is interesting that total intake of isoflavones in Japanese women exceeded
that of other dietary antioxidants, such as flavonoids, carotenoids (3.5 mg/d) and
vitamin E (8.2 mg/d), and was approximately one half of the vitamin C intake
(109 mg/d) (Arai et al., 2000a). In that study the total intake of flavonoids was
inversely correlated with the plasma total cholesterol concentration and plasma
LDL cholesterol concentration. Therefore, similarly to two other ideal diets
Japanese diet contain quite high levels of phytochemicals possessing antioxidant
activity which in combination with n-3 fatty acids could have health-promoting
properties. For example genistein acted in synergism with eicosapentaenoic acid
as growth inhibitor for human breast cancer MCF-7 cells (Nakagawa et al., 2000).
One more interesting feature of the Japanese diet is a specific emphasis to
animal protein. For example, the relationship of nutrient intakes to life expectancies
in Japan since the Second World War has demonstrated that sufficient intakes of
animal protein and fat are crucial for attaining longevity (Shibata, 2001). It is
interesting that low serum cholesterol was deleterious for higher levels of functional
capacity and accelerated depressive status in the community dwelling elderly.
When the relationship of nutritional status to further life expectancy and health
status in the Japanese elderly was examined, it was shown that high intakes of
milk and fats and oils had favourable effects on 10-year (1976-1986) survivorship
in 422 urban residents aged 69-71 (Shibata et al., 1992). The survivors revealed
a longitudinal increase in intakes of animal foods such as eggs, milk, fish and
meat over the 10 years. These data indicate that diet should be considered as a
whole entity, not just by individual nutrients, since nutrient interactions could be
important elements of health-promoting properties of diet.
However healthy-eating traditions in Japan are changing. For example, from
1957 to 1982, the average daily intake of cholesterol by Japanese rose 2.1-fold
from 183 to 376 mg while that of phytosterol remained at about 373 mg. At the
same time daily intakes of total fatty acid increased by more than 2.5-times, PUFAs
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by 1.5 times and SFA more than 2.5 times and the ratio PUFA/SFA decreased
almost twice (Hirai et al., 1986). Therefore, despite of highest life expectancy, in
Japan, nutrition and diet are considered to play important roles in the emerging
problems of obesity, diabetes mellitus, hypertension etc., because of excessive
energy intake and deficiency or excessive intake of certain nutrients (Matsumura,
2001). Therefore internationalisation of food with a fashion for fast food make it
difficult to preserve the typical Japanese or Mediterranean diet. In fact, today
there is no substantial difference in the incidence of ischemic heart disease between
Greece and the UK or USA (Hoshi, 2000).
The main conclusion which can be made from analysis of ideal diets is that
natural antioxidants are among major players in these diets. Recent research on
phytochemicals has changed a view on dietary factors affecting our health. In
particular it seems likely that there are hundreds and even thousands of such
factors and now we are dealing just with the top of iceberg. However, emerged
information can help us to understand and to some extent explain a beneficial
effect of various vegetables and fruits in our diet.
It seems likely that omega-3 fatty acids, vitamin E, carotenoids and selenium
are nutrients, which are commonly present in all three ideal diets and are lacking
in our everyday diets. Vitamin E and carotenoids will be considered in the chapter
13.
Omega-3 PUFAs
Major advances have been made in recent years in our understanding the molecular
mechanisms whereby dietary fatty acids influence the bodys metabolism. It has
become clear that in addition to well defined roles in energy metabolism and as
constituents of biological membranes, PUFAs also have specific regulatory
functions. For example, PUFAs are precursors of different biologically active
compounds, including eicosanoids. These compounds are considered to be
secondary messengers (Graber et al., 1994) as well as take part in the direct
regulation of gene expression by altering the transcription of specific genes (Clarke
and Jump, 1994).
PUFA may be subdivided into families depending on the position of the double
bond in the molecules. Two of the important groups of PUFA in human nutrition
are the, n-6 or omega-6 fatty acids and the n-3 or omega-3 fatty acids. The
precursors of the n-6 and n-3 family of fatty acids are linoleic acid (LA, 18:2n-6)
and alpha-linolenic acid (ALA, 18:3n-3) respectively. These PUFA have to be
supplied by the diet (Nair et al., 1997). The n-3 and n-6 PUFAs are not interconvertible in the human body and affect eicosanoid metabolism, gene expression
and intercellular cell-to-cell communication (Simopoulos, 2000).
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Did you know that two of the important groups of PUFA in
human nutrition are the, n-6 or omega-6 fatty acids and the
n-3 or omega-3 fatty acids?
LA and ALA can be elongated and desaturated into their longer-chain active
metabolite arachidonic acid (20:4n-6) and eicosapentaenoic acid (EPA; 20:5n-3)
and docosahexaenoic acid (DHA; 22:6n-3) (Roche, 1999). These two classes of
PUFA are metabolically and functionally distinct and in many cases have opposing
physiological effects (Simopoulos, 2000). Therefore, the absolute level and the
balance between n-6 and n-3 PUFA in the diet is considered to be an important
determinant of many metabolic functions in the human body (Table 11.4; Surai
and Sparks, 2001).
Table 11.4 Beneficial effects of n-3 fatty acids for human in prevention and management of human
diseases (Adapted from Surai and Sparks, 2001).
Disease
Disease

Hypertension
Trombosis
Type 2 diabetis
Renal disease
Rheumatoid arthritis
Ulcerative colitis
Crohns disease
Depression
Embolism
Allergic problems
Chronic obstructive pulmonary disease
Prostate and breast cancer
Immunological functions
Autoimmune disease
Fetal brain and visual development
Improvement of learning ability
Positive effects on longevity
Industrial production of grain-based feeds (rich in n-6 PUFAs) for farm animals
results in production of meat which is rich in n-6 fatty acids and poor in n-3
PUFAs. While wild and free range animals tend to contain higher proportions of
n-3 PUFAs in their muscles or eggs (green grass and leaves are rich sources of
ALA) they are not the major sources of the fat and protein in human diet. As a
result of this shift to the n-6 PUFAs the ratio n-6 PUFAs/n-3 PUFAs in modern diet
comprises about 1:10-50 while evolutionary human diet was rich in n-3 fatty
acids and the n-6/n-3 ratio was at the level 1:2 (Simompoulos, 2000). Evidence is
accumulating that the metabolism of omega-3 and omega-6 fatty acids requires
the same desaturation enzymes, resulting in competition between these two families
(Sargent, 1997). Therefore, high proportions of n-6 fatty acids in the diet inhibit
conversion of ALA to EPA and DHA exacerbating the n-3 fatty acid deficiency
(Connor, 1999).
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Realisation that the long chain n-3 PUFAs are also important came to light
when the diets in the developed countries were already rich in n-6 PUFA (Sargent,
1997). The main PUFA in the Western diet is LA, found mostly in vegetable
oils such as safflower oil, sunflower oil, corn oil, soybean oil, etc. (Nair et al.,
1997). Because the typical Western diet, provides high levels of n-6 PUFAs
and low levels of n-3 PUFAs it is considered to be imbalanced and to be associated
with the increased incidences of certain diseases (Shahidi and Vanasundara, 1998).
This balance could be improved by reversing the trend for fish consumption
decrease (oily fish being an important source of DHA; Sargent, 1997). A survey
conducted in Nebraska (USA) indicated that fish was consumed only once per
two weeks (Lewis et al., 1995). Fish consumption in European countries averages
34 g/day and varies from 19 g/day (Netherlands) up to 92 g/day (Portugal)
providing n-3 fatty acids in a range 0.2-1.84 g/day (Gibney, 1997). Therefore
there is a recommendation to people who do not eat fish to obtain 200 mg of very
long chain n-3 PUFA daily from other sources (De Deckere et al, 1998).
It has been shown that inadequate intakes of n-3 polyunsaturated fatty acids
per se, particularly DHA, adversely affects cardiovascular function (Kinsella et
al, 1990) and, in neonates a deficient supply of this polyunsaturate may impair
the development and functioning of the retina and brain (Green and Yavin, 1998;
Uauy et al, 1996; Wainwright et al, 1994). The interest in this subject was
associated with observations in 1970s that the low incidence of heart attacks
among native fishing populations in Greenland, Japan and Alaska who ate very
large amounts of fish were associated with an increased level of omega-3 fatty
acids in their diet. More recently a significant inverse relationship was found
between fish consumption and the risk of a fatal myocardial infarction (Daviglus
et al., 1997). Similarly, an intake of 5.5 g of n-3 PUFA per month was associated
with a 50% reduction in the risk of primary cardiac arrest compared with no
dietary intake of n-3 PUFA (Siscovick et al, 1995).
There are a number of potential solutions to improve n-3/n-6 fatty acid balance
in our diet. For example:
1.
2.
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Increase consumption of fish and fish products. However, world stocks of

cold water fish, which are rich in n-3 PUFAs, are decreasing and their farmed
equivalent usually contains much lower levels of DHA compared to wild
fish (Van Vilet and Katan, 1990). Furthermore, a large proportion (up to
65%) of the population, do not eat fish regularly (Gregory et al., 1990).
Use of a capsulated form of DHA or other n-3 fatty acids. This option has
the advantage of being able to delivering a known quantity of n-3 PUFA
but the capsules can be relatively expensive and some individuals do not
like swallowing capsules.
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3.
Produce designer or so-called functional foods, enriched with n-3 fatty acids.
This option has been embraced during the last few years and there are now
many products in the market that are enriched with n-3 fatty acids, including
various oils, bakery products, infant formula milk, mayonnaise, margarine,
salad dressings and others (Simopoulos, 2000). Among them, eggs, as shown
in the chapter 12, could have a special place as an ideal delivery system for
n-3 fatty acids. In addition, chicken meat enriched with n-3 fatty acids could
also be a valuable option (Phetteplace and Watkins, 1989). Furthermore, it
is possible to alter fatty acid composition of pork, lamb, beef (Wood and
Enser, 1997; Wood et al., 1999) and milk (Mansbridge and Blake, 1997). It
is interesting, that recent survey in the UK revealed that poultry consumption
in 1999 was 236 g/person per week which is equal to total consumption of
beef, veal, lamb and pork (236 g; National Food Survey, 1999). This is
probably associated with lower chicken meat prices and healthier chicken
meat image from the point of view of fatty acid profile. However, chicken
meat enrichment with n-3 PUFA is associated with decreased quality in
terms of storage and flavour (Manilla and Husveth, 1999) and dietary
linolenic acid is not always effective in transferring to muscles (LopezFerrer et al., 2001). Indeed n-3 enriched meat is characterised by increased
susceptibility to lipid peroxidation (Li et al., 1996). Antioxidants are found
to be effective in decreasing lipid peroxidation in such meat (Li et al., 1996;
Wood and Esner, 1997). It seems realistic that a combination of increased
dietary antioxidant supplementation with new sources of high quality fish
oil could help produce meat with increased DHA concentration and without
off-flavour.
Selenium: global perspective

As it was shown in previous chapters, Se is a key component of a number of
functional selenoproteins required for normal health. It is provided in the Western
European diet from bread and cereals, fish, poultry and meat (Reilly, 1998).
Selenium enters the food chain through incorporation into vegetable proteins as
the amino acids selenomethionine and selenocysteine and some others.
A great body of evidence indicates that European intakes of selenium are falling
(Ryman, 2000). For example, in 1978 selenium intake in Britain was 60 g/day
(Thorn et al., 1978), 7 years later it was only 43 g/day, and in 1990 fell to 30 g/
day (MacPherson et al., 1993). Even in 1997, average reported selenium intake
was only 43 g/d (Shortt et al., 1997; Figure 11.1). Dietary intakes of selenium in
other countries vary considerably but in some of them intake is still lower than
the RDI. These include such countries as Egypt (29 g/day); Belgium (30 g/
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day); Turkey (32 g/day), Sweden (38 g/day), Slovak Republic (38.2 g/day),
France and Germany (47 g/day) and Italy (49 g/day) (Reilly, 1996; Kadrabova
et al., 1998; Table 11.5).
70
Se intake (mcg/day)
60
50
40
30
20
10
0
1974
1986
1991
1994
1995
RDA
Figure 11.1 Selenium intake in the UK (Adapted from MAFF Food Surveillance Information sheet,
October 1997).
Did you know that selenium enters the food chain through
incorporation into vegetable proteins as the amino acids
such as selenomethionine
The decline in selenium intake is reflected in decreased serum and whole blood
selenium concentrations (Alfthan and Neve, 1996; MacPherson et al., 1997).
Indeed, in 1991 a French study showed a large-scale deficit in micronutrients,
including Se affecting 3040% of the healthy population (Hercberg et al., 1991).
This was confirmed by many other studies showing low plasma concentrations of
selenium. Plasma selenium concentrations are decreasing progressively in the
healthy European population since the 1980s, reflecting lower nutritional selenium
intake due to decreased nutrient Se content (Rayman, 1997; 2002; 2003; 2004).
The mean plasma concentration in various European areas (4085 g/l) is
substantially lower than the selenium concentration associated with a cancer
prevention activity according to the American Nutritional Cancer Prevention Study
or Se levels that required for maintenance of an optimal plasma GSH-Px activity
(Rayman, 2004).
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Table 11.5 Low daily selenium intakes in selected countries, g/day
Country
g/day
Reference
China, Keshan disease area

China, Keshan disease area
New Zealand, low-Se area
Saudi Arabia
Poland
UK
2-36
7-11
11
15
11- 40
12-43
New Guinea
Czech republic
Nepal
Finland before selenium
fertilization
India,vegan low income
Egypt
Serbia
Slovenia
China
Croatia
Slovakia
Belgium
20
15- 50
23
26
27
29
30
30
26.0 - 37.2
27.3-33.9
27-38.2
28.4-61.1
Brazil
New Zealand
28.4-37.0
29-38
Luo et al., 1985

Combs, 2001
Robinson and Thomason, 1984
Al-Saleh et al., 1997
Rayman, 2000; Wasowicz et al., 2003
Barclay et al., 1995; MAFF 1997; Church et
al., 1998; Jackson et al., 2003; Barclay and
MacPherson, 1992
Donovan et al., 1992
Kvicala et al, 2003
Moser et al., 1988
Koivistoinen and Varo, 1987; Mutanen, 1984;
1985
Mahalingam et al., 1997
Reilly, 1996; Maxia et al., 1972
Djujic et al. (Cited by Combs, 2001)
Pokom et al., 1998
Hu et al., 2002; Chen et al., 2000
Klapec et al., 1998, Matek et al., 2000
Kadrabova et al., 1996; 1998
Robberecht and Deelstra, 1994; Oster and
Prellwitz, 1989
Maihara et al., 2004
Duffield et al., 1999; de Jong et al., 2001;
McLachlan et al., 2004
Becker and Kumpulainen, 1991; Rayman,
2000; Nydhal et al., 2003
Pelus et al., 1994; Lamand et al., 1994
Reilly, 1996; Foster and Sumar, 1997; Giray
and Hincal, 2004
MacPherson et al., 1997
MAFF, 1997
Brown et al., 2000
Diaz-Alarcon et al., 1996
Oster and Prellwitz, 1989; Rayman, 2000
Reis et al., 1990
Rayman, 2000
Allegrini et al., 1985
Mahalingam et al., 1997
Sima and Pfannhauser (Cited by Combs, 2001)
Murphy et al., 2002
Sweden
29-44
France
Turkey
29-48
30-36.5
UK, 1994
UK, 1995
England
Spain
Germany
Portugal
Denmark
Italy
UK, 1985
India, conventional diet
Austria
Ireland
UK, 1974
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32
33
35
35
35-48
37
38-47
43
43
48
48
50
60
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Se distribution and reserves in human body

Selenium enters the human body in several forms including selenomethionine, derived
mainly from plants, and selenocysteine as an integral part of animal selenoproteins.
As mentioned in previous chapters, SeMet is not specifically recognized as a selenium
compound and is metabolized in the methionine pool and non-specifically
incorporated into body proteins. Indeed, such incorporation of Se into the muscle
proteins could build Se reserves in the body. These reserves are made available for
specific use when proteins are catabolised releasing free SeMet. This SeMet can be
degraded to selenide and then be incorporated into selenoproteins.
Did you know that SeMet is a storage form of Se
in the human body?
It seems likely that absorption does not play any role in the homeostatic regulation of
selenium with SeMet to be absorbed almost completely and efficiency of absorption
of sodium selenite and selenate is somehow lower (Burk and Levander, 1999). In
fact, Se absorption in free-living males on self-selected diets was shown to be 682%
(Levander and Morris, 1984) or 626% (Levander et al., 1981) while in females it
was 612% (Levander and Morris, 1984), 555% (Stewart et al., 1978) or 71-76%
in healthy white girls aged 11 to 14 years (Holben et al., 2002). Recently using
labelled sodium selenite it was shown that its absorption in young men comprised
only 15.3 4.7%, while sodium selenate was absorbed at 68.5 2.5 % (Finley, 1999;
Table 11.6). Similarly, Christensen et al. (1983) using a triple stable-isotope method,
found that the absorption of Se from selenite was 36% and that from intrinsically
labelled poultry meat was 71%. The absorption of dietary Se in the form of SeMet in
solution was >95% (Thomson et al., 1978), selenate >90% and selenite only about
60% (Thomson et al., 1986).
Table 11.6 Absorption of stable isotopes of Se fed to young men, % administered dose (Adapted from
Finley, 1999).
Low dietary Se, 32.6 g/day
Absorption
Urine Se
Retained Se
High dietary Se, 226.5 g/day
Broccoli
Selenate
Selenite
Broccoli
Selenate
Selenite
66.4
7.9
58.5
68.5
28.6
39.9
15.3
5.1
12.7
74.4
14.6
59.8
76.0
37.5
37.8
38.0
14.2
26.3
Labelled Se (infused as sodium selenite) was incorporated predominantly in the

plasma selenoprotein P fraction and the half-life of Se in this fraction was
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determined to be 1.9 to 2.9 days (Janghorbani et al., 1999). In fact equilibrium

distribution of plasma Se corresponded to (as % of plasma Se): 43.5 4.3 for SeP,
29.6 4.3 for GSH-Px and 37.1 9.6 for albumin. Similar data on Se distribution
in plasma of Chinese men were reported by Deagen et al. (1993) with 58-60% Se
found in SeP, 17-23% in GSH-Px and 17-32% in albumin.
Selenium accumulation in the human body depends on many factors including Se
content in the diet. For example, the amount of selenium in humans in Poland was
approximately 5.2 mg, while in New Zealand 3.0-6.1 mg, Germany 6.6 mg and in
the United States 13.0-20.3 mg (Zachara et al., 2001). The authors obtained tissue
samples taken at autopsy from 46 healthy individuals killed in Poland to analyse
selenium levels. The per-weight-unit basis selenium levels (ng/g wet tissue) in tissues
decreased in the following descending order: kidney (469 ng/g) > liver > spleen >
pancreas > heart > brain > lung > bone > skeletal muscle (51 ng/g). Most of the whole
body selenium was found in skeletal muscles (25-50%), which appears to act as a Se
storage organ; much less selenium was found in bones (16%), blood (7-10%) and
kidney (4%, Oster et al., 1988; Zachara et al., 2001).
The pharmacokinetics of Se supplementation was studied in six adults receiving a
single dose of 200 g of 74Se as SeMet (Swanson et al., 1990). The authors showed
that an average turnover time in the plasma was 0.01-1.2 days, in the liver 1.6-3.1
days and in peripheral tissues 61-86 days. The estimated half-life was found to be
200-300 h. Urinary and fecal selenium excretion composes the primary routes of
disposal, and it has been shown that urinary selenium excretion decreases with lower
dietary selenium intakes (Robinson et al., 1985). Therefore, under physiological
conditions urinary excretion is considered to be the primary route of body selenium
regulation and humans appear to adjust their selenium excretion for maintenance of
selenium status. In fact, in healthy North American adults approximately 80% of
excreted selenium was via urine, whereas 20% was via feces (Veillon et al, 1990).
However, at very high Se intakes, volatile forms of selenium, mainly dimethylselenide,
are exhaled and the breath becomes a significant route of excretion. Estimates of the
daily excretion of selenium with the bile indicate that the amounts are three times
higher than the daily urinary losses and in the same order of magnitude as the daily
dietary selenium intakes (Oster et al., 1988). It is interesting to note that most excretory
metabolites of selenium appear to be methylated including methylselenol and
trimethylselenonium ion (Burk and Levander, 1999).
Selenium deficiency
Selenium essentiality for human is proven and based on the following (Burk and
Levander, 1999):
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Selenium is a component of about 25 selenoproteins identified in human

body and playing important roles in regulation of various physiological
functions
Selenium supplementation of depleted patients undergoing long-term total
parenteral nutrition had favourable responses
Selenium deficiency has been implicated in certain human diseases and Se
supplementation was shown to be cancer-protective and immunomodulatory
in human
The signs and symptoms of selenium deficiency closely simulate each other for
animals and man. Deficiency states have been demonstrated for inhabitants of
regions where selenium supply is limited, in protein-energy malnutrition, and in
patients maintained on total parenteral nutrition without selenium supplementation.
Severe deficiency is characterized by cardiomyopathy while moderate deficiency
results in less severe, myodegenerative syndromes such as muscular weakness
and pain as well as a variety of other selenium-associated diseases (Koller and
Exon, 1986). For example, a suspected Se deficiency syndrome has been
demonstrated in a few patients treated with parenteral nutrition without added Se
(Rannem et al, 1996). Muscular pain and muscular and cardiac dysfunction have
been demonstrated in some patients, but no uniform symptomatology has been
described. A case of fatal cardiomyopathy caused by selenium deficiency during
parenteral nutrition for 6 consecutive years was also described (Fleming et al.,
1982). Furthermore, a case of fulminant heart failure in a middle aged woman
with a complex medical and surgical history including documented malabsorption
and selenium deficiency was presented where pathological examination of the
heart showed features consistent with Keshan disease (Burke and Opeskin, 2002).
Clinical manifestations of many of these disorders require contributory factors,
such as stress, to precipitate symptoms which are documented for animals and
implicated for humans.
Se deficiency in human population is associated with two diseases (Keshan
disease and Kaschin-Beck disease) reported in areas of China and some other
countries characterised by extremely low Se content in the soil and food. In
particular, the diseases occur in a geographic belt stretching from Heilonjiang
Province in north-east China to Yunnan Province in the south-west (Fordyce et
al., 2000).
Keshans disease is a cardiomyopathy, which mainly affects young children and
women of child-bearing age, has occurred in some areas of China where the soil
is low in Se (Chen et al., 1980). In fact, Keshan disease was first described in
Chinese medical literature more than 100 years ago, but not until 40 years after its
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widespread occurrence in 1935 was it discovered that Se deficiency was an

important factor in its aetiology (FAO/WHO, 1996). The earliest identification of
this disease was in the Keshan area of the north eastern provinces of China and
this gave the name to the disease. In the period between 1959 and 1970 peak
Keshan disease rates exceeded 40 per 100,000 (approximately 8500 new cases
per annum) with 1400-3000 death recorded each year (Fordyce et al., 2000). For
example, Keshan Disease occurred in 6 communities in the north-west of Lichuan
County. In total, 213 people have suffered KD in the County. From those with
KD, 136 recovered, 163 died and 13 persons continue suffering. Children between
the ages of 3-8 accounted for 83.4% of the total cases and 80% of the children
affected by the disease died (Fordyce et al., 2000). The acute form is characterized
by sudden onset of insufficient heart function, whereas individuals with chronic
disease exhibit moderate-to-severe heart enlargement with varying degrees of
heart insufficiency (Ge et al., 1983). Typical manifestations are fatigue after even
mild exercise, cardiac arrhythmia and palpitations, loss of appetite, cardiac
insufficiency, cardiomegaly, and congestive heart failure (FAO/WHO, 1996).
Pathologic changes include a multifocal myocardial necrosis and fibrosis. The
coronary arteries are essentially unaffected. The additional symptoms of the disease
also include: coughing, difficulty on breathing, vomiting/or tendency to vomiting,
chest uncomfortable, low desire for food; swollen body, severe lung enlargement
and pass out.
Ultrastructural studies showed that membranous organelles, such as
mitochondria or sarcolemma, are affected earliest, while the histopathological
features include:
multifocal necrosis
fibrous replacement of the myocardium
myocytolysis
It is important to note that there are other factors that affect this disease development
including (Burk and Levander, 1999):
marginal-to-deficient vitamin E status

other complicating nutritional deficiencies and disbalances (e.g., protein,
polyunsaturated fatty acids, etc. )
presence of a cardiotoxic agent, such as a virus; in fact data presented in
Chapter 4 related to Se and immunity indicate that viruses can mutate in the
Se-deficient host. It seems likely that this is the case for areas with low Se
level in China where Keshan disease occurs.
A series of intervention trials with Se supplementation proved to be effective in

prevention of Keshan disease. As a result of Se inclusion in table salt, Se status of
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general population in those high risk areas was improved, there was also
improvement in general diet and as a result Keshan disease practically disappeared
from endemic areas (Yang et al., 1984). In a large, prospective, placebo-controlled
study conducted in China (Keshan Disease Research Group, 1979a), the incidence
of Keshan disease in selenium-supplemented children fell from 2.2% in 1974 to
1% in 1975, to 0.32% in 1976 and in 1977 no cases of the disease were reported.
Did you know that Keshan disease and Kashin-Beck diseases
are related to Se deficiency?
KaschinBeck disease, is an osteoarthropathy, a generative articular disease caused
by oxidative damage to cartilage that leads to deformation of bone structure (Tan,
1989; Ge and Yang, 1993). The disease is endemic in Tibet and other areas of
China, Siberia, and North Korea areas where Se deficiency is also endemic
(Sokoloff, 1989). This Se-responsive bone and joint disease has been detected in
children aged 5-13 years in China and less extensively in south-east Siberia. Indeed,
the disease occurs during preadolescence or adolescence (Allander, 1994). The
disease is characterised by joint necrosis - epiphyseal degeneration of the arm
and leg joints resulting in structural shortening of the fingers and long bones with
consequent growth retardation and stunting (FAO/WHO, 1996). Therefore,
affected subjects have varying degrees of joint deformation and limited joint
mobility. In the most severe cases, there is necrosis of growth plates and joint
cartilage (Moreno-Reyes et al., 1998). Therefore, necrotic degeneration of the
chondrocytes is the most striking pathologic feature of this disease. Dwarfism
and joint deformation result from these cartilage abnormalities (Tan, 1989; Burk
and Levander, 1999). As mentioned above, Kaschin-Beck disease occurs in areas
where the availability of soil Se for crop growth is low. The selenium contents of
hair and of whole blood are abnormally low and the blood content of GSH-Px is
reduced. The disease has been reported in white migrants to the areas of endemic
disease, and clinical improvement was observed in children who move to areas
where the disease is not endemic (Sokoloff, 1990). In addition to Se deficiency,
a number of other etiologic factors have been suggested for this condition,
including mycotoxins in grain, mineral imbalance, organic contaminants in
drinking water and some others. It has also been hypothesized that iodine deficiency
and KaschinBeck disease might be associated (Moreno-Reyes et al., 1998). A
spontaneous decrease in incidence from 1970 (44 percent) to 1980 (14 percent)
to 1986 (1 percent) has been attributed to general improvements in the nutritional
status of Chinese rural communities (FAO/WHO, 1996). However, the efficacy of
Se supplements in the prevention of KaschinBeck disease is still controversial.
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When establishing Se requirement for human there are the same problems as
mentioned above in relation to animals. The main problem is to decide what kind
of measurements/tests to use for such an assessment. On the one hand, Se dietary
doses providing maximal activity of GSH-Px have been actively used to establish
human Se requirement. On the other hand, GSH-Px is only one from at least 25
selenoproteins identified in human body. Furthermore, maximal expression of
GSH-Px does not mean that all other selenoproteins will have maximum expression
at the same Se dose. For example, recently it has been shown that expression of
SeP is not maximum at the Se levels providing maximum activity of GSH-Px
(Burk and Hill, 2005; Xia et al., 2005). Furthermore, TR activity is increasing
with Se doses much higher than those needed for maximal expression of GSH-Px
(Gromer et al., 2004). Since there are analytical difficulties to assess expression
and activities of all selenoproteins at certain Se doses there is always uncertainty
if a used parameter would be adequate. However, the bigger problem is related to
functional changes in the body depending on Se status. For example,
immunomodulating properties of Se are shown (see chapter 4) at doses much
higher than those considered to be adequate to maintain growth and development.
Similarly, anticancer properties of Se are also shown at doses much higher than
RDI.
There are also other factors affecting Se requirement:
diet composition
antioxidant concentration in the diet
level of stress
health status
Therefore, RDI would not be considered as an optimal Se requirement, since it

would be changeable depending on aforementioned factors.
In 1980, the National Research Council (USA) established an estimated safe
and adequate daily dietary selenium intake for adults of 50 to 200 g (NRC,
1980). This recommendation was the first dietary standard for selenium and was
based primarily on data extrapolation from animal experiments because few human
data were available at that time. Later balance human studies and dietary surveys
in areas with different Se status helped to further clarify Se requirement. However,
balance studies were shown to be of little use for this purpose. Therefore first
Recommended Dietary Allowance (RDA) for selenium was proposed in 1989
(FNB, 1989). By using the dietary Se intake needed to maximize the activity of
GSH-Px in Chinese people living in Keshan disease area. It was calculated that
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plasma GSH-Px was maximised at Se supplementation of 30 g/day. By adding

11 g/day from the food it was calculated that 41 g/day Se would provide
maximum GSH-Px activity (Yang et al., 1987). By taking into account body
weight and safety factors Se requirement was calculated to be 70 and 55 g/day
for adult men and women respectively (Burk and Levander, 1999). It should be
noted that the daily requirements of selenium remains controversial. Thus, although
dietary selenium intake of 40 g/day is considered as adequate for prevention of
Keshan disease, higher intake of 50-200 g/day (Badmaev et al., 1996) or even
400-600 g/day (Yang and Xia, 1995) have been recommended for treating active
conditions. An intake of 40 g/day was suggested as the minimum Se amount
required for humans (Whanger, 1998).
An expert consultation of the WHO produced dietary selenium standards that
were considerably lower than the RDAs produced in the United States (FAO/
WHO, 1996). The basal requirement was defined as the intake needed to prevent
pathologically relevant and clinically detectable signs of impaired function
attributable to inadequacy of the nutrient, whereas the normative requirement
was defined as the intake that serves to maintain a level of tissue storage or other
reserve that is judged by the Expert Consultation to be desirable. (Levander,
1999). Moreover, the WHO advisory group presented the values in their report in
the form of safe ranges of population mean intakes, which meant that their
recommendations were applicable only to large groups of people and not to
individual persons (Burk and Levander, 1999). The population minimum mean
intake of selenium likely to meet basal requirements was given as 21 and 16 g/
day for adult males and females, respectively, The population mean intake of
selenium that would meet the normative requirements was given as 40 and 30 g/
day for adult males and females, respectively. These figures were calculated from
Chinese experimental data that showed the amount of ingested selenium needed
to achieve two-thirds of the maximum attainable activity of plasma GSH-Px. This
is in contrast to the RDA approach in the United States, which favoured the dietary
intake needed to achieve full activity of the enzyme (Burk and Levander, 1999).
Indeed, an approach using Se doses providing two-third of the maximum GSHPx activity (which is necessary for blood cells to effectively metabolise hydrogen
peroxide) has limited values, since GSH-Px has a variety of functions in the body
and it is one from about 25 selenoproteins identified to date in the human body.
Did you know that a daily RDA of 55 g for adults including
men and women was set by Institute of Medicine (USA) and the
British governments defined reference nutrient intake is
75 g/day for men and 60 g/day for women?
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Recently a human study was carried out in New Zealand by Duffield et al. (1999).
In the study men and women were given Se supplements (placebo, 10,20, 30 or
40 g) for 20 weeks and they had an average dietary Se intake of 28 g/day. The
authors showed that GSH-Px activity reached a plateau only in the group with the
maximal Se supplementation and they proposed an intake of 90 g Se as
recommended daily allowance. The Institute of Medicine (USA) used two studies
(Chinese study of 1983 and aforementioned New Zealand study) to establish
RDA in selenium. As a result of this analysis the recommendations of the Panel
was for a daily RDA of 55 g for adults including men and women (Institute of
Medicine, 2000; Table 11.7). The British governments defined reference nutrient
intake is 75 g/day for men and 60 g/day for women (Rayman, 2000; Department
of Health, 1991).
Table 11.7 The 2000 Reference Dietary Intakes (RDI) for Selenium
Life stage
Age
Infants
0 6 months
7-12 months
1-3 years
4-8 years
9-13 years
14-70 years
>70 years
9-13 years
14-70 years
>70 years
Children
Males
Females
Pregnancy
Lactation
RDI for Se, g/day

15
20
20
30
40
55
55
40
55
55
60
70
The Reference Daily Intake suggested by other countries are as follows (Levander,
1999):
The Nordic countries: 30 to 60 g/d for adult males and females (Standing
Nordic Committee on Food, 1989).
The German Estimated Value for Adequate Supply: 20 to 100 g/d for adults
(Committee on Nutrient Requirements, 1991).
The Australians: 85 and 70 g/d for men and women, respectively (Truswell
et al., 1990; Table 11.8).
Se status assessment
The definition of the exact role of selenium in human homeostasis has been
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Table 11.8 Australian recommended dietary intakes for selenium (Adapted from Tinggi, 2003)
Age group
Infants
0-6 months
7-12 months
Children
8-11 years
12-18 years
g/day
Age group
g/day
10
15
Adults
Males
Females
85
70
50
85
Pregnancy
Lactation
+10
+15
Due to the limited data available for Se dietary survey in Australia, the estimation for the
Australian RDI is based on the data from other countries.
complicated by the lack of a sensitive parameter, usable in routine investigation,

to assess selenium status. Measurements of plasma and urinary Se levels, although
useful in clinical practice, are inadequate indicators. The only true evidence of Se
deficiency lies in a positive response to selenium therapy (Neve et al., 1985).
Therefore, to evaluate Se status of human is a very difficult task. In fact, all
advantages and disadvantages of various methods described for animals above
are the same for human. The most frequent tests used to date include Se level in
plasma, serum or total blood, Se concentration in toenail as well as GSH-Px activity
in plasma or whole blood. It seems reasonable to agree with a conclusion of Burk
and Levander (1999) that at present, there are no suitable clinical methods for
evaluating selenium status.
Selenium and health

When considering health-related properties of Se it is important to concentrate on
those diseases which are considered to be the major cause of mortality in modern
society. Indeed, in the USA cardiovascular diseases and cancer are responsible
for more than 50% of total death (Table 11.9). Therefore, in this chapter a special
emphasis will be given to the role of Se in prevention of these two diseases.
Reilly (1998) presented more than 40 human diseases and conditions associated
with selenium deficiency. These include aging, arthritis, cancer, cardiovascular
disease, cataracts, cholestasis, cystyc fibrosis, diabetes, immunodeficiency,
Kaschin-Beck disease, Keshan disease, lymphoblastic anemia, macular
degeneration, muscular dystrophy, stroke and some others. Adequate Se is also
essential for immune function and can protect the immune system from oxidative
damage (McKenzie et al., 1998). The results clearly indicate that selenium plays
an important role in human health and prevention disease. The mechanisms of Se
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Table 11.9 Major Causes of Death in United States in 1995 and 2002(Adapted from Knight, 1999 and
Cancer Statistics, 2005)
1995
Cause of death
Heart disease
Cancer
Cerebrovascular
Chronic pulmonary diseases
Accidents
Pneumonia/influenza
Diabetes mellitus
HIV infection
Suicide
Chronic liver disease and
cirrhosis
2002
Rank
Number
died
Percent of
total
1
2
3
4
5
6
7
8
9
10
738,781
537,969
158,061
104,756
89,703
83,528
59,085
42,506
30,893
24,848
32.0
23.3
6.8
4.5
3.9
3.6
2.6
1.8
1.3
1.1
Number
died
Percent of
total
696,947
557,271*
162,672
124,816
106,742
65,681
73,249
58,8661
40,9742
33.8653
28.5
22.8
6.7
5.1
4.4
2.7
3.0
2.41
1.72
1.43
1
/Alzheimer disease; 2Nephritis; 3Septicemia
*In 2005 estimated number of new cancer cases in the USA is 1,372,910 and 580,280
Americans are expected to die of cancer, more than 1500 people a day (Cancer Facts and
Figure, 2005)
participation in such conditions needs further elucidation but it appears that in

many cases the antioxidant role of selenoproteins is the driving force. Clearly, Se
appears as the key micronutrient in prevention of cancer, cardiovascular,
inflammatory and infectious diseases.
Cancer
Cancer is a major public health problem in the developed countries. It has been
calculated by Parkin et al (2005) that globally in 2002, there were:
10.9 million new cases of cancer

6.7 million deaths
24.6 million persons alive with cancer (within three years of diagnosis).
The most commonly diagnosed cancers were (Figures 11.2 and 11.3):
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lung (1.35 million, accounting for 12% of all cases),

breast (1.15 million),
colorectal (1 million).
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1400000
1200000
1000000
800000
600000
400000
Bladder
Oesophagus
Cervix
Liver
Prostate
Stomach
Bowel
Breast
Lung
200000
Figure 11.2 The most commonly diagnosed cancers worldwide in 2002 (Adapted from http://
info.cancerresearchuk.org/cancerstats/world/commoncancers
1200000
1000000
800000
600000
400000
Prostate
Pancreas
Cervix
Oesophagus
Breast
Bowel
Liver
Stomach
Lung
200000
Figure 11.3 The most common causes of death from cancer worldwide, 2002 estimates (Adapted from
http://info.cancerresearchuk.org/cancerstats/world/commoncancers
Overall, cancers of the lung, breast, bowel, stomach and prostate accounted for
almost half of all cancer diagnosed worldwide. Since 1975 the number of people
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being diagnosed with lung cancer worldwide has doubled, it is now the most
common cancer. The incidence rate for male lung cancer varies by 12-fold between
the different regions of the world, while the female rate varies by more than 30fold.
Did you know that the most common causes of cancer death
are lung cancer, stomach cancer and liver cancer?
The most common causes of cancer death are:
lung cancer (1.18 million deaths; five-year survival rates for lung cancer
are consistently poor at between 7-14% (Parkin et al., 1999) so mortality
patterns follow incidence patterns closely),
stomach cancer (700,000 deaths),
liver cancer (598,000 deaths).
Therefore lung cancer is the most common cause of death from cancer accounting
for 18% of all deaths from cancer. The most prevalent cancer in the world is
breast cancer (4.4 million survivors up to 5 years following diagnosis; Parkin et
al., 2005). One in ten of all new cancers diagnosed and almost one in four cancers
diagnosed in women worldwide is a cancer of the female breast. More than 1.1
million women are diagnosed each year and the numbers of women being
diagnosed annually worldwide has almost doubled since 1975. Incidence rates of
breast cancer are increasing in most countries, and the changes are usually greatest
in areas where rates were previously low. Breast cancer is the main cause of death
from cancer in women globally.
Each year an estimated one million people are diagnosed with bowel cancer
and it accounts for 9% of all cases. There have been steady increases worldwide
in the numbers of people being diagnosed with bowel cancer over the last 25
years. Bowel cancer is the fourth most common cause of death from cancer
worldwide accounting for 8% of deaths from cancer. Prostate cancer is the fifth
most commonly diagnosed cancer worldwide, it accounts for 6% of all cases, and
there have been large increases in the incidence of prostate cancer over the last
25 years (Parkin et al., 2005).
Currently, one in four deaths in the United States is due to cancer (Tables 11.10
and 11.11). In accordance with cancer statistics in the USA in 2005 (Jemal et al.,
2005):
Did you know that one in four deaths in the United States
is due to cancer?
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Table 11.10 Cancer incidence rates in the US in 1997-2001 (Adapted from American Cancer Society, 2005)
Males
Cancer
African American
rate
Prostate
Breast
Lung and bronchus
Colon and rectum
Oral cavity and pharynx
Ovary
Urinary bladder
Stomach
Kidney and renal pelvis
Non-Hodkin lymphoma
Pancreas
Leukemia
Myeloma
Larynx
Liver and intrahepatic bile duct
Esophagus
Brain and other nervous system
All malignant neoplasms
274.3
120.7
73.1
20.1
20.0
19.4
18.9
18.4
17.4
13.0
12.9
12.1
11.6
11.3
4.9
697.3
Females
White
rate
African American
rate
White
rate
171.2
82.3
64.4
15.9
41.2
10.2
16.5
24.5
12.6
17.0
6.7
6.8
6.7
8.1
8.8
568.3
118.6
55.3
56.1
5.9
10.3
7.8
9.8
10.0
11.2
14.2
8.0
10.2
2.7
3.9
3.9
3.2
400.2
143.2
53.5
46.8
6.6
15.0
10.2
4.5
8.2
16.9
9.5
10.1
4.2
1.5
2.6
2.0
6.1
435.1
Rates are per 100,000 and age-adjusted to the 2000 US population standard
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A total of 1,372,910 new cancer cases and 570,280 deaths are expected
corresponding to more than 1,500 deaths per day. Cancer accounted for
approximately 23% of all deaths, ranking second only to heart disease. Similarly,
in 2004 the American Cancer Society estimates 1,368,050 new cancer cases
and 536,700 deaths from the disease. Although cancer rates for different sites
vary widely, the 5-year relative survival rate for all cancers combined is estimated
at 63% (Borek, 2004).
The lifetime probability of developing cancer is 46% for men and 38% for
women. This means that during the lifetime almost one in two men and more
than one in three women will develop cancer.
Among men, cancers of the prostate, lung and bronchus, and colon and rectum
account for more than 56% of all newly diagnosed cancers
Prostate cancer alone accounts for approximately 33% (232,090) of incident
cases in men
The three most commonly diagnosed cancers among women in 2005 will be
cancers of the breast, lung and bronchus, and colon and rectum, accounting
for approximately 55% of estimated cancer cases in women
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Table 11.11 Cancer mortality rates in the US in 1997-2001 (Adapted from American Cancer Society, 2005)
Males
Cancer
African American
rate
Lung and bronchus

Prostate
Breast
Colon and rectum
Pancreas
Urinary bladder
Stomach
Esophagus
Liver and intrahepatic bile duct
Ovary
Leukemia
Myeloma
Oral cavity and pharynx
Non-Hodkin lymphoma
Kidney and renal pelvis
Larynx
Brain and other nervous system
All malignant neoplasms
Females
White
rate
104.1
70.4
34.3
16.0
5.6
13.3
11.7
9.3
76.6
28.8
24.8
12.0
7.9
5.8
7.4
6.1
9.0
9.0
7.5
7.4
6.3
5.4
3.3
352.2
10.5
4.4
3.9
10.8
6.2
2.3
6.0
250.5
African American
rate
White
rate
39.9
41.6
35.4
24.5
12.8
2.9
6.3
3.2
3.8
9.2
5.4
6.6
2.0
4.6
2.7
0.9
2.3
200.4
26.4
17.1
8.9
2.3
2.8
1.7
2.7
7.5
6.0
2.9
1.6
7.2
2.8
0.5
4.0
169.1
Rates are per 100,000 and age-adjusted to the 2000 US population standard
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Breast cancer alone is expected to account for 32% (211,240) of all new
cancer cases among women
Cancers of the lung and bronchus, prostate, and colon and rectum in men
and cancers of the lung and bronchus, breast, and colon and rectum in women
are the most common fatal cancers. These four cancers account for 50% of
the total cancer deaths among men and women. Indeed, colorectal cancer
is the second leading cause of mortality in the United States. In the United
States, the cumulative lifetime risk of developing colorectal cancer for both
men and women is 6%. Despite advances in the management of this disease,
the 5-year survival rate in the United States in only 62%. Four classes of
chemopreventive compounds have demonstrated efficacy in reducing
recurrent colorectal adenomas and/or cancer in randomized, controlled trials.
They are selenium, calcium carbonate, hormone replacement therapy, and
nonsteroidal anti-inflammatory drugs (Hawk and Levin, 2005).
When age-adjusted death rates are considered cancer is the leading cause of
death among men and women under age 85. Indeed, cancer is the leading
cause of death among women aged 40 to 79 and among men aged 60 to 79.
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For example, a total of 476,009 people under age 85 died from cancer in
the United States in 2002 compared with 450,637 deaths from heart disease
Among men, cancer of the lung and bronchus predominates in men aged
40 years and older. Colorectal cancer is the second most common cause of
cancer death among men 40 to 79 years old, and prostate cancer is the
second most common among men aged 80 and older.
Among women, breast cancer ranks first at ages 20 to 59 years, and lung
cancer ranks first at age 60 years and older. Indeed, lung cancer surpassed
breast cancer as the leading cause of cancer death in women in 1987. Lung
cancer is expected to account for 27% of all female cancer deaths in 2005.
Did you know that genetic damage, leading to the accumulation
of specific mutations, is a prerequisite for the cells
transformation from a normal into a malignant phenotype?
It is generally accepted that genetic damage, leading to the accumulation of specific

mutations, is a prerequisite for the cells transformation from a normal into a
malignant phenotype. Therefore antioxidant compounds preventing genetic
damage possess anti-cancer properties.
Selenium and cancer

Doll and Peto (1981) and Doll (1992) estimated that 35% of cancer deaths in the
United States were attributable to diet, with no specific reference to obesity or physical
inactivity. The importance of diet on the development and progression of prostate
cancer was initially suggested by epidemiological studies. Compelling evidence now
provides hope that evidence-based dietary alterations may markedly alter the natural
history of this disease. It seems likely that among many nutritional factors affecting
cancer Se has a special place. There are several lines of evidence indicating protective
effect of various selenium compounds against cancer. They include:
Untitled-2
epidemiological observations and prospective studies where an inverse

correlation between Se levels in food and blood and risk of cancer and
cancer mortality were observed
case-control studies showing that Se levels in blood, serum, hair or toenails
are lower in cancer patients than in controls
laboratory animal studies showing a protective effect of various forms of
Se against cancer initiation and development
human intervention trials showing Se supplementation to be effective means
of decreasing risk of cancer
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672

Did you know that there are several lines of evidence
indicating protective effect of various selenium
compounds against cancer?
Epidemiological observations and prospective studies

There is convincing evidence in the literature suggesting a cancer protective effect
of selenium in humans. For example, correlation studies conducted in different
regions worldwide and in the U.S. have shown an inverse association between
selenium levels in forage crops or diet and cancer mortality rates (Shamberger et
al. 1976; Schrauzer et al. 1977; Yu et al. 1985; Clark et al., 1991; Table 11.12).
Table 11.12 Age-specific (55-64 year group) cancer death rates in 1968 for white males in states different
in Se status (Adapted from Shamberger et al., 1976)
No of states
6
19
11
20
Se level, ppm
Cancer death rates/100,000
Probability
Very high,
0.26+
High,
0.1+
Medium,
0.06-0.09
Low,
0.01-0.05
392.0
P<0.001
429.7
P<0.001
450.0
P<0.001
516.0
Furthermore, several prospective and case-control studies also confirmed that

people with low blood selenium had an increased risk of cancer (Clark et al.
1984; 1993, Salonen et al. 1984; 1985, Willett et al. 1983; Tables 11.13-11.15).
Did you know that correlation studies conducted in different
regions worldwide and in the U.S. have shown an inverse
association between selenium levels in forage crops or diet
and cancer mortality rates?
Untitled-2
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673
Table 11.13 Ecological studies showing an inverse relationship between dietary intake of Se and cancer
risk (Adapted from Whanger, 2004; Meuillet et al., 2004; Combs and Gray, 1998)
Effects
References
Human cancer mortalities were lower in high- than in low-Se regions

of the USA
Shamberger and Frost,

1969; Schrauzer and
Rhead, 1971
Statistically significant inverse associations were obtained when

cancer mortalities were correlated with blood-Se levels of US
healthy donors
Shamberger and Willis,

1971
Inverse associations of cancer mortalities with the apparent

dietary Se intakes in different countries (a 27-country comparison)
Schrauzer et al., 1977
Inverse associations of cancer mortalities with the apparent dietary

Se intakes in China
Yu et al., 1985
Inverse correlation between geographic distribution of liver cancer

incidence and the Se contents of whole blood and grains in Qidong
county, Jiangsu province, a high liver cancer area of the Peoples
Republic of China
Yu et al., 1988; 1988a
Cancer mortality rates were significantly lower in intermediateselenium and high-selenium counties in comparison to low-selenium
counties for total cancer and cancers of the lung, colon, rectum,
bladder, esophagus, pancreas, breast, ovary and cervix
Clark et al., 1991
Table 11.14 Observational studies examining the relationship between Se status and cancer risk (Adapted
from Whanger, 2004; Meuillet et al., 2004; Combs and Gray, 1998)
Effects
References
In a U.S. study of 11,000 hypertensives followed for 5 years showed a Willett et al., 1983
two-fold increase in risk for all cancers in those in the lowest
(<115 ng/ml) quitile compared to those in the highest quintile
(<154 ng/ml) of plasma selenium. The association between low
selenium level and cancer was strongest for gastrointestinal and
prostatic cancers. Serum levels of vitamins A and E compounded
the effect of low selenium; relative risks for the lowest tertile of
selenium were 2.4 and 3.9 in the lowest tertiles of vitamins E and A,
respectively
A matched-pair analysis was conducted with data based on a
prospective six-year follow-up of a random population sample with
8,113 persons in eastern Finland. Cases were 31- to 59-year-old men
and women initially free of cancer. One control was matched to each
case according to age, gender, daily tobacco consumption, and serum
Untitled-2
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674
Table 11.14 Contd.
Effects
References
cholesterol concentration. The mean serum selenium of the 128 cases

was 50.5 g/l and that of the controls was 54.3 g/l (p = 0.012).
When possible confounders were taken into account serum selenium
of less than 45 g/l was associated with a relative risk of cancer of
3.1
Salonen et al., 1984
Patients who died of cancer during the follow up period had a 12%
lower mean serum selenium concentration than the controls. The
adjusted risk of fatal cancer was 5.8-fold among subjects in the lowest
tertile of selenium concentrations compared with those with higher
values. Subjects with both low selenium and low alpha-tocopherol
concentrations in serum had an 11.4-fold adjusted risk.
Sera from 43 persons who developed thyroid cancer on an average
Glattre et al., 1989
4.8 years after blood sampling were compared with sera from controls.
Cases were significantly lower in serum Se than controls, and the
estimated odds ratio of thyroid cancer increased from 1 for levels
greater than or equal to 1.65 mol/l, to 6.1 for levels 1.26-1.64 mol/l,
to 7.7 for levels less than or equal to 1.25 mol/l
Low serum and plasma Se concentrations were associated with
increased risk of malignant oral cavity lesions. Mean serum Se levels
were 105, 101, and 77 ng/ml in the precancerous, controls, and
malignancy groups, respectively
Toma et al., 1991
Compared with the lowest quartile of selenium (range 82-107 ng/ml),

the odds ratios of the second (108-118), third (119-132) and fourth
(133-182) quartiles were 0.15, 0.21 and 0.24 respectively. Therefore,
low plasma selenium was associated with a 4 to 5-fold increased risk
of prostate cancer.
Brooks et al., 2001
Low serum and plasma Se concentrations were associated with

increased risk of oesophageal and gastric cancers. The RR for
comparison of highest to lowest quartile of serum selenium was 0.56
for esophageal cancer and 0.47 for gastric cardia cancer
Mark et al., 2000
Cervical cancer mortality rates in China correlated negatively and

significantly with serum levels of selenium (r = -0.26, P < 0.05)
Guo et al., 1994
The mean plasma selenium concentrations for cases with colorectal

Russo et al., 1997
adenomas (n = 37) and noncases (n = 36) were 107 and 120 g/l,
respectively (p = 0.06). Those in the fourth quartile of plasma Se
level had 0.24 times the risk for colorectal adenomas of those in the
first quartile. The adjusted odds ratio for colorectal adenomas was 0.58
for a 30 g/l increase in plasma selenium level. Furthermore, lower
plasma Se levels were associated with multiple adenomas
Untitled-2
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675
Table 11.14 Contd.
Effects
References
A 10-year prospective study of 1,738 Americans found initial plasma Clark et al., 1993
Se levels inversely correlated to both non-melanoma skin cancer and
colonic adenomatous polyps. Those with plasma levels below the U.S.
population median of 128 ng/ml plasma were four times more likely to
have one or more polyps than those with levels above 128 ng/ml.
The association between selenium and prostate cancer risk was in the
protective direction with individuals in the top four fifths of the
distribution having a reduced risk of prostate cancer compared with
individuals in the bottom fifth
Helzlsouer et al., 2000
In an eight-year retrospective trial in Washington County, Maryland,

there was a nearly linear increase in risk of bladder cancer with
decreasing serum Se levels (P = 0.03). When examined by tertiles, the
odds ratio associated with the lowest tertile of selenium compared to
the highest tertile was 2.06.
Helzlsouer et al., 1989
Serum selenium was associated with a decreased risk of ovarian cancer Helzlsouer et al.,
among case participants diagnosed 4 or more years after blood
1995, 1996
collections (P for trend =0.02). Women in the highest tertile compared
with the lowest tertile were 4 times less likely to develop ovarian cancer
In Dutch patients the mean Se levels were significantly lower than
Kok et al., 1987
that of control values in men. The adjusted risk of death from cancer for
men in the lowest quintile of serum selenium (below 100.8 ng/ml) was
more than twice that of subjects with higher levels (relative risk = 2.7)
The association between toenail selenium and lung cancer was
van den Brandt et al.,
investigated in a cohort study of diet among 120,852 Dutch men and 1993a
women aged 55-69 years. The rate ratio of lung cancer for subjects in
the highest compared to the lowest quintile of toenail selenium, after
controlling for age, gender, smoking, and education, was 0.50 with a
significant inverse trend across quintiles. The protective effect of Se was
concentrated in subjects with a relatively low dietary intake of betacarotene or vitamin C.
In a multivariate analysis, the relative rates (RRs) of stomach cancer
van den Brandt et al.,
for subjects in increasing quintiles of toenail selenium level were 1.00, 1993b
0.44, 0.59, 0.84, and 0.64
Higher toenail Se levels were associated with a reduced risk of
Yoshizawa et al.,
advanced prostate cancer, OR for comparison of highest to lowest
1998
quintile was 0.49. After additionally controlling for family history of
prostate cancer, body mass index, calcium intake, lycopene intake,
saturated fat intake, vasectomy, and geographical region, the OR was 0.35
Untitled-2
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676
Table 11.14 Contd.
Effects
References
Low selenium status was associated with increased risk for lung cancer Hartman et al., 2002
The OR for men with adjusted toenail selenium concentrations at the
75th percentile compared to those with the lowest selenium
concentrations ranged between 0.20 and 0.61
The association between cancer of the pancreas and serum Se was
significant when the data were analyzed as a whole but its effect was
seen principally in men
Burney et al., 1989
Case-control differences in prediagnostic serum levels of selenium

were compared for 10 cancer sites in 10 study populations. The
majority of results showed lower levels among persons who
subsequently became cases than among controls
Comstock et al., 1992
After 7.5 years, low-dose antioxidant supplementation (120 mg of

ascorbic acid, 30 mg of vitamin E, 6 mg of beta-carotene, 100 g of
selenium, and 20 mg of zinc) lowered total cancer incidence and
all-cause mortality were observed in men but not in women
Hercberg et al., 2004
Selenium concentrations were measured from blood specimens from

a total of 1763 trial participants (3 trials), and quartiles of baseline
selenium were established from the pooled data. Individuals whose
blood Se values were in the highest quartile (median = 150 ng/ml)
had significantly lower odds of developing a new adenoma
compared with those in the lowest quartile (OR = 0.66)
Jacobs et al., 2004
The serum Se level of non-cancerous patients (n=2730) who later

Ujiie et al., 1998
developed cancer during the 3 years was determined and compared
with that of the non-cancerous patients (n=2127). A high incidence of
cancer was observed in the lower serum Se patients of the non-cancerous
group. The serum Se levels of cancerous patients were significantly lower
than in non-cancerous patients
In rural blacks living in areas with moderate to high esophageal cancer Jaskiewicz et al., 1988
(EC) incidence rates in southern Africa, the whole blood Se levels were
found to be significantly lower (58 to 71 ng/ml) than those of rural and
urban populations living in low EC rate areas (114 to 177 ng/ml). Mean
Se levels of subjects with premalignant or malignant esophageal
cytological changes (54 ng/ml) were significantly lower than those of
subjects without such lesions (68 ng/ml). An inverse relationship was
found between Se status and the degree of cytological abnormality
In Venezuela, a reduction in cancer was observed in the districts with
high serum selenium content. Further, serum selenium was lower in
male patients with cancer disease.
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676
Burguera et al., 1990
1/8/2007, 6:58 PM
677
Table 11.14 Contd.
Effects
References
In China, the relation between baseline serum selenium and the

subsequent risk of death over 15 y of follow-up was studied.
Significant inverse associations between baseline serum Se and
death from esophageal squamous cell carcinoma and gastric cardia
cancer was reported. Trends toward inverse associations were noted
for death from heart disease
Wei et al., 2004
The association between toenail selenium status and subsequent

Zeegers et al., 2002
bladder cancer incidence was investigated in a prospective cohort
study among 120,852 men and women aged 55-69 years at baseline.
The multivariable case-cohort analysis was based on 431 bladder cancer
cases and 2,459 subcohort members, for whom toenail selenium levels
were available. The age-, sex- and smoking-adjusted rate ratios for increasing
quintiles of toenail selenium were 1.00 (reference), 1.09 , 0.55 , 0.63,
and 0.67, respectively. An inverse association between toenail selenium
and bladder cancer risk was most pronounced among ex-smokers
Serum selenium was measured in 212 cases of prostate cancer and 233 Vogt et al., 2003
controls (black and white men aged 40-79 years). Serum Se was
inversely associated with risk of prostate cancer (comparing highest to
lowest quartiles, OR = 0.71) with similar patterns seen in both blacks and
whites. A reduced risk of prostate cancer above concentrations of 135 ng/ml
was shown.
Using plasma samples obtained in 1982 from healthy men enrolled in Li et al., 2004
the study, a nested case-control study among 586 men diagnosed with
prostate cancer during 13 years of follow-up and 577 control subjects was
conducted. Pre-diagnostic plasma selenium levels were inversely associated
with risk of advanced prostate cancer (5th versus 1st quintile OR = 0.52),
even among men diagnosed after 1990 (5th versus 1st quintile OR = 0.39).
The inverse association with prostate cancer risk was observed only for case
subjects with elevated baseline PSA levels (PSA >4 ng/ml, 5th versus
1st quintile OR = 0.49).
A nested case-control study in a cohort of 9345 Japanese-American
Nomura et al., 2000
men examined between 1971 and 1977 was conducted. After a
surveillance period of more than 20 years, 249 tissue-confirmed incident
cases of prostate cancer were identified. Their stored sera and those of
249 matched controls were measured for selenium levels. The
multivariate odds ratio for the highest quartile was 0.5. The inverse
association was more notable for cases with advanced disease and for
cases diagnosed 5-15 years after phlebotomy. However, the association
was mainly present in current or past cigarette smokers rather than non-smokers.
Untitled-2
677
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678
Table 11.14 Contd.
Effects
References
Serum samples from 9,101 cancer-free individuals were collected and Knekt et al., 1998
stored at -20 degrees C by the Finnish Mobile Clinic in 1968-1971
and 1973-1976. During follow-up until the end of 1991, 95 cases of
lung cancer were diagnosed. Selenium concentrations were determined
from the serum samples of the cases and 190 controls. Mean levels of
serum selenium in cases and controls were 53.2 ng/ml and 57.8 ng/ml,
respectively. The relative risk of lung cancer between the highest and
lowest tertiles of serum selenium, was 0.41. The association was
stronger at lower levels of alpha-tocopherol. The association was also
pronounced among current smokers.
In a prospective study of 39,268 men and women in Finland, risk for
several cancers (stomach, pancreas and lung) was significantly
elevated in men who had the lowest level of serum selenium
(53-63 g/l) , RR=2.5
Knekt et al., 1990
A prospective study of 4,857 Japanese patients followed serum Se

levels in those who developed cancers over 36-month period. Those
who developed cancer had significantly lower serum Se levels at
baseline than those who were cancer free
Ujiie et al., 1998;

Patrick, 2004
Studies in Chinas Qidong Province demonstrated a significant

inverse association between the incidence of primary hepatocellular
carcinoma and plasma Se levels
Blot et al., 1995
A meta-analysis examined nine studies that assessed prediagnostic

levels of antioxidants and 10 specific types of cancer. The levels of
serum Se in those who developed cancer of the stomach, pancreas,
lung and bladder were significantly lower than controls
Burney et al., 1989
In 70 colorectal cancer patients and 70 sex, age-matched subjects

without cancer history: diet history and detailed nutrient intake,
alcohol, smoking, physical activity, co-morbidities and body mass
index were evaluated. Age-adjusted Relative Risks were calculated.
Reduced colorectal cancer risk for the highest vs the lowest intake
quartile was found for selenium (0.36)
Ravasco et al., 2005
In a population study, 10,000 men plasma selenium was significantly

lower in cancer cases than in controls (means: 1.06 vs. 1.12 mol/l).
The proportion of cases increased significantly from the highest to
the lowest quintile of plasma selenium, and the relative risk for
cancer death was 3.8 times higher in the lowest quintile compared
with the highest
Fex et al., 1987
In a nested case-control study of a stratified sample from the

Kornitzer et al., 2004
Belgian population, serum selenium was shown to be an independent
Untitled-2
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679
Table 11.14 Contd.
Effects
References
predictor of cancer mortality in male subjects only. Unadjusted ORs

of cancer deaths taking the highest tertile of serum selenium as a
reference: in male subjects T1/T3 is 2.2, whereas in female subjects
a nonsignificant OR of 0.8 was observed
Smoking-adjusted risks of lung cancer were elevated only among
those in the lowest quartiles of serum selenium (OR = 1.8). Little or
no excess risk of stomach cancer was observed among those with
lowest levels of selenium (OR = 1.0). These was little association
with either lung or stomach cancer across normal selenium ranges in
this Japanese population.
Kabuto et al., 1994
Toenail selenium level was not associated with cancer at any major
site, including uterine cancer, colorectal cancer, melanoma, ovarian
cancer, or lung cancer
Garland et al., 1995
The association between serum selenium concentration and the risk

Virtamo et al., 1987
of cancer was studied in 1110 men aged 55 to 74 years in two rural
areas of Finland. The men were followed-up prospectively for 9 years.
The patients had a slightly lower adjusted mean serum selenium
than the subjects without cancer at the end of the follow-up
53.9 vs 55.3 g/l, respectively. The relative risks of cancer were
the same when these were calculated in the tertiles of the serum
selenium distribution.
Case control pairs came from a population of 9364 people in Norway. Ringstad et al., 1988
The mean serum selenium concentration of 123.2 g/l in patients
was not different from that in controls 128.7 g/l. The risk of
developing adenocarcinomas was not influenced by serum selenium
concentration.
In South Carolina coastal region, a study of 130 cancer patients who Peleg et al., 1985
developed a malignancy 2-12 years after baseline examination showed
no dose response relationship between baseline serum selenium levels
and risk of subsequent cancer.
In Northwest Washington State, 156 cases of cancer were identified in Coates et al., 1988
patients from whom specimens had been previously obtained for Se
analysis. Levels of selenium were unassociated with the incidence of
cancer of all sites combined
In the USA, a nested case-control study of squamous cell skin cancer
(SCC) was conducted. Those who developed a new SCC during the
3-5-year follow-up period were selected as cases (n = 132). There was
no consistent pattern of SCC risk associated with Se. The odd ratio
for the highest versus the lowest quartiles of selenium was 0.86
Untitled-2
679
Karagas et al., 1997
1/8/2007, 6:58 PM
680
Table 11.14 Contd.
Effects
References
A case-control study of cervical cancer in five areas of the US was

conducted. Values of serum Se ranged from 67.5 to 185.0 ng/ml.
Adjusted odds ratios for invasive cervical cancer by quintile were:
1.0 (highest selenium), 1.1, 1.0, 0.8, and 1.0 (lowest selenium), p for
trend = 0.82. There was no association between Se level and cervical
cancer risk
Thompson et al., 2002
A case-control study among 656 British men was conducted. There

