1

TITLE

BIOCHEMICAL CHANGES ASSOCIATED WITH

PROTEIN ENERGY MALNUTRITION (PEM)
AMONG PREGNANT WOMEN IN ENUGU
METROPOLIS
A DISSERTATION SUBMITTED IN PARTIAL FULFILMENT
OF THE REQUIREMENTS FOR AWARD OF DEGREE OF
MASTER OF SCIENCE (M.Sc) IN MEDICAL
BIOCHEMISTRY, UNIVERSITY OF NIGERIA,
NSUKKA

BY

IKEYI, ADACHUKWU PAULINE

(PG/M.Sc/02/33502)

DEPARTMENT OF BIOCHEMISTRY
UNIVERSITY OF NIGERIA
NSUKKA

SUPERVISOR: DR. E. O. ALUMANAH

JULY, 2008
 2

CERTIFICATION

Ikeyi, Adachukwu Pauline, a postgraduate student of the Department of Biochemistry with the Reg. No
PG/MSc/02/33502, has satisfactorily completed the requirement of research work, for the degree of Master of
Science (M.Sc.) in Medical Biochemistry. The work embodied in this project (dissertation) is original and has not
been submitted in part or full for any other diploma or degree of this or any other university.

DR. E. O. ALUMANAH PROF I. N. E. ONWURAH

(Supervisor) (Head of
Department)

EXTERNAL EXAMINER
 3

DEDICATION

This work is dedicated to my husband James O. N. Ikeyi and children Chiesonu, Nneoma, Kenechukwu, Ekhdnma

and Nkechinyere. Also to all pregnant women with protein

energy malnutrition (PEM) in Nigeria.

 4

ACKNOWLEDGEMENT

I owe my profound gratitude to my Supervisor Dr. E. Alumanah for his tolerance, patience and fatherly
encouragement throughout the period of my studies. May God bless and reward him abundantly. The Dean,
Faculty of Biological Sciences, Prof. O. U. Njoku is worthy of mention for the unquantifiable assistance.
The present Head, Department of Biochemistry, Prof. I. N. E. Onwurah, is well-acknowledged for his
encouragement. Also included in the list of acknowledgement are Mr. Onwusi and Mrs. Ikekpeazu, both of the
school of medical laboratory technology UNTH Enugu for their assistance and guidance during the course of this
work.
I will not fail to express my appreciation to all the technical and medical staff of Park Lane Specialist
Hospital Enugu, Mother of Christ Maternity Hospital and UNTH Enugu, especially Mr. Obiora, the Chief medical
lab scientist Park Lane Specialist Hospital Enugu.
Many thanks to my Head of Department, Mr. Raymond Ofoezie and all my colleagues in the Department
of Science Technology I. M. T. Enugu for their encouragement, understanding and moral support.
I must express my gratitude to Dr. Parker Elijah Joshua and my friends, especially postgraduate students
of the 2002 and 2004 session for all their assistance, encouragement and friendship.
My lecturers in the Department of Biochemistry are not left out. They are Prof. O. Obidoa, Prof. I. C.
Ononogbu, Prof. O. F. C. Nwodo, Prof. L. U. S. Ezeanyika, Prof. F. C. Chilaka, Dr. B. C. Nwanguma, Dr. V.
Ogugua and Dr. S. O. Eze.
I thank in a special way my family, especially my husband James O.N. Ikeyi who encouraged me all the
way; all my children Chiesonu, Nneoma, Kenechukwu, Ekhdnma and Nkechinyere; my father Dr. F. M. O.
Agbo, my mother Mrs. T. U. Agbo, and siblings Ijeoma Agbo, Nkem Onodingene, Nkasi Ogakwu and Cheta
Agbo, and my parents in law.
Finally I thank the Almighty God for his Divine mercy, guidance and protection.
 5

ABSTRACT
Serum total protein, albumin, urea, total cholesterol, creatinine and calcium were evaluated in three groups of
female subjects as part of an investigation on the biochemical changes associated with protein energy malnutrition
(PEM) in pregnant women. The first group were 52 pregnant women with low total protein (<52g/l), the second
group were 50 pregnant women with normal total protein (>52g/l) while the third group were 50 non-pregnant,
non-lactating, apparently healthy women with normal total protein (>63kg). All the subjects were resident in
Enugu metropolis and aged between 20 to 40 years. The pregnant subjects were in different gestational stages of
pregnancy, having different parity and attending the antenatal clinic of Parklane Specialist Hospital, Enugu. The
results show that there was no significant difference between the mean serum total protein of the different age
groups (p>0.05). Parity (ie the number of children had by mother) correlated negatively (p<0.05) with serum total
protein, urea, total cholesterol, creatinine and calcium. Gestational stage of pregnancy in trimesters correlated
negatively and significantly with serum total protein and serum calcium (p<0.05). The results also revealed that
serum total cholesterol did not correlate significantly with serum total protein ,urea, total cholesterol, creatinine
and calcium (p>0.05 in each case). Urea levels correlated significantly and positively with serum total protein (r =
+ 0.246, p<0.05), and creatinine (r = +0.275,p<0.05). creatinine correlated positively with serum total protein (r =
+0.497,p<0.05), urea (r = +0. 275, p<0.05) and calcium ( r = + 0.356, p<0.05). Calcium negatively and
significantly correlated with gestational stage of pregnancy in trimesters (r = -0.288, p<0.05) and correlated
positively and significantly with serum total protein (r = + 0.681, p<0.05) and creatinine (r = + 0.0356, p<0.05).
While parity and gestational stage of pregnancy were implicated in this study as factors in PEM, this study does
not implicate age as a factor in PEM. However, serum level of cholesterol, urea, creatinine and calcium were
implicated in this study as factors in PEM?

TABLE OF CONTENTS

PAGE
Title Page .. .. .. .. .. .. .. .. .. .. i
Certification .. .. .. .. .. .. .. .. .. .. ii
Dedication .. .. .. .. .. .. .. .. .. .. iii
Acknowledgements .. .. .. .. .. .. .. .. .. iv
Abstract .. .. .. .. .. .. .. .. .. .. v
Table of Contents .. .. .. .. .. .. .. .. .. vi
List of Figures .. .. .. .. .. .. .. .. .. .. x
List of Tables .. .. .. .. .. .. .. .. .. .. xi
List of Abbreviations .. .. .. .. .. .. .. .. .. xii
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CHAPTER ONE: INTRODUCTION

1.2 Protein energy malnutrition 2

1.2.1 Types of protein energy malnutrition 3
1.2.1.1 Marasmus 3
1.2.1.2 Kwashiokor 3
1.2.1.3 Marasmic kwashiokor 3
1.2.2 History of concept of protein energy malnutrition 4
1.2.3 Classification of protein energy malnutrition 5
1.2.3.1 Welcome classification 5
1.2.3.2 Gomez classification 6
1.2.3.3 Water low classification 7
1.2.4 The centile chart 9
1.2.5 Aetiology of protein energy malnutrition (PEM) 10

1.2.5.1 Social and economic factor

10
1.2.5.2 Biological factors 10

1.2.5.3 Environmental factors

11
1.2.5.4 Age of host
11
1.2.6 Epidemiology of protein energy malnutrition
12
1.2.7 Nutritional assessment 13
1.2.7.1 Anthropometric measurements
14
1.3 Biochemical methods 16
1.3.1 Serum biochemical markers
16
1.3.1.1 Serum proteins
16
1.3.1.1.1 Serum albumin and globulin
16
 7

1.3.1.1.2 Other serum proteins

17
1.3.2 Serum urea and creatinine
17
1.3.3 Serum total cholesterol 17
1.3.4 Electrolytes Calcium 18
1.4 Recommended daily allowances (RDA)
18
1.5 Diagnosis of protein energy malnutrition
20
1.5.1 Mild and moderate protein energy malnutrition
20
1.5.2 Severe protein energy malnutrition
20
1.6 Pathophysiology and biochemical changes associated with
protein energy Malnutrition 21
1.6.1 Energy changes 21
1.6.2 Protein changes 22
1.6.3 Endocrine changes 22

1.6.4 Haematology and oxygen transport

23
1.6.5 Cardiovascular and renal function 23
1.6.6 Immune system 24
1.6.7 Monokines 24
1.6.8 Electrolytes 24
1.6.9 Gastro intestinal functions 24
1.7 Clinical features and presentation of protein energy malnutrition 25
1.8 Effects and complications associated with
protein energy malnutrition (PEM) 25
1.9 Management and Treatment of Protein Energy Malnutrition (PEM) 26
1.9.1 Mild and Moderate Protein Energy Malnutrition 26
1.9.2 Severe Protein Energy Malnutrition 27
 8

1.9.2.1 Resolving life threatening conditions 27

1.9.2.2 Restoring nutritional status without disrupting homeostasis 29
1.9.2.3 Ensuring nutritional rehabilitation 29
1.10 Prevention of PEM 29
1.10.1 Availability of food 30
1.10.2 Reducing infections 30
1.10.3 Education 31
1.10.4 Prognosis 31
1.11 Aim of Research 32
CHAPTER TWO: MATERIALS AND METHODS
2.1 Materials 33
2.2.1 Equipment used 33
2.1.2 Chemicals and reagents used 33
2.1.3 Study subjects 33
2.2 Methods 34
2.2.1 Collection of blood samples and preparation of serum 34
2.2.2 Preparation of reagents 34
2.2.2.1 Reagents for the determination of serum total protein concentration 34
2.2.2.2 Reagents for the determination of serum albumin concentration 34
2.2.2.3 Reagents for the determination of serum total cholesterol concentration 35
2.2.2.4 Reagents for the determination of urea concentration 35
2.2.2.5 Reagents for the determination of creatinine concentration 35
2.2.2.6 Reagents for the determination of calcium concentration 35
2.2.3. Determination of samples 36
2.2.3.1 Determination of serum total protein 36
2.2.3.2 Determination of serum albumin concentration 37
2.2.3.3 Determination of serum total cholesterol 38
2.2.3.4 Determination of serum urea concentration 40
2.2.3.5 Determination of serum creatinine concentration 41
2.2.3.6 Determination of serum calcium concentration 42
2.2.3.7 Statistical analysis 43

CHAPTER THREE: RESULTS

3.1 Distribution of Mean values of measured parameters in the subjects 44
3.2 Effect of age on the different parameters measured 46
3.3 Effect of parity (Number of children had by mothers) on the different
Parameters measured 51
3.4 Effect of gestational stage of pregnancy (in trimesters) on the
different parameters measured 55
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CHAPTER FOUR: DISCUSSION

Discussion 59
Recommendations 62
References 63
Appendices 70

LIST OF FIGURES
 10

Fig. 1.1 Distribution of height in 100 children 9

Fig. 1.2 The major chemical determinants of body weight and how they
relate to total body energy content 15
Fig. 2.1 Biuret Reaction: the coordination complex formed in alkaline
solution between cupric ions and the nucleophilic nitrogen atoms
of four Moles of Biuret 36
Fig. 3.1 Distribution of mean values of measured parameters in the subjects 45
Fig. 3.2 Concentration of serum total cholesterol of both pregnant and
non-pregnant subjects with different levels of serum total protein 47
Fig. 3.3 Concentration of serum urea of both pregnant and non-pregnant
subjects with different levels of serum total protein 48
Fig. 3.4 Concentration of serum creatinine of both pregnant and non-pregnant
subjects with different levels of serum total protein 49
Fig. 3.5 Concentration of serum calcium of both pregnant and non-pregnant
subjects with different levels of serum total protein 50
Fig. 3.6 Concentrations of some biochemical parameters determined
for mothers with 1 to 2 children 52
Fig. 3.7 Concentrations of some biochemical parameters determined
for mothers with 3 to 4 children 53
Fig. 3.8 Concentrations of some biochemical parameters determined
for mothers with 5 or more children 54
Fig. 3.9 Concentrations of some biochemical parameters determined
for mothers in the first trimester 56
Fig. 3.10 Concentrations of some biochemical parameters determined
for mothers in the second trimester 57
Fig. 3.9 Concentrations of some biochemical parameters determined
for mothers in the third trimester 58

