STUDY OF OXIDATIVE STRESS IN SICKLE CELL TRAIT AND

SICKLE CELL ANEMIA PATIENTS IN SCHOOL CHILDREN OF

CHHATTISGARH
A Dissertation submitted in partial fulfilment for the

Degree of

MASTER OF SCIENCE

IN

MEDICAL BIOTECHNOLOGY

Ayush and Health Science University of Chhattisgarh,

Raipur (C.G.)

Session: 2010 – 2011

UNDER THE GUIDANCE OF SUBMITTED BY

Dr. B. K. Talukdar PARITOSH SONEKAR
Assistant Professor M.Sc. Medical Biotechnology
Department of Biochemistry Pt. J.N. M. Medical College
Pt. J.N. M. Medical College Raipur (C. G.)
Raipur (C. G.)
 CERTIFICATE

This is to certify that the work embodied in this project

entitled “Study of oxidative stress in sickle cell trait and
sickle cell anemia patients of school children of
Chhattisgarh ” is bonafide work done done by Mr.
Paritosh Sonekar under the guidance and constant
supervision of Dr.Bipul Kr Talukdar. The results reported by
him are genuine and the script of the project has been
written by the candidate himself. His project therefore,
being forwarded for acceptance in partial fulfilment for the
award of MASTER OF SCIENCE in MEDICAL
BIOTECHNOLOGY at Pt. J. N. M. Medical College, Raipur
(C.G.).

Dr.P.K.Patra

M.D. (Biochemistry)
Professor and Head of
Department
Department of
Biochemistry,
Pt. J.N.M. Medical
College &
Dr B.R. Ambedkar
Memorial
Hospital, Raipur-
492001
 ACKNOWLEDGEMENT

The list of people who I want to thank is long, but,

few of them who have helped me a lot in this dissertation are;
very first of all, I would like to acknowledge Dr. P. K. Patra Head
of Department of Biochemistry, Pt. J.N.M. Medical College for
letting me do this dissertation work in his laboratory. I would
like to thank and express my earnest gratitude to Dr.
Sanvidhan G. Suke who was Assistant Professor in department
of Biochemistry in Pt. J. N. M. Medical College, who helped me
from the very beginning of the dissertation work and gave
valuable inputs for shaping up of this dissertation.

I am grateful to Dr. Bipul Talukdar , Assistant

Professor of Biochemistry in Pt. J. N. M. Medical College, who
after Dr. Suke helped me to complete this dissertation on time
and with minimum flaws.

I would also like to thank Mrs. Anjana Kar, Assistant

Professor of Medical Biotechnology in Pt. J.N. M. Medical
College, for inspiring us when I have lost hope and giving us
direction so as to achieve my goal. I would also like to
acknowledge Miss. Sanjana Bhagat, our nodal officer of our
curriculum, for allowing me to do this dissertation work in
Biochemistry laboratory and helping me out whenever I needed
one.

I would like to give my hearthiest gratitude to Dr. S.

Patel Associate Professor of Biochemistry in Pt. J.N. M. Medical
College and Dr. S. Tripathi Assistant Professor of Biochemistry
in Pt. J.N. M. Medical College for helping me out in my
dissertation work.

I am thankful to all laboratory members Mrs. Silvia and

Miss. Sheikh Hasina, Mr. Lileshwar Sahu and Mr. Deshmukh who
helped me out in making me comfortable with laboratory
surroundings and giving me inputs so as to complete my
dissertation work with ease.