Allen et al., 2004
was no association between Se levels in nails and prostate cancer risk:
men in the highest quartile of nail selenium had a slightly increased
risk compared with men in the lowest quartile (OR 1.24); for advanced
prostate cancer, men in the highest quartile had a slightly reduced risk
compared with men in the lowest quartile (OR 0.78).
The association between prediagnostic levels of Se in toenails and subsequent

risk of advanced prostate cancer by use of a nested case-control design within the
Health Professional Follow-Up Study was assessed. In 1986, 51529 male health
professionals aged 40-75 years responded to a mailed questionnaire to form the
prospective study (Yoshizawa et al., 1998). In 1987, 33737 cohort members
provided toenail clippings for Se analysis. From 1989 through 1994, 181 new
cases of advanced prostate cancer were reported. When matched case-control
data were analyzed, higher Se levels were associated with a reduced risk of
advanced prostate cancer (odds ratio, OR, for comparison of highest to lowest
quintile=0.49). After additionally controlling for family history of prostate cancer,
body mass index, calcium intake, vasectomy, and geographic region, the OR was
0.35. The relationship between toenail Se and cases of prostate cancer is shown
in the Figure 11.4 (Yoshizawa et al., 1998). Therefore, these data support the
hypothesis that a higher intake of selenium may reduce the risk of advanced
prostate cancer. In general, the majority of epidemiological studies provide
convincing evidence for selenium as a chemopreventive agent for specific cancers
(Patick, 2004) including:
Untitled-2
prostate (Combs, 2004; Hardell et al., 1995)

lung (Piccinini et al., 1996; Zachara et al., 1997; Knekt et al., 1998)
colorectal (Ghadirian et al., 2000; Salonen et al., 1985)
stomach (Nelson et al., 1995)
multiple cancers (Fex et al., 1987; Comstock et al., 1992; Willet et al.,
1983)
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681
Table 11.15 Prospective studies examining the role of selenium in cancer prevention (Adapted from
Donaldson, 2004)
Study
No.
No.
Outcomes
cases controls
Comments
Reference
Se = risk of
Result only in men
advance prostate
with PSA 4 ng/ml;
cancer (OR = 0.52,
13 years of follow up
95% CI = 0.280.98)
Physicians
586
Health Study
577
Li et al., 2004
Netherlands 540
Cohort Study
1,211
Baltimore
52
Longitudinal
Study of
Aging
96
Se = risk prostate
cancer (OR for
quartiles of Se = 1.0,
0.15, 0.21, 0.24
Low plasma selenium Brooks et al.,

is associated with a
2001
4 to 5-fold increased
risk of prostate cancer
Washington
County,
Maryland
117
233
Top 4/5 of Se had

reduction in prostate
cancer risk; statistically significant result
for Se only when
-tocopherol levels
were high
Men in top quintile of Helzlsouer et al.,

serum -tocopherol had 2000
5-fold reduced risk of
prostate cancer compared
to lowest quintile
Health
181
Professional
Follow-up
Study
181
Se = risk of
advanced prostate
cancer
Adjusted OR = 0.35
Yoshizawa et al.,
(95% CI = 0.160.78) 1998
Prospective
study
136
238
Se = risk of gastro- Results not statistically

intestinal and prostate significant
cancer
A case
control
study
212
233
Se = risk of
prostate cancer
Se = risk prostate Results greatest in ex- van den Brandt

cancer (OR for
smokers; 6.3 years of et al., 2003
quintiles of Se = 1.0, follow up
1.05, 0.69, 0.75, 0.69;
95% CI = 0.480.99)
Criqui et al.,
1991
OR=0.71 (95%CI
Vogt et al.,
0.39-1.26) comprising 2003
highest to lowest
quartiles
Recent data (Woodson et al., 2003) suggest an effect of the MnSOD ala/ala
genotype on the development of prostate cancer. In fact, men homozygous for
the MnSOD ala allele had a 70% increase in risk of prostate cancer over men
Untitled-2
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682

60
50
40
30
20
10
0
0.66
0.76
0.82
0.88
1.14
Se levels in nails (ppm)

Figure 11.4 Cancer cases depending on Se level in toenails (total number of cases was 181 from 33737
individuals; adapted from Yoshizava et al., 1998)
homozygous for the val allele. Furthermore men homozygous for MnSOD ala
(compared to MnSOD val/val or val/ala) showed a three-fold risk increase for
high-grade tumors. It is interesting to mention that among men with the AA
genotype, high selenium level (4th versus 1st quartile) was associated with a
relative risk (RR) of 0.3 for total prostate cancer; for clinically aggressive prostate
cancer, the RR was 0.2. In contrast, among men with the VV/VA genotype, the
RRs were 0.6 and 0.7 for total and clinically aggressive prostate cancer (Li et al.,
2005). The inverse association between baseline plasma selenium levels and risk
of advanced prostate cancer, even among men diagnosed during the post-PSA
era, suggests that higher levels of selenium may slow prostate cancer tumor
progression (Li et al., 2004).
However, there is a number of studies which failed to show an association between
Se in the diet and cancer incidence (Coates et al. 1988; Knekt et al. 1988; Menkes et
al. 1986; Nomura et al. 1987; Ringstad et al. 1988). In general, of the 36 observational
studies that were reviewed, approximately half supported an association with total
cancers and half did not (Trumbo, 2005). It was noted, that the greatest consistency
was observed for breast and prostate cancer.
Case-control studies of Se levels in tissues of cancer patients

It has been shown that cancer patients were characterised by lower Se levels in
body fluids than do control subjects (Tables 11.16-11.17). Indeed, Se
concentrations in erythrocytes and plasma (Zachara et al., 2005; 1997), serum
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683
(Salonen et al., 1984; 1985; Wilett et al., 1983; Kim et al., 2003) and toenails
(Sakoda et al., 2005) were significantly lower in cancer patients than in control
subjects. However, there are quite a few cases when Se concentrations in body
fluids of cancer patients are not different from those in control groups. It is necessary
to mention that the reported reduction of Se levels in body fluids of cancer patients
in comparison with control subjects ranged between 5% and 35% (Alaejos et al.,
2000).
Table 11.16 Selenium concentrations in cancer patients (Adapted from Whanger, 2004; Combs and Gray,
1998)
Effects
References
Median toenail Se was lower for individuals with hepatocellular

carcinoma in comparison to healthy controls in China
Sakoda et al., 2005
Selenium was lowered in patients with carcinoma of the uterine cervix Bhuvarahamurthy et
al., 1996
Se concentration in whole blood and plasma in prostate cancer
patients was lower in comparison to healthy controls
Zachara et al., 2005
Se concentrations in whole blood and plasma in lung cancer patients

were lower by 23% (p < 0.001) compared with healthy controls
Zachara et al., 1997
Se concentrations in whole blood and plasma, and the activity of red

cell and plasma GSH-Px were measured in patients with breast cancer,
gastric cancer and colorectal cancer and they were significantly lower
than in healthy controls
Pawlowicz et al., 1991
In an area with high esophageal cancer mortality In China, there was

a significant difference in the erythrocyte Se concentration of the
normal and severe dysplasia groups and erythrocyte Se concentration
and GSH-Px activity were significantly lower in the cancer group
than in the normal group
Li et al., 1991
Significantly lower serum Se levels and GSH-Px activity were found

in Korean women who were cancer patients compared with the
healthy controls.
Kim et al., 2003
The serum levels of selenium were significantly lower in patients

with transitional cell carcinoma than in controls
Yalcin et al., 2004
For the half of the New Zealand population with Se level below
Karunasinghe et al.,
97.8 ng/ml, lower serum selenium levels showed a statistically
2004
significant inverse relationship with overall accumulated DNA damage.
Plasma concentrations of selenium was significantly lower in colorectal Musil et al., 2005
cancer patients than in healthy controls over the monitored period
Untitled-2
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684
Table 11.16 Contd.
Untitled-2
Effects
References
The mean serum Se values in cancer patients were significantly

lower than in non-cancer patients. This difference, however, failed to
exist in women 50 years of age and less
Ujiie and Kikuchi,

2002
The cancer patients had a slightly lower adjusted mean serum

selenium than the subjects without cancer at the end of the follow-up
(+/- standard error of mean) 53.9 and 55.3 g/l, respectively
Virtamo et al., 1987
Blood Se levels in the Japanese healthy women, patients with benign

breast disease and with breast cancer were 0.286; 0.200 and
0.195 ng/ml respectively
Schrauzer et al., 1985
Serum selenium levels in patients (n = 59) with different types of

cancer from southeastern Spain (54.4 g/l) were significantly lower
(P < 0.001) than those determined in control groups [healthy subjects
from the same area (n = 130) and institutionalized elderly people
(n = 93)].
Navarro-Alarcon et
al., 1998
Mean serum concentrations of selenium were 81.1 g /l in women

with breast cancer and 98.5 g /l in women with non-tumoral disease
(p < 0.001)
Lopez-Saez et al.,
2003
The concentration of selenium in blood serum in breast cancer

patients (76 g/l) was significantly lower than in healthy controls
(119 g/l)
Chaitchik et al., 1988
The concentration of selenium in serum of women with breast

cancer was significantly lower than in healthy controls
McConnell et al.,
1980
In the patients with cervical and endometrial cancer serum

concentrations of selenium were significantly lower than the age-,
weight- and place of residence-matched paired control women
Sundstrom et al., 1984
In China, the serum levels of Se were lower in patients with cervical

cancer than in healthy subjects
Cunzhi et al., 2003
Se deficiency is common among newly diagnosed pediatric cancer

patients and Se levels are lower in widespread disease than localized
disease
Postovsky et al., 2003
Among subjects aged < or = 60 yr, mean serum selenium levels were
significantly lower in both patient groups (adenoma, 57.9 g /l;
cancer, 43.7 g /l) than in healthy controls (88.9 g /l) (p = 0.0001).
Subjects with higher selenium status had a lower probability to be in
the adenoma group than subjects with lower selenium status.
Fernandez-Banares et
al., 2002
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Table 11.16 Contd.
Effects
References
Serum selenium levels were significantly lower in lung cancer patients Miyamoto et al., 1987
than in the controls. Among families of adenocarcinoma patients, the
mean level was significantly lower (111 g /l) than that (122 g /l )
in age-ratio matched controls who did not have cancer patients among
their second-degree relatives.
In the nested case-control study of a stratified sample from the Belgian Kornitzer et al., 2004
population, serum selenium was an independent predictor of cancer
mortality in male subjects only
In Poland, Se concentrations in gastrointestinal cancer patients
(37.0 g /l or 38.4 g /l in stomach or colon cancer patients,
respectively) were significantly lower as compared to the healthy agematched control group (51.4 g /l)
Scieszka et al., 1997
In India, patients with head and neck cancer had serum selenium levels Yadav et al., 2002
significantly lower as compared with controls, and these levels
decreased further as tumour burden increased
In India, plasma selenium level of 75.4 ng/ml in cancer patients was
significantly less than control values (117 ng/ml) in normal healthy
individuals. The strongest association of plasma selenium level and
cancer was found in breast cancer (70.5 ng/ml) and gastrointestinal
tract cancer (73.1 ng/ml). Selenium level decreased with the progress
of disease and recurrence of disease.
Gupta et al., 1994
In Belgium, in patients with acute non-lymphocytic leukemia serum

Se levels were lower (82 ng/ml) than in controls (97 ng/ml) Serum Se
correlated inversely with the absolute peripheral blast cell count and
other measurements of the tumor burden.
Beguin et al., 1989
In Greece, patients either with newly diagnosed cancers or with

Bratakos et al., 1990
metastases or who are undergoing therapy have statistically significant
less blood, urine and hair Se than age- and sex-matched healthy controls
In Taiwan, Se levels were lower in the serum of the tumor patients
(benign and malignant) compared to the control group
Kuo et al., 2002
In patients with upper gastrointestinal and other malignancies Se

Pothier et al., 1987
levels were 28% lower than those found in normal controls. Se levels
were substantially decreased by recent radiotherapy or sepsis, by
regional tumor spread and increased tumor burden, and by intravenous
and/or enteral hyperalimentation and intravenous lipids
In Yugoslavia, serum Se levels in patients with colorectal cancer was
lower (46.8 ng/ml) than that in healthy controls (64.2 ng/ml).
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Mikac-Devic et al.,
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686
Table 11.16 Contd.
Effects
References
Mean serum Se levels were 101, 105 and 77 ng/ml in the controls,
precancerous and in patients with malignant oral cavity lesions,
respectively.
Toma et al., 1991
In patients with various osteosarcomas, the mean selenium content

in serum decreased to 71% in comparison to healthy controls
Golubkina et al., 1995
Table 11.17 Mean pre-diagnostic serum selenium concentrations in cancer cases and matched controls
from prospective cohort studies (Adapted from Waters et al., 2004)
Cases
Serum Se concentration,
g/l
P-value
Cohort
Case
Control
49.3
49.9
59.5
59.1
63.6
63.5
58.4
60.5
62.5
63.9
<0.05
<0.05
>0.05
<0.001
>0.05
Japan
35 males
38 females
105.2
97.4
112.8
102.7
0.18
0.25
Ujiie and Kikuchi, 2002
Netherlands
40 males
29 female
116.7
130.6
126.4
129.3
0.04
0.83
Kok et al., 1987
Norway
26 male
34 female
124.8
123.2
130.3
127.9
0.08
0.36
Ringstad et al., 1988
USA
60 male
51 female
127.0
132.0
137.0
134.0
0.008
0.57
Willett et al., 1983
Finland
16 male smokers
14 male non-smokers
21 female non-smokers
597 males
499 females
Knekt et al, 1990
Did you know that cancer patients were characterised by

lower Se levels in body fluids than do control subjects?
Furthermore, it is not clear at present if decreased Se concentrations in cancer
patients is a risk factor for cancer or if they are just a consequence of changes in
metabolisms in cancer patients. For example, it was suggested that low Se status
of cancer patients was more likely a consequence of their illness than the cause of
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687
the cancer (Robinson et al., 1979; Lopez-Saez et al., 2003). The confirmation of
this idea came from the study showing that decreased Se levels came within normal
range after 1 year of radiotherapy in 10 patients with head and neck cancer who
were cured but in the remaining patients who had residual disease, levels remained
persistently low (Yadav et al., 2002). Similarly, low serum Se levels in patients
with acute leukemia are mostly dependent on tumor activity (Beguin et al., 1989).
On the other hand, data reported by Ujiie et al (1998) suggest that the low serum
Se level in cancerous patients may not be induced by the tumor but it was more
likely already present before the tumor. Results of Fernandez-Banares et al. (2002)
suggest that high selenium status may decrease the risk of large size adenomas in
a low selenium region, and that this preventive effect seems to be exclusive to
subjects < or = 60 yr
Laboratory animal studies of anticancer effect of Se

Selenium compounds have been found to inhibit tumorigenesis in a variety of
animal models (Table 11.18). Indeed, laboratory animal models have been used
to demonstrate selenium inhibition of many types of cancer, including liver, skin,
pancreas, mammary gland, and colon (Kise et al., 1990). Despite the fact that
animal data, whatever the species, cannot be applied directly to humans, these
experiments help in the understanding of the mechanisms of cancer development
and action of anticancer compounds. Indeed, anticarcinogenic effects of different
forms of selenium have been demonstrated in several animal models (Combs and
Gray, 1998; Ip, 1998; El-Bayoumy 1991; Medina and Morrison 1988). The effects
are not organ-specific, since tumor inhibition has been achieved in various tissues
including mammary gland, liver, skin, pancreas, esophagus, colon and in a few
other tissues. However, protective effects of Se are dose-dependent. For example,
7,12-Dimethylbenz[a]anthracene (DMBA), a polycyclic aromatic hydrocarbon,
has been used extensively as a tool to initiate mammary carcinogenesis and
subsequent chemoprevention. Recently, in an experiment, rats were administered
a single, oral dose of DMBA (12 mg). In the Se group, rats received 20 g Se
daily via gavage, starting 2 wk before the DMBA administration and continued
for 1 wk. One hundred twenty days after DMBA administration the rats were
sacrificed and toxicity was evaluated using histopathological and biochemical
criteria. Five rats (30%) died in the DMBA group within the study period, whereas
no death occurred in the DMBA-Se-treated group. Malignant tumor frequency
was 33% in the DMBA group, while no malignant tumors occurred in the DMBASe-treated group (Kocdor et al., 2005).
It is commonly accepted that carcinogen-induced genetic damage via the
formation of covalent DNA adduct is necessary, but not sufficient, for the initiation
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Table 11.18 Effect of Se on cancerogenesis in animal models
Effect of selenium
References
Development of DMBA*-induced breast cancer was highly reduced

(fewest breast tumours) in rats fed selenium-enriched garlic
Ip et al., 1992
Selenium inhibited colon cancer incidence (tumours/animal) in rats
Reddy et al., 1992
Administration of selenium in drinking water reduced cervical cancer

incidence from 72% in control mice to 37% in selenium treated mice
as well as decreased hyperplasia and dysplasia
Hussain and Rao, 1992
Mice treated with selenium had reduced skin inflammation,

Burke et al., 1992
pigmentation, later onset and lesser incidence of skin cancer following
UV irradiation
Selenium inhibited the incidence of breast cancer in rats probably
due to seleniums inhibition of dimethylbenz(a)anthracene (DMBA)DNA binding in breast tissue
El-Bayoumy et al.,
1992
Selenium reduced liver and bile duct tumours in hamsters (incidence

rate and number per animal), and significantly inhibited bile duct
proliferation
Kise et al., 1991
In rats dietary selenium, in the form of sodium selenite in drinking

water, inhibited formation of DMBA-DNA adducts and decreased the
incidence of mammary tumors
Birt et al., 1986
In hamsters, sodium selenite inhibited N-nitrosobis(2-oxopropyl)

amine (BOP)-induced pancreatic cancer, in a dose-dependent fashion
Birt et al., 1988
Inhibitory activity of diphenylmethylselenocyanate was shown in

azoxymethane-induced colon carcinogenesis in rats: reduction
(by 63.3%) in incidences of aberrant crypt foci in the treated groups
Ghosh et al., 2005
Malignant tumor frequency was 33% in the DMBA-treated rats,

while no malignant tumors occurred in the DMBA-Se-treated group
Cocdor et al., 2005
DMBA-induced carcinoma in situ was detected in 25% of control

Yilmaz et al., 2003
rabbits, while none of the rabbits had carcinoma in situ in the seleniumfed (4 ppm sodium selenite) group
Selenite treatment (5 ppm in water) of rats during the cancer promotion Bjrkhem-Bergman et
phase decreased the volume fraction of pre-neoplastic liver nodules
al., 2005
from 38% to 14% an the cell proliferation within the nodules decreased
from 42% to 17%
Significant inhibition in the incidence of DMBA-induced papilloma
Das et al., 2004
formation (58-80%) as well as in the cumulative number of papilloma
per papilloma-bearing mouse were observed in the diphenylmethyl selenocyanate treated groups as compared with the carcinogen control group
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Table 11.18 Contd.
Effect of selenium
References
When fed at supplemental levels (1ppm) in the diet, Se in dairy protein McIntosh et al., 2004
was very effective at reducing tumours of the colon in azoxymethaneinduced rats
Significant inhibition in the incidence of DMBA-induced papilloma
formation (53-80%) and the cumulative numbers of papillomas per
papilloma bearing mouse were observed in the diphenylmethyl
selenocyanate-treated (2-3 mg/kg b.w.) groups as compared to the
carcinogen control group
Das and Bhattacharya,

2004
Antimetastatic effect of the high-Se soy proteins was shown in the

mice model
Li et al., 2004a; Yan et

al., 2002
Dietary supplementation of SeMet (2.5 or 6 ppm) reduced

experimental metastasis of melanoma cells in mice and inhibited the
growth of metastatic tumors that formed in the lungs
Yan et al., 1999
1,4-phenylenebis(methylene)selenocyanate at 5 and 10 ppm doses

significantly reduced lung tumor induction by 4-(methylnitrosamino)
-1-(3-pyridyl)-1-butanone from 10.4 to 2.7 and 1.8, respectively
Das et al, 2003
Topical L-SeMet application was shown to protect against UV-induced Burke et al., 2003
skin cancer in a mice model
Pregnant CBA mice were administered selenium during the last week Popova, 2002
of pregnancy and for ten days following parturition. Selenium
significantly reduced the incidence of spontaneous hepatomas in adult
male progeny
Sodium selenite, selenocysteine and SeMet have been compared at
Mukhopadhyay-Sardar
4, 6, 8 and 10 ppm, in terms of their bioavailability and prolongation et al., 2000
of survival of Daltons lymphoma (DL) bearing mice. SeMet, at a dose of
8 ppm, was found to be the most bioavailable and least-cytotoxic form
that was capable of increasing the life span of the tumour bearing hosts
maximally (almost two-fold)
Rats receiving esophagoduodenal anastomosis surgery plus iron
Chen et al., 2000
supplementation were supplemented with sodium selenate at 1.7 mg/kg
Histopathological analysis showed that Se supplementation increased
the incidence of esophageal adenocarcinoma and the tumor volume
Supranutritional amounts of Se (1-2 ppm) supplied as high Se broccoli Finley et al., 2000;
to carcinogen-injected rats significantly decreased the incidence of
2001
aberrant crypts and aberrant crypt foci (preneoplastic lesions indicative
of colon cancer) compared with control animals
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Table 11.18 Contd.
Effect of selenium
References
Five-week-old heterozygotic male multiple intestinal neoplasia mice Davis et al.., 2002
were fed a basal diet containing either low-selenium broccoli or highselenium broccoli for 10 wk. Mice fed the selenium-enriched broccoli
had fewer small intestinal and large intestinal tumors than those fed an
equivalent amount of unenriched broccoli
In N-methylnitronitrosoguanidine-induced colorectal carcinogenesis Mukherjee et al., 2001
model treatment with SeMet either on initiation or on selection/
promotion, or during the entire experiment showed that SeMet was most
effective in regulating the cellular antioxidant defence systems, DNA
chain break control and reducing aberrant crypt foci in the colorectal
tissues of rats
Sodium selenite (2.5 ppm) given in drinking water to 8 months old
OF1 mice, for 4 consecutive months, reduced significantly the
mortality of mice with 6% and 50% mortality rate for Se and control
groups, respectively. In addition 80% of control deaths resulted from
a lymphoid cell neoplasma, while no one of Se supplemented mice
produced tumor
Wong et al., 2001
Selenite inhibited invasion of HT1080 human fibrosarcoma cells and

adhesion of HT1080 cells to the collagen matrix was also inhibited
by treatment with selenite
Yoon et al., 2001
Supplementation of triphenylselenonium chloride to carcinogenIp et al., 2000

treated rats at a level of 30 ppm Se suppressed mammary tumorigenesis
by approximately 50% regardless of dietary selenium nutritional status
*DMBA- 7,12-Dimethylbenz[a]anthracene
of carcinogenesis (El-Bauomy, 2001). Therefore, several studies were conducted

in vitro in rodents to examine the effects of various levels and forms of selenium
on carcinogen DNA adduct formation (Table 11.19). In the studies various tumor
initiators, selenium compounds and model systems were used. It is necessary to
mention that not all animal experiments were successful (El-Bayoumy, 2001).
However, in most of them protective effect of selenium was obvious.
Selenium was shown to be effective in cancer prevention only prior to, or in
early phases of, malignant transformation and the anticancerogenic action of Se
depended on (Schrauzer, 2000):
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its chemical form

its dosage
nature of carcinogenic agent
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Table 11.19 Protective effects of selenium on carcinogen-induced covalent DNA adduct formation in
rodents (Adapted from El-Bayoumy, 2001).
Form of Se
Carcinogen
Sex, species, organ
References
Selenite
DMBA
F, SD rat, mammae
Selenite, selenateDMAB
Selenite
2-AAF
Selenite
AFB1
p-XSC
DMBA
M, F344 rat, colon

Male, CD rat, liver
M, F344 rat, liver
F, SD rat, mammae
p-XSC
o-,m-, p-XSC
RSeCN
BSC
F, A/J mouse
F, SD rat, mammae
F, SD rat, mammae
M, F344 rat, colon
Liu and Milner, 1991; Liu et al.,

1991; Ejadi, et al., 1989
Davis et al., 1999
Wortzman et al., 1980
Chen et al., 1982; 1982a
El-Bayoumy et al., 1992;
Upadhyaya and El-Bayoumy et al.,
1998
Prokopczyk et al., 1996
Chae et al., 1997
Ip et al., 1995
Fiala et al., 1991
NNK
DMBA
DMBA
AOM
1,4-phenylenebis(methylene)selenocyanate (p-XSC)
4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)
azoxymethane (AOM)
3,2'-dimethyl-4-aminobiphenyl (DMAB)
7,12-dimethylbenz(a)anthracene (DMBA)
2-acetylaminofluorene 2AAF
benzylselenocyanate- BSC
Cancer is a disease that develops slowly. Indeed, for most solid human tumors,
there is a 20-year interval from carcinogen exposure to clinical detection. During
this time, cancer cells acquire the capacity to divide, invade, and metastasise (Loeb
et al., 2003). Since, it is difficult to predict timing of carcinogen action and its
nature in real life, it is recommended Se to be supplemented continuously over
the entire lifespan. Indeed, decreased carcinogen-induced damage to DNA supports
a role for selenium inhibition of cancer at the stage of initiation, and increased
latency to tumor development suggests inhibition of tumor promotion (Birt et al.,
1986; 1988). The results of Bjrkhem-Bergman et al. (2005) suggest that selenium
is able to reduce the risk for liver cancer even when it is used only during a short
period of time covering the promotion phase of the carcinogenic process. Therefore
the carcinogenetic process may be prevented by selenium supplementation both
during the promotion and the progression phase. Similarly, most significant
beneficial effect of selenium during hepatocarcinogenesis was exerted potentially
in long-term continuous and/or before the initiation phase of carcinogenicity, rather
than in the promotion phase (Thirunavukkarasu and Sakthisekaran, 2003).
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Did you know that Combs and Combs (1986) estimated that Se
was used in more than 100 animal carcinogen studies and twothird showed reduction in the incidence of cancer, with half
showing a substantial (50% or more) reduction?
Rat, mouse, and hamster models have been used to study liver, breast, colon,
skin, and pancreatic cancers. In general Combs and Combs (1986) estimated that
Se was used in more than 100 animal carcinogen studies and two-third showed
reduction in the incidence of cancer, with half showing a substantial (50% or
more) reduction. This was substantiated in later reviews (Combs and Gray, 1998;
El-Bayoumy, 1991; Whanger, 2004). It is necessary to mention that more than
90% of the selenium cancer chemoprevention experiments have used either sodium
selenite or selenomethionine as the test reagent because they are commercially
available (Ip, 1998). In animal models using chemically-induced cancerogenesis
selenite was more effective than SeMet irrespective of Se concentration in tissues.
Therefore it was suggested that the anticancer effect was due to various metabolic
forms of Se including methylated forms of selenium (Ip, 1998). In particular, it
was considered that anticancer effect of Se is independent on H 2 Se and
selenoprotein formation. Therefore, the effect of high-level Se supplementation
in cancer prevention studies is most likely due to direct chemical properties of Se
and its metabolites rather than being mediated through selenoproteins. The
methylated selenium products, particularly the monomethylated species, are
proposed to have the greatest anticarcinogenic potential (Szarka et al., 1994). In
particular, such Se analogues as selenobetaine and Se-methylselenocysteine, have
been successfully tested for anticarcinogenic potential (Reddy et al., 1994). In
particular, the authors showed that the partially methylated species have greater
efficacy as antitumorigenic agents in animal models than the fully methylated
species. Furthermore, doses of sodium selenite used in cancer prevention studies
using various animal models were quite high and in many cases almost toxic.
Therefore, to avoid Se toxicity various organo-selenocompounds were synthesized
and tested. For example, 1,4-phenylene-bis(methylene)selenocyante or xylene
selenocyanate (p-XSC), have been found to be very effective inhibitors of
mammary and colon carcinogenesis (Ip et al., 1994). At the same time this
compound was characterised by dramatic (almost 30-fold) decrease in toxicity
(el Bayoumy et al., 1992). Different forms of selenium supplementation, either
as inorganic Se (sodium selenite) or as organic forms such as selenomethionine,
methylseleninic acid, and other unspecified seleno-compounds in yeast and
broccoli, may have differing efficacies in cancer prevention (Chu et al., 2004).
Up to date there have been hundreds of chemicals that have been tested for their
anticancer activities in both in vivo and in vitro models. However, it is difficult to
translate all those data into specific recommendations for cancer prevention.
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There are few questions to answer:
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chemically-induced carcinogenesis used in animal models is in many cases

different from those observed in human. In particular chemicals, which
were used to induce cancer in model systems in many cases are different
from those causing cancer in natural situations. There is a need for deeper
understanding molecular mechanisms of cancer-initiation and progression.
Se doses used in the studies (1-5 ppm) are substantially higher than those
tested in human trials. In fact, in experimental animals, anti-carcinogenic
effects have been consistently associated with Se at supranutritional intakes
that are at least 10 times those required to prevent clinical signs of Se
deficiency and to support near-maximal tissue activities of selenoenzymes
(Combs, 2005). The pharmacological approaches (chemoprevention) should
be assessed against nutritional approaches (balanced diet) in terms of their
efficacy in cancer prevention. Indeed, in most of human trials protective
effect of Se against cancer was seen only in those participants who were
initially Se-deficient or at least were characterised by comparatively low Se
status. It is interesting to mention that in some animal model systems
physiological Se doses were shown to have anticancer effect when compared
to Se deficient animals. For example, chemoprotective effect of dietary
selenium (as sodium selenite or as Se-rich egg) on mouse skin tumor induced
by topical application of 2'-(4-nitrophenoxy)oxirane (NPO) as tumor initiator
and 12-O-tetradecanoylphorbol-13-acetate (TPA) as tumor promoter was
evaluated in relation to the dietary source and levels of selenium. Selenium
supplementation (0.3 ppm) to the basal diet (0.07 ppm Se) as sodium selenite
(SS) or as Se-rich egg (SE) brought about a 40 or 37% reduction respectively,
in the incidence of papilloma formation at 12 weeks after NPO treatment
(Oh et al., 1995). Furthermore, tumor yield (number of papillomas per
mouse) at 14 weeks after NPO treatment in the basal diet group, SS group
and SE group were 7.5, 2.7 and 4.1 respectively. It is, therefore, concluded
that a moderate level of dietary selenium (0.3 ppm) has a chemopreventive
activity at the promotional stage of carcinogenesis and that dietary selenium
rich egg as well as dietary selenite exerted antitumor activity.
form of selenium. Most of successful experiments showing anticancer effects
of selenium were conducted with inorganic selenium compounds, while
Se-yeast was a preparation of the choice tested in human trials
selenium and other antioxidants and general diets. Protective effect of Se
can differ depending on the diet composition especially on the presence
and concentrations of other antioxidants and pro-oxidants
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Human intervention trials with selenium

Up to date there were 10 human trials to test protective effect of Se against cancer
(Whanger, 2004; Table 11.20). Six trials were conducted in China, a country
characterised by a number of Se-deficient regions.
Table 11.20 Clinical trials employing selenium alone or its combination with other minerals and vitamins
(Adapted from El-Bayoumy, 2001).
Level and form of Se
Country Population
Type of cancer References

(outcome)
200 g Se/day for

2 years as Se-Yeast
China
Hepatitis surface
antigen carriers
Liver cancer Yu et al., 1991;

1997
Table salt fortified with

15 mg/kg Se as sodium
selenite
China
Hepatitis surface
antigen carriers
Liver cancer Yu et al., 1991;

1997
50 g Se/day for 2 years

as Se-Yeast
China
Double-blind,
Stomach
placebo controlled, cancer
general population
Blot et al., 1993
50 g Se/day for 2 years

as selenite
China
Double-blind,
Esophageal
placebo controlled, cancer
displasia
Li et al., 1993
100 g Se/day for 6

India
months and 50g Se/day
for 6 months
Reverse smokers
Prasad et al.,
1995
200 g Se/day
Italy
Patients with prior New

resected adematous adematous
polyps, randomised, polyps
double blind
Bonelli et al.,
1998
200 g Se/day for

4.5 years as Se-Yeast
with 7.3-7.6 years of
follow-up
US
Patients with prior

skin cancer;
randomised, double
blind,placebocontrolled
Clark et al.,
1996; 1998;
Duffield-Lilico
et al., 2003
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Oral lesions
Basal cell or
squamous cell
carcinoma
lung, colon,
prostate
The first trial in China. In the Qidong county of China, the incidence of
hepatocellular carcinoma (HCC) is particularly high. It is interesting to note
that in this region, about 15% of adults carry the hepatitis B surface antigen
and these individuals are 200 tines more likely to develop HCC (Rayman,
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2002). Subjects were given table salt fortified with 15 g Se as sodium

selenite/g, which provided about 30-50 g Se for 8 years (n=20,847; Yu et
al., 1991; 1997). Se-supplementation resulted in a drop of the primary liver
cancer incidence from 50.4 per 100,000 population consuming ordinary
salt to 27.2 per 100,000 population consuming Se-enriched salt. It is
important to underline that after withdrawal of Se from the treated group
the primary liver cancer incidence began to rise. Therefore a protective
effect of Se against liver cancer was clearly proven.
Did you know that there were at least 10 human trials to test
protective effect of Se (singly or in a combination with other
antioxidants) against cancer?
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The second trial in China. In the study of Yu et al. (1997) 2474 members
of families with high risk of primary liver cancer (PLC) were consuming
200 g of selenium per day in the form of Se-yeast or a placebo. During the
2-year period of study, 1.26% of the controls developed PLC vs 0.69% in
the placebo group. Furthermore, the authors studied 226 hepatitis B surface
antigen-positive subjects randomly assigned to either a placebo or Seenriched bakers yeast (200 g Se/d) for a 4-year period. They showed a
lower PLC incidence in the Se group compared with the placebo group (0
vs 7 cases, P<0.05).
The third trial in China. There were 3698 subjects randomly assigned to
an array of mixed treatments. The treatment consisting of Se-enriched bakers
yeast (providing 50 g Se/d) in combination with vitamin E and -carotene
was associated with modest reduction in stomach cancer mortality (by 21%)
and total mortality (by 9%; Taylor et al., 1994; Blot et al., 1995). Results
from the same study, published recently (Wei et al., 2004) showed a
significant inverse association between baseline serum Se and death from
oesophageal squamous cell carcinoma and gastric cancer.
The fourth trial in China. It was conducted from 1984 to 1991 in Linxian,
a rural county in Henan Province, characterised by exceptionally high
mortalities from esophageal cancer, a total of 29,584 adults were used to
assess the effect of vitamins and minerals on cancer (Blot et al., 1993).
During study 2127 subjects died from cancer. It was found that a
combination of Se (50 g Se/d as Se-yeast) with -carotene and vitamin A
significantly lowered total mortality and cancer mortality. There was a 13%
reduction in total and cancer mortality in the group treated with Se-enriched
yeast plus vitamin E and -carotene. The protective effects of this antioxidant
mixture was evident against cancers of the stomach, esophagus, and lung.
In particular, rate of lung cancer was substantially decreased (Blot, 1997).
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The fifth trial in China. Li et al. (1993) used randomly assigned 3318 subjects
with confirmed esophageal dysplasia who received a similar array of mixed
treatments of vitamins and minerals, including 50 g Se as selenate per day. In
general vitamin and mineral levels used were 2-3 times the recommended dietary
allowance levels. No significant treatment effects were observed on any cancerrelated end point. However, it is necessary to mention that justification of various
mixtures and doses of individual compounds in those mixtures are always a
problem. Therefore, it is difficult to answer the question if the trial was not
successful because of lack of effects of individual compounds, including
selenium, or because of possible synergistic-antagonistic interactions of those
compounds. Indeed, it seems likely that Se is more effective in cancer prevention
rather in reversing tumours as well as Se dose was not high enough to give the
best protection (Whanger, 2004).
The sixth trial in China. A double-blind intervention study was performed on
the participants aged 35 -74 years, who had matched at least one of the following
criteria: a medical history of stomach disorder, a family history of tumour, or
smoking and/or alcohol consumption. A total of 2,526 and 2,507 persons were
randomly enrolled into intervention group and control group respectively from
288 natural villages of seven communities in Qixia County, Shandong Province,
China. Each person of the intervention group orally took 200 mg synthetic
allitridum, an active principle of garlic, every day and 100 g selenium every
other day for one month of each year during November 1989 to December
1991 (Li et al., 2004). At the same time, people in control group were given 2
placebo capsules containing corn oil with the identical appearance to that in
the intervention group. In the first follow-up five years (1992 - 1997) after
stopping the intervention, after adjusting for age, gender, and other potential
confounders, relative risks (RRs) for all tumours and gastric cancer of the whole
population were 0.67 and 0.48, respectively, and for male group they were
0.51 and 0.36, respectively. No significant protective effect was found for the
female subgroup (Li et al., 2004).
The seventh trial in India. The trial was conducted with 298 rolled tobacco
smokers with pre-cancerous lesions in the oral cavity randomly assigned to a
placebo or a twice weekly supplement containing vitamin A, riboflavin, zinc,
and Se (Se-enriched yeast providing 100 g/d for 6 months, followed by 50
g/d for 6 months). After 1 year of treatment, subjects with precancerous oral
lesions in the treatment group had a significantly greater rate of complete
remission of lesions compared with those in the control group (57 vs 8%).
Subjects without lesions in the treated group also had significantly fewer new
lesions than those in the placebo group (12 vs 38%; Krishnaswamy et al.,
1995; Prasad et al., 1995).
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The eighth trial in Italy. A group of 304 patients with previous resected
adenomatous polyps who received a mixed treatment containing Se (200
g Se/d as L-SeMet) plus zinc, vitamin A, and riboflavin for 5 years had a
lower incidence of recurrent polyps than those receiving a placebo (5.6 vs
11%; Bonelli et al., 1998). However, this report was not published in peerreviewed journals.
The ninth trial in France. The SUpplementation en VItamines et
MinerauxAntioXydants (SU.VI.MAX) study was a randomized doubleblind, placebo-controlled, primary prevention trial designed to test the
efficacy of daily supplementation with antioxidant vitamins (vitamin C, 120
mg; vitamin E, 30 mg; and beta-carotene, 6 mg) and minerals (selenium,
100 g; and zinc, 20 mg), at nutritional doses (one to three times the daily
recommended dietary allowances), in reducing the frequency of major health
problems in industrialized countries, and especially the main causes of
premature death (cancers and cardiovascular diseases; Hercberg et al., 1998).
The study involves 12,735 eligible subjects (women aged 35 to 60 years;
men aged 45 to 60 years) included in 1994 in France. Sex-stratified analysis
showed a protective effect of antioxidants in men (relative risk, 0.69, but
not in women (relative risk, 1.04). A similar trend was observed for allcause mortality (relative risk, 0.63) in men vs 1.03 in women. Therefore,
after 7.5 years, low-dose antioxidant supplementation lowered all-cause
mortality in men but not in women (Hercberg et al., 2004). In the
SU.VI.MAX trial 5,141 men took either a placebo or a supplement daily for
8 years. During the follow-up, 103 cases of prostate cancer were diagnosed.
Overall, there was a moderate nonsignificant reduction in prostate cancer
rate associated with the supplementation (hazard ratio = 0.88). However,
the effect differed significantly between men with normal baseline PSA (<
3 g/l) and those with elevated PSA (p = 0.009). Among men with normal
PSA, there was a marked statistically significant reduction in the rate of
prostate cancer for men receiving the supplements (hazard ratio = 0.52).
However, in men with elevated PSA at baseline, the supplementation was
associated with an increased incidence of prostate cancer of borderline
statistical significance (hazard ratio = 1.54). The supplementation had no
effect on PSA or IGF levels (Meyer et al., 2005).
The tenth trial in the USA. The Nutrition Prevention of Cancer Trial (NPC
Trial) was designed in 1983 to test the hypothesis that an increase in Se
status would reduce the incidence of non-melanoma skin cancers (primary
endpoints) in a high-risk population. This was the first double-blind placebocontrolled intervention trial in the western population of this kind (Rayman,
2002). The participants with a history of non-melanoma skin cancer (1312
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subjects; 63.2 10.1 y; range: 1880 y; Americans living along the eastern
seaboard) in the NPC trial received 200 g/d of selenium supplements (as
selenized yeast) or placebo. In 1990, the trials Safety Monitoring and Advisory
Committee established a number of formal secondary endpoints (all-cause and
cancer mortalities, incidences of cancers of the lung, prostate, and colon-rectum,
and total non-skin cancers (Combs, 2005). First results of this trial were rather
disappointing. No significant effects of Se-treatment on the incidences of either
basal or squamous cell carcinomas of the skin were found. Indeed, there was
no effect of selenium on the primary end point of non-melanoma skin cancer.
However, significant differences between treatment groups in risks of total cancer
incidence, total cancer deaths, and incidences of carcinomas at sites other than
skin, namely, lung, prostate, colon-rectum, and total non-skin cancers were
observed. Indeed, male participants receiving 200 g/day selenium in the form
of selenized yeast (in addition the average daily intake in the U.S. of about
100 g/day) had 63% reduced risk of prostate cancer incidence (Clark et al.,
1998). Furthermore, there was 56% reduction in total cancer mortality and
37% lower total cancer incidence with 63% fewer cases of cancer of the colon
and 46% fewer cases of cancer of the lung (Clark et al., 1996; Table 11.21;
Figure 11.5). There were 29 cancer death in the selenium group and 77 in the
placebo group. The selenium supplement was most effective in ex-smokers
and for those who began the study with the lowest levels of serum selenium. It
was concluded that of all the human cancer intervention studies that have been
completed to date, the selenium trial was by far the most successful (Ip, 1998).
To include a further 25 months of blinded treatment and follow-up in the NRP
trial, data were reanalysed. There was a significant reduction in total cancer
incidence (105 vs 137 cases, P = 0.03), prostate cancer (22 vs 42 cases, P =
0.005), a marginally significant reduction in colorectal cancer incidence (9 vs
19 cases, P = 0.057), and a reduction in cancer mortality, all cancer sites (40 vs
66 deaths, P = 0.008; selenium versus control group cases reported, respectively;
Duffield-Lillico et al., 2002). Therefore a 7.9-y follow-up of this trial evaluated
the relative risk of the secondary cancer endpoints and found that the significant
reduction in lung and colorectal cancer were no longer observed. However, as
in previous observations, the protective effect of Se was strongest in those
individuals with lower baseline plasma Se concentrations (<121.6 g/l).
Therefore, in this trial the protective effect of Se depended on the initial level of
Se in the blood of participants at time of entering the study. Those with plasma
Se < 106 g/l, i.e., in the lowest tertile of that cohort, showed the strongest
effect of Se-treatment (RR = 0.14, P = 0.002) in reducing the risk of being
diagnosed with prostate cancers over the subsequent years of follow-up (Combs,
2005). Se supplementation reduced the risk of cancer in this tertile by 48%
(Rayman, 2002).
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Table 11.21 Cancer incidence (numbers of cancers) in selenium- and placebo-treated subjects (Adapted
from Clark et al., 1996)
Site
Skin carcinomas
Basal cell
Squamous cell
Lung
Prostate
Colorectal
Total carcinomas other than skin
Total non-carcinomas
Total cancer
Selenium
Placebo
Relative risk
P value
377
218
17
13
8
59
19
77
350
190
31
35
19
104
16
119
1.10
1.14
0.54
0.37
0.36
0.55
1.17
0.63
0.2
0.15
0.08
0.002
0.025
<0.001
0.65
0.001
Did you know that the Nutrition Prevention of Cancer Trial

showed 63% reduction in risk of prostate cancer incidence, 56%
reduction in total cancer mortality and 37% lower total cancer
incidence as a result of consumption of 200 g Se daily for 4.5
years?
35
Selenium
30
Placebo
25
20
15
10
5
0
Lung
Prostate
Colorectal
Figure 11.5 Cancer incidence (numbers of cancers) in selenium- and placebo-treated subjects (Adapted
from Clark et al., 1996)
Furthermore, participants in the middle tertile of plasma Se, 107123 g/l, were
characterised by a more modest, but still protective effect of Se-treatment (RR =
0.39, P = 0.03). In contrast to participants with low Se level, those subjects in the
top tertile at entry to the trial, whose plasma Se concentration was >121 g/l had
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no additional reduction in cancer risk due to Se supplementation. That subjects

entering the trial with plasma Se levels < 120 g/l showed the greatest risks of
subsequent cancer as well as the strongest protective effects of Se-yeast
supplementation would suggest that level to be an appropriate upper limit for
eligibility for future cancer prevention trials using Se (Combs, 2005). It should be
mentioned, that Se concentration in plasma of those who benefited most of all in
the NRP trial (<106 g/l) is a common concentration in European population
(Rayman, 2000). Therefore, beneficial effects of similar to NRP Se-supplementation
in Europe is very likely and awaits investigations. Indeed, the Prevention of Cancer
by Intervention with Se (PRECISE) trial, where Denmark, Sweden and UK are
participants is being planned (Rayman, 2002) and its pilot studies are already
well advanced.
Therefore, from 10 clinical trials selenium was used as a single compound
only in 3 of them, 30-50 g as sodium selenite (1 trial) or 200 g as selenized
yeast (2nd and 10th trials). There was also a double-blind randomised, placebocontrolled trial with 36 healthy adult males (19-43 years of age) who consumed
Se-yeast for 9 months. In a supplemented subjects some positive biochemical
and physiological responses were observed: there was an increase (by 32%) in
blood GSH levels and a significant decrease in prostate-specific antigen (ElBoyoumy et al., 2002).
When choosing a combination of various antioxidants and other cancerpreventive agents there is always a risk to have synergistic or antagonistic
interactions between them and therefore a final result could be different from that
obtained with a single compound. For example, a combination of DHA and
selenium was shown to be more effective than individual compounds in their
effect on cancer cells. In fact, a 48 h incubation of CaCo-2 cells with 5 microM
each DHA or p-XSC (a synthetic organoselenium compound) induced cell growth
inhibition and apoptosis and altered the expression of the above molecular
parameters. However, the modulation of these cellular and molecular parameters
was more pronounced in cells treated with low doses of DHA and p-XSC (2.5 M
each) in combination than in cells treated with these agents individually at higher
concentrations (5.0 microM each; Narayanan et al., 2004).
In accordance with recent evaluation of trials only the Nutrition Prevention of
Cancer Trial (USA) was well designed to answer the question related to protective
effect of Se against cancer. The main conclusion from the trial is that Sesupplementation of subjects who are characterised by comparatively low Se-status
is associated with a significant protection against cancers, in particular against
prostate cancer. As mentioned above low Se status is a common situation in most
of European countries (Rayman, 2000).
Aforementioned data provide a strong incentive to design a definite trial for
selenium and vitamin E with prostate cancer as a primary end point. Therefore
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there are new human trials under way to further substantiate protective effects of
Se against cancer:
The Selenium and Vitamin E Cancer Prevention Trial (SELECT) was

designed as a largest prostate cancer prevention trial ever conducted with
32,000 participants without evidence of prostate cancer from more than
400 participating study sites in the USA, Puerto Rico and Canada. Eligibility
criteria include age 50 years for AfricanAmericans, 55 years for
Caucasians, serum PSA 4 ng/ml, and normal blood pressure. The study is
designed as a prospective, randomised, double-blind, placebo-controlled
multicenter trial with 2x2 factorial design (Figure 11.6). The main aim of
the study is to determine a protective effect of Se and vitamin E on the risk
of prostate cancer (Greenwald, 2004; Pak et al., 2002). Patients will either
receive placebo, selenium (200 g/day as L-selenomethionine), vitamin E
(400 mg/day of all rac alpha-tocopheryl acetate) or a combination of Se
and vitamin E for a minimum of 7 years (maximum of 12 years).
SELECT trial
Pre-Randimization period
Calendar Year
2001-2006
Randomized
Vit.E+
Se
20012013
Vit.E+
Placebo
Selenium +
Placebo
Placebo+
Placebo
Follow-up
Prostate cancer, other cancers, death
Figure 11.6 SELECT trial design (Adapted from Klein et al., 2003)
Each SELECT participant undergoes routine clinical evaluations, including

yearly digital rectal examinations and prostate-specific antigen test. The
primary endpoints will be the diagnosis of histologic cancer or the diagnosis
of clinically evident prostate cancer with an elevated TPSA>50 ng/ml and
positive bone scan study (Pak et al., 2002). A minimum of 7 years and a
maximum of 12 years of follow up will be reported from the trial. As
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secondary goals SELECT will assess the effect of selenium and vitamin E
on the incidence of lung and colon cancer and on survival rates of participants
diagnosed with lung and colon cancer. SELECT is a large, simple trial that
conforms as closely as possible with community standards of care. Enrolment
began in 2001 and initial data analysis is anticipated in 2006 with final
results anticipated in 2013. The study design will permit detection of a 25%
reduction in the incidence of prostate cancer for selenium or Vitamin E
alone, with an additional 25% reduction for the combination of selenium
and Vitamin E compared to either agent alone (Table 11.22). However, there
is a potential problem with this study, since in previous studies a natural
product, Se-enriched yeast, was used, while in the SELECT trial, purified
SeMet is used, and so there is some questions as to how results of this study
can be extrapolated to intakes of Se through food (Finley, 2005).
Table 11.22 Expected incidence of prostate cancer in the SELECT trial (Adapted from Klein et al., 2003)
Group
Number
at risk
Proportion with
prostate cancer
Number with
prostate cancer
8100
8100
8100
8100
0.066
0.05
0.05
0.038
533
403
403
304
Placebo
Selenium
Vitamin E
Se + Vitamin E
Further prostate cancer studies at the Arizona Cancer Center and in Europe
and Australia using Se doses of 200-800 g/d are in progress (Rayman,
2004):
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Negative Biopsy trial: The hypothesis is that Se may prevent the progression
of cancer in 25% of men in this population who have early cancer missed
biopsy (Patrick, 2004). In the trial 700 men with persistently-elevated
prostate-specific antigen (PSA), but negative biopsies are randomly allocated
to placebo or 200 or 400 g Se/day with follow up period of 57 months
(Stratton et al., 2003a). Primary study endpoints include development of
prostate cancer and PSA velocity. Secondary biochemical endpoints include
change in chromagranin A and alkaline phosphatase. The trial will evaluate
the ability of selenium to halt or slow the preclinical progression of prostate
cancer and to decrease the incidence of clinical disease. The trial will be
directly generalizable to those patients at high risk of prostate cancer, but
who have not yet been diagnosed (Clark and Marshall, 2001). Randomization
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scheme was effective for selected parameters including age, body mass
index, smoking status, baseline PSA and baseline plasma selenium level.
Various data, including medical history, family history, and urological
symptoms and specimens (including blood and subsequent prostate biopsy
samples) had been collected at baseline, and throughout both the intervention
and follow-up stages of the protocol (Stratton et al., 2003a).
High-grade Prostatic Intraepithelial Neoplasia Trial: The trial will evaluate
the ability of Se to prevent the transformation of neoplasia to invasive cancer
(Patrick, 2004). For this purpose 470 men with high-grade prostatic
intraepithelial neoplasia, who are at risk for subsequent prostate cancer and
have had 2 or more biopsies that indicate no invasive prostate cancer, but
have not been treated with surgery or irradiation will be assigned to placebo
or to 200 g Se as selenomethionine. They will be followed for at least 3
years. The primary endpoint in the trial is the diagnosis of biopsy-proven
prostate cancer. Secondary endpoints include apoptosis and proliferation.
This study will assess the anticancer activity of Se immediately before
neoplastic growth becomes transformed into invasive growth.
Preprostatectomy trial: This trial will evaluate biomarkers of inflammation
and tissue selenium level. The study will examine prostate tissue from initial
biopsies and from prostate surgery. This will enable to examine tissue before
and after the administration of selenium. Eligible patients will be randomly
placed into one of the three groups. One hundred and ten men with localised
prostate cancer will be supplemented with 200 or 400 g Se/day between
biopsy and prostatectomy (Marshall, 2001). The third group will be given a
placebo pill that looks and smells exactly like a selenium pill, but contains
no selenium. This placebo group will serve as the control group so that the
effect of selenium in the other groups can be clearly determined. Participants
in this study will have a 66% chance of taking selenium.
Watchfull Waiting trial, a phase II, multi-center, randomized, double-blind,
placebo-controlled clinical intervention study testing the effects of two doses
of selenized yeast on progression of prostate cancer. Participants are men with
biopsy-proven prostate cancer who have elected to forgo therapy and be closely
followed by watchful waiting that includes quarterly prostate-specific antigen
(PSA) screening. The trial will evaluate 264 men with localised prostate cancer
and life expectancy <10 years and they will receive 200 or 800 g Se/day as
Se-yeast or matched placebo daily. Endpoints include time to disease progression
and PSA velocity. Secondary endpoints include time to initiation of therapy as
well as biochemical markers of disease progression including chromagranin A
and alkaline phosphatase. Immunohistochemical analyses for indicators of
apoptosis, proliferation and differentiation will be performed on baseline and
subsequent prostate biopsy specimens. (Stratton et al., 2003b).
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PRECISE trial (Denmark, Sweden and UK are participants) will use Se

doses of 0, 100, 200 and 300 g/d in 42,000 volunteers recruited through
the Internet.
A Pilot study for the UK PRESICE trial was initiated in October 1999
(Rayman, 2004). This is double-blind placebo-controlled pilot study with
500 male and female volunteers aged 60-74 years with four treatment
groups: placebo, 100, 200 and 300 g/d as Se-yeast
A pilot study for the Danish PRESICE trial was initiated in December
1998. The design is the same as in aforementioned UK Precise
Australian Prostate Cancer Prevention Trial Using Selenium (APPOSE) is
going to test the hypothesis that daily dietary supplementation with selenium
will reduce prostate cancer incidence in a population of men who are at increased
risk because of a first-degree relative with prostate cancer (Costello, 2001).
This is a randomized, controlled chemoprevention trial in which men at risk of
prostate cancer are given 200 g selenium daily while placebo is given to a
matched group. The cohort size will be 2000 subjects in each arm. Sample size
calculations suggest that two groups of 2000 would be sufficient at 3 years to
detect a relative risk (RR) of 0.5, with 94% power, and an RR of 0.70 with 50%
power. The study would be powered to detect a specified RR by size of control
cohort of 0.75 with 80% power at 10 years.
Selenium and secondary lymphedema

The incidence of secondary lymphedema after cancer treatment varies widely,
depending on its location and origin. Reported incidences of lymphedema after
breast cancer treatment vary from 6% to 30%, after pelvic cancer therapy from
1% to 47% and after treatment for head and neck cancer, from 22% to 56% (Bruns
et al., 2003).
It has been suggested that supplementation with selenium could be a valuable
and safe extension of the physical decongestive therapy of secondary lymphedema
(LE). To test this hypothesis, to patients with LE of the arm sodium selenite was
administered orally in isotonic solution at oral dosages of 800 g Se/day on days
1 through 4 and 500 g Se/day on days 5 through 28 (Kasseroller and Schrauzer,
2000). The authors showed a spontaneous reduction in LE volume and normalized
blood parameters in a manner consistent with diminished oxygen radical
production as a result of Se supplementation. Indeed, this effect of Se could be
attributed to its antioxidant, anti-inflammatory properties and ability to reduce the
expression of endothelial cell adhesion molecules. It seems likely that selenium
has anti-edematous effect. In fact, clinical studies have shown that oral Se
supplementation (Bruns et al., 2003):
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caused a spontaneous reduction in lymphedema volume

increased the efficacy of physical therapy for lymphedema
reduced the incidence erysipelas infections in patients with chronic
lymphedema at various sites
significantly reduced edema volume and showed improvement in skin-fold
index in subjects with arm edema; reduced incidence of erysipelas
(Kasseroller, 1998)
positively affected radiation-associated secondary lymphedema showing a
reduction in edema characteristics; 65% of patients with interstitial grade III
and IV endolaryngeal edema, who normally would require tracheotomy
for treatment, could avoid surgical intervention (Micke et al., 2003)
A few mechanisms of the beneficial effect of Se on lymphedema have been

suggested (Bruns et al., 2003):
Se may suppress the expression of adhesion molecules and thus prevent

activated lymphocytes from attaching to and clogging lymphatic
microcapillaries
Se can unclog lymph capillaries invaded by leukocytes and induce
spontaneous reduction in edema volume
Se can enhance activity of the immune system stimulating macrophage
degradation of the excess tissue proteins and lowering the incidence of erysipelas
Therefore, administration of selenium (300-500 g/day; sometimes initial treatment

up to 1,000 g/day for the first week) appears to be a biochemically sound,
promising treatment option for patients with secondary lymphedema, with little
risk. It has the potential to increase the efficiency of combined physical
decongestive therapy (CPDT) and appears to prevent or diminish the incidence
of erysipelas infections (Bruns et al., 2003).
Selenium and cancer treatment

Organic selenium was shown to be effective in reducing side effects of chemotherapy
of ovarian cancer in 62 women with ovarian cancer undergoing chemotherapy. Indeed,
after 2 and 3 months of Se administration (200 g/day as Se-yeast) a significant
decrease of hair loss, flatulence, abdominal pain, weakness, malaise and loss of appetite
was observed (Sieja and Talerczyk, 2004; Table 11.23). Similarly, Se was shown to
alleviate nausea, vomiting, abdominal pain and body weight loss (Kosmider et al.,
1991).
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Table 11.23 Effect of Se (50 g/day as Se-yeast) on side effects in patients with ovarian cancer
undergoing chemotherapy (Adapted from Sieja and Talerczyk, 2004)
Studied parameter Sequence of

study
Nausea
Vomiting
Stomatitis
Hair loss
Flatulence
Abdominal pain
Weakness
Malaise
Loss of appetite
I
II
III
I
II
III
I
II
III
I
II
III
I
II
III
I
II
III
I
II
III
I
II
III
I
II
III
Experimental Se-supplemented
group, n=31
Control group,
n=31
2.19
1.19
0.97
1.74
1.07
0.97
0.35
0.10
0.32
1.83
1.87
2.12
2.10
1.16
0.61
0.93
0.39
0.45
2.13
1.26
0.97
1.93
0.84
0.87
2.39
1.00
0.84
1.81
1.93
2.03
1.68
1.87
2.16
0.45
0.45
0.58
1.55
2.22
2.55
1.58
1.71
1.97
1.32
1.22
1.45
2.19
2.26
2.35
2.19
2.45
2.54
2.13
2.35
2.26
Scale of evaluation: 0- without signs, 1-mild signs, 2-moderate signs, 3-severe signs;
I-study after 1 month; II-study after 2 months, III- study after 3 months.
Furthermore, in vitro studies showed that the use of yeast Se in combination with
chemotherapeutic drugs can reduce the effective dosage of either Taxol and/or
Adriamycin in several different cancer cells, including ovarian cancer cells in
culture (Vadgama et al., 2000). Supplementation with Se improved the apoptotic
response. A combine selenium (200 g/day) and zinc (21 mg/day) treatment was
used with 30 patients during the course of chemotherapy treatment of colorectal
carcinoma (Federico et al., 2001). At the end of the chemotherapy course, 70% of
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those receiving Se and Zn showed no signs of deterioration of their nutritional

status and had an improvement in appetite and energy level while 80% of those
on chemotherapy without nutritional supplements were characterised by worsening
in nutritional status and symptoms related to malnutrition. Advanced squamous
cell carcinoma of the head and neck is associated with significant
immunosuppression. Patients with this type of cancer were given 200 g sodium
selenite starting simultaneously with surgery or radiation treatment and followed
for 60 days (Kiremidjan-Schumacher and Roy, 2001). Compared to a placebo
group, the Se-supplemented group was characterised by a significant increase in
immune activity. There are some results suggesting that selenium-based dietary
compounds may help to overcome resistance to Tumor necrosis factor-related
apoptosis-inducing ligand (TRAIL)-mediated apoptosis in prostate cancer cells.
It was demonstarated that concomitant administration of TRAIL and
methylseleninic acid (MSA) produced synergistic effects on the induction of
apoptosis in androgen-dependent cells and androgen-independent DU-145
prostate cancer cells (Yamaguchi et al., 2005).
Did you know that selenium was shown to be effective in reducing
side effects of chemotherapy of ovarian cancer and improving
efficiency of chermotherapy of other cancers?
MSA rapidly and specifically downregulates expression of the cellular FLICE
inhibitory protein, a negative regulator of death receptor signalling. Furthermore,
the synergistic effects of MSA and TRAIL resulted from the activation of the
mitochondrial pathway-mediated amplification loop. It was suggested that
selenium supplementation by decreasing the COX-2 protein and PGE-2 levels in
cancer cells may increase efficacy of cetuximab in advanced colorectal cancer
patients (Altundag et al., 2005). Recent results suggest a novel use of methylated
selenium for improving the chemotherapy of prostate cancer (PCA). In particular,
MSeA enhanced apoptosis induced by cancer therapeutic drugs in androgenindependent PCA cells (Hu et al., 2005). Indeed, in DU145 cells, the enhancing
effect was primarily through interactions between MSeA and JNK-dependent
targets to amplify the caspase-8-initiated activation cascades. Both androgendependent LAPC-4 and androgen-independent DU 145 cells pre-treated with
selenite showed increased sensitivity to gamma-irradiation as measured by
clonogenic survival assays (Husbeck et al., 2005). Importantly, selenite-induced
radiosensitization was observed in combination with a clinically relevant dose of
2 Gy. Furthermore, results of experiments conducted by Schueller et al. (2004)
suggested a radiosensitizing effect of selenite in glioma cells at concentrations of
2-3 M. Indeed, irradiated cells exposed to increasing selenite concentrations
showed a lower plating efficiency and, for doses > 2 Gy, a lower survival than the
control.
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The use of selenium, in the form of 5-methylselenocysteine (MSC), or selenoL-methionine (SeMet) as selective modulators of anticancer drugs is considered
as a novel way of improvement of cancer therapy. Indeed, results from Roswell
Park Cancer Institute (USA) have demonstrated that MSC and SeMet are highly
effective modulators of irinotecan cure rates in de novo sensitive and resistant
human tumor xenografts (Fakih et al., 2005). It is interesting to note that the
observed effects were not drug-specific or species-specific and are currently being
confirmed in ongoing clinical trials.
Molecular mechanisms of anticancer effects of selenium