LIST OF TABLES

1.1 Welcome classification of malnutrition 6

1.2 Gomez classification of malnutrition 6
1.3 Waterlow classification of malnutrition 7
1.4 Seoane and Lathams classification of malnutrition 8
1.5 Recommended daily allowance (RDA) chart for pregnant,
 11

non-pregnant and lactating women 19

LIST OF ABBREVIATIONS

FAO - Food and Agricultural Organization

PCM - Protein Caloric Malnutrition

PEM - Protein Energy Malnutrition

RDA - Recommended Dietary Allowance

WHO - World Health Organization

 12

CHAPTER ONE
INTRODUCTION
Worldwide, an estimated 852 million people are undernourished with most (815
million), living in developing countries (WHO, 2002; FAO, 2004) Poverty is the main
underlying cause of malnutrition and its determinants (Sachs and McArthur, 2005). The degree
and distribution of Protein Energy Malnutrition (PEM) in a given population depends on many
factors the political and economic situation, level of education and sanitation, the season and
climate conditions, food production, cultural and religious food customs, breastfeeding habits,
prevalence of infectious diseases, the existence and effectiveness of nutrition programmes and
the availability and quality of health services (FAO, 2004; Salama et al., 2004).
Malnutrition continues to be a major health burden in developing countries. It is
globally the most important risk factor for illness and death with hundreds of millions of
pregnant women and young children particularly affected (Muller and Krawinkel, 2005). Poor
nutrition in pregnancy in combination with infections is a common cause of maternal and infant
mortality and morbidity, low birth weight and intrauterine Growth Retardation (IUGR) (Pena
and Bacalao, 2002). In Nigeria, maternal death per 100,000 births is put at 800 while
percentage low birth weight stands at twenty (Enwonwu et al., 2004)
Low birth weight babies have increased risk of mortality, morbidity and development of
malnutrition. Children who suffer from malnutrition are more likely to have slowed growth,
delayed development, difficulty in school and high rates of illness and they may remain
malnourished to adulthood (Scrimshaw, 1998; Abidoye and Eze, 2000). IUGR is associated
with poor cognitive and neurological development for the infant and in adulthood,
susceptibility to cardiovascular disease, diabetes and renal disease (De Onis et al., 1998).
Malnutrition remains one of the worlds highest priority health issues not only because
its effects are so widespread and long lasting but also because it can be eradicated. Eradication
is best carried out at the preventive stage. Hence the need to identify groups of pregnant
women at greater risk of developing PEM. Such high-risk groups can be targeted in any
planned intervention programme.
 13

1.2 PROTEIN ENERGY MALNUTRITION

Protein Energy Malnutrition (PEM) results when the bodys need for protein, energy or
both cannot be satisfied by the diet. It includes a wide spectrum of clinical manifestations
conditioned by:
i) The relative severity of protein or energy deficit
ii) The severity and duration of the deficiencies.
iii) The age of the host
iv) The cause of the deficiency
v) The association of the deficiency with other physiological problems such as infectious
diseases and pregnancy (Torun and Chew, 1994).
Pregnancy is a normal physiological process associated with major alterations affecting
every maternal organ, system and metabolic pathway (McGanity et al., 1994). This
physiological process results in increased plasma volume and red blood cells, decreased
concentration of circulating nutrient-binding proteins and other micronutrients (Ladipo, 2000).
Values of these biochemical parameters are significantly altered and may change as the
pregnancy advances from first to third trimester and to delivery and then return towards normal
during post partum period (McGanity et al., 1994).
These physiological changes are however aggravated by malnutrition leading to adverse
biochemical consequences for both mother and newborn.
Nutrition may be defined as the sum of the process by which a living organism receives
nutrient materials from the environment and uses them to promote its own vital activities
(Barker and Lees, 1996). Malnutrition on the other hand is when the diet contains an incorrect
amount of one or more nutrients (Barker and Lees, 1996).
Protein Energy Malnutrition (PEM) or protein calorie malnutrition (PCM) generally
referred to simply as malnutrition is an imbalance between the supply of protein and energy
and the bodys demand for them to ensure optimal growth and function (WHO, 1997).
The World Health Organization (WHO) defines Protein Energy Malnutrition as the cellular
imbalance between the supply of nutrient and energy and the bodys demand for them to ensure
growth, maintenance and specific function (Lin and Santoro, 2003).
Protein Energy Malnutrition (PEM) or Protein calorie malnutrition is also a deficiency
syndrome caused by inadequate intake of macro-nutrients as well as micro-nutrients (Smith,
2002). It is a syndrome that represents one of the various levels of inadequate protein and or
energy intake between starvation (no food intake) and adequate nourishment
 14

1.2.1 Types of Protein Energy Malnutrition

Clinically PEM has three forms. These forms depend on the balance of non-protein and
protein sources of energy. The origin of these three forms can be primary, when it is the result
of inadequate food intake or secondary, when it is the result of other diseases that lead to low
food ingestion, inadequate nutritional absorption or utilization and or increased nutrient losses.
Also these forms of PEM can be graded as mild, moderate or severe.

1.2.1.1 Maramus
This is the dry, thin desiccated form of PEM. It results from near starvation with
deficiency of energy, protein and non protein nutrients. The marasmic individual consumes
very little food. In children it is often because the mother is unable to breastfeed. Marasmus is
characterised by stunted growth. Usually the children are thin from loss of muscle and body fat.
It develops in children between 6-12 months who have been weaned from breast milk or who
are suffering from weakening conditions like chronic diarrhoea.

1.2.1.2 Kwashiokor
This is the wet oedematous and swollen form. Kwashiokor is a Ghanaian word
meaning first child-second child. It refers to the observation that this is a disease the first
child develops when the second child is born and replaces the first child at the breast. This is
because the weaned child is fed with a thin gruel of poor nutritional quantity compared with
breast milk and as a consequence the child fails to thrive. This condition is marked with protein
deficiency more marked than energy deficiency, and Oedema results. Children with
Kwashiokor tend to be older than those with marasmus and tend to develop the disease after
weaning.
Adults develop kwashiokor as a result of under-nutrition from diets rich in carbohydrate
than protein. This may be as a result of poverty, wars, famine etc. Kwashiokor is characterised
by fluid retention, oedema, dry peeling skin, hair discolouration, etc (Lin and Santoro, 2003).

1.2.1.3 Marasmic Kwashiokor

This is the combined form of the Protein Energy Malnutrition. It is a combination of
chronic energy deficit and chronic or acute protein deficiency. Children with this form of PEM
have some oedema and or body fat than those with maramus. The clinical manifestation is a
combination of maramus and kwashiokor (Stanfield et al., 1978).
 15

1.2.2 History of Concept of Protein Energy Malnutrition

It has long been recognised that inadequate food intake produces weight loss and
growth retardation and, when severe and prolonged leads to body wasting and emaciation.
Nutritional disorders date back as far as the sixteenth century and were recognised under such
names as maries, atrophy, atrepsy, etc. In Ireland, William Benette of the society of friends
described hunger oedema in March 1847 as that horrid disease the result of long continued
famine and low living in which limbs and body swell most frightfully. Specific interest in
what we know today as Protein Energy Malnutrition began early in this century (Maclaren and
Burman, 1976).
Cicely Delphine Williams, a Jamaican paediatrician working in the African Gold Coast
now Ghana introduced the world KWASHIOKOR into modern medicine. It is the name used
by the Ga tribe of Accra Ghana to describe sickness of the weaning child. The term translates
literally First child second child and refers to the sickness the older child develops when
the next baby is born. She noted same condition has been described in 1906 in Germany, 1924
in Indo-China,1926 in Mexico and 1928 in East Africa. Williams first report on Kwashiokor
was in 1931 Annual Medical Report of the Gold Coast colony which was reprinted in 1973.
This was only read locally, but a paper soon followed in an international journal in 1933
(Davidson et al., 1979).
There was a long incubation period before the disease became generally recognized.
This was partly because international communication was disrupted by the Second World War
(1938 1948). Also in Africa the disease was confused with pellagra. At the first session of the
FAO/WHO expert committee on Nutrition in 1949 there was no place for Protein Energy
Malnutrition on the agenda. The subject was raised indirectly under the heading of pellagra, the
committee asked WHO to conduct an enquiry into the various features of kwashiokor.
In Uganda Trowell in 1954 wrote the classic textbook on kwashiokor. Only after this
did kwashiokor start to appear in textbooks. A few years after, protein malnutrition was
considered an important nutritional disease in the world. Descriptive names for this syndrome
which have been used in the past by clinicians include- infertile pellegra, malignant
malnutrition, syndrome depigmentation oedema, distrofia, pluricarential. Jelliffee in 1959
introduced the term protein caloric malnutrition (PCM) because of the close association
between kwashiokor and marasmus. This was later changed to Protein Energy Malnutrition.
 16

The term malnutrition is usually used in lay language for PEM (Davidson et al., 1979;
Stanfield et al., 1978).

1.2.3 Classification of Protein Energy Malnutrition

The classification scheme for PEM is useful for diagnosis and treatment as well as the
application and evaluation of public health measures. Several methods have been suggested for
the classification of PEM. The choice of classification depends on the purpose for which it is
used, e.g. clinical studies or community surveys.
There are three main classifications of PEM based on clinical and anthropometric assessments.
a) The welcome classification
b) The water-low classification
c) The Gomez classification
In order to understand these classifications, it is necessary to have a knowledge of the
central chart system on which they are based.

1.2.3.1 Welcome Classification

This was proposed by the Welcome Working Party. In this classification reduction in
body weight below 80 percent of the Harvard Standard (50th Centile) is considered
malnutrition. There is also the presence and absence of oedema as well as deficit in body
weight. Therefore children with oedema with weight 60-80 percent of the expected weight for
age are classified as suffering from kwashiokor. Those without oedema and who weigh less
than 60 percent of the standard are considered as marasmic. Those with oedema and body
weight less than 60 percent of the standard are diagnosed marasmic kwashiokor. However,
children without oedema weighing 60-80 percent of the standard weight are classified as
underweight. The Welcome classification is the most generally accepted and widely used for
clinical purposes (See Table 1.1).
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Table 1.1: Welcome Classification of Malnutrition

MALNUTRITION BODY WEIGHT % OF OEDEMA
STANDARD
Underweight 80-60 -
Marasmus <60 -
Kwashiokor 80-60 +
Marasmic kwashiorkor <60 +

50th Centile of Harvard Standard (Welcome Trust Working Party, 1970).

1.2.3.2 Gomez Classification

The Gomez classification is based on the deficit in weight for age and the 90 percent of
the Harvard Standard is used as cut-off point from normal to malnourished. Malnutrition is
subdivided into three degrees, viz first, second and third degree malnutrition.
First degree malnutrition is defined as 75-90 percent, second degree is defined as 60-
75% while third degree is defined as less than 60% of expected weight as illustrated Table 1.2.
All cases of oedema are included in third degree malnutrition regardless of body weight.
The Gomez classification is useful for community surveys and helps to access the
magnitude of the problem in a community. However it does not indicate the duration or types
of malnutrition (Table 1.2).

Table 1.2: Gomez Classification of Malnutrition (Gomez, 1956)

MALNUTRITION BODY WEIGHT (% of standard)
First degree 75-90
Second degree 60-75
Third degree <60
 18

1.2.3.3 Water Low Classification

Waterlow described a classification of malnutrition using both weight and height for
age. This classification is useful in that it distinguishes those children with acute malnutrition
(wasting) from those with chronic under-nutrition who are stunted.
It also assesses the relationship between weight and height in early childhood which is
reasonably constant as indicated in Table 1.3. Waterlow suggested the terms wasting for a
deficit in weight and stunting for a deficit in height for age.
Therefore patients fall into four categories:
1) Normal
2) Wasted but not stunted (suffering from acute PEM)
3) Wasted and stunted (suffering from acute and chronic PEM)
4) Stunted but not wasted (nutritional dwarfs with past PEM with present adequate
nutrition)
The disadvantage of this method is that, although height is a far more accurate
reflection of growth in the long term, it is often difficult to measure accurately in community
surveys. There is also the tendency to place the genetically or constitutionally small child or
premature infants into the category of malnutrition.

Table 1.3: Waterlow Classification of Malnutriton

(Stanfield et al., 1978)
Height for age >80% Weight for Age <80%
>90% Normal Wasted
<90% Stunted Stunted and wasted

Other classifications of malnutrition are based on the FAO/WHO Joint Expert

committee on Nutrition. It emphasized that height deficit in a population in relation to age may
be regarded as a measure of the duration of malnutrition. The expression of weight in relation
to height gives a measure of nutritional status which is independent of age and of age related
external standards.
 19

The Seoane and Lathams classification is based on this concept (see Table 1.4). They
proposed that weight for height for age gives a picture of past nutritional history. In this
system malnutrition was classified into three major categories:
1) Current short term or acute malnutrition
2) Current long-term or chronic malnutrition
3) Past malnutrition or nutritional dwarfs.