Paritosh
Sonekar

ABBREVIATION

1. AA – Control Patients
2. AS – Sickle cell Trait Patients
3. DTNB – 5 – 5’ dithiobis
4. EDTA – Ethylene diamine tetraacetic acid
5. FRAP – Ferric Reducing Ability of Plasma
6. MDA – Malondialdehyde
7. mM – Milli Mole
8. nM – Nano Mole
9. SCD – Sickle Cell Disease
10. SS – Sickle cell Disease Patients
11. TBA – Thio - Barbituric Acid
12. TCA – Tri - chloro Acetic Acid
13. TPTZ – 2,4,6 – tripyridyl – s – triazine
14. T-Sh – Total thiol

CONTENTS

1. Introduction 22
2. Aims and Objective 22

3. Review of literature 22

4. Materials and Methodology 22

5. Result 22

6. Discussion 22

7. Conclusion 22

8. References 22
Introduction

INTRODUCTION

Historical background:
The clinical findings of sickle cells was unknown until the explanation of the it in
1910 by the Chicago cardiologist and professor of medicine James B. Herrick (1861–1954),
whose intern Ernest Edward Irons (1877–1959) found "peculiar elongated and sickle-shaped"
cells in the blood of Walter Clement Noel, a 20-year-old first-year dental student from
Grenada, after Noel was admitted to the Chicago Presbyterian Hospital in December 1904
suffering from anaemia.

Noel was readmitted several times over the next three years for "muscular
rheumatism" and "bilious attacks". Herrick's published account included illustrations, but the
earliest available slide showing sickle cells is that of a 1918 autopsy from a solider with
sickle trait, initially reviewed only 92 years later.

The disease was named "sickle-cell anaemia" by Verne Mason in 1922, then a
medical resident at Johns Hopkins Hospital. However, some elements of the disease had been
recognized earlier. A paper in the Southern Journal of Medical Pharmacology in 1846
described the absence of a spleen in the autopsy of a runaway slave. The African medical
literature reported this condition in the 1870s, when it was known locally as ogbanjes
("children who come and go") because of the very high infant mortality rate caused by this
condition.

Linus Pauling and colleagues were the first, in 1949, to demonstrate that sickle-cell
disease occurs as a result of an abnormality in the haemoglobin molecule. This was the first
time a genetic disease was linked to a mutation of a specific protein, a milestone in the
history of molecular biology, and it was published in their paper "Sickle Cell Anemia, a
Molecular Disease".

The origin of the mutation that led to the sickle-cell gene was initially thought to be in
the Arabian Peninsula, spreading to Asia and Africa. It is now known, from evaluation of
chromosome structures, that there have been at least four independent mutational events,
three in Africa and a fourth in either Saudi Arabia or central India. These independent events
occurred between 3,000 and 6,000 generations ago, approximately 70,000–150,000 years.

Sickle cell disease:

Sickle cell disease is a group of disorders that affects haemoglobin, the molecule in
red blood cells that delivers oxygen to cells throughout the body. People with this disorder
have atypical haemoglobin molecules called haemoglobin S, which can distort red blood cells
into a sickle, or crescent shape.

Signs and symptoms of sickle cell disease usually begin in early childhood.
Characteristic features of this disorder include a low number of red blood cells (anemia),
repeated infections, and periodic episodes of pain. The severity of symptoms varies from
person to person. Some people have mild symptoms, while others are frequently hospitalized
for more serious complications.

The signs and symptoms of sickle cell disease are caused by the sickling of red blood
cells. When red blood cells sickle, they break down prematurely, which can lead to anemia.
Anemia can cause shortness of breath, fatigue, and delayed growth and development in
children. The rapid breakdown of red blood cells may also cause yellowing of the eyes and
skin, which are signs of jaundice. Painful episodes can occur when sickled red blood cells,
which are stiff and inflexible, get stuck in small blood vessels. These episodes deprive tissues
and organs of oxygen-rich blood and can lead to organ damage, especially in the lungs,
kidneys, spleen, and brain. A particularly serious complication of sickle cell disease is high
blood pressure in the blood vessels that supply the lungs (pulmonary hypertension).
Pulmonary hypertension occurs in about one-third of adults with sickle cell disease and can
lead to heart failure.

Sickle cell disease affects millions of people worldwide. It is most common among
people whose ancestors come from Africa; Mediterranean countries such as Greece, Turkey,
and Italy; the Arabian Peninsula; India; and Spanish-speaking regions in South America,
Central America, and parts of the Caribbean. Sickle cell disease is the most common
inherited blood disorder in the United States, affecting 70,000 to 80,000 Americans. The
disease is estimated to occur in 1 in 500 African Americans and 1 in 1,000 to 1,400 Hispanic
Americans.