The relation between the trace element selenium and the etiology of cancer in
humans remains elusive and intriguing, despite the number of epidemiological
studies, animal model experiments and human trials published on the topic. The
anticancerogenic actions of Se occur at the systemic, cellular and nuclear level
and therefore cannot be interpreted by a single mechanism (Schrauzer, 2000).
Potential mechanisms of the antitumorigenic effects of selenium include (Klein et
al., 2003; 2003a; Schrauzer, 2000; Whanger, 2004).
ANTIOXIDANT EFFECTS
Among the many possible causes of cancer ROS have been implicated in the
development of the disease. Indeed oxidative stress may induce gene mutation
and promote carcinogenesis. It has been shown that free radical tissue damage is
increased in proportion to prostate cancer (PCa) severity (Malins et al., 2001;
1997). In fact, the age-related increase in the proportion of hydroxyl radicalinduced DNA damage was a significant factor in the development of PCa.
Aforementioned studies have demonstrated that free radical-induced DNA damage
is greater in cancerous than in non-cancerous prostate tissues and lends support
to the hypothesis that the mechanism by which antioxidants may reduce the risk
of PCa is by their effect on reducing the production of free radicals. Furthermore,
ROS can modulate the apoptotic program, disregulation of which has a role
particularly in gastrointestinal cancer (Bjelakovic et al., 2004).
Experimental studies support this idea in part by showing that antioxidants that
prevent ROS damage can act as cancer preventive agents. However, once cancer
has occurred, radiation therapy and some forms of chemotherapy rely on ROS
formation and toxicity to eradicate tumor cells. It seems likely that at least some of
the 25 mammalian selenoproteins are involved in the mechanism of protection
against cancer by selenium. There is evidence that several of these proteins may
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be involved with the mechanism by which selenium provides its anticancer effects
(Diwadkar-Navsariwala and Diamond, 2004). First of all, GSH-Px is responsible
for removal of H2O2 and lipid hydroperoxides and in this way prevents formation
of hydroxyl radical responsible for DNA damages and carcinogenesis. Both TR
and Trx are critical for redox control at the cellular level; together, they are involved
in several biologic processes including antioxidant defense, cell proliferation, and
inhibition of apoptosis (Mustacich and Powis, 2000). Therefore in some cases
changes in activities of selenoproteins and redox status of the cell may encourage
cancerous cells to undergo apoptosis. Components of the TR-TRx system may
play an important regulatory role in the function of p53 (Pearson and Merrill,
1997; Ueno et al., 1999; Moos et al., 2003), a tumor suppressor gene that is either
deleted or suppressed in several human cancers. It seems likely that methionine
sulfoxide reductase B, responsible for prevention of SH-group oxidation in proteins
could also be involved in prevention of carcinogenesis.
Did you know that molecular mechanisms of anticancer effects of
selenium include: antioxidant effects, stimulation of DNA-repair
enzymes and activation of the tumor suppressor protein p53, effect
on gene expression, enhancement of immune function, induction
of apoptosis, inhibition of cell proliferation, alteration of
carcinogen metabolism, influence on estrogen and androgen
receptor expression, inhibition of angiogenesis and interactions
with heavy metals involving in cancer promotion?
Genetic data indicate functional polymorphisms in the genes for several
selenoproteins and the association of allelic variants with cancer risk. Furthermore,
allelic loss of selenoprotein genes during tumor evolution supports the possibility
that the deletion of these genes promotes cancer development. Indeed, allelic loss
at the GSH-Px-1 locus is an early event in the development of cancer of the head
and neck (Hu et al., 2004). Therefore, reduced levels of one or more selenoproteins
increase the likelihood of cancer development (Diwadkar-Navsariwala and
Diamond, 2004). The authors suggested that individuals with lower selenium
intake, attenuating polymorphisms, or haploinsufficiency may be those who will
benefit most from additional dietary selenium intake. Therefore, an optimal Se
status is an important factor responsible for selenoprotein synthesis and an effective
antioxidant defence. In this way Se can prevent damages to DNA leading to
cancer.
STIMULATION OF DNA-REPAIR ENZYMES AND ACTIVATION OF THE
TUMOR SUPPRESSOR PROTEIN P53
The presence of thousands of mutations in cancer cells suggests that among the
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108 cells in a human tumor at the time of diagnosis there are billions of different
mutations, and that gene mutations are present in one or more cells within a tumor
(Loeb et al., 2003). The hypotheses was proposed and the evidence was provided
showing that selenium supplementation can inhibit carcinogen-induced covalent
DNA adduct formation and can retard oxidative damage as a result of chemical
and physical insult to DNA, lipids, and proteins (El-Bayoumy, 2001). It is known
that carriers of BRCA1 mutations are characterised by significantly greater mean
frequencies of induced chromosome breaks per cell than did healthy non-carrier
relatives. It is known that such mutations are responsible for greatly increased
risks of breast and ovarian cancer. Indeed, the product of the BRCA1 gene is
involved in the repair of double-stranded DNA breaks and therefore increased
susceptibility to DNA breakage contributes to the cancer phenotype. In women
who are born constitutional heterozygous mutations of the BRCA1 gene the
frequency of chromosome breaks was greatly reduced following 1 to 3 months of
oral selenium supplementation (Kowalska et al., 2005). In the New Zealand
population characterised by comparatively low serum selenium levels (97.8 ng/
ml) a statistically significant inverse relationship (P = 0.02) with overall accumulated
DNA damage in blood leukocytes was shown. The authors suggested that the
selenium intake in half of this population is marginal for adequate repair of DNA
damage, increasing susceptibility to cancer and other degenerative diseases
(Karunasinghe et al., 2004). To evaluate the effects of dietary selenium
supplementation on DNA damage in prostate tissue and on apoptosis in prostate
epithelial cells an in vivo canine model was used (Waters et al., 2003). Sexually
intact elderly male beagle dogs were randomly assigned to receive an
unsupplemented diet (control group) or diets that were supplemented with selenium
(treatment group), either as SeMet or as high-selenium yeast at 3 g/kg or 6 g/kg
body weight per day for 7 months. The extent of DNA damage in prostate cells
and in peripheral blood lymphocytes was lower among the selenium-supplemented
dogs than among the control dogs but was not associated with the activity of the
antioxidant enzyme GSH-Px in plasma. Similarly, in human formation of DNA
adducts, an indicator of carcinogenicity, was significantly lower in the Sesupplemented subjects than in those receiving placebo (Liu et al., 1991).
It is important to mention that different forms and levels of selenium compounds
are critical factors with regard to cellular responses. SeMet-mediated redox
regulation of p53 and activation of the DNA-repair branch of the p53 pathway
has been reported (Seo et al., 2002). Similarly sodium selenite and methyl-seleninic
acid (MSA) also can affect p53 activity defined as trans-activation of a p53dependent reporter gene. It seems likely that specific mechanisms of p53 activation
differ among selenium chemical forms that may differentially modify p53 for
DNA repair or apoptosis in conjunction with a given level of endogenous or
exogenous DNA damage. In particular, it was shown that SeMet did not affect
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p53 phosphorylation, while MSA caused phosphorylation of one or more p53

threonine residues and sodium selenite caused phosphorylation of p53 serines
20, 37 and 46 (Smith et al., 2004). Selenite-induced p53 Ser15P appeared to be
important for activating the caspase-mediated apoptosis involving both the caspase8 and the caspase-9 pathways in the LNCaP cells. (Jiang et al., 2004; 2004a). The
authors suggested that post-translational modifications of p53 are determinants
of p53 activity and probably affect the threshold for p53-mediated functions.
Indeed, protective effect of selenium against DNA damage in many cases related
to organic selenium and using other forms of Se could give different results. For
example, selenite treatment during the cancer initiation phase, when the first critical
DNA-damage occurs, did not have any effect (Bjrkhem-Bergman et al., 2005).
Furthermore, using cell culture direct evidence on the inhibitory effect of inorganic
Se on cellular DNA repair capacity was demonstrated (Abul-Hassan et al., 2004).
Taking into account the importance of maintaining an efficient DNA repair
pathways for sustainable genetic stability, their inactivation by selenite exposure
suggests Se in this form may contribute to cancer risk by limiting the capacity of
cells to repair DNA damage. This could explain experimental results showing
that dietary sodium selenite delivered to rats by pipe drinking aggravated and
caused gastric erosion, hemorrhage and promoted intestinal metaplasia of gastric
mucosa (Su et al., 2005). Therefore a choice of Se compounds for cancer
chemoprevention is of great importance and SeMet, a natural form of selenium,
deserves more attention.
EFFECT ON GENE EXPRESSION

Using gene expression profiling a similar whole transcriptome gene expression
patterns in prostate cancer cells from human and rat prostate cancer cell lines
both at baseline expression levels and after treatment with physiologic
concentrations of selenium was demonstrated (Schlicht et al., 2004). Furthermore,
a subset of one hundred and fifty-four genes that demonstrate a similar level of
differential expression to selenium treatment in both species was identified. In
particular two genes, the Insulin like Growth Factor Binding protein 3, and Retinoic
X Receptor alpha, are considered prime candidates for explaining the mechanism
of seleniums chemopreventive effect in prostate cancer (Schlicht et al., 2004).
Indeed, a downregulation of these genes is associated with cancer and Se can
induce these genes and inhibit tumorigenesis. Osteopontin (OPN) is another
potential target gene in the inhibition of mammary tumorigenesis by selenium. In
fact, cDNA array analysis showed a reduced expression of OPN in mammary
tumors of mice fed with the 3 ppm selenium diet in comparison with OPN
expression in tumors arising in 0.1 ppm selenium-fed mice (Unni et al., 2004).
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Furthermore, a 24-hour treatment of TM6 cells with Se-methylselenocysteine

significantly inhibited their migration and also reduced their OPN expression in
comparison with untreated cells. The plethora of effects of Se on almost all cellular
functions and pathways has just been exemplified in an analysis of changes in the
transcriptome of human prostate cancer cells treated with methylseleninic acid in
vitro (Zhao et al., 2004).
ENHANCEMENT OF IMMUNE FUNCTION (SEE CHAPTER 4)

Selenium is characterised by immunomodulating properties, which are important
for cancer prevention and maintenance of general health. The evolution of a tumor
is monitored by immunologic defences that recognize tumor antigens. Therefore
new tumor clones are probably those that have overcome the hosts immunologic
defenses that eliminate mutant cells. Random mutations affecting proteins within
cells that have not undergone clonal proliferation may not have tested the hosts
immunologic defenses and yet may be able to elicit a strong immunologic response.
On the other hand, selenium inhibits production of prostaglandins that cause
inflammation. Since chronic inflammation can lead to cancer development antiinflammatory action of Se could be protective against cancer development. It is
important to mention that supplementation with selenium, even in seleniumreplete individuals, has marked immunostimulating effects, including an
enhancement of proliferation of activated T cells (clonal expansion). In fact,
lymphocytes from volunteers supplemented with selenium (as sodium selenite)
at 200 micrograms per day showed an enhanced response to antigen stimulation
and an increased ability to develop into cytotoxic lymphocytes and to destroy
tumor cells. Natural-killer-cell activity was also increased. Supplementation
resulted in a 118% increase in cytotoxic-lymphocyte-mediated tumor cytotoxicity
and an 82% increase in natural-killer-cell activity compared with baseline
(Kiremidjian-Schumacher et al., 1994). Twenty-two adult UK subjects with plasma
selenium concentrations <95 g/l received 50 or 100 g Se (as sodium selenite)
or placebo daily for 15 wk in a double-blind study. Selenium supplements
augmented the cellular immune response through an increased production of
interferon gamma and other cytokines, an earlier peak T cell proliferation, and an
increase in T helper cells (Broome et al., 2004). Selenium-supplemented subjects
also showed more rapid clearance of the poliovirus, and the poliovirus reverse
transcriptase-polymerase chain reaction products recovered from the feces of the
supplemented subjects contained a lower number of mutations. Se-enriched yeast
was shown to have immunostimulatory properties in elderly humans. For example,
the effect of selenium supplementation on lymphocyte-proliferation responses to
mitogens was investigated in 22 elderly institutionalized subjects. Subjects were
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assigned to a 6-month trial with either 100 micrograms Se/d or a placebo (Peretz
et al., 1991). During selenium supplementation the proliferative response to
pokeweed mitogen increased significantly (+79% of baseline concentrations after
4 months, P<0.01) and reached the upper limit of the usual range for adults after
6 mo (+138%, P <0.001). Furthermore, Se positively affected the immune status
of advanced cancer patients. For example, the ability of selenium dioxide (SeO2)
to enhance the lymphocyte progression through the cell cycle in patients with
advanced (stage IV) cancer was studied (Mantovani et al., 2004). The addition
into culture of 1.5 microM SeO2 enhanced significantly the progression into S
phase of lymphocytes (an essential prerequisite for the physiological functioning
of the immune system) isolated from cancer patients, whilst no significant effect
was observed on lymphocytes isolated from controls.
It is important to mention that Se has a protective effect against age-related
immunosuppression and Se supplementation provides significant improvement
in elderly patients by increasing the humoral response after vaccination and could
have considerable public health importance by reducing morbidity from respiratory
tract infections (Girodon et al., 1999). Free radicals cause a cascade of intracellular
events resulting in liberation in cytoplasm of nuclear transcription factor kappa B
(NF B) from its inhibitory protein IB (Flohe et al., 1997) which permits its
translocation into the nucleus, where it binds to DNA, enabling the initiation of
the transcription process. NF B controls the production of the acute phase
mediators such as TNF-, lIL-2, and IL-2 receptors, which in turn activate NFB,
amplifying the inflammatory cascade (Berger, 2005). Selenium has been shown
to down regulate NFB and thereby to limit the extension of the inflammatory
response (Flohe et al., 1997; Kim and Stadtman, 1997). Indeed effect of Se on
human immunity is difficult to overestimate.
INDUCTION OF APOPTOSIS
Specific induction of apoptosis in pre-neoplastic/neoplastic lesions thereby
preventing the development of manifest tumors is considered as an important
mechanism of anticancer Se activity (Combs and Gray, 1998; Whanger, 2004).
The ability of cells to program their own death provides a mechanism to control
mutations. In fact, inhibition of cell growth can be accomplished by either a
decrease in cell proliferation or an increase in apoptosis or both. Apoptosis is
therefore an important cellular mechanism for growth regulation. Se was shown
to inhibit growth and stimulate programmed cell death in a variety of cell culture
systems. For example, it has been shown that selenium compounds can cause
apoptosis in cultured cells (Krishnaswamy et al., 1995). Both caspase-dependent
and -independent selenite-mediated apoptosis have been reported (Jnsson-
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Videster et al., 2004; Jiang et al., 2001) and it is associated with the inhibition of
several important intracellular signalling pathways (Gopee et al., 2004). In
particular, it was shown that p38 mitogen-activated protein kinases is a key
upstream mediator for the methylselenol-specific induction of vascular endothelial
caspase-dependent apoptosis, which is principally executed by caspase-3-like
activities (Jiang et al., 2004). Selenium can cause irreversible apoptosis with DNA
strand breaks (Spallholz, 1994; Davis et al., 1998; Spallholz et al., 2001) as well
as cell arrest and/or apoptosis independent of DNA strand breaks (Wilson et al.,
1992; Lu et al., 1995; Finley, 2005).
The effects of inorganic Se compounds on induction of apoptosis by which
Se compounds exert cancer chemopreventive activity have been studies recently
(Takahashi et al., 2005). With the use of HSC-3 human oral squamous cell
carcinoma cells, it has been shown that treatment with Se for 72 h, in the form of
SeO 2 and Na 2SeO 3, but not Na 2SeO 4, markedly induced apoptosis in a dosedependent manner. In particular, the authors suggested that modulation of the
mitochondrial redox equilibrium by Se contributes to the mitochondrial pathway,
regulating caspase-9-mediated apoptosis without a concurrent increase in ROS.
On the other hand, Se-methylselenocysteine (MSC), one of the most effective
selenium compounds at chemoprevention, induced apoptosis in HL-60 and ROS
played a crucial role in MSC-induced apoptosis (Jung et al., 2001). Recent data
of Jiang et al. (2004) support p38 MAPK as a key upstream mediator for the
methylselenol-specific induction of vascular endothelial caspase-dependent
apoptosis, which is principally executed by caspase-3-like activities. The signal
transduction pathways affected by Se compounds in biopsies of normal human
oral mucosa cells and human oral squamous carcinoma cells (SCCs) were
investigated (Ghose et al., 2001). Two Se compounds were tested. Firstly,
selenodiglutathione (SDG), the primary metabolite of selenite and the most
commonly used cancer-protective Se compound in animal models. Secondly, the
synthetic Se compound, 1,4-phenylenebis(methylene)selenocyanate (p-XSC), one
of the most potent chemopreventive pharmacological Se compounds. The authors
showed that:
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SCCs were significantly more sensitive to induction of apoptosis by SDG

than normal human oral mucosa cells
both Se compounds induced the expression of Fas ligand (Fas-L) in oral
cells to a degree that correlated with the extent of apoptosis induction
both SDG and p-XSC induced the stress pathway kinases, Jun NH2-terminal
kinase (JNK) and p38 kinase, at concentrations causing apoptosis;
p-XSC, and to a lesser extent SDG, activated extracellular regulated kinases
1&2 (ERKs 1&2) and protein kinase-B or Akt.
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INHIBITION OF CELL PROLIFERATION

In vitro studies have demonstrated significant and complex effects of
selenocompounds on prostate cancer cell proliferation, differentiation, and
signalling related to the initiation, progression, and regression of the cancer (Willis
and Wians, 2003; Berger, 2005). Cell growth inhibition was invariably seen with
selenite treatment in a dose-dependent manner. This was accompanied by decreases
in DNA synthesis and a block in the cell cycle at the S/G2-M phase (Ip, 1998).
Treatment with Se-methylselenocysteine was associated with a lower rate of cell
growth and DNA synthesis and cell cycle progression was blocked at the G1 phase.
Furthermore a marked elevation in DNA single strand breaks that occurred within
a few hours was seen in selenite-treated cells. Such an outcome was absent with
exposure to the methylated selenium compounds. Cell death by necrosis or acute
lysis was another hallmark of the selenite effect. After the initial wave of cell
swelling and lysis, some visible signs of apoptosis were evident in the longer
cultures (Ip, 1998). It has been shown that low Se status increases angiogenesis
by inducing the production of vascular endothelial growth factor (VEGF). This
could be mediated via changed in TrxR activity. In particular, it was found that
chemical inhibition of TrxR in Se-sufficient endothelial cells (ECs) was associated
with increases in VEGF and VEGF receptor expression, cell migration,
proliferation, and angiogenesis to levels similar to those seen in Se-deficient Ecs
(Streicher et al., 2004). It is interesting that the TrxR-overexpressing cells grew
slower over a wide range of selenium concentrations (Nalvarte et al., 2004).
ALTERATION OF CARCINOGEN METABOLISM

Data reviewed by Combs and Gray (1998) indicate that Se can affect enzymes
involved in carcinogen metabolism and in that way affect cacinogenicity of various
compounds. Indeed, in many cases carcinogens require metabolic activation and
Se can interfere with this process decreasing chances of carcinogens to cause
cancer initiation. In fact, P450 enzymes in the liver may be induced by selenium,
leading to detoxification of some carcinogenic molecules. For example,
diphenylmethyl selenocyanate was found to upregulate significantly different
phase II detoxifying enzymes such as glutathione-S-transferase and superoxide
dismutase in skin cytosol when measured after 15 days and also after 12 weeks of
the first DMBA treatment of mice (Das and Bhattacharya, 2004). A significant
increases in glutathione S-transferase, quinone reductase and GSH-Px activities
in liver of dams were observed following administration of selenium-enriched
yeast (Kramer et al., 2003).
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INFLUENCE ON ESTROGEN AND ANDROGEN RECEPTOR EXPRESSION

(Lee et al., 2005; Dong et al., 2004)
Selenium (methylseleninic acid, MSA) decreased the levels of expression

of ERmRNA and protein and reduced the binding of labelled estradiol to
estrogen receptor in MCF-7 cells;
MSA inhibited the trans-activating activity of estrogen receptor in MCF-7
cells expressing functional estrogen receptor
MSA decreased the binding of estrogen receptor to the estrogen responsive
element site
MSA suppressed estrogen induction of the endogenous target gene c-myc.
MSA increased ER mRNA expression in MDA-MB231 human breast cancer
cells.
MSA was able to significantly down-regulate the expression of prostatespecific antigen (PSA) transcript and protein within hours in the androgenresponsive LNCaP cells. Decreases in androgen receptor (AR) transcript
and protein followed a similar dose and time response pattern upon exposure
to selenium. Indeed, androgen receptor signalling has been documented
extensively to play an important role in the development of both androgendependent and -independent prostate cancer. The reduction of AR and
PSA expression by selenium occurred well before any significant change
in cell number. Indeed, MSA decreases the expression of androgen receptor
and PSA in five human prostate cancer cell lines, irrespective of their
androgen receptor genotype (wild type versus mutant) or sensitivity to
androgen-stimulated growth (Dong et al., 2005).
MSA inhibited the trans-activating activity of AR in cells transfected with
the wild-type AR expression vector.
MSA suppressed the binding of AR to the androgen responsive element site
INHIBITION OF ANGIOGENESIS (Lu and Jiang, 2001; Lipinski, 2005).

Low Se status was shown to increase angiogenesis by inducing the production of
vascular endothelial growth factor (VEGF). At the same time Se-dependent
thioredoxin reductase regulates angiogenesis by increasing endothelial cell-derived
vascular endothelial growth factor (Streicher et al., 2004). The chemopreventive
effect of increased Se intake on chemically induced mammary carcinogenesis
has been associated with reduced intratumoral microvessel density and an
inhibition of the expression of vascular endothelial growth factor. The in vitro
data also show that monomethyl Se potently inhibits cancer epithelial expression
of vascular endothelial growth factor (Lu and Jiang, 2001). In an animal model
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mice with PC3 tumors in the prostates were fed baseline selenium replete diet
(0.07 ppm), supplemented with different forms of selenium (sodium selenate,
selenomethionine, methylselenocysteine and selenized yeast) at 2 different
concentrations (0.3 and 3 ppm) in drinking water. It was shown that sodium
selenate significantly retarded the growth of primary prostatic tumors and the
development of retroperitoneal lymph node metastases, which was associated
with a decrease in angiogenesis (Corcoran et al., 2004). Furthermore, selenite
inhibited invasion of HT1080 human fibrosarcoma cells and adhesion of HT1080
cells to the collagen matrix (Yoon et al., 2001).
INTERACTIONS WITH HEAVY METALS INVOLVING IN CANCER

PROMOTION
Se is known to interact with heavy metals, including Cd and As, and it has been
suggested that its cancer protective action could be attributable in part to its
interaction with Cd, a toxic and suspected carcinogenic element, which is found
in many foods, in drinking water, and in the environment (Drasch et al., 2005).
Indeed, Cd is considered a significant prostate cancer risk factor as it stimulates
the growth of prostate epithelial cells and promotes their malignant transformation.
The pathogenesis of prostatic cadmium carcinogenesis might include aberrant
gene expression resulting in stimulation of cell proliferation or blockage of
apoptosis. Furthermore, activation of transcription factors such as the
metallothionein gene and activation of some protooncogenes may enhance cell
proliferation with damaged DNA. Suppression of DNA repair would add to the
population of cells with damaged DNA (Goyer et al., 2004). Therefore, prostate
cancer risk is determined by many different factors including the degree of Cd
exposure. In this case a complex between Cd and Se would inactivate cancerpromoting properties of Cd and decrease Se status of the body. The associated
physiological inactivation of Se could account for the increase of the prostate
cancer risk with advancing age. In particular, the excessive accumulation of Cd
in the prostates of smokers along with sub-optimal Se intakes could explain why
smokers develop more aggressive and lethal forms of prostate cancer than
nonsmokers (Drasch et al., 2005). Similarly, through epidemiological studies, As
exposure has been associated with increased risks to cancers of the lung, skin,
bladder, and liver and the US Environmental Protection Agency has classified As
a known human carcinogen (Category A; Zeng et al., 2005). Indeed antagonistic
effects or mutual detoxification between As and Se have been confirmed in many
animal species including humans. It is generally accepted that uptake of one of
these elements causes release, redistribution, or elimination of the other element
by urinary, biliary, and/or expiratory routes (Zeng et al., 2005). The authors
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suggested that Se may function as an endogenous stop signal for As-induced

carcinogenic cell signalling such as the activation of AP-1 and NFB. At low
dietary Se intakes, interactions with heavy metals may produce states of latent Se
deficiency as well as increased susceptibility to cancer development. For example,
in experiments with female mice, exposures to low levels of the Se-antagonistic
elements As, Pb and Cd in the drinking water abolish the cancer-protecting effects
of Se (Schrauzer, 1987). At higher exposure levels, these elements may act as
inhibitors or promotors of malignant transformation and tumor growth.
From the analysis of results of animal studies showing anticancer effect of various
Se compounds it is clear that doses used in those studies (>1.5 ppm) are several times
higher than those required for maximum selenoprotein expression (<0.5 ppm; Combs
and Gray, 1998). Therefore it has been suggested that various Se metabolites (in
particular methylated selenium metabolites, e.g. methyl selenol) can directly affect
tumour cells. Selenium properties relted to anticancer effect of its various compounds
are shown in Tables 11.24-11.26. Indeed inorganic selenium compounds as well as
organic selenium and some synthetic preparations can be metabolised producing
chemicals toxic for tumour cells. However, molecular mechanisms of such protective
effects of Se require further investigation. For example, selenite by virtue of oxidizing
cell membrane thiols, can prevent the formation of the coat and consequently makes
cancer cells vulnerable to the immune surveillance and destruction (Lipinski, 2005).
Table 11.24 Selenium actions relevant to its anticancerogenic properties (Adapted from Schrauzer, 2000)
Se action
References
Prevention of DNA methylation

Prooxidant properties of selenite
Slowing cell growth
Selective destruction of tumor cells
Partial downregulation, improved contact inhibition and
cell adhesion
Inhibition of cell colony formation
Induction of p53 and of apoptosis
Cox, 1985
Spallholz, 1994
Vogl et al., 1987
Yu et al., 1988
Pung et al., 1987
Inhibition of microtubular formation

Partial retransformation of human hepatoma cells
Inhibition of the expression of MAZ, the c-myc-activating
zinc-finger protein, which regulates the activation of the
oncogene c-myc
Antagonistic effects to metals-cancerogens
nhibition of the activation of the nuclear transcription factor
nF-B
Immunomodulation properties
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Caffrey and Frenkel, 1991

Lanfear et al., 1994;
Gallegos et al., 1997
Leynadier et al., 1991
Yu et al., 1990
Nelson et al., 1996
Sunderman and Barber, 1988

Macropoulos and Bruening,
1996
see chapter 4
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Table 11.25 Antitumorigenic effect of different selenium compounds (Adapted from Whanger, 2002)
Compound
Dose of Se for 50% inhibition,

ppm
Se-methylselenocysteine
Selenobetaine
Selenobetaine methyl ester
Selenite
SeMet
Selenocysteine
2
2
2-3
3
4-5
4-5
Table 11.26 Growth inhibition of human tumor cell lines and normal diploid fibroblasts by
selenomethionine (adapted from Redman et al., 1998)*
Cell line
Concentration (IC50)
MCF-7/S (breast cancer)

UACC-375 (melanoma)
HT-29 (colon cancer)
DU-145 (prostate cancer)
A-549 (lung cancer)
Fibroblast (normal diploid)
45 mM
50 mM
130 mM
40 mM
65 mM
1 mM
*While micromolar concentrations of selenomethionine inhibited the growth of the tumor

cell lines tested, selenomethionine in millimolar concentrations was necessary to inhibit
normal fibroblast cell growth
New clinical trials including SELECT and PRECISE are designed to further explore
the possibilities of cancer-prevention properties of selenium. However, it is necessary
to mention that a choice of selenium compounds for clinical trials is of great
importance. Indeed, from the point of view of general nutrition, Se-yeast providing a
natural mixture of selenocompounds in organic form would be the most beneficial.
Indeed, a profile of Se compounds of the yeast is similar to other natural Se sources,
such as grains and forages. Therefore, a choice of SeMet for the SELECT trial seems
to be risky. The problem is that SeMet is the main compound of the Se-yeast, but not
the only one. When results are obtained with Se-yeast it is not possible to be 100%
sure that they are just due to SeMet. As mentioned above, Se-yeast was used in
previous clinical trials and will be used in some other trials. Indeed, ideally increasing
Se consumption to have Se concentration in the blood >121 g/l would be a way to
address Se deficiency issues and simultaneously provide natural protection against
cancer. Therefore, there are two approaches to this:
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nutritional: consumption of Se-enriched food, including eggs, meat, milk

as well as various vegetables. This could also include dietary supplements
in the form of Se-yeast or SeMet (if efficiency is proven). There is a great
body of evidence to support this approach, including epidemiological
studies and clinical intervention studies.
pharmacological: consumption of Se-tablets in the form of sodium selenite,
SeMet, various chemically synthesised organo-selenium compounds, etc.
There is strong evidence indicating that those Se compounds show cancerprotective effects in model systems and in animal experiments. However,
results of experiments with chemically induced cancer models are difficult
for interpretation and transfer for humans.
Selenium and cardiovascular diseases

Cardiovascular diseases are the leading cause of death and disability in many
industrialized countries accounting for more than 50 per cent of the deaths and
they are increasing in the developing world. The principal cardiovascular diseases
are related to atherosclerosis: coronary heart disease, stroke, and peripheral vascular
disease (Luepker, 2002). The patterns on distributions of these diseases vary in
different regions, however, coronary heart disease is assuming pre-eminence in
many areas. Primary risk factors of CVD include:
hypertension
hyperlipidaemia
smoking
Predisposing factors of CVD include:
poor quality diet

obesity
lack of physical inactivity
diabetes, hyperglycaemia, and hyperinsulinaemia
genetics
maleness
Did you know that cardiovascular diseases are the leading cause
of death and disability in many industrialized countries?
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A great body of evidence indicates that a nutritionally balanced diet plays an

important role in maintaining a healthy weight and can have a favourable impact
on blood pressure (Chobanian et al., 2003) and plasma lipids (National Cholesterol
Education Program, 2002). There are at least three major hypothesis of
atherosclerosis (Keaney, 2000):
Response-to injury hypothesis

Oxidative modification hypothesis
The response-to-retention hypothesis of early atherogenesis
Accumulating data confirmed the hypothesis involving oxidized LDL as a major

factor in atherogenesis and protection being offered by natural antioxidants. Dietary
deficiency of selenium has been incriminated in the etiology of cardiovascular
diseases (CVD). There are several lines of evidence for this (Huttunen, 1997).
Cardiomyopathy associated with low selenium intake has been described

in areas of exceptionally low selenium intake (Keshan disease) and in patients
receiving total parenteral nutrition. For example, dietary selenium deficiency
associated with an endemic form of juvenile cardiomyopathy has been
reported in China, and is called Keshan disease (Li et al., 1985; Keshan
Disease Research Group, 1979; 1979a). In western countries, a small number
of cases of dilated cardiomyopathy complicating selenium deficiency have
been reported mainly in patients receiving long-term parenteral nutrition
(Johnson et al., 1981; Quercia et al., 1984; Lockitch et al., 1990; Sando et
al., 1992; Markus et al., 1993; Levy et al., 1994) or in association with
other disease states. Selenium deficiency has also been documented in black
African women with peripartum cardiomyopathy (Cenac et al., 1992). The
importance of identifying selenium deficiency as the underlying cause of
dilated cardiomyopathy is underscored by the demonstrated ability of dietary
selenium supplementation to prevent the development of the disease, and
to occasionally reverse its deleterious effects on the myocardium (Levy et
al., 1994; Kavaneau-McHugh et al., 1991).
Did you know that there are at least three major hypothesis of
atherosclerosis: response-to injury hypothesis, oxidative
modification hypothesis and the response-to-retention
hypothesis of early atherogenesis ?
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Several studies have suggested that dietary selenium deficiency may be

associated with an increased risk of coronary heart disease (CHD). Indeed,
epidemiological studies have provided some evidence for the role of
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selenium deficiency in the etiology of atherosclerotic disease (Table 11.27).
Dietary selenium deficiency has also been reported to be associated with
increased risk of coronary artery disease. In particular, for humans,
ecological and epidemiological results are reported that show a relationship
between the serum selenium concentration and cardiovascular disease in
populations where low serum selenium concentrations are found, e.g., in
Eastern Finland. From clinical studies done in Germany, Finland, and
Sweden, subnormal serum selenium and partially whole blood selenium
concentrations are reported in patients with acute myocardial infarction
(Oster and Prellwitz, 1990). An inverse relationship between blood Se
concentrations and cardiovascular disease has been also reported (Kok et
al., 1989). Epidemiological studies indicate an association between low
nutritional selenium status and increased risks of cardiomyopathy,
cardiovascular disease, and carcinogenesis in various sites of the body
(Badmaev et al., 1996). A cohort study including 4419 individuals was
carried out in Northern Italy, analysing the 7-year temporal distribution of
deaths due to coronary disease (Vinceti et al., 1994). It was shown that
exposure to high Se drinking water (7 g/l vs <1 g/l) for at least 5 years
was associated with a substantial decrease in coronary disease mortality. A
prospective epidemiological study in Finland provided evidence of an inverse
relation between plasma selenium concentration and the risk of cardiovascular disease (Salonen et al., 1982). Case-control pairs came from a
population of 11,000 persons examined in 1972 from two counties in eastern
Finland, an area with an exceptionally high mortality from cardiovascular
diseases. The mean serum selenium concentration for all cases was 51.8
g/l and for all controls 55.3 g/l (p<0.01). The results indicated that serum
Se of less than 45 g/l was associated with an adjusted relative risk of
coronary heart disease (CHD) death of 2.9, a relative risk of CVD death of
2.2, and a relative risk of fatal and nonfatal myocardial infarction of 2.1.
The association between serum selenium concentration and five-year risk
of cardiovascular disease was studied in 1,110 men aged 55 to 74 years in
two rural areas of Finland. In the total cohort, all-cause and cardiovascular
deaths were associated significantly with serum selenium of less than 45
g/l, an adjusted relative risk of 1.4 and 1.6 respectively (Virtamo et al.,
1985). Among men free of stroke at the outset, low serum selenium was
associated significantly with stroke mortality, an adjusted relative risk of
3.7. An inverse association between the incidence of ischaemic heart disease
and selenium intake has been described in population comparisons. Recently
it has been shown that dietary intake of non-fish Se in rural community in
Japan had a positive correlation with HDL cholesterol, and an inverse
correlation with the atherogenic index in human (Miyazaki et al., 2002). In
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fact, the atherogenic index was significantly higher in the low selenium
intake group than in the middle selenium intake group and in the high
selenium intake group.
Table 11.27 Epidemiological follow-up studies of selenium status and CHD (Adapted from Alissa et al.,
2003)
Study
Region
No. of
cases
Age
Follow Serum Adjusted RR, 95% CL

up, years Se level,
g/l
Salonen et al.,
1982
Eastern
Finland
11,000
persons
35-59
<45
Miettinen et al., Southern 1222

1983
Finland males
48
5-7
50-105 No significant risk
Salonen et al.,
1985
Eastern
Finland
30-64
<45
1.3 from IHD
Virtamo et al.,
1985
Eastern
1110 men 55-74
and
Southwestern
Finland
<45
1.4 from IHD, 1.6 from

CVD, 3.7 from CRVD
Ringstad and
Thelle, 1986
Norway
6706 men 20-49
59-197 No significant risk
10,532
persons
37-87
<105
1.1 from CHD,

1.2 3.2 from stroke
Suadicani et al., Denmark 3387

1992
males
53- 74
<79
1.6 from CHD
Salvini et al.,
1995
USA
>80
No significant risk
Kardinaal
et al., 1997
EURA1412
MIC study subjects
<70
Toenail 0.63 from MI

Se 0.55
g/g
Kok et al., 1987 Netherlands
92
persons
251
subjects
2.9 from CHD, 2.2

from CVD,
2.1 from MI
CHD- coronary heart disease; CVD- cardiovascular disease; IHD- ischemic heart disease;
MI- myocardial infract; CRVD- cerebrovascular disease
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Association has been shown between cardiovascular death, myocardial

infarction and serum Se by Salonen et al. (1982), Kok et al. (1989) and
Korpela et al. (1989). Indeed, ischemic heart death correlated inversely with
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blood Se in 25 cities of 22 states of the USA (r = -.70; p<0.01; Jackson,
1988).
The decrease in serum levels Se in patients with myocardial damage was
shown implying that Se may play a role in the pathogenesis of ischemic
heart disease. In fact, levels of Se, activities of GSH-Px and C-reactive protein
(CRP), a marker of inflammation, were partly interrelated in acute coronary
syndromes (ACS) and their relationship to cardiac troponins and creatine
kinase-MB mass (CK-MBm), an important prognostic markers was shown
(Altekin et al., 2005). Mean serum Se concentrations measured in patients
with acute myocardial infarction (n = 32) or with ischemic cardiomyopathy
(n = 50) were significantly lower than those determined in control groups
(Navarro-Alarcon et al., 1999). In 54 hospitalized patients with clinical
diagnosis of acute myocardial infarction, serum selenium levels were 670
nmol/l, as compared to 981 nmol/l in 93 healthy controls. In 32 patients
with general arteriosclerosis, the serum Se level was 375 nmol/l, in 64 patients
with arteriosclerotic occlusional disease in the leg region, 366 nmol/l,
respectively (Koehler et al., 1988). In a Sahelian area of Niger, plasma
selenium concentration was measured by neutronic activation and particle
induced X-ray emission in 35 black African women with peripartum
cardiomyopathy and 36 breast-feeding women without cardiac failure as
controls. The plasma selenium concentration in patients was significantly
lower (48 ng/ml) than in controls (77 ng/ml) (Cenac et al., 1992). Moreover,
40% (14/35) patients with peripartum cardiomyopathy had very low plasma
selenium concentrations, below 45 ng/ml, versus none in controls.
From animal experiments, it is known that selenium protects against
cardiotoxic elements, cardiotoxic xenobiotics, and viral infections that affect
the heart (Oster and Prellwitz, 1990). Evidence is presented to suggest that
selenium is preventing oxidative damage of heart cell membranes by lipid
peroxidation (Koehler et al., 1988).
Did you know that there are several lines of evidence indicating
protective effect of selenium against CVD ?
However, the results of longitudinal studies within populations are conflicting

with some investigations observing a relationship between low serum-selenium
levels and the risk of coronary disease, while others did not. In general, dietary Se
supplement may be considered anti-atherosclerotic (Hampel et al., 1989).
Moreover, at least eight Se-containing proteins, including glutathione peroxidase
and thioredoxin reductase, were identified from the rat arterial wall, although the
functions of some of them, such as 69 kDa Se-containing protein, yet remained
unclear (Qu et al., 2000a).
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Mechanisms whereby selenium protects against CVD diseases include (Neve,

1996; Table 11.28):
increased resistance of low-density lipoproteins against oxidative

modification,
modulation of prostaglandin synthesis and platelet aggregation,
protection against toxic heavy metals.
Table 11.28 Protective functions of Se in prevention of CVD (Adapted from Alissa et al., 2003)
Reduction LDL levels, possibly by increasing peripheral catabolism, through its effect on
thyroid hormone metabolism
Inhibition of the oxidative modification of LDL in vitro
Increase resistance of LDL to oxidation in vitro and inhibition of foam-cell formation
Modification of cytokine-induced expression of ICAM-1 and VCAM-1 by human
endothelial cells
Se deficiency may induce a shift in prostaglandin production from prostacyclin to
thromboxanes
Serum Se levels are closely related to platelet aggregability, and possibly related to effects
on thromboxanes A2
Se deficiency is associated with increased lipid hydroperoxides that may cause endothelial
injury
Se has the potential to inhibit production of chemotactic products of LDL oxidation
Se deficiency is associated with impaired cell-mediated immune function, including
reduced circulating T-cells and reduced lymphocyte responsiveness
Selenoenzymes inhibit foam-cell formation and hence the release of growth factors that
stimulate vascular smooth muscle cell proliferation
To enunciate the mechanisms whereby Se protects against cardiovascular diseases,

weanling male Wistar rats were fed deficient (0.022 mg/kg diet) and adequate
(0.159 mg/kg diet) Se diets for 14 and/or 39 wk. The results give some experimental
supports to the hypothesis that low Se status and lipid peroxidation are involved
in the etiology of cardiovascular diseases (Qu et al., 2000; Huang et al., 2002).
In particular, Se deficiency was associated with:
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decreased Se content, GSH-Px activity and total antioxidant capacity in

blood and the arterial wall, heart, liver, and kidney.
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decreased SOD activity in rats arterial wall (Wu and Huang, 2004). In fact,
there is an inverse relationship between dietary Se and antioxidant capacity
of rat cardiovascular tissues (Wu et al., 2003; Wu and Huang, 2004).
increased injuries to arterial endothelium by cholesterol oxides in comparison
to the Se adequate animals (Huang et al., 2002). It seems likely that the
mechanism of selenium inhibition of cell apoptosis induced by oxysterols
in rat vascular smooth muscle cells was related with the antioxidant activities
of selenoproteins (Tang et al., 2005).
increased lipid peroxide levels in blood. In fact, dietary selenium is a potent
antioxidant against plasma and LDL lipid peroxidation and may thus be
considered antiatherogenic (Hussein et al., 1997).
decreased plasma nitric oxide content and vascular nitric oxide synthase
activity.
increased platelet aggregation, thromboxane B2 production and synthesis
of the lipoxygenase-derived compounds (Vitoux et al., 1996).
increased the collagen and ADP-stimulated platelet aggregation rate and
intensity in the Se-deficient rabbits (Turan et al., 1997). In human platelets,
the activity of GSH-Px is particularly high and is very sensitive to the
requirement of selenium. In these deficient subjects, Se administration
increases platelet GSH-Px activity and inhibits platelet hyperaggregation
and leukotriene synthesis.
increased serum total plasma cholesterol, low-density lipoprotein cholesterol
levels and very low density cholesterol (Stone et al., 1994 and decreased
serum high-density lipoprotein cholesterol level. Raised concentration of
LDL cholesterol is regarded as one of the risk factors associated with
atherosclerosis and ischaemic heart disease, whereas HDL cholesterol is
thought to protect against these diseases. Hypercholesterolaemia associated
with Se deficiency was related to increased 3-hydroxy-3-methylglutarylcoA (HMG-CoA) reductase (EC 1.1.1.34) activity in liver microsomes as
compared with control animals (Nassir et al., 1997). On the other hand,
supplemental intake of Se was associated with lower plasma concentrations
of total cholesterol and low-density lipoprotein cholesterol plus very low
density lipoprotein cholesterol (Poirier et al., 2002). Furthermore, Se
supplementation could increase the level of good cholesterol in HDL.
For example, serum Se levels in 26 healthy subjects were positively correlated
with high-density lipoprotein cholesterol concentrations (Luoma et al.,
1984). The high-density lipoprotein cholesterol/cholesterol ratio increased
during Se supplementation in a group excluding subjects with low
cholesterol. The results suggest a link running from low serum selenium to
reduced high-density lipoprotein cholesterol, and further to high coronary
risk. Therefore, in subjects with low selenium its supplementation may reduce
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the risk of coronary heart disease. Indeed, selenium supplementation

modulates the sequences favoring pathogenesis of atherosclerosis. For
example, after three months of high fat diet, there were significant increases
in serum cholesterol and triglycerides in rats. Additional Se supplementation
of those animals significantly decrease the levels of serum cholesterol and
triglycerides in comparison to high-fat diet group (Kang et al., 1998).
Selenium seems to be involved with triglycerides metabolism, eventually
improving the triglycerides status of aged animals. For example, a decrease
in triglycerides concentration was observed in rats fed a diet supplemented
with 0.25 or 0.50 ppm selenium, for 12 months (Crespo et al., 1995). These
data are in agreement with a suggestion that Se may have recuperative effects
for hypercholesterolemia. In fact, Se supplementation of rats fed high
cholesterol diet suppressed the amounts of triglyceride (TG), total cholesterol
(CH) and free fatty acid in the serum. Furthermore Se also decreased the
amounts of low-density lipoprotein cholesterol in the serum and inhibited
the amount of liver TG and CH (Iizuka et al., 2001). Women with the
lowest tertile of selenium concentration had significantly higher atherogenic
index and lower HDL-cholesterol levels compared to those with the highest
tertile (Lee et al., 2003). Furthermore, it has been concluded that Se or
selenoproteins in the vascular wall played an important role in cytoprotection
against cholesterol oxide-induced vascular damage in rats. It is well accepted
that cholesterol oxides are important contributor to endothelial cytodamage
and atherogenesis In fact, the luminal surface of Se-deficient cholesterol
oxide-treated group exhibited numerous crater-like defects and appeared
sponge-like as well as platelets adhering followed by thrombus formation
in focal area of extensive endothelial damage (Huang et al., 2002).
Furthermore, the endothelium of aorta of Se deficient cholesterol oxidetreated group had more frequent lesion where endothelial cell plasma were
swelling with profuse intracellular edema and some vacuoles were seen in
cytoplasm. In severely injured areas, endothelial integrity was completely
destroyed and smooth muscle cells were proliferating and migrated to the
endomembrane. Indeed, Se supplementation can protect human endothelial
cells from oxidative injury (Thomas et al., 1993), while the toxicity of oxLDL was enhanced in Se-depleted endothelial and smooth muscle cells
(Colles et al., 1996).
In liver phospholipids (PL), the conversion of linoleic acid (LA) to
arachidonic acid was reduced and the ratio of n-6/n-3 fatty acids was
increased. As in liver PL, an increase in the n-6/n-3 ratio was also observed
in the mucosal total fatty acids of the small intestine (Schafer et al., 2004).
Increased plasma thromboxane A(2) (TXA(2)) content and decreased plasma
6-keto-PGF1alpha concentration. In contrast, Se-supplemented cells released
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less thromboxane B(2) and more 6-keto-prostaglandin F(1alpha) than Sedepleted cells (Ricetti et al., 1999);
decreased plasma prostacyclin (PGI(2)) concentration. There was also a reduction
in the formation of 6-oxoprostaglandin F1 alpha and monohydroxy products
formed by cyclooxygenase (Funk et al., 1987). In general, Se might be a potent
nutritional modulator of aortic prostacyclin synthesis and lipoprotein metabolism
(Meydani, 1992; Qu et al., 2000). It is important to mention that PGI2 synthesized
and released by endothelial cells has vasodilative effects on smooth muscle
cells and anti-aggregating action on the platelets; meanwhile, TXA2 synthesized
and released by platelets has completely opposite activity. In particular, after
peroxide stimulation, the PGI2 release was significantly lower in the Se-deficient
group compared to the control group and the release ratio of PGI2/TXA2
decreased under peroxide stress in Se-deficient animals (Haberland et al., 2001).
improved Se status of elderly patients could be beneficial in terms of cardiac
functions. It is well accepted that cardiovascular ageing is associated with an
increase in cardiac susceptibility to ischaemia and reperfusion and it seems
likely that increased Se supplementation could improve the prognosis of
cardiovascular diseases in old patients. In fact, the crucial role of selenium in
determining the vulnerability of the heart to ischemia and reperfusion was
demonstrated (Toufektsian et al., 2000). The ability of selenium supplementation
to protect aged cardiomyocytes from hypoxia/reoxygenation damage underlines
the importance of an optimal selenium dietary intake, particularly in the elderly.
For example, the protective effect of selenium, as sodium selenite (SS) or seleno
methionine (SM), in cultured rat cardiomyocytes aged in vitro was studied. It
was shown that Se supplementation, particularly as SM, was able to increase
GSH-Px activity, and consequently total antioxidant activity, and to decrease
lipid peroxidation (Bordoni et al., 2005). Furthermore, in an experiment
conducted in France, 22 months old male Wistar rats received either a highselenium (1.5 mg Se/kg diet) or a low-selenium (0.05 mg Se/kg diet) diet for
10 weeks. At the end of the diet, hearts were submitted to ischaemia and
reperfusion ex vivo (Tanguy et al., 2003). It was shown that high-selenium
supply increased cardiac total, mitochondrial and cytosolic GSH-Px activities.
This diet induced a significant improvement of cardiac post-ischaemic functional
recovery and the preservation of cardiac function was associated with a
significant limitation of ultrastructural alterations of sarcomeres and
mitochondria. Indeed, hearts from selenium deficient animals were more
susceptible to ischemia-reperfusion injury when compared to normal controls.
Selenium supplementation increased the endogenous activity of TR and GSHPx and resulted in improved recovery of cardiac function post ischemia
reperfusion (Venardos et al., 2004).
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From data presented above it is clear that Se could provide protective effect against
cardiovascular diseases. However, the therapeutic benefit of selenium
administration in the prevention and treatment of such diseases still remains
insufficiently documented and convincing proof of such an association can only
be obtained from large, controlled, prospective prevention trials.
Selenium and other diseases

REPRODUCTIVE DISORDERS
Role of free radicals and antioxidants in male reproduction was addressed elsewhere
(Surai, 2002; Surai et al., 2003) and selenium involvement in sperm quality
maintenance was considered in the Chapter 5. Indeed Se deficiency is related to male
subfertility (Bleau et al., 1984). The administration of selenium to sub-fertile patients
(100 g per day) significantly increased sperm motility (MacPherson et al., 1993)
and chance of successful conception (Scott et al., 1998). A significant inverse
correlation was observed between serum selenium level and sperm count in Nigerian
men. Similarly, seminal plasma selenium correlated with spermatozoa motility, viability,
and morphology, while serum selenium level showed positive correlation with the
serum testosterone level (Akinloye et al., 2005). Significant positive correlation was
observed between semen concentration of Se and sperm density, as well as sperm
count, sperm motility and viability in Chinese men. Furthermore, 8-OHdG levels in
sperm DNA inversely correlated with semen Se concentrations in fertile and infertile
subjects (Xu et al., 2001; 2003).
It has also been suggested that free radicals play an important role in female
reproduction with involvement in the pathophysiology of preeclampsia, hydatidiform
mole, free radical-induced birth defects and other situations such as abortions,
endometriosis, tubal and peritoneal factor infertility and unexplained infertility
(Agarwal et al., 2005). Indeed, a prevention of the oxidantive stress is an important
task for diet optimization. In particular, antioxidant-rich food and antioxidant
supplements are shown to have a positive effect on reproduction.. The low selenium
concentration in the blood is associated with increased risks of spontaneous abortions
in women (Barrington et al., 1996) Se is required not only for sperm motility but also
may reduce risk of miscarriage (Rayman, 2000). For example, pre-eclampsia (PE) is
a major complication of pregnancy that is associated with high maternal and perinatal
morbidity and mortality and it is one of the major indications for elective premature
delivery because there is currently no other cure (Rayman et al., 2003).
Did you know that Se deficiency is implicating in human
reproductive disorders development?
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It seems likely that oxidative stress is involved in this human pathology. In

particular, recent findings suggest that lipid and protein oxidation may be an
important factor in the pathogenesis of preeclampsia (Sedar et al., 2003). Indeed,
oxidative stress and subsequent lipid peroxidation were shown to accompany the
complications of hypertension, preeclampsia and diabetes mellitus in pregnancy
(Orhan et al., 2003). It has been shown that median toenail selenium
concentrations in the preeclamptic subjects were significantly lower than in their
matched controls. Furthermore, the authors showed that being in the bottom tertile
of toenail selenium was associated with a 4.4-fold greater incidence of the condition
(Rayman et al., 2003). Within the preeclamptic group, lower selenium status was
significantly associated with more severe expression of disease, as measured by
delivery before 32 weeks. However, an earlier study indicated that women with
preeclampsia had significantly higher median leukocyte selenium concentrations
than normotensive controls and there was evidence of a linear increase in risk of
preeclampsia with increasing concentrations of selenium in leukocytes (Mahomed
et al., 2000). Serum levels of MDA was markedly higher (P < 0.001) and serum
level of Se was markedly lower (P < 0.001) in PE women compared with healthy
pregnant or non-pregnant women (Atamar et al., 2005). Selenium deficiency in
pregnant rats lead to symptoms similar to those seen in human PE (Vanderlelie et
al., 2004). There was a significant reduction in the mean hair selenium level in
the recurrent miscarriage group compared with the control group (0.14g/g vs
0.34 g/g; Al-Kunani, et al., 2001). However, in an earlier study there was no
association between unexplained recurrent miscarriage and reduced selenium status
(Nicoll, et al., 1999).
The effect of Se supplement on pregnancy was studied in 52 pregnant women
with high risk factors of pregnancy induced hypertension (PIH). They were given
natural Se dietetic liquid (100 g/d) for 6-8 weeks during late pregnancy, and 48
controls were given placebo. The results revealed that Se supplement on the
pregnant women prevented and decreased the incidence of PIH and gestational
edema (Han and Zhou, 1994).
It is well known that neural tube defects are important causes of infant mortality
and childhood morbidity. Maternal Se deficiency during pregnancy was thought
to be one of the factors responsible for fetal-neural-tube defects (NTDs). The
relationship between selenium concentration and neural-tube-defect occurrence
in women with a second-trimester termination due to fetal-neural-tube defects
(NTDs) was investigated in the case-control study (Cengiz et al., 2004). It was
shown that cases had significantly lower serum selenium levels (55.2 vs 77.4 ng/
ml respectively, p<0.001). However, the lowered serum and hair Se concentrations
may be secondary manifestations of an abnormal pregnancy and did not contribute
to its production (Guvenc et al., 1995).
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From data presented above it is clear that Se has a specific role in human
reproduction by maintaining high semen quality and noncomlicating pregnancies.
SELENIUM AND ASTHMA

Asthma bronchiale is a multifactorial disease process with genetic, allergic,
environmental, infectious, emotional and nutritional components. Indeed, asthma
is a chronic inflammatory disorder of the respiratory airways characterized by
bronchial airway inflammation resulting in increased mucus production and airway
hyper-responsiveness (Miller, 2001), however, the origin of pathogenesis has not
been elucidated in details (Gazdik et al., 2004). It is believed that oxidative stress
contributes significantly to its inflammatory pathology (Greene 1995; Fenech
1998). In fact, many decades of research have produced a significant amount of
data indicating increased oxidative stress in asthma and suggesting a potential
role for oxidants in the pathogenesis of disease and antioxidants in its prevention
and treatment (Caramori and Papi, 2004).
Did you know that an optimal Se balance could be important
element to control asthma?
A link between asthma and Se deficiency has been hypothesised (Omland et al.,
2002). Indeed, asthma is associated with reduced circulatory selenium status and
lowered activity of GSH-Px (Hasselmark, 1993; Malmgren et al., 1986). For
example, in 49 asthmatics Stone et al. (1989) reported significantly decreased
concentration of Se in plasma and whole blood in comparison with the healthy
control group. There was 3.5- and 5.1-fold increase in asthma probability in the
lower range plasma and whole blood Se concentrations, respectively. New Zealand
asthmatics were also characterised by decreased Se status (Flatt et al., 1990).
There are other similar indications of decreased Se status of asthmatics (Hasselmark
et al., 1993; Pearson et al., 1991; Kadrabova et al., 1996; Misso et al., 1996;
Powell et al., 1994; Shaw, 1994).
It was reported that selenium supplementation might be beneficial to patients
with intrinsic asthma (Kadrabova 1996). A population-based case-control study
was carried out in South London, UK to investigate whether asthma is less common
and less severe in adults who consume more dietary antioxidants. Participants
were aged 16-50 yr and registered with 40 general practices (Shaheen et al.,
2001). After controlling for potential confounding factors and total energy intake,
Intake of selenium was negatively associated with asthma (OR per quintile increase
0.84). The analysis was based on 1471 individuals including 607 asthmatics and
864 non-asthmatics. Participants with the highest Se intakes (54-90 g/day) were
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only about half as likely to have asthma as those who consumed the least Se (2330 g/day). Clinical improvements were observed in non-allergic asthmatics after
Se supplementation (100 g/day) for 14 days (Hasselmark et al., 1993).
Significantly decreased consumption of corticoids by asthmatics was reported
after supplementation with 200 g/day of Se for 96 weeks (Gazdik et al., 2002).
It has been shown that children aged 4-16 who increased their intake of selenium
were 20% less likely to develop asthma (Ellwood et al., 2001). Similarly, for
children exposed to second-hand smoke, the decreased prevalence of asthma
with increased Se was 50% (Rubin et al., 2004). Furthermore, there is some
evidence indicating that Se-rich foods, such as whole-grain cereals and fish may
protect against asthma in children (Ellwood et al., 2001). However, not all trials
related to Se and asthma were successful. For example, a short-term Se
supplementation did not show any significant benefit in patients with intrinsic
asthma (Baker and Ayres, 2000).
It seems likely that optimal Se status is an important factor helping asthmatics.
Indeed, immunomodulating and antioxidant properties of selenoproteins are of
major importance in preventing/treatment of asthma.
SELENIUM AND RHEUMATOID ARTHRITIS

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are actively
involved in clinical expression of the inflammatory arthritis. In fact free radicals
play an important role in the chronicity of the inflammatory reaction contributing
to cartilage and bone destruction (Hadjigogos et al., 2003). Furthermore, ROS
and RNS can also alter signalling system in the cell, including NFkB and AP-1,
and induce gene expression causing apoptosis in cartilage (Kuhn et al., 2004)
and joint inflammation (Pelletier and Martel-Pelletier, 2003). Therefore, it is
reasonable to suggest that optimal Se status would be beneficial for arthritis patients.
Did you know that Se deficiency could be associated with
increased severity of rheumatoid arthritis?
In a prospective study, 28 rheumatoid arthritis (RA) patients were followed for
7.3 years (Tarp et al., 1989). It was shown that low Se levels were related to high
disease activity and normal Se levels were observed in periods of low disease
activity. Indeed plasma and synovial fluid Se concentration were found to be
significantly lower in patients with RA than those of healthy subjects (Yazar et
al., 2005). Similarly, serum selenium was low in RA and this was associated with
disease activity parameters (Honkanen et al., 1991). It was suggested that Se
could be beneficial for RA patients as anti-inflammatory agent (Tarp, 1995).
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Furthermore, Se can downregulate cytokine signalling (McCarthy and Russel,

1999) improving arthritis symptoms.
Se supplementation of RA patients was shown to be beneficial in terms of
improvement of RA symptoms (Peretz et al., 1992; Aaseth and Teigen, 1993;
Heinle et al., 1997). However, there are earlier reports indicating no change in
RA symptoms after Se supplementation (Tarp et al., 1985; Jantti et al., 1991;
Petersson et al., 1991). These differences could be a reflection of variables in
study design in terms of duration, Se doses and state of the disease. It should be
mentioned that the largest study (n=70) which was a double-blind, randomised
control trial where patients were supplemented with 200 g sodium selenite for 3
months, showed a significant decrease in RA symptoms and biochemical indicators
of inflammation (Heinle et al., 1997). A double blind multi-centric placebocontrolled study was carried out to investigate the effects of selenium
supplementation in rheumatoid arthritis (RA). Fifty-five patients with moderate
RA received during 90 days either capsules containing selenium-enriched yeast
(200 g/d) or a placebo (Peretz et al., 2001). There was no effect of Se
supplementation on the visual analog scale, the Ritchie index, the number of
swollen and painful joints, and morning stiffness. However, when examining the
quality of life a significant (p<0.01) improvement in arm movements and health
feeling was evidenced in selenium-treated patients. However, not all attempts of
using selenium in arthritis treatment were successful. For example, in a rat model
of adjuvant arthritis, markers of inflammation and destructive changes associated
with the disease (levels of serum albumin, serum nitrite/nitrate concentrations,
hind paw swelling, arthrogram scores, whole-body bone mineral density and bone
erosions) were not affected by sodium selenite treatment (Rovensky et al., 2005).
Therefore, it seems likely that Se can affect rheumatoid arthritis by
immunomodulation as well as by changing cell signalling. However, there is a
need for well designed, placebo-controlled studies to prove Se efficiency in antiarthritis therapy.
SELENIUM AND DIABETES

Diabetes is a prevalent systemic disease affecting a significant proportion of the
population worldwide. The morbidity and mortality associated with diabetes is
the result of the myriad complications related to the disease. It has been suggested
that enhanced production of free radicals and hyperglycemia-induced oxidative
stress are central events to the development of diabetic complications (Rahimi et
al., 2005; Niedowicz and Daleke, 2005). This suggestion has been supported by
demonstration of oxidative stress in diabetic individuals suffering from
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complications. Therefore, it seems reasonable to expect that antioxidants could

have a protective role in the improvement of diabetes.
Did you know that optimal Se balance could help in preventing
diabetis complications?
Selenium was shown to play an important role in reducing the oxidative stress
associated with diabetes. The effect of oral administration of sodium selenite on
streptozotocin (STZ)-induced diabetic mice was studied (Mukherjee et al., 1998).
Diabetes caused hyperglycemia (2.8-fold increase) with a significant increase in
the MDA levels (89% in liver and 83% in blood) and glutathione S-transferase
(GST) activity (55%) and marked decreases in GSH levels (approximately 73% in
blood and 79% in liver) in the 5th week after STZ treatment as compared to
normal control animals. Treatment of STZ-induced diabetic mice with sodium
selenite significantly reduced oxidative stress changing aforementioned
parameters to near control values in almost all cases. Furthermore, treatment of
diabetic rats with sodium selenite had beneficial effects on both antioxidant system
and the ultrastructure of the liver tissue. In an experiment conducted in Turkey,
both diabetic and normal rats were treated with sodium selenite (5 mol/kg/d,
intra peritoneally) for 4 wk following 1 wk of diabetes induction. This treatment
of diabetic rats improved significantly diabetes-induced alterations in liver
antioxidant enzymes (Can et al., 2005). Moreover, treating diabetic rats with
sodium selenite prevented primarily the variation in staining quality of hepatocytes
nuclei, increased density and eosinophilia of the cytoplasm, focal sinusoidal
dilatation and congestion, and increased numbers of mitochondria with different
size and shape.
Selenium showed insulin-mimic properties in vitro and in vivo (Ghosh et al.,
1994) restoring glucose and glycogen concentrations affected by diabetis. In
particular, it has been shown that selenite treatment of diabetic mice with an effective
dose is beneficial for the antioxidant system of liver and brain although it exerts a
toxic effect on the liver of normal mice (Sheng et al., 2005). Indeed, selenite
treatment for diabetic mice reduced the TBARS levels in red blood cells (RBC)
compared to the normal and significantly improved GSH-Px activity in RBCs
compared to the diabetic control. Similarly, intraperitoneally administered vitamin
E and Se to rats had significant protective effects on the blood, liver, and muscle
against oxidative damage of diabetes (Naziroglu and Cay, 2001).
Therefore, promising data obtained in animal models with sodium selenite
need further clarification before testing various Se compounds in clinical trials.
Indeed, organic selenium in the form of Se-yeast deserves more studies in diabetic
animal models as well as in clinical conditions related to diabetes.
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SELENIUM AND HIV

Patients infected with HIV are characterised by compromised antioxidant system
as a result of excessive production of ROS during the development of the disease
(Pace and Leaf, 1999). Indeed, cells infected with HIV can substantially enhance
production of superoxide radical (Kameoka, et al., 1993). Furthermore, a
deficiency in key antioxidant enzymes SOD and catalase, and a decreased
concentrations of the antioxidant vitamins (Jaruga et al., 2002) lead to severe
oxidative stress in HIV-infected patients. In addition, modified DNA bases that
were found at high levels in the lymphocytes of HIV-infected patients may lead
to apoptosis of immune cells (Olinski et al., 2002). Low serum micronutrient
levels are common in HIV-positive individuals and have been associated with
immune impairment, HIV disease progression, and increased mortality (Lanzillotti
and Tang, 2005). However, studies of micronutrient supplementation have yielded
conflicting results, although the results suggested that antioxidant supplementation
may decrease markers of oxidative stress in individuals with HIV, while selenium
may enhance immune function by modulating cytokine production. Selenium
appears important in reducing virulence of HIV and slowing disease progression
(Singhal and Austin, 2002). Indeed, there is a substantial body of observational
evidence and several small trial results implicating Se inadequacy as an independent
predictor for accelerated progression of HIV disease. For example, in a prospective
study on HIV-infected drug-users low plasma selenium levels were associated
with a 10-fold higher risk of death compared with individuals with normal Se
status (Baum et al., 1997). Furthermore, low plasma selenium was associated
with 6-fold higher risk of mortality in HIV-1-infected children (Campa et al.,
1999) and low plasma selenium levels were significantly associated with an
increased risk of mortality in a cohort of HIV-infected women in Tanzania (Kupka
et al., 2004). It has been suggested that selenium status may have a profound
impact on the pathogenesis of mycobacterial disease. In fact, in HIV-1-seropositive
drug users low plasma selenium was associated with a 3-fold increased risk for
development of mycobacterial disease (Shor-Posner, et al., 2002).
Did you know that Se deficiency is related to increased
death rate of HIV-infected patients?
Several small intervention studies of HIV-infected individuals provide evidence
showing beneficial effects of selenium supplementation. For example,
supplementation with 100 g of selenium daily for 1 y resulted in decreased
oxidative stress (higher GSH-Px activity and GSH levels) among 14 HIV-infected
persons (Delmas-Beauvieux et al., 1996). In a prospective, randomised trial with
partial crossover a combination of sodium selenite and N-acetylcysteine in 13
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HIV-infected outpatients was associated with improvement of immunocompetence.