Table 1.4: Seoane and Lathams Classification of Malnutrition (Stanfield et al., 1978)

Malnutrition Weight for Height for Weight for

age age height
1) Acute or current short term malnutrition Low Normal Low
(wasted)
2) Chronic or long-term malnutrition (wasted Low Low Low
and stunted)
3) Past malnutrition or nutritional dwarfs Low Low Normal
(stunted)
 20

1.2.4 The Centile Chart

The Welcome, Waterlow and Gomez classifications are based on the Boston 50th centile
chart. In Boston in 1950s and 1960s 100 normal children were measured by weight and height
from birth to 18 years and their growth curve plotted (Fig. 1.1). It was found that 50 children
(50 percent) had heights below and 50 children (50 percent) above 108cm. This was therefore
referred to as the 50th centile or median. Three percent had heights less than 99cm and this was
referred to as 3rd centile. Similarly, 97th, 90th, 75th, 25th and 10th centile points were identified.
The lowest acceptable centile for normal growth was the 3rd centile and the highest, the 97th
centile.

120
..97th centile

115
..90th centile
Height (stature) (cm)

..75th centile
110 Mean
..50 th
centile

..25th centile
105
..10th centile
100
..3rd centile

Fig. 1.1: Distribution of heights of 100 children (Welcome Trust Working Party, 1970).
 21

1.2.5 Aetiology of Protein Energy Malnutrition (PEM)

Adequate food and nutrition are essential from conception to adulthood, for proper
growth and physical development, to ensure optimal work capacity and normal reproductive
performance and also to ensure the adequacy of immune mechanism and resistance to
infections and diseases. Undernutrition or malnutrition increases susceptibility to infection and
disease and reduces work capacity and productivity among adults. The nutritional status of a
person depends on food consumption and not solely the production and availability of food.
Dietary energy supply measurement assume that available food is distributed and consumed in
relation to requirement which is often not the case (WHO, 2001).
Social, economic, biologic and environmental factors have been implicated as
underlying causes of the insufficient food intake or ingestion of food without proteins or of
poor nutritional quality that lead to PEM.

1.2.5.1 Social and Economic Factor

Poverty that results in low food availability, over crowded and unsanitary living
conditions and improper childcare are frequent causes of PEM. Ignorance by itself or
associated with poverty, leads to poor infant and child rearing practices, misconceptions about
the use of certain foods, inadequate feeding conducts during illnesses and improper food
distribution within family members, inadequate breast feeding and weaning practice and
duration of breast feeding, combined with when breast milk is withdrawn or when it can no
longer provide sufficient dietary energy and protein to the infant, are associated with growing
rates of infantile PEM.
Social problems, child abuse, maternal deprivation, abandonment of the elderly,
alcoholism and drug addiction can result in PEM. Cultural and social practices, food taboos,
some food and diet fads, particularly popular among adolescents and women, migration from
traditional rural settings to urban slums and hard labour performed within and outside their
homes lead to a situation of chronic deficit in caloric intake vis a vis their caloric expenditure
(WHO, 2001).

1.2.5.2 Biological Factors

Maternal malnutrition and other additional biological demands on women prior to and
or during pregnancy, menstruation and lactation further accentuate PEM in women within child
bearing age. This intrauterine malnutrition leads to underweight babies which is compounded
 22

after birth by insufficient food to satisfy the infants need, resulting in PEM. Infectious diseases
are also major contributing and precipitating factors in PEM. Diarrhoea, measles, respiratory
and other infections frequently result in PEM. Anorexia, vomiting, decreased absorption and
catabolic processes also contribute to PEM (Torun and Chew, 1994). Diet with low
concentrations of proteins and energy as occur with over diluted milk formula, or bulky
vegetable foods with low nutrient density, diets poor in protein and rich in carbohydrate also
contribute to PEM.
PEM also occurs as a result of malabsorption found in disease conditions among the
elderly in nursing homes, surgical patients and other hospitalized patients with chronic or
terminal diseases (Smith, 2002; Lin and Santoro, 2003).

1.2.5.3 Environmental Factors

Over crowded and/or unsanitary living conditions lead to frequent infections. This is an
important cause of PEM, especially among weanings who develop severe or frequent
diarrhoea. Agricultural patterns, droughts, floods, wars and forced migration lead to cyclic,
sudden or prolonged food scarcities and can cause PEM among whole populations. Post harvest
losses of food due to bad storage conditions and inadequate food distribution systems
contribute to PEM even after periods of agricultural plenty.

1.2.7.4 Age of Host

PEM affect all age groups. However, it is more frequent in infants and young children
whose growth is dependent on increases in nutritional requirements. Also they cannot obtain
food by their own means especially when living under poor hygienic conditions. The infants
who are weaned prematurely from the breast or who are breast fed for a prolonged time without
adequate complementary feeding plan become malnourished due to lack of protein and energy.
In older children, there is the milder form of PEM because they can cope better with
social and food availability constraints. Infections are less severe. In pregnant and lactating
women, PEM is due to increase in nutritional requirement. Under nourishment combined with
early pregnancy and poor weight gain in pregnancy lead to life-threatening consequences. Also,
the consequences of dietary deficiencies affect mainly the growth and nutritional status and
survival of fetuses, newborn babies and infants.
The elderly tend to suffer PEM because they are unable to care properly for themselves.
Gastrointestinal alterations in the elderly are also a contributing factor. Terminal and chronic
 23

illnesses found in the elderly is also a factor. Adolescents, adult men, non-pregnant and non-
lactating women usually have the lowest prevalence and the mildest form of PEM. This is
because they have greater opportunities to obtain food. Food cultural practices protect male
members of the family. However weight reducing fads and diets, food fads, etc., can predispose
or actually cause PEM (Torun and Chew, 1994).

1.2.8 Epidemiology of Protein Energy Malnutrition

Protein Energy malnutrition (PEM) is the most important nutritional disease in
developing countries because of its high prevalence and its relationship with child mortality
rates, impaired physical growth and inadequate social and economic development. Associated
deleterious effects on mental growth and maturation have been demonstrated in experimental
animals and they seem to occur in humans. However, it has not been possible to dissociate
completely the nutritional factors from other environment factors or to ascertain the
irreversibility of the nutritional mental damage. PEM occurs more frequently when infections
impose additional demands, induce greater losses of nutrients or produce metabolic alterations.
Most malnourished persons live in developing countries of the world. About 30% each
in Africa and the Far East and 15% each in Latin America and Near East. In 1990, it was
estimated that one in every three children under five years of age in the developing world or
177 million children were or had been malnourished. This prevalence ranged from 14% in
America to 47% in South Asia. The United Nations Food and Agricultural Organization
(FAO) estimated that in the mid 1980s more than 20% of the population of 98 developing
countries was undernourished and that 512 million persons or 21% of the people in the
developing World were affected (FAO, 1962). This proportion ranged from 11% in the Near
East to 32% in Africa.
In 2000, WHO estimated that malnourished children numbered 181.9 million (32%) in
developing countries. Also an estimated 149.6 million children younger than 5 years are
malnourished when measured in terms of weight for age. In South Central Asia and Eastern
Africa about one half of children have growth retardation due to PEM. This figure is five times
the prevalence in the Western world (Lin and Santoro, 2003).
Marasmus is the predominant form of PEM in the urban areas of most developing
countries. It is associated with early abandonment or failure of breast feeding with consequent
infections most notably those causing infantile gastroenteritis. These infections result from
improper hygiene, inadequate knowledge of infant rearing and poverty with consequent and
 24

persistent infections resulting from improper hygiene prevalent in the growing slums of
developing countries.
Kwashiokor is more prevalent in rural areas of the developing countries. It develops
early in babies about 12 months when breast feeding is being discontinued and mothers lack
proper weaning techniques. The child usually is weaned on to a diet excessively starchy with
low protein content like cassava, corn and plantain porridge (Morley, 2002).
Marasmic kwashiokor manifests mainly in adults and its effect is less severe because
the relative protein requirement is lower. It tends to be confined in parts of the world where the
staple foods are protein deficient and excessively starchy, such as yam, cassava, plantain, sweet
potatoes etc. Marasmic kwashiokor is also as a result of environmental factors such as drought,
wars etc (Torun and Chew, 1994, Barker and Lees, 1996).
In industrialized countries PEM is not prevalent in the general population. It is seen
mainly among children of the lower socio-economic groups. Also it is often found among the
elderly who live in nursing homes, surgical patients and other hospitalized patients with
chronic or terminal diseases such as chronic renal failure, cancer etc. (Lin and Santoro, 2003).
It is also found in adults addicted to alcohol and drugs, psychiatric patients, etc. (Torun
and Chew, 1994). A long term analysis shows a trend for a decade-by-decade gradual
improvement in the prevalence of child malnutrition, if the countries are not disturbed by
natural and man-made disasters such as droughts, desertification, wars, and economic crisis.
However, total number of malnourished children has not decreased because of the rise in
population in the countries where malnutrition is highly prevalent.

1.2.9 Nutritional Assessment

Maintaining optimum health requires adequate tissue levels of essential nutrients and a
source of energy. More than 40 syndromes develop if tissue levels of these components are
either too low or too high. The depletion of body nutrient stores and ultimately loss of specific
cellular functions are common to many acute and chronic diseases. Therefore, the sequence of
changes in progressive Protein Energy Malnutrition involve negative nutrient balance, loss of
cellular functions and ultimately clinical complications (See Fig. 1.2).
Nutritional assessment is an examination which involves the use of anthropometric
measurements and serum biochemical markers of PEM in the evaluations of acutely and
chronically ill malnourished patients. With nutritional assessment examination, loss of nutrients
 25

can be prevented or reversed and ultimately, the risk of clinical complication can be minimized
or eliminated (Heymsfield et al., 1994).

1.2.7.1 Anthropometric Measurements

The anthropometric measurements in general use body weights and stature, skinfolds,
arm circumferences and bone breadth. These body measurements are used directly or indirectly
in nutritional assessment.
(1) Body Weight and Stature: The body weight is a fundamental component of nutritional
assessment. This is because the body weight under most conditions is an indirect marker of
protein mass and energy stores. A loss or gain in body weight is usually assumed to reflect
changes in protein mass and/or energy. The body weight is the sum of all compartments of
body composition. The body weight varies less than 0.1 kg per day in healthy adult. A
significant correlation exists between loss of body weight and change in total body protein.
(Heymsield et al, 1994).
A weight loss of more than 0.5 kg per day indicates negative energy or water balance or
a combination of the two. Rate of weight loss in total starvation is 0.4 kg per day. A relative
decrease in weight of more than 10% over a time interval of 6 months is considered a clinically
significant weight loss.
The minimal survival body weight in humans is between 48%-50% of desirable body
weight or a BMI of between 13 and 15. However, an absolute body weight loss of 55% to 60%
places the individual at or near survival limits of semi starvation.
Body weight is measured within 0.1 kg on a calibrated physicians scale.
 26

MINERALS

H2O
INTRACELLULAR
WATER

FAT FREE MASS

BODY WEIGHT

ENERGY
EXTRACELLULAR
WATER

GLYCOGEN

PROTEIN

FAT

Fig. 1.2: The major chemical determinants of body weight and how they relate to total body
energy content (Heymsfield et al., 1994)

(2) Skinfold and Circumferential Measurement.

Skinfold and circumferential measurement are methods used to asses fat available in the
body. Fats in healthy individuals vary greatly. There is a relatively small amount in trained
athletes and relatively large amount in later stage of pregnancy. The adequacy of fat depends
on the total body triglycerides and energy. Very little fat is sufficient in healthy individuals. In
nutritional assessment, fat measurement provides indirect guide to energy balance.
The skinfold measurement is carried out with a skinfold caliper. The skinfold represents
a double layer of subcutaneous tissue, including a small and relatively constant amount of skin
and variable amount of adipose tissue.
The six sites of skinfold measurement are:
(1) Biceps skinfold thickness
(2) Triceps skinfold thickness
(3) Subscapular skinfold
(4) Suprailiac skinfold
(5) Thigh skinfold
(6) Calf skinfold
 27

The circumferential measurement is carried out with a tape measure. The tape should
have an accuracy of 0.1 cm.
The three circumferential measurement sites are:
(1) Mid-upper arm
(2) Mid thigh
(3) Mid calf
(Heymsfield et al., 1994).