Mutations in the HBB gene cause sickle cell disease. Haemoglobin consists of four
protein subunits, typically, two subunits called alpha-globin and two subunits called beta-
globin. The HBB gene provides instructions for making beta-globin. Various versions of
beta-globin result from different mutations in the HBB gene. One particular HBB gene
mutation produces an abnormal version of beta-globin known as haemoglobin S (HbS). Other
mutations in the HBB gene lead to additional abnormal versions of beta-globin such as
haemoglobin C (HbC) and haemoglobin E (HbE). HBB gene mutations can also result in an
unusually low level of beta-globin; this abnormality is called beta thalassemia.

In people with sickle cell disease, at least one of the beta-globin subunits in
haemoglobin is replaced with haemoglobin S. In sickle cell anemia, which is a common form
of sickle cell disease, haemoglobin S replaces both beta-globin subunits in haemoglobin. In
other types of sickle cell disease, just one beta-globin subunit in haemoglobin is replaced
with haemoglobin S. The other beta-globin subunit is replaced with a different abnormal
variant, such as haemoglobin C. For example, people with sickle-haemoglobin C (HbSC)
disease have haemoglobin molecules with haemoglobin S and haemoglobin C instead of beta-
globin. If mutations that produce haemoglobin S and beta thalassemia occur together,
individuals have haemoglobin S-beta thalassemia (HbS Beta Thiol) disease.

Abnormal versions of beta-globin can distort red blood cells into a sickle shape. The
sickle-shaped red blood cells die prematurely, which can lead to anemia. Sometimes the
inflexible, sickle-shaped cells get stuck in small blood vessels and can cause serious medical
complications.

Chhattisgarh, one of the Indian states is worst-affected with the sickle-cell blood
disorder. Around 15-18 percent of the Chhattisgarh’s 20.08 million population is affected by
the disease and more than 50 percent of the affected children in the state die before the age of
five. Since the majority of the population of this state lives in rural areas where the awareness
of such disease is very less and so the disease is seen so prevalent in this state and studies on
sickle cell could improve lives of thousands of people of this state. The expected outcome of
this thesis could be used to determine a novel method to prepare a drug or in personalizing
medicine for sickle cell disease.
 Ai
ms & Objective

AIMS AND OBJECTIVE

The present study was undertaken to evaluate oxidative stress in patients with homozygous
(SS) or heterozygous (AS) sickle cell gene carriers and in normal control with
haemoglobin (AA).

1. To estimate the plasma malondialdehyde (MDA) level in the Sickle cell trait subject
and Sickle cell anemia patients and in the control.

2. Total thiol (T-SH) level in the Sickle cell trait subject and Sickle cell anemia patients
and in the control group.

3. Ferric reducing ability of plasma (FRAP) level in the Sickle cell trait subject and
Sickle cell anemia patients and in the control group.
 Re
view Literature

REVIEW OF LITERATURE

Sickle cell disease (SCD) was first described in 1910, by a dental student who
presented it with pulmonary symptoms (Frenette et al, 2007) 1. Herrick coined the term
“sickle-shaped” to describe the peculiar appearance of the RBC of this patient. Shortly
thereafter, Hahn and Gillespie suggested that anoxia caused RBC sickling by demonstrating
that shape changes could be induced by saturating a cell suspension with carbon dioxide
(Frenette et al, 2007) 1. Seminal studies were noted by Linus Pauling, who was the first to
hypothesize in 1945 that the disease might originate from an abnormality in the haemoglobin
molecule. This hypothesis was validated in 1949 by the demonstration of the differential
migration of sickle versus normal haemoglobin as assessed by gel electrophoresis. That same
year, the autosomal recessive inheritance of the disease was elucidated. Around the same
time, Watson et al. predicted the importance of fetal haemoglobin (Hb F) by suggesting that
its presence could explain the longer period necessary for sickling of new born RBC
compared with those from mothers who had “sicklemia”. Ingram and colleagues
demonstrated shortly thereafter that the mutant sickle haemoglobin (Hb S) differed from
normal haemoglobin Hb A by a single amino acid. This was followed by studies that
analyzed the structure and physical properties of Hb S, which formed intracellular polymers
upon deoxygenation (Stamatoyannopoulos, 2005) 2. These studies placed SCD at the leading
edge of investigations to elucidate the molecular basis of human diseases.