In particular there was a trend towards an increase in the percentage of CD4+
lymphocytes after 6 weeks (P = 0.08), and a significant increase in the CD4/CD8
ratio and a significant decrease in the absolute CD8/CD38 count and percentage
of lymphocytes after 6 and 12 weeks of supplementation (Look et al., 1998). In
another randomized trial from Florida (n = 186), selenium (200 g/d) resulted in a
significant decrease in total hospital admission rates and in the percentage of
hospitalizations due to infections (Burbano et al., 2002). The relation between
selenium status and child mortality and morbidity among children born to HIVinfected mothers was evaluated at Muhimbili National Hospital, Dar es Salaam,
Tanzania, a tertiary-care hospital (Kupka et al., 2005). A total of 117 (19%) of the
610 study children died during follow-up. In a multivariate model, child plasma
selenium levels were inversely associated with risk of all-cause mortality. Baseline
plasma selenium measurements from HIV-positive pregnant women (n = 670)
were obtained between 12-27 weeks of gestation and mother-child pairs were
followed prospectively until 24 months after delivery (Kupka et al., 2005a). It
was shown that low plasma selenium levels were associated with increased risks
of fetal death, child death, and HIV transmission through the intrapartum route.
Because plasma selenium concentrations are very low, their replenishment by
high dosages is urgent and mandatory particularly in advanced HIV infections
bordering on acrodermatitis enteropathica (Stehbens, 2004).
Recently it has been shown that HIV-1 encodes for one of the human GSH-Px
and as a consequence, as it is replicated, it deprived HIV-1 seropositive individuals
not only of GSH-Px, but also of the four basic components of this selenoenzyme,
namely selenium, cysteine, glutamine, and tryptophan (Foster, 2004). The author
hypothesised that this depletion process causes severe deficiencies of all these
substances leading to the major symptoms of AIDS which include immune system
collapse, greater susceptibility to cancer and myocardial infarction, muscle
wasting, depression, diarrhea, psychosis and dementia. Therefore, it is compulsory
for HIV/AIDS treatments to address those deficiencies. Indeed, preventing those
deficiencies in a selenium and amino-acid-supplementation field trial in Botswana
was shown to be effective in reversing AIDS in the patients (Foster, 2004).
Future research priorities include examining further roles of minerals, including
selenium, in HIV infection, as well as determining the safety and the efficacy of
micronutrient supplements among individuals who are advanced in their disease
and who are receiving antiretroviral therapy (Fawzi et al., 2005).
SELENIUM AND PANCREATITIS

Free radical and oxidative stress involvement in the development of pancreatitis
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have been postulated (Braganza , 1998; Schulz et al., 1999). Indeed, free radicals
play a role in the etiopathogenesis of acute experimental pancreatitis (Lankisch et
al., 1989; Sanfey et al., 1985) and human pancreatitis of various origins, namely
ischemic, alcoholic and cholelithiasic (Sanfey et al., 1986). There is evidence
that patients with chronic pancreatitis have enhanced levels of free radical
production, cytochrome P450 induction and antioxidant deficiencies, in particular
selenium. The mean serum selenium levels were lower in chronic pancreatitis
patients than in control (Vaona et al., 2005). Indeed, in patients with acute
pancreatitis Se levels in toenails and in RBCs were significantly reduced (Musil et
al., 2005). Low GSH-Px activity and selenium concentration in severe acute
pancreatitis reflect the lower antioxidative ability in this form of disease
(Wereszczynska-Siemiatkowska et al., 2004). However, there are also publications
indicating no difference in Se and other antioxidant levels between healthy control
and pancreatitis patients. For example, there were no significant differences
between the antioxidant profiles of patients with chronic pancreatitis due to alcohol
excess and patients with idiopathic chronic pancreatitis, or between the antioxidant
profiles of patients with recurrent acute pancreatitis and control subjects (MorrisStiff et al, 1999). The pancreatic juice concentration of selenium is similar in
patients with chronic pancreatitis compared with age-matched controls (Scolapio
et al., 2004). The results therefore suggested that the effects of selenium on
pancreatic injury might be systemic rather than local tissue effect.
Did you know that free radicals are involved in the development
of pancreatitis and optimal Se supplementation could help to
control the disease?
The limited published literature in this field suggests that dietary antioxidant
supplementation may ameliorate the pain associated with chronic pancreatitis,
diminish the frequency of acute exacerbations and reduce the requirement for
pancreatic surgery (Bowrey et al., 1999). The selenium therapy caused a significant
increase in selenium, a moderate increase in activity of GSH-Px, a significant
decrease in MDA, whereas SOD remained unchanged (Wollschlager et al., 1997).
In a 20-week double-blind double-dummy crossover trial active treatment was
given as two types of tablets providing daily doses of 600 micrograms organic
selenium, 9000 IU -carotene, 0.54 g vitamin C, 270 IU vitamin E and 2 g
methionine. In the trial of 28 patients enrolled, 20 adhered to the full protocol. Six
patients had an attack whilst on placebo but none whilst on active treatment.
Analysis of visual analogue score sheets to compare background pain in the 10week period before entry and during each phase of the trial, endorsed the beneficial
effect of active treatment (Uden et al., 1990). The same trend emerged from
analysis of pain-score diaries by conventional and time series methods. An
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improvement in the prognosis of acute pancreatitis can be achieved if antioxidative

selenium therapy is introduced in time. For example, a substantial reduction in
mortality following intravenous administration of selenium to patients with acute
pancreatitis was observed (Kuklinski and Schweder, 1996). In Germany
immediately after making the diagnosis 200 micrograms of Se were given as a
bolus, 800 micrograms in the following 24 hours. From the second day on 500
micrograms of selenite were administered daily. With a well-timed selenium
therapy the rates of letality, complications and operation dropped drastically
(Kuklinski et al., 1995). The same authors treated 99 patients affected with acute
pancreatitis of different genesis by antioxidants in hospital in Germany. Nearly
80% of these illnesses were ethanol-induced, 12% were of biliary origin.
Furthermore 90 patients were submitted to an adjuvant antioxidant therapy with
selenium and D--tocopherol. The average lethality rate of 34% (before treatment)
fell to 1.1% (after treatment). Clinical courses proceeded more easily under adjuvant
antioxidant therapy, surgical treatment was not necessary (Kuklinski et al., 1992).
Supplementation of patients with chronic and recurrent pancreatitis in the UK
with antioxidant mixture containing 600 g Se/day has been shown to reduce
pain and frequency of attacks (McCloy, 1998). However, it is difficult to prove
efficiency of antioxidant therapy of pancreatitis. Indeed, recent two attempts were
not successful. For example, in an experiment 70 patients with acute pancreatitis
were prospectively allocated to a selenium (as sodium selenite) or a placebo group.
The clinical course of the two treatment groups did not show statistically significant
differences (Lindner et al., 2004). Therefore, substitution of sodium selenite has
no beneficial effect on the clinical outcome of patients with acute pancreatitis.
Similarly, case-control analyses do not appear to demonstrate any benefit from
the multiple anti-oxidant combination of sodium selenite, N-acetylcysteine and
ascorbic acid intravenous injection in severe acute pancreatitis (Virlos et al., 2003).
It seems likely that one of the problems of combine use of sodium selenite and
ascorbic acid is a possibility of selenite reduction to metalic unavailable form by
ascorbate. This could be a reason of low efficiency of such a mixture in pancreatitis
patients.
Taking into account results presented above it is possible to conclude that Se
supplementation is an important tool for improvement of pancreatitis clinical
outcome, however, there is a need for further research in this field.
SELENIUM, BRAIN FUNCTION, MOOD AND NEURODEGENERATIVE

DISEASES
Oxidative stress causes neurodegeneration and has been recently shown to be
also involved in the early stages of the pathogenesis of various neuronal and
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neuromuscular disorders, including stroke and cerebrovascular disease,

Alzheimers disease (AD), Parkinsons disease, familial amyotrophic lateral
sclerosis, and Duchenne muscular dystrophy (Behl, 2005; Chen and Berry, 2003)
and therefore the concept of neuroprotection by antioxidants has been developed.
However, the exact molecular mechanisms and the impact of oxidative stress on
the development and progression of various neurodegenerative disorders,
including AD are of great importance for future research. For example, in the
histopathology of AD many signs of oxidative reactions can be found building
the basis of the oxidative stress hypothesis of AD. In fact, in AD amyloid-beta
protein can induce oxidative changes rendering nerve cells more vulnerable to
additional insults and inflammatory mediators attracted by amyloid deposits could
further speed up the generation of an oxidative micro-environment (Behl, 2005).
It has been suggested that free radical-associated lipid peroxidation could initiate
the chain polymerization of amyloid peptides and other biomolecules found in
Alzheimers disease (Tappel and Tappel, 2004). Therefore antioxidant nutrients
could protect against free radical oxidant damage, thereby delaying or preventing
the onset of Alzheimers disease and other neurodegenerative diseases. The
evidence is quickly accumulating to show that the use of a combination of various
antioxidants might indeed be effective in preventing AD.
Did you know that human brain is characterised by
exceptionally high proportion of PUFAs and needs an
adequate antioxidant protection?
The relationship between AD and Se concentration in tissues is not straightforward.
For example, concentrations of Se in isolated subcellular fractions (whole brain,
nuclei, mitochondria, microsomes) of temporal lobe from autopsied AD patients
and normal controls were determined revealing decreased Se concentration in
AD microsomes (Wenstrup et al., 1990). However, in other studies increased
level of Se in AD patients was reported. For example, trace elements were measured
in seven brain regions from 58 AD patients and 21 control subjects and Se was
shown to be significantly elevated only in AD amygdala (Cornett et al., 1998).
Increased plasma levels of selenium were shown in 24 subjects with dementia of
the Alzheimer type (DAT) in comparison to 28 healthy volunteers (Basun et al.,
1991). Similarly a significant increase in GSH-Px and Se levels in plasma from 40
patients with DAT in comparison to 34 aged control subjects with normal cognitive
function were shown (Ceballos-Picot et al., 1996). It seems likely that cerebrospinal
fluid (CSF) selenium concentrations are apparently unrelated with the reported
oxidative stress processes in patients with AD. Indeed, there was no difference in
CSF and serum selenium levels in 27 patients with AD without major clinical
signs of undernutrition, and 34 matched controls (Meseguer et al., 1999).
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Did you know that human neurodegenerative diseases are
associated with overproduction of free radicals and oxidative
stress?
Selenium has been shown to protect the brain in models of focal cerebral ischemia
(Imai et al., 2001). Increasing evidence demonstrates that accumulation of
oxidation of DNA, proteins, and lipids by free radicals are responsible for the
functional decline in aged brains. A positive correlation between cognitive function
and Se serum levels in elderly was found (Smorgon et al., 2004). It was suggested
that Se may protect cognitive function and the oxidative stress in the brain could
be associated with some cases of depression. Five studies have reported that a
low selenium intake was associated with poorer mood (Benton, 2002). The
possibility that a sub-clinical deficiency of the trace element selenium might exist
in a sample of the British population was examined by giving a selenium
supplement for 5 weeks (Benton and Cook, 1990). Using a double-blind crossover
design 50 subjects received either a placebo or 100 g selenium on a daily basis.
On three occasions they filled in the Profile of Moods state. Mood did not change
when taking the placebo, whereas when taking the selenium the subjects reported
a substantial improvement after both 2.5 and 5 weeks. The lower the initial level
of selenium in the diet the more reports of anxiety, depression, and tiredness,
decreased following 5 weeks of selenium therapy (Benton and Cook, 1991). It is
interesting to note that in the trial subjects with baseline diets lower in selenium
evidenced a greater improvement. More recent results suggest that persons with
low selenium status might experience relatively depressed moods and support
the idea that selenium plays a particular role in the brain (Hawkes and Hornbostel,
1996). Indeed, metabolic feeding trials have shown that individuals fed marginal
selenium diets reported more symptoms of depression and hostility than individuals
fed higher selenium diets (Finley and Penland, 1998; Finley, 2005; Hawkes and
Hornbostel, 1996). In a randomized, double-blind, placebo-controlled selenium
therapy (200 g/day) trial with HIV+drug users the impact of selenium on anxiety,
depression, and mood state was determined (Shor-Posner et al., 2003). At the 12month evaluation, participants who received selenium reported increased vigor
(p = 0.004) and had less anxiety, compared to the placebo-treated individuals.
The risk for state anxiety was almost four times higher, and nearly nine times
greater for trait anxiety in the placebo-treated group.
Did you know that Se deficiency is associated with decreased
mood state and depression?
In a placebo controlled, double blind prospective study, 16 elderly subjects (3
males, 13 females) with a mean age of 63 years and Beck Depression Inventory II
(BDI) scores indicative of mild to moderate depression were randomly assigned
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to a placebo group or to receive 200 g Se/day as SeMet for 7 weeks (Spallholz et

al., 2005). After treatment, the Se group had a statistically significant decrease in
BDI scores of 10.4 points (p<0.001) whereas the placebo had a non-significant
decrease of 3.0 points in the mean BDI score. The mean BDI score (14.7) in the
Se groups was indicative of mild to moderate depression before supplementation
and decreased to normal (4.3) after treatment. The underlying mechanisms of
mood-modulating properties of Se are unclear although a response to
supplementation was found with doses greater than those needed to produce
maximal activity of the selenoprotein GSH-Px. It is important to mention that
childbearing-aged women are particularly vulnerable to the adverse effects of
poor nutrition on mood because pregnancy and lactation are major nutritional
stressors to the body. The depletion of nutrient reserves throughout pregnancy
and a lack of recovery postpartum may increase a womans risk of depression.
The mechanism by which selenium affects mood is not certain. However, it seems
likely that this effect can be mediated via regulation by Se of thyroid hormone
metabolism, immunity and antioxidant defences (Bodnar and Wisner, 2005).
As a consequence of the growing recognition of the important biological role
of selenium, a number of novel pharmaceutical agents, either selenium-based or
which target specific aspects of selenium metabolism, are under development.
Among these are orally active selenium-based antihypertensive agents, anticancer,
antiviral, immunosuppressive and antimicrobial agents, and organoselenium
compounds which reduce oxidative tissue damage and oedema (May, 1999).
Furthermore, more attention should be given to general diet improvement in order
to supply substantial amount of food-derived selenium. Functional Se-enriched
food as well as Se-Yeast supplements could help solving this problem improving
various brain functions and preventing neurodegenerative diseases especially in
elderly.
SELENIUM AND RADIATION

It is believed that radiation damages cells by direct ionization of DNA and other
cellular targets and by indirect effect through ROS. In particular, exposure to
ionizing radiation is associated with a production of oxygen-derived free radicals
in the tissue environment. Cell response to radiation depends on the type and
dose of radiation, inherent tissue sensitivity and repair, and modulating intracellular
factors that include position in the cell cycle, oxygen concentration, and levels of
thiols and other antioxidants (Borek, 2004a). The potential role of natural
antioxidants in reducing the cellular damage induced by ionising radiation has
been studied in animal models for more than 50 years. However, the application
of antioxidant radioprotectors to various human exposure situations has not been
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extensive although it is generally accepted that endogenous antioxidants provide

some degree of protection (Weiss and Landauer, 2003). In particular, results from
animal experiments indicate that selenium is protective against lethality and other
radiation effects. For example, both sodium selenite and SeMet, enhanced the
survival of irradiated mice (60Co, 0.2 Gy/min) when injected IP either before (24 hr and -1 hr) or shortly after (+15 min) radiation exposure (Weiss et al., 1992).
In particular, when administered at equitoxic doses (one-fourth LD10; SeMet =
4.0 mg/kg Se, sodium selenite = 0.8 mg/kg Se), both selenocompounds enhanced
the 30-day survival of mice irradiated at 9 Gy. However, survival after 10-Gy
exposure was significantly increased only after SeMet treatment. Indeed, the major
advantage of SeMet is lower lethal and behavioural toxicity (locomotor activity
depression) compared to sodium selenite, when they are administered at equivalent
doses of Se (Weiss et al., 1992). Similarly, a Se-enriched diet enhanced nonspecific resistance to ionising radiation doses capable of shortening the lifespan
and lessening the body-weight increase (Knizhnikov et al., 1991). On average, a
32% reduction in radiation-induced carcinogenesis in rats by Se treatment was
documented and optimal Se dose was considered to be 30 g/rat/day (Tutelyan et
al., 2002). The effect of the antioxidant mixture (AM) containing -carotene, tocopherol, ascorbic acid, rutin, and microelements zinc and selenium on mouse
resistance to acute and chronic irradiation was studied (Fomenko et l., 1997).
Micronucleus test demonstrated that a daily dietary supplement of AM reliably
decreased the rate of chromosomal damage, namely, X-ray-induced micronuclei
in the bone marrow polychromatophilic erythrocytes of aging mice. Furthermore,
AM significantly decreased the rate of gene mutations in mouse splenocytes after
chronic irradiation.
Did you know that major mechanisms of the detrimental
effects of radiation are related to overproduction
of free radicals?
There is also convincing evidence that administration of Se and vitamin E could
exerts a protective effect against liver radiation damage. In an experiment conducted
in Turkey the liver tissue of rats irradiated with a single dose of 1,000 cGy 60Cogamma-irradiation was examined for morphological changes after the
intraperitoneal (i.p.) administration DL--tocopherol acetate and sodium selenate
as compared to controls. Also, the amounts of blood glutathione and serum alanine
transaminase, aspartate transaminase, alkaline phosphatase, lactate dehydrogenase,
and total protein were determined by spectrophotometric methods (Yanardag et
al., 2001). Degenerative changes were observed under light and electron
microscopy in the liver tissue of the control (radiation only) group. In the group
receiving radiation and i.p. doses of sodium selenate and vitamin E, the damage
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to the liver tissue was minimal or absent. Furthermore, in the radiation-only group,
a reduction of the blood glutathione level and increases in serum values of
aforementioned enzymes activity were observed, whereas in the irradiation-treated
group, the reverse was found to occur. In an experiment conducted in Egypt,
animals were categorized into eight groups, receiving -tocopherol and/or Se
with or without whole-body gamma-irradiation (6.5 Gy). The results indicated
that antioxidant pre-treatments before irradiation had beneficial effects against
irradiation-induced injury (Noaman et al., 2002).
It seems likely that antioxidant pre-treatments prior to irradiation may also
have some beneficial effects against irradiation-induced intestinal injury. For
example, the effects of selenium and vitamin E (separately or in combination)
pre-treatments prior to whole abdominal irradiation on intestinal injury were
investigated in rats (Mutlu-Turkoglu et al., 2002). It was shown that irradiation
caused increased lipid peroxide and decreased GSH levels in the intestine. Selenium
and/or vitamin E pre-treatments ameliorated these disturbances in prooxidantantioxidant balance. Selenium pretreatment can also have protective effect against
damaging effects of radiation on the developing foetuses. A single dose of sodium
selenite (0.5 mg Se/kg b.w.) was injected intraperitoneally into mice on day 9 of
pregnancy, either 30 min or 2 h before 1.75 Gy whole body irradiation. The
results showed that administration of selenite 2 h (but not 30 min) before irradiation
resulted in a significant decrease in the number of malformed foetuses (Cekan et
al., 1985). The decrease in foetal malformations occurred proportionally for all
the major malformations observed, i.e. short or kinked tail, rib and vertebral
malformations, coloboma and deformation of retina and iris. Furthermore, selenium
pretreatment also protected against radiation-induced retardation of the sternum
of the foetus. In a different experiment the same authors used a dietary Se
supplementation of female mice to test the same hypothesis. The mice were fed a
standard pellet diet (containing 0.2 ppm Se) or the same diet supplemented with
0.8 ppm (low dose) or 3.4 ppm (high dose) of SeMet for 10 weeks. After mating
with males the mice were subjected, on the 9th day of pregnancy, to whole body
roentgen irradiation of 1.75 Gy. On day 18 of gestation the frequency of
resorptions, mortality and the incidence of fetal malformations were studied (Cekan
et al., 1985a). It was found that supplementation with Se-Met resulted in a
significant decrease of the number of malformed fetuses (from 62 per cent in the
irradiated controls to 47 per cent in the Se-supplemented groups). However, there
was no difference between two Se doses. Furthermore, the number of total
malformations as well as fetal resorptions were significantly decreased in a doseindependent manner in the supplemented groups. Similar to the previous
experiment described above, the decrease in fetal malformations occurred
proportionally for all the major malformations observed. GSH-Px activity in whole
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blood of SeMet-fed mice was significantly increased. These data indicated that Se
supplementation at 0.8 ppm was adequate to provide a protection against radiation
and further increase in Se dose was not required. Se-supplementation could also
improve immunity in radiation-exposed animals. For example, supplementation with
200 g/day of sodium selenite during therapy for squamous cell carcinoma of the
head and neck, e.g., surgery, radiation, or surgery and radiation, resulted in a
significantly enhanced cell-mediated immune responsiveness (KiremidjianSchumacher and Roy, 2001).
It seems likely that Se may provide an extended window of protection against
low-dose, low-dose-rate irradiation, including therapeutic potential when administered
before or after irradiation as well as can decrease toxicity of various radioprotective
compounds. The effect of Se pretreatment on the acute toxicity and radioprotective
effect of an effective radioprotector (WR-2721) was studied in male CD2F1 mice.
Injection of 1.6 mg/kg Se 24 hr before WR-2721 (800-1200 mg/kg, IP) decreased
the lethality of WR-2721 significantly (Weiss et al., 1987). At the same time, Se
injection alone (1.6 mg/kg) 24 hr before cobalt-60 irradiation increased the survival
significantly. Furthermore, a synergistic effect on post-irradiation survival was observed
when Se was injected 24 hr before WR-2721 (200-600 mg/kg IP 1/2 before irradiation).
Indeed, after exposure to 22 Gy (1 Gy/min), 30-day survival was 100% when mice
were treated with both Se and 600 mg/kg WR-2721, and was 13% with WR-2721
alone (Weiss et al., 1987). Similarly, glucan, WR-2721, and selenium were evaluated
in C3H/HeN mice for survival-enhancing and hemopoietic-regenerating effects when
administered alone or in combinations before exposure to 60Co radiation. At LD50/
30 radiation doses (radiation doses lethal for 50% of mice within 30 days postexposure),
dose reduction factors varied from 1.02 to 1.66 and were highest when all three
compounds were combined in a single treatment (Patchen et al., 1990).
To better understand molecular mechanisms of radiation-protective effects of Se,
in vitro cell culture experiments were conducted using normal human skin fibroblasts
and squamous cell carcinoma cells. Combined treatment, i.e. radiation exposure +/sodium selenite in single-dose (0 to 7 Gy) and multiple fractionated-dose (5 x 2 Gy)
were used (Rodemann et al., 1999). The results indicate that sodium selenite under
both radiation exposure conditions provided the radioprotective effect. In contrast,
selenite treatment of human tumor cells did not affect their radiation sensitivity. In a
cell culture, protection against radiation-induced mutation was observed for both 30
nM sodium selenite or 4 mM aminothiol (WR-1065; Diamond et al., 1996). In
addition, the protection against mutation induction provided by the combination of
these agents appeared additive. However, in this experiment sodium selenite did not
provide protection against radiation toxicity when provided either alone or in
conjunction with WR-1065. Molecular mechanisms of protective effects of Se against
radiation injuries are not clarified. The authors of the aforementioned study suggested
that selenium and WR-1065 offer protection via independent mechanisms and that
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GSH-Px stimulation remains a possible mechanism of the anti-mutagenic effect of

selenium. In contrast, the results of Sandstrom et al. (1989) suggested that seleniumdependent GSH-Px did not contribute significantly to the radiation resistance of
cultured mammalian cells. The single and combined effects of selenium and vitamin
E on cell transformation induced in C3H/10T-1/2 cells by x-rays, benzo[a]pyrene, or
tryptophan pyrolysate and on the levels of cellular scavenging systems and peroxide
destruction were investigated. Incubation of C3H/10T-1/2 cells with 2.5 M sodium
selenite or with 7 M -tocopherol succinate 24 hr prior to exposure to x-rays or the
chemical carcinogens resulted in an inhibition of transformation by each of the
antioxidants with an additive-inhibitory action when the two nutrients were combined
(Borek et al., 1986). Cellular pretreatment with selenium resulted in increased levels
of cellular GSH-Px, catalase, and nonprotein thiols (glutathione) and in an enhanced
destruction of peroxide.
Did you know that Se supplementation before the radiation or
shortly after the exposure could substantially increase
resistance to radiation and survival?
The main application from the above-presented data is usage of the Se-therapy for
people exposed to radiation. For example, a long-term experiment in 400 rats exposed
to radiation following Chernobyl pattern showed that Se-enriched diet (0.5, 1.5 or 5
ppm Se as Se-yeast) started after exposure caused a longer average lifespan
(Knizhnikov et al., 1996; Table 11.29).
Table 11.29 Effect of organic selenium on the radiation-exposed rats (Adapted from Knizhnikov et al.,
1996)
Percentage of dead animals at certain period

Se dieting, months
Control
7
0
9
2.5
11
4.0
15
21
Average lifespan, days 578
Malignant tumour
8.5
number
Latent period of tumours, days
Breast cancer
562
Lung cancer
584
Radiation (R) R+0.5 Se*
R+1.5Se*
R+5Se*
14
21
35
80
448
66.2
6
13
25
61
503
26.2
10
19
31
81
461
20.0
6
16
29
61
489
23.6
461
462
480
572
407
481
466
612
Selenium was added to the rat diets as Se-enriched yeast at 0.5, 1.5 and 5 ppm, a fortnight
after radiation exposure
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The treatment also enhanced rat resistance to radiation and reduced (1.5-3.5-fold) the
incidence of radiation-induced leukaemias and other malignancies, including breast,
thyroid and lung cancers. In the group with Se supplementation, latent periods were
longer than in exposed control without Se supplementation. According to these results
Se can reduce the risk of cancer when added to the diet long after the radiation.
SELENIUM AND AGEING

Aging is a very complex, multifactorial process, and numerous aging theories
have been proposed; the most important of these are probably the genomic and
free radical theories. In particular, the free radical theory of aging postulates that
the production of intracellular reactive oxygen species is the major determinant
of life span (Harman, 1956). Numerous cell culture, invertebrate, and mammalian
models exist that lend support to this half-century-old hypothesis.
Did you know that numerous aging theories have been
proposed; the most important of these are probably the
genomic and free radical theories?
It has been shown that elderly individuals have a higher risk to develop trace
element deficiencies due to modified dietary habits and requirements, age related
physiological changes, drug therapy, and chronic diseases leading to or associated
with enhanced consumption or excretion of trace elements (Ekmekcioglu et al.,
2001). There is a great body of evidence indicating decreased Se status in elderly
populations. For example, in aged individuals aged blood Se levels were shown
to be negatively correlated with age. A study was conducted in a representative
sample of non-institutionalized individuals aged > or = 65 years living in
Southwestern France. Plasma and erythrocyte selenium was measured in 239
volunteers (mean age 73.7 years). It was shown that plasma Se decreased
significantly with age. A similar but non-significant trend was found for erythrocyte
selenium (Berr et al., 1993). The analysis of the relationship between Se and
GSH-Px activity conducted by the authors suggests that low Se values were
associated with decreased GSH-Px activity. In Denmark old women had a lower
Se status and higher TBARS level in the blood than the young women (P< 0.01),
irrespective of their vitamin E status which was fully adequate (Schafer and
Thorling, 1990). Selenium levels in plasma of healthy individuals were found to
be low in the oldest group (41-69 year old) in comparison to younger people and
a negative correlation between age and selenium levels was found (Erden-Inal et
al., 2002). A decreasing levels of selenium with age was shown in Korea. In fact,
the overall proportion of women having Se deficiency, with less than 80.0 g/l of
the Se concentrations in the serum, was 18.3% (Lee et al., 2003). The serum Se
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levels in the young-adult, middle-aged and elderly groups were 120.6 g/l, 97.2
g/l, and 90.8 g/l, respectively. Subjects with the lowest tertile of Se concentration
had significantly higher atherogenic index and lower HDL-cholesterol levels
compared to those with the highest tertile. However, only the serum HDLcholesterol level showed the dependency on the Se status as determined by stepwise analysis and this dependency was significant only in the subjects below the
age of 40. Similar relationship between Se and age was shown in animal models
as well. In an experiment conducted in Turkey male Wistar rats at ages of 1, 6 and
12 months were used. The activity of Se-GSH-Px and the level of GSH in rat
erythrocytes were decreased with age and the correlation between age and SeGSH-Px activity (r=-0.376, p<0.05) was negative (Ozturk and Gumuslu, 2004).
It is generally accepted that a well-preserved function of the immune system is
an excellent marker of health and longevity. Indeed, in ageing populations
immunity is often compromised. The elderly suffer a decline in immune function
that increases their vulnerability to infections. For example, detrimental changes
were observed in prematurely aging mice (PAM). The effect of ingestion during 5
weeks of a diet supplemented with antioxidants (vitamin C, vitamin E, -carotene,
zinc, and selenium) on several immune functions of peritoneal leukocytes from
PAM was studied (Alvarado et al., 2005). The results showed that, in macrophages,
chemotaxis and phagocytosis as well as the intracellular free radical levels (which
are depressed in PAM) increased after antioxidant supplementation. An increase
also occurred in lymphocyte chemotaxis, proliferative response to the mitogen
concanavalin A, and interleukin-2 release, as well as in natural killer cell activity.
However, the release of tumor necrosis factor-alpha, which increases with aging,
decreases after 5 weeks of supplementation. The effect of dietary (2.00 ppm for 8
weeks) supplementation of aged (24-month-old), mice with selenium (as sodium
selenite) on the lymphocytes functions was studied and the results suggested that
selenium restores the age-related defect in cell proliferation through an increase
in the number of high-affinity IL-2 receptors (Roy et al., 1995). Indeed, the
supplementation with selenium resulted in:
a significant increase in the ability of spleen lymphocytes from aged animals

to undergo blastogenesis;
a restoration of the ability of the cells to respond to stimulation by nuclear
DNA synthesis and cell proliferation;
increased numbers of cytotoxic lymphocytes, which resulted in an enhanced
capacity to destroy tumor cells
In another animal experiment it was shown that selenium and vitamin E facilitated
recovery from lipopolysaccharide-induced sickness in aged mice. Male mice were
fed diets that were low, adequate, or high in both -tocopherol (10, 75, or 500
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mg/kg) and selenium (0.05, 0.15, or 2 mg/kg) from 18 to 21 months of age.

Sickness was quantified by measuring time in social exploration. The
lipopolysaccharide treatment reduced social exploration by 74% at 2 h, regardless
of diet. (Berg et al., 2005). By 4 h, aged mice fed the low Se diet were 88% less
social, whereas mice fed the adequate and high Se diets displayed only
approximately 40% reductions due to LPS treatment. Mice fed the low Se diet had
greater LPS-induced weight loss than mice fed the high diet.
Supplementation with low doses of trace elements is able to rapidly correct
corresponding deficiencies in the institutionalized elderly and improve their
resistance to various diseases. For example, a double-blind controlled trial was
performed on 81 elderly subjects in a geriatric center during a 2-year period
(Girodon et al., 1997). Subjects were randomly assigned to one of four treatment
groups, and received daily: placebo, trace elements (zinc 20 mg and selenium
100 g), vitamins (E, C and -carotene) or a combination of trace elements and
vitamins. Low serum values in vitamin C, Zn and Se were observed in more than
two thirds of the patients before the experiment. However, after 6 months of
supplementation, a significant increase in Se and Zn serum levels were obtained
in the corresponding treatment groups. Furthermore, subjects who received trace
elements (Zn and Se) alone or associated with vitamins had significantly less
infectious events during the 2 years of supplementation.
The accumulating evidence indicates that dietary supplementation of Se and
Zn provides significant improvement in elderly patients by increasing the humoral
response after vaccination and could have considerable public health importance
by reducing morbidity from respiratory tract infections. For example, a randomized,
double-blind, placebo-controlled intervention study included 725 institutionalized
elderly patients (>65 years) from 25 geriatric centers in France. Patients received
an oral daily supplement of nutritional doses of trace elements (zinc and selenium
sulfide) or vitamins (-carotene, ascorbic acid, and vitamin E) or a placebo within
a 2 x 2 factorial design for 2 years (Girodon et al., 1999). Correction of specific
nutrient deficiencies was observed after 6 months of supplementation and was
maintained for the first year, during which there was no effect of any treatment on
delayed-type hypersensitivity skin response. Antibody titers after influenza vaccine
were higher in groups that received trace elements alone or associated with
vitamins. The number of patients without respiratory tract infections during the
study was higher in groups that received trace elements (P = 0.06).
Optimal Se status and effective antioxidant defences potentially could improve
life span and quality of life. In order to investigate the effects of Se on the activity
of GSH-Px and the life-span of drosophila melanogaster, newly enclosed flies
were divided randomly into 4 groups and fed with mediums containing different
concentrations of sodium selenite (Zhang et al., 2000). The results indicated that
the activity of GSH-Px in flies were increased by the increase of sodium selenite
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in the medium (P < 0.05). More importantly, the average life-span and the average
maximum life-span of flies in Se groups were increased significantly as compared
with control group (P < 0.05). It is interesting to note that the response of GSH-Px
activity and life-span of male flies to Se intake was better than that of females. It
seems likely that antioxidant supplementation could affect longevity of animals
and human. For example, the effect of daily dietary supplements of an antioxidant
mixture (AM) consisting of -carotene, -tocopherol, ascorbic acid, rutin,
selenium, and zinc on the survival of male mice starting at 2, 9, 16, and 23 months
of age was investigated. The results indicated that the survival of mice given AM
starting at 2 and 9 months of age increased significantly (from 86 to 108 days)
compared to the control (Bezlepkin et al., 1996). The times, of 50, 90, and 100%
mortality in mice given AM starting at 2 and 9 months of age increased by 169.5% compared to the control. It has been shown that beneficial effect of antioxidant
supplementation depended on the age when the supplementation started, since in
mice given AM, starting at 16 and 23 months of age, there was no effect of AM
supplementation.
Did you know that an optimal Se supplementation could improve
quality of life of elderly by improving their immunity and
preventing degenerative diseases?
The spatial distribution of the elderly, those aged 80 years or more, in 2408
counties, was compared with the prevalence of Kaschin-Beck and Keshan diseases,
both of which involve extreme Se deficiency. It was shown that far fewer people
of advanced age reside in those counties with Se deficiency than in unaffected
counties (Foster and Zhang, 1995). The authors suggested that possible reasons
for this include elevated mortality from endemic and chronic diseases in Se deficient
areas and accelerated ageing due to excessive cellular damage caused by free
radicals. To examine the hypothesis that inadequate plasma Se can adversely
affect the maintenance of optimal health the relationships between plasma Se and
mortality in an elderly population was studied. During the 2-year period from
1991 to 1993, 1389 men and women born between 1922 and 1932 were recruited.
In this 9-year study baseline plasma Se was higher (1.10 mol/l) in individuals
who were alive at the end of follow-up than in those who died during the followup (1.01 mol/l , P <0.0001). Furthermore, mortality rates were significantly higher
in individuals with low Se (relative risk (RR) = 1.56 ). When the underlying causes
of death were considered, an association with cancer-related mortality was found
(adjusted RR = 1.79; Akbaraly et al., 2005).
Dementia is one of the most pressing public health problems with social and
economic implication. The form called cognitive impairment non-dementia (CIND)
represents a subclinical phase of dementia. A range of various studies have shown
a possible effect of antioxidants and trace minerals on cognitive function. In a study
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conducted in Italy possible correlations between serum trace element concentrations

and cognitive function in a group of subjects with CIND and manifest dementia were
evaluated and compared them with a control group. A positive correlation between
cognitive function, expressed as the Milan overall dementia assessment (MODA)
score, and Se serum levels was found (Smorgon et al., 2004). It was suggested that
Se could have a role in protecting cognitive function. Indeed, increased levels of
oxidative stress and/or antioxidant deficiencies may pose risk factors for cognitive
decline. For example, a total of 1166 high cognitive functioning subjects aged 60 to
70 in the Etude du Vieillissement Arteriel (EVA) cohort with a 4 year follow-up were
participating in a longitudinal population-based study (Berr et al., 2000). Subjects
completed a baseline interview and a global cognitive test (Mini-Mental Status
Examination, MMSE). Blood samples were obtained at baseline to determine plasma
levels of antioxidants. Subjects with the highest levels of TBARS showed an increased
risk of cognitive decline (OR = 2.25). Furthermore, subjects with low levels of Se had
an increased risk of cognitive decline (OR = 1.58) after adjustment for various
confounding factors.
A randomized, double-blind, placebo-controlled trial was design to determine
whether supplementation with vitamins and trace elements in modest amounts
influences cognitive function in apparently healthy, elderly subjects. Ninety-six,
apparently healthy, independent men and women older than 65 y of age were recruited
and randomized to receive a supplement of trace elements (including 20 g Se) and
vitamins or a placebo daily for 12 months (Chandra, 2001). Eighty-six subjects
completed the 1-y trial. The supplemented group showed a significant improvement
in all cognitive tests except long-term memory recall. Furthermore, those with bloodnutrient levels below the reference standard showed lower responses on cognitive
tests. A clinical study was conducted with an aim to determine whether use of
supplemental antioxidants (vitamins A, C, or E, plus selenium or zinc) was associated
with a reduced risk of development of cognitive impairment or cognitive decline in a
representative sample of the community-dwelling elderly (Gray et al., 2003). At
baseline, 224 (10.8%) subjects were currently taking a supplement containing
antioxidants. During the follow-up period (3-7 years), 24.0% of subjects developed
cognitive impairment and 34.5% experienced cognitive decline. It was shown that
antioxidant users had a 34.0% lower risk of developing cognitive impairment compared
with non-antioxidant users (adjusted relative risk RR=0.66) and a 29.0% lower risk
of experiencing cognitive decline (adjusted RR= 0.71). However, there are some
published results which do not support the hypothesis that antioxidant supplement
use is associated with cognitive function (Mendelsohn et al., 1998).
From the data presented above it is possible to conclude that free radicals are
involved in ageing process and Se, as an integral part of a range of selenoproteins,
could have a positive effect on life span and life quality. In particular, cognitive
function of elderly subjects could be improved by correction of their antioxidant
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status and Se is shown to be most important among others. Indeed cognitive impairment
is an important component of age-related dementing diseases. In particular,
observational studies of relationships between cognitive impairment and antioxidant
status are based on the evaluation of dietary intake or on the levels of various
antioxidants and trace elements in plasma. Despite some limitations, the comparison
between results obtained in various populations is becoming increasingly informative
and these studies argue for a protective effect of antioxidants on cognitive performance
(Berr, 2002).
Epidemiological data suggest that antioxidants may have a beneficial effect on
many age-related diseases including atherosclerosis, cancer, some neurodegenerative
and ocular diseases. The beneficial impact of antioxidants on various age-related
degenerative diseases may forecast an improvement in life span and enhance quality
of life. However, the widespread use of supplements is hampered by several factors
(Bonnefoy et al., 2002):
the lack of prospective and controlled studies;

insufficient knowledge on the pro-oxidant, oxidant and anti-oxidant properties
of the various supplements;
growing evidence that free radicals are not only by-products, but also play an
important role in cell signal transduction, apoptosis and infection control.
Adverse health effects of excess selenium in humans

Chronic toxicity of selenium in humans results in a condition termed selenosis. Field
studies conducted in high Se areas of Venezuela (Jaffe, 1976) and conclusions drawn
from outbreaks of endemic selenium poisoning in humans in China (Yang et al.,
1983) and in the United States (Burk and Levander, 1999; Goldghaber, 2003) indicate
that most common signs of Se intoxication in human are:
hair and nail loss and brittleness,

gastrointestinal problems (diarrhea),
skin rash (dermatitis and irritability),
nausea,
fatigue,
garlic breath odor,
lowered hemoglobin levels,
mottled teeth,
nervous system abnormalities
In China, selenosis was reported to occur in people who consumed selenium regularly
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at levels above 0.85 mg/day (Yang and Zhou, 1994) or 5 mg of selenium daily from
food or 1 mg/day as sodium selenite for a long period of time (Yang et al., 1983).
Epidemiologic studies and case reports have shown that chronic exposure to selenium
compounds is associated with several adverse health effects in humans (Vinceti et
al., 2001):
An early toxic effect of selenium is on endocrine function, particularly on the

synthesis of thyroid hormones following high dietary Se exposure and on the
metabolism of growth hormone and insulin-like growth factor-1.
Other adverse effects of selenium exposure can be the impairment of natural
killer cells activity and at higher levels, hepatotoxicity and gastrointestinal
disturbances.
The problem in addressing Se overdose is that no sensitive and specific biochemical

test is currently available to indicate overexposure to selenium (Levander, 1982).
However, it should be mentioned that toxic Se doses are more than 10-fold higher
than physiological requirement. For example, no signs or symptoms of selenium
overexposure were observed among residents of seleniferous ranches in South Dakota
or Wyoming whose dietary intake was as high as 724 g/day and signs of selenosis
(nail changes) were seen in susceptible patients in China at intakes of 910 g/day or
more (Burk and Levander, 1999). In an experiment, conducted in the USA, cancer
patients received Se at doses 1.6 and 3.2 mg per day and only mild toxicity symptoms
were observed (Table 11.30). The general characteristic of Se consumption in relation
to its requirement and toxicity is shown in Table 11.31.
Table 11.30 Frequency of reported symptoms of potential selenium toxicity (Adapted from Reid et al.,
2004)
Reported symptoms
1600 g group (n=8)
3200 g group (n=16)
Garlic breath
Brittle nails
Brittle hair
Stomach upset
Dizziness
Serious Se-related toxicities
Blood chemistry and hematology
Patients characteristics
Plasma Se level, ng/ml*
Months on high Se treatment
Mean
Range
Age in years
0
1
0
1
1
0
Within normal limits
6
3
1
2
3
0
Within normal limits
492.2
639.7
14.2
5.5-22.7
73.6
11.5
1.0-23.6
70.5
*There was not relationships between Se toxicity symptoms and Se concentration in plasma
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Table 11.31 Selenium requirement and status (Adapted from Yang and Xia, 1995; Whanger, 2004).
Se requirement
g/day
Prevalence of Keshan disease and Kaschin-Beck

<11
disease
The minimum dietary selenium requirement
17
for the prevention of Keshan disease
Minimum requirement
21
Suggested adequate dietary Se requirement (to
40
maintain the plasma GSH-Px activity at plateau)
RDA USA
55
RDA UK for women
60
RDA Australia for women
70
RDA UK for men
75
RDA Australia for men
85
Oral Reference Dose (RfD)*
350
Maximum Safe Dietary Se Intake
400
Individual daily maximum safe dietary
600
Se intake (individual NOAEL)
Maximum safe dietary Se intake (mean NOAEL)
819
No-observed adverse effect level (NOAEL)
853
Mean low adverse effect level (LOAEL) of dietary 900
Se intake
Toxic levels
>:900
Individual LOAEL
1540
The toxic dietary selenium intake (adverse effect
1600
level), which would maintain the characteristic
fingernail changes
Adverse effect level (AEL) of dietary Se intake
1660
Occurrence of selenosis with hair and nail loss
4990
Reference
Whanger et al., 2004
Yang and Xia, 1995
Levender, 1997
Yang and Xia, 1995;
Food Nutrition Board, 2000
Department of Health, 1991
Tinggi, 2003
Department of Health, 1991
Tinggi, 2003
Poirier, 1994
Burk and Levander, 1999
Yang and Xia, 1995
Yang and Xia, 1995

*an estimate of a daily exposure to the human population that is likely to be without an
appreciable risk of deleterious effects during a lifetime
It seems likely that there is a need for more research addressing upper range of
dietary Se intake. From the one hand, high Se exposure could be detrimental. On
the other hand, Se doses close to toxic are shown to be effective in cancer prevention
and treatment. The form of Se is also of great importance. As mentioned above
sodium selenite could be considered as a drug which can damage DNA, cause
pro-oxidant reactions and having other adverse health-related effects and therefore
should be used with extra caution. In contrast Se-enriched yeast is a food/feed
additive and can help meeting Se requirement of general population. It seems
likely that long-term consumption of organic selenium in the form of Se-yeast at
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doses 100-300 g/day for a long period of time is safe and could be beneficial for
those who live in areas with low Se status.
Conclusions
The main conclusion which can be drawn from this chapter is that an optimal Se
balance in the human body is an essential element of health maintenance. Indeed,
selenium is the most controversial trace element. Depending on the dose and
form it can be beneficial or detrimental for human health. Recent findings in the
field of molecular biology indicating a presence in human body of at least 25
selenoproteins can change our way of addressing Se requirement. Indeed, the
human Se requirement is developed based on the effect of Se on GSH-Px, an
important member of the selenoprotein family, but not the only one and probably
not the main one. In some instances, such as anticancer activity and
immunomodulation, Se requirement is much higher than needed to maintain
growth and development. It seems likely that we need to learn from nature a way
of Se supplementation. Indeed, food ingredients contain a range of
selenocompounds with Se-Met comprising 60-80% total selenium. Therefore, this
kind of organic selenocompound mixture could be an ideal for dietary
supplementation to meet Se requirement. There is also a scope for sodium selenite
and some synthetic selenocompounds as specific drugs to address specific
problems. Nevertheless, the global Se deficiency awaits a scientifically-based
solution. It seems likely that production of Se-enriched eggs, meat and milk could
be considered as a way forward in solving the Se deficiency. On the other hand,
anticancer activity of various Se compounds needs further investigation.
Diet is closely related to our life style, national traditions, preferences and
habits so the likelihood of the three ideal diets finding their way to our tables is
minimal. Since habit is a second nature it would be almost impossible to introduce
such diets in Western countries like the UK or USA. However, improvement of
the diet by balancing essential nutrients via designer/functional food without
changing peoples food preferences would bring health benefit. In this respect,
natural antioxidants have become a wonder remedy of the 21st century.
We are what we eat!
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Se-enriched Eggs, Milk and Meat as Functional Foods
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12
SELENIUM-ENRICHED EGGS, MILK AND MEAT AS FUNCTIONAL
FOODS AND A SOLUTION TO SELENIUM DEFICIENCY
An egg a day keeps diseases at bay
Introduction
Relationships between diet and human health have received substantial attention
in the last few years with the realisation that unbalanced diets can cause serious
health-related problems. However not everyone eats the same foods and people
meet their nutritional needs in the various ways. In fact there is a range of groups
and factors affecting food choice (Tables 12.1 and 12.2; Blades, 2001). From
many food ingredients commonly present in our diet the natural antioxidants are
considered particularly important. It is well known that free radicals produced
under both normal and abnormal physiological conditions can have damaging
effects on polyunsaturated fatty acids, DNA and proteins. Antioxidant protection
is vital for either prevention or substantial reduction of the damage caused by free
radicals and products of their metabolism. Our food provides a major part of
natural antioxidants including vitamin E, carotenoids, flavonoids and selenium.
Particular interest in selenium was generated as a result of clinical studies showing
that dietary supplementation with organic selenium in the form of yeast grown on
a media enriched with this trace element decreased cancer mortality 2-fold.
Additionally, there are data indicating that inadequate selenium consumption is
associated with poor health and development of various viral and bacterial diseases.
Unfortunately, in many countries all over the world human food ingredients do
not provide sufficient selenium. As a result, finding solutions to this public health
problem are on the agenda of many government bodies. Results of various research
studies conducted over the last few years indicate that enrichment of animalderived foods with selenium via special supplements of food animal diets could
be an effective way of increasing human selenium status in countries where
selenium consumption falls below the Recommended Daily Allowances (RDA).
For example, selenium consumption in the UK is shown to be about 50% of
RDA.
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Table 12.1 Main groups affecting food choice (adapted from Blades, 2001).
Groups
Effect
Government
Economic effects, food laws, import, export

tariffs
Cultural (ethnic and religious)

groups
Avoidance of some products due to various

religion restrictions
Regulators of food laws
Environmental and quality issues of food

production and availability
Advertisers and media
Promotion of specific food items
Health professionals, e.g. doctors,

nurses, dieticians, etc.
Advises related to food choice
Educators: teachers and health

promotion officers
Explanations of beneficial or detrimental

effects of food
Caterers and cooks
Quality, taste, presentation
Food suppliers
Food availability and price
Parental choices of food and

cooking methods
Formation of family diets
Table 12.2 Factors influencing food choice (adapted from Blades, 2001).
Factor
Effect
Quality: flavour, appearance,

Very often is a major deciding factor
texture, odour and food presentation
Socio-economic factors
Availability, price and culture
Biological factors (energy and

nutrient requirement)
Vary depending on age and life phase
Psychological factors: behaviour,

mood and attitude toward eating
Depend on educational, cultural and other

levels as well as on age
Different strategies to address Se deficiency in human

Since selenium content in plant-based food depends on its availability from soil,
the level of this element in human (or food animal) foods varies among regions.
In general eggs and meat are considered to be good sources of Se in human diet
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(Table 12.3). When considering ways to improve human selenium intake, there
are several potential options. These include direct supplementation, soil fertilisation,
supplementation of food staples such as flour and production of functional foods.
Table 12.3 Selenium concentrations in various European food sources (adapted from Brown and Arthur,
2001).
Food
Selenium content, g/100 g fresh weight

19.4a
7.6
14.0
3.8
18.5a
36.0a
42.0
145.0
1.5
3.2a
4.5
11.0
1.0
2.0
254.0
Eggs
Beef
Pork
Lamb
Poultry
Fish
Liver
Kidney
Milk
Dairy produce
Bread
Cereals
Fruit
Vegetables
Brazil nuts
a
/ BNF, 2001
Did you know that selenium consumption in the UK

is about 50% of RDA?
In some countries selenium supplements in tablet form are available to healthconscious consumers. Some supplemental Se is supplied as inorganic salts, but
the most common supplement uses high-Se yeast that contains SeMet and some
other organic selenocompounds (Finley, 1999). Many prominent scientists have
championed use of such natural antioxidants. For example, Professor Schrauzer,
known for his pioneering work related to medical application of organic selenium,
regularly consumes Se supplements. In fact, various dietary supplements are used
by more than one-half (more than 100 million people) of the adult US population
(Halsted, 2003; Davidson and Geohas, 2003). However, there are many people
who either do not like to swallow capsules, cannot afford them or are simply
unaware of the need to increase daily selenium intake.
Table salt fortified with 15 mg/kg sodium selenite is used as a daily Se supplement
to reduce the incidence of primary liver cancer in Se-deficient areas of China (Hu
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et al., 2002). However, since Se in high doses could be toxic and selenite is not
the optimal form of Se dietary supplementation, this approach is limited to the
specific areas of China and did not find a great support in other countries.
Soil selenium availability is dependent on both selenium content and soil pH.
Low crop selenium content owing to low soil pH is quite common situation in
various countries all over the world. For example, in Finland the availability of
soil Se for plants is poor owing to the relatively low Se concentration, low pH and
high iron content of the soil. In areas where soil selenium content is low (Finland
and New Zealand) sodium selenate was added to fertilizers used for both grain
and forage production (Alfthan, 1993; Oldfield, 1999; Arthur, 2003). Therefore,
since 1984 multimineral fertilizers have been supplemented with Se (16 mg/kg to
fertilizers for grain production and 6 mg/kg to those for fodder production) in the
form of sodium selenate. Within two years a three-fold increase of mean Se intake
was observed. The supplementation affected the Se content of all major food
groups with the exception of fish (Aro et al., 1995). As a result, there was a rapid
increase in the Se content of the crops and the foodstuffs derived from animals
consuming the crops, as well as the human Se status was improved (Arthur, 2003).
For example, the mean Se concentration in young adults and children in Finland
increased from 1.04 and 0.87 mol/l in 1985 to 1.59 and 1.31 mol/l in 1990
respectively (Wang et al., 1998). The Se level in human maternal milk was also
significantly increased (Kantol and Vartiainen, 2001). In 1990 the amount of Se
that was supplemented was reduced to 6 mg/kg for all fertilizers. This reduced the
mean Se intake by 30% and the serum Se concentration decreased by 25% from
the highest levels observed in 1989 (Aro et al., 1995). In fact, three different
methods of Se application were tested: seed pre-treatment, fertiliser enrichment,
and foliar application (Gissel-Nielsen, 1986). Seed pre-treatment had some
disadvantages while the two other methods proved to be efficient in a series of
experiments and in tests on a large number of farms all over Denmark. In general,
foliar application of selenium provided a higher efficiency for increasing the
selenium content of soybean than soil application (Yang et al., 2003) and
successfully used for production of Se-enriched rice in China (Hu et al., 2002).
This approach has been successful in Finland and New Zealand, but has had
limited interest in other countries because of environmental issues. For example,
in the USA, the use of Se fertilizers caused run-off of the element, resulting in its
accumulation in the aquatic biota (Maier et al., 1998). Furthermore, even in
Finland, simultaneous increase of total nitrogen, phosphorus, and selenium levels
in consecutive samples from some ground water pools indicated leaching of
selenium from the fertilizers into the ground water in certain areas (Makela et al.,
1995).
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Did you know that by adding sodium selenate to fertilizers

used for both grain and forage production Se status
of animals and humans in Finland and New Zealand was
significantly improved?
Supplementation of staple foods such as bread flour is another approach to
improving selenium status of the human population (Rayman, 1997). Alternatively,
selenium-enriched yeast may be used to produce bread. For example, in Hungary
bread produced with selenium-enriched yeast was given to ten volunteers with
the daily Se dose being approximately 100 g. After two weeks of supplementation,
the subjects mean whole blood selenium level increased significantly (from 52.2
to 66.1g/l; Rumi et al., 1994). Four New Zealand women were supplemented
with 200 g Se in the form of high-Se wheat bread daily for 8-13 weeks. GSH-Px
activities increased in whole blood, erythrocytes, plasma and platelets of all subjects
but increases were considerably less than those of Se concentrations in whole
blood, plasma and erythrocytes (Thomson et al., 1985). It is interesting, that during
the post-dosing period Se concentrations and GSH-Px activities fell to levels,
which were in most cases somewhat higher than baseline values probably reflecting
Se reserves, build during organic selenium consumption. In the Netherlands six
healthy subjects were supplemented with 200 g Se as Se-rich bread for 6 weeks
and another six subjects received low-Se bread and served as controls (Van
Dokkum et al., 1992). Platelet GSH-Px activity increased significantly in the
supplementation group indicating availability of Se from the bread. In another
experiment, twenty-four Dutch men received 55, 135 or 215 g Se/d as Se-rich
meat or bread for a 9-week period (van der Torre et al., 1991). Except for the
higher erythrocyte Se levels after supplementation with high-Se meat, there were
no differences in bioavailability of Se between meat and wheat products. Similarly
to above mentioned data, at 10 weeks after supplementation ended, plasma Se
levels and platelet GSH-Px were still higher than the baseline values whereas
erythrocyte Se levels continued to increase. This could also indicate usage of Se
from the body reserves accumulated as a result of organic selenium consumption.
Several weeks supplementation with high-Se bread increased Se concentration
in plasma of New Zealand subjects from 50-70 ng/ml up to 120-175 ng/ml and
plasma Se remained elevated when supplementation ceased (Robinson et al.,
1985). It seems likely that wheat Se has high bioavailability and is the main
determinant of blood Se levels in Norway. For example, eighteen healthy,
Norwegian women were given Se-rich bread providing 100, 200 and 300 g Se
daily for 6 weeks. Serum Se increased by 20, 37 and 53 g/l respectively, in the
three groups (Meltzer et al., 1992). Furthermore, wheat was shown to be a
relatively available source of Se (73-86%) to rats regardless of whether its Se
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content is naturally high or is increased by fertilisation or spraying (Mutanen et

al., 1987).
This approach deserves close consideration owing to its practical ability to
reach wide segments of the population and previous success with other trace
element deficiencies such as iron. For example, in China Se-enriched wheat flour
is produced by its fortification with a Se-enriched mushroom extract (Combs,
2000). The UK supermarket chain ASDA sells bread produced from Canadian
high Se flour and Brazil-nut bread is also available on the market (Rayman, 2002).
A fourth strategy is production of functional foods enriched with selenium
(Surai, 2000; Surai, 2002). Common foods fortified with Se are shown in Table
12.4.
Table 12.4 Common foods fortified with selenium (Combs, 2001; Surai and Sparks, 2001; Surai, 2002).
Selenium-enriched products by direct fortification

Table salt, margarine, cereal gruel, beverages
Plant-derived foods, enriched with Se
Brussels spouts, brocoli, brassica vegetables, garlic, onions, celery, mint, chamomille, tea,
vinegar, beer, yeast, mushrooms and bread
Animal-derived foods enriched with Se
Eggs, beef, lamb, pork, chicken, turkey, milk and milk products
Selenium-enriched products
Several important factors must be considered when choosing the best food
supplementation strategy for a given population. Such factors are shown in Table
12.5. In general, main sources of dietary selenium could differ between different
countries. For example, in the UK meat and meat products provide 32% daily Se
consumption and dairy products and eggs are responsible for 22% Se consumption
(BNF, 2001; Figure 12.1). In contrast, in Russia about 50% Se in the diet originates
from bread and cereals and meat, milk and eggs provide about 20%, 10% and 5%
daily Se consumption respectively (Golubkina et al., 2002; Figure 12.2). In the
USA beef, white bread, pork, chicken and eggs account for half of the Se in the
diet (Schubert et al., 1987). In Ireland, meat and meat products (30%), bread and
rolls (24%), fish/fish products (11%), and milk and yoghurt (9%) were the main
contributors to mean daily Se intake (Murphy et al., 2002).
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Table 12.5 Some characteristics of food choice for Se-enrichment (adapted from Yaroshenko et al.,
2003a).
The food should be
Comments
A part of traditional
meals for the population
It would be counter-productive to attempt a change in

culturally-based food habits by introducing a new type of food.
Emphasis should be given to the possibilities of changing
composition of existing foods such as by selenium enrichment.
Consumed regularly in
a moderate amount
Since the objective is to deliver the amount of selenium needed

to meet RDA it is necessary to choose food which is consumed
regularly in moderate amount. Over-supplementation is
unnecessary and undesirable.
Consumed by the
This is particularly important given that immune function is
majority of the population more likely to be compromised in groups such as children and
the elderly.
Affordable
Affordability of food would play an important role in the

consumer choice.
Enriched with other

health-promoting
nutrients that are in
short supply in the
same population
Examples of minerals critical to health that are frequently

deficient include iron and iodine. Vitamin E and lutein are also
in short supply in the human diet. This can give a greater
improvement in the diet.
Supplying a meaningful
amount of the nutrient
(e.g. at least 50% RDA)
This is an important point that distinguishes true functional

foods from products that include tag-dressing amounts of
nutrients for advertising purposes.
Did you know that in the UK meat and meat products provide
32% daily Se consumption and dairy products and eggs are
responsible for 22% Se consumption?
Among animal-derived products, the egg is ideally suited to meet the list of
requirements mentioned in Table 12.5. The egg is a traditional and affordable
food in most countries and is consumed by people of all ages more or less regularly
and in moderation. It is also a very safe vehicle for supplementation given that a
toxic dose of selenium from eggs would require consumption of 30 eggs per day
over time, an impossible situation to imagine. There is an option of simultaneous
enrichment of eggs with several important nutrients, including omega-3 fatty acids,
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Other foods
Vegetables
Meat and meat products
Fish
Bread and cereals
Dairy products and eggs
Figure 12.1 Estimated intake of Se from different food in the UK in 1997 (adapted from BNF (2001).
Vegetables
Potato
Fruit
Eggs
Milk and milk products
Fish
Bread and cereals
Meat
Figure 12.2 Estimated intake of Se from different food in the Russia (Ural region) (adapted from
Golubkina et al., 2002).
vitamin E, carotenoids (Surai and Sparks, 2002; Surai, 2002) and with a single
egg it is possible to deliver at least 50% of the RDA for selenium. It seems likely
that pork, beef, chicken and milk can also be enriched with selenium.
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Improving the image of the egg

Chickens eggs have been used as a food by human beings since antiquity.
Compared with the hens egg, no other single food of animal origin is eaten by so
many people all over the world and none is served in such a variety of ways. Its
popularity is justified not only because it is so easy produced and has so many
uses in cookery, but also because of its nutritive excellence. From the nutritional
point of view, the wide use of the egg in the human diet is well deserved. Of the
three most important dietary essentials - proteins, fats, and carbohydrates - the
egg is composed largely of the first two (Table 12.6). The nutritive excellence of
the egg enhances the value of any food in which it is incorporated. Its wide use
in cookery for purposes of leavening, thickening, binding, and emulsifying
considerably improves the human diet (Stadelman, 1999).
Table 12.6 Major lipids (% weight of total) in the yolk (adapted from Noble, 1987).
Lipid
Phospholipid
Cholesterol esters
Triglycerides
Free fatty acids
Free cholesterol
Phospholipids
1.3
63.1
0.9
4.9
29.7
Phosphatidyl ethanolamine
Phosphatidyl serine
Phosphatidyl choline
Sphingomielin
Others
23.9
2.7
69.1
3.2
3.2
Did you know that compared with the hens egg, no other single
food of animal origin is eaten by so many people all over the
world and none is served in such a variety of ways?
Its proteins are highly digestible and remarkably complete, containing the most
important essential amino acids. The egg amino acid profile is similar to the ideal
balance of amino acids needed by men and women. It also supplies various
minerals, some in significant amounts, and contains a range of vitamins. For
example, in recent study in the USA it has been shown that eggs contributed 10%
to 20% of daily intake of folate and total, saturated and polyunsaturated fat, and
20% to 30% of daily intake of vitamins A, E and B12 (Song and Kerver, 2000). In
this respect, an egg can deliver (as % the daily value for a nutrient) protein: 10,
riboflavin: 15, vitamin B12: 8, vitamin A: 6, vitamin D: 6, folate: 6, vitamin B6: 4,
vitamin E: 3, thiamine 2, zinc 4 and iron 4. Many of those nutrients in the egg can
be manipulated by dietary means, however, a real value for improvement of human
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diet could have only those nutrients which are usually in short supply with other
products or have a positive effect on human health when consumed in excess.
Among them n-3 fatty acids, vitamin E, carotenoids and selenium have attracted
a lot of attention in nutritional sciences (Figure 12.3).
Designer egg production