1.3 Biochemical Methods

Serum biochemical markers are primarily proteins and non proteins used in establishing
the nutritional status of patients. They are used to determine whether they are at risk of
complications and also in monitoring their nutritional treatment (Heymsfield et al., 1994).
Most pregnant women are normal, healthy females without disorders that interfere with
the ingestion, digestion, absorption, metabolism or utilization of a normal well balanced diet.
Therefore, it is unlikely that they will have evidence of either biochemical or clinical
manifestation of any nutritional disorder. However, to provide proper management of their
physiological condition, it is necessary to conduct biochemical studies at suitable intervals to
monitor organ and hormonal functions as well as measurement of direct nutritional status
(Heymsfield et al., 1994).

1.3.1 Serum Biochemical Markers

1.3.1.1 Serum Proteins
Blood serum contains a large amount of proteins. A total serum protein test measures
the total amount of protein in blood serum. It helps to determine whether an individuals diet
contains adequate protein or to estimate the risk of developing disease due to protein
deficiency. It is also used to evaluate nutritional status and also to evaluate some congenital or
rare diseases of the blood e.g. multiple myeloma, etc (Payne, 2004).
There are two major groups of protein in the blood. They are albumin and globulin.

1.3.1.1.1 Serum Albumin and Globulin

The total serum protein test not only determines the amount of total proteins, but the
amount of albumin present in blood is considered a classic marker of nutritional status (Toigo
et al., 2000). Albumin is synthesized primarily in the liver and the biosynthetic rate is
 28

determined by dietary protein intake. The rate of synthesis of albumin falls as well as its
catabolic rate with low protein intake resulting in negative albumin balance, thereby reducing
whole body albumin loss to preserve serum levels. Serum globulin is made up of different
proteins that can be separated into alpha, beta, gamma types. Some globulins are formed by the
liver while others are formed by the immune system. Certain globulins bind with hemoglobin
called haptoglobins. Other globulins transport metal, e.g. iron into the blood.
A decrease in globulin levels have been implicated in leukemia, blood clotting disorder
and PEM (Payne, 2004).

1.3.1.1.2 Other Serum Proteins

Other serum protein such as prealbumin, transferin, creative protein, fibronectin, retinol
binding protein, ribonuclease and cholinesterase have been implicated as markers of nutritional
status (Toigo et al., 2000). The synthesis and the concentrations of these proteins decrease
under condition of PEM. Under ideal conditions these proteins are more sensitive indices of
dietary protein and energy intake. This is because they have lower biosynthetic rates, smaller
total body pool, shorter half-lives and lower serum levels (Heymsfield et al., 1994).

1.3.2 Serum Urea and Creatinine

A major portion of tissue function can be attributed to proteins that are activated by
energy derived from metabolism of organic fuels. During periods of nutritional deprivation,
approximately half the total body protein mass can be used as metabolic fuel. The end product
of oxidation of its amino acids is urea. Urea is not metabolised further and is excreted
unchanged in the urine. Therefore when food intake is less than nutrient losses, amino acids
from proteins are oxidized to provide energy. A low protein intake will result in low serum
urea concentration (Heymsfield et al., 1994).
Creatinine is related to muscle mass and is related to protein catabolic rate. Serum
creatinine concentration is used to assess creatinine production and therefore will determine
dietary protein intake. Creatinine levels are low in individuals with PEM (Toigo et al., 2000).

1.3.3 Serum Total Cholesterol

The main sources of non-protein energy are glycogen and fat or triacylglycerols. Fats
are found almost entirely within the adipose tissues or fat cells. During periods of under-
 29

nutrition all but a small amount of fat is used as metabolic fuel. Thus the measurement of fat
provides an indirect guide to energy balance.
In laboratory investigation, total serum cholesterol has been found to be a useful marker
for energy intake but not for protein intake. Low total serum cholesterol level is an indication
of negative energy balance in PEM (Toigo et al., 2000).

1.3.4 Electrolytes Calcium

The Recommended Dietary Allowances (RDA) for calcium increase during pregnancy
over that for non-pregnant, non-lactating adult women, primarily for foetal skeletal
development. Calcium deficiency is rare in pregnancy except in cases of hyperparathyroidism
and severe dietary deficiency (Ladipo, 2000).
Calcium is the structural material in bones and teeth. It is also required by cells to
conduct specialized functions. It acts as second messenger, enabling cells to respond to stimuli,
initiates cell division, secretion and movement, blood-clotting, etc (Allen and Wood, 1994).

1.4 Recommended Daily Allowances (RDA)

Recommended Dietary/Daily Allowances are recommended intakes that are based on
the amount of dietary nutrients required to replace losses in endogenous intestinal secretions,
urine, sweat and allowing for the efficiency of intestinal absorption (McGanity et al., 1994).
Pregnant women are encouraged to make sure they get all the calories and nutrients
they need. Although pregnancy requires additional nutrient and caloric intake, it does not
require a drastic change in caloric intake. The best way to pump up the diet is by eating a
variety of healthy foods and the daily prenatal vitamin supplement recommended by the doctor
or midwife (Chen, 2001).
Some of the most important nutrients, vitamins and minerals required during pregnancy
are iron, folic acid, calcium, phosphorus, vitamin A, vitamin B6, vitamin B12, vitamin C,
vitamin K, vitamin D, proteins and carbohydrates. The Recommended Dietary Allowances
(RDA) for pregnant women, non-pregnant and lactating women is shown in Table 1.5.
These nutrient requirements can be taken in by choosing the right kinds of food;
For example, green leafy vegetables are rich both in folic acid and iron. Calcium can be
obtained through dairy products, salmon, beans, etc. Dairy products are also rich sources of
nutrients such as phosphorus, riboflavin, vitamin A and vitamin D. Meat, fish and chicken are
rich in protein while cereals are rich in carbohydrates (Chen, 2001; Pizzorno, 2004).
 30

Table 1.5: Recommended Dietary Allowances (RDA) chart for pregnant, non-pregnant and
lactating women (Chen, 2001)
Nutrients Non Pregnant Lactating Functions
pregnant women women
women
Vitamin A (g) 800 800 1,300 Aids vision, growth of bones and
teeth.
Vitamin B6 (g) 1.6 2.2 2.1 Formation of red blood cells.
Vitamin B12 (g) 2.0 2.2 2.6 Formation of red blood cells.
Vitamin C (g) 60 70 95 Healing of wounds, Resistance to
infection and collagen formation.
Vitamin D (g) 10 10 12 Growth of bones and teeth.
Vitamin E (g) 8 10 12 Formation and use of red blood cells
and muscles.
Vitamin K (g) 55 65 65 Prevents a rare bleeding disorder in
the newborn.
Calcium (g) 1200 1200 1200 For bone and teeth formation. Proper
muscle and nerve function.
Folate (g) 180 250 280 Prevents neural tube defect. For
blood and protein formation and cell
division.
Iodine (g) 150 200 200 Required for hormone production.
Iron (mg) 15 15 15 For formation of hemoglobin
Magnesium (mg) 280 355 355 For proper nerve and muscle
function.
Niacin (mg) 15 20 20 Promotes healthy skin, nerves and
digestion.
Phosphorus (mg) 1200 1200 1200 For health and growth of bones and
teeth.
Proteins (g) 55 65 65 For overall health and growth, aids
in blood formation, supplies
building block for babys body.
Carbohydrate 2200 2500 2600 Provides energy for body function.
(kilocalories)
Riboflavin (mg) 1.3 1.5 1.5 Aids in energy release to cell.
Thiamin (mg) 1.1 1.5 1.6 Helps to digest carbohydrate.
Zinc (mg) 12 19 19 Aids in the production of enzymes
and insulin.
 31

1.5 Diagnosis of Protein Energy Malnutrition

The differential diagnosis for PEM is done by identifying by the clinical, biochemical
and physiological characteristics. They vary however, according to the severity of the disease,
age of patient, presence of other nutritional deficits, infection and the predominance of energy
or protein deficiency.

1.5.1 Mild and Moderate Protein Energy Malnutrition

The main diagnostic feature of mild and moderate PEM is weight loss. A decrease in
subcutaneous adipose tissue may become apparent in both adults and children. There is a
reduction of adiposity below 12% and 20% in men and women, respectively. There may be
other functional alterations not yet well characterised (Torun and Chew, 1994).
Biochemical information is not consistent in mild and moderate PEM. Laboratory data
related to low protein intakes may include low urinary excretion of creatinine, low urinary urea
nitrogen and hydroxyproline excretion, altered plasma patterns of free amino acid with
decrease in branched chain essential amino acids, slight decrease in serum albumin, transferrin
and circulating lymphocytes (Heymsfield et al., 1994).

1.5.2 Severe Protein Energy Malnutrition

Principal diagnosis for severe PEM in based on dietary history and clinical features. In
marasmus, there is generalised muscular wasting and absence of subcutaneous fat which give
the patient severe nonodematous PEM, a skin and bone appearance. They usually have 60%
or less of their expected weight for height. The hair is sparse, thin and dry without sheen; it is
easily pulled out without causing pains. The skin is dry, thin with little elasticity and wrinkles.
Patients have a look of apathy, anxiety with sunken cheeks caused by disappearance of the
Bichats fat pads, the last subcutaneous adipose depot to disappear. Some patients are anorexic
while some are hungry but they seldom tolerate large amount of food. Vomiting and diarrhoea
may be present. There is marked weakness, and heart rate, blood pressure and body
temperature may be low with tachycardia. Hypoglycemia may occur with hypothermia. The
viscera are usually small, abdominal distention may be present and lymph nodes easily
palpable. Dietary history plays an important role to differentiate PEM from other body wasting
diseases such as AIDS (Smith, 2002).
In kwashiokor there is soft, pitting, painless oedema, usually in the feet, legs and
extending to the perineum and the face. Patients may have skin lesions often confused with
 32

pellagra. In the areas of oedema, there is continuous pressure and irritation with zones of
dryness, hyperkeratosis and hyperpigmentation. The epidermis peels off exposing tissues for
easy infection. Subcutaneous fat is preserved and muscle wasting is not as severe as in
marasmus. The hair is brittle, without sheen and can be pulled out without pains. Pigmentation
usually changes it to dull brown, red or yellowish white. Patients may be pale. Anorexia,
vomiting and diarrhoea is common. There is hepatomegaly with protruding abdomen.
Tachychardia, hypothermia and hypoglycemia may occur.
In Marasmic- kwashiokor there is a combination of the clinical manifestation of
kwashiokor and marasmus. The main diagnostic feature include oedema as in kwashiokor with
or without skin lesions, muscle wasting and decreased subcutaneous fat as in marasmus. All the
biochemical features of both marasmus and kwashiokor are manifested. (Torun and Chew,
1994).
In severe PEM, the most common biochemical features are as follows:
(1) Serum concentrations of total proteins and albumin are markedly reduced in
kwashiokor but are normal or low in marasmus.
(2) Hemoglobin and hematocrit are usually low more so in kwashiokor than in marasmus.
(3) The ratio of nonessential to essential amino acids in plasma is elevated in kwashiorkor,
but usually normal in marasmus
(4) Serum levels of fatty acids are elevated in both, particularly in kwashiokor.
(5) Blood glucose level is normal or low
(6) Urinary excretion of ceatinine, hydroxyproline, 3 methyl histidine and urea are low.
(7) Plasma levels of other nutrients e.g. iron vary but tend to be low (Heymsfield et al.,
1994).

1.6 Pathophysiology and Biochemical Changes Associated With Protein Energy

Malnutrition

1.6.1 Energy Changes

In PEM, a decrease in energy intake is quickly followed by a decrease in energy
expenditure, accounting for shorter periods of play in children and shorter physical activities
and longer rest periods in adults. When the decrease in energy expenditure cannot compensate
for the insufficient intake, body fat is mobilized with a decrease in adiposity and resulting in
weight loss. Body mass diminishes gradually due to muscle protein catabolism with increased
 33

influx of amino acid as energy source. As the cumulative energy changes become severe
subcutaneous fat is markedly reduced and protein catabolism leads to muscle wasting. Visceral
protein is preserved longer especially in marasmus (Torun and Chew, 1994).
In Marasmus these alterations in body compositions lead initially to increased basal
oxygen consumption (ie basal metabolic rate) per unit of body weight and decreases in more
severe stages. In Kwashiorkor, the severe dietary protein deficit leads to an earlier visceral
depletion of amino acids that affects visceral cell function and reduces oxygen consumption.
Therefore, basal energy expenditure decreases per unit of total body mass.
Blood glucose concentrations may remain normal due to gluconeogenesis from amino
acids, glycerol and fats. However, it falls in severe PEM or when complications set in (Torun
and Chew, 1994).