As mentioned above, pioneering studies by Pauling et al. established that SCD results
from a defect in the haemoglobin molecule. During the same year, the mode of inheritance of
the disease was shown to be autosomal recessive. The sickle mutation was characterized
several years later by Ingram et al. as a glutamine to valine substitution at the sixth residue of
the β-globin polypeptide. Several decades later, the human globin genes were cloned, their
DNA sequence was determined, the organization of the globin gene clusters was
characterized, and a great deal of insight was provided into the mechanisms of their regulated
expression (Bank A,2006) 3. Human haemoglobin is a tetrameric molecule that consists of
two pairs of identical polypeptide subunits, each encoded by a different family of genes. The
human α-like globin genes (ζ, α1, and α2) are located on chromosome 16, and the β-like
globin genes (ε, Gg, Ag, d, and b) are located on chromosome 11. Interestingly, the genes are
present on both chromosomes in the same order in which they are expressed during
development (Figure 1).
Figure 1
Chromosomal organization of the α- and β-globin gene clusters. (A) The genes of the β-globin gene cluster (ε,
Gg, Ag, d, and b) are present on chromosome 11 in the same order in which they are expressed during
development. The β–locus control region (b–LCR) is a major regulatory element located far upstream of the
genes of the cluster that is necessary for the high level of expression of those genes. (B) The genes of the α-
globin gene cluster (ζ, a1, and a2) are present on chromosome 16, also in the same order in which they are
expressed during development. HS-40 is a major regulatory element located far upstream of the genes of the
cluster that is necessary for their high level of expression. (C) During fetal life, Hb F (a2g2) is the predominant
type of haemoglobin. Haemoglobin switching refers to the developmental process that leads to the silencing of
g-globin gene expression and the reciprocal activation of adult β-globin gene expression. This results in the
replacement of Hb F by Hb A (a2b2) as the predominant type of haemoglobin in adult life. Figure modified
from ref. (Stuart Marie et al, 2004) 4.

During fetal life, the predominant type of haemoglobin is Hb F (α2γ2). During the postnatal
period, Hb F is gradually replaced by Hb A (α2β2). Hb A2 (α2δ2) is a minor adult-type
haemoglobin that accounts for less than 2.5% of the circulating haemoglobin in normal
individuals in adult life. Upon completion of the switch from Hb F to Hb A, patients with
disorders of the β- globin genes start manifesting the clinical features of their diseases. The
prospect of therapeutic reactivation of Hb F production in adult life has been in large part
responsible for the tremendous interest in the elucidation of the molecular mechanisms of the
switch from fetal to adult haemoglobin production. This field of investigation, which has
recently been reviewed (Bank A, 2005)5, led to approval by the FDA of the use of
hydroxyurea for the treatment of patients with SCD.