Designer eggs
Omega-3 +
other nutrients
Omega-3
Linolenic
acid
DHA
Columbus
EggsPlus
Iodine
Egglands
best
Vitamin E
Others
Se
Lutein
Super Eggs
Figure 12.3 Designer egg production scheme (adapted from Yaroshenko and Surai, 2002).
The main problem with eggs consumption is that the image of the egg in the
mind of the consumer is not necessarily always good. Despite the nutritious
qualities of the egg, its comparatively high content of cholesterol has been the
driving factor causing declining egg consumption for the last 20 years. Indeed,
purchases of eggs for household consumption in the UK fell by 54% between
1975 and 1993 and by 9% between 1993 and 2000 (Buttriss, 2002). The problem
started when cholesterol was implicated as an important risk factor of coronary
heart disease (CHD), the leading cause of death in developed countries. The direct
relationship between the levels of plasma cholesterol and atherosclerosis was
experimentally demonstrated in an animal model (rabbits) 92 years ago
(Anischenkow and Charlatov, 1913). Since that time numerous studies have been
devoted to that subject. In particular, a consistent correlation was found between
levels of total cholesterol and CHD in populations living in different countries
(Keys, 1980). Therefore, dietary cholesterol became the centre of attention because
of its association with heart vascular disease development. That information
provided the stimulus for mass media blaming cholesterol for many pathological
conditions in the human body. As a result, many dieticians and doctors started to
recommend lower egg consumption to avoid excess of cholesterol in the diet.
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Similarly, it was initially believed that egg consumption was associated with a
rise in blood cholesterol (Yaffee et al., 1991) and as a consequence was deleterious
to health and life expectancy. The situation deteriorated when problems with egg
contamination with Salmonella were reported publicly. In the wake of health fears,
whole egg consumption in Britain has fallen by almost half in the past five years,
from more than three eggs a week to 1.70 eggs. Similar decreases in egg
consumption have been reported in other countries, including the US (Stadelman,
1999; Lewis et al., 2000). The proliferation of magazine articles and public
statements by organisations such as the American Heart Association relating eggs
to blood cholesterol and heart disease caused a downward trend in egg
consumption (McIntosh, 2000).
Did you know that misconception about relationship between
egg consumption and heart diseases was responsible for
sharp declining in egg consumption in the USA and many
European countries?
However, our understanding of cholesterol metabolism has substantially improved
since the initial furore over cholesterol, food animal products and heart disease.
Knowledge of reverse cholesterol transport by HDL from the vessel wall to the
liver for the catabolism (Lacko and Pritchard, 1990) was an important milestone
changing the current views of cholesterol intake. Dietary cholesterol is not regarded
anymore as the major determinant of the cholesterol level in the blood. There are
other more important determinants of this parameter such as saturated fat intake.
More is also known about the development of coronary heart disease (Shaper,
1987). Furthermore, detailed analyses of many cholesterol-lowering trials showed
that a decreased coronary morbidity did not reflect changes in total mortality
(Libbi et al., 2000), which was not changed. Furthermore, after analysing results
from 27 studies involved 30,902 person-years of observation, Hooper et al. (2001)
concluded that alterations of dietary fat intake had a positive effect on
cardiovascular events but practically no effect on total mortality.
Correlations drawn between nutrient intakes and longevity in the Japanese
since WWII illustrate this point. It was found that sufficient intakes of animal
protein and fat are crucial for attaining longevity (Shibata, 2001). It is interesting
that low serum cholesterol was deleterious for higher levels of functional capacity
and accelerated depressive status in the elderly in the community. High intakes of
milk and fats and oils had favourable effects on 10-year (1976-1986) survivorship
in 422 urban residents aged 69-71 (Shibata et al., 1992). The survivors revealed
a longitudinal increase in intakes of animal foods such as eggs, milk, fish and
meat over the 10 years. Furthermore, an increase in total cancer mortality in lung,
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prostate and colon was observed in men >60 years of age with low plasma
cholesterol in Switzerland (Eichholzer et al., 2000).
Results of many recent studies have shown that large numbers of eggs can be
consumed over lengthy periods without adverse changes to plasma cholesterol or
other lipid components. Furthermore, it is a general misconception that elevated
plasma cholesterol represents the main atherogenic stimulus. It seems likely that
the time has come to reconsider the eggs image and to distinguish between
scientific fact and conjecture.
Ginsberg et al. (1994) showed that consuming two eggs per day did not change
serum cholesterol; and consumption of four eggs per day had only a slight effect
on this parameter. Similarly, consumption of up to 14 eggs per week by students
did not alter serum cholesterol level (Vorster et al., 1992). Analyses of the results
from over 30 years in more than 2750 patients indicated that for the majority of
individuals modest changes in dietary cholesterol have little if any effect on plasma
lipoprotein cholesterol level (McNamara, 1995). McNamara also calculated that
a reduction in dietary cholesterol intake by 33% (from 450 to 300 mg/day) would
lower plasma cholesterol level by 3.7 mg/dl (approximately by 1.85%) and a 1%
decrease in energy intake from saturated fat decreases plasma cholesterol by 3
mg/dL (approximately 1.5%). Since dietary cholesterol has little effect on plasma
cholesterol in most individuals, the population-wide restriction on egg
consumption is not justified (McNamara, 1997). The same conclusion was drawn
by Dawber et al. (1982), who showed that differences in egg consumption were
unrelated to blood cholesterol level or to the incidence of coronary heart disease.
After adjusting for demographics (age, gender and ethnicity) and lifestyle variables
(smoking and physical activity), dietary cholesterol was found to be unrelated to
serum cholesterol concentration (Song and Kerver, 2000). When 116 male
volunteers between the ages 32 and 62 years consumed two whole fresh eggs
daily for 3 months no significant changes in mean serum cholesterol were recorded
and there was no significant association between dietary cholesterol intake and
either serum cholesterol or triglyceride (Flynn et al., 1979). Similarly a recent
study involving 37,850 men and 80,082 women concluded that the consumption
of up to one egg per day was unlikely to have a substantial overall impact on the
risk of cardiovascular disease or stroke among healthy men and women (Hu et
al., 1999).
Interestingly, recently it has been shown that people who reported eating four
or more eggs per week had significantly lower mean serum cholesterol than those
eating one egg or less per week (193 mg/dL vs. 197 mg/dl, p < 0.01) (Song and
Kerver, 2000). Furthermore, when dietary confounding factors were considered,
no association was found between egg consumption at levels up to one egg per
day and the risk of coronary heart disease in non-diabetic men and women
(Kritchevsky and Kritchevsky, 2000). Recent conclusions from a review of
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epidemiological and clinical data published by McNamara (2000) was that for
the general population, dietary cholesterol makes no significant contribution to
atherosclerosis and risk of cardiovascular disease. These suggestions are in line
with a recent finding that consumption of three eggs per day for 30 days by premenopausal women did not increase the risk of developing an atherogenic
lipoprotein profile (Herron et al., 2002). From another analysis of data
summarising 166 cholesterol-feeding studies conducted over the past 40 years in
3500 subjects, it was concluded that there was little justification for restriction in
egg consumption for general healthy individuals (McNamara, 2000a).
Did you know that recently it has been proven that there was
little justification for restriction in egg consumption for
general healthy individuals?
Once the public image of the egg is changed for the better, there are many
opportunities to produce designer eggs enriched with various nutrients with
health-promoting properties (Figure 12.3).
Selenium-enriched eggs as a route toward improving human selenium

status
Before the advent of commercially available organic selenium for food animal
diets, the main problem as regards the enrichment of eggs with selenium was the
low efficiency of transfer of inorganic selenium (selenite or selenate) to the egg.
In fact, even high doses of selenite in the diet of laying hens were not able to
substantially enrich eggs with this trace element (for review see Surai, 2002).
Did you know that development and commercialisation of the
organic form of selenium (Sel-PlexTM, Alltech Inc. USA)
opened a new era in production of selenium-enriched products?
Studies in our laboratory showed that egg selenium content can be easily increased
when Sel-Plex is included in the diet at a level to provide 0.4 ppm Se (Table 12.7;
Surai, 2000; 2000a; 2000b). In fact Se content in the egg was increased from 7.1
g up to 30.7 g as a result of dietary supplementation with organic selenium.
This finding is in agreement with recent data of Paton et al. (2002) indicating that
whole egg Se is directly affected by the level of the organic selenium in the diet of
the laying hen. As a result, the technology for production of eggs delivering ~50%
of selenium RDA was developed and successfully tested (Surai et al., 2000).
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Table 12.7 Selenium in the egg (adapted from Surai, 2000b).
Organic Se
added to the
feed, ppm
0
0.2
0.4
0.8
Se in egg
yolk, ng/g
298.3
605.3
854.0
1087.3
Se in egg
white, ng/g
50.7
193.7
403.7
621.7
Se per egg,
g
RDA from
one egg
7.10
18.04
30.67
43.35
11.4
28.9
49.1
69.4
Our investigation of the commercial characteristics of Se enriched eggs

(Yaroshenko et al., 2003; Dvorska et al., 2003; Surai et al., 2003) indicate that:
Inclusion of increased levels of organic selenium or their combination with

vitamin E for 12 weeks did not affect egg production, egg weight, ratio of
yolk/white, feed consumption, FCR or body weight of laying hens
There was no significant difference in carotenoid and vitamin A levels in
the egg yolk
Increasing diet Se increased vitamin E in the egg yolk and total Se in a
single egg reached 35-40 g, providing 55-73% RDA
During egg storage at 20C for 7-14 days lipid peroxidation occurred in the
egg yolk and MDA concentration significantly increased (Figure 12.4).
Egg yolk enrichment with selenium was associated with a significant decrease
in MDA accumulation, which was related to increased GSH-Px activity
(Figure 12.5). When yolk was incubated at 37C, lipid peroxidation was
enhanced and the protective effect of increased diet Se was significant
(Figure 12.6).
These data clearly indicate that enrichment of eggs with Se and vitamin E is
beneficial not only from the point of view of nutritional value of the eggs
but also as an important technological solution to decrease lipid peroxidation
in eggs during storage.
Did you know that Se-enriched eggs are also characterised
by improved lipid stability during storage?
Lipid peroxidation is considered an important detrimental factor affecting food

deterioration during storage and cooking procedures. In fact, eggs are a rich source
of cholesterol, and its oxidation products are a human health concern. It has been
shown that cholesterol is easily oxidized when eggs are cooked, in particular as a
result of egg powder preparation (Galobart et al., 2002). On the other hand,
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30
Fresh
1 week, 20 C
25
20
15
10
5
0
Control
0.2 Se
0.4 Se
0.8 Se
Figure 12.4 Effect of Se on lipid peroxidation in egg yolk, MDA ng/g (adapted from Yaroshenko et al.,
2003).
3.0
Yolk
White
2.5
2.0
1.5
1.0
0.5
0.0
Control
0.2 Se
0.4 Se
0.8 Se
Figure 12.5 Effect of Se on GSH-Px activity in egg yolk and albumin, U/g (adapted from Yaroshenko et
al., 2003).
cholesterol oxides are considered detrimental to human health (Savage et al.,

2002). Therefore a decrease in products of lipid peroxidation as a result of egg
enrichment with antioxidants has far-reaching health-related applications for food
industry. Similarly, enrichment of eggs with vitamin E can also substantially
decrease lipid peroxidation (Surai et al., 2000) and cholesterol oxide formation
(Galobart et al., 2002). A combination of vitamin E with organic Se is shown to
be a route to achieving this goal.
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90
2 week 20 C
80
3 h, 37 C, in vitro
70
60
50
40
30
20
10
0
Control
0.2 Se
0.4 Se
0.8 Se
Figure 12.6 Effect of Se on lipid peroxidation in egg yolk during egg storage, MDA ng/g (adapted from
Yaroshenko et al., 2003)
It seems that Se in eggs is highly available. For example, a recent clinical trial
conducted in the Ukraine showed that consumption of two Se-enriched eggs per
day for eight weeks significantly increased the Se level of the plasma of volunteers
(Surai et al., 2004). In fact sixty volunteers (30 in control and 30 in experimental
group) successfully finished the trial. Eggs consumed in the control group contained
7-9 g Se/egg and experimental eggs were enriched with selenium (28-32 g Se/
egg). Blood was collected before the beginning and at the end of experimental
period and Se was determined in plasma by hydride generation atomic absorption
spectrometry with fluorometric detection. The level of selenium in plasma of
volunteers living in the Kiev area of Ukraine (0.055-0.081 g /ml) was on the low
side of the physiological range and was somehow lower than we reported earlier
in volunteers in Scotland (Surai et al., 2000). Consumption of commercially
available eggs for eight weeks only slightly increased Se in plasma, which reached
physiological level (0.075-0.085 g/ml). In contrast, consumption of two eggs
daily, which together delivered the daily requirement of 55-65 g Se, for eight
weeks was associated with a significant increase in Se concentration in plasma.
Plasma Se reached 0.09- 0.14 g/ml, indicating improving Se status of volunteers
(Surai et al., 2004). This is the first clinical trial to prove that selenium-enriched
eggs could be used as an important vector to improve selenium status in countries
with low Se consumption like Scotland or Ukraine. Se availability from eggs for
human needs further elucidation and effect of different dietary sources of Se on
its concentration in plasma probably depends on the Se status of the human. For
example, in our previous clinical study in Scotland a consumption of one Se-
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enriched egg per day for 8 weeks did not change Se concentration in blood which
was in a physiological range (Surai et al., 2000). In contrast, in the case of low
selenium status, the consumption of eggs enriched with Se during 3 months
significantly increased serum and hair Se level (Yu et al, 1996). Similarly, in
Chinese children from the area endemic for Keshan disease, Se content in hair
increased due to consumption of Se-enriched eggs during 3 years (Yu et al, 1998).
Did you know that recently in a clinical trial it has been
proven that consumption of Se-enriched eggs can improve
Se status of people?
After the successful clinical trial with Se-enriched eggs in Ukraine, the production
of such eggs, enriched with Se and vitamin E, started commercially and now the
eggs called Basket of life can be found in the supermarkets in Ukraine. This
development is very important for this region. From the one hand Se deficiency
was documented in people working in Chernobil area (Tutelian et al., 2002;
Golubkina et al., 2002). On the other hand selenium and other antioxidants can
be especially beneficial for people living in radionuclide-contaminated areas of
the Ukraine. Currently various companies all over the world market Se-enriched
eggs including Mega-Eggs in Ireland, NutriPlus eggs in Malaysia, as well as
selenium enriched eggs in Russia, Thailand, Australia and the US (Table 12.8).
Prices for those eggs varied from country to country and are similar to those for
free-range eggs.
In the UK the only designer egg available through the major supermarkets is
the Columbus egg produced by the Belgium company Belovo. These eggs,
enriched in n-3 fatty acids and vitamin E, first appeared in Belgium in 1997, and
since then they have been sold in the UK (1998), Netherlands (1999) and India,
Japan and South Africa (2000). Currently production of the Columbus egg exceeds
50 million eggs per year in Europe. These eggs are characterised by a balanced
nutritional lipid composition (C18, n-6:n-3=1:1) and a favourable structural lipid
ratio (long-chain PUFA, n-6:n-3 = 1:3). When fed to selected groups of people,
Columbus eggs have been shown to improve the circulating cell membrane fatty
acid composition by favourably altering the n-6:n-3 ratio (De Meester et al., 1998).
The level of alpha-linolenic acid in Columbus eggs is about 12.6% while DHA
comprises about 2% of total fatty acids in the egg yolk. Therefore, these eggs
could adjust a diet toward recent nutritional recommendations regarding dietary
fatty acid profiles. In particular it is recommended linoleic acid:alpha-linolenic
acid ratio in the diet to be 5:1-10:1 with n-3 PUFA to provide 0.4-2% of total
energy (FAO, 1998).
In this respect it is worth mentioning that recent n-3 PUFA consumption in the
UK provides only 0.23% of total energy and in the Netherlands, Belgium,
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Table 12.8 Some examples of Se-enriched eggs produced in various countries.
Trade name
Countries
Columbus
UK, Belgium, Netherlands, France, Spain, USA,

Japan, South Africa, India, Israel, Korea,
Australia
Origin
Northern Ireland
Mega-Eggs
Ireland
Vita-eggs
UK
NutriPlus
Malaysia
Selen Egg
Thailand
Organic Selenium Egg
Singapore
Heart Beat eggs
New Zealand
Tavas Yumurta
Turkey
Seker Yumurta
Turkey
Selenyum eggs
Turkey
SelPlex eggs
Switzerland
Nutriplus
Portugal
Bounty Eggs
Philippines
Bag of Life (Koshik zhitja)
Ukraine
Rejuvenating (Molodiljnije)
Russia
Aksais sun (Aksaiskoye solnishko)
Russia
Spring of cheerfulness (Rodnik bodrosti) Russia
Universal (vSELENskoye)
Russia
Mettlesome eggs (Molodetskoye)
Belarus
Germany, Ireland and Italy this figure is even lower (Roche, 1999). Recently,
Columbus eggs were also enriched with selenium, delivering with a single egg
more than 50% RDA (35 g) of this trace element as well as 10 mg vitamin E
(66% RDA) and 75 g iodine.
Did you know that after long discussions about selenium
deficiency in the UK, a product (Columbus eggs) is available
on the supermarket shelves that can substantially improve
Se status of the major part of population?
Since these types of designer eggs find their way to supermarket shelves in other
countries the selenium status in those countries can also be improved. This could
be a result of successful knowledge transfer from developer and producer of the
feed additive (Alltech, Inc., USA) via scientific evaluation and development of
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the effective technology of designer egg production (Surai, 2002) to the egg
producer and food retailers to consumers. To satisfy consumer demand in the
UK, free range Columbus eggs enriched with n-3 PUFAs, vitamin E and selenium
are also on the supermarket shelves.
It is important to mention that in many countries there are several different
competitive companies producing Se-enriched eggs. For example in Russia
Seimovskaya poultry farm (Nizni Novgorod region) produces about 65,000 Seenriched eggs per day, while Magnitogorskaya (Magnitogorsk city), Aksaiskaya
(Rostov region), Voktinskaya (Izhevsk region) and Volzhaninskaya (Yaroslavl
region) poultry farms produce 40,000; 30,000; 15, 000 and 10,000 Se-enriched
eggs daily. In Belarus, Soligorskaya (Minsk region) poultry farm produces about
40,000 Se-enriched eggs daily. Se concentration in those eggs in Russian and
Belarus varies in a range of 20-30 g/egg. Similar range of Se in Se-enriched
eggs can be found in other countries with a target to achieve a 50% RDA in Se
with a single egg. There is also a possibility to produce eggs with much higher Se
content. For example, Combs (2000a) reported that using selenium in the form of
high-quality Se-enriched yeast in the chicken diet at the level 1.2 ppm it is possible
to achieve Se content in the egg up to 200 g.
If egg selenium content is enhanced along with increased levels of omega-3
PUFAs and vitamin E (Columbus) or vitamin E and specific carotenoids (Super
eggs, Table 12.9; Surai, 2001) in levels comparable with RDA, this could further
widen opportunities for producers and consumers to meet specific nutrient demands
that aid in maintaining a healthy life style. There is also an opportunity to produce
Se-enriched quail eggs (Figure 12.7) which can find their way into niche market.
Did you know that Se-enriched eggs are on the supermarket
shelves in more than 20 countries all over the world?
Se-enriched meat and milk

Various types of meat are important natural sources of Se in human nutrition.
Indeed in 2003 world pig meat production reached 95.8 million metric tons, while
poultry meat rose to 75.2 million tons and the beef volume was 61.9 million tons
(Best, 2004). In particular in 2000, an estimated 56 billion was spend on household
food in the UK (Buttriss, 2002). In general, meat is a good source of selenium.
However, Se concentration in the meat varies substantially depending on
geographical origin of the country and Se supplements used. For example, pork
produced in the UK, Australia and USA contains Se at the levels of 14, 9.4-20.5
and 14.4-45.0 g/100g respectively (McNaughton and Marks, 2002). In Sweden,
pork contained Se at the level of 11.3 g/100g (Daun et al., 2001: Table 12.10).
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Table 12.9 Major nutrients in a super egg (Adapted from Surai, 2001).
Nutrient in
the egg
Amount,
mg
% Recommended
dietary allowances
Vitamin E
19.3
129
Lutein
1.91
RDA not known
Selenium
0,032
50
100g wheat bread

150g brown bread
500g meat
1 kg vegetables
209
100
49 g sardine
165 g Atlantic cod
170 g haddock
180 g carp
DHA
Similar amount provided by:
100 g corn oil

150 g margarine
300 g peanuts
1 kg butter
10 kg meat
50 g celery
100 g green peas
200g asparagus
200g green pepper
200 g yellow pepper
Selenium/egg, g
Control
Experimental
4
3
2
1
0
5 designer quail eggs = 50% RDA
Figure 12.7 Quail egg enrichment with selenium (adapted from Karadas et al. 2004).
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Table 12.10 Selenium content in meat from different countries, g/100g (adapted from Diaz-Alarcon et
al., 1996; McNaughton and Marks, 2002;Daun et al., 2001; Tutelyan et al., 2002; Golubkina et al., 2002;
Schubert et al., 1987).
Country
Pork
Year reported
Chicken
Year reported
USA (Ohio)
USA (Dakota)
USA
USA
Canada
France
Spain
Spain
Spain
Russia
Sweeden
Australia
UK
New Zealand
4,0
31.0
33
14.4-45.0
30.0
10.7
6.0
4.0
6.1-11.6
15.0-20.0
11.3
9.4-20.5
14.0
1.93-15.0
1980
1984
1987
1999
1982
1991
1983
1987
1996
2002
2001
2002
1991
2000
20
19.0-27.6
16.0
5.4
2.0
16.0
5.8-8.4
20.0
11.6-28.0
6.0-7.0
13.7-14.5
1987
1999
1982
1991
1983
1987
1996
2002
2002
1991
2000
Indeed, it is well established that selenite or selenate dietary supplementation is

not effective in increasing Se concentration in the meat. However, organic selenium
in the form of Sel-PlexTM can be added into diets fed to pigs to produce seleniumenriched meats. At high Se supplementation its content in loin of grower pigs was
more than twice compared to selenite group (Mahan, 1999). For example, adding
selenite to the pig diet at the level of 0.5 ppm increased Se concentration in loin
from 0.114 up to 0.189 ppm, while adding Sel-Plex into the pig diet in the same
amount increased Se level up to 0.362 ppm (Mahan and Parrett, 1996). Therefore
100 g of such pork could provide about 66% RDA in selenium. The opportunity
of producing Se-enriched pork is clearly shown by Kim and Mahan (2001)
studying comparative effects of high dietary levels of organic and inorganic
selenium on selenium content in tissues of growing-finishing pigs. In fact, dietary
supplementation with organic selenium at the level of 5 ppm for 12 weeks caused
Se concentration in loin to be increased from 0.154 ppm up to 3.375 ppm (Table
12.11). This extremely high Se dose caused only a slight (by 2.5%) decrease in
body weight without changing feed intake (Kim and Mahan, 2001) showing
comparatively low toxicity and commercial opportunities in using organic selenium
in high doses to produce Se-enriched pork. Indeed, Se-enriched pork can be sold
separately or can be included into various meat-based products to enhance their
Se content. These data were a background for the development of a technology
of pork enrichment with Se and this food is already on the market in Korea. It is
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interesting that in addition to increased Se level in the meat there are other important
improvements in pork quality (Cole, 2000):
Tender and chewy

Good colour
Low fat
Low drip loss
Reduced pig odour
Table 12.11 Effect of dietary Se source and level on loin Se content of growing-finishing pigs, g/g
(adapted from Kim and Mahan, 2001)
Se doses
Selenite
Sel-Plex
0
0.154
0.154
10
15
20
0.333
3.375
0.277
5.927
0.323
10.311
0.322
7.648
Did you know that in Korea Se-pork is served in special

restaurants where information about benefits of Se is widely
available to educate customers and increase demand for this
functional meat?
Se-pork is served in special restaurants where information about benefits of Se is
widely available to educate customers and increase demand for this functional
meat.
Beef is considered to be a major source of dietary Se, but the concentration of
Se in edible beef products is very variable and depends on the geographical origin
(Finley, 1999; Table 12.12). For example, in the review of McNaughton and Marks
(2002) Se concentration in the beef was reported to be 3.0-7.6 g/100g, 2.2-8.3
g/100g, 7.2-12.1 g/100g and 13.4-19.0 g/100g in the UK, New Zealand,
Australia and USA respectively. In North Dakota, Se concentration in beef was
27; 38; 40; 47 and 67 g/100g in Southeast, Central, Southwest, South central
and Northwest respectively (Hintze et al., 2001). Taking into account an average
red meat consumption in the USA (57 kg/year) Finley (1999) calculated that daily
intakes of Se from beef in various USA areas would range from 40 up to 100 g/
day. Venison and Ohio-raised beef contained respectively 7.52 and 7.65 g/100g
Se, while in nonregion-raised USA beef Se concentration was 20.51 g/100g
(Holben, 2002) which was similar to 22 g/100g reported earlier (Schubert et al.,
1987). On the other hand, Se in beef raised in North Dakota could reach even
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higher levels of 67 g/100 g (Finley et al., 1996; Hintze et al; 2001; 2002). The
opportunity of substantial enrichment of beef by Se is shown by analysis of beef
produced in a seleniferous area of South Dakota (Hintze et al., 2002). Indeed Se
levels in ribeye, sirloin, clod and round were 1.20; 1.19; 1.21 and 1.22 mg/g
respectively. After 14 weeks on high selenium diet (11.9 ppm from seleniferous
wheat and hay) the Se level in muscles further increased up to 2 g/g (Hintze et
al., 2002). Indeed, inclusion of such beef in any meat foods could substantially
enrich it with selenium. It is also interesting to mention that in the study high Se
intake for 14 week of the experimental period did not affect feed intake, average
daily gain and final weight of steers.
Table 12.12 Selenium content of common meats in a grocery store in North Dakota (adapted Finley,
1999).
Item
Concentration, Standard
g/g
serving, g
Beef, ground, raw, eastern North Dakota

Beef, ground, raw, southwestern Missouri
Beef, ground, raw, central Virginia
Beef, ground, raw, local supermarket
Venison, uncooked, western North Dakota
Venison, uncooked, central South Dakota
Ham, 95% fat free, deli
Lamb, raw, eastern North Dakota
0.37
0.06
0.08
0.33
0.20
0.45
0.35
0.30
85
85
85
85
85
85
85
85
Se in
serving, g
%
RDA
31.5
5.1
7.1
28.3
16.9
38.1
30.1
25.3
57.3
9.3
12.9
51.5
30.7
69.3
54.7
46.0
In European and some Asian countries Se concentration in beef is much lower

than in the USA. However, it is possible to substantially increase Se content of
beef by inclusion of organic selenium into the diet of cattle. For example, when
Holstein bulls at the age of 22 months were supplemented with organic selenium
in the form of Sel-Plex at the level of 4 mg/head/day for 30 days, Se concentration
in the Longissimus dorsi muscle increased from 0.107 g/g up to 0.223 g/g
(Simek et al., 2002). At the same time enrichment of beef with Se was associated
with decreased drip loss and fluid loss during beef storage (Figure 12.8). Similarly,
supplementing one-month-old calves with 0.3 ppm organic selenium in the form
of Sel-PlexTM for 2 months increased Se concentration in striated muscle from
0.092 g/g up to 0.263 g/g (Pavlata et al., 2001). Therefore, 100 g of such Seenriched beef could provide about 40-50% RDA in selenium. Lamb meat can
also be substantially enriched in Se (MacPherson et al., 1994; Molnar et all.,
1997). Shoulder and thigh Se exhibited a dose-response relationship to dietary
supplementation and lamb meat could contribute to increased human dietary
intakes of the element (Molnar et al., 1996).
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1.0
Control
0.9
Se-supplemented
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
Drip loss
Fluid loss
Figure 12.8 Drip loss (%) and fluid loss (%) in beef during storage (adapted from Simek et al., 2002).
Selenium concentration in commercial chicken meat varies substantially (Table

12.10). Inclusion of selenite in the chicken diet even in high concentrations (up
to 8 ppm) only moderately increased Se level (up to 23-26 g/100 g) in chicken
meat (Arnold et al., 1973). On the other hand, as in the case with pork and beef,
there is an opportunity to increase Se content in chicken meat by inclusion into
the diet of organic selenium. For example, Selen Chicken is a premium chicken
brand, offering added value to producers and retailers. The development of this
kind of meat started in Korea and now is under the development in other countries.
For example, under commercial conditions in the Ukraine, the RozDon company
produced chicken meat enriched with Se and vitamin E by dietary inclusion of
organic Se in the form of Sel-Plex TM (Alltech Inc., USA) and increased doses
(250-500 mg/kg) of vitamin E (Figure 12.9; Yaroshenko et al., 2004). The results
indicated that dietary inclusion of organic Se from day old to slaughter significantly
increased selenium level in the breast (from 85.2 to 284.3 ng/g) and leg (from
72.2 to 274.2 ng/g) muscle in comparison to the chickens fed a commercial diet
supplemented with selenite. Increased dietary vitamin E (250-500 mg/kg) during
the last four weeks of growth also significantly increased vitamin E concentration
in muscle tissue. A combination of increased concentrations of selenium and
vitamin E was responsible for substantial decreases in lipid peroxidation in the
meat during storage at 4C and at 20C. In fact, concentration of malondialdehyde
in the experimental meat was significantly decreased as a result of increased
antioxidant concentrations in the meat. Therefore our data indicate that
consumption of approximately 100 g Se-enriched chicken meat, which can be
produced commercially, could deliver about 50% of the RDA for Se and could
help in solving problems of Se deficiency. Furthermore, the combination of
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increased selenium and vitamin E concentration in chicken meat could improve

meat quality during storage. There is also a valuable option to produce Se-turkey,
where growth period is substantially longer. Indeed, commercial turkey meat
produced in the USA contained Se at the level of 34 g/100g (Schubert, 1987)
reflecting high Se soils in the USA and showing a possibility of increasing Se
content of turkey in other regions by using organic selenium in the diet.
300
Control
Se-enriched
250
200
150
100
50
0
Breast Muscle
Leg muscle
Figure 12.9 Se-enriched chicken meat ng/g (adapted from Yaroshenko et al., 2004).
The bioavailability of Se in meat from domestic animals has been evaluated. In

the experiment with rats after 9 wk of the dietary Se repletion, it has been shown
that relative Se availability (based on liver GSH-Px) was as follows: pork 86%,
sodium selenite 81%, SeMet 80%, beef 80%, chicken 77%, veal 77%, and lamb
58% (Wen et al., 1997). Similarly it has been shown that beef is highly bioavailable
sources of dietary Se when compared with selenite or selenomethionine (Shi and
Spallholz, 1994). Using similar tests the authors have found that after Se depletion
the recovery of liver GSH-Px activity compared to the control animals (set at
100%) were selenite (98%), selenate (117%), raw beef (127%) and cooked ground
beef (139%). The data suggest that bioavailability of Se from ground beef is greater
than that from either selenite or selenate. Therefore meat as a source of selenium
is characterised by high availability.
Did you know that pork, beef and chicken are characterised
by high Se bioavailability?
For the last few years consumer demands regarding aspects meat quality have
substantially increased. Therefore a challenge to the meat industry is to enhance
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the image of meat purchased at the supermarket (Janssens, 1998). There are many
meat quality characteristics that attract consumer attention. They include
appearance, texture and flavour (Liu et al., 1995) as well as tenderness, juiciness,
aroma (Janssens, 1998) and other subjective characteristics. Among these
appearance has a major impact on the initial decision of the customer to purchase
or reject the product (Sheehy et al., 1997). Consumers prefer fresh meat with a
minimum loss of water during handling and cooking. Therefore water-holding
capacity of the meat (Mahan and Kim, 1999) as well as colour (Froning, 1995)
and absence off-flavours (Sheehy et al., 1997) are considered among most
important meat quality characteristics.
It has been shown that sensory quality of meat is affected by muscle
biochemistry and modern processing technologies (Ouali, 1991). For example,
grinding increases oxygen incorporation into muscle and cooking releases proteinbound iron into the intracellular pool (Chan and Decker, 1994). In this process
free radical production and lipid peroxidation cause membrane structure and
quality disruption, which leads ultimately to significant losses in food quality,
including off-flavour, off colours, poor texture etc. (Stanley, 1991).
One approach to enhancing oxidative stability of meat is to add antioxidants
either into the animal diet or directly during processing (Decker, 1998). For
example, an increasing body of evidence indicates that increased vitamin E
supplementation is an effective means of meat quality improvement in chickens,
turkeys, cattle, pigs and lambs (Sheehy et al., 1997; Wulf et al., 1995; Buckley et
al., 1995; Liu et al., 1995). Based on the data presented above and taking into
account antioxidant interactions, it is possible to suggest that synergism between
Se and vitamin E could work to enhance meat quality. In fact, it has been shown
that GSH-Px activity in muscles did not change significantly over 8-day storage
of beef (Renerre et al., 1996). This means that once GSH-Px activity is elevated it
is maintained post-mortem. Therefore we might expect a stabilising effect of dietary
Se supplementation during meat storage. Indeed supplementing broiler diets with
0.25 ppm Se substantially increased GSH-Px activity in breast (2.1-fold) and leg
(4.1-fold) muscle; and as a result decreased lipid peroxidation was detected (2.5fold in breast muscle and 3.3-fold in leg muscles) after 4 days storage at 4C
compared to the control group (DeVore et al., 1983). These data clearly indicate
that GSH-Px significantly contributes to the overall antioxidant defence of muscle,
decreasing tissue susceptibility to lipid peroxidation and that increasing oxidative
stability of skeletal muscle can be accomplished by organic Se supplementation
of the diet. Additionally, Avanzo et al. (2001) have shown that deficiencies of tocopherol and Se caused multiple alterations in the antioxidant system and
adversely affected the redox state of chicken superficial pectoralis muscle.
Protective effects of Se may be not direct, but are mediated via improvement of
other chains of antioxidant defence. For example, Se in combination with vitamin
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E increased activity of SOD in chicken serum (Tras et al., 2000). A stabilising

effect of Se in combination with other antioxidants on meat quality would be a
great advantage in producing so-called designer meat. For example, meat enriched
with n-3 PUFAs was shown to have increased TBA values during storage; and the
same meat from antioxidant-supplemented (Se + vitamins E and C) chickens
showed lower TBA values and greater colour stability during storage (Ahn et al.,
1998). In our experiment (Surai and Dvorska, 2002; 2002a) inclusion of organic
selenium in the commercial breeder diet increased Se-GSH-Px activity in muscles
more than two-fold. A combination of organic selenium and vitamin E
supplementation was associated with the highest Se-GSH-Px activity in muscles.
The highest level of the final product of lipid peroxidation in muscle after 2-year
storage at 20C was found in muscles from laying hens fed on a semi-synthetic
diet and characterised by the lowest vitamin E and Se-GSH-Px activity. The lowest
initial level of MDA was found in muscles from birds fed diets supplemented with
either 200 ppm vitamin E or 100 ppm vitamin E in combination with 0.4 ppm
organic selenium.
Did you know that meat enrichment with selenium could have
a triple benefit: increasing lipid stability during storage,
maintaining attractive appearance/colour and providing
selenium in amounts comparable with daily requirement?
Indeed a combination of increased omega-3 fatty acids, vitamin E and Se in the
meat is a way forward to the functional food category.
Milk and milk products fulfil optimal nutrition requirements providing a range
of important nutrients for humans. However, between 1975 and 2000 there was a
progressive reduction in milk consumption in many developed countries. For
example, in the UK milk consumption decreased from 2733 ml/person/week in
1975 to 1802 ml in 2000 (Buttriss, 2002). Nevertheless, new scientific evidence
on the milk importance in human nutrition is emerging and this will help to change
negative trends in milk consumption. In particular, milk contains Se in various
concentrations depending on country studied (Table 12.13) and it was calculated
that in Australia milk provide about 4.4-5.4 g, 8.6-10.5 g and 10.0-12.2 g Se
daily for adults, pre-school children and infants (Tinggi et al., 2001). Therefore,
infants received almost 100% RDA and pre-school children had about 50% RDA
in selenium with their daily consumption of milk. In general, comparatively low
Se levels in the milk are associated with cows diets. In fact, supplemental inorganic
selenium is poorly transferred to the milk. In contrast, selenized yeast (Sel-PlexTM)
as a source of organic selenium was 2 to 3 times more effective than selanate at
increasing milk Se concentration (Knowles et al., 1999).
It has been shown that dairy proteins, caseins and whey proteins, are rich in
methionine and cysteine and therefore they could provide a potentially useful
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Table 12.13 Selenium content in cows milk in various countries (adapted from Tinggi et al., 2001)
Country
Se, g/l
Year reported
Finland
7.9
1981
Germany
23.2
1978
Norway
10.1
1983
Netherlands
16.4 (winter)
1989
Belgium
10.1 (summer)
11.3 (summer)
17.3 (winter)
1989
1988
1988
Poland
10.5
1992
UK
15.0
1995
Israel
73.0
1990
Canada
28.0
1980
USA (Ohio)
8.0
1979
USA (South Dakota)
32-138
1984
Japan
23.1
1982
New Zealand
2.9-9.7
1973
Australia
15.8
1983
vehicle for dietary selenoprotein enrichment (McIntosh and Royle, 2002). The
authors showed that using organic selenium in the cows diet it is possible
substantially increase Se concentration in milk. In fact there was a significant and
rapid increase in milk Se concentration with the introduction of 2 and 6 mg Se
daily supplements as Sel-PlexTM. Therefore, Se concentration in the milk increased
from 6.9 up to 25.2 g/l. It seems likely that this concentration is still too low to
substantially affect Se consumption of the humans. However, in the same study it
was shown that when dietary Se content was increased to supply 25 mg Se/day,
milk concentrations plateaued at about 150 g/l without any clinically detectable
signs of Se excess (McIntosh and Royle, 2002). It is possible to suggest, that at
longer period of organic Se supplementation (in the reported study it was only 16
days), it is possible to achieve similar Se concentration with lower Se doses.
Therefore, about 200 ml of such Se-enriched milk could provide about 50% RDA
in Se. Since milk-processing products would also be enriched with Se, they
potentially could be a substantial source of Se for improvement of human diet
especially in regions low in natural Se level in the soil, feeds and foods. Therefore,
Se-enriched milk is under the consideration in Australia. In 2003 one of the
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biggest raw milk producers in Korea, Seoul Dairy Co-op. launched their Seenriched milk under the SELK name. SELK is enriched with Se, Ca, vitamins A,
D and E. In general, Se concentration SELK is about 50 g/l being 2.5fold
higher than commercial milk and they sell Se-enriched milk on premium price.
The co-op is running an educational program in a weekly magazine and in daily
newspapers on the importance of selenium for human health. Indeed, education
is a key for further development and successful marketing of animal-derived
products enriched with selenium. Unfortunately in many cases in public eyes Se
is a pollutant and toxicant and it will take some time and a lot of efforts from
scientist to change this gloom picture.
Did you know that recently Se-enriched milk has reached
supermarket shelves in Korea?
It seems likely that Se bioavailability from milk is quite high and comparable with
that for sodium selenite (Mutanen et al., 1986). It is interesting that Se availability
from milk depends on the dietary source of Se for cows. For example, the
experimental milks had their Se content increased by feeding cows either sodium
selenite (selenited milk) or Se-enriched barley (barley milk). The Se in the barley
milk was significantly more available than that in the selenited milk when the
plasma Se level was the response criterion (Mutanen et al., 1986). Absorption of
selenium from milk protein and isolated soy protein formulas in pre-school children
was evaluated using stable isotope tracer 74Se (Solomons et al., 1986). In the
experiment, mean fractional absorption of selenium from the formulas based on
milk, isolated soy protein, and milk-soy were 64.2, 73.4 and 45.0%, respectively,
with the combined formula having a significantly lower intestinal uptake for added
selenite than the casein formula. It is also interesting that the apparent digestibility
coefficient and retention of selenium in rats were higher for the goat milk diet
than for the cow milk-based diet (Barrionuevo et al., 2003).
Se-enriched eggs, meat and milk as functional food

The concept of healthy food additives arrived from Japan in the 1970s and the
term Functional foods appeared in 1984 (Harris, 2000). At this time consumers
began to view food from a radically different point. This changing face of food
led to the development of a new area in the food and nutrition sciences known as
functional foods (Hasler, 2000). The Food and Nutrition Board of the National
Academy of Sciences defines a functional food as one that encompasses potentially
healthy products providing health benefits beyond that of the traditional nutrients
it contains (Milner, 2000). This is in agreement with the data of the 1998 USA
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study from written questionnaires, completed by 2,074 respondents indicating

that most shoppers believe foods can offer benefits beyond basic nutrition to
functional nutrition for disease prevention and health enhancement (Gilbert, 2000).
However, a recent USA survey reported that taste is the primary influence on
food choice, followed by cost (Glanz et al., 1998). Similarly, in a survey in
Ireland, quality/freshness of food was the most frequently selected food choice
factor (51%) followed by taste (43%) and trying to eat a healthy diet (36%)
(Kearney et al., 2000).
Today, functional foods have received substantial attention (Mazza, 1998; Reilly,
1998) and represent one of the fastest growing segments of the world food industry
(Harris, 2000). For example, dairy products and other processed foods, including
mayonnaise, margarine, dressings containing DHA (Takahata et al, 1998) as well
as n-3 enriched eggs (Van Elswyk, 1997; Leskanich & Noble, 1997) are already
on the market in different countries. Antioxidant-fortified margarine is shown to
be effective in the delivery of vitamins E and C as well as - and -carotene to
human (van het Hof et al, 1998). In the USA annual sales of functional food
products comprise around $50 billion (Harris, 2000). In total, Functional Food
have a market share of around 2% in the US food market and quickly growing
(Menrad, 2003).
Did you know that in the USA annual sales of functional food
products comprise around $50 billion?
There are three major reasons for the increased interest in functional foods (Milner,
2000):
increased health care costs;

recent legislation; and
scientific discoveries
Recently, six major targets in relation to functional food science have been
identified (Roberfroid, 2000):
gastrointestinal functions;
redox and antioxidant systems;
metabolism of the macronutrients;
development in foetal and early life;
xenobiotic metabolism and its modulation;
mood and behaviour or cognition and physical performance.
In the same review the author has stated that the health benefit of a functional
food will be limited if the food is not part of the diet. Therefore functional foods
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must remain foods and they must achieve their effects in amounts normally
consumed in a diet (Contor, 2001).
Eggs have not traditionally been regarded as a functional food, primarily due
to concerns about their adverse effects on serum cholesterol levels (Hasler, 2000).
However recent finding described above indicating that there is little if any
connection between dietary cholesterol and blood cholesterol levels as well as
between moderate egg consumption and heart diseases could help changing a
bad image of eggs. In this respect, eggs enriched with selenium as well as with a
combination of Se, DHA, vitamin E and lutein, ideally fit in the category of
functional food enabling to substantially improve the diet. Eggs are consumed
regularly by the most of the population and are served as an integral part of
traditional English or Mexican breakfast. The development of super egg
enriched with these 4 nutrients received a lot of publicity through mass media
including radio, TV and newspapers. Some of newspaper titles are shown in Table
12.14. They clearly show great public interest in improvement of egg quality and
in creation healthy eggs.
Table 12.14 Some newspaper titles related to the super egg development (adapted from Surai, 2002)
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Title
Newspaper
Date
Super egg that could poach the vitamin

pill market
Daily Mail
June 12, 1998
Science goes to work on an egg
The HERALD
June 12, 1998
Super egg
Cambridge Evening
News
June 12, 1998
Good health is no yolk
The EXPRESS
June 12, 1998
Scientists go to work on creating

super-egg
The TIMES
June 12, 1998
Experts crack the super eggss secret
The EXPRESS
August 2, 1998
Scientists develop a natural panacea:

New super egg bid to allay killer diseases
The HERALD
April 5, 1999
Scientists go to work on a super-egg
The GUARDIAN
April 7, 1999
Scots to market life-saving eggs
The SUNDAY TIMES
April 5, 1999
Super-eggs to help fight against cancer
METRO
April 7, 1999
Could SUPEREGG save your life?
The EXPRESS
April 8, 1999
The egg that goes to work on your health
Daily Mail
April 7, 1999
New enriched egg could bring health

benefits
Farmers Guardian
April 9, 1999
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When considering possibilities of egg enrichment with antioxidants it is necessary

to take into account the following (Yarosheno and Surai, 2002):
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Efficiency of nutrient transfer from the feed to the egg.

For example, vitamin E and lutein are effectively transferred to the egg,
efficiency of vitamin A transfer is much lower and ascorbic acid is not
accumulated in the egg at all. There are no data available on the flavonoid
transfer to the egg.
Form of nutrient in the diet.
Inorganic selenium in the form of selenite or selanate has low efficiency of
transfer to the egg. However, organic selenium in the form of selenized
yeast is transferred to the egg much more easily. This makes it possible to
enrich eggs with selenium. Either oil form or dry form of vitamin E are
suitable for inclusion into the diet.
Availability of commercial sources of effective feed forms of antioxidants.
Vitamin E (ROCHE, BASF, etc), organic selenium (Alltech, Inc), lutein
(Kemin) are available on the market
Possible toxic effects of nutrients for the laying hens.
Vitamins A and D are toxic for chickens when added into the diet at very
high levels. Vitamin E and lutein can be added into the chicken diet at high
doses without any side effects. When organic selenium is used the toxicity
is not a problem since doses used are far lower than toxic levels of selenium.
Amount of nutrient delivered with an egg in comparison with RDA.
If an egg can deliver an amount of a nutrient comparable with RDA this
could be an ideal situation for marketing strategy. On the other hand, if this
amount is lower than 30-50% RDA it would be difficult to build such a
strategy.
Established health-promoting properties of nutrients and their shortage in
a modern diet
The justification for inclusion of vitamin E into the egg is clear. It is important
component of antioxidant defences, in many cases diets are deficient in this
antioxidant and consumption of high doses of vitamin E (higher than RDA)
is beneficial. The same is true for lutein and selenium.
Possible interactions with assimilation of other nutrients from the egg.
When egg is enriched simultaneously with vitamin E and/or lutein lipids of
egg yolk could help antioxidant assimilation. In fact, amount of lipids in
the egg yolk (about 6 g) and their composition (saturated, monounsaturated
and polyunsaturated fatty acids) could provide an ideal mileu for vitamin E
and/or lutein absorption in human intestine. On the other hand, vitamin E,
lutein and Se can prevent omega-3 peroxidation during absorption.
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Stability during egg cooking.

Vitamin E, lutein and Se are quite stable during egg boiling or frying.
Effect on appearance and taste.
Vitamin E, carotenoids and Se do not affect egg taste, but might help in
prevention of fishy taste appearance in omega-3 eggs. Egg enrichment with
lutein could be beneficial (in some countries) in terms of consumer
preference of deep coloured egg yolk.
Possibilities to claim health benefit.
Health claim regulations differ substantially from country to country. For
example, in Malaysia there are no restrictions on health claims and such
claims as delays the onset of ageing or increases fertility can be found
on egg box.
Did you know that antioxidant-enriched eggs could be
considered as Functional Food?
Therefore, from information provided above, it is clear that vitamin E, lutein and
selenium are major antioxidants to consider for egg enrichment. An egg has also
a great potential in terms of improvement of human diet. These and other options
of egg use in the human diet need further investigation. Indeed, most of studies,
including our own 8 week study, with designer eggs have been conducted during
a limited length of time and it is not clear what kind of benefit we could expect
when, for example, super eggs are in the diet during 6-12 month of study.
The data indicate that a designer egg enriched in vitamin E, lutein, DHA and
Se can be not only a good nutritional product but also a good vector for the
delivery of four essential nutrients vital for human health. A crucial feature of
these designer eggs is the synergistic combination of n-3 fatty acids with major
antioxidants, vitamin E, lutein and Se, as an important approach to the improvement
of the human diet. These eggs will not be able to replace vegetable and fruits as a
major source of natural antioxidants and fish products as a source of DHA.
However, they can substantially improve the diet, especially in countries like
Scotland, significantly contributing to the recommended daily intake of vitamin
E, lutein, DHA and Se. Commercially, it is possible to produce designer eggs
enriched with 4 nutrients as mentioned above or with 3, 2 or 1 nutrient depending
on the consumer demand. It seems likely, that egg yolk can also be enriched with
vitamin D (Mattila et al., 2003). As a result, price for the production of such eggs
could substantially vary. Therefore the way of egg to the functional food category
started successfully and now it is consumer education which is needed to fulfil
the idea of using eggs as functional food. Indeed, many categories of people will
benefit from using designer eggs as part of their everyday diet. However, more
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research should be done in this fascinating area to convince costumes to go for

designer eggs. Further development of various types of designer eggs could be
an important contribution to functional food development with a consequent
improvement of the human diet.
Did you know that production of Se-enriched eggs, meat and
milk could solve a problem of the global Se deficiency
and help maintaining human health?
For example, designer eggs could contribute to several aforementioned categories:
redox and antioxidant systems (an egg delivers three antioxidant components,
including 150% RDA in vitamin E, 50% RDA in Se and a substantial amount of
lutein); development in foetal and early life (an egg deliverers a minimal RDA for
DHA which is an essential element of the baby brain development) and mood
improvement (an egg deliveries 50% of RDA in Se which is considered as having
an effect on human mood). Indeed, increased omega-3 PUFA levels in pork and
chicken, simultaneously with enrichment with Se and vitamin E could have a
multiple benefit. As in the case of designer eggs, meat enriched with omega-3
PUFAs needs increased antioxidant protection. This protection could be provided
by increased Se and vitamin E concentrations (See Chapter 7). In fact there are
currently a number of vitamin E-enriched meat products on the market, including
sausages and cooked ham (Jimenez-Colmenero et al., 2001). It is necessary to
underline that Se-enriched eggs, meat and milk could have a positive effect on
gastrointestinal function. Lipid peroxidation in the gut is believed to be one of the
major contributing factors in the development of various gastro-intestinal disorders,
which are associated with enterocyte damage and the development of
malabsorption (Chapter 13).
Let food be your medicine and medicine be your food (Hippocrates).
Conclusions: back to nature?

Analysis of literature presented above clearly indicate that new trends in the egg
and meat production are associated with quality issues. Indeed, for many years
animal industry was driven mainly by consumer demands on quantity and price.
Quality issues were overshadowed by increasing demands. However, time has
come to reconsider several important issues. It has been suggested that for the
past 150 years our diet has changed substantially, while our genes have not been
changed. In particular, animal-derived food composition has been dramatically
changed as a result of using cheap feed ingredients. The meat from animals in the
wild, chicken eggs produced under complete natural conditions contain higher
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amounts of omega-3 fatty acids compared to cultivated ones (Simopoulos, 2002).

Indeed, analyses of egg composition from various avian species in wild conducted
at the SAC (Speake et al., 1998; Surai, 2002) indicate a great difference in fatty
acid profile of eggs between commercial laying hens and wild birds. In fact,
designer eggs enriched with omega-3 fatty acids are very similar in fatty acid
profile to eggs produced in wild or so called Greek eggs, produced by free range
birds having unlimited access to various greens, worms, insects etc. The most
interesting development in this area is the Columbus Concept. It stands for a
return of alpha-linolenic acid into the ration of land-based bred animals to such
an extent that fat depots exhibits balanced fatty acid ratios, characteristic for fat
depots in wild animals or game (De Meester and Sim, 2002).
Did you know that enrichment of eggs, meat and milk by Se
due to inclusion of organic selenium in the diet is a way of
returning back to nature?
The same is true for selenium. Indeed, decreased Se levels in feeds and foods in
many cases reflect consequences of our agricultural practises. For example, usage
of inorganic fertilizers rich in sulphur interferes with Se assimilation from soil as
well as soil acidification decreases Se availability. Therefore, eggs or meat
produced by free-range poultry/animals fed on natural feed sources grown on
well-balanced soils 100-200 years ago would contain much higher Se concentration
than we currently have in many European and Asian countries. Again, by
supplementing animal diet with natural organic sources of Se we are returning
back to nature. Therefore Se-enrichment of eggs, meat and milk is nothing else
but production of naturally-designed food ingredients. Indeed, production and
commercialisation of such organic Se sources as selenized yeast (for example
Sel-PlexTM) opened a new era in Se supplementation of animals and gave a real
chance for producers to meet growing requirements of consumers. What is more,
production of these kind of animal-derived foodstuffs is a natural way to health
promotion.
Indeed, it is possible to provide consumers with a range of animal-derived
products with nutritionally modified composition in such a way that they can
deliver substantial amount of health-promoting nutrients to improve general diet
and help to maintain good health. Therefore, without changing habits and traditions
of various populations (habit is a second nature) it is possible to solve problems
related to deficiency of various nutrients, in particular selenium. The consumer
will go to the same supermarket to buy the same animal-derived products (egg,
milk and meat), cook and consume them as usual. The only difference will be in
the amount of specific nutrients delivered with such products.
Diets cures more than lancet
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Antioxidant-Prooxidant Balance in the Intestine
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13
ANTIOXIDANT-PROOXIDANT BALANCE IN THE INTESTINE: FROM
HEALTHY GUT TO GENERAL HEALTH
A good beginning makes a good ending
Introduction
A delicate balance between antioxidants and pro-oxidants in cells is an important
determinant of various physiological processes and maintenance of this balance
is the main aim of so called an integrated antioxidant system built in the human
body. This system was developed during evolution to provide an antioxidant
defence and give a chance for animals to survive in oxygenated atmosphere.
Recent data suggest that the antioxidant-pro-oxidant balance starts in the intestine.
Relationship between food and human health has attracted attention of scientists
for many years. It is now widely accepted that fruits and vegetables are important
dietary components responsible for maintenance of good health. However,
molecular mechanisms of protective effects of fruits and vegetables have not
been fully elucidated. One of the attractive ideas is that various antioxidant
compounds of fruits and vegetables are responsible for prevention of oxidative
damage in digestive tract. In fact, antioxidant-prooxidant balance in digestive
tract is considered to be a major determinant of human and animal health (Surai et
al., 2003; 2004).
The gastrointestinal tract (GIT) as a major site of antioxidant action

As mentioned above some antioxidants, such as antioxidant enzymes and
glutathione can be synthesised in the body, but the diet is the major provider of
nutrients possessing antioxidant properties directly (vitamin E, vitamin A,
carotenoids, ascorbic acid, flavonoids etc.) (Table 13.1) or essentials for the
synthesis of antioxidant enzymes: Se is an essential part of a range of
selenoproteins performing antioxidant functions (GSH-Px, thioredoxin reductase,
selenoproteins P and W, etc.); Mn is an integral part of mitochondrial Mn-SOD;
Zn and Cu are integral parts of cytosolic Cu,Zn-SOD; Fe is an integral part of
catalase.
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Table 13.1 Natural food sources of some antioxidants (Adapted from Shahidi, 1997).
Compounds
Source
Vitamin E (tocopherols and

tocotrienols
Oilseeds, vegetable oils, nuts, whole grains, cereals,

margarine etc.
Vitamin C
Fruits and vegetables, berries, citrus fruits, green papers
Carotenoids
Dark leafy vegetables, carrots, sweet potatoes, tomatoes,

apricots, citrus fruits, kale etc.
Flavonoids/isoflavonoids
Fruits and vegetables, oilseeds, berries, peppers, citrus fruits,

tomatoes, onions etc.
Phenolic acids/derivatives
Oilseeds, cereals, grains, etc.
Catechins
Green tea, berries, certain oilseeds, etc.
Extracts/essential oils
Green tea, rosemary, sage, clove, oregano, thyme, oat, rice

bran etc.
When food is consumed and appears in the stomach and then in the small intestine,
it contains a range of antioxidants but may also contain a range of potentially
dangerous substances (Figure 13.1). To keep a balance between antioxidants and
prooxidants in the intestine is a very important task in which diet plays a crucial
role. In general, prooxidants which can be found in the digestive tract could be
summarised as follows (Surai et al., 2003).
tocopherols and tocotrienols
coenzyme Q
carotenoids
flavonoids
ascorbic acid
spices, essential oils
vitamin A
selenomethionine
BOA, BOT
Antioxidant system
maintained,
good health
oxidized PUFAs
oxysterols
persistent organic pollutants
Fe2+ and Cu+
nitrites
heavy metals
mycotoxins
alcohol
Antioxidant system
compromised, poor
health
Figure 13.1 Antioxidant-prooxidant balance in the intestine (adapted from Surai et al., 2003).
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Prooxidants in the GIT

PEROXIDIZED PUFAS
It is generally accepted that in modern food technology low-level oxidation of
lipids in meat, poultry and milk during storage and processing is practically
unavoidable (Cohn, 2002). In fact, lipid stability in meat and meat products depends
on many factors. They include species, muscle type, the amount and type of fat in
the diet, the nutritional status of the animal at slaughter, the presence of absence
of disease or infection and the type of processing to which the meat is subjected
(Morrissey et al., 1998). Most fried meat products contain lipid peroxides in various
concentrations and if cooked meat is stored, the level of peroxidized PUFAs
increases dramatically. For example, in heated turkey red muscle (a fast food) the
level of lipid peroxides was found to be about 100-fold higher than in fresh turkey
red meat (Kanner and Lapidot, 2001) and this comprised only part of total lipid
peroxides consumed every day. Fish and fish oil are especially susceptible to
peroxidation due to presence of highly unsaturated docosahexaenoic (DHA, 22:6n3), docosapentaenoic (DPA, 22:5n-3) and eicosapentaenoic (EPA, 20:5n-3) fatty
acids.
Lipid hydroperoxides (LOOH) derived from unsaturated fatty acids are
important intermediates of peroxidative reactions induced by ROS. As mentioned
in the chapter 1, lipid hydroperoxides are not stable and in the presence of transition
metal ions can decompose producing new free radicals and cytotoxic aldehydes
(Diplock, 1994). Lipid oxidation yields a very complex group of by-products
that include hydroxy and dihydroxy fatty acids, hydroperoxides, volatile
aldehydes, and alkyl and olefinic radicals that contribute to the flavor deterioration
of foods and that are implicated in biological oxidation and can cause oxidative
stress (Frankel, 1987). Oxidised lipids are partly absorbed in the digestive tract
(Staprans et al., 1999) and incorporated into membrane phospholipids altering
their structure and properties (Hayam et al., 1994; Staprans et al., 1994). For
example, chylomicrons isolated from subjects consuming oxidized fat are more
susceptible to lipid peroxidation ex vivo, suggesting that at least some oxidised
fatty acids are absorbed (Vine et al., 1998). In animal models it has been shown
that oxidised lipids in the diet can suppress growth (Lin et al., 1989; Calabotta
and Shermer, 1985), reduce vitamin E level in tissues increasing their susceptibility
to lipid peroxidation (Sheehy et al., 1994), increase tissue protein oxidation (Hayam
et al., 1997) and increase the number of aberrant crypts in the intestine (Yang et
al., 1998). In particular, in rats consuming thermally oxidized corn oil, increased
concentrations of lipid peroxides were observed in the liver and kidney, in
association with a decreased growth rate, food and protein efficiency ratio
(Nwanguma et al, 1999; Lopez-Varela et al., 1995). The gastrointestinal epithelium
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of swine and chickens responded to oxidant stress imposed by oxidized fat by

increased enterocyte turnover and the gut associated immune system was
compromised (Dibner et al., 1996). The consumption of oxidized fats is associated
with diarrhoea, liver enlargement, growth depression and histological changes in
tissues of experimental animals (Andia and Street, 1975; Cutler and Schneider,
1973; Koshio et al., 1994; Shibata et al., 1992). Heated oils also showed potent
teratogenic actions in experimental animals (Indart et al., 2002).
One of the biggest contributors to the consumption of lipid peroxides are fast
foods, since the typical American consumes approximately three hamburgers and
four servings of fries per week (Saguy and Dana, 2003). They could provide a
significant amount of potentially hazardous peroxides as well as trans fatty acids.
The percentage of fat from fast foods and ethnic foods increased from 1% in
1965 to 11% in 1996 (Dobarganes and Marquez-Ruiz, 2003). In particular oils,
which are used for deep-fat frying (e.g. chips and French fries preparation) are
heated to very high temperature and decomposition products are formed (Chang
et al., 1978). In fact most of the oxidised lipids in foods come from fats and oils
heated at high temperature in particular from frying fats (Dobarganes and MarquezRuiz, 2003). During frying, the oil undergoes three deleterious reactions: hydrolysis
caused by water, oxidation, and thermal alteration caused by oxygen and heat
(Saguy and Dana, 2003). These reactions cause the formation of polymerisation
products, of which over 400 have been identified (Paul and Mittal, 1997).
Furthermore, decomposition products, which are formed as a result of reactions
between food ingredients and oil comprise another large group of potentially
toxic compounds (Takeoka et al., 1996). It is generally accepted that oxygen
plays a major role in the deterioration of the oil during frying and selective
absorption may occur, enriching the food product with breakdown oil compounds
(Saguy and Dana, 2003). Products of lipid oxidation formed in the food depend
on the temperature. For example, at low or moderate temperature hydroperoxides
are the major products formed while in high temperature treated products secondary
oxidised triacylglycerol monomers and polymers are more common compounds
(Dobarganes and Marquez-Ruiz, 2003). Therefore food frying in fast food
restaurants may be problematic due to lengthy oil exposure to extreme conditions
and the lack of adequate oil replenishment and discarding. In particular a significant
number of oils and fats from fast-food outlets contain more than 25% newly formed
compounds (Dobarganes and Marquez-Ruiz, 2003).
Lipid peroxidation is associated with the formation of a wide range of secondary
aldehyde products such as n-alkanals, trans-2-alkenals, 4-hydroxy-trans-2-alkenals
and MDA (Lynch et al., 2001). While linoleic, gamma-linolenic and arachidonic
acids found in different foods were precursors of hexanal, propanal was the
dominant aldehyde formed from the breakdown of alpha-linolenic,
eicosapentaenoic and docosahexaenoic acids (Shahidi, 2001). For example,
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propional, pentenal, hexanal and 4-hydroxynonenal were the primary aldehydes

formed during lipid oxidation in beef (Lynch et al., 2001). Those products are
shown to be comparatively stable and can readily diffuse into cells causing
toxicological effects (Szweda et al., 1993). Therefore prolonged frying caused a
substantial rise in MDA concentration (Saguy and Dana, 2003) which is shown to
be toxic and mutagenic. Furthermore, MDA can damage proteins and
phospholipids by covalent bonding and cross-linking (Aubourg, 1993). Rats fed
a diet containing MDA suffered from retarded growth, irregular intestinal activities,
enlarged liver and kidneys, anaemia and low serum and liver vitamin E (Esterbauer,
1993). Similarly, the results of Raza et al. (2002) showed that 4-hydroxynonenal
(HNE), a reactive by-product of lipid peroxidation, caused mitochondrial oxidative
stress leading to a decrease in the GSH pool and increased membrane lipid
peroxidation.
It is necessary to underline that heat treatment of the food can also cause
heterocyclic amine formation. For example, approximately 20 heterocyclic amines
of high mutagenesis were isolated and identified from protein-rich foods (Saguy
and Dana, 2003). Furthermore, mutagenic activity was found in beef cooked in
regular domestic conditions (Felton et al., 1997). It has been shown that
heterocyclic amines can cause oxidative stress leading to DNA adduct formation
and oxidative DNA damage (Murata and Kawanishi, 2002). Therefore heterocyclic
amines formed during the cooking of meat and fish and possessing mutagenic,
genotoxic and carcinogenic properties can be detected in burgers, steaks, pork
ribs (Knize et al., 1998) and Italian sausages (Abdulkarim and Smith, 1998).
Acrylamide in food products-chiefly in commercially available potato chips,
potato fries, cereals, and bread was determined by liquid chromatography-tandem
mass spectrometry in thirty food samples (Becalski et al., 2003). Concentrations
of acrylamide varied from 14 ng/g (bread) to 3700 ng/g (potato chips) and the
WHO estimates the average consumer ingests about 0.8 g/kg body weight daily
(Mitka, 2002). It is necessary to take into account that acrylamide caused an
increase in lipid peroxidation and decrease in glutathione contents and activity of
glutathione-S-transferase in the rat liver in a dose dependent manner (Srivastava
et al., 1983).
From data presented above it is clear that lipid peroxidation in the food is an
important source of toxic products and a potential source of free radicals in the
human digestive tract.
OXYSTEROLS
Cholesterol oxidation products (oxysterols) are commonly found in foods of animal
origin and they are formed during food processing, storage and cooking. In fact,
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more than 60 cholesterol oxides have been identified (Schroepfer, 2000; Savage
et al., 2002). Recent findings suggest that oxysterols may modulate cytotoxicity
by inducing apoptosis. Furthermore, the generation of an oxidative stress may be
a significant event occurring during 7-beta-oxysterol-induced apoptosis
(OCallaghan et al., 2002) simultaneously with a significant decrease in GSH
levels and an increase in the activity of SOD and caspase-3. When cytotoxicity of
various oxysterols was examined in a human monocytic blood cell line
(OCallaghan et al., 2001), it was shown that they are cytotoxic and the mode of
cell death was by apoptosis. However, mode of cytotoxic action of various
oxysterols varied substantially, since some oxysterols induced apoptosis without
changes in the GSH concentration or the SOD activity in the U937 cells. It is
interesting that 7-ketocholesterol is considered to induce apoptosis and the
substantial overproduction of superoxide radical was obtained with 7-betahydroxycholesterol and 7-ketocholesterol (Miguet-Alfonsi et al., 2002).
Furthermore, oxidation of triglyceride-rich lipoproteins was significantly greater
in rabbits fed oxidized cholesterol compared to the pure cholesterol-fed animals
(Vine et al., 1998). Oxysterols can also replace cholesterol in membranes,
perturbing permeability, stability and other membrane properties (Guardiola et
al., 1996), which are extremely important in relation to enterocyte membranes in
the intestine.
Sources of cholesterol oxides in the diet are represented mainly by processed
animal-derived products including egg products, dairy products and meat products.
For example, fresh egg yolk contains negligible levels of cholesterol oxide
(Paniangvait et al., 1995), however, spray-dried egg yolk powder is a rich source
of cholesterol oxides. Similarly, fresh meat is almost free of cholesterol oxides
but cooked meat products are considered to be a substantial source of cholesterol
oxides in the diet (Savage et al., 2002). Cholesterol was oxidised in meat samples
during household cooking and the rate of oxidation differed according to the
cooking conditions (Pie et al., 1981). In general, cholesterol oxide consumption
could be substantial in many populations. For example, the average diet in the
Netherlands provides about 1 mg of 7--hydroxycholesterol and 0.5 mg of
cholesterol--epoxide per day (Van de Bovenkamp et al., 1988). It is necessary
to underline that certain oxidation products of cholesterol are carcinogenic
(Pearson et al., 1983).
Therefore, cholesterol oxides can be found in the diet and they are cytotoxic
and potentially can be involved in lipid peroxidation in the intestine.
IRON IONS
Iron is recognised as an essential nutrient and its reference nutrient intakes (RNI)
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in the UK are 8.7 mg/day for men and postmenopausal women and 14.8 mg/day
for premenopausal women with USA RDA being 8 mg/day and 18 mg/day
respectively (Kelly, 2002). However, iron absorption from a diverse diet has been
shown to be about 15% (Department of Health, 1991). The main problem with
iron nutrition is its reactivity and possible involvement in free radical generation.
Iron ions are considered to catalyse the formation of the hydroxyl radical and
accelerate the decomposition of lipid hydroperoxides (Davies and Slater, 1987;
Donovan and Menzel, 1978) and to stimulate lipid peroxidation (Braughler et al.,
1986, Minotti and Aust, 1987). In fact the potential risks of an excess intake of
iron, associated with an increase in free radical generation were first proposed
more than 40 years ago (Richmond, 1959). Numerous compounds, including
heme and nonheme iron, have been reported to act as catalysts of lipid oxidation
in food systems (Pearson et al., 1983). In fact muscle tissue is rich source of iron
bound to proteins in the form of myoglobin, Furthermore, meat can also contain
some hemoglobin residues from residual blood and iron-containing cytochromes.
Post-slaughter events such as early post-mortem pH drop, carcass temperature
and tenderising techniques such as electrical stimulation are involved in disruption
of cellular compartmentalisation and release of catalytic metal ions (Morrissey et
al., 1998). The next step of lipid peroxidation in the meat is associated with meat
handling, processing, storage and cooking. It is widely believed that during heat
treatment of the meat products nonheme iron is released from high molecular
weight sources such as haemoglobin, myoglobin, ferritin and haemosiderin
becoming the major pro-oxidant in cooked meat (Pearson et al., 1983; Morrissey
et al., 1998).
Iron fortification of various foods could represent another source of potentially
dangerous free iron in the digestive tract. Fortification of cereal products with Fe
has been in practice for a number of years throughout the developing world. In
fact thirty countries throughout the developing world now have a range of products
fortified with iron including wheat flour, maize flour, milk, rice and weaning foods
(Darnton-Hill and Nalubola, 2002). Ferrous sulfate was considered to be the first
choice for food fortification and used to fortify infant formula, bread and pasta
and wheat flour (Hurrell, 2002). At the same time this form of iron is the most
active one in promoting lipid peroxidation. Therefore, iron supplements could
also be involved in lipid peroxidation in the intestine. In fact, iron supplements
are commonly prescribed for pregnant women at doses ranging from 30 mg/d in
the United States to as high as 240 mg/d where prevalence of anemia is high
(Knutson et al., 2000). However, undesirable gastrointestinal side effects of iron
supplementation are high, suggesting iron-related oxidative stress in the intestine
(Hollan and Johansen 1993). For example, iron supplementation in relatively high
doses resulted in abnormal iron accumulation and increased lipid peroxidation in
rats (Knutson et al., 2000). Furthermore faecal iron available to the Fenton reaction
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was increased in a dose-dependent manner (Carrier et al., 2001). Chronic feeding

of iron to rats was associated with an increase in free radical generating capacity
in the colon and increased lipid peroxidation, particularly in the cecum (Lund et
al., 2001).
Iron supplementation amplified the inflammatory response and enhance the
subsequent mucosal damage in a rat model of colitis (Reifen et al., 2000). In the
conditions of dietary iron overload in rats, in vivo hydroxyl radical generation
(ultimately responsible for iron-induced injury) was demonstrated (Kadiiska et
al., 1995). It is necessary to mention that other mineral supplements can also be
involved in stimulation of lipid peroxidation in the intestine. For example, recently
it has been appreciated that selenite can generate free radicals when present in
combination with reduced glutathione (for review see Surai, 2002). Iron can also
interact with other nutrients stimulating free radical production. For example, it
has been shown that iron in combination with the secondary bile acids, lithocholic
and deoxycholic acids and the vitamin K group can generate free radicals
(Blakeborough et al., 1989).
Clearly meats and meat-containing products are an important source of free
iron which can be involved in generation of free radicals and lipid peroxidation in
the digestive tract. Furthermore food iron fortification and iron supplements
represent another possible source of catalytic iron in the GIT.
NITRITES AND NITRATES