1.6.2 Protein Changes

The poor availability of dietary proteins reduces protein synthesis. Adaptation leads to
sparing of body proteins and preservation of essential protein-dependent functions. The gradual
and inevitable loss of body protein as a result of long term dietary protein deficit is primarily
from skeletal muscle. Some visceral proteins are lost in early PEM but then become stable until
the non-essential tissue proteins are depleted, the loss of visceral protein then increases and
death may be imminent. When protein intake is reduced there is a decrease in total nitrogen and
amino acid turnover and a proportional decrease in amino acid catabolism and synthesis. This
leads to a markedly reduced urea synthesis and urinary nitrogen excretion. The half life of
several proteins increase. The rate of albumin synthesis decreases and in severe conditions, the
rate of breakdown falls and its half life increases. In addition to this, there is a shift of albumin
from extravascular to the intravascular pool to maintain adequate albumin levels due to reduced
synthesis. When protein depletion becomes too severe, the serum protein and albumin
concentrations decrease. This ensuing reduction in intravascular oncotic pressure and outflow
of water into the extravascular space contribute to the development of the oedema in
Kwashiokor (Torun and Chew, 1994).

1.6.3 Endocrine Changes

Hormones are important in the adaptive metabolic process. They contribute to the
maintenance of energy homeostasis through increased glycolysis, lipolysis, amino acid
 34

mobilization, preservation of visceral proteins, decreased storage of glycogen, fats and protein,
and decreased energy metabolism. Endocrine changes in PEM can be summarized as follows:
(1) The decreased food intake tends to reduce plasma concentrations of glucose and free
amino acids which in turn reduce insulin secretion and increase glucagon and
epinephrine release.
(2) The low plasma amino acid levels seen in kwashiokor, also stimulate the secretion of
human growth hormone and reduces somatomedin activity. These ultimately influence
the reduction in urea synthesis and favours amino acid recycling.
(3) The stress induced by low food intake and further amplified by fever, dehydration and
infections that accompany PEM also stimulate epinephrine release and corticosteroid
secretion more in Marasums than in Kwashiorkor due to greater severity in energy
deficit.
(4) Low levels of circulating insulin and high levels of circulating cortisol may further
reduce the secretion of somatomedins.
(5) Decrease in the activity of 51 monodiiodinase reduces the production of triodotyronine
with a concomitant increase in the inactive T3. Tyroxine levels are also reduced due to
decrease in iodine uptake. This decreases oxygen consumption and conserves energy
(Torun and Chew, 1994).

1.6.4 Haematology and Oxygen Transport

Reduction in haemoglobin concentration and red cell mass is an adaptation that
accompanies PEM. The reduction in lean body mass and reduced physical activity of
malnourished patients lead to lower oxygen demands. The simultaneous decrease in dietary
amino acids results in reduced haematopoietic activity which spares amino acids for synthesis
of other more necessary body proteins. When tissue synthesis, lean body mass and physical
activity improve there is a rise in oxygen demand calling for accelerated haematopoiesis
(Torun and Chew, 1994).

1.6.5 Cardiovascular and Renal Function

In PEM cardiac output, heart rate and blood pressure decrease, central circulation takes
precedence over peripheral circulation, cardiovascular reflexes are altered leading to
hypotension. In severe PEM peripheral circulatory failure may occur. Renal plasma flow and
 35

glomerular filtration rates are reduced as a consequence of decreased cardiac output. However,
water clearance and urine concentrations are not impaired (Torun and Chew,1994).

1.6.6 Immune System

In severe PEM, the major defects of the immune system involve the T lymphocytes and
the complement system. There is a marked depletion of lymphocytes from the thymus and
atrophy of the gland occurs. Also, cells of the T-lymphocyte regions of the spleen and lymph
nodes are depleted (Lin and Santoro, 2003). The production of complement components, the
functional activity of the complement system are depressed in severe PEM. Phagocytosis,
chemotaxis and intracellular killing are also impaired. There are also defects in antibody
production. The overall consequences are a greater predisposition to infections and a high
susceptibility to gram-negative bacterial sepsis (Torun and Chew, 1994).

1.6.7 Monokines
Monokines or cytokines are peptides/glycoproteins mediators of the bodys response to
injury. They are synthesized from monocytic and phagocytic cells lining the liver and spleen.
They activate neighbouring tissues and enter circulation to exert distant effects (Lin and
Santoro, 2003). Examples include 1L-1 and cachectin or tumor necrosis factor (TNF). In severe
PEM, there is low or decreased activity of IL-1 and might contribute to poor febrile response
and low leukocyte count in infections (Torun and Chew, 1994)

1.6.8 Electrolytes
In PEM, total body potassium decreases due to loss of intracellular potassium and
reduction in muscle protein. There is increased intracellular sodium due to alteration in cellular
exchange of sodium and potassium. Water accompanies sodium influx and although total body
intracelluar water is decreased because of loss in lean body mass there may be over hydration.
Other electrolytes are not affected in PEM unless there is a super imposed depletion as a result
of dietary deficiency e.g. anorexia, diarrhoea, pregnancy, etc (Torun and Chew, 1994).

1.6.9 Gastro Intestinal Functions

Impaired intestinal absorption of lipids and disaccharides and a decreased rate of glucose
absorption occur in severe protein deficiency i.e. kwashiorkor. The greater the protein
 36

deficiency, the greater the impairment. There is a decrease in gastric, pancreatic and bile
production. These further impair absorptive functions. Consequently, these may lead to
diarrhoea because of irregular intestinal motility and gastro-intestinal bacterial overgrowth
(Torun and Chew, 1994).

1.7 Clinical Features and Presentation of Protein Energy Malnutrition

Clinical features and presentation of PEM range from mild to severe. The type and
intensity of symptoms depend on the patients prior nutritional status and on the nature of the
underlying disease and the speed at which it is progressing (Lin and Santoro, 2003).
In mild and moderate PEM, the main clinical feature is weight loss. There is a decrease
in physical activity and energy expenditure in both adults and children. Capacity for prolonged
physical work is reduced especially in persons engaged in energy-demanding occupations.
Women have a higher probability of giving birth to infants with low birth weights. (Torun and
Chew, 1994).
In severe marasmus, there is gross weight loss giving a skin and bone appearance and
in children they have the appearance of a monkey or an old mans face (Torun and Chew,
1994). There is wasting of the subcutenous fat and muscles. The skin is wrinkled and loose
(inelastic). There is no clinical dermatosis however, the hair is brittle, and does not grow. It is
thin, dry and can be pulled out without pain. There is also retardation in growth.
In kwashiorkor, there is a failure to thrive moon face in children. There is generalized
oedema with swollen abdomen, flaky paint-dermatosis which split open when stretched. There
is depigmentation of the hair which causes it to be easily pulled out without pains. The nail
plates are thin, soft and fissured. There is a petulant apathy in addition to growth and mental
retardation (Lin and Santoro, 2003). Infection occurs in all forms of PEM with a variety of
bacteria infections producing pneumonia, diarrhoea, otitismedia, genito-urinary disease and
sepsis (Torun and Chew, 1994).

1.8 Effects and Complications Associated with Protein Energy Malnutrition (PEM)
PEM develops gradually in weeks and months. This allows a series of metabolic
adjustments that result in decreased nutrient demand and a nutritional equilibrium compatible
with a lower level of cellular nutrient availability. When the nutrient supply becomes
persistently lower, the patient can no longer adapt and eventually dies. PEM results in clinical
 37

complications and mortality. This is as a result of metabolic disruptions due to severe nutrient
deficit, infections or inadequate treatment.
In Marasmus the common complications are: gastroenteritis, dehydration, respiratory
infections e.g. pneumonia and tuberculosis, eye lesions due to hypovitaminosis A and anaemia.
Systemic infections lead to septic shock or intravascular clotting with high mortality rates.
In kwashiorkor, the same complications occur as in marasmus, but diarrhoea,
respiratory and skin infections are more frequent and severe. In marasmic kwashiokor. there is
a combined effect of marasmus and kwashiokor. There is oedema with or without skin lesions
(Lin and Santoro, 2003).
The most common cause of death in PEM are pulmonary oedema with broncho
pneumonia, tuberculosis, septicemia, measles, gastroenteritis and water and electrolyte
imbalance (Torun and Chew, 1994).
The long term effects of PEM abound especially when treatment is inadequate and
socioeconomic status is not improved. After PEM, liver cirrhosis, malabsorption and pancreatic
deficiency may persist. Humoral immunity may be impaired and cell mediated
immunocompetence may be markedly compromised. The degree of mental impairment is
related to duration, severity and age at the outset of malnutrition. Some relatively mild degree
of mental retardation may persist into school age in children. In adults it may result in
morbidity, in some cases mortality and organ failure. Patients generally have stunted growth
and are small for their age (Morley, 2007).
In women of child bearing age, PEM leads to stunted growth, poor weight gain during
pregnancy, anaemia, obstructed labour during delivery and they give birth to low birth - weight
babies. Baby girls born with low-birth weight are in addition likely to go through childhood
and adolescence with a disadvantage in terms of feeding and care and grow up to be severally
undernourished adult women in poor health. The vicious cycle of under nourishment and ill
health is set in motion of malnourished mothers giving birth to low-birth weight babies (Smith,
2002).

1.9 Management and Treatment of Protein Energy Malnutrition (PEM)

1.9.1 Mild and Moderate Protein Energy Malnutrition
Mild and moderate PEM are uncomplicated PEM and is treated in an ambulatory
setting such as outside the hospital or in the home. The patient is given home diet
supplemented with easily digested food that contains proteins of high biologic value and high
 38

energy density and adequate amounts of micronutrients. Therapy is also achieved by

instructing the adult patient about adequate eating habits and a better use of food resources.
Also by instructing mothers in improved feeding practices and more nutritious, culinary habits.
Breast feeding of infants should also be encouraged. Adequate amounts of vitamins and
minerals are ensured and supplemented by use of fortified foods e.g. iron-enriched bread or
sugar fortified with retinol. For pregnant, lactating and non-lactating women, there is added
protection in the ingestion of a daily mineral/ vitamin supplement that contain only the
essential minerals and vitamins (Lin and Santoro, 2003; Smith, 2002).

1.9.2 Severe Protein Energy Malnutrition

Patients with severe PEM are hospitalized to ensure adequate medical and nutritional
treatment. Treatment strategy is divided into three:
(1) Resolving life-threatening conditions
(2) Restoring nutritional status without disrupting homeostasis.
(3) Ensuring nutritional rehabilitation.

1.9.2.1 Resolving Life Threatening Conditions

Restoration of nutrition starts as soon as possible, but is delayed until life-threatening
conditions are resolved. The most frequent life-threatening conditions are as follows:-

(a) Fluid and Electrolyte Imbalance:

The first step in the treatment of PEM is to or correct fluid and electrolyte
abnormalities. The most common electrolyte abnormalities are hypokalemia, hypocalcemia,
hypophosphatemia and hypomagnesemia. The oral dehydration salt (ORS) recommended by
(WHO) World Health Organisation is used (WHO, 1990). One litre of ORS contains: 3.5g
NaCl, 2.5g Na2Co3, 1.5g KCl, and 20g glucose or 40g sucrose. It has osmolarity of 310m
Osm/L. It contains 90mmol/L of Na, 20mmol/L of K and 111mmol/L of glucose.
Other solutions successfully used include:-
1) 1:1 mixture of 10% dextrose in water (D/W) either with isotonic saline (i.e, 5% glucose
in 0.5N saline)
2) Darrows solution A 1:2:3 mixture of 0.17 sodium lactate: isotonic saline: 10% D/W
(glucose).
 39