Genetic epidemiology of the sickle gene

The sickle gene has a genetic advantage; it protects heterozygous carriers from
succumbing to endemic Plasmodium falciparum malaria infection (Bookchin et al, 2002) 6.
However, with the increased premature death rate of homozygous individuals, the sickle gene
is an example of balanced polymorphism (Gibson et al, 2002) 7. Globin haplotypes of the
gene are based on a series of restriction endonuclease defined polymorphisms in the globin-
gene cluster on chromosome 11.The S-globin gene is present on three major distinct African
haplotypes, all localised exclusively to one of three separate geographical areas (figure 2).
The vagaries of war and Atlantic and Arab slave trades have been responsible for gene
dissemination in the diaspora. A fourth major Indo-European sickle mutation (Arab-India)
probably originated in the Indus Valley Harappa culture, and by gene flow it was distributed
to Saudi Arabia, Bahrain, Kuwait, and Oman. This haplotype is also linked to the sickle gene
in populations from the eastern oasis of Saudi Arabia and the Adivasis tribe of India. Whether
gene conversion explains haplotype diversity because of their strict geographic segregation is
unlikely (Brugnara, 2001) 8. Sickle-cell disease denotes all genotypes containing atleast one
sickle gene, in which HbS makes up at least half the haemoglobin present. In addition to the
homozygotic Hb SS disease (sickle-cell anaemia), five other major sickle genotypes are
linked to the disease. Generation of HbS is a monogenic event, determining the
polymerisation of the deoxygenated conformer of sickle haemoglobin. The process is an
indispensable but insufficient determinant of phenotype. By contrast, the phenotype of sickle-
cell anaemia is multigenic. Other genes, unlinked to the globin locus, participate in relevant
pathological events (e.g., rapid destruction of sickle cells, dense cell formation and adhesion
to endothelium) that are controlled
Figure 2: Geographical distribution and schematic representation of the sickle gene.
(A) Map identifies the three distinct areas in Africa and one in the Arab-India region where the sickle gene is
present (dotted lines). Numbers of individuals with sickle-cell disease (red lines) in Senegal, Benin, and Bantu
are higher near the coast, and falls concentrically inland. (B) The globin gene cluster haplotype is determined by
DNA polymorphic sites (boxes) that are identified by endonuclease enzymes. With this information, haplotypes
are constructed as shown.

by many genes, known as pleiotropic or secondary effector genes. Severity of sickle cell
anaemia varies greatly between individuals, since not all patients have identical pleiotropic
genes (Brittain et al, 2001) 9. Some carriers have mutated genes that can either ameliorate or
exacerbate the phenotype. Expression microarrays are being used to identify up regulated or
down regulated genes in several organs affected by the disease in man and in sickle
transgenic mice. After pleiotropic genes are located, polymorphisms can be searched for, to
identify epistatic or modifier genes that will help to define individual risk, allowing for
rationale-based interventions before the onset of organ damage (Manodori et al, 1998) 10. The
presence of epistatic effects is not theoretical, although our knowledge of these factors is far
from complete. Known epistatic or modifier genes include: co-presence of thalassaemia that
ameliorates the phenotype (by reduction of mean corpuscular haemoglobin concentration,
dense cell numbers, and haemolytic rate); the 158C→T mutation upstream of the gene (that
enhances HbF expression, especially in the Senegal and Arab-Indian globin-cluster
haplotypes) and the female population, in whom as yet unidentified epistatic genes ameliorate
phenotype (Setty et al, 2002) 11.

Cation homeostasis
Abnormal cation homoeostasis is implicated in a pathogenic pleiotropic event. It leads
to formation of dehydrated dense sickle cells (in general), and short-lived, irreversibly sickle
cells (in particular). Irreversibly sickled cells are the densest, and are fixed in their deformed
shape, and do not return to normal contours even when oxygenated because of irreversible
membrane damage. In addition to their role in the initiation of vaso-occlusion, these cells are
a crucial causative factor for anaemia and a raised haemolytic rate. In-vivo dehydration of
sickle erythrocytes takes place mainly by activation of one or more of the cation transport
pathways. Pro-inflammatory molecules induce activation of the Gardos channel, which might
explain the association between inflammation, vaso-occlusion, and increased haemolysis
sometimes seen during infection. Cation homoeostasis is relevant to therapy, since inhibition
of these transporter channels prevents sickle erythrocyte dehydration, formation of dense and
irreversibly sickled cells, with ameliorative effects on both haemolysis and adhesion. Table 1
shows a selection of ion-channel blockers being investigated in sickle-cell treatment (Hines
et al, 2003) 12.
Adhesion reactions