Nitrite is consumed in the diet, through vegetables and drinking water and it is
also added to meat products as a preservative (Cammack et al., 1999). In fact,
nitrates and nitrites, are common antimicrobial agents used in the food industry
and have been used for centuries as preservatives in a number of red meat, poultry,
and fish products. Nitrite contributes to the flavour, reacts with myoglobin producing
the characteristic pink colour of cured meat and inhibits the growth of food spoilage
bacteria (Cammack et al., 1999). The currently allowable daily intakes for nitrites
and nitrates are 8 and 220 mg/person per day, respectively (Joint FAO/WHO Expert
Committee on Food Additives, 1974). In addition to added nitrate and nitrite to
the food as preservatives, vegetables (e.g. lettuce, potato, etc.) comprise a major
source of nitrates (Cammack et al., 1999). Therefore, humans are subjected to
significant nitrate and nitrite levels in foods and water, as well as those formed in
vivo.
Nitrites and nitrates formed from nitrogenous sources by microorganisms in
saliva and intestine, are considered to be the major source of human exposure
under physiological conditions (Chow and Hong, 2002). It is generally accepted
that nitrate is concentrated in the saliva and rapidly converted to nitrite by facultative
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anaerobic bacteria. Benjamin et al. (1994) showed that nitrite is converted to NO

under the highly acidic conditions (pH 3) which occur in the lumen of the stomach.
It was observed that the generation and accumulation of NO from typical nitrite
concentrations found in biological tissues increases 100-fold when the pH falls
from 7.4 to 5.5 (Zweier et al., 1999). Therefore nitrate and nitrite can generate
NO* radical by either direct disproportionation or reduction under the acidic and
highly reduced conditions (Chow and Hong, 2002). More importantly, NO* can
be a source of another reactive free radical peroxynitrite (ONOO-), which is 1000
times more oxidizing than H2O2 and has half-life in solution about 12 seconds
(Van Dyke, 1997). It is well accepted that peroxynitrite is a potent cytotoxic agent
(Pryor and Squadrito, 1995; Squadrito and Pryor, 1998). Peroxynitrite can also
affect antioxidant system by changing activities of antioxidant enzymes. In fact
peroxynitrite has been shown to inhibit the activities of catalase (Keng et al.,
2000), GSH-Px (Padmaja et al., 1998) and promote lipid peroxidation (Shi et al.,
1999). Moreover, it appears that ONOO can oxidize a variety of essential
molecules (e.g., sulfhydryls, thiols, ascorbate, proteins, DNA) and trigger injurious
processes, including lipid peroxidation (Patel et al., 1999, Hogg and Kalynaraman,
1999). On the other hand, peroxynitrite may be decomposed forming highly
reactive hydroxyl radical (*OH) and nitrogen dioxide (Beckman et al., 1990;
Augusto et al., 1994).
The amount of nitrates and nitrites in the diet vary substantially. However, in
combination with other prooxidants in the digesta they can be involved in free
radical formation and lipid peroxidation.
HEAVY METALS
Agricultural uses of phosphate fertilizers and sewage sludge and industrial uses
of cadmium have been identified as a major cause of widespread dispersion of
the metal at trace levels into human foodstuffs. Approximately, 1/3 of cadmium
dietary intake is attributed to the ingestion of animal products, while plant products
provide the remaining 2/3 (Nasreddine and Parent-Massin, 2002). Recently it has
been calculated that daily cadmium intake to be 30 g being within the safe intake
level of 70 g per day recommended by WHO/FAO (Satarug et al., 2003). The
dietary intakes of lead for European countries vary between 16 g/day (Spain)
and 280 g/day (Italy) and intakes of arsenic and mercury comprise 38-286 g/
day and 0.7-13.5 g/day respectively (Nasreddine and Parent-Massin, 2002).
Mercury is a common contaminant of fish. For example, fish with a total mercury
content between 0.5 and 1.5 ppm include fresh and frozen tuna, swordfish and
shark. Mercury levels in freshwater fish varies, but in general bass, pike,
muskellunge and walleye have high levels (Wooltorton, 2002).
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It is well known that heavy metals can cause oxidative stress and stimulate
lipid peroxidation. For example, cadmium increased lipid peroxidation in liver,
kidney, and testes of rats and reduced metallothionein and total sulphydryl in
liver and kidney (Khandelwal et al., 2002). In renal tubular epithelial cells of rats
significant decrease in activities of GSH, GSH-Px, SOD and increased MDA
formation were observed as a result of the treatment with lead and cadmium (Wang
et al., 2002). In fact prooxidant effect of cadmium is much more complex.
Cadmium depletes GSH and protein-bound sulfhydryl groups, resulting in
enhanced production of reactive oxygen species such as superoxide, hydroxyl
radicals, and hydrogen peroxide (Stohs et al., 2001). The ROS production is
associated with lipid peroxidation, enhanced excretion of urinary lipid metabolites,
modulation of intracellular oxidized states, DNA damage, membrane damage,
altered gene expression, and apoptosis (Stohs et al., 2001). On the other hand,
reduced glutathione (GSH) and -tocopherol offer significant protection against
cadmium toxicity in rats by diminishing oxidative stress via raising GSH
concentration and reducing lipid peroxidation (Singh and Rana, 2002).
It is believed that lead can alter certain membrane bound enzymes and may
cause oxidative stress. For example, exposure of HepG2 cells to lead ions decreased
cell viability and stimulated lipid peroxidation of cell membranes decreasing the
fluidity in the polar surface of cell membranes (Chen et al., 2002). Levels of lipid
peroxidation products such as MDA, conjugated diene and hydroperoxide were
increased in liver, lung and kidney of lead-treated rats. Consumption of lead in
drinking water by rats imposed oxidative stress increasing lipid peroxidation in
peripheral blood mononuclear cells and liver (Ercal et al., 2000). Administration
of exogenous antioxidants in the lead treated animals significantly reduced the
prooxidant effect of the toxicant (Upasani et al., 2001). Mercury is also a strong
prooxidant able to increase lipid peroxidation and decrease GSH content in liver
of Swiss albino mice. In particular, mercury treatment enhanced lipid peroxidation
in kidney, testis and epididymus of rats (Mahboob et al., 2001). Mercury can
interact with other potential prooxidants. For example, mercury or selenite when
injected alone, did not alter hepatic or renal lipid peroxidation in mouse, however,
simultaneous exposure to these compounds significantly increased lipid
peroxidation (Farina et al., 2003).
Did you know that our food could contain a range of prooxidants in various concentrations, including oxidised lipids,
oxysterols, iron, nitrites, nitrates, heavy metals, persistent
organic pollutants and mycotoxins? A combination of those
compounds could promote lipid peroxidation in the gut.
Heavy metal concentrations in major food sources are quite low, however, in
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combination with other prooxidants they potentially can be involved in generation

of free radicals and causing oxidative stress in the GIT.
PERSISTENT ORGANIC POLLUTANTS

Persistent organic pollutants (POPs) comprise a class of chemicals that are among
the most insidiously dangerous compounds and it includes many organochlorine
pesticides. Examples of persistent organic pollutants found in food include dioxins,
polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers, and some
pesticide chemicals (Jensen et al., 2001). Recently, typical daily diets were
constructed for four regions in the US, reflecting foods typically eaten in each
region. The data were then analysed to determine the number and concentrations
of POPs residues found in each food item for each of these daily meal plans.
Analysis of the data showed that POPs residues are present in virtually all categories
of food, including baked goods, fruit, vegetables, meat, poultry and dairy products
(Schafer and Kegley, 2002). For example DDT and its metabolites were found in
21% of samples tested in 1998 and 22% in 1999 and dieldrin was found in 10%
of samples tested in 1998 and 12% in 1999. Therefore a typical daily diet in the
western US could potentially deliver 66 hits (hit is defined as the occurrence of
one POP chemical in a single food item) of POPs exposure (Schafer and Kegley,
2002). Residues of organochlorine pesticides were detected in 21% of total diet
samples in Spain (Lazaro et al., 1996) with eggs, legumes, fish and meat being
major sources of those contaminants. About 60% of the samples of organic
vegetables in the USA are found to contain pesticides and contaminated with
organochlorine insecticide residues (Benbrook, 2002). Market samples (60) of
six seasonal vegetables were monitored during 1996-1997 in India to determine
the magnitude of pesticidal contamination by insecticide residues representing
four major chemical groups i.e. organochlorine, organophosphorous, synthetic
pyrethroid and carbamate. The tested samples showed 100% contamination with
low but measurable amounts of residues (Kumari et al., 2002). In 1998, oranges,
peaches, carrots and spinach were analysed for 20 pesticides in the EU and about
32% of samples contained residues of pesticides at the levels legally permitted in
specific food items and 2% samples exceeded that level (Nasreddine and ParentMassin, 2002). Similarly in 1999 residues of pesticides were found in melons
(32% samples), peppers (24%), wheat grains (21%) and cauliflower (17%;
Nasreddine and Parent-Massin, 2002). On the other hand, organochlorines can
cause oxidative stress. For example, the adverse effect of organochlorine pesticide
methoxychlor on the male reproductive system was shown to be due to induction
of oxidative stress in testis (Latchoumycandane and Mathur, 2002;
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Latchoumycandane et al., 2002). Similarly, a pesticide hexachlorocyclohexane

(HCH) compromised antioxidant defence and induced oxidative stress in rat
cerebral hemisphere (Sahoo et al., 2000).
Dieldrin exposure caused depolarization of mitochondrial membrane potential
in a dose-dependent manner, stimulated generation of ROS and significantly
increased lipid peroxidation (Kitazawa et al., 2001). The effect of the cyclodiene
organochlorine pesticides aldrin, dieldrin and endosulfan was assessed on CHOK1 cultures. Unlike oxidised glutathione, the content of total glutathione declined
significantly after exposure to cyclodiene insecticides. Membrane leakage and
peroxide production were significantly enhanced by the pesticides (Bayoumi et
al., 2001). Recent experimental studies indicate that dieldrin-induced production
of reactive oxygen species, depletion of hepatic antioxidant defences (particularly
-tocopherol), and peroxidation of liver lipids (Stevenson et al., 1999).
Furthermore, dieldrin-induced oxidative stress could be associated with modulation
of gene expression. Oral administration of DDT to rats dose dependently increased
TBARS in serum after 8 wk of treatment. In addition, such DDT exposure markedly
suppressed the humoral immune response as assessed by anti-sheep RBC antibody
titres (Koner et al., 1998). Simultaneous treatment with ascorbic acid (100 mg/
kg) markedly attenuated the effects of DDT on lipid peroxidation and humoral
immune suppression indicating the possible involvement of free radicals in
organochlorine-induced immunotoxicity. Similarly, in chickens hydrogen peroxide
and lipid peroxide formation significantly increased in kidney, lung and intestine
as a result of their treatment with DDT (Hatolkar and Pawar, 1995). The results of
Hassoun et al. (1993) demonstrated that the four structurally dissimilar
polyhalogenated hydrocarbons (lindane, DDT, chlordane and endrin) could
produce oxidative stress in rats with a significant increases in hepatic lipid
peroxidation and DNA damage. In general, in all those studies POPs concentrations
tested were much higher than in contaminated food, however, if they are present
in combination with each other or with other prooxidants their detrimental effects
could be multiplied. Even if the levels of those are in the accepted limits, they are
still able to participate in lipid peroxidation. Therefore persistent organic pollutants
represent an important health hazard for humans and they also can be involved in
promotion of lipid peroxidation in the GIT.
MYCOTOXINS
At least 25% of worlds grain production is contaminated with mycotoxins, which
are a worldwide problem (Fink-Gremmels, 1999). For example, aflatoxins are
considered to be unavoidable contaminants of food, since they cannot be prevented
or eliminated by current agricultural practice (Peraica and Domijan, 2001). The
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intake of aflatoxin M1 from milk was calculated to vary from 0.1 ng/person/day
in Africa up to 12 ng/person/day in Far Eastern countries (Creppy, 2002). Milk
and dairy products purchased at Egyptian markets and breast milk from lactating
mothers were analysed. Three of 15 cows milk samples were found positive for
AFM1 with mean value 6.3 mg/kg. Twenty percent of hard cheese samples
contained detectable levels of AFM1 and one sample from ten contained AFB1
and AFG1. For breast milk, two of ten samples were positive for AFM1 with mean
value 2.75 g/kg, while 3 of 10 samples were positive for OA (Ei-Sayed et al.,
2000). In Turkey, the incidence of AFM1 in cheese was 89.5% with the highest
concentration to be 810 ng/kg (Huseyin and Sonal, 2001). From fifty four samples
of fresh full cream and skimmed milk, powdered milk, yoghurt and infant formula
collected in Kuwait, 28% were contaminated with AFM1 with 6% being above
the maximum permissible limit of 0.2 g/l (Srivastava et al., 2001).
Therefore the chance of getting traces of mycotoxins in our diet are very high.
For example, the results of survey of 313 UK retail foods and 153 UK cereal
samples showed that ochratoxin A was detected in 25% samples with 27 samples
containing OA at concentrations above 4 ng/g (Atkins and Norman, 1998). OA
was found in a number of samples of feed and food from various countries with
its detection in human blood (Peraica and Domijan, 2001). In fact, OA can be
detected at levels greater than 0.1 ppb in more than 90% of human and swine
blood samples in central European countries (Petzinger and Weidenbach, 2002).
In fact, OA has been found in human blood samples in number of countries in
cool temperate areas of the Northern Hemisphere (Creppy, 2002). When blood
analyses were performed in Scandinavian blood donors the mean plasma levels
of OA was 0.18 g/l in Oslo and 0.21 g/l in Visby (Thuvander et al, 2001). In
particular in Croatia OA consumption was estimated to be 0.4 ng/kg body weight
(Peraica and Domijan, 2001). In the UK composite duplicate diet samples from
50 individuals and corresponding plasma and urine samples were obtained over
30 days. Average intake of OA varied in a range of 0.24-3.54 ng/kg body weight/
day and OA was detected in all plasma samples and in 92% of urine samples
(Gilbert et al., 2001). In Germany the absolute OA intake was approximately
28.7-290.8 ng/day in 1996-1999 and the calculated total daily intake of OA varied
between 0.9 ng/kg body weight in Germany and 4.6 ng/kg body weight in Italy
(Petzinger and Weidenbach, 2002). It is necessary to mention that OA altered
both barrier and absorption function of the intestinal epithelium causing intestinal
injuries, including inflammation and diarrhea (Maresca et al., 2001). Ochratoxin
in combination with aflatoxin showed a synergistic toxicity (Campbell et al., 1983).
For ochratoxin the elimination half-life in human was calculated to be 840 hours
(Creppy, 2002).
Consumption of mouldy sorghum or maize containing high levels of fumonisin
B1 caused an outbreak a human disease in India involving gastrointestinal
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symptoms (Creppy, 2002). Estimated intake of fumonisin in Europe, Far East,

Latin America, Middle East and Africa comprises (g/kg body weight) 0.2, 0.7,
1.0, 1.1 and 2.4 respectively (Creppy, 2002). Many outbreaks of acute human
disease involving gastrointestinal upset and diarrhoea have been resisted in Asia
due to consumption of Fusarium-contaminated grains (Creppy, 2002). DON was
detected in various food products (bread, breakfast cereals, beer, baby and infant
foods) in Europe and Northern America (Creppy, 2002). When 88 commercially
available samples of wheat-based breakfast cereals were randomly collected from
different supermarkets in Lisbon, 72.8% samples contained levels of DON between
103 and 6040 g/kg with mean level of 754 g/kg (Martins and Martins, 2001). It
is interesting that inhibition of protein synthesis and induction of apoptosis are
the main mechanisms of DON toxicity in intestinal cells (Maresca et al., 2002).
Patulin, a common contaminant of apples and apple products, is shown to affect
the barrier function of the intestinal epithelium by inducing epithelial cell
degeneration, inflammation, ulceration and hemorrhages (Mahfoud et al., 2002).
In general, main mycotoxin contaminants of the food including AFB1, fumonisins,
T-2 toxin, DON, zearalenone and OA are shown to compromise antioxidant system
and stimulate lipid peroxidation in vivo and in vitro (Surai, 2002).
Mycotoxins are considered to be unavoidable contaminants of the most food
and feed ingredients and they are potent prooxidants. Therefore even in
comparatively low concentrations (lower than officially allowed limits) they still
represent an important source of free radical generation in the GIT.
ALCOHOL
Exposure of the intestinal mucosa to ethanol leads to morphological injuries and
impairs the absorptive capacity creating an oxidative stress in the small intestine
(Kaur et al., 1998). The diet seems to affect this process since enrichment of the
rat diet with fish oil increased the detrimental effect of alcohol. The in vivo formation
of the -hydroxyethyl free radical metabolite of ethanol has been demonstrated
(Knecht et al., 1990). Free radical formation in ethanol-treated rats has been
detected (Knecht et al., 1995). In the study of Reinke et al. (1997) ascorbyl radicals
and spin adducts of dietary alcohol or endogenous compounds, such as lipids,
were detected with higher frequency in bile from alcohol-fed rats than in
corresponding samples from rats fed control diets. Furthermore, formation of lipid
radicals was enhanced after acute alcohol administration. Liver microsomes from
alcohol-fed rats also metabolized ethanol to the 1-hydroxyethyl radical at higher
rates than controls. During ethanol metabolism the antioxidant system is
compromised, with reduced levels of vitamin E (Kawase et all, 1989), selenium
(Aaseth et al., 1980; Dworkin et al., 1985) and GSH (Trimble et al., 1993).
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Therefore it has been suggested that the underlying mechanism of intestinal

epithelial barrier dysfunction induced by alcohol is due to oxidative injury to the
cytoskeleton (Banan et al., 2000). This hypothesis includes following points:
Ethanol causes iNOS up-regulation.

Activation of the iNOS enzyme leads to intracellular increases in NO and
ONOO that causes oxidative stress oxidising cellular proteins such as tubulin.
Tubulin oxidation causes disruption in the microtubule cytoskeleton, leading
to a loss in integrity of the intestinal barrier, and predispose the organism to
inflammation.
Therefore, alcohol can be involved directly or indirectly in the free radical

production and lipid peroxidation in the intestine. Furthermore its effects on
intestinal immune system and on inflammation in particular need further
investigation. It seems likely that alcohol in combination with other prooxidants
can be a source of free radicals in the GIT.
IMMUNE SYSTEM
The immune system is considered to be an important source of ROS in the human
body (Surai, 2002) and intestinal immunity is not an exception. Indeed, the
intestinal tract is considered to represent the largest immune organ of the human
body responding to the challenge of bacteria or food antigens by production of
ROS (Halliwell et al., 2000). Mucosal surfaces covered by a layer of epithelial
cells represent the most critical interface between the organism and its environment
since the mucosal interstitia of the intestine is continuously exposed to large
amounts of dietary and microbial antigens. Therefore epithelial cells engage in
cross talk with luminal bacteria and their products and produce mediators and
signals that are key components of host innate and acquired mucosal immunity
(Maaser and Kagnoff, 2002). The mucosal immune system is a first line of defence
against foreign antigens, including microbial and dietary antigens and under
normal circumstances it employs tightly regulated dynamic mucosal intra- and
internets consisting of inductive (e.g. Peyers patch) and effector (e.g. intestinal
lamina propria) tissues and maintains an appropriate immunological homeostasis
between the host and mucosal environments (Kiyono et al., 2001). Therefore the
mucosal immune system has evolved efficient mechanisms to distinguish potentially
pathogenic from nonpathological antigens. For example, the mucosal immune
compartment must be able to choose the appropriate effector function (e.g.,
tolerance vs. clearance) necessary to deal with each encountered antigen whether
it be innocuous or pathogenic in nature (Laroux et al., 2001).
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However, abrogation of these mucosal defence mechanisms may alter

immunological homeostasis in the gastrointestinal tract and induce pathological
changes including chronic active inflammation, mucosal atrophy and tissue
injuries (Nagura et al., 2001). It is important to stress that under inflammatory
conditions in the intestine the maintenance of the epithelial barrier could be broken.
Diet is a potent mechanism for altering the environment of cells of most organs,
particularly the gastrointestinal tract. Nutrient metabolism provides an essential
stimulus for the induction, differentiation, and maintenance of the mucosal immune
system (Cunningham-Rundles, 2001). Therefore changes in diet, through the
composition of the lumen environment, alter the expression of genes encoding
for proteins that signal to the mucosal immune system (Sanderson and Naik, 2000).
In fact enterocytes act as immune cells having receptors for bacterial products
and expressing a variety of molecules on their surface responsible for interaction
with immunocytes within the intestine (Sanderson, 2001). Macrophage
inflammatory protein 2 (MIP-2) is a chemokine that attracts neutrophils, and its
secretion from intestinal epithelial cells is enhanced by inflammatory stimuli such
as IL-1 and the production of MIP-2 by epithelial cells is responsible for increase
leukocyte migration into the intestine (Ohtsuka et al., 2001). In particular, increased
neutrophil migration into the intestine and activation of myeloperoxidase were
shown to be a result of induced inflammatory bowel disease-like colitis in transgenic
mice (Ohtsuka and Sanderson, 2003). Inflammatory responses involve the
recruitment of specific leukocyte subsets. In this process nonpathogenic, resident
bacteria have a specific role in the pathogenesis of inflammation in the small
intestine releasing butyrate, a normal bacterial metabolite, and modulating
chemokine secretion by epithelial cells (Ohno et al., 1997). Therefore, epithelial
cells, apart from their participation in absorptive, digestive and secretory processes
perform more than a passive barrier function and are directly involved in immune
processes (Tlaskalova-Hogenova et al., 1995). Moreover, in specific pathological
conditions enterocytes could become a target of mucosal immune factors. For
example, the intestinal immune response to enteric antigens includes production
of T helper-1-type cytokines which may affect the gut epithelium directly and/or
activate a resident macrophage to release large amounts of proinflammatory
mediators: cytokines as well as reactive oxygen species (ROS) and nitric oxide
(NO). The net result is the recruitment of additional leukocytes and subsequent
tissue injury (Laroux et al., 2001).
Macrophage migration inhibitory factor (MIF) inhibits macrophage migration
and has pleiotropic activities on immune and inflammatory responses, cell growth,
and glucose metabolism. MIF is produced by T cells, macrophages, and endothelial
cells. It has been shown recently that human intestinal epithelial cells are a major
source of MIF (Maaser et al., 2002). It has been also suggested that paracrine
factors of intestinal epithelium increased the phagocytic capacity of intestinal
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monocytes/macrophages to be ready for immune and inflammatory responses

(Kanzato et al., 2001). A characteristic feature of inflammation is the concomitant
peroxidation of lipids and formation of bioactive lipid peroxidation products, some
of which are considered to be potent chemoattractants, pro-inflammatory
eicosanoids and other signalling molecules. Recent studies demonstrated a principal
role for myeloperoxidase (MPO), a heme protein secreted by activated neutrophils,
monocytes, and some macrophages, in promotion of oxidant stress at sites of
inflammation. It has been shown that this enzyme serves as a major enzymatic
catalyst of lipid peroxidation by formation of NO-derived oxidants (Zhang et al.,
2002). Furthermore, in response to various stimuli intestinal epithelial cells can
produce nitric oxide (Witthoft et al., 1998) and gastric surface mucous cells
possessed a phagocyte NADPH oxidase-like system and secreted abundant
superoxide anion (Rokutan, 1999).
It is necessary to stress that the nutrient requirement to maintain a highly active
immune system in the digestive tract could be quite high. In fact different sources
of injury to the intestinal mucosa (nutritional, infectious or allergic) act via a
common mechanism of cell-mediated immune damage and nutrient repletion is
required for restoration of immune function (Cunningham-Rundles, 2001). In
particular, the immunomodulating properties of natural antioxidants (Surai, 2002)
could be of great advantage for the intestinal immunity. Furthermore, intestinal
epithelium can modulate the level of immune activity in the mucosal immune
system according to the environment of the intestinal lumen (Sanderson, 1999).
Data presented above indicate that intestinal immune system can generate free
radicals in response to various antigens including microbes and some food
allergens.
Did you know that immune system of the gut could also be a
source of free radical production in the intestine?
COMBINATIONS OF PROOXIDANTS IN THE GIT AND THEIR
DETRIMENTAL EFFECTS
Physicochemical environment of the gastrointestinal tract depends on many factors
with diet, bacterial metabolites and body secretion being major determinants
(Sanderson, 1999). There is a delicate balance between the environment of the
lumen and epithelial cell functionality and dietary factors are responsible for gene
expression in the intestine and its adaptation. In this regard, oxidative stress could
cause changes in this balance affecting absorption of nutrients. Even if each of
those lipid peroxidation promoters is present at a very low concentration, their
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combination could be much more powerful. For example, as mentioned above

lipid hydroperoxides can produce peroxyl radicals in presence of iron or copper
ions and once chain reaction of lipid peroxidation started many other food-derived
PUFAs can be oxidized. It was calculated that pH and temperature, as well as
presence of oxygen in stomach could be favourable for lipid peroxidation (Kanner
and Lapidot, 2001).
Data provided above indicate that average diet contains a range of various
prooxidants. They include oxidized polyunsaturated fatty acids, oxysterols, nitrites,
nitrates, heavy metals, mycotoxins, persistent organic pollutants, alcohol, etc. In
many cases those contaminants are found in the food in low or very low
concentrations, however, their various combinations in the food could be an
important source of free radical production in the gut.
Antioxidant defences in the GIT

An effective antioxidant protection in the gastrointestinal tract is needed to maintain
gut health and this protection is based on food derived antioxidants (Surai et al.,
2004).
VITAMIN E
Vitamin E is main biological chain-breaking antioxidant, found in food in the
form of 4 tocopherols and 4 tocotrienols. Biological activity of vitamin E in tissues
is mainly due to -tocopherol, but in food the main form of vitamin E is tocopherol. It is possible that the gut is a special place for -tocopherol and
tocotrienols to play their antioxidant role. Vitamin E is not stable and is easily
oxidized during food processing. Synthetic antioxidants added to the food can
inhibit vitamin E oxidation. Vitamin E (-tocopherol) in the tablet or capsular
form is mainly produced in the stable esterified form or as a mixture of tocopherols.
The major vitamin E sources in the diet are vegetable oils (wheat, soybean,
sunflower and corn) and some other plant-derived foods. For example, in middleaged Japanese, vitamin E was mainly of vegetable origin with main contributions
coming from spinach, safflower oil and pumpkin (Imaeda et al., 1999). In the
UK, the average daily vitamin E intake is 11.7 mg in men and 8.6 mg in women.
Similar consumption was reported in the USA and other countries (Weber et al,
1997) with margarines and mayonnaise supplying 23% of total vitamin E
consumed. These levels are in the line with the RDA. In fact the Food and Nutrition
Board of the Institute of Medicine recently published dietary reference intakes for
vitamin E, which is 15 mg for adults being 50% greater than the generous
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allowance in the 10th edition of Recommended Dietary Allowances published in

1989 (Horwitt, 2001). It has been concluded that, according to the RDA, the
intake of antioxidants is adequate in healthy subjects (Diplock et al, 1998).
However, the recent data of Bodner et al. (1998) indicate that vitamin E intake in
Scotland is 6.6 mg/day for women and 7.3 mg/day for men, comprising only
50% of the RDA and being lower than that in other European countries. In addition,
there are several categories of people whose vitamin E consumption is lower than
the RDA. For example it is generally believed that elderly people have inadequate
vitamin E consumption (Cid-Ruzafa et al, 1999). Rudman et al (1995) showed
that more than 60% of institutionalised elderly people consume less than 50% of
the ideal dietary intake of vitamin E.
Vitamin E deficiency is associated with a development of a range of specific
diseases involving major tissues of the organism including immune system
incompetence, impairment of lipid metabolism, fertility problems and increased
susceptibility to common and specific diseases (Machlin, 1991). There are also
several clinical conditions where vitamin E deficiency states are described: vitamin
E malabsorption syndrome, abetalipoproteinemia, chronic childhood cholestasis,
cystic fibrosis, total parenteral nutrition and prematurity (VERIS, 1998). Vitamin
E can not be synthesised in the human and its adequate intake relies upon adherence
to a well balanced diet (Thurman and Mooradian, 1997). It has been suggested
that by enhancing the intake of vitamin E by fortification of foods or by dietary
supplements it may be possible to reduce the risk of many common, yet disabling
human diseases (Diplock, 1997). Furthermore, there are many studies suggesting
that intake of vitamin E in amounts much higher than RDA are associated with
reduced risk of various diseases (Bendich et al, 1997; Weber et al., 1997; Diplock
et al, 1998; Chan, 1998) and with enhancement of certain immune responses
(Meydani, 1995). During the past decade, the health benefits of vitamin E have
been shown in several epidemiological studies (Meydani, 2000). For example,
epidemiological evidence shows a lower incidence of infectious disease in subjects
with high plasma tocopherol concentrations (Ayres and Mihan, 1978; Nockels,
1979; Chevance et al., 1985). In this respect, Lachance (1996) has shown that the
optimal daily antioxidant intakes are 23 mg for vitamin E and 3.2 mg for carotene.
It is interesting that about one half of American cardiologists take supplemental
vitamin E (Pryor, 2000) even so results of clinical trials with vitamin E
supplementation were not as successful as expected.
Vitamin E is considered to be not toxic for humans and a daily dosage of 100300 mg vitamin E can be considered harmless from a toxicological perspective
and therapeutic vitamin E doses start at several hundred mg/day and end at
approximately 1,600 mg/day (Kappus and Diplock, 1991). Clearly, vitamin E
can be considered as a main contributor to the antioxidant potential of the digesta.
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COENZYME Q
Coenzyme Q, known also as ubiquinone, was discovered in 1957. The name
ubiquinone is related to its ubiquitous presence in all cells and the name coenzyme
Q reflects the chemical structure of the compound containing one quinone group
and 10 isoprenyl units. These two names have been used interchangeably for
more than 40 years. Coenzyme Q10 (CoQ10) exists both in an oxidised and a reduced
form, ubiquinone and ubiquinol, respectively (Overvad et al., 1999). It was found
that the ratio of ubiquinol-10 to ubiquinone-10 was about 95/5 in human plasma
from healthy donors. A significant increase in the oxidized form (ubiquinone-10)
content was observed in plasmas of patients with hepatitis, cirrhosis, and hepatoma
when compared with normal subjects, reflecting increased oxidative stress in these
patients (Yamamoto and Yamashita, 1997). Therefore the ubiquinol/ubiquinone
ratio is considered to be a sensitive marker of oxidative stress and an altered
ubiquinol/ubiquinone ratio is the first sign of lipoprotein exposure to oxidative
stress (Lagendijk et al, 1997).
The highest concentrations of CoQ10 in human tissues are found in heart, liver
and kidney (70-110 g/g) and the lowest concentration (8 g/g) was detected in
the lung. In human plasma CoQ10 was found in the range of 0.75-1,00 g/ml with
the total content in the body to be about 1.0-1.5 g (for review see Overvad et al.,
1999). A statistically significant circadian rhythm in human plasma CoQ 10
concentration was demonstrated, but no differences were found in CoQ 10
concentration between male and female subjects. On the other hand, some racial
differences were demonstrated with lower plasma CoQ levels in Caucasian
compared to African subjects (Reis et al., 2002). Coenzyme Q is the only fatsoluble antioxidant synthesised in the body. Therefore, tissue CoQ originates from
endogenous synthesis and from food. In fact, there are two major forms of CoQ
in the human tissues namely CoQ10 comprising 95-97% in heart, kidney, liver
and spleen and 92% in brain and 87% in lung (Dallner and Sindelar, 2000), the
rest of this compound is represented by CoQ 9 . In accordance with CoQ 10
concentration human tissues can be placed in the following descending order:
muscle>>heart>kidney> liver>>spleen>>brain> lung (Dallner and Sindelar, 2000).
Contents of CoQ10 in foods varied from 157.9 g/g to below the detection limit
with meat, meat products (55%) and fish (9%) to be major sources of this
compound. Vegetable oils, dairy products, vegetables and fruits plus berries provide
18%, 8%, 6% and 4% of daily CoQ10 consumption respectively. In the same foods
CoQ9 is also found varying from 8.5 g/g (reindeer), 0.4 g/g (beef and chicken)
down to lower levels in other foods. Daily intake of CoQ10 was calculated to be
5.4 mg for men and 3.8 mg for women and CoQ9 intake was shown to be 0.6 mg/
day for men and 0.4 mg/day for women (Mattila and Kumpulainen, 2001). It is
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believed that CoQ is quite stable during food cooking loosing only 15-30% its
initial value (Weber et al., 1997).
CoQ10 is characterised by comparatively slow absorption in humans with blood
values reaching a maximum after 6 h following intake of 100 mg CoQ10 with a
secondary peak been shown in the blood 24 h after intake with half-life in the
plasma to be about 33 h (Overvad et al.,1999). Potentially, slow absorption of
CoQ could be an advantage for antioxidant protection in the GIT. Animal studies
showed that the total uptake of dietary CoQ supplement comprised only 2-3%
(Zhang et all, 1995). However, high consumption of CoQ10 is associated with a
substantial (2.8-fold) increased concentration in human plasma, but tissue response
was shown to be much less pronounced. In particular, only liver and spleen
responded to CoQ supplementation in rats (Dallner and Sindelar, 2000).
Administration of the labelled CoQ10 to rats intraperitoneally resulted in an efficient
uptake into the circulation, with high concentrations found in spleen, liver, and
white blood cells; lower concentrations in adrenals, ovaries, thymus, and heart;
and practically no uptake in kidney, muscle, and brain (Bentinger et al., 2003). In
liver homogenate most CoQ appeared in the organelles, but it was also present in
the cytosol and transport vesicles. In particular mitochondria had a very low
concentration of labelled CoQ, which was mainly present in the lysosomes. All
organs that took up the labelled lipid also contained water-soluble metabolites
(Bentinger et al., 2003).
In general, dietary supplementation of CoQ does not affect the endogenous
synthesis of CoQ in tissues. However, oxidative stress (physical exercise, thyroid
hormone treatment, cold adaptation, vitamin A deficiency, etc.) is associated with
increased CoQ synthesis reflecting a cellular adaptation (Ernster and Dallner, 1995).
Therefore, CoQ synthesis is considered to be an adaptive mechanism in response
to stress conditions when other antioxidants are depleted. For example, in vitamin
E and Se deficient rats CoQ concentration elevated and CoQ-dependent reductase
system is activated (Navarro et al., 1998).
It is believed that exogenous CoQ, protects cells from oxidative stress by
conversion into its reduced antioxidant form by cellular reductases. In particular
cytosolic NADPH-CoQ reductase is responsible for cellular CoQ redox cycle as
an endogenous antioxidant (Kishi et al., 1999). The plasma membrane
oxidoreductase and DT-diaphorase are two such systems, likewise, they are
overexposed under oxidative stress conditions (Genova et al., 2003). In addition,
the selenoenzyme thioredoxin reductase is an important ubiquinone reductase
and can explain how selenium and coenzyme Q, by a combined action, may
protect the cell from oxidative damage (Xia et al., 2003).
Possible mechanisms of action of dietary CoQ 10 administration on organ
function include (Dallner and Sindelar, 2000):
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Direct CoQ organ uptake

Modulation of signal transduction system
Influence on organ circulation/vasculature
Antioxidant effects in the gastrointestinal system
Effects of CoQ metabolites
Specific protective effects in GIT
Since CoQ is an essential part of oxidative phosphorylation complex in

mitochondria the majority (molar amounts) of endogenous CoQ is found in these
organelles. However, exogenous CoQ is usually found in the extramitochondrial
fractions including lysosomes and Goldgi vesicles (Dallner and Sindelar, 2000).
Catabolic pathways of CoQ have not been fully elucidated and it was shown that
two major catabolic derivatives of CoQ in both urine and feces had an intact
quinone ring and shortened side chain and they were in conjugated (disulphated
or glucuronidated) form (Dallner and Sindelar, 2000).
Coenzyme Q function in the cell include (Nohl et al., 2001):
Proton-motive Q cycle of oxidative phosphorylation in mitochondria

Electron and proton carrier in plasma membranes, lysosomes and Goldgi
vesicles
Antioxidant in mitochondria and LDL
NO2- reduction to NO in mitochondria
Considering antioxidant function of CoQ it is necessary to mention its unique

position in antioxidant system (Ersner and Dallner, 1995; Lass and Sohal, 1998;
Stoyanovsky et al., 1995; Crane and Navas, 1997):
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Membrane localisation at sites of lipid peroxidation

Endogenous synthesis providing an adaptive response to stress conditions
Enzymatic powerful recycling maintaining CoQ in a reduced active
antioxidant form
Effective synergistic interactions with vitamin E and possibly with other
antioxidants enhancing protective activities of the antioxidant system
Presence in molar amounts in mitochondria, a main source of free radicals
in the cell, sufficient to effectively participate in antioxidant defence
Possibilities of preventing the formation of lipid peroxyl radicals by reducing
the initiating perferryl radical
Possible direct quenching L*
Direct or indirect eliminating LOO*
Presence in all parts of the GIT
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The presence of high concentrations of CoQ10 in all membranes provides a basis

for antioxidant action either by direct reaction with radicals or by regeneration of
tocopherol and ascorbate. In fact, a protective effect of CoQ against lipid
peroxidation was shown in fatty acid emulsions, mitochondria, submitochondrial
particles and other model systems (Ersner and Dallner, 1995). CoQ10 protects
efficiently not only membrane phospholipids from peroxidation but also
mitochondrial DNA and membrane proteins from free-radical-induced oxidative
damage (Pobezhimova and Voinikov, 2000).
It is generally accepted that stress-induced apoptosis is mediated by the
activation of plasma membrane-bound neutral sphingomyelinase with ceramidedependent caspase activation being an important part of the apoptosis pathway. It
was hypothesised that CoQ in the plasma membrane prevents both lipid
peroxidation and sphingomyelinase activation resulting in the prevention of
ceramide accumulation and caspase 3 activation with a consecutive apoptosis
inhibition (Villalba and Navas, 2000). It has also been suggested that CoQ10 is a
gene regulator and consequently has wide-ranging effects on over-all tissue
metabolism (Linnane et al., 2002). In particular it was hypothesised that CoQ10
plays a major role in the determination of membrane potential of main sub-cellular
membrane systems and that H2O2 arising from the activities of CoQ10 acts as a
second messenger for the modulation of gene expression and cellular metabolism
(Linnane et al., 2002). Evidence for a function in redox control of cell signalling
and gene expression is developing from studies on coenzyme Q stimulation of
cell growth, inhibition of apoptosis, control of thiol groups, formation of hydrogen
peroxide and control of membrane channels (Crane, 2001).
Did you know that the diet provides a range of antioxidants
necessary to balance the prooxidants in the intestine and to
maintain the healthy gut?
Antioxidant properties of CoQ are directly related to the protection in the
gastrointestinal tract. For example, in rats treated per os with sodium nitrite
increases TBARS in small intestinal mucosa and liver were observed. Pre-treatment
of nitrite-poisoned rats with CoQ10 mitigated lipid peroxidation and increased
total antioxidant status in animal blood (Grudzinski and Frankiewicz-Jozko, 2001).
The protective effect of administered CoQ10 against small intestinal damage caused
by ischemia reperfusion was also shown (Matsusaka et al., 1992). In rat intestine,
administration of CoQ10 normalised a sharp gamma-irradiation-induced inhibition
of transformation of phosphatidylcholine from phosphatidylethanolamine
(Novoselova et al., 1985). Compared to paired noninflamed mucosa, concentration
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of CoQ10 was significantly decreased in inflamed mucosa (Buffinton and Doe,

1995). The decreased antioxidant defences may severely compromise the inflamed
mucosa, rendering it more susceptible to oxidative tissue damage, hindering
recovery of the mucosa and return of epithelial cell layer integrity. Therefore,
antioxidant and other regulating functions of CoQ10 could be extremely important
in the GIT.
CAROTENOIDS
Carotenoids comprise a family of more than 600 compounds responsible for a
variety of bright colours in fall leaves, flowers (narcissus, marigold), fruits
(pineapple, citrus fruits, paprika), vegetables (carrots, tomatoes), insects (ladybird),
bird plumage (flamingo, cock of the rock, ibis, canary) and marine animals
(crustaceans, salmon) (Pfander, 1992). These pigments provide different colours
from light yellow to dark red and when complexed with proteins they can produce
green and blue colorations (Ong and Tee, 1992). Yellow, orange and green fruits
and vegetables provide a range of carotenoids. -carotene, -carotene and cryptoxanthin are the major provitamin A carotenoids in human and lutein,
zeaxanthin and lycopene are major carotenoids in the diet which are not converted
to vitamin A. Biological functions of these natural pigments in relation to animals
or humans are not well defined but their antioxidant properties seem to be of
major importance. In mixture with other antioxidants they could be much more
effective than on their own, and the GIT could be a major place for these
compounds to exert their activity. In some conditions, carotenoids can be
prooxidants. However, it is well recognised that this possibility is not likely to be
the case in physiological conditions, including in the GIT when an array of other
antioxidants is present.
Approximately 40 carotenoids are commonly consumed in the U.S. diet and
approximately 20 can be detected in human serum and tissues (Cooper et al.,
1999). Most nutrition research was concentrated on the six carotenoids found in
the highest concentrations in human blood: -carotene, lycopene, -carotene,
lutein, zeaxanthin and -cryptoxanthin. The major dietary lutein sources in the
human diet are green vegetables and fruits, including spinach, squash, grapes
(Sommerburg et al, 1998), broccoli, parsley, peas etc. (Hart and Scott, 1995).
Carotenoid consumption and their serum profile vary substantially depending on
the origin of the population studied. For example France presents the highest
levels of serum lutein and -carotene and Spain shows the lowest level of carotene, along with the highest levels of -cryptoxanthin (Olmedilla et al, 1997).
American women consume approximately 6 mg of total carotenoids per day (Chug-
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Ahuja et al, 1993), the average daily intake of major carotenoids in Spanish
population is 3.5 mg/day (Olmedilla et al, 1997) and in Germany total carotenoid
intake amounts to 5.33 mg/day with average lutein intake being 1.91 mg/day
(Pelz et al, 1998). Daily consumption of lutein and zeaxanthin in American elderly
subjects was 2.7 mg for men and 3.09 mg for women (Tucker et al, 1999). In
general, the recommended daily intake of carotenoids can only be achieved by
consuming 100-200 g/day of vegetables and fruits with a particularly high
carotenoid content (Muller, 1996).
Low lutein consumption reflects low consumption of fresh vegetable and fruits,
changes in nutritional habits and use of highly processed food. According to
National Health Interview Surveys, the intake of lutein declined among different
categories of people in the USA between 1987 and 1992 (Nebeling et al, 1997).
There were also significant seasonal differences in plasma carotenoid
concentrations in the UK reflecting a higher intake of lutein during the spring
compared with summer and autumn (Scott et al, 1996). It is interesting to mention
that there is also a high positive correlation of lutein (r=0.889) between maternal
plasma concentrations and cord plasma (Yeum et al, 1998) indicating that the
nutritional status of mothers is the major determinant of the lutein status of their
babies. In addition it has been shown that breast milk is the major source of lutein
to the infants (Thurnham et al, 1997). An increased intake of another carotenoid,
-carotene, by lactating women increases the supply of milk -carotene available
to their breast-fed infants (Canfield et al, 1998).
In general carotenoids are not toxic for humans. There is no evidence that
conversion of -carotene to vitamin A contributes to vitamin A toxicity. Probably
this process is metabolically regulated. However, extremely high consumption of
carotenoids for a long time could cause hypercarotenemia. It can lead to yellowing
of the skin which eventually returns to the norm after carotenoid exclusion from
the diet. Diabetes mellitus, hypothyroidism, hypothalamic amenorrhoea, anorexia
nervosa, liver disease, and some other metabolic disorders can increase carotenoid
absorption, leading to hypercarotenemia (Dawson, 2000).
Carotenoid assimilation from the diet varies significantly depending on many
various conditions, however, it seems likely that a substantial proportion of ingested
carotenoids could be found in all segments of the digestive tract. Therefore, in
combination with other dietary antioxidants carotenoids could promote antioxidant
defence in the GIT. Furthermore carotenoid activities related to the promotion of
cell differentiation, regulation of cell proliferation and intracellular communication
via gap junctions, as well as regulation of the detoxifying enzymes and
enhancement of immune system (Surai, 2002) could also be of great importance
in the GIT.
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VITAMIN A
Vitamin A is a generic term combining all-trans-retinol or its esters (acetate,
palmitate, etc.), and all-trans-retinal. Most of food products are poor source of
vitamin A, however it can be efficiently synthesised in the GIT from carotenoids.
Vitamin A is present in foods mainly in the form of esters with long-chain fatty
acids. The richest food source of this vitamin is liver. Biological functions of
vitamin A are diverse (vision, testicular function, development, bone growth,
differentiation, hematopoiesis, pattern formation during embryogenesis, etc.), but
its antioxidant properties can be also of importance (Livrea et al., 1996) especially
in combination with other antioxidants in the GIT. Intake requirements were
calculated to amount to 1000 retinol equivalents (RE) for men, 800 RE for women
(Gerster, 1997). One RE is equal to 1 g retinol or 6 g -carotene. Unlike most
carotenoids, vitamin A and most retinoids are highly toxic when taken in excessive
amounts (Bates, 1995). The US NRC Committee on Dietary Allowances has
recommended that adults should not consume more than 7500 retinol equivalents
daily (Dawson, 2000).
ASCORBIC ACID (AA)

Vitamin C is referred to as L-ascorbic acid and its two-electron reduction product
dehydro-L-ascorbic acid. Most animal species synthesize AA from glucose, but
human subjects are not able to synthesize it. Therefore AA is an essential dietary
component playing an important role in many physiological processes and it is a
hydrophilic antioxidant functioning in an aqueous environment and possessing
high free-radical-scavenging activity. It can participate in vitamin E recycling
thus maintaining efficient antioxidant defence. Fresh green fruits and vegetables
are good sources of AA. However, during food processing AA is easily oxidized
and as a result AA concentration in such foods is substantially decreased. Due to
its high reducing potential, in combination with iron ions AA can be a prooxidant.
However, it is believed that in physiological conditions and in the GIT ascorbic
acid performs mainly its antioxidant functions. In fact ascorbic acid inhibits
chemical synthesis of nitrosamines (animal carcinogens) in the gastric contents
and there are suggestions that intakes of ascorbic acid much higher than RDA
may reduce the risk of such diseases as heart disease and cancer (Hathcock, 1997).
RDA for ascorbic acid for humans is 60 mg/day and the most common adverse
effects of high vitamin C intakes (>2 g/day) are gastrointestinal symptoms such
as nausea, abdominal cramps, and diarrhea (Hathcock, 1997). After exclusion of
the vitamin supplements the symptoms usually disappear within a week or two
with no further consequences.
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GLUTATHIONE
Glutathione (GSH) is the most abundant non-protein thiol in mammalian cells,
and is considered to be an active antioxidant in biological systems providing cells
with their reducing milieu. Cellular GSH plays a key role in many biological
processes and can be synthesised in the human body. GSH is abundantly distributed
in the mucosal cells of GIT in man and its highest concentration is found in the
duodenum (Loguercio and Pierro, 1999). Cellular GSH plays a key role in many
biological processes: the synthesis of DNA and proteins, including cell growth
and proliferation, regulation of programmed cell death, immune regulation, the
transport of amino acids, xenobiotic metabolism, redox-sensitive signal
transduction (Sen and Packer, 2000). Furthermore, GSH thiolic group can react
directly with H 2 O 2 , superoxide anion, hydroxyl radicals, alkoxyl radicals,
hydroperoxides (Lenzi et al., 2000; Meister and Anderson, 1983). Therefore, a
crucial role for GSH is as free radical scavenger, particularly effective against the
hydroxyl radical (Bains and Shaw, 1997), since there are no enzymatic defences
against this species of radical. Usually decreased GSH concentration in tissues is
associated with increased lipid peroxidation (Thompson et al., 1992). Furthermore
in stress conditions GSH prevents the loss of protein thiols and vitamin E
(Palamanda and Kehrer, 1993) and plays an important role as a key modulator of
cell signalling (Elliott and Koliwad, 1997). Animals and human are able to
synthesise glutathione. In addition to body synthetic activity food also provide
GSH.
FLAVONOIDS
Flavonoids are low molecular weight polyphenolic substances based on the flavan
nucleus. They are widespread in nature, occurring in all plant families, and are
found in considerable quantities in fruits, vegetables, grains, cola, tea, coffee,
cocoa, beer and red wine (Skibola and Smith, 2000). The list of flavonoids
substantially increased from more than 4000 in 1996 (Cook and Samman, 1996)
to over 8000 individual compounds known in 2000 (Pietta, 2000). The major
flavonoid classes include flavonols, flavones, flavanones, flavanols (catechins),
anthocyanidins, isoflavones, dihidroflavonols and chalcones (Cook and Samman,
1996). Representatives of major groups of flavonols were characterised as having
antioxidant properties in vitro and in vivo (Pietta, 2000; Bors et al., 1996; Cook
and Samman, 1996; Larson, 1988).
These compounds have received substantial attention in recent years. The major
driving forces of research in the field were positive effects of fruits and vegetables
on human health and their preventive role in the development of various diseases,
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especially cancers. The flavonoid content in fruits and vegetables can be as high
as 300 mg/kg fresh weight (Ishige et al., 2001). The major problem with
antioxidant properties of these compounds is their low availability from the dietary
sources. For example, in human blood or urine polyphenol concentrations was
shown to be in a range 1-2 M (Bell et al., 2000; Lapidot et al., 1998) in
comparison to the general concentration of antioxidants in human plasma to be
about 1000 M (Benzi and Strain, 1999). Therefore it has been suggested that the
digestive tract is the major site of antioxidant defence afforded by polyphenolic
compounds such as flavonoids (Kanner and Lapidort, 2001; Halliwell et al., 2000).
Daily intake of flavonoids varies substantially between different countries and
in Asian population and in vegetarians it is the highest. In particular, average
daily intake of flavonols in Asian countries comprised about 68 mg and isoflavones
20-240 mg (Skibola and Smith, 2000). In contrast the mean intake of flavonols of
the German population was about 11.5 mg, mainly derived from fruits and
vegetables, but also from black tea and red wine (Bohm et al., 1998). Indeed,
naturally occurring polyphenolic compounds may play a role in the protective
effects of fruits and vegetables against cancers in general, and they appear to
have considerable potential as chemopreventive agents against neoplastic changes
in the alimentary tract (Gee and Johnson, 2001). In general flavonoids can prevent
LDL oxidative modification by scavenging ROS, chelating transition metal ions
or inhibiting lipoxygenase and this leads to the prevention of atherosclerosis. For
example, a number of studies have shown that consumption of soy is
antiatherogenic and that the isoflavones genistein, diadzein and biochanin, which
inhibit lipoprotein oxidation in vitro and suppress formation of plasma lipid
oxidation products in vivo, are most likely responsible for this effect (Patel et al.,
2001).
Did you know that flavonoids and other phenolics could provide
efficient antioxidant protection in the lower gut due to their
comparatively low efficiency of absorption in the small intestine?
However, there are no data available on the long-term effects of flavonoid dietary
supplementation on humans. A serious problem with flavonoids is that, depending
on conditions, they could be antioxidants or prooxidants, antimutagens or
promutagens. Therefore unregulated use of flavonoid-containing supplements
can have a detrimental effect on human health. For example, the results obtained
by Silva et al. (2000) suggest that there is a range of flavonols whose genotoxicity
in eukaryotic cells depends on their autooxidation. These flavonols can autooxidize
when the pH value is slightly alkaline, such as in the intestine, and therefore can
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induce genotoxicity in humans. Clearly more research is needed to clarify health

benefit and potential dangers of these compounds.
Comparatively low bioavailability and antioxidant potential of various
flavonoids could be beneficial for the human providing antioxidant protection in
various parts of the digestive tract, including the large intestine where levels of
other antioxidants would be quite low.
OTHER POLY(PHENOLICS)
Cereal brans contain significant quantities of the phenolic ferulic acid and diferulic
acid and their potential health benefits (protection of LDL from oxidative
modification and reduction in atherogenesis as well as inhibitory effects on tumor
promotion and chemopreventive properties) have been related mostly to their
antioxidant activity (Andreasen et al., 2001).
SPICES AND ESSENTIAL OILS

Addition of spices to food is a common procedure in most cultures. The seasonings
contribute a pleasant flavour and recently it has been shown that they contain a
range of antioxidant compounds and it seems likely that only a small proportion
of them have been isolated and identified (Madsen et al., 1997). They include
such phenolic diterpenes as carnosoic acid, carnosol, rosmaridiphenol and
rosmariquinone from rosemary sage and summer savory. In other spices a range
of flavonoids have been identified. In general, spices and herbs have been shown
to have over a hundred compounds with high antioxidant activity including 26
active comopounds from the Labiatae family, Rosmarinus officinalis, Thymus
vulgaris, Origanum vulgare and O. majorana, over 40 antioxidative compounds
from Zingiber officinale and 26 compounds from Curcuma domestica (Nakatani,
2000). Spices are effective in prevention food deterioration during storage and
this explains why traditional diets in countries with high temperature (India,
Thailand, Mexico etc.) are usually rich in spices.
Essential oils from aromatic and medicinal plants have been shown to have
antibacterial, antimycotic and antioxidant properties. Recently the essential oils
from black pepper, clove, geranium, melissa, nutmeg, oregano and thyme and 33
phytoconstituents have been assessed in vitro (Dorman et al., 2000). All the
compounds demonstrated antioxidant capacities superior to the water-soluble tocopherol analogue Trolox with the exception of the essential oil melissa and
three phytoconstituents. The best results were obtained with clove, oregano and
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thyme oils and their corresponding phytoconstituents namely eugenol, carvacrol

and thymol. Again in the GIT antioxidant properties of various compounds from
spices and herbs would contribute to total antioxidant potential.
SELENOMETHIONINE
In most diets, the main food sources of Se are cereals, meats and fish (Combs,
2001). Selenomethionine (SeMet) represents the major natural form of selenium
in feed and food ingredients. It seems likely that SeMet has specific functions on
its own beyond being an important source of Se for selenoprotein synthesis. In
fact, there is strong evidence that this compound itself can be considered as an
antioxidant (for review Surai, 2002). Therefore it is most likely that SeMet can
contribute to the total antioxidant potential of the GIT.
SYNTHETIC ANTIOXIDANTS
Antioxidants in foods may be endogenous origin or may be added externally to
preserve their lipid components from peroxidation. Synthetic antioxidants such
as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), propyl
gallate (PG) and tert-butylhydroquinone (TBHQ) are commonly used in food
formulations. However, due to safety concerns, public interest shifted from
synthetic to natural antioxidants. As a result mixed tocopherols, herbal extracts
such as those of rosemary and sage, as well as tea extracts have been
commercialised for food and nutraceutical applications (Shahidi, 2000).
SPECIFIC PLACE FOR SELENIUM-DEPENDENT ENZYMES IN

ANTIOXIDANT DEFENCE OF GIT
Food derived antioxidant enzymes would be inactivated during thermal food
processing. However GIT contains internally-originated antioxidant enzymes SOD,
GSH-Px and CAT and they represent an important mechanism of the enterocyte
defence from oxidative damage. A specific gastrointestinal GSH-Px (GI-GSHPx) have been described in 1993 (Chu et al., 1993). GI-GSH-Px activity was
present in both the villus and crypt regions of rat mucosal epithelium and its
activity nearly equalled that of classical GSH-Px throughout the small intestine
and colorectal segments (Esworthy et al., 1998). GI-GSH-Px could be considered
to be a barrier against hydroperoxide resorption (Brigelius-Flohe, 1999).
Furthermore, in the gastrointestinal tract there are at least three more selenoproteins
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including plasma GSH-Px, selenoprotein P and thioredoxin reductase (Mork et

al., 1998). The data on GI-GSH-Px presented in Chapter 2 clearly indicate that
this enzyme should be considered as a major antioxidant defence in the intestine.
In fact many other antioxidant compounds could improve antioxidant defence by
up-regulating the expression of various GSH-Px in the gut.
Did you know that selenium has a special place in antioxidant
defences in the gut being an integral part of a range of
selenoproteins expressed in the intestine?
Glutathione and glutathionedependent enzymes contribute significantly towards
intestinal antioxidant defences. In fact, an important peroxide detoxification
pathway in the intestine is based on the GSH redox system (LeGrand and Aw,
1998). In this system GSH-Px reduces peroxides at the expense of GSH oxidation.
Oxidised glutathione is reduced back to the active form by glutathione reductase
utilizing reducing potential of NADPH which is produced in the pentose phosphate
pathway. It is known that exogenous GSH provided rat small-intestinal epithelial
cells with significant protection against injury induced by t-butyl hydroperoxide
or menadione (Lash et al., 1986). Thus, rat small-intestinal epithelial cells can
utilise plasma GSH to support intracellular detoxication systems that function in
protection against chemically induced injury. On the other hand, decreased
intestinal GSH concentration was associated with an increased susceptibility to
oxidative injury, metal intoxication and various intestinal pathologies (Iantomasi
et al., 1997).
OTHER ANTIOXIDANT MECHANISMS