Rehydration is intravenous or by nasogastric tube in vomiting or anorexic patients. It can also

be administered orally in small quantities in children during rehydration. (Torun and Chew,
1994).
(b) Infections:
PEM patients are particularly prone to infections which are frequently the cause of
death in severe PEM. Septicemia and infection are treated with appropriate antibiotics that do
not affect protein synthesis and taking into consideration drug metabolism which may have
been altered by the impaired metabolic system.
Other supportive and related medical treatments may be necessary, such as treatment for
respiratory distress, hypothermia, hypoglycemia etc. (Smith, 2002).
(c) Anaemia:
Severe anaemia is usually associated with PEM. Usually haemoglobin levels improve
with proper dietary treatment. However, in severe cases of PEM blood transfusion is necessary.
Whole blood is used with marasmic patients but packed red blood cells are better in
kwashiorkor or Oedematous PEM. IM Iron is also used to treat mild anaemia in adults with
PEM. This can be administered orally (Smith, 2002).
(d) Hypothermia and Hypoglycemia:
Hypothermia as low as 35.50C and plasma glucose level as low as 3.3 mmol/l
(60mg/dl) is found in PEM and can be due to reduced fuel substrate and infections.
Hypoglycemia is treated by intravenous or oral administration of 10ml to 20ml of 50% glucose
followed by 25ml 50ml of 5% glucose. Body temperature usually gradually rises after
administration of glucose-containing diets or solutions.
(e) Vitamin Deficiency:
Severe PEM is often associated with vitamin deficiency, especially vitamin A. Water-miscible
vitamin A as retinol is given orally or intramuscularly. Corneal ulcerations are treated with
ophthalmic drops and antibiotic ointment or drops.
For other vitamins, a daily multivitamin supplement is given (Smith, 2002).
(f) Hemodynamic Alterations:
Cardiac failure may develop during rehydration process in severe PEM. This is usually due to
impaired cardiac and membrane function.
Diuretics e.g. furosemide are administered intravenously or intramuscularly to correct the
alterations (Torun and Chew, 1994).
 40

1.9.2.2 Restoring Nutritional Status Without Disrupting Homeostasis

The second step in the treatment of PEM is to supply macronutrients by dietary therapy.
Milk based formulas are treatment of choice at the beginning of dietary therapy (Morley,
2002).
The main objective of dietary therapy is to replace nutrient-tissue deficits as rapidly and
safely as possible. This starts as soon as life-threatening conditions have been managed.
Regimen provides a diet that meets daily maintenance requirements for a few days, followed
by a gradual increase in nutrient delivery. For children and adults with good appetite, liquid
formula is substituted gradually by solid food of high density and easily digestible nutrients.
However, oedematous PEM patients will require diet which contains vegetable oil to increase
the diet energy density.
The protein source must be of high biologic value and easily digestable. Cows milk,
goats, ewes buffalos and carmels milk are frequently used. Eggs, meat, fish, soy isolates and
some vegetable protein mixtures are also sources of protein. Additional water is given to
provide at least 1ml of total fluid per kilocalorie in the diet. Multivitamin is also added as
dietary supplements (Torun and Chew, 1994).

1.9.2.3 Ensuring Nutritional Rehabilitation

The last stage of treatment is to ensure that the patient must continue to eat adequate
amounts of protein, energy and other nutrients especially when traditional foods are introduced
in the diet. At this stage emotional stimulation is provided, persistent diarrhea, infections and
other complications are treated in both adults and children. Children are vaccinated during this
period as well. Adult patients may undergo physical therapy or made to exercise regularly to
improve deteriorated muscles and cardio respiratory system. Full recovery is often experienced
as nutritional status improves and stabilizes (Torun and Chew, 1994).

1.10 Prevention of PEM

Poverty, Ignorance, frequent infection, cultural norms/customs, severe cyclic climatic
conditions, natural and man made disasters are among the main causes of PEM. Therefore, its
control and prevention require multi-sectoral approaches that include food production and
distribution, preventive medicine, education, social development and economic improvement.
At a national or regional level, control and prevention can only be achieved through short-term
 41

and long-term political commitments and effective actions to enforce the measure to eradicate
the underlying causes of malnutrition.
The most likely victims of PEM are children and women, especially those within child-
bearing age from low socioeconomic strata. Children whose parents have misconceptions
concerning the use of food, who come from broken or unstable families, whose families have a
high prevalence of violence, alcoholism and drug abuse, who live under poor sanitary
conditions in urban slums or in rural areas frequently subject to droughts or floods, whose
societal beliefs prohibit the use of nutritious foods. Special attention must be given to the
following for the prevention of PEM.

1.10.1 Availability of Food

Food can be made available directly during periods of famine as a short-term solution,
but a more long term solution is needed which will include agricultural development
programmes
Animal foods are the best source of protein but they tend to be expensive, not always
available and/or prohibited by religious practices. Under such circumstances staple vegetable
foods can be complemented with other foods combined in culturally accepted ways to permit a
good essential amino acid complementation and improve the biologic value of dietary protein.
Energy density can be increased by adding fats or carbohydrates.
Parents should also be convinced about safety and benefits of using foods which in
some cultures are fed only to adults and older children. This is especially true of foods used to
complement mothers milk or to wean infants from the breast.
Children who are fully weaned or only occasionally breast fed must receive adequate
amounts of energy and protein-rich staple food and ideally animal foods to satisfy their
nutritional needs and allow adequate growth. Also foods should not be withheld during
diarrhoea for it may shorten the duration of diarrhoea.
Breastfeeding a baby for at least 6 months is considered the best way to prevent early
childhood malnutrition (Torun and Chew, 1994).

1.10.2 Reducing Infections

Because young children are at a greater risk of malnutrition, high priority must be given
to immunization, sanitary measure to reduce fecal contamination and early oral rehydration and
feeding of children with diarrhea (Torun and Chew, 1994).
 42

1.10.3 Education
Nutritional and public health education programmes, especially programmes that
monitor growth and development as well as provide nutritional information and supplements,
and improved food distribution system (Smith, 2002).
Such programmes must emphasize promotion of breastfeeding, distribution of child
instant formulas, appropriate use of weaning foods, nutritional alternatives using traditional
foods, personal and environmental hygiene, feeding practices during illness, and
convalescence, early immunization, and early treatment of diarrhoea (Torun and Chew, 1994).
Hospitalized patients should be screened for the presence of illness and condition that
could lead to PEM. The nutritional status of patients at higher than average risk should be more
thoroughly assessed and periodically re-evaluated during extended hospitalization or in nursing
home residence.
Women especially women of child-bearing age should be educated on the benefits of
many, if not all micronutrients, trace elements, vitamins and minerals, the consequences of
their deficiency in pregnancy and the functional effects of their supplementation. Also on the
deficiencies that exist because of losses or malabsorption associated with PEM or inadequate
intakes, lack of knowledge about adequate prenatal nutrition or dietary taboos associated with
pregnancy, with potential adverse consequences for both mother and newborn (Ladipo, 2000).

1.10.4 Prognosis
Mortality rate in severe PEM can be as high as 50% but adequate treatment and dietary
therapy can reduce it to less than 10%. The immediate cause of death is usually infections but
with prevention and adequate treatment of infections and other complications together with
adequate dietary therapy mortality rate can be reduced. (Lin & Santoro, 2003). In children,
mortality rate due to PEM varies between 5% - 40%. Lower mortality are observed in children
under intensive care. Death in the first days of treatment is usually due to electrolyte
imbalance, infection with sepsis, hypothermia and heart failure. Stupor, Jaundice, Petechiae
and persistent diarrhoea are all effects of PEM (Smith, 2002).
Treatment of mild and moderate PEM corrects the acute signs of severe PEM.
Childrens catch up growth in height may take a long time or might never be achieved. Weight
for height can be restored easily but the child may remain stunted and a small body size may
 43

influence maximal working capacity as adult. They may have residual behavioural and mental
problems in terms of creativity and social interactions (Torun and Chew, 1994).
The disappearance of apathy, oedema and anorexia in severe PEM is a sign of
favourable recovery. Recovery is more rapid in kwashiokor than in marasmus. There is no
evidence that damage done by PEM cannot be corrected in a good stimulating environment.

1.11 Aim of Research

The aim of this study is to investigate some of the biochemical changes associated with
protein energy malnutrition (PEM) in pregnant women in Enugu metropolis.
 44

CHAPTER TWO
MATERIALS AND METHODS
2.1 Materials
2.1.1 Equipment Used
The major equipment used in this study were:
Water Bath Gallenkamp, England
Chemical Balance Gallenkamp, England
Test-tubes Pyrex, England
Conical Flasks Pyrex, England
Hotbox Gallenkamp, England
Centrifuge (3,500 rpm) PIC, England
Syringe (1ml and 5ml) DANA JET, Nigeria
Microscope Slides Unescope, U.S.A
Digital Photo Calorimeter EI (312 Model), Japan
Adjustable Micropipette PERFECT, U.S.A
Refrigerator Kelvinator, Germany
Beakers Pyrex, England
pH Meter Pye, Unicam 293, England
Stirrer SWARD, England

2.1.2 Chemicals and Reagents Used

All the chemicals used in this study were of analytical grade. Sample analysis was
carried out using high performance enzymatic colorimetric commercial kits (All from Bio-
systems Reagents and Instruments, Barcelona, Spain).

2.1.3 Study Subjects

Three groups of female volunteers were involved in this study. The first group were 52
pregnant women with low total protein (<52g/l), the second group were 50 pregnant women
with normal total protein (>52g/l), while the third group were 50 non pregnant, non lactating,
apparently healthy women (>52g/l). All the subjects were between 20-40 years of age. All the
pregnant subjects were attending antenatal clinic of Parklane Specialist Hospital, were in
 45

different gestational stages of pregnancy with different parity. Subjects with complications
such as hypertension, diabetes, HIV/AIDS on admission were excluded.

2.2 Methods
2.2.1 Collection of Blood Samples and Preparation of Serum
Blood (2.5mls) was collected from each volunteer by venepuncture and delivered into
clean and duely labelled specimen containers. The blood was allowed to clot and then
centrifuged at 5000 rpm for 10 minutes. Using a Pasteur pipette serum was separated from the
cells and delivered into a clean and dry bottle. It was stored frozen at 200C until it was used.

2.2.2 Preparation of Reagents

The reagents used were high performance enzymatic colorimetric commercial analytical kits
(Biosystems Reagents and Instruments, Barcelona, Spain). These commercial kits were
purchased and used according to the manufacturers direction for all the parameters assayed.

2.2.2.1 Reagents for the determination of serum total protein

The commercial serum total protein kit contained:-
A. Reagent A made up of copper II acetate (6 mmol/l), potassium iodide (12mmol/l), and
sodium hydroxide (1.15mol/l).
B. Protein standard made up of bovine albumin concentration of standards provided were
ready for use.

2.2.2.2 Reagents for the determination of serum albumin

The reagents for the determination of serum albumin contained:-
A. B.C.G. Concentrate Made up of succinate buffer (75mmol/l, pH 4.2), bromocresol
green (0.15mmol/l) and Brij 35.
B. Albumin Standard Made up of human serum albumin 45g/L (4.5g/dl), and Tris buffer
(100mmol/l, pH 7.3).
Reagents and standard were provided ready for use.
 46

2.2.2.3 Reagents for the determination of serum total cholesterol assay

Commercial cholesterol kit contained:-
A: Reagent A Made up of Pipes buffer (pH 6.8) 35mmol/L, sodium cholate (0.5mmol/l),
phenol (28mmol/l), cholesterol esterase (0.2/ml), cholesterol oxidase (0.1/mL,
Peroxidase 0.8u/mL, 4-amino-anitpyrine 0.5 mmol/L (pH 7.0).
B: Cholesterol standard Cholesterol 200mg/dl (5.18mmol/L).
Reagents and standards were provided ready for use.

2.2.2.4 Reagents for the determination of urea

Commercial kit for the determination of urea contained:-
A1: Reagent - Sodium salicylate 62mmol/L, sodium nitroprusside 3.4mmol/L
and phosphate buffer 20mmol/L (pH 6.9).
A2: Reagent - Urease 500U/mL.
B: Reagent - sodium hypochlorite 7mmol/L and sodium hydroxide
150mmol/L.
S: Urea standard - Urea 50mg/dl (8.3mmol/L)
Reagent B and standard (S) were provided ready for use.
For working reagent, the contents of Reagent A2 was transferred into reagent A1 bottle
and mixed thoroughly. The mixture was stored at 2oC till use.

2.2.2.5. Reagents for the determination of creatinine

The commercial kit for the determination of creatinine contained:-
A: Reagent - Picric acid 25mmol/L
B: Reagent - Sodium hydroxide 0.4mmol/L
S: Creatinine Standard - Creatinine 2mg/dl (177mmol/L)
Standard was provided ready for use
For working reagent, equal volumes of reagent A and reagent B were mixed thoroughly and
stored at 2oC till use.

2.2.2.6. Reagents for the determination of calcium

The commercial kit for the determination of total calcium contained.
A: Reagent - Potassium cyanide 7.7mmol/L and ethanolamine 1.5mmol/L.
B: Reagent - Methylmol blue 0.1mmol/L, Hydrochloric acid 10mmol/L,
Hydroquinoline 17mmol/L.
 47

S: Calcium standard - calcium 10mg/dl (2.5mmol/L)

Calcium standard was provided ready for use.
For working reagent, equal volumes of reagent A and reagent B were mixed thoroughly but
gently. It was stored at 8oC and used within 2 days.