To adapt to environmental changes and survive different types of injuries,
eukaryotic cells have evolved networks of different responses which detect and
control diverse forms of stress. To deal with various aggressive factors including
free radicals and toxic products of their metabolism, the gastrointestinal mucosa
has a variety of defence mechanisms consisting of functional (mucus-alkaline
secretion, mucosal microcirculation), humoral (prostaglandins and nitric oxide)
and neuronal (capsaicin sensitive sensory neurones) factors (Tsukimi and Okabe,
2001). Furthermore, oxidative stress at the cellular level is reduced by enzymatic
and nonenzymatic antioxidant mechanisms. Recently, new mechanisms of such
adaptive defences of the gastrointestinal mucosa at the intracellular level have
been characterised. One of these responses, known as the heat shock response is
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considered to be a universal fundamental mechanism necessary for cell survival

under a variety of unfavourable conditions (Santoro, 2000). As mentioned above,
intestinal cells are challenged with a great variety of potentially toxic compounds
and their protection is a vital part of the strategy to maintain human health. In
mammalian cells, the induction of the heat shock response requires the activation
and translocation to the nucleus of one or more heat shock transcription factors
which control the expression of a specific set of genes encoding cytoprotective
heat shock proteins (Santoro, 2000).
Did you know that endogenously synthesised antioxidants in
human body include GSH, coenzyme Q, uric acid and a
range of antioxidant enzymes?
Therefore heat shock proteins (HSP) function as molecular chaperones in
regulating cellular homeostasis and promoting survival. However, if the stress is
too high, a signal that leads to programmed cell death, apoptosis, is activated,
thereby providing a finely tuned balance between survival and death (Kopecek et
al., 2001). In addition to extracellular stimuli, several nonstressfull conditions
induce HSPs during normal cellular growth and development. In particular, the
HSP family is activated under oxidative stress and provides an important protection
against protein denaturation and modifications by capping and refolding, or drives
damaged proteins into appropriate proteolytic pathways (Yenari , 2002). In fact
HSPs have been assigned to multiple subcellular sites and implicated in multiple
functions ranging from stress response, intracellular trafficking, antigen processing,
control of cell proliferation, differentiation, and tumorigenesis (Wadhwa et al.,
2002). Therefore in response to environmental or physiological stresses including
ethanol and heavy metals cells increase synthesis of HSP (Tsukimi and Okabe,
2001). It has been suggested that the conserved heat shock protein HSP33 functions
as a potent molecular chaperone with a highly sophisticated regulation. In fact, at
the transcriptional level, the HSP33 gene is under heat shock control; at the
posttranslational level, the HSP33 protein is under oxidative stress control (Graf
and Jakob, 2002). Therefore redox-regulated chaperone activity of HSP33
specifically protects proteins and cells from the detrimental effects of reactive
oxygen species.
ROS-mediated damage has been implicated in the pathophysiology of the
gastrointestinal mucosa and HSPs are suggested to play an important role in
cytoprotection against oxidative stress-induced injury (Prabhu and
Balasubramanian, 2002). For example, the mammalian intestinal epithelial cells
respond to heat stress by producing heat shock proteins that provide protection in
stress conditions, which would otherwise lead to cell damage or death. The
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protective effects of HSP are seen in heat stress, infection, and inflammation
(Malago et al., 2002). Similarly, glucocorticoid protection of rat intestinal cells
against oxidant-induced stress was mediated by HSP72 (Urayama et al., 1998).
The molecular mechanisms of heat shock response-induced cytoprotection
are beyond this review. However, they involve inhibition of proinflammatory
cytokine production and induction of cellular proliferation for restitution of the
damaged epithelium (Malago et al., 2002). HSPs play an important role in gastric
mucosal defence under conditions of stress. For example, exposure of rats to
restraint and water-immersion stress caused rapid HSP70 mRNA expression and
HSP70 accumulation in gastric mucosa and the extent of HSP70 induction inversely
correlated to the severity of mucosal damage (Rokutan, 1999). Therefore HSP70
is involved in repair of partially damaged proteins and substantially contributes to
protection of the gastrointestinal mucosa against various necrotising factors
(Tsukimi and Okabe, 2001).
Heme oxygenase (HO-1), known as HSP 32, can be induced by various stresses.
It has been shown that HO-1 induction and the maintenance of its appropriate
activity is critical in protecting the intestinal epithelial cells from oxidative injury
(Fujii et al., 2003). It is interesting that in the aforementioned experiment HO-1
was markedly induced following LPS treatment in the mucosal epithelial cells in
the upper intestine (duodenum and jejunum) but not in the lower intestine (ileum
and colon). It seems likely, that there is a delicate interaction between HSPs and
other antioxidant defence mechanisms to maintain mucosal integrity and repair
of acute mucosal damage.
Site-specificity in antioxidant-prooxidant balance in the intestine

Presence of antioxidants in the GIT is an essential factor preventing lipid
peroxidation in the stomach, small intestine and colon. In fact total antioxidant
activity and activities of SOD and catalase were higher in the rat mucosa/submucosa
of the small intestine than in the colon (Blau et al., 1999). It was shown that
antioxidant enzymes play a key role in rendering the intestinal mucosal cells
resistant to iron induced oxidative damage in rats (Srigiridhar and Nair, 1997). It
is interesting that in the rat small intestine, activities of SOD and GSH-Px and
lipid peroxidation were not affected by age or strain difference (Jang et al., 2001).
On the other hand a differential modulating effect of flavonoids on antioxidant
enzyme activities in liver, colon, heart and red blood cells (Breinholt et al., 1999)
is of great importance in understanding the antioxidant interactions in the GIT
and human tissues. Furthermore naringin, a citrus bioflavonoid, plays an important
role in regulating antioxidative capacities by increasing the SOD and catalase
activities, up-regulating the gene expressions of SOD, catalase, and GSH-Px, and
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protecting the plasma vitamin E in high cholesterol-fed rabbits (Jeon et al., 2001).
In addition, synergistic effect of different antioxidants is also of great importance.
For example, lycopene acts synergistically, as an effective antioxidant against
LDL oxidation, with several natural antioxidants such as vitamin E, the flavonoid
glabridin, the phenolics rosmarinic acid and carnosic acid, and garlic (Fuhrman
et al., 2000). Similarly synergistic interactions between isoflavones and ascorbic
acid have been shown (Patel et al., 2001). It is believed that rat intestine and
mesenteric lymph possess efficient antioxidant defences against preformed lipid
hydroperoxides and (peroxyl) radical mediated lipid oxidation (Mohr et al., 1999).
It was also shown that supplementation of vitamin E alone or in combination
with ascorbic acid protects the GIT of Fe-deficient rats against Fe-mediated
oxidative damage during Fe repletion (Srigiridhar and Nair, 2000). Since flavonoids
are consumed in concentrations usually much higher than other antioxidant
compounds, their protective effect during digestion is of great importance. For
example, recently it has been shown that plant antioxidants such as flavonoids
not only prevented an accumulation of peroxidized lipids but also could switch
prooxidant properties of heme-proteins to antioxidant ones (Kanner and Lapidot,
2001). Dietary polyphenols can also modulate in vivo oxidative damage in the
gastrointestinal tract of rodents (Giovannelli et al., 2000) supporting the hypothesis
that dietary polyphenols might have both a protective and a therapeutic potential
in oxidative damage-related pathologies.
The dietary concentration of linoleic acid significantly affected oxidation of
pig jejunal mucosa and vitamin E has a protective effect. In particular, a histological
study of the jejunal mucosa of pigs showed lower cell desquamation in groups
supplemented with -tocopheryl acetate and a higher cell desquamation was found
in the groups fed diets containing the higher concentration of linoleic acid (Lopez
Bote et al., 2001). In the same study in vitro-induced oxidation of jejunal mucosa
homogenates was lower in pigs fed diets supplemented with -tocopheryl acetate.
Vitamin E also protects the rat colon from oxidative stress associated with
inflammation. In fact, vitamin E supplementation, which resulted in increased
colonic vitamin E levels, reduced colonic weight and damage score, prevented
lipid peroxidation and diarrhea, reduced interleukin-1 beta levels and preserved
glutathione reductase activity and total glutathione levels (Gonzalez et al., 2001)
There are other inducable antioxidant enzymes in the intestine. For example,
feeding mice with BHA induces phase II detoxifying enzymes and intestinal and
hepatic peroxiredoxin I, a stress-inducible antioxidant, in a manner similar to the
induction of glutathione S-transferases (GST, Ishii et al., 2000) and it was suggested
that the induction of this antioxidant may be important to protect the cells and
tissues against toxic electrophiles and reactive oxygen species. Similarly, in small
intestine GST activity changed in response to the different fatty acid supplemented
diets and increased as a result of the elevated oxidative stress (Giron et al., 1999).
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There are other antioxidant applications in relation to GIT health. For example,
irradiation caused increased lipid peroxide and decreased GSH levels in the
intestine. Intestinal SOD and GSH-Px activities were increased, but GST activity
decreased following irradiation of rats. Selenium and/or vitamin E pre-treatments
ameliorated these disturbances in prooxidant-antioxidant balance in the GIT
(Mutlu-Turkoglu et al., 2000). This amelioration has been confirmed with
histopathological findings. Common mucosal immune responses in orally
immunized aged mice were depressed and dietary supplementation with vitamin
E restored their mucosal and systemic humoral immune responses to mature adult
levels (Enioutina et al., 2000).
When the antioxidant system of the GIT is compromised, various diseases can
be observed. For example, in rats at weaning it was shown that early chronic
diarrhea and severe protein-energy malnutrition impair the antioxidant defence
system in both the small and large intestine (Nieto et al., 2000). Similarly, ulcerative
colitis in rats induced by trinitrobenzenesulfonic acid damages the intestinal mucosa
and is accompanied by a shift in the antioxidant enzyme activities, and low levels
of glutathione (Nieto et al., 2000a).
Indeed, the antioxidant-prooxidant balance in various parts of the intestine
would ultimately depend on the level of antioxidants and prooxidants provided
with the diet and released by cells themselves as well as on the level of absorption
of both antioxidants and prooxidants. Therefore in the stomach the conditions are
favourable for lipid peroxidation since major antioxidants and prooxidants are
there before absorption or major metabolism, and the pH is also favourable (Kanner
and Lapidot, 2001). In the small intestine this balance will be more variable since
many antioxidants, mainly vitamins E, A, C and carotenoids will be effectively
absorbed. However efficiency of absorption of iron is quite low (Department of
Health, 1991) therefore it will be available for stimulation of peroxidation. On the
other hand, after lipid absorption, concentrations of substrates of peroxidation
will be substantially decreased. Furthermore, various flavonoids will also be
available for antioxidant defence. Finally, in the colon/rectum, levels of vitamin
E, A, C are low, although various flavonoids and some carotenoids are present
(Halliwell et al., 2000) as well as iron, but the lipid concentration would be low.
Therefore in each part of the digestive tract there is a possibility of oxidative
stress and damage to various biological molecules. Indeed, it seems likely that
the protective effects of vegetables and fruits against development of various
cancers, especially cancers related to the digestive tract, are based on provision
of a variety of antioxidants which are not always well absorbed, thus providing
antioxidant protection in the large intestine and preventing oxidative damage and
possible mutagenic effect. It is possible to suggest that there is a reason for some
antioxidants not to be absorbed completely and in that way providing antioxidant
protection in lower parts of the intestine. For example, tocotrienols are common
compounds of major food and feed ingredients and possess high antioxidant
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activity (Surai, 2002). It is well appreciated that they are not well absorbed from
the food and feed and would be found in the digesta in colon providing antioxidant
protection in the lower intestine. However, this idea needs further investigation.
What should be changed to have a healthy diet?

Dietary factors are considered to be major contributors to the leading causes of
death of Americans, namely coronary heart disease and certain types of cancer
(Milner, 2000). Epidemiological findings, supported by animal studies, have led
to recommendations that people should consume at least two servings of fruit and
three servings of vegetables daily (Diplock et al., 1998) in addition to at least two
servings of fish weekly (Krauss et al., 2001). While findings and reports such as
these have had an impact on the type and quantity of the food that many of us eat
(Margetts et al., 1998) the majority of adults in developed countries fall well short
of meeting healthy eating guidelines. At the same time from information provided
above it is quite clear that a decreased risk of heart disease and cancers of the
breast, prostate, lung, colon and stomach is associated with increased consumption
of fruits, vegetables and soy products (Skibola and Smith, 2000). A range of
antioxidants can be found in our diet and they are essential part of antioxidant
defence in the intestine and further in human tissues. However, it seems most
likely that the average diet of Americans or Europeans contains restricted amounts
of antioxidant-rich fruits and vegetables and this can at least partly account for
the poor health records in some developed countries. Se-deficiency is also a
problem of great concern in many countries all over the world.
From information presented above it is clear that in most cases producers and
consumers themselves are responsible for increased levels of prooxidants in the
diet and ultimately in the GIT. Therefore there is a need for changes in ways our
food is produced, prepared, stored and eaten (Table 13.2). It is possible to improve
the situation both at the producer and consumer levels.
AT THE PRODUCER LEVEL
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To enrich meats and meat-related food with vitamin E. Indeed there is a

great body information accumulated indicating that chicken (Morrissey et
al., 1997), turkey (Ahn et al., 1998, Mercier et al., 2001), pork (Buckley et
al., 1995), beef (Liu et al., 1995), lamb (Macit et al., 2003) as well as meat
from other species (Oriani et al., 2001) can be enriched with vitamin E and
this technological solution can substantially decrease lipid peroxidation in
meat during processing, storage and cooking. It seems likely that fish
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Table 13.2 Healthy meals via antioxidant enrichment and decreased lipid peroxidation (adapted from
Surai et al., 2004).
Meat
Egg
Fish
+++
+++
+
+++
+++
+++
+++
+
+++
Food preparation
Cooking oil
Boiling vs frying
Spices and herbs
Olive
+
+++
Olive
+++
+++
Olive
+
+++
Food serving
With vegetables
+++
+++
+++
Meal composition
Fruit
Fruit juice
Red wine
Tea
+++
+++
+++
+++
+++
+++
+
+++
+++
+++
+
+++
Technological improvement
at the producer level
Vitamin E enrichment
Selenium enrichment
Carotenoid enrichment
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enrichment with vitamin E will also be beneficial in terms of prevention of

lipid peroxidation (Ruff et al., 2003). Similarly enrichment of eggs with
vitamin E can also substantially decrease lipid peroxidation (Surai and
Sparks, 2001) and cholesterol oxide formation (Galobart et al., 2002). It is
also possible to enrich eggs with vitamin E to such extent that they will
provide substantial (daily requirement) amounts of vitamin E which will be
beneficial during digestion as well as for general health (Surai et al., 2000;
Surai and Sparks, 2001). It is worth mentioning that vitamin E in the egg
yolk is in free tocopherol highly available form which could be a major
benefit at the level of digestion (Surai, 2002).
To enrich meat, milk and eggs with organic selenium. This could be
beneficial in terms of preventing lipid peroxidation in meat (Surai and
Dvorska, 2002; 2002a; Krska et al., 2001) or eggs (Surai and Sparks, 2001,
Surai, 2002) but more importantly, selenium is absolutely essential for
expression of GI-GSH-Px, the main defence against lipid peroxide absorption
in GIT (Brigelius-Flohe, 1999). Selenium is also important for expression
of other selenoproteins (e.g. thioredoxin reductase etc.) which play an
important role in antioxidant defence in the intestine and in other tissues. In
fact, Se-enriched eggs, meat and milk are already on the market in various
countries in the world (Surai, 2002).
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To enrich eggs with carotenoids. These natural antioxidants will be a part of

the complex antioxidant defence system interacting with other antioxidants
in the GIT and they per se can also provide antioxidant protection (Surai,
2002).
To improve product storage by decreasing oxygen availability and lipid
peroxidation. To minimise storage of cooked products, which are especially
susceptible to peroxidation.
In the fast food restaurants, to change frying oils more often and use olive
oil which is less sensitive to peroxidation (Quiles et al., 2002) and to enrich
frying oils with natural antioxidants (e.g. tocopherol mixture). It would be
advantage to serve fast food with bigger portions of salads and use more
sauces providing additional antioxidants. Some other oils (e.g. rapeseed
oil) enriched with tocopherols can also be considered to be useful.
To produce antioxidant-enriched sources, especially for fast food restaurants
Did you know that at producer level there is a range of simple

measures, which can help in maintaining our diet healthy?
Therefore it is possible to provide consumers with a range of animal-derived
deliver substantial amount of health-promoting nutrients to improve the general
diet and help to maintain good health. In fact, in the UK in main supermarkets
(Tesco, Safeway, etc.) Columbus eggs delivering 70% of RDA in Se and vitamin
E with a single egg are already available. Therefore, without changing habits and
traditions of various populations it is possible to solve problems related to deficiency
of various nutrients, in particular selenium. The consumer will go to the same
supermarket to buy the same animal-derived products (egg, milk and meat), cook
and consume them as usual. The only difference will be in amount of specific
nutrients delivered with such products.
AT THE CONSUMER LEVEL
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To choose olive oil for frying food. This will decrease accumulation of
oxidation products (Quiles et al., 2002) and will be beneficial in terms of
decreasing omega-6 PUFA consumption and increasing MUFA consumption
in accordance with recent health-related findings (Tuck and Hayball, 2002).
Rapeseed oil enriched with tocopherols is also an important choice.
To use more spices and herbs during cooking. This will decrease oxidation
and prevent accumulation of the lipid peroxides.
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To choose boiled eggs instead of fried. If using frying, it is necessary to

make sure that duration of the process is minimal.
To choose antioxidant-enriched eggs, which are already on the market (Surai,
2002).
To decrease usage of cooked meals after storage.
To decrease consumption of fast food, prepared by current technology of
deep-frying. Use more sauces, which can provide additional antioxidants.
To make sure that meat meals are served with plenty of vegetables, providing
necessary antioxidants.
To increase vegetable and fruit consumption on everyday basis.
Therefore, since we cannot avoid pro-oxidants in our food we need to make sure
that they are compensated by consumption of increased levels of natural
antioxidants. For this reason, it would be advantage if our meat and fish meals are
served with plenty of vegetables. Various sauces (e.g. tomato sauce) could provide
additional antioxidants. Red wine could also add additional flavonoids as a source
of antioxidants. Various juices are also good sources of natural antioxidants as
well as fruits. If a meal is finished with tea, this will also add to antioxidant potential
of the digesta.
Did you know that simple changes in our diet and ways of
the food preparation could help in preventing prooxidant
formation in the digestive tract?
All these suggestions are in a line of traditional meals serving in various countries
of the worlds. Antioxidants compensate prooxidants and a positive balance in the
digestive tract is the first step to healthy life. All the future exists in the past.
ANTIOXIDANT-PROOXIDANT BALANCE IN THE INTESTINE AND

ANIMAL HEALTH
Data presented above clearly indicate, that antioxidant-prooxidant balance in
human intestine is an important determinant of the intestinal integrity and efficiency
of nutrient absorption and assimilation. From the one hand, it was suggested that
a compromised antioxidant-prooxidant balance in the intestine could lead to the
development of various diseases. On the other hand, consumption of dietary
antioxidants, including those, which are not well absorbed in the small intestine,
could improve intestinal health and help in maintaining general health.
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It is necessary to answer an important question if the mentioned balance is

important for animal nutrition. Unfortunately this question is not addressed in
animal and poultry production. However, a list of prooxidants, which can be
found in animal diet could be quite long. Similarly to humans, animals obtain
with feed oxidised PUFAs, iron supplements, nitrites and nitrates, heavy metals,
persistent organic pollutants, mycotoxins and some other compounds possessing
prooxidant properties. For most of mentioned prooxidants there are legal limits
established. However, a combination of several prooxidants, even in concentrations
not exceeding legal limits, could lead to promotion of peroxidation in the intestine.
Taking into account recent information on the effect of redox status of the cell on
the apoptosis, it is possible to suggest, that apoptosis of enterocytes could be
triggered by a compromised antioxidant-prooxidant balance in the intestine. This
could lead to inflammatory reactions, decreased efficiency of absorption of various
nutrients and decreased productive and reproductive performances of farm animals
and poultry. Therefore, development of various animal diseases where intestinal
inflammation and necrosis/apoptosis are involved could be affected by antioxidantprooxidant balance in the intestine. This question awaits investigations.
Conclusions
Recent achievements in biochemistry and molecular biology, together with
epidemiological data have changed our thinking about food. It became
increasingly clear that our diet plays a pivotal role in maintenance of our health
and a disbalanced diet can cause serious health-related problems. It seems likely
that antioxidants are among the major regulators of many physiological processes
and therefore a balance between antioxidants and prooxidants in the diet, GIT,
plasma and tissues is an important determinant of the state of our health. Plants
consumed by humans contain thousands of phenolic compounds. Among them
the effects of dietary polyphenols are of great current interest due to their higher
consumption in comparison to other antioxidants such as vitamin E and their
antioxidant and possible anticarcinogenic activities. It could well be, that some
dietary constituents which are not well absorbed could have health-promoting
properties by maintaining antioxidant-prooxidant balance in the large intestine,
where concentration of other antioxidants (vitamin E, carotenoids, ascorbate) could
be low, but prooxidants (iron) and substrates of oxidation are still present. This
protective effect in the large intestine could be responsible for bowl cancer
prevention. Therefore, there could be a biological reason for some nutrients not
to be absorbed, but be involved in antioxidant protection in the lower gut.
Realising an importance of the food choice issue fundamental solutions for
food improvement should be found and production of functional food is one of
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them. Therefore, improvement of the diet by balancing essential nutrients via

designer/functional food without changing peoples food preferences would bring
health benefit. In this respect, natural antioxidants have become a wonder remedy
of the 21st century.
We are what we eat!
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14
CONCLUSIONS: LOOKING AHEAD
All is well that ends well
Selenium is considered to be one of the most controversial trace elements. From

the one hand, it is toxic at high doses and there is a great body of information
related to environmental issues of Se contamination. On the other hand, Se
deficiency is a global problem related to increased susceptibility to various diseases
of animals and human, decreased productive and reproductive performances of
farm animals.
Importance of Se is related to the quickly developing understanding of the role
of free radicals in biology and medicine. Indeed, detrimental effects of free radicals
are well established and their involvement in the development of various diseases
is proven. It is also generally accepted that mitochondria are the major source of
free radicals in biological systems and therefore even in physiological conditions
free radicals are produced and they can damage all types of biological molecules
including DNA, lipids and proteins. In fact, lipid peroxidation is shown to be an
important detrimental process in biological systems. Indeed, various calculations
of electron escape from electron-transport chain in mitochondria gave an estimation
of about 200 billion of free radicals in every cell every day to be produced in
physiological conditions. Therefore, an old perception that free radicals are
produced in stress conditions was corrected to confirm that the stress conditions
increase free radical production, which is already substantial in physiological
conditions. In recent years it has been established that damages to DNA and
proteins are also of great importance from the point of view of understanding
pathology of many different changes in the body caused by free radicals. An
integrated antioxidant system was developed in the body and in particular in
every cell to deal with those free radicals. In fact, there are three major levels of
antioxidant defence and Se participates in all of them. The first level is based on
the activity of antioxidant enzymes with SOD to be the main device to detoxify
superoxide radical, the most important radical produced in the biological systems.
Se-dependent GSH-Px, catalase and metal-binding proteins are also integral part
of the first level of antioxidant defence. It is important to underline, that GSH-Px
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also belongs to the second level of antioxidant defence, since it is responsible for
detoxification of lipid hydroperoxides formed as a result of reaction of lipid
peroxides with such antioxidants as vitamin E. Indeed, an optimal interaction
between vitamin E and GSH-Px provides an important mechanism of antioxidant
defence. Recently it has been shown that SeMet could affect activity of DNArepairing enzymes. This means that Se also belongs to the third level of antioxidant
defence. The understanding that all antioxidants in the body are working as a
team, called the antioxidant system, creates an additional interest in antioxidant
interactions. Indeed, vitamin E recycling is shown to be the most important
mechanism of the antioxidant defences. This means that if the recycling is effective,
even low levels of vitamin E could provide a substantial antioxidant protection.
For example, in the brain the vitamin E level is usually comparatively low and
levels of polyunsaturated fatty acids are high but in physiological conditions it is
very difficult to detect any products of peroxidation there. On the other side, if
the recycling is broken probably even high vitamin E supplementation would not
provide the adequate antioxidant protection. There are various additional
mechanisms of the antioxidant protection including redox signalling and changes
in gene expression, stress-protein synthesis as well as apoptosis. Recent
understanding of essentiality of free radicals in various physiological processes
shifted research to antioxidant-prooxidant balance rather than studying simply
the lipid peroxidation. For example, free radicals are synthesised in phagocyte
cells and used as a weapon to kill pathogens. This process is under strict metabolic
control, since an excess of the free radical production could escape from the
phagosome and damage various biological molecule including damages to
phagocytes themselves. It seems likely that some toxicants, such as mycotoxins
could affect this regulation causing an excessive free radical production and
creating an oxidative stress. In general, ingestion of excessive amounts of
antioxidants is presumed to shift the oxidant-antioxidant balance toward the
antioxidant side. This is supposed to be beneficial; however, this may also
adversely affect key physiological processes that are dependent on free radicals,
including prostaglandin production, cell division and differentiation. Free radicals
are important signalling molecules in spermatozoa capacitation, they are involved
in neurone communications, blood vessel tone maintenance and in some others
important physiological processes. Furthermore, recent evidence suggests that
cellular oxidation can induce changes in gene expression during normal
development. Conversely, antioxidants such as ascorbate, glutathione, tocopherol or carotenoids are inhibitory to differentiation in many types of cells.
New data coming from understanding important role of antioxidant-prooxidant
balance in gene expression and it seems likely, that antioxidants can affect gene
expression by mechanisms independent on their antioxidant activity. For example,
it has been demonstrated that GSH, in addition to its antioxidant and protective
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function against oxidative stress, has a specific signalling role in redox regulation.
Indeed, recently it has been shown that 2,016 genes regulated by H2O2 (Fratelli et
al., 2005). Of these, 215 genes showed GSH-dependent expression changes,
classifiable into four clusters displaying down- or up-regulation by H2O2, either
potentiated or inhibited by GSH depletion. In particular, the biological process
categories overrepresented in the largest cluster (genes whose up-regulation was
inhibited by GSH depletion) were NF-kappaB activation, transcription, and DNA
methylation. Furthermore, this cluster also included several cytokine and
chemokine ligands and receptors, the redox regulator thioredoxin interacting
protein, and the histone deacetylase sirtuin. The cluster of genes whose upregulation was potentiated by GSH depletion included two heat shock proteins
(HSP40 and HSP70) and the AP-1 transcription factor components Fos and FosB
(Fratelli et al., 2005).
In 1973 GSH-Px was shown to be a selenoprotein and antioxidant role of Se
was established. Since then a family of selenoproteins in human and animals was
shown to include at least 25 different selenoproteins. In fact, many of new
selenoproteins have been described only recently and therefore the knowledge of
their functions and regulation is far from being complete. Traditionally
selenoproteins have been considered to function as antioxidants. This is particularly
true for GSH-Px, but also for TR and MSR. In fact there are six different forms of
GSH-Px and they are located in different parts of the cell, in different tissues and
in some cases are differently regulated. It seems likely that this is an adaptive
mechanism to more effectively deal with free radical production. For example,
PH-GSH-Px is specifically located in membranes and is able to deal with lipid
peroxides directly without necessity to release them from membranes by
phospholipases. GI-GSH-Px is considered to be the most important defensive
mechanism against lipid hydroperoxide absorption. In fact, GI-GSH-Px destroys
lipid peroxides in GI tract preventing them from absorption. However, low GIGSH-Px activity could potentially fail to destroy all peroxides and some of them
can be absorbed and incorporated into lipoproteins. This incorporation could be
a triggering mechanism of the lipid peroxidation in LDL leading to changes related
to CVD development. The role of GI-GSH-Px in the maintenance of antioxidantprooxidant balance in the GI tract needs further investigation. Furthermore, sperm
nuclear GSH-Px is also of great importance for sperm quality maintenance. For
the question if GSH-Px is the main selenoprotein in the human and animals my
answer would be, probably not. A great body of information related to the role
and functions of GSH-Px accumulated for the last 30 years put this enzyme at the
top of the selenoprotein list. However, TR, described as a selenoprotein 23 years
later than GSH-Px and methionine sulfoxide reductase B (MSR-B), described as a
selenoprotein only in 2003 are going to change that view on the importance of
different selenoproteins. TR is involved in regulation of such processes as DNA
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synthesis, cell proliferation and apoptosis as well as participates in vitamin E

recycling. At the same time understanding functions of MSR is going to change
our perception to lipid peroxidation. Indeed, free radicals induce oxidation not
only PUFAs, but also oxidise proteins and DNA. It is generally accepted now that
MSR is an enzyme responsible for prevention of protein oxidation. Indeed,
methionine molecules located in active centre of various enzymes are considered
to be bodyguards for active cysteine molecules. When free radicals attack
proteins methionine would be oxidized first protecting cysteine from oxidation
and in this way maintaining enzymatic activity. MSR is responsible for reducing
oxidised methionine back to an active form. Therefore, the more we understand
functions of MSR, the more we realise an importance of protein oxidation and its
prevention in biological systems. Taking into account possible role of all 25 known
selenoproteins it was suggested that Se is the chief executive of the antioxidant
system. This means that Se is responsible for regulation of major antioxidant
protections via direct antioxidant activities of GSH-Px, TR or MSR or indirectly
via vitamin E recycling or other important interactions. Therefore, in Se deficiency
many important physiological functions could be compromised as a result of the
detrimental changes in the antioxidant defence system.
The selenium cycle in the food chain of land animals and humans starts from
soils and includes plant and animal sources ultimately dependent on its assimilation
from the soil. Indeed, soils are the major source of Se for plants and therefore for
animals eating those plants and humans consuming plant and animal-derived
foods. Considering food and feed sources of Se it is necessary to mention that Se
level is greatly varied in different foods as well as in the same foods grown in
different areas. In fact it seems likely that low Se availability from various soils is
a result of agricultural practises. Firstly, usage of inorganic fertilizers containing
sulphur decreased Se availability. Secondly, soil acidification also substantially
decreased Se availability. Furthermore, decreased soil aeration also decreasing Se
availability. There were several attempts to solve this problem by using Se
fertilization. In particular, recently there was a workshop devoted to 20-year
experience in Se fertilization in Finland (Eurola, 2005). There were several
important conclusions from the workshop:
Finland used to be a country with low Se status before 1980's

In 1984 the Ministry of Agriculture and Forestry made a decision to include
sodium selenate into fertilizers at 16 mg/kg for cereals and horticulture and
6 mg/kg for grassland
In 1990 the level of Se in the fertilizers was lowered to 6 mg/kg
In 1998 the level of Se in fertilizers was increased up to 10 mg/kg

Twenty years of research and practical applications showed the following:
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Only about 10% of the added Se was utilized by plants

Se level in grains significantly increased and this was associated with
increased Se concentrations in eggs, milk and meat
There was no effect of the Se fertilization on rates of heart diseases and
cancer in Finland
It was difficult to prove the Se accumulation in soils after 20 years' Se
fertilization. The main problem was the technical difficulties with analysis
as well as a high variability in the Se concentration in the soil
There was no research on the effect of the Se fertilization on the microbial
population of soil, which could be the biggest obstacle for wider usage of
the technology
There are quite a few questions to answer before the technology could be
used worldwide
The plant absorbs Se from the soil in the form of selenite or selenate and synthesises
selenoamino acids with SeMet representing more than 50% of the Se in cereal
grains and other feed ingredients. In many cases the variability in results of Se
specification investigations of plant material reflects analytical difficulties. Recently
strong cation-exchange chromatography (SCX-HPLC) was used in conjunction
with inductively coupled plasma mass spectrometry (ICP-MS) to investigate cationic
selenium species present in leaf extract of wild-type Brassica juncea supplemented
with selenite. Total amount of Se accumulated by the leaves was found to be 352
g/g (Yathavakilla et al., 2005). Major cationic selenium species identified in the
present study were methylselenomethionine and methylselenocysteine while SeMet
was found in minor quantities. By high performance liquid chromatography with
inductively coupled plasma mass spectrometry (HPLC-ICPMS) it was shown that
the main Se species in bulbs, leaves or flowers of the Se-enriched garlic, onions,
cabbage and ashitaba were selenate, Se-methylselenocysteine or gamma-glutamylSe-methylselenocysteine, while those in fruit bodies of the peppers and pumpkin
were selenomethionine bound to protein (Yoshida et al., 2005).
Recent advances in Se biochemistry have provided a deeper understanding of
the principal differences in metabolism of the two forms of Se namely inorganic
Se (sodium selenite or selenate) and organic Se (mainly SeMet). Selenite is taken
up by red blood cells within several minutes, reduced to selenide by glutathione,
and then transported to the plasma, bound selectively to albumin and transferred
to the liver (Suzuki and Ogra, 2002). Contrary to selenite, intact selenate is either
taken up directly by the liver or excreted into the urine. The chemical speciesspecific metabolic pathway for Se was explained by the metabolic regulation
through selenide as the assumed common intermediate for the inorganic and
organic Se sources and as the checkpoint metabolite between utilization for the
selenoprotein synthesis and methylation for the excretion of Se (Suzuki and Ogra,
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2002). In particular, organic Se, which can be found in grains, forages and other
feed ingredients, is primarily in the form of SeMet and is metabolised in the same
way as methionine. It is actively transported through intestinal membranes during
absorption and actively accumulated in such tissues as liver and muscle. It is well
known that methionine is not synthesised by animals or humans and therefore it
is an essential amino acid. The same is true for SeMet, which is not synthesised in
animals or humans and must be derived from feed sources. In fact SeMet is
considered to be a storage form of Se in the body. Indeed, when organic Se is
used in the diet, the Se reserve is built in muscles in the form of SeMet. These
reserves can be used in stress conditions when the Se requirement increases but
feed consumption decreased. The skeletal muscles are the major Se-storage organ,
accounting for about 46.9% of the total Se in the human body, while kidney
contains only 4% of Se reserves. In humans, whole body Se depends on the
regional location and varies from 3-6 mg up to 13-20 mg. In stress conditions
protein catabolism by proteasomes can release SeMet, which could serve as a
source of Se for newly synthesied selenoproteins, such as GSH-Px, thioredoxin
reductase and methionine sulphoxide reductase. Those enzymes can deal with
overproduction of free radicals and prevent decrease in productive and
reproductive performance of farm animals. It was proven that Se from both selenite
and SeMet is readily available for synthesis of the selenoenzyme GSH peroxidase
in rat tissues. There are also several lines of evidence confirming the idea that Se
accumulated in tissues in the form of SeMet can be available for selenoprotein
synthesis (see relevant references).
Studies in our laboratory indicated that chicks hatched from eggs enriched
with Se by means of using Se yeast had higher liver and muscle selenium and
GSH-Px activity not only at hatching, but more importantly, even at 21-28 days
posthatch (Surai, 2000; Surai et al., 2005; Pappas et al., 2005). This could be
explained by usage of SeMet accumulated in tissues as a result of Se transfer from
the egg during embryogenesis. In experiments with rats supplemented with high
doses of selenium in the form of selenite or SeMet it was shown that half-life of
decay of GSH-Px to be 4.2 and 9.1 days, respectively indicating that SeMet was
used for the GSH-Px synthesis at time of Se deprivation. Furthermore, in SeMet
or Se-yeast supplemented mice, liver GSH-Px activities declined more slowly
during Se depletion than in mice given selenite. Similarly, in human supplemented
with organic selenium, during Se deprivation the decline in the level of
selenoprotein P was slower than in individuals who had been supplemented with
selenite. When wheat and selanate were used as Se sources in a supplementation
study in Finnish men it was shown that once the supplements were withdrawn,
platelet GSH-Px activity declined less in the group given wheat Se. After several
weeks supplementation with high-Se bread, plasma Se remained elevated when
supplementation ceased. Furthermore, in Finish children Se-yeast provided a longer
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lasting body pool of Se (see references in Chapter 3).

It should be noted that only a small proportion of the methionine pool can be
replaced by SeMet, since only part of methionine could be replaced by SeMet in
the diet. Furthermore, the protein turnover prevents accumulation of SeMet to
toxic levels in the organism. There is a great discrepancy in results of comparative
evaluation of Se bioavailability from various sources. The problem is that it is
difficult to choose a proper end point for such evaluation. A range of techniques
has been used for this purpose, including prevention of specific Se-responsive
diseases in animals, measurement of the uptake of Se in different tissues after
ingestion of a test compound, activity of GSH-Px in plasma, RBC, whole blood,
liver and other tissues and selenium excretion with urine. However, none of those
techniques can give an ultimate answer as for Se bioavailability or Se status of the
animal or human. It seems likely, that a combination of various techniques has to
be used for such evaluation. In general, absorption of various forms of Se is quite
high. However, the main difference between organic and inorganic forms of Se is
in its accumulation in tissues and transfer to the egg, milk or meat. Since SeMet is
the main form of Se in muscle, milk, colostrum and egg and animals cannot
synthesise it, only organic selenium can substantially increase Se concentrations
in those substrates. This can give advantages to the developing animals or human
in terms of improvement of their Se status as well as can be an important tool for
the production Se-egg, Se-meat and Se-milk.
For the last few years an effective technology of producing of organic selenium
in the form of Se-Yeast has been developed and commercialised. Indeed, yeast as
other plants can take selenite from the medium and synthesise SeMet and other
organic Se-compounds. Since Se and S are very similar chemically, yeast cannot
distinguish between them and in the medium deficient in S, but supplemented
with Se they synthesise SeMet instead of Met. As a result such yeast are enriched
with organic selenium to such extent (1,000-2,000 ppm) that it can be used as a
selenium source for human and animal consumption. The main advantage of
such yeast is that the composition of organic Se compounds there is the same as
in major feed ingredients (e.g. grains) with SeMet comprising more than 60-70%
of total selenium. Therefore such yeast is used effectively as a natural source of
Se for human and animal nutrition. Recent results indicate that not all yeast are
the same and they are different in terms of Se-compound composition, which is a
reflection of the conditions of Se growth, genetics and some other factors. This
could affect Se availability from various yeasts and clearly more research should
be done in terms of comparison between different Se-yeasts. Considering
importance of organic Se in animal nutrition there is a question regarding the best
source of organic selenium. From the one hand, pure SeMet could be a choice of
organic Se for supplementation. However, SeMet is not a stable compound per se
and can be easily oxidised during feed storage and processing. This could restrict
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technological advances of feed production and usage in animal industry. On the

other hand, Se-Yeast provides SeMet in the protected form as a part of yeast
protein. During protein digestion SeMet is released and can be effectively
assimilated in the digestive tract. The great body of evidence, presented in previous
13 chapters of the book, clearly indicate advantages of organic selenium in the
form of Se-Yeast in comparison with inorganic form of Se in animal and human
nutrition. The data presented above also suggested that selenite be considered as
a drug, which should be used accordingly. For example, when the Se deficiency
is diagnosed based on clinical signs, selenite would be the preparation of the
choice. Using it via feed, water or injection will solve the short-term or acute Se
deficiency, which has been demonstrated under various experimental conditions
with chickens, pigs and cattle. However, when the goal is meeting physiological
requirements of animals in order to maintain high productive and reproductive
performance, optimum food animal product quality and immunocompetence, a
Se supplement that allows tissue reserves is needed. Indeed, Se-yeast is such a
supplement.
The most important application of the dietary Se supplementation is related to
its immunomodulating properties. In fact immune system is the most complex
system in the body and until now it is still not known how this system is regulated
on the molecular level. It seems likely, that an effective communication between
different types of the immune cells (e.g. neutrophils, macrophages, B- and Tlymphocytes) is a key element in immunocompetence. It has been suggested that
receptors on the surfaces of these cells are working like mobile phones receiving
and sending various signals using so called signalling molecules (e.g. cytokines,
eicosanoids, etc.). These receptors are extremely sensitive to low concentrations
of communicating molecules, but they are also sensitive to free radicals, which
could damage those receptors. In fact, in stress condition free radical production
can exceed ability of antioxidant system to detoxify them leading to an oxidative
stress. Those radicals can damage communication between immune cells and
those many millions of immune cells will be useless without effective
communication and immunocompetence would be substantially decreased. In
fact, phagocytic cells produce free radicals and use them as a weapon to kill
pathogen. This process is very strictly regulated. It could be comparable to the
nuclear power station where control chain reaction produces energy. However, if
this control is broken (e.g. Chernobyl power station) the power station could
become an atomic bomb. Something similar could happen when the regulation in
phagocytic cells is broken. It seems likely that an example of such process would
be related to mycotoxins action on immune cells. The free radicals, produced by
phagocytes could be considered as chemical weapon used to kill pathogens.
However, the problem is that the chemical weapon is not specific and could kill
anything and anywhere. A protection of the immune cells in general and the
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receptors in particular from their own weapon by antioxidants is of great importance

for the immunocompetence. Therefore selenoproteins expressed in immune cells
could be considered as a protective mechanism maintaining an integrity of receptors
and preventing declining immunocompetence in stress conditions.
It has been shown that selenium affects all components of the immune system,
including the development and expression of non-specific, humoral, and cellmediated responses. In general, a deficiency in Se appears to result in
immunosuppression, whereas supplementation with low doses of Se appears to
result in augmentation and/or restoration of immunologic functions. In fact, a
deficiency of Se has been shown to inhibit resistance to microbial and viral
infections, neutrophil function, antibody production, proliferation of T and B
lymphocytes in response to mitogens, and cytodestruction by T lymphocytes and
NK cells. On the other hand, Se supplementation has been shown to stimulate the
function of neutrophils, production of antibodies, proliferation of T and B
lymphocytes in response to mitogens, production of lymphokines, NK cellmediated cytodestruction, delayed-type hypersensitivity reactions and allograft
rejection, and the ability of a host to reject transplanted malignant tumors.
When considering immuno-facilitating properties of selenium it is necessary
to take into account several important points. Individual antioxidants in the body
interacts with each other (vitamin E, C, carotenoids, Se) and with pro-oxidants
(iron, high level of PUFAs, mycotoxins) which themselves have
immunostimulating or immunosuppressive effects. Therefore in every experiment
the results reflect a sum of all these interactions and if background dietary
concentrations of those nutrients differ, results could be completely different. This
could explain inconsistency of some results published for the last 20 years.
Furthermore antioxidants can suppress respiratory burst, however, it is not clear
at present if there is a limit of this suppression after which the phagocyte antimicrobial activity would be compromised. Immunostimulating effects of
antioxidants are shown to be maximal at their supplementation usually above the
requirement for maximal growth and maintenance of reproduction. It could well
be that the Se and vitamin E doses that are adequate for maximal productivity in
healthy, unchallenged birds are not optimal for immunocompetence and disease
resistance. There is no data available on the effect of antioxidants on intestinal
immune structures, which could be crucial barriers to external pathogens. Since
free radicals can be damaging to intestinal structures antioxidant functions of
selenium are responsible for prevention damages to intestinal lymphoid structure
as well as damages to intestinal enterocyte membranes. This could be especially
important in relation to digestive immunosuppression caused by toxins/
mycotoxins, nutritional deficiencies and infectious agents. Selenium source
(organic vs inorganic) seems to be an important element of its immunostimulating
properties. Organic selenium appears to be at an advantage because it is better
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assimilated from feed and better accumulated in tissues. Indeed with the same
dose of supplementation organic selenium can deliver more element to the target
tissues and because of toxicity of high selenium levels this could be a solution to
avoid adverse effect of selenium overdose. If the body has selenium reserves in
the form of SeMet in muscles, during acute phase response it would be liberated
as a result of skeletal muscle protein catabolism by proteosome action and would
be used for re-synthesis of new selenoproteins which could decrease oxidative
stress. In chickens, first two weeks posthatch represent most important period of
immune system development and maternal diet is shown to have a profound
effect on this process. In particular, the first week of chick life is a period of rapid
expansion of leukocyte population, seeding of lymphoid organs and other events
ultimately leading to the production of unique clones of lymphocytes that will
mediate immunity in postnatal development. In this respect effects of various
combinations of natural antioxidants and n-3 PUFA await investigation. As a result
of antioxidant (selenium) deficiency increased oxidative stress of a host can lead
to increased virus mutation rate and change in a viral pathogen resulting in
emerging viral pathogens with new pathogenic properties. Therefore, Se deficiency
was associated with a change to the viral genotype, converting the virus from a
benign to a virulent strain. This possibility was not exploited in animal/poultry
production, but seems important to study more extensively. There is a need to
study antioxidant composition and fatty acid profile of immunocompetent tissues
depending on animal age and nutritional supplementation of antioxidants and n3 PUFAs.
The second important application of Se nutrition of animals and human is
related to its effect on reproduction. Animal and human spermatozoa are unique
in structure and chemical composition and are characterized by high proportions
of PUFAs in the phospholipid fraction of their membranes. This feature of these
highly specialized cells is a reflection of the specific needs of their membranes
for high levels of fluidity and flexibility, which are necessary for sperm motility
and fusion with the egg. This functional advantage conferred by PUFAs is, however,
associated with disadvantages in terms of the susceptibility of sperm to free radical
attack and lipid peroxidation. Therefore antioxidant protection is a vital element
in maintaining sperm membrane integrity, motility and fertilizing ability. It has
been suggested that natural antioxidants (vitamin E, ascorbic acid and glutathione)
together with antioxidant enzymes (superoxide dismutase and glutathione
peroxidase) build an integrated antioxidant system in semen capable of protecting
it against free radicals and toxic products of their metabolism. The delicate balance
between free radical production and antioxidant defense is considered to be an
important determinant of semen quality and in particular its fertilizing ability. The
essentiality of selenium for mail fertility was shown in the early 1980s. For many
years sperm capsular selenoprotein (SCS) was considered as the major protein
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expressed in spermatozoa. However, recently it has been establishes that, PHGSH-Px can be converted into structural selenoprotein and it is the protein which
was previously named SCS. The role of PH-GSH-Px as a structural protein may
explain the mechanical instability of the mitochondrial midpiece that is observed
in selenium deficiency. Recently a fifth member of Se-dependent glutathione
peroxidases: a specific sperm nuclei GSH-Px (sn-GSH-Px), has been characterized.
In particular, in Se-depleted rats the concentration of sn-GSH-Px decreased to a
third of the normal level and chromatin condensation was severely disturbed and
it seems likely that the main function of sn-GSH-Px is protamine thiol crosslinking during sperm maturation.
There are species-specific differences in the Se level in semen. For example,
the level of Se in seminal plasma was lowest in the human and the stallion, higher
in ram and boar, with the highest levels in the bull. Selenocysteine is shown to be
the main form of Se in rat sperm and selenocysteine and selenomethionine were
found in ovine sperm. It has been shown that Se concentration in chicken semen
depends on the Se in the diet and by inclusion of organic selenium in the diet it is
possible to significantly increase Se level in the spermatozoa (Pappas et al., 2005a).
GSH-Px in the sperm is considered to be the main enzyme, which removes
peroxides and thereby protects cells against damage caused by free radicals and
the products of lipid peroxidation in vivo.
There is species and tissue-specificity in GSH-Px expression. For example,
GSH-Px activity has been found to be expressed in the semen of several mammalian
species including ram, dog, human, goat, bull. It is interesting to note that in
bulls, for example, GSH-Px is exclusively associated with the seminal plasma
and not found in spermatozoa, while GSH-Px activity in seminal plasma was low
in man and ram and not detectable in boar and stallion. Furthermore, bull and ram
seminal plasma GSH-Px activities per mg protein were comparable, but when
expressed per ml seminal plasma, activity of the bull was more than 7 times the
activity of ram seminal plasma. It has been shown that approximately two thirds
of GSH-Px activity in bull semen was non-Se-GSH-Px. Among avian species, in
seminal plasma total GSH-Px activity was the highest in turkey and lowest in
duck and goose. In spermatozoa, on the other hand, the highest GSH-Px activities
were found for goose and duck and much lower GSH-Px activity was recorded
for guinea fowl, turkey or chicken. Recently, it has been shown that despite a
high proportion of PUFAs and a low level of vitamin E, duck spermatozoa have
the same susceptibility to lipid peroxidation as chicken spermatozoa. It has been
suggested that an increased activity of Se-GSH-Px in duck semen compensates
for the relatively low concentrations of other antioxidants. It was proven that
inclusion of Se in the chicken diet increased GSH-Px activity in semen and
improved the antioxidant defences. In boars it was shown that low Se diet had a
greater detrimental effect on semen quality that diets inadequate in vitamin E.
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Detailed studies on comparison between organic and inorganic selenium in

the diet of cockerels were conducted at the North Carolina State University. In
particular, it was shown that, when cockerels were fed on a basal diet containing
0.28 ppm Se without additional dietary supplementation of this trace element, the
percentage of normal spermatozoa was only 57.9% and two major abnormalities
seen were bent midpiece (18.7%) and corkscrew head (15.4%). When this diet
was supplemented with an additional 0.2 ppm Se in the form of selenite, the
percentage of normal spermatozoa increased to 89.4% and abnormalities in the
form of bent midpiece and corkscrew head were decreased down to 6.2 and 1.8%
respectively. However, when organic selenium was included in the cockerels
diet in the same amount, semen quality was further improved and those
abnormalities decreased down to 0.7 and 0.2% and the percentage of normal
spermatozoa increased up to 98.7%. These results clearly showed that the form of
dietary Se supplementation is a crucial factor of its efficiency, with organic selenium
being much more effective in comparison to selenite. There was also evidence of
increased fertilizing ability of spermatozoa from cockerels fed an organic source
of selenium in comparison to sodium selenite. Therefore, Se supplementation of
the male diet is needed to maintain sperm membrane integrity during in vitro
sperm manipulation including artificial insemination as well as during sperm
transport and storage (avian species) in the female reproductive tract. However,
the form of dietary selenium is a key for success in optimising Se supplementation.
In fact organic selenium can help maintaining reproductive performance in farm
animals and poultry.
The next application of Se is related to mycotoxins. Indeed, problem of
mycotoxin contamination of the feed and food is a global one. There are several
unresolved questions in this regard. Firstly, more than 25% of world grain
production is contaminated by mycotoxins. In particular, Fusarium mycotoxins
(so called field mycotoxins) contaminate up to 100% of the grain. Since these
mycotoxins are coming from the field it is difficult to deal with them and various
technological approaches including plant selection for mycotoxin resistance did
not produce any significant results. Secondly, in nature there are more than 300
mycotoxins, but analytical techniques for routine mycotoxin analysis are
developed only for about 20 major mycotoxins. Therefore, if there is a conclusion
from the analytical lab that the mycotoxins are not found this means that 10-20
mycotoxins analysed were not found. As for others, there is no answer. Thirdly,
sampling for mycotoxins analysis is extremely difficult and is an important source
of errors. Fourthly, there are no safe levels of mycotoxins, because of synergistic
interactions of many mycotoxins: several mycotoxins in low concentrations could
cause more problems than a single mycotoxin at higher dose. The data presented
in chapter 6 indicate that by using absorbents, such as Mycosorb it is possible to
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substantially decrease detrimental effect of mycotoxins and a combination of

absorbents with an organic selenium and possibly with other antioxidants could
be the next step in solving mycotoxin-related problems.
Selenium has important applications in poultry nutrition. In particular
commercial poultry production uses modern chickens characterised by high egg
production and high growth rate. However, the price for such improvements is
related to high sensitivity of birds to various stresses. Therefore, in the modern
poultry production there is an important move from preventing Se deficiency to
meeting Se requirement. The data presented in chapter 7 indicate that organic
selenium is a choice for diets designed to maintain high productive and
reproductive performance of poultry. In particular, replacement of sodium selenite
by organic selenium in the form of Se-Yeast (Sel-Plex) in the breeder diet is related
to improvement of fertility, hatchability and viability of chicks in early postnatal
development. Indeed, organic selenium (SeMet) is more effectively transferred
from the diet to the egg and further to the developing embryo. This improves
antioxidant defences and helps chickens to overcome an oxidative stress of
hatching leading to improvement of hatchability. The data are accumulating to
show similar positive effect of organic Se on goose reproduction (Table 14.1). It
is well known that when chicken is hatched many physiological systems, including
immune system, are not mature and continue developing during at least 2 weeks
posthatch. Therefore this is the most vulnerable period of ontogenesis of the chicks.
Our data indicate that Se transferred from the egg to the embryo as a result of
organic Se supplementation of the maternal diet had positive effect on Se status
of the developing chicks up to 4 weeks posthatch (Surai et al., 2005; Pappas et
al., 2005). These data showed an importance of maternal diet not only for newly
hatched chick but also for the developing chicken in early postnatal life. Taking
into account recent data presented by Koutsos et al. (2003) showing similar effect
of carotenoids in the maternal diet on the carotenoid concentration in 4-week-old
chickens, it is possible to suggest that Se and probably carotenoids could affect
gene expression during the embryonic development. As a result, Se/carotenoid
assimilation in postnatal development could be affected. Alternatively, antioxidant
system could be affected and less Se/carotenoids being used for metabolic needs
and higher concentrations of these compounds were observed in tissues. Indeed,
this hypothesis needs further clarification, however, it is clear that maternal effect
is seen beyond newly hatched chicks. In general this could be an example of
maternal programming responsible for various changes in postnatal life of chicken.
Indeed, chicken egg could be an ideal model to study this phenomenon and more
research should be done in this area.
Advantages of organic selenium for commercial laying hens are related to
better shell quality and improvement of egg production. Our data on Se content
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Table 14.1 Effect of selenium on goose reproduction (Adapted from Tverdochlebov et al., 2005)
Maternal diet supplementation

No
Selenite,
Sel-Plex,
supplementation
0.3 ppm
0.3 ppm
Items
Fertility, %
Hatchability of fertile eggs, %
Hatch of eggs set, %
Weight of day-old gosling, g
94.4
67.2
63.4
93.9
96.7
71.3
68.9
96.4
97.1
75.0
72.8
98.8
in the shell and possibility its manipulation by inclusion of organic Se in the

laying hen diet are a background for further research. Indeed, it is well recognised
that eggshell consists of about 95% of mineral and 5% of organic matrix. Recent
evidence indicates that organic matrix is responsible for regulation of crystal
formation in the developing shell. This means that 5% of the organic matrix
determines shell quality. Since organic Se is an integral part of the organic matrix
it was suggested that organic Se could affect shell quality and information is
accumulating to substantiate this claim. The second advantage of organic selenium
for laying hens is related to egg production maintenance at the peak of production.
The problem is that even low stresses in commercial egg production could affect
peak of egg production and once egg production is decreased it is almost
impossible to return it back. Since Se provides an additional antioxidant protection,
this could help to overcome those small stresses and maintain high egg production
at the peak. An additional benefit of organic Se for commercial layers is related to
egg freshness during storage. Indeed organic selenium transferred from the diet
to the egg stimulate GSH-Px in the egg yolk, white and probably perivitelline
membrane leading to decrease lipid and protein oxidation and helping to maintain
Hough units high during egg storage.
Advantages of organic Se for broilers include improvement of growth rate,
FCR, decreased mortality and decreased drip loss during meat storage. This could
be related to antioxidant Se action, activation of thyroid hormone as well as
improvement of immunity. Indeed, it is very expensive to maintain activated
immune system. Many nutrients are distributed from growth and development to
the immune system. Therefore immunomodulating properties of Se could help to
use nutrients properly and not loosing them for unnecessary stimulated immune
system.
Importance of Se in pig nutrition was described in the Chapter 8. In particular,
there is a problem in the antioxidant defences of newly born piglets. Since placenta
restricts antioxidant transfer to the foetus, antioxidant defences of the piglet is
quite weak. Therefore Se transfer via placenta, colostrum and milk is of great
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importance for piglet growth and development. The data presented clearly showed
that only organic Se could solve this problem. Indeed, Se transfer via placenta, to
the colostrum and milk are shown to be much higher when organic selenium was
in the diet. Furthermore, usage of sodium selenite in the sows diet could potentially
have a detrimental effect on the level of splay leg and stillborn piglets. In contrast,
organic Se can improve the situation. Recently published data of the experiment
conducted in the commercial conditions in Iowa (USA) confirmed a positive effect
of organic selenium in the form of Sel-Plex on the sows and piglets (Tables 14.2
and 14.3). Substitution of inorganic selenium in diets fed commercial sows with
Sel-Plex (0.3 ppm added Se) resulted in more piglets weaned with lower preweaning mortality (9.76 vs 11.3%; Gourley et al., 2005). Furthermore, culls were
reduced in nursery pigs weaned from sows given organic selenium. Se
accumulation in the pig muscles as a result of organic Se inclusion into the diet
was a background information for the development of the technology for Sepork production.
Table 14.2 Effect of sow selenium source on litter parameters (Adapted from Gourley et al., 2005)
Sow selenium source, 0.3 mg/kg

Selenite
Sel-Plex
Item
Number of sows
Litter size, number
Total
Live
Stillbirths
Weaned
Pre-wean mortality, %
383
377
11.32
10.44
0.95
9.26
11.3
11.49
10.65
0.76
9.61
9.76
Table 14.3 Effect of sow and nursery diet selenium source on growth performance (Adapted from
Gourley et al., 2005)
Sow diet:
Selenite
Wean diet:
Number of pigs
Daily gain, g
Death, number
Culled, number
Total removal, %
Pig sold, number
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Sel-Plex
Selenite
Sel-Plex
Selenite
Sel-Plex
250
390
3
12
6.0
235
250
390
3
9
4.8
238
250
390
1
8
3.6
241
250
376
0
7
2.8
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The main problem with Se nutrition of ruminants is an ability of rumen bacteria to

reduce inorganic selenium into the metallic Se which is not available for further
metabolism. It should be mentioned that the second problem is related to that Se
which is incorporated into protein synthesised by rumen bacteria. As some data
showed Se has low availability from the rumen protein. On the other hand, it has
been shown that low Se status in cows is characterised by increased somatic cell
counts, mastitis, metritis, retained placenta and cystyc ovaries. Furthermore,
thermoregulation of the newly born calves could be compromised due to low Se
status and low iodothyronine deiodinase activity. Therefore a common practise is
to inject animals with selenium preparations to compensate for the low Se
availability from the diet.
In recent years data have been accumulated to indicate several important
advantages of organic Se for ruminants. In fact, similar to pigs, the Se transfer via
placenta, colostrum and milk was substantially improved when organic selenium
was included into the cows diet in comparison to sodium selenite. There are also
commercial data to substantiate the positive effect of organic selenium on animal
health. Indeed, somatic cell counts, incidence of mastitis and metritis were
decreased as a result of organic Se supplementation. In fact, when organic Se is
used in the cows diet there is no need for Se injections. The same data on the
efficiency of Se transfer to the milk from organic sources built a strategy for
commercial production of Se-enriched milk.
When considering Se nutrition of horses, the most important issue is an oxidative
stress during exercising. It was shown that Se supplementation could decrease
those stresses and organic Se to be more effective than inorganic one. It seems
likely that transfer to colostrum and milk of the organic forms of selenium could
also be important advantages. It was shown that in cats and dogs Se metabolism
has several important features. In particular the level of Se in cat blood is severalfold higher than that in other animals. Again, organic Se sources could be of great
importance for companion animal nutrition. In fish nutrition Se also plays an
important role. In particular, in several fish species diet is extremely high in
polyunsaturated fatty acids and needs an adequate antioxidant supply. Similar to
other animal species, organic Se in the fish diet is more effectively assimilated
than sodium selenite. Selenium dietary supplementation is shown to be important
to maintain fish reproduction and progeny viability as well as can help to maintain
fish flesh coloration.
Problems and solutions of Se nutrition in human were considered in the Chapter
11. It was identified that Se requirement in the USA is 55 g per day and in the
UK it is 75 g /day for men and 60 g /day for women. However, in many countries
all over the world human diet is deficient in Se. On the other hand, there is a great
body of evidence to show health-promoting properties of selenium. It seems
likely that absorption does not play any role in the homeostatic regulation of
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selenium with SeMet to be absorbed almost completely and efficiency of absorption

of sodium selenite and selenate is somehow lower. Labelled Se (infused as sodium
selenite) was incorporated predominantly in the plasma selenoprotein P fraction
with the comparatively short (1.9-2.9 days) half-life of Se in this fraction. An
average Se turnover time in the plasma was 0.01-1.2 days, in the liver 1.6-3.1
days and in peripheral tissues 61-86 days. Most of the whole body selenium was
found in skeletal muscles (25-50%), which appears to act as a Se storage organ;
much less selenium was found in bones (16%), blood (7-10%) and kidney (4%).
Recently metabolic pathways of Se in human have been re-evaluated and it
has been shown that selenosugar 1 is the major urinary metabolite after increased
selenium intake, and it was suggested that previously accepted pathways for human
metabolism of selenium involving trimethylselenonium ion as the excretory end
product may need to be re-evaluated (Kuehnelt et al., 2005). In the study selenium
speciation analysis by HPLC/ICPMS was used on samples of human urine from
one volunteer over a 48-hour period after ingestion of selenium (1.0 mg) as sodium
selenite, L-selenomethionine, or DL-selenomethionine. The major species in
background urine were two selenosugars, namely methyl-2-acetamido-2-deoxy1-seleno-beta-D-galactopyranoside (selenosugar 1) and its deacylated analog
methyl-2-amino-2-deoxy-1-seleno-beta-D-galactopyranoside (selenosugar 3).
Indeed, in all experiments, the major metabolite was selenosugar 1, constituting
either approximately 80% of the total selenium excreted over the first 24 hours
after ingestion of selenite or L-selenomethionine or approximately 65% after
ingestion of DL-selenomethionine. Selenite was not present at significant levels
(<1 g Se/L) in any of the samples; selenomethionine was present in only trace
amounts (approximately 1 g /L) following ingestion of L-selenomethionine, but
it constituted about 20% of the excreted selenium (first 24 hours) after ingestion
of DL-selenomethionine. Trimethylselenonium ion, a commonly reported urine
metabolite, could not be detected (<1 g/L) in the urine samples after ingestion of
selenite or selenomethionine (Kuehnelt et al., 2005).
The amount of volatile dimethylselenide (DMSe) in breath has been monitored
after ingestion of sub-toxic amounts of selenium (300 g 77Se, as selenite) by a
healthy male volunteer (Kremer et al., 2005). Dimethylselenide was the only
selenium species detected in breath samples before and after the ingestion of
77
Se-enriched selenite. It was also shown that the high Se dose led to a significant
increase of DMSe and renal excretion of background selenium. These data
confirmed the idea that selenium ingested as selenite is homeostatically controlled
by excretion. Overall excretion as DMSe was calculated to be 11.2% from the
ingested selenite within the first 10 days whereas urinary excretion accounts for
nearly 18.5% (Kremer et al., 2005).
An evaluation of Se status of human subjects is as difficult as it was shown for
animals and this question needs further research. Se deficiency in human
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population is associated with two diseases (Keshan disease and Kaschin-Beck

disease) reported in areas of China and some other countries characterised by
extremely low Se content in the soil and food. In human Se deficiency is associated
with immune system compromised and increased susceptibility to various diseases,
including arthritis, cancer, cardiovascular disease, cataracts, cholestasis, cystyc
fibrosis, diabetes, immunodeficiency, lymphoblastic anemia, macular
degeneration, muscular dystrophy, stroke and some others.
The most compelling evidence exists in relation to cancer-protective effect of
Se. Firstly in epidemiological observations and prospective studies an inverse
correlation between Se levels in food and blood and risk of cancer and cancer
mortality were observed. Secondly, there are case-control studies showing that Se
levels in blood, serum, hair or toenails are lower in cancer patients than in controls.
Thirdly, laboratory animal studies showed a protective effect of various forms of
Se against cancer initiation and development. Finally, there are human intervention
trials showing Se supplementation to be effective means of decreasing risk of
cancer.
There are several questions related to protective effect of Se against cancer,
which need to be addressed. First of all, chemically-induced carcinogenesis used
in animal models is in many cases different from those observed in human. In
particular chemicals, which were used to induce cancer in model systems in many
cases are different from those causing cancer in natural situations. There is a need
for deeper understanding molecular mechanisms of cancer-initiation and
progression. Secondly, Se doses used in the studies (1-5 ppm) are substantially
higher than those tested in human trials. In fact, in experimental animals, anticarcinogenic effects have been consistently associated with Se at supranutritional
intakes that are at least 10 times those required to prevent clinical signs of Se
deficiency and to support near-maximal tissue activities of selenoenzymes. The
pharmacological approaches (chemoprevention) should be assessed against
nutritional approaches (balanced diet) in terms of their efficacy of cancer
prevention. Indeed, in most of human trials the highest protective effect of Se
against cancer was seen in those participants who were initially Se-deficient or at
least were characterised by comparatively low Se status. Thirdly, in animal models
most of successful experiments showing anticancer effects of selenium were
conducted with inorganic selenium compounds, while Se-yeast was a preparation
of the choice tested in human trials. Fourthly, protective effect of Se can differ
depending on the diet composition especially on the presence and concentrations
of other antioxidants and pro-oxidants. Finally, there is sex-related differences in
cancer-preventing properties of selenium.
Recent data on relationship between Se and cancer reinforced an importance
selenium as cancer-preventing agent. For example, MEDLINE, EMBASE and
Cochrane Library between 1966 and May 2005 have been searched to conduct a
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systematic review and meta-analysis of the literature. Sixteen studies (eleven cohort
studies and five case-control studies) that examined the association between intake
of selenium and the risk of prostate cancer were included in the final analysis.
The results of the systematic review suggest that selenium intake may reduce the
risk of prostate cancer (Etminan et al., 2005). In fact, the pooled relative risk of
prostate cancer for any intake of selenium was 0.72 for cohort studies and 0.74
for case-control studies. The pooled relative risk of moderate intake was 0.74 for
cohort studies and 0.74 for case-control studies. Furthermore, prediagnostic
selenium concentrations measured in archived toenails were inversely associated
with bladder cancer risk in women (P for trend=0.02), but not in men, in a nested
case-control study of 338 cases and 341 matched controls (Michaud et al., 2005).
Similarly, a strong association of selenium with breast cancer in the Indian
population was shown. In particular a hospital based case-control study to examine
the associations of vitamin C, vitamin E and selenium with breast cancer was
conducted in India (Singh et al., 2005). One hundred and sixty breast cancer
patients and an equal number of normal healthy individuals constituted the study
population. The mean vitamin C, vitamin E and selenium levels were lower in
patients as compared to the controls. There was a 7% lower risk of breast cancer
if the level of selenium was increased by 1 unit.
Up to date there were 10 human trials to test protective effect of Se against
cancer and six of them were conducted in China, a country characterised by a
number of Se-deficient regions. The main outcome of the mentioned trials is a
protective effect of Se (in most cases Se-Yeast) against cancer. Recently, an
investigation evaluated the association between selenium supplementation and
prevalent and incident colorectal adenomas and colorectal cancers detected during
the Nutritional Prevention of Cancer trial follow-up. In addition to being associated
with a reduced risk of incident colorectal cancers, selenium supplementation was
associated with a significantly reduced risk of prevalent adenomas, but only among
subjects with either a low baseline selenium level or among current smokers (Reid
et al., 2005).
Aforementioned data provided a strong incentive to design a definite trial for
selenium and vitamin E with prostate cancer as a primary end point. Therefore
there are new human trials under way to further substantiate protective effects of
Se against cancer, including SELECT trial employing 32,000 participants without
evidence of prostate cancer from more that 400 participating study sites in the
USA, Puerto Rico and Canada. Further prostate cancer studies at the Arizona
Cancer Center and in Europe and Australia using Se doses of 200-800 g/d are in
progress.
There is a strong evidence to suggest a protective effect of Se supplementation
against side effects of cancer chemotherapy. Recently twenty patients have been
enrolled in the study. Each of the participants received 1 mg sodium selenite
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intravenously or orally daily for 3 wk during the pre-, intra-, and postoperative
period (Zimmermann et al., 2005). There was an inverse correlation between the
severity of the lymphedema and the whole-blood/plasma selenium concentration
and GSH-Px activity. In addition, a positive correlation between the ROS
concentration and the extent of lymphedema was observed. A significant reduction
of lymphedema occurred in the sodium selenite-treated group.
Molecular mechanisms of anticancer effect of Se are diverse and include
antioxidant effects of selenoproteins, stimulation of DNA-repair enzymes and
activation of the tumor suppressor protein p53, effect on gene expression,
enhancement of immune function, induction of apoptosis, inhibition of cell
proliferation, alteration of carcinogen metabolism, influence on estrogen and
androgen receptor expression, inhibition of angiogenesis, interactions with heavy
metals involving in cancer promotion and some others. Indeed, redox modification
of thiol/disulfide interchange in proteins by selenium could lead to protein
unfolding. Recent data supported the idea that unfolded protein response could
be an important mechanism in mediating the anticancer activity of selenium (Zu
et al., 2005) and endoplasmic reticulum stress response is shown to be important
in mediating the anticancer effect of selenium (Wu et al., 2005).
It has been shown that, ideally, increasing Se consumption to have Se
concentration in the blood >121 g/l would be a way to address Se deficiency
issues and simultaneously provide natural protection against cancer. It is not clear
at present how protective effect of increased Se concentrations in plasma happened.
It seems likely that in those people with the highest Se concentration in plasma
activity of GSH-Px is not much different from those who are in the lower Se
groups. The only difference would be the level of SeMet in the plasma. This
could also indicate higher Se reserves in the body of those patients. Therefore, it
could well be that the Se reserves in the body which could be protective in stress
conditions may play a crucial role in anti-cancer effects. Indeed, various stresses
are important in mutagenesis and further cancer initiation. Therefore, if SeMet
accumulated in tissues could be used for additional synthesis of selenoproteins,
this could provide an essential defence dealing with those stress conditions and
overproduction of free radicals leading to mutagenesis. Furthermore, SeMet per
se is shown to up-regulate DNA repairing enzymes increasing defences against
possible mutagenesis. Therefore, this could explain why those patients with
comparatively high Se in the blood and respectively high proportion of SeMet
(this could be indicative of Se reserves in the body) are more protected from
cancer than those with lower Se level in tissues and plasma.
In general, as mentioned above there are two approaches to achieve Se
protective effect against cancer. Firstly, nutritional approach including consumption
of Se-enriched food, such as eggs, meat, milk as well as various vegetables. This
could also include dietary supplements in the form of Se-yeast. There is a great
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body of evidence to support this approach, including epidemiological studies

and clinical intervention studies. Secondly, pharmacological approach, including
consumption of Se-tablets in the form of sodium selenite, SeMet, various
chemically synthesised organo-selenium compounds, etc. There is strong evidence
indicating that those Se compounds show cancer-protective effects in model
systems and in animal experiments. However, results of experiments with
chemically induced cancer models are difficult for interpretation and transfer for
humans.
The importance of deeper understanding of molecular mechanisms of anticancer
effect of various selenocompounds was underlined by the recent data suggesting
that changes in selenoprotein expression may either suppress or promote
tumorigenesis depending on cell type and genotype (Novoselov et al., 2005).
These authors investigated, the role of Se and selenoproteins in liver tumor
formation in TGFalpha/c-Myc transgenic mice, which are characterized by
disrupted redox homeostasis and develop liver cancer by 6 months of age. Under
the experimental conditions, both Se deficiency and high levels of Se compounds
suppressed hepatocarcinogenesis. In particular, it has been shown that both
treatments induced expression of detoxification genes, increased apoptosis and
inhibited cell proliferation (Novoselov et al., 2005). Clearly, these data indicate
that not all subjects will benefit from Se supplementation.
Dietary deficiency of selenium has been incriminated in the etiology of
cardiovascular diseases. However, the results of longitudinal studies within
populations are conflicting with some investigations observing a relationship
between low serum-selenium levels and the risk of coronary disease, while others
did not. In general, dietary Se supplement may be considered anti-atherosclerotic.
Mechanisms whereby selenium protects against CVD diseases include increased
resistance of low-density lipoproteins against oxidative modification; modulation
of prostaglandin synthesis and platelet aggregation and protection against toxic
heavy metals. Indeed, it is clear that Se could provide protective effect against
cardiovascular diseases. Selenium can inhibit the expression of MCP-1 by
repressing P38MAPK signaling pathway and therefore prevent the development
of atherosclerosis (Li et al., 2005). Furthermore, selenium can protect the heart
against ischemia-repefusion (I/R) injury due to its action on the redox state and
deactivation of NF-kappaB in I/R hearts (Turan et al., 2005). In fact selenium
modulates the cardiac mRNA expression of thioredoxin and glutathione related
enzymes post ischemia-reperfusion, and impacts on tolerance to ischemiareperfusion (Venardos et al., 2005). However, the therapeutic benefit of selenium
administration in the prevention and treatment of such diseases still remains
insufficiently documented and convincing proof of such an association can only
be obtained from large, controlled, prospective prevention trials.
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Data are actively accumulating to indicate that from the one hand, Se deficiency
is related to reproductive disorders in human, including poor semen quality and
pregnancy complications and from the other hand, the Se dietary supplementation
could potentially have prevent those changes. Human and animal studies suggest
that the developing fetus is susceptible to metabolic programming throughout
gestation (Maloney and Rees, 2005). Although there are still controversial areas,
there is at present sufficient scientific evidence for foetal programming to be
regarded as an additional risk factor for chronic disease, in interaction with genetic
and lifestyle risk factors (Delisle, 2002). Indeed, evidence from both human and
animal studies suggests that diseases of adult life are induced by the fetal
environment (Haimov-Kochman, 2005). Evidence is also emerging that suggests
programming of hormonal systems in response to an adverse fetal environment
may be one of the mechanisms underlying these long-term consequences of early
life events. In particular, alterations in the neuroendocrine response to stress may
play an important part (Phillips, 2004). The available evidence suggest that nutrient
sensing regulatory systems are present in many tissues during early development
(Maloney and Rees, 2005). Programming agents seem to include growth factors,
cytokines and hormones, all of which can be altered by stress. As a consequence,
such stress-modified systems of the offspring are more susceptible to
environmental influences during later life, e.g. the development of atopic diseases
upon exposure to antigens (Knackstedt et al., 2005). It seems likely that oxidative
stress is also involved in programming. Indeed, oxidative stress may be a common
link underlying the superficial programming associations between adverse fetal
growth or preterm birth and elevated risks of certain chronic diseases. The
mechanisms of oxidative stress programming may be through directly modulating
gene expression or indirectly through the effects of certain oxidized molecules
(Luo et al., 2005). Experimental investigations have well demonstrated the role
of redox balance in modulating gene expression, and recent studies indicate that
both the insulin functional axis and blood pressure could be sensitive targets to
oxidative stress programming. For example, reduced lifespan in rats subject to
prenatal protein restriction is a consequence of enhanced oxidative processes
promoting apoptosis and loss of tissue function (Langley-Evans and Sculley, 2005).
Furthermore, maternal treatment with cholesterol-lowering agents or antioxidants
greatly reduces fetal and postnatal atherogenesis, indicating a pathogenic role of
lipid peroxidation and a potential involvement of oxidation-sensitive signaling
pathways (Palinski and Napoli, 2002). Taking into account that Se can affect
concentration of the above-mentioned programming agents, it could well be that
Se plays an important role in the foetal programming.
An optimal Se status is shown to be beneficial in asthma, rheumatoid arthritis,
diabetes, HIV, pancreatitis, brain and neurodegenerative disorders. It is also
protective against radiation and can be considered as an anti-ageing agent. For
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example, a recent study with rats has shown that a period of 5-week diabetes was
enough to cause some important and degenerative changes in the structure of the
bone tissues, and it has demonstrated that selenium treatment of the diabetic rats
could normalize these alterations (Ozdemir et al., 2005). Furthermore, it has been
shown that oxidative stress is involved in the etiology of diabetes-induced downregulation of heart function via depressed endogenous antioxidant defense
mechanisms and Se supplementation could prevent such detrimental changes
(Ayaz and Turan, 2005). Further clarification of the Se role in diabetes came from
the data indicating that diabetic ischemia increased neuronal death by augmenting
the production of reactive oxygen species, such as superoxide radical and by
suppressing the antioxidant selenoprotein P (Tagavilla and Li, 2005). Further
studies of effect of selenium on arthritis are related to gene expression. For
example, the effects of SeMet on gene expression, activation of mitogen-activating
kinases, and DNA binding of nuclear factor-kB (NF-kB) and apolipoprotein-1
(AP-1) in articular chondrocytes have been recently determined
(Andriamanalijaona et al., 2005). Pretreatment with 0.5 M SeMet prevented IL1beta-induced matrix metalloproteinases (MMP-1) and aggrecanase-1 expression,
and reduced the cytokine inhibitory effect on type II collagen, aggrecan core
protein, and transforming growth factor-beta receptor II mRNA levels. Therefore,
SeMet stimulated the signalling pathway. Effects of Se on pancreatitis are also of
great interest. For example, intravenous selenium given 24 hours after induction
of experimental acute pancreatitis was associated with a reduction in the histological
stigmata of pancreatic injury and a dramatic reduction in broncho-alveolar lavage
protein content (Hardman et al., 2005). To further examine the hypothesis on
relationship between Se status and optimal health the relationships between plasma
selenium and mortality in an elderly population was studied in the EVA (Etude du
Vieillissement Arteriel) study (Akbaraly et al., 2005). The EVA study was a 9year longitudinal study with 6 periods of follow-up. Baseline plasma selenium
was higher in individuals who were alive at the end of 9 year follow-up (1.10
mol/L) than in those who died during the follow-up (1.01 mol/L). Furthermore,
mortality rates were significantly higher in individuals with low selenium (relative
risk (RR) = 1.56). After various potential confounding factors were taken into
account, this association remained significant (RR = 1.54). When the underlying
causes of death were considered, an association with cancer-related mortality
(adjusted RR = 1.79) was found (Akbaraly et al., 2005). Indeed, it is clear that
more research should be done in an area related to health-promoting properties of
various Se compounds.
The analysis of current literature indicates that an enrichment of eggs, meat
and milk with Se is a valuable option to improve Se status of the general population.
Such eggs are currently being produced in more than in 25 countries worldwide
delivering approximately 50% RDA in Se with a single egg. There are also various
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other combinations of egg enrichment including omega-3 PUFAs, vitamin E,

carotenoids, iodine, etc. Commercial technologies of the production of Se-meat
and Se-milk are under the development in various countries. As it was mentioned
above it seems likely that the major form of Se in Se-enriched egg, meat and milk
is SeMet and it is just a matter of time when it will be proven analytically. For
example, it has been shown recently that after organic Se supplementation SeMet
represented main form of Se in cow blood and milk (Palacios et al., 2005).
It has been suggested that for the past 150 years our diet has changed
substantially, while our genes have not been changed. In particular, animal-derived
food composition has been dramatically changed as a result of using cheap feed
ingredients. The meat from animals in the wild and chicken eggs produced under
complete natural conditions contains higher amounts of omega-3 fatty acids
compared to cultivated ones. Indeed, analyses of egg composition from various
avian species in wild conducted at the SAC indicate a great difference in fatty
acid, vitamin E and carotenoid profile of eggs between commercial laying hens
and wild birds. In fact, designer eggs enriched with omega-3 fatty acids are very
similar in fatty acid profile to eggs produced in wild or so called Greek eggs,
produced by free range birds having unlimited access to various greens, worms,
insects etc. The same is true for selenium and carotenoids. Indeed, decreased Se
levels in feeds and foods in many cases reflect consequences of our agricultural
practises. Therefore, eggs or meat produced by free-range poultry/animals fed on
natural feed sources grown on well-balanced soils 100-200 years ago would
contain much higher Se concentration than we currently have in many European
and Asian countries. Again, by supplementing animal diet with natural organic
sources of Se we are returning back to nature. Our recent data on the Se profile of
eggs from various avian species in wild (Table 14.4) confirmed this idea: Se
concentration in eggs collected in wild nature in many cases much higher than
that in commercial poultry production. Our results may imply that the Se
requirement for birds breeding in captivity will vary among species. An appropriate
guideline could be provided by considering the yolk Se concentration displayed
by the free-living counterparts of that species. The Se level in the chicken eggs
even after organic Se supplementation (Surai, 2000) only raised the yolk Se level
into the lower end of the range achieved by avian species in the wild, suggesting
there may be scope for much higher levels of supplementation for poultry. It
seems likely that Se level which is considered to be the norm for the commercial
eggs is too low to be physiological and this should be studied more in detail in the
future. Similar evidence of high Se concentrations in wild water birds were related
to eggs of little egrets, black-crowned night herons and bridled terns from coastal
areas of Hong Kong (Lam et al., 2005). In tissues of the seabirds from the Barents
Sea (Savinov et al., 2003), from Alaska and arctic Russia (Stout et al., 2002) as
well as in bald eagles from Adak Island (Alaska; Stout and Trust, 2002) selenium
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Table 14.4 Selenium concentration (ng/g wet yolk) in egg yolks of free-living avian species (Adapted
from Pappas et al., 2005)
Common name
Scientific name
Country
Yolk Se (ng/g)
Gull
Moorhen
Black coot
American coot
Canada goose
American crow
House sparrow
Barn swallow
Tree swallow
Yellow headed
blackbird
Brewers blackbird
Blackbird
Song thrush
Starling
Larus fuscus
Gallinula chloropus
Fulica atra
Fulica americana
Branta canadensis
Corvus brachyrhynchos
Passer domesticus
Hirundo rustica
Tachycineta bicolour
Xanthocephalus
xanthocephalus
Euphagus cyanocephalus
Turdus merula
Turdus philomelos
Sturnus vulgaris
UK
UK
UK
Canada
Canada
Canada
Canada
Canada
Canada
744
430
394
503
732
1190
939
Canada
Canada
New Zealand
New Zealand
New Zealand
1033
1026
1081
989
1373
34
34
39
36
124
153
75
1896
2238
65
11
95
54
85
Values are means S.E.M
levels were also several-fold higher in comparison do domestic chickens.

Furthermore, high Se concentrations were reported in eggs from tree swallow,
bank swallow and house wren (Dickerson et al., 2002). Therefore Se-enrichment
of eggs, meat and milk is nothing else but production of naturally-designed food
ingredients. Indeed, production and commercialisation of such organic Se sources
as selenized yeast (for example Sel-PlexTM) opened a new era in Se supplementation
of animals and gave a real chance for producers to meet growing requirements of
consumers. What is more, production of these kind of animal-derived foodstuffs
is a natural way to health promotion.
Indeed, it is possible to provide consumers with a range of animal-derived
deliver substantial amount of health-promoting nutrients such as selenium to
improve general diet and help to maintain good health. Therefore, without
changing habits and traditions of various populations (habit is a second nature)
it is possible to solve problems related to deficiency of various nutrients, in
particular selenium. The consumer will go to the same supermarket to buy the
same animal-derived products (egg, milk and meat), cook and consume them as
usual. The only difference will be in the amount of specific nutrients delivered
with such products.
Relationship between food and human health has attracted attention of scientists
for many years. It is now widely accepted that fruits and vegetables are important
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dietary components responsible for maintenance of good health. However,

molecular mechanisms of protective effects of fruits and vegetables have not
been fully elucidated. One of the attractive idea is that various antioxidant
compounds of fruits and vegetables are responsible for prevention of oxidative
damage in digestive tract. In fact, antioxidant-prooxidant balance in digestive
tract is considered to be a major determinant of human and animal health. Analysis
of the literature related to human diet showed that from the one hand, there is a
range of harmful compounds in the human diet including oxidized PUFAs,
oxysterols, traces of mycotoxins, heavy metals and other environmental pollutants.
A combination of such compounds potentially could stimulate lipid peroxidation
and cause DNA damages in the intestine leading to mutagenesis and cancerogenesis.
On the other hand, the diet contains a range of antioxidant compounds including
vitamin E, carotenoids, vitamin A, ascorbic acid, coenzyme Q, flavonoids etc.
which potentially can deal with those free radicals produced in the digestive tract
as a result of aforementioned toxic compounds action. Therefore a balance between
antioxidants and pro-oxidants in the digestive tract is a key for general health.
Furthermore, some nutrients which are not absorbed could have health-promoting
properties. For example, it is well known that various flavonoids are not well
absorbed in the small intestine and as a result many of them will be available in
the large intestine providing there an important antioxidant protection, preventing
lipid peroxidation, damages to DNA and cancer.
There is a specific place for selenocompounds in the digestive tract. From the
one hand sodium selenite is a reactive compound which in combination with
reduced glutathione (which can be found in the digestive tract) could potentially
produce free radicals contributing to lipid peroxidation there. On the other hand,
organic selenium in the form SeMet possesses antioxidant properties per se and
could have completely different protective effect. Furthermore, a beneficial effect
of SeMet on the DNA repairing enzymes could be relevant to anticancer effect of
Se in the intestine. Furthermore, specific gastro-intestinal GSH-Px located in the
intestine and responsible for decomposition of lipid peroxides. In fact, if activity
of this enzyme is optimal the lipid peroxides found in the digestive tract will not
be able to be absorbed and as a result will not be found in the blood. However, if
the activity of this enzyme is low lipid peroxides can escape pre-absorptive
detoxication leading to their incorporation into plasma lipoproteins. Indeed, lipid
peroxidation in LDL is an important factor of atherosclerosis. However, it is not
clear at present what is the trigger of that peroxidation. It could well be that traces
of oxidised lipids incorporated into the lipoproteins could be triggering factors
for further lipid peroxidation. This could explain a relationship between Se status
and CVD. Clearly healthy gut is one of the most important determinants of the
general health of human and animals. Until now major attention to the gut was
related to the bacterial population and various prebiotics and probiotics were used
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to maintain beneficial microbes there. However, the antioxidant-prooxidant

balance in the digestive tract should be studied more in detail. In fact, recently
antioxidant potential of the chicken intestine has been characterised (McLean et
al., 2005).
Selenium is shown in high doses to be toxic. In fact, historically Se toxicity
was discovered earlier than its essentiality. That is why Se toxicity for many years
was first to consider in different textbooks. There are also various environmental
issues related to accumulation of Se in various water reservoirs up to toxic levels.
This lead authorities in many countries to restrict Se supplementation in the animal
diets. For example, in the EU countries legal Se level in the complete feed should
not exceed 0.5 ppm. In the US legal limit of Se supplementation is set up at 0.3
ppm of the supplemental Se. In recent years a great body of information has been
accumulated to show, firstly, that Se toxicity started at doses, which are at least
10-fold higher than those commercially relevant. Secondly, in most of cases
organic selenium is less toxic than sodium selenite, independently on the Se
concentration in tissues. This means that Se concentration in tissues is not
necessarily related to Se toxicity. The form of Se is a more important factor and
promotion of lipid peroxidation by high doses of sodium selenite is considered to
be an important mechanism of the Se toxicity. When organic Se is included into
the diet more Se retained in the body and less is released with manure. This means
that legal limits of dietary Se supplementation, developed based on data with
sodium selenite, should be reconsidered and increased. This would give more
flexibility for feed/premix producing companies as well as for poultry, pig and
dairy producers who wish, for example, to produce Se-enriched eggs, meat and
milk.
The analysis of the literature presented in this volume reinforced importance
of Se in animal and human nutrition and health. Indeed, the global Se inadequacy
is responsible for increased susceptibility to various diseases, including major
modern killers namely the cancer and CVD. Optimisation of Se nutrition of poultry
and farm animals will allow to increase efficiency of egg, meat and milk production
and what is more important, to increase their quality. From the data presented
above it is clear that organic selenium is an effective choice to achieve this goal.
Really we are not what we eat, we are what our animals eat.
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Turan, B., Saini, H.K., Zhang, M., Prajapati, D., Elimban, V. and Dhalla, N.S. (2005).
Selenium improves cardiac function by attenuating the activation of NF-kappaB
due to ischemia-reperfusion injury. Antioxidants and Redox Signalling 7: 13881397
Tverdochlebov, A.A., Papazyan, T.A., Davtyan, D.A. and Surai, P.F. (2005). Effect
of organic selenium on goose reproduction. In: Eurola M. (Ed) Proceedings:
Twenty Years of Selenium Fertilization. September 8-9, 2005, Helsinki, Finland.
MTT Agrifood Research Finland, Data and Information Services, p.96
Venardos, K., Ashton, K., Headrick, J. and Perkins, A. (2005). Effects of dietary
selenium on post-ischemic expression of antioxidant mRNA. Molecular and
Cellular Biochemistry 270: 131-138
Wu, Y., Zhang, H., Dong, Y., Park, Y.M. and Ip, C. (2005). Endoplasmic reticulum
stress signal mediators are targets of selenium action. Cancer Research 65:
9073-9079
Yathavakilla, S.V., Shah, M., Mounicou, S. and Caruso, J.A. (2005). Speciation of
cationic selenium compounds in Brassica juncea leaves by strong cationexchange chromatography with inductively coupled plasma mass spectrometry.
Journal of Chromatography A. (In Press)
Yoshida, M., Sugihara, S., Inoue, Y., Chihara, Y., Kondo, M., Miyamoto, S. and
Sukcharoen, B (2005). Composition of chemical species of selenium contained
in selenium-enriched shiitake mushroom and vegetables determined by high
performance liquid chromatography with inductively coupled plasma mass
spectrometry. Journal of Nutritional Science and Vitaminology 51: 194-199
Zimmermann, T., Leonhardt, H., Kersting, S., Albrecht, S., Range, U. and Eckelt, U.
(2005). Reduction of postoperative lymphedema after oral tumor surgery with
sodium selenite. Biological Trace Element Research 106: 193-204
Zu, K., Bihani, T., Lin, A., Park, Y.M., Mori, K. and Ip, C. (2005). Enhanced selenium
effect on growth arrest by BiP/GRP78 knockdown in p53-null human prostate
cancer cells. Oncogene (In Press)
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954
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Index 955
INDEX
Absorption, see selenium

Acetonitrile 133
Acute phase protein 96, 214, 215, 221, 261, 603
Adhesion molecules 255, 256, 704, 705
Adipose tissue 31,80, 89, 92, 105, 172, 223, 367, 541
Adriamycin 11, 706
Adjuvant 30, 733, 738
Aflatoxin, see Mycotoxins
Albanian paradox 648
Albumin 12, 157, 163, 167, 233, 375, 392, 417, 507, 555, 560, 658, 733, 823, 927
Alkoxyl radical see free radicals
Aluminosilicate 344
Alzheimers disease 8, 666, 739
Aminomalonic acid 6
Aminotransferase, see Enzymes
Androgen 289, 707, 709, 716, 942
Anorexia 221, 321, 603, 604, 605, 954
Antibiotics 26, 224, 322, 489, 506
Antibodies 68, 214, 220, 229, 242, 253, 260, 339, 399, 931
Titre 229,230, 231, 232, 249, 250, 599, 868
Antigen recognition 221, 253
Antioxidant defence
In embryonic tissues 374-385
In semen 286-301
Three levels 7-27
Antioxidant enzymes
Catalase 7, 11, 12, 13, 25, 26, 56, 58, 79, 109, 112, 325-329, 368, 374-382, 595, 596,
735, 745, 857, 865, 889, 924
In embryonic tissues 374-385
GSH-Px 45-116
Classical GSH-Px 45- 67
GI-GSH-Px 74
GSH-Px6 76
non-Se-GSH-Px 79-82
955
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956
p-GSH-Px 73-74
PH-GSH-Px 67- 72
snGSH-Px 74-76
Role 76- 79
SOD 7, 9
Cu,Zn-SOD 10
Extra-cellular Cu,Zn-SOD 10
Fe-SOD 10
Mn-SOD 10
Antioxidant-prooxidant balance 487, 589, 599, 601, 857, 889, 891, 895, 897, 924, 948, 954
Antioxidant protection
Brain 325-329
Kidney 331
Liver 322-325
Lung 330
Muscle 331-332
Seminal plasma 415-422
Spermatozoa 404-415
Yolk 311-316
YSM 329-330
Antioxidant recycling 25, 87, 422
Antioxidant system 1, 7, 9, 14, 16, 23, 25, 27, 30, 101, 102, 106, 248, 258, 259, 279, 286,
303, 304, 324, 344, 347, 363, 368, 373, 377, 381, 383, 385, 392, 397, 400, 414, 422, 446,
451, 474, 540, 550, 595, 610, 615, 624, 734, 834, 838, 857, 865, 870, 878, 891, 923,
926, 930, 935
Apoptosis 18, 19, 25, 28, 33, 51, 61, 62, 64, 70, 71, 72, 77, 83, 86, 101, 108, 111, 112, 116,
250, 256, 258, 259, 318, 322, 342, 347, 373, 374, 492, 612, 613, 614, 700, 703, 707,
709, 710, 711, 713, 714, 715, 717, 718, 726, 732, 751, 862, 866, 870, 879, 888, 896, 924,
926, 942, 943, 944
HIV 735
Mycotoxin 326-330
Asbestosis 8
Ascaridia galli 244
Ascorbic acid
Antioxidant defence 13, 279, 286
Antioxidant properties 14-15
Cancer 676
Embryonic tissues 372-385
GIT 116, 857, 858, 882,890
Horses 596
Mycotoxins 325-326, 344
NE 373
Se absorption 183
Semen 279,286
Sodium selenite 112, 114, 192, 197
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Index 957
TR 87,107
Vitamin E recycling 23, 24, 25, 106, 372
Ascites 410, 423, 447
Aspergillus 317, 318, 319, 602
Asthenospermia 69
Ataxia 19, 96, 370, 617
Atherosclerosis 8, 644, 720, 721, 726, 727, 751, 818, 821, 884, 943
Autoimmune disease 8, 215, 259, 652
Barley 176, 177, 190, 296, 319, 320, 321, 322, 459, 554, 837
Beta-carotene 31, 601, 676, 697
Bile 384, 608, 617, 658, 669, 670, 678, 688, 864, 870
Bilirubin 25, 595
Bioavailability
Se 66, 154, 162, 163, 169, 171-181, 197, 478, 526, 527, 547, 548, 607, 611, 625, 627,
689, 813, 833, 837, 929
Brain
Antioxidant protection 25, 378
CoQ 876, 877
GSH-Px 57, 58, 59, 62, 69, 80, 183, 376, 380
ID 89, 90
Lipid peroxidation 372, 373, 377, 609
Mycotoxins 326
NE 106
Se 50, 166, 180, 390, 391, 419, 658, 739, 740
SeMet 164
SeP 95, 96, 97
SeW 92, 93, 94
TR 88
Broccoli 157, 158, 657, 689, 690, 692, 880
Brucella 233, 249, 332, 954
Bursa of Fabricius 88, 221, 234, 243, 334, 336, 341, 399, 410
Butylated hydroxytoluene (BHT) 886
Butylated hydroxyanisole (BHA) 373, 603, 886
Calcium 104, 154, 254, 365, 492, 528, 534, 537, 608, 614, 646, 670, 675, 680
Cancer 666-671
Se 671- 729
Carnosine 377
Carotenoids
Antioxidant system 7, 9, 13, 14, 26, 29, 102, 260
Chick embryonic development 29, 374, 375
Liver 376
Other tissues 377, 379, 380
Diet and food 650, 651, 809, 816, 827, 841, 857, 858, 880- 882, 894, 948
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Feed 115, 116

Fish pigmenting 622, 623
Functional food 615
Immunity 612, 615
Mycotoxins 324, 345, 346
Vitamin E recycling 23, 24
Catalase, see Antioxidant enzymes
Cataracts 7, 8, 104, 611, 618, 665, 940
Catechin 23, 112, 858, 883
Cerebellum 56, 93, 105, 370, 371, 372, 378
Ceruloplasmin 12, 13, 31, 595
Chemotaxis 228, 236, 241, 254, 747
Chicken 363-424
Egg 363-424
Embryo 374-385
Immune system 213-262
Mycotoxins 317-347
Semen 279-305
See also vitamin E, carotenoids, selenium
Cholesterol
Diet and food 819
Egg 818-821
Esters 73, 817
HDL 722, 726, 727, 747, 819
Heart disease 818-821
LDL 648, 650, 727
Oxides 73, 823, 861-862, 893
Plasma and serum 727, 819, 839
Choline hydroperoxide 67, 71, 73, 74
Chromatin 6, 75, 76, 77, 285, 289, 329, 933
Cobb broilers and breeders 386, 403, 407, 408
Coccidia and Coccidiosis 235
Coccidiostats 31
Coenzyme Q 14, 858, 876-880, 948
Columbus eggs 818, 825, 826, 827, 843, 894, 954
Complement 20, 214, 215, 216, 222, 223, 258, 332, 333, 340
Concanavalin A (ConA) 223, 335, 508, 612, 747
Conjugated diene 327, 866
Copper 468, 519, 546, 596, 621
Binding protein 12, 13
SOD 10
Corn 156, 157, 158, 174, 184, 185, 190, 319, 320, 322, 403, 446, 450, 460, 874
Coronary heart disease 250, 644, 647-649, 652, 720-729, 819-820, 892
Crohns disease 652
Cysteine 16, 19, 22, 23, 24, 45, 59, 83, 85, 198, 366, 368, 608, 926
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Index 959
Cystic fibrosis 665, 940
Cytokines 10, 106, 365, 539, 603, 944
Immunity 214-262, 712, 872, 930
Mycotoxins 339
Selenoproteins 65, 78
Cytotoxicity 11, 64, 65, 94, 111, 112, 113, 114, 712, 862
Immunity 216, 221-223, 237-241, 250
Mycotoxins 330-331, 343-344
Daidzein 650
Deficiency of
Selenium 250-252
Cattle 487-521
Diagnosis 534
Dogs 604
Fish 616
Horses 589, 604
Human 659-661, 665, 725-735, 740, 746, 749, 810, 825, 832, 842, 892, 923-932,
935-945
Pigs 446-455, 471, 476
Poultry 363-374, 394, 418
Vitamin E 23, 102, 104, 105, 106, 107, 229, 234, 237, 241, 256
Cat 604
Fish 617
Human 875
Pigs 446- 453
Poultry 364 - 372
Ruminants 488-506
Dehydroascorbic acid 14, 87
Delayed-type hypersensitivity (DTH) 222, 240, 254, 332, 334, 612, 613
Dendritic cells 214, 222
Deoxynivalenol, see Mycotoxins
Depression 652, 740, 741, 742
Diet
Hunter-gatherer 644-650
Ideal 644, 645, 650, 651, 754
Japanese 644-650
Mediterranean 644, 645, 646, 647, 648, 651
Paleolithic 645-650
Western 653, 654
Digestive system 151, 383, 419, 470, 646
Diketo-L-gulonic acid 24
Dioxine 30
DNA
Adduct formation 112, 324, 325,342, 343, 687, 688, 690, 691, 710,861, 870
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Damage 4, 5, 17, 18, 19, 61, 62, 111, 114, 115, 197, 320, 343, 649, 683, 708, 709, 710,
711, 861, 866, 868, 948
Repair system 17, 18, 19, 25, 115
Strand breaks 6, 17, 18, 31, 61, 111, 114, 326, 714, 715
Drip loss 115, 197, 408, 414, 422, 423, 473, 474, 478, 556, 560, 830, 831, 832, 936
Drosophila melangaster 46, 50, 68, 748
Duck 105, 106, 365, 366, 367, 369, 394, 400, 401, 411, 455, 604, 933
Embryo 384
Mycotoxins 319, 326
Sperm 279, 280, 284, 292, 293
Duodenum 163, 521, 883, 889
E coli 11, 83, 243, 338, 409
E tenella 243, 251
Egg, see Vitamin E, Carotenoids, Selenium
Eggs
Designer 817-827
Greek 843, 946
n-3 (Omega-3) designer 818-841
Super 818-841
Eicosanoids 214, 218, 248, 254, 257, 279, 371, 651, 873, 930
Embolism 652
Embryonic degeneration 105
Emphysema 8
Encephalomalacia 102, 105, 106, 321, 364, 367, 370-373, 385
Endoplasmic reticulum 84, 90, 97, 100, 101, 617, 942
Endotoxin 8, 62, 614
Environmental pollutants 858, 867, 868, 874, 896, 948
Enzymes
5I-apurinic endonuclease 17, 329
Aconitase 12
Cyclooxygenase 77
DNA glycosylase 17
DNA polymerase 17
Gastrointestinal GSH-Px 46, 50, 60, 74, 78, 107, 886, 887, 893, 925
Glucose-6 phosphate dehydrogenase 24, 286
Glutamic-oxalacetic transaminase (GOT) 449, 450
Glutathione reductase 9, 16, 23, 24, 53, 56, 57, 82, 85, 87, 165, 368, 595, 610, 887, 890
Glutathione-S-transferase 56, 79, 81, 82, 617, 715, 734, 861,890
Iodothyronine 5-deiodinase 46, 50, 60, 89-92, 177, 178, 470, 511, 541, 938
Lactate dehydrogenase (LDH) 71, 111, 286, 449, 490, 492, 742
5-lipoxigenase 254
Mieloperoxidase 228, 613, 872, 873
NADPH oxidase 28
Phospholipase A2 (PLA2) 82, 371
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Index 961
Phospholipid hydroperoxide GSH-Px (PH-GSH-Px) 46, 49, 50, 54, 59, 67-72, 75, 77, 78,
92, 96, 99, 107, 289, 304, 458, 531, 925, 933
Plasma GSH-Px 46, 73-74
Proline hydroxylase 12
Protein kinase 86, 714
Protein phosphatase 86
Ribonucleotide reductase 12, 51, 83, 86
Selenophosphate synthetase 47, 97
Specific sperm nuclei glutathione peroxidase 67, 74, 75, 289, 925, 933
Succinate dehydrogenase 12
Thioredoxin 9, 19, 21, 51, 82, 83, 84, 87, 96, 227, 290, 291
Thioredoxin peroxidase 9, 11, 83, 85
Thioredoxin reductase 9, 12, 14, 19, 21, 23, 50, 51, 82-89, 103, 1106, 107, 168, 253, 258,
374, 418, 716, 724, 857, 877, 887, 893, 928
Tyrosine hydroxylase 12
Xanthine oxidase 2, 10, 26, 65, 597
Epididymis 89, 409, 427, 433
Epilepsy 52, 81, 289, 290, 291, 296, 299
Erythrocyte 51, 52, 55, 56, 57, 80, 84, 93, 105, 165, 166, 169, 170, 172, 177, 179, 181, 182,
183, 230, 296, 299, 405, 409, 449, 452, 498, 531, 535-537, 547, 549, 557, 560, 592, 595,
603, 609, 624, 683, 742, 746, 813
Essential oils 26, 858, 885
Ethanol 8, 56, 57, 610, 738, 870, 871, 888
Ethoxyquin 26
Exudative diathesis 102, 104, 105, 172, 174, 183, 256, 364-366, 367, 385, 617
Faeces 162, 164, 180, 197, 448, 524, 525
Fatty acids
Arachidonic (20:4n-6) 5, 280, 282, 370, 371, 372, 375, 379, 380, 601, 645, 652, 727,
860
Docosatetraenoic acid (22:4n-6) 280, 282
Docosapentaenoic acid (DPA, 22:5n-3) 280, 282, 859
Eicosapentaenoic acid (EPA, 20:5n-3) 650, 652, 859, 860
Linoleic (LA, 18:2n-6) 370, 371, 372, 651, 727,825, 890
-Linolenic (ALA, LNA, 18:3n-3) 371, 372, 651, 654, 818, 825, 843, 860
Monounsaturated 648, 840
n-3 (omega-3) 615, 646, 651-654, 815, 818, 827, 835, 840-843, 946
n-6 (omega-6) 646, 651-654, 894
Polyunsaturated 4, 25, 78, 259, 279, 280, 341, 374, 375, 377, 397, 492, 546, 601, 616,
620, 649, 653, 660, 809, 817, 840, 924, 938
Saturated 4, 25, 399, 449, 646, 817, 819, 820, 840
Favism 8
Fenton reaction 3, 12, 863
Ferritin 12, 863
Fertility, see spermatozoa
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Fetal resorption 743

Fibre 367, 491, 589, 590, 645
Fish 645-654, 732, 812-819, 859, 861-867, 876, 886, 892-895
Health and immunity 621-622
Human food 626-627
Meal 173, 174, 175, 190, 619, 623, 626, 895
Oil 388, 389, 392, 403, 654, 859, 870
Quality 622 623
Se deficiency 616-618
Se requirement 620-621
Se toxicity 618-620
Fishy taste 841
Flavonoids 26, 115, 116, 369, 615, 648, 649, 650, 809, 857, 858, 883-885, 890, 891, 895,
948
Free fatty acids (FFA) 817
Free radicals
Alkoxyl radical 2, 6, 12, 15, 25, 883
Damages to DNA see DNA damage
Damages to proteins 6, 19,100, 106, 546,649, 730, 884, 926
Damages to PUFA 4-7, 447
External sources 2
Hydroperoxyl 2
Hydroxyl radical 3-6, 12, 15, 16, 87, 114, 217, 218, 324, 708, 863-866, 883
Internal sources 2
Peroxyl radical 5, 6, 12, 13, 113, 874, 878, 890
Phenoxyl radical 23
Superoxide radical 3, 4, 5, 9, 10, 12, 13, 26, 107, 109, 110, 111,218, 385, 499, 735, 862,
923, 945
Tocopheroxyl radical 13, 24
Fruit 185, 186, 189, 190, 644, 645, 646, 647, 648, 651, 811, 816, 841, 857, 858, 867, 876,
880, 881, 882, 883, 884, 892, 893, 895, 947, 948
Protective effects against cancer 891
Fumonisins, see Mycotoxins
Functional foods 197, 615, 654, 809, 811, 814, 835, 837, 838, 839, 841, 842, 896
Furazolidone 32
Fusarium graminearun 320,
Fusarium moniliforme 321
Fusarium species 317, 318, 322
Gap junction 113, 881
Gene expression 11, 19, 25, 33, 58, 97, 101, 108, 342, 382, 392, 394, 396, 612, 651, 709,
711, 717, 732, 866, 868, 873, 879, 889, 924, 935, 942, 944, 945
Genistein 649, 650, 884
Gizzard myopathy 367
Glucocorticoids 51, 539, 889
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Index 963
Glucomannan 344, 245
Glucose 24, 101, 183, 184, 192, 226, 609, 612, 649, 743, 872, 882
Glutaredoxin 9, 14, 52, 82
Glutathione
Antioxidant 15-16
In semen 286
Glutathione peroxidase, see Antioxidant enzymes
Glutathione reductase, see Enzymes
Guinea fowl 280, 292, 293, 933
Gull 417, 947
Haber-Weiss reaction 3
Haemagglutination inhibition (HI) 229
Haemoglobin 475, 504, 863
Haptoglobin 12
Hatchability 105, 302, 322, 323, 367- 370, 402-404,415, 416, 421-423, 935, 936
Heavy metals 8, 30, 96, 173, 177, 182, 602,709, 717-718, 725, 858, 865-867, 874, 888, 896,
942, 943, 948
Hemopexin 12
Hemorrhages 104, 365, 370, 448, 450
Heart
Antioxidants 380
Fatty acids 380
Heat shock protein 25, 410, 888, 925
Hemaglutination (HA) 222
Hepatotoxicity 319, 320, 752
Herbicides 30
Heterophils 30, 335, 399
Hormones 49, 89, 219, 287, 498, 538, 542, 558, 603, 752, 944
Hydrogen peroxide 2, 3, 6, 11, 53, 60, 72, 73, 76,77,80, 87, 110, 217, 228, 249, 290, 291,
329, 376, 546, 663, 866, 868, 879
Hydroperoxides 13, 14, 15, 31, 52, 53, 54, 69, 70, 73, 74, 79-82, 87, 102, 107, 115, 173,
254, 286, 288, 290, 859, 860, 883
Lipid hydroperoxides 12-13, 67, 7682, 96, 376, 709, 725, 859-863, 874, 890
5-hydroxyeicosatetraenoic acid 254
15-hydroxyeicosatetraenoic acid 372
Hypertension 651, 652, 720, 730
Hypochlorous acid 2, 15, 16,217, 613
Ileum 163, 889
Immune system 214-262
Immunity
Adaptive (acquires, specific) 214-262
Cell mediated 214-262
Humoral 214-262
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964
Natural 214-262
Immunoglobulin 214-262
Immunomodulation 51, 102, 262, 410, 608, 612, 718, 733, 754
Immunosuppression 213,225,259, 260, 261, 320, 323, 324, 330, 331, 340,341, 516, 707,
713, 931
Industrial solvents 2
Infertility 247, 263, 285, 289, 303, 488, 495, 505, 512, 729
Inflammation 2,3,8, 30, 83, 108, 216, 223, 245, 254, 255, 259, 488, 499, 504, 505, 506,
597, 608, 688, 703, 712, 724, 731, 732, 733, 869-873, 889,890, 896
Interferon (IFN) 20, 215, 253, 255, 256, 331, 340, 712
Intestine
Antioxidant-prooxidant balance 857-897
Interleukin (IL) 215, 253, 341
IL1 65, 218, 227, 261, 603, 790
IL2 237, 242, 335, 747
IL6 20, 218, 227
Iodothyronine deiodinases, see Enzymes
Iron 8, 55, 57, 155, 379,380, 449, 453, 468, 475, 476, 528, 546, 612, 621, 689, 812, 814,
815, 834, 862,-864, 866, 891, 896
Lipid peroxidation inducer 12, 260, 413, 422, 492, 615, 874, 882, 889, 931
Isoflavonoids 858
Jejunum 164, 521, 889
Kidney
Antioxidants 380-381
Fatty acids 380-381
Klebsiella pneumoniae 247
Lactoferrin 12
Lameness 247, 367, 475, 488, 493, 512, 591
Laying hens 105, 184, 365, 375, 387, 391, 404, 405, 412, 416, 417, 418, 419, 423, 821,
822, 835, 840, 843, 935, 936, 946
Leukocytes 3, 106, 215, 217, 224, 234, 235, 255, 365, 498, 499, 614, 705, 710, 730, 747,
872
Leukotrienes 76, 77, 217, 218, 254
Linseed oil 372, 451
Lipase 9, 17, 82, 384, 398, 473
Lipid classes
Phospholipid 23, 82, 727, 879
Chicken embryo 375, 377, 379, 380, 381
Egg yolk 817
Free fatty acids 817
Semen 280, 281, 282, 284, 285, 932
Triacylglycerol 280, 860
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Index 965
Lipid peroxidation 3, 4, 27-32 397, 399, 420, 421
Egg 416, 822, 823, 824
Embryo 378-385
GIT 615, 842, 859-874
Horses 597
Human 654, 724, 725, 726, 728, 730, 739
Immune system 241, 248, 250, 253
Initiation 4-5
Meat 411- 415, 832, 834, 835, 892
Mycotoxins 318-330
Petfood 603
Pig 451, 459, 473
Propagation 5
Ruminants 492
Sperm 279, 281-295
Stomach 891
Taurine 609, 610, 611
Lipofuscin 8, 491, 605
Lipoic acid 14, 23, 87
Lipopolysaccharide (LPS) 223, 227, 748
Lipoproteins
HDL 166, 819
Cholesterol 648, 722, 726, 727, 747
LDL 73, 166, 649, 725, 878
Cholesterol 648, 650, 726
Oxidation 62, 648, 721, 725, 726, 884, 885,890, 925, 948
Lymphocyte 18, 58, 113, 114,214,216, 219, 221-260 , 366, 399, 491, 501, 504, 597, 612,
613, 618, 705, 710, 735, 747
B 214, 215, 220-260, 930
T 214, 215, 220-260, 930
Mycotoxins 326- 342
Lymphocyte proliferation assay 223
Macrophages 4, 55, 214-228, 250- 261, 451, 501, 504, 613, 622, 747, 872, 873, 930
Mycotoxins 331-342
Macular degeneration 7, 8, 644, 665, 940
Maize 446, 863
Diet 389
Mycotoxins 318, 320, 321, 322, 323, 869
Malabsorption 213, 659, 842, 875
Malaria 8, 243
Malondialdehyde (MDA) 16, 822-824, 860-866
Chicken embryo 378, 396, 397
Human 730, 734, 737
Muscles 412, 413, 414, 835
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Mycotoxins 325-346
Semen 283-298
Taurine 609, 610, 611
Manganese 10
Mareks disease (MD) 243, 250, 410
Mast cells 215, 216, 222
Meat quality 26, 103, 107, 411-415, 473-476, 833-835
Membrane fluidity 233, 281, 337
Metallothionenin 717, 866
Metal-binding proteins 9, 12, 25, 924
Mieloperoxidase, see enzymes
Migration inhibitory factor (MIF) 236, 240, 249, 872
Mitochondria 63,70, 71, 112, 228, 286, 366, 368, 450, 451, 491, 492, 617, 660, 714, 739, 868
A source of free radicals 2, 26, 589, 923
CoQ 877, 878, 879
Electron transport chain 4
GSH-Px 52, 59, 60, 67, 68, 69, 72, 75, 289, 728, 933
MCS 98-99
MsrA 20
ROS 329, 861
Semen 296, 304
SOD 10, 857
TPx 85
TR 46, 86, 88, 89
Trx 83, 84
Mitogen 28, 86, 223, 225, 226, 233-243, 250, 260, 333,335, 336, 338, 339, 341, 612, 712,
714, 747, 931, 945
Molting 30
Moulds 213, 319
Multiple sclerosis 8
Mutation 11, 61, 100, 219, 251, 252, 262, 671, 708-713, 742, 744, 932
Mycosorb 344-347
Mycotoxicosis 107, 318, 330, 342, 346
Mycotoxins
Aflatoxin 230, 317, 318, 319, 322, 323, 327, 331, 332, 341, 343, 344, 362, 602, 604,
868-870
Aurofusarin 326-328
Deoxynivalenol (DON) 317, 318, 320-344, 870
Fumonisins 317-347
Ochratoxin A 317 347, 602, 869
Patulin 870
T-2 toxin 317, 318, 320, 323-331, 337, 341, 344, 345-347, 870
Trichothecenes 318, 320, 331, 340
Zearalenone 317-347, 870
Myoglobin 10, 12, 863, 864
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Index 967
Natural killer cells (NK cells) 214, 216, 221, 222, 239, 253, 259, 260, 931
Neutrophils 15, 214-262, 338-339, 383, 499, 501-508, 614, 873, 930
Newcastle disease/virus (NDV) 229, 230
Nitric oxide 2, 6, 28, 101, 217, 255, 337, 726, 872, 973, 887
Nitrogen dioxide 2, 865
3-nitrotyrosine 6
Nitrous acid 2
Oats 175, 176, 186, 190, 225, 236
Mycotoxin 319, 322
Ochratoxin A, see Mycotoxins
Ocular haemorrhage 8
Oedema 104, 321, 364, 490, 741
Oestrogens 650
Oligoasthenospermia 69
Olive oil 114, 644-648, 894
Oxidative stress 10-11, 20-32, 56, 62-65, 72-116,252-262, 289, 320, 341, 345, 372-383,
409, 445-470, 495, 539, 589, 595, 597, 602-627, 649, 708, 730, 731, 733, 734-750, 859891, 924, 925, 930, 932, 944
3-oxohistidine 6
Oxidised fat 859
Oxygen 1, 4-25
Radicals 382
Ozone 2, 56, 342
Pancreas 105, 164, 170-175, 395, 448, 461-473, 612, 658, 667-687
Pancreatic atrophy 184, 366, 367, 373
Parkinsons disease 8, 82, 739
Penicilium species 317-319
Perforins 221
Peritoneal exudate cells 240, 333
Peroxidability Index 280
Peroxidation, see lipid peroxidation
Peroxinitrite 15, 54, 217
Peroxisomes 2, 11, 59
Peroxyl radical, see free radicals
Pesticides 15, 30-31, 867-868
Phagosome 217, 218, 257, 340, 924
Phenolic acids 858
Phospholipids, see lipid fractions
Phosphatidylcholine 285, 379, 381, 879
hydroperoxide 54, 71, 73, 74, 82
Phosphatidylinositol (PI) 285
Phosphatidylserine (PS) 285
Phosphatidylethanolamine (PE) 285
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Phospholipase A2 (PLA2), see enzymes

Phytochemicals 646, 647, 649, 650, 651
Phytohemagglutin (PHA) 223,613
Plaque-forming cell test (PFC test) 224
Pokeweed mitogen 223, 225, 238, 240, 243, 612, 713
Pollution 2, 455
Polychlorinated biphenyls 31
Polymorphonuclear cells, neutrophils (PMN) 225, 504, 614
Polyphenols 648, 649, 890, 896
Polyunsaturated fatty acids (PUFA) see Fatty acids
Prostaglandins 76, 77, 214, 218, 253, 254, 712, 887
Proteasomes 48, 66, 168, 421, 928
Pulmonary hypertension syndrome, see ascites 410, 423, 447
Quail 55, 105, 169, 319, 324, 326, 345, 346, 347, 370, 389, 391, 401, 418
Diet 344, 417
Egg 326, 389, 390, 827, 828
Embryo 326, 328, 390
Radiation 2, 3, 5, 8, 19, 26, 72, 250, 342, 688, 703, 705, 708, 741-746, 891, 944
RDA 655, 662-664, 753, 809, 811, 815-822, 826-832, 835, 836, 840, 842, 863, 875, 882,
894, 945
Reactive nitrogen species 1, 3, 4, 7, 17, 27, 64, 214, 218, 732
Reactive oxygen species (ROS) 1-33, 64, 65, 88, 102, 109-115,214, 217, 220-262
Receptor 12, 19, 28, 217, 218, 220, 221, 234, 236, 237, 238, 239-243, 252-259, 322, 331,
341, 342, 347, 400, 614, 650, 707-716, 872, 925
Expression 237
Redox cycle of vitamin E 24
Redox signalling 25, 102, 924
Reperfusion 8, 16, 62, 63, 609, 728, 979, 943
Retinal degeneration 608
Retinol 31, 325, 610, 882
Rheumatoid arthritis 7, 8, 652, 732-733, 944
Rosemary extract 23
Saccharomyces cervisiae 50
Safflower oil 372, 653
Selenate 48, 109-113, 151-198
Selenite 151-198
Prooxidant properties 109-116
Selenium
Absorption 182, 183, 244, 390, 422, 521-530, 605, 657
Ageing 746- 751
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Index 969
Availability 49, 87, 88, 154, 155, 172, 174, 175, 182, 191, 287, 405, 446, 527, 528, 538,
606, 626, 627, 824, 833, 837, 843, 926, 929, 938
Brain function 738-741
Cancer 671-729
Case-control studies 682-682
Epidemiology 672-682
Human intervention trials 694-707
Laboratory animal studies 687- 693
Molecular mechanisms 708-720
Secondary lymphedema 704-705
Treatment 705-708
Cow
Practical application of Se nutrition 538-546
Route of Se supplementation 537-538
Se-yeast vs selenite 546-559
CVD 720-729
Deficiency
Chickens 91, 183, 364- 374
Cow 244, 487, 491-559
Dogs 604
Fish 616
Goats 236
Horse 589
Human 250, 659, 660-661
Mouse 22, 100, 237
Pig 446-454
Rats 89, 180
Diabetes 733-736
Egg freshness 415-424
Enriched eggs 153, 390, 418, 424, 754, 809-843
Feathering 407, 414, 423
Human health 665-753
Immunomodualtion 213-262
Metabolism
Cats and dogs 606
Effect of mycotoxins 345
Horses 593
Pigs 471
Plants 155, 161
Ruminants 521-526
Mood enhancement 738-741
Mycotoxins 317-347
Pancreatitis 736-738
Radiation 741-746
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970
Reproductive disorders 729-731

Asthma 731-732
Requirement
Breeders 400-405
Broilers 405
Cows 528-531
Human 662- 664
Pigs 454-459
Rheumatoid arthritis 732- 733
Se chicken 827-837
Se milk 827-837
Se pork 827-837
Soil 151-156, 188, 191, 446, 520, 521, 538, 659, 810-812, 836, 843, 926, 927
Status assessment
Cow 531-537
Human 664-665
Toxicity
Human 751-754
Selenocysteine 45
Selenoproteins 46-117
Selenomethionine
Absorption 163-164
Availability 174, 175-198
Feed and food 151-161
Incorporation into proteins 66
Metabolism 166, 167
Plants 156
Se source 18, 363-897
Storage form of Se 168-169
Selenoproteins
Cytosolic GSH-Px, see antioxidant enzymes
GI-GSH-Px, see antioxidant enzymes
Iodothyronine 5-deiodinase, see enzymes
p-GSH-Px, see antioxidant enzymes
PH-GSH-Px, see antioxidant enzymes
Selenoprotein W 46, 92-95
Selenoprotein P 46, 49, 60, 74, 78, 95, 96, 103, 166-170, 304, 531, 555, 657, 887, 928,
939, 945
Selenoprotein synthetase-2 97
Selenoproteins R, T, N and X 99-101
Specific sperm nuclei GSH-Px, see antioxidant enzymes
Thioredoxin reductase, see antioxidant enzymes
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Index 971
Selenosis, see Se toxicity
Sel-Plex 88, 151, 168, 194, 197, 259, 301, 346, 388-424, 461-478,528-561
Seminal plasma
GSH-Px 291, 292, 293, 297, 299, 933
Se 287, 291, 297, 298, 729, 933
Selenoproteins 303
Taurine 612
Sheep red blood cells (SRBC) 222, 229, 332, 334, 336-339
Signal transduction 15, 19, 20, 28, 33, 76, 77, 83, 255, 303, 714, 751, 878, 883
Singlet oxygen 2, 15, 71, 217
Soya 174, 446, 645, 649
Soybean meal 173, 174, 190, 225, 403, 446, 450, 460, 471, 626
Soybean oil 653
Sperm
Abnormality 284
Axoneme 286, 291
Capacitation 28, 303, 608, 924
Freezing 286, 304
Midpiece 68, 69, 75, 99, 284, 288, 289, 296, 301, 302, 933
Sperm-oocyte fusion 286
Spermatozoa 68, 279
Antioxidants 286
Avian 280
Boar 295-298, 299, 449
Bull 284
Chicken 280, 301-303
Dogs 280
Duck 280
Fertilizing capacity 28, 103, 304
GSH-Px 292, 294
Guinea fowl 292
Human 281, 284, 729
Lipid peroxidation 284, 286
Mammalian 281
PH-GSH-Px 68, 69, 77, 99, 289
Rat 299
Se 933
Selenoproteins 286
Turkey 284, 285
Sperm storage tubules (SST) 303
Spinach 867, 874, 880
Spleen 12, 80, 84, 88, 93, 164, 166, 170, 226-241, 323-339, 490, 658, 747, 876, 877
Splenocytes 335, 337, 742
Stomach 448, 450, 615, 667-696, 752, 858-892
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Sunflower oil 653

Superoxide
Dismutase (SOD) 7-33, 56, 304, 325, 329, 374, 595, 624, 681, 737, 867-891
Radical see free radicals
Tardive dyskinesia 8
Testes 52, 68, 74, 80, 86, 93, 94, 99, 105, 164-168, 181, 287-304, 323, 612, 866
Tetanus toxoid 232, 233, 339, 599
Thiobarbituric acid reactive substances (TBARS) 31, 56, 110-113, 284, 324-328, 415, 492,
609, 611, 734, 746, 750, 868, 879
Thioredoxin 19, 21, 51, 52, 73, 82-89, 290-291
System 9, 13, 22, 83-89, 102
Thioredoxin peroxidase, see enzymes
Thioredoxin reductase, see enzymes
Thrombosis 451
Thromboxane 76, 725-728
Thymocytes 240
Thymus 59, 80-90, 221, 234, 336-341, 877, 885
Thyroxine 89, 91, 511, 542
Tocopherols, see Vitamin E
Tocotrienols, see Vitamin E
Tocopheroxyl radical, see free radicals
Tomato 858, 880
Toxicity of
Selenium 591-592, 618-620, 752
Transferrin 12
Trichothecenes, see Mycotoxins
Triglycerides, see lipid fractions
Trioeponema hyodysenteriae 244
Trolox 112, 114, 885
Turkey 153, 365, 814
Egg 384
Embryo 384
GSH-Px 411, 747, 933
Mycotoxins 319-343
Peroxides 859
Selenium 105, 172, 189, 191, 367-384, 400-404, 423-424, 814, 833
Sperm 279-293
Vitamin E 384, 834, 892
Tumour necrosis factor a (TNF-a) 11, 216-223, 250, 255, 333, 339, 713
Ubiquinols (Coenzyme Q) 9, 13, 23, 25, 26, 86, 377, 876-880
Ulcerative colitis 652, 891
Uric acid 7, 9, 13-26, 595, 597, 888
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Index 973
Urine 163, 164, 167, 171, 173, 180, 185, 197, 334, 340, 472, 524-526, 594, 685, 869, 878,
884, 927, 929, 939
Uterus 74, 90-96, 104, 322, 454, 494, 498-501, 514
Vaccination 30, 220-249, 332, 334, 383, 399, 614, 713, 748
Vaccine 30, 221, 229-250, 334, 488, 612, 613, 748
Vegetables 185-190
Human health 644-655, 720, 811-895, 942-948
Oils 653, 874, 876
Vero cells 326
Viability
Calf 487, 540, 559
Cell 62, 86, 111, 114, 194, 242, 610, 866
Chicks 370, 385, 422, 935
Embryo 385, 397, 398
Fish 627
Foal 597
Lymphocyte 234, 237
Macrophage 225, 228
Piglet 477
Spermatozoa 103, 283-298, 729
Vitamin E 58
Antioxidant 13, 23
Companion animals 601-615
Cow 488-559
Deficiency 104-106, 364-374,
Egg yolk 312-316, 393, 818-827, 841-842, 893
Embryo 378-385
Excess 102
Fish 616- 627
Gene expression 108
GIT 874- 875
Horses 589-599
Human 648-753
Diet 816-817
Immunity 224-262
Meat 832-836, 892
Mycotoxin 326, 343-347
Pig 449-478
Recycling 14, 23,24, 83, 87, 102
Se interactions 103-109, 446
Spermatozoa 292-305
Stress 27-33
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Vitamin C, see ascorbic acid

Vitamin D 418, 601, 841
Vomitoxin, see deoxynivalenol (Mycotoxins) 455, 482
Wheat 151, 153-193, 870, 874, 928
Mycotoxins 318-322
Se-wheat 813, 814, 831
Xanthine oxidase, see enzymes
Xanthophylls, see carotenoids
Xenobiotic metabolism 15, 108, 838, 883
Yolk 186, 328, 374-417
Fatty acids 372
Yolk sac membrane (YSM) 375-383
Zearalenone, see mycotoxins

Zeaxanthin, see carotenoids
Zeolite 344-345
Zinc 102, 378, 417, 468, 621, 707
Deficiency 182
Human 748
SOD 10, 26, 381, 589, 605, 857
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Selenium in Nutrition and Health

SELENIUM IN NUTRITION AND

Nottingham University Press

Typeset by Nottingham University Press, Nottingham

1. Antioxidant systems in animal body

2. Molecular mechanisms of Se action: Selenoproteins

3. Selenium in food and feed: selenomethionine and beyond

4. Selenium and immunity

5. Selenium and semen quality

6. Selenium and mycotoxins

7. Selenium in poultry nutrition

8. Selenium in pig nutrition

Se for companion animals

Selenium in fish nutrition

11. Selenium and human health

12.Se-enriched eggs, milk and meat as functional foods and a solution to

To my wife Helen, my daughter Katie and my son Anton

Free fatty acids

Tumor necrosis factor-related apoptosis-inducing ligand

Antioxidant Systems in Animal Body

ANTIOXIDANT SYSTEMS IN ANIMAL BODY

Free radicals and reactive oxygen and nitrogen species

Selenium in Nutrition and Health

Hydrogen peroxide, H2O2

Antioxidant Systems in Animal Body

nitration of tyrosine or triptophan residues or oxidation of methionine or

Selenium in Nutrition and Health

transport chain in the mitochondria is responsible for major part of superoxide

Antioxidant Systems in Animal Body

At this stage a relatively unreactive carbon-centered radical (L*) is converted to a

Therefore lipid peroxidation is a chain reaction and potentially large number of

Selenium in Nutrition and Health

In general the accumulation of oxidized proteins depends on the balance between

Antioxidant Systems in Animal Body

biological activity of the modified proteins would be compromised. The degree

Three levels of antioxidant defence

natural fat-soluble antioxidants (vitamins A, E, carotenoids, ubiquinones,

Selenium in Nutrition and Health

Heart and cardiovascular system

Brain and nervous system

Antioxidant Systems in Animal Body

thiol redox system consisting of the glutathione system (glutathione/

The protective antioxidant compounds are located in organelles, subcellular

Superoxide dismutase, glutathione peroxidase, catalse,

Selenium in Nutrition and Health

Superoxide dismutase was discovered by McCord and Fridovich in 1969 as an

Antioxidant Systems in Animal Body

systems, is well controlled. In fact, exposure to increased pO2, increased

1 Inactivation of Mn-SOD genes in

Increases mutation frequency in aerobic

5 Transfection of Mn-SOD cDNA into

Dilated cardiomyopathy and neonatal lethality

The hydrogen peroxide formed by SOD action can be detoxified by GSH-Px or

Catalase (EC 1.11.1.6) is a tetrameric enzyme consisting of four identical subunits

Selenium in Nutrition and Health

Transition metal ions also accelerate the decomposition of lipid hydroperoxides

LO* + Fe3+ + OHLOO* + Fe2+ + H+

Therefore, metal-binding proteins (transferrin, lactoferrin, haptoglobin, hemopexin,

Antioxidant Systems in Animal Body

(LOO* is lipid peroxyl radical; Toc - tocopherol, Toc* - tocopheroxyl radical,

ROH (non-toxic)+ H2O + GSSG

Selenium in Nutrition and Health

GSH-Px and thioredoxin reductase) to complete the second part of antioxidant

Antioxidant Systems in Animal Body