2.2.3 Determinations of Sample

2.2.3.1 Determination of serum total protein.
Principle: The serum total protein was determined using the Biuret method. The protein in the
sample reacted with copper II ion in alkaline mediun forming a coloured complex that was
measured spectrophotometrically (Gornall et al., 1949).

O=C-NH2 N2N-C=O

HN C
O=C C=O

NH2 NH2
NH2 C2+ NH2

O=C C=O

HN NH

O=C C=O
NH2 NH2
Fig. 2.1: Biuret Reaction: The co-ordination complex formed in alkaline solution between
cupric ions and the nucleophilic nitrogen atoms of four moles of Biuret.

Procedure

The following aliquot of water, protein standard, test sample and reagent were pipetted into
different test tubes.
Blank Standard Sample
Distilled water 20 l - -
Protein standard - 20 l -
Test sample - - 20 l
Reagent 1.0 ml 1.0 ml 1.0 ml
 48

They were mixed thoroughly and allowed to stand for 10 minutes at room temperature. The
absorbance (A) of the test sample and standard were measured against the reagent blank within
60 minutes at 545nm.

Calculation:-

The protein concentration of the sample was calculated using the following general formula:
Absorbance of Sample
Concentration of Standard
Absorbance of S tan dard

Reference Values:

Non pregnant women - 63-78 g/L

1st Trimester - 58-72 g/L

2nd Trimester - 56-64 g/L

3rd Trimester - 52-65 g/L

(Berg et al., 1984)

2.2.3.2 Determination of Serum Albumin Concentration

Principle:
The measurement of serum albumin was by the quantitative method using (B.C.G)
Bromocresol green. This method is based on the quantitative binding of albumin in the sample
to the indicator 3, 3, 5, 5 tetra bromo-m cresol sulphonephthalein (BCG) (Bromocresol
Green). This will form a complex known as the Albumin BCG complex. This complex
absorbs maximally at 578nm, the absorbance (A) being directly proportional to the
concentration of Albumin in the sample (Doumas et al., 1971).
 49

Procedure:-
The following aliquots of distilled water, Albumin standard, test sample and reagent
were pipetted into different test tubes.
Blank (ml) Standard (ml) Sample (ml)
Distilled water 0.01 ml - -
Albumin standard - 0.01 ml -
Test sample - - 0.01 ml
Reagent 3.00 ml 3.00 ml 3.00 ml

They were mixed thoroughly and incubated in a water bath for five minutes at 25oC.
The absorbance (A) of the test sample and standard were measured against the reagent blank
within 60 minutes.

Calculation:-
The albumin concentration in the sample was calculated using the following formula:
Absorbance of Sample
Concentration of Standard
Absorbance of S tan dard

Where concentration of albumin standard was given as 45 g/l (4.5 g/dl).

Reference values:
Non pregnant women - 36-46 g/L
Pregnant women:-
1st Trimester - 33-43 g/L
2nd Trimester - 29-37 g/L
3rd trimester - 28-36 g/L
(Berg et al., 1984)

2.2.3.3 Determination of Serum Total Cholesterol

Principle:
The total cholesterol concentration of the test individuals was determined using
cholesterol enzymatic endpoint method. Cholesterol is determined after enzymatic hydrolysis
and oxidation. The indicator quinoneimine is formed from hydrogen peroxide and 4-
aminoantipyrine in the presence of phenol and peroxide. The free and esterified cholesterol in
 50

the sample originates by means of the coupled reactions described below, a coloured complex
is formed that can be measured spectrophotometrically (Allain et al., 1974),
(Meiattini et al., 1978).

Cholesterol-
esterase
Cholesterol ester +H20 Cholesterol + fatty acid

Cholesterol-
esterase
Cholesterol + 02 + H20 Cholesterol + H202

2H2O2 + 4-Aminoantipyrine + Phenol Peroxidase Quinoneimine + 4H20

Procedure:
The following aliquots of distilled water, test sample, cholesterol standard and reagent were
pipetted into different test tubes.
Blank Standard Sample
Distilled water 10 l - -
Protein standard - 10 ul -

Test sample - - 10 ul
Reagent 1.0 ml 1.0 ml 1.0 ml

They were mixed thoroughly and incubated for 10 minutes at room temperature. The
absorbance (A) of the sample and the standard were measured at 500nm against the blank
within 60 minutes.
Calculation:-
The total cholesterol concentration in the sample was calculated using the following formula.
Absorbance of Sample
Concentration of Standard
Absorbance of S tan dard
Where concentration of standard was given as 200mg/dl or 5.18mmol/L cholesterol.
 51

Reference values:
Non pregnant women - 3.13 - 6.87mmol/L(120-200mg/dl)
Pregnant women
1st Trimester - 3.03 - 5.94mmol/L (120-200mg/dl)
2nd Trimester - 3.19 7.47mmol/L (120-200mg/dl l)
3rd trimester - 4.71 8.55mmol/L (120-200mg/dl)
(Berg et al., 1984)

2.2.3.4 Determination of serum urea concentration

The urea concentration of the test individuals were determined by the urease
salicylate enzymatic method. This uses the enzyme urease to hydrolyze urea. The ammonia
produced reacts with alkaline hypochlorite and phenol in the presence of a catalyst to form
indophenol. The coloured complex is measured spectrophotometrically.

Urea +H20 2NH4+ + CO2

NH4+ + Salicyalate + Naclo Nitroprusside Indophenol

(Cheestbrough, 1998).

Procedure:

The following aliquots of distilled water, test sample, standard and (reagent A) working reagent

were pipetted into different test tubes.

Blank Standard Sample

Distilled water 10 ul - -
Urea standard - 10 ul -
Test sample - - 10 ul
Reagent A (working 1.0 ml 1.0 ml 1.0 ml
reagent)
They were mixed thoroughly and the tubes were incubated for 10 minutes at room temperature

Then 1.0ml each of reagent B was pipetted into the different test tubes.

Reagent B 1.0ml 1.0ml 1.0ml.

 52

They were mixed thoroughly and the tubes were incubated for 10 minutes at room

temperature. The absorbance (A) of the test sample and standard were measured against the

blank at 600nm within 60 minutes.

Calculation:-

The urea concentration in the sample was calculated using the general formular below:
Absorbance of Sample
Concentration of Standard Sample Dilution Factor
Absorbance of S tan dard

Where concentration of urea standard is given as 50mg/dl (8.3mmol/L) urea.

Reference values:
Non pregnant women - 2.5 - 6.5mmol/L (15-45mg/dl)
st
1 Trimester - 1.6 5.0mmol/L (9.6 - 300mg/dl)
2nd Trimester - 1.6 4.3mmol/L (9.6-25.8mg/dl)
3rd trimester - 0.9 4.5mmol/L (5.4 -27mg/dl)
(Cheesbrough, 1998).

2.2.3.5 Determination of serum creatinine concentration

Principle:
Creatinine in the sample reacted with picrate in alkaline medium to form a coloured
complex. The coloured complex is measured spectrophotometrically. It is measured within a
short period to avoid interference from non creatinine substances.
(Bartels and Bohmer, 1971) and (Fabiny and Ertingshausen, 1971)

Procedure:-
The following were pipetted into a cuvette; the working reagent, standard and or sample.
Cuvette (A) Cuvette (B)
Working reagent 1.0 ml 1.0 ml
Standard 1.0 ml -
Test sample - 1.0 ml
 53

They were mixed thoroughly and the cuvette was inserted into the spectrophotometer.
The absorbance for the sample and standard were measured and recorded at 500nm after 30
seconds (A1) and after 90 seconds (A2).
Calculation:-

The creatinine concentration in the sample was calculated using the following general
formula:-
Absorbance of Sample
Concentration of Standard Sample Dilution Factor
Absorbance of S tan dard

Where concentration of standard is given as 2mg/dl (177 mol/L) creatinine

Reference values:
Non pregnant women - 50 90umol/L (0.6-1.0mg/dl)
Pregnant women
1st Trimester - 25 79umol/L (0.3-0.9mg/dl)
2nd Trimester - 25 74umol/L (0.3-0.8mg/dl)
rd
3 trimester - 23 93umol/L (0.3-1.1mg/dl)
(Berg et al., 1984)

2.2.3.6 Determination of serum calcium concentration

Principle:

Calcium in the sample reacted with methylthymol blue in alkaline medium to form a

coloured complex. The coloured complex was measured spectrophotometrically.

Hydroxyquinoline was included in the reagent to avoid magnesium interference.

(Gindler and King, 1972; Barnett et al., 1973)

 54

Procedure:-
The following aliquots of distilled water, calcium standard, test sample and working
reagent were pipetted into different test-tubes.
Blank Standard Sample
Distilled water 1.0 l - -
Calcium standard - 1.0 l -
Test sample - - 10 or 1.0 l
Working Reagent 1.0 ml 1.0 ml 1.0 ml

They were mixed thoroughly and left to stand for 2 minutes at room temperature. The
absorbance (A) of the sample and standard were measured at 610nm against the blank within
60 minutes.
Calculations:-
The calcium concentration in the sample was calculated using the following general formula:-
Absorbance of Sample
Concentration of Standard Sample Dilution Factor
Absorbance of S tan dard

Where concentration of calcium standard was given as 20mg/dl (5mmol/L) calcium.

Reference values:
Non pregnant women - 2.17 2.43mmol/L (8.7-9.7mg/dl)
Pregnant women
1st Trimester - 2.12 2.44mmol/L (8.5-9.8mg/dl)
2nd Trimester - 2.04 2.36mmol/L (8.2-9.5mg/dl)
3rd trimester - 2.05 2.37mmol/L (8.2-9.5mg/dl)
(Berg et al., 1984)

2.2.3.7 Statistical Analysis

The data were analysed using the SPSS package of windows version 11.00 (SPSS
Corporation, IL). Differences between the means were separated and analysed for statistical
difference using the one way ANOVA while correlations between parameters were calculated
using the Pearsons correlation coefficient. Difference in means with p values < 0.05 were
accepted as significant. Data were presented as means standard deviations.
 55

CHAPTER THREE
RESULTS
Three groups of female subjects were involved in this study. The first group represented
52 pregnant women with low total protein, the second group represented 50 pregnant women
with normal total protein while the third group represented 50 non pregnant, non lactating
apparently healthy women. All the subjects were aged between 20 and 40 years. All the
pregnant subjects were in various gestational stages of pregnancy, and different parity. The
mean age was 28.90 5.31 for all the subjects studied.

3.1 Distribution of Mean Values of Measured Parameters in the Subjects

Fig. 3.1 shows the means values of serum total protein, albumin, Globulin, Cholesterol,
urea, creatinine and calcium of all the subjects studied
 56

200

180

160
Pregnant Low S.T.P
Pregnant Normal S.T.P
Non-pregnant Normal S.T.P
140
Mean Conc. (mg/dl)

120

100

80

60

40

20

0
Serum total Albumin Globulin Serum Urea Creatinine Calcium
Protein Cholesterol

Group

Fig. 3.1: Distribution of mean values of measured parameters in the

subjects
 57

3.2 Effect of Age on the Different Parameters Measured

Figs. 3.2 to 3.5 show the results of all parameters measured for mothers in different age
groups divided according to their level of serum total protein. There was no significant
difference in the means of the serum total protein of mothers of different age ranges and other
parameters measured (p>0.05). Therefore age may not affect the level of serum total protein of
a mother and also other parameters measured. A test of correlation showed that age of mother
did not correlate significantly with serum total protein. However age of mother correlated
positively and significantly with parity only (r = + 0.545) (p<0.05) and no other parameter
measured (p>0.05 in each case).
 58

200

< 25
180
25 - 33
> 33

160

140
Mean Cholesterol Conc. (mg/dl)

120

100

80

60

40

20

0
Pregnant low STP Pregnant normal STP Non-pregnant Normal
STP
Group

Fig. 3.2: Concentration of serum total cholesterol of both pregnant

and non-pregnant subjects with different levels of serum total
protein
 59

30
< 25 Years
25 - 33 Years
> 33 Years

25
Mean Serum Urea Conc. (mg/dl)

20

15

10

0
Pregnant low STP Pregnant normal STP Non-pregnant Normal
STP
Group

Fig. 3.3: Concentration of serum urea of both pregnant and non-

pregnant subjects with different levels of serum total protein
 60

1 < 25 Years
25 - 33 Years
> 33 Years
0.9

0.8
Mean Serum Creatinine Conc. (mg/dl)

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0
Pregnant low STP Pregnant normal STP Non-pregnant Normal
STP
Group

Fig. 3.4: Concentration of serum creatinine of both pregnant and

non-pregnant subjects with different levels of serum total protein
 61

< 25 Years
12 25 - 33 Years
> 33 Years

10
Mean Serum Ca Conc. (mg/dl)

0
Pregnant low STP Pregnant normal STP Non-pregnant Normal
STP
Group

Fig. 3.5: Concentration of serum calcium of both pregnant and non-

pregnant subjects with STP
 62

3.3 Effect of Parity (Number of Children Had By Mother) on the Different

Parameters Measured
Fig. 3.6-3.8 show results of all parameters measured for mothers with different parity
divided according to their level of serum total protein.
A significant difference was seen between the mean serum total protein levels of
mothers who have 1-2, 3-4 and 5 or more children at 95% confidence internal, using analysis of
variance test (p<0.05).
A test of correlation revealed that number of children a mother had (parity), correlated
negatively with serum total protein. (r = - 0.226, p<0.05).
This suggests that serum total protein levels significantly decreased as number of
children increased and vice versa. Age of mother also correlated positively and significantly
with parity (r = + 0.545, p<0.05).
 63

180

160

Pregnant low STP

Pregnant normal STP
140
Non-pregnant Normal STP

120
Mean Conc. (mg/dl)

100

80

60

40

20

0
Total Protein Cholesterol Urea Creatinine Calcium

Parameters determined

Fig. 3.6: Concentrations of some biochemical parameters

determined for mothers with 1 to 2 children
 64

180

160

Pregnant low STP

Pregnant normal STP
140
Non-pregnant Normal STP

120
Mean Conc. (mg/dl)

100

80

60

40

20

0
Total Protein Cholesterol Urea Creatinine Calcium

Parameters determined

Fig. 3.7: Concentrations of some biochemical parameters

determined for mothers with 3 to 4 children
 65

180

160

Pregnant low STP

Pregnant normal STP
140
Non-pregnant Normal STP

120
Mean Conc. (mg/dl)

100

80

60

40

20

0
Total Protein Cholesterol Urea Creatinine Calcium

Parameters determined

Fig. 3.8: Concentrations of some biochemical parameters

determined for mothers with 5 or more children
 66

3.4 Effect of Gestational Stage of Pregnancy (in Trimesters) on the Different

Parameters Measured
Figs. 3.9 to 3.11 show results of all parameters measured for mothers in different
gestational stage of pregnancy (in trimesters) tabulated according to their level of serum total
protein. The serum total protein levels of mothers at different gestational stage of pregnancy
were found to be significantly different from a statistical point (p<0.05).
The stage of pregnancy was found to correlate negatively and significantly with serum
total protein (r = - 0.402, p<0.05) and serum calcium (r = - 0.288, p<0.05). This suggests that as
the stage of pregnancy increases in trimesters, the serum total protein decrease significantly.
Also as the stage of pregnancy increases in trimesters, the level of serum calcium significantly
decreases.
 67

180

160

Pregnant low STP

Pregnant normal STP
140
Non-pregnant Normal STP

120
Mean Conc. (mg/dl)

100

80

60

40

20

0
Total Protein Cholesterol Urea Creatinine Calcium

Parameters determined

Fig. 3.9: Concentrations of some biochemical parameters

determined for mothers in the first trimester
 68

180

160

Pregnant low STP

Pregnant normal STP
140
Non-pregnant Normal STP

120
Mean Conc. (mg/dl)

100

80

60

40

20

0
Total Protein Cholesterol Urea Creatinine Calcium

Parameters determined

Fig. 3.10: Concentrations of some biochemical parameters

determined for mothers in the second trimester
 69

180

160

Pregnant low STP

Pregnant normal STP
140
Non-pregnant Normal STP

120
Mean Conc. (mg/dl)

100

80

60

40

20

0
Total Protein Cholesterol Urea Creatinine Calcium

Parameters determined

Fig. 3.11: Concentrations of some parameters determined for

mothers in the third trimester
 70

CHAPTER FOUR
DISCUSSION
Pregnancy is a physiological condition severely aggravated by protein energy
malnutrition. This makes protein energy malnutrition (PEM) the most widespread and disabling
public health problem among women especially in developing countries like Nigeria.
The demand for both energy and nutrient is increased during pregnancy and for well
nourished women only a small amount of additional energy is required (WHO, 1999).
Pregnancy is also associated with major alterations in every maternal organ, system and
metabolic pathway. Values of biochemical parameters may change as the pregnancy advances
from first to third trimester and to parturition and then return towards normal during post
partum period. The two major physiological forces driving these changes are:
(1) The increase in plasma volume, increase in red blood cells and decreased
concentrations of circulating nutrient-binding proteins and micronutrients.
(2) The ever increasing levels of estrogen and progesterone as well as other placental
related hormones, which have particular impact on maternal lipids (cholesterol)
(McGanity et al., 1994).
These two physiological modifications result in two dominant effects: the first reduces
levels of biochemical substances such as albumin and haemoglobin which return to normal
8-10 weeks post partum. The second causes lipids to rise during pregnancy and return to
normal at post partum. The major consequences of protein energy malnutrition (PEM) are
mainly poor weight gain in pregnancy, anaemia leading to high risk delivery and low birth-
weight babies that fail to thrive.
The result of this investigation showed that all the biochemical parameters measured viz
serum total protein, Albumin, urea, total cholesterol, creatinine and calcium were significantly
reduced in pregnant PEM individual compared with pregnant and non-pregnant controls.
This agrees with the studies of Onyeneke et al. (2003) which suggests that
abnormalities in serum levels of biochemical parameters occur in any form of PEM and are
related to the severity of the condition (Fig. 3.1).
The result of this investigation also showed there was no significant difference in the
mean serum total protein in the different age groups (see Figs. 3.2-3.5). Therefore age of
mother may not be a factor and may not affect the level of serum total protein and other
parameters measured. This agrees with the study of Okwu et al. (2007), which showed that the
 71

lower age groups (below 20years and 20-24years) presented higher prevalence of PEM than
other age groups,with the effect more prominent in rural areas than in urban areas.
The result also showed a negative correlation between parity, serum total protein, urea,
total cholesterol, creatinine and calcium (Figs. 3.6 3.8). This suggests that serum total protein
significantly decreased as number of children increased and vice versa. This implies that parity
therefore, may be a contributing factor to PEM. This agrees with the views of McGanity et al.
(1994) that PEM is manifests in many mothers because their nutritional status has not improved
from their last birth and so they are not nutritionally prepared for the next pregnancy.
In addition, Lapido (2000) suggested that many pregnancies in developing countries
are unplanned, coupled with inadequate dietary intake due to dietary taboos associated with
pregnancy, gender and other cultural beliefs.
The result of the experiment on the relationship between stages of pregnancy and on
serum protein level further showed that the stage of pregnancy in trimesters correlated
negatively and significantly with serum total protein level and serum calcium level
(Figs. 3.9 3.11).
This suggests that as stage of pregnancy in trimesters increases, the serum total protein
levels and calcium levels decrease significantly. The implication is that stage of pregnancy in
trimesters affects serum total protein and calcium levels and may therefore be a factor in
Protein Energy Malnutrition. This agrees with the studies of Sanchez et al. (1997) that serum
calcium levels fall gradually during pregnancy and towards the end of gestation seem to
become calcium deficient. However, Kazzi et al. (1998) reported that calcium deficiency in
pregnancy appears in severe dietary inadequacy and in individuals who are unable to eat diet
rich in dairy products. Earlier researchers like Stabile et al. (1995) agreed with this. Maine
(2000) also suggested that low calcium deficiency has been associated with hypertensive
disorders of pregnancy.
The results from assessment of biochemical parameters showed that serum total
cholesterol did not significantly correlate with serum total protein or any other parameters
measured (p> 0.05 in each case). Serum total cholesterol therefore may not be a factor in
changes associated with low serum total protein and protein energy malnutrition. These
findings are in consonance with the view of Toigo et al. (2000) that low serum cholesterol in
PEM may reflect energy imbalance. Total serum cholesterol therefore is a useful marker for
energy intake but not for protein intake.
 72

The serum urea levels were found to correlate significantly and positively with serum
total protein (r = + 0.246, p< 0.05) and serum creatinine level (r = + 0.275, p< 0.05) only. Its
correlation with other parameters were not significant (p>0.05 in each case). This suggests that
as the serum total protein increases, serum urea and creatinine levels increase with it and vice
versa.
A test of correlation further revealed that serum creatinine correlated positively and
significantly with serum total protein level (r = + 0.497, p < 0.05), serum urea level (r = +
0.275, p < 0.05) and serum calcium levels (r = + 0.356, p < 0.05). This suggests that as serum
total protein increases, serum creatinine, serum urea and serum calcium levels increase and
vice versa.
The result of this study further showed that serum calcium levels were negatively and
significantly correlated with stage of pregnancy in trimester (Table 12) (r = - 0.288, p < 0.05).
However, serum calcium was found to positively and significantly correlate with serum
total protein level (r = + 0.681, p<0.05) and serum creatinine level only (r = + 0.0356, p <
0.05). This suggests that as serum total protein increases, serum calcium levels and serum
creatinine levels increase with it and vice versa. However, serum calcium levels decrease as
stage of pregnancy increases in trimester. The reverse is also true.
These factors are pointers to Protein Energy Malnutrition (PEM).
Toigo et al. (2000) reported that in individuals with PEM, there is low serum concentration of
creatinine, suggesting a decreased skeletal muscle mass or a low dietary protein intake. In the
same way Ladipo (2000) reported gradual decline in serum calcium levels from first trimester
to third trimester. The researcher suggested that in PEM individuals, homeostatic control of
calcium depletion is maintained by a complex interaction of vitamin D, parathyroid hormones
and calcitonin.
In summary, the serum total protein which is known to correlate positively and
significantly with protein energy malnutrition was found to also correlate positively and
significantly with serum levels of urea, creatinine and calcium. Serum total cholesterol did not
correlate significantly with serum total protein or any other parameter measured (p>0.05 in
each case). Serum total protein was also found to correlate negatively and significantly with
parity (i.e. the number of children had by the mother) and the gestational stage of the
pregnancy in trimesters (r and p values are the same as those given earlier for the respective
parameters). Also there was no significant difference between the age of mothers and serum
total proteins.
 73

The serum levels of urea, creatinine, calcium and cholesterol have been implicated in
this study as Biochemical indices or Biochemical Markers of PEM in pregnant women. Serum
total cholesterol may however be a useful marker for energy intake and not for protein intake.
This agrees with the work of Toigo et al. (2000) which listed urea, creatinine, calcium and
cholesterol as serum Biochemical indices or serum biochemical markers of PEM.

4.2 Recommendations
Most pregnant women are normal healthy females without disorders that interfere with
ingestion, digestion, absorption metabolism and utilization of a normal balanced diet. General
obstetrics care therefore requires apart from physical examination and anthropometric
assessment, a biochemical assessment using serum Biochemical markers of protein energy
malnutrition PEM. This will establish the nutritional status of the mother, identify the
biochemical manifestation of PEM, determine whether they are at risk of complications and
also help in monitoring nutritional status during treatment. Also serum biochemical markers of
PEM each have specific applications in the nutritional assessment of hospitalized patients with
other chronic illnesses such as chronic renal failure.
It is therefore recommended that as a component of prenatal care, multiple
micronutrient supplements should be given to pregnant women as prophylaxis in developing
countries free of charge or at lowest cost possible by the health authorities. These multiple
micronutrient supplements are often taken by pregnant women in developed countries. The
multiple micronutrient supplementation will reduce maternal morbidity and mortality directly
by treating pregnancy related PEM and indirectly by lowering risk of complications at delivery.
In addition there should be a well defined government policy on maternal health and nutrition
reflecting adequate funding for reproductive health care programs and access to well-staffed
primary healthcare centres.
There should be a social support including health and nutrition education to teach
pregnant women about pregnancy related issues, to provide information and close their
knowledge gap about nutritional values of locally available foodstuffs which they eat and can
actually eat; and other socio-cultural issues especially where women do not make decisions
about their own health.
Finally for further studies enlarging sample size and including population from the rural
areas surrounding Enugu metropolis may help corroborate these results.
 74

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