The seminal observation that sickle erythrocytes adhere to endothelium in vitro, and
that this adherence was thought to correlate with disease severity, made necessary the
delineation of the mechanisms involved. These adhesion reactions are mainly mediated by
interaction between receptors on erythrocytes endothelial cells. Interactions have also been
shown between sickle cells and immobilised extracellular matrix components (exposed after
vascular injury, or thrombo-induced endothelial retraction (figure 3). Two of the earliest red-
cell adhesion molecules to be identified were very-late-activation-antigen-4 (VLA- 4/4-1) and
CD36. The integrin-4-1 binds to its endothelial ligand vascular cell adhesion molecule-1
(VCAM-1). Agonist-induced alterations in 4-1 conformation also allows for additional
binding to fibronectin (figure 3). Although VCAM-1 is not constitutively expressed on the
endothelial surface, expression takes place after exposure to several agonists, including
cytokines and hypoxia. Hypoxia also increases VCAM-1 adhesion to the endothelium via 4-
1(Setty et al, 2000) 13.
Figure 3: Adhesive interactions between sickle erythrocytes and the endothelium, subendothelial matrix,
and plasma ligands.
SO4 glycolipid=sulphated glycolipids. PS=phosphatidylserine. TSP=thrombospondin. FN=fibronectin.
LM=laminin. VWF=von Willebrand factor. SE matrix=sub endothelial matrix. HSPG=heparan sulphate
proteoglycans. EPI=epinephrine. CD62P=P-selectin. The red cell receptors associated with adhesion are either
present in increased numbers or sickle reticulocytes (_4 _1 and CD36), on mature sickle cells, when compared
with normal erythrocyte (B-CAM/LU) or exhibit abnormal structure-function activity in sickle red cells (B-
CAM/LU and integrin-associated protein). Phosphatidylserine levels are also raised on sickle when compared
with control HbAA red cells.
Figure taken from reference (Setty et al, 2000) 13.

Another well characterised mechanism is the bridging role of the soluble ligand
thrombospondin between erythrocyte CD36 and several constitutively expressed endothelial
receptors, including v-3 (vitronectin receptor), CD36, and heparan sulphate proteoglycans.
Sickle red cells also bind to immobilised thrombospondin via the integrin associated protein
CD47, a molecule that is associated with the rhesus complex. High-molecular-weight
multimers of von Willebrand factor promote red-cell adhesion to endothelial v-3, and the
glycoprotein Ib (GPIb)-IX-V complex, although the interactive site on the erythrocyte is
unknown (Matsui et al, 2001)14. Non-receptor mechanisms include pro-adhesive roles for
both red-cell sulphated glycolipids and phosphatidylserine. Of particular interest, laminin
binds strongly to sickle erythrocytes via B-CAM/Lu, the protein that carries Lutheran blood-
group antigens (Titus J, 2004, Das SK, 1980)15, 16. Epinephrine increases this adhesion,
concomitant with rises in intra erythrocyte cAMP concentrations and with BCAM/Lu as the
target receptor for cAMP signalling (De Jong et al, 2001)17. Since stress is a potential
initiation factor for vaso-occlusion, epinephrine modulation of adhesion provides a powerful
biological link between intra erythrocytic signalling pathways and the external milieu.
Another important interaction is mediated via thrombin, which causes endothelial retraction
with exposure of pro-adhesive extracellular matrix components and the endothelial
expression of P-selectin, involved in erythrocyte, white-cell, and platelet endothelial
interactions (Miller et al, 2000)18. The present availability of transgenic and knockout animal
models have modernised sickle-cell treatment, since individual adhesive interactions can be
better delineated in vivo, thus forming the foundation for future anti-adhesion therapy (Yasin
et al, 2003) 19.

Lipid bilayer dysfunction

In steady state, the choline-containing phospholipids, sphingomyelin and

phosphatidylcholine, are mostly located on the outside of the membrane bilayer whereas the
amino-phospholipids are either mainly (phosphatidylethanolamine) or exclusively
(phosphatidylserine) located in the inner monolayer. This membrane structure is maintained
by an active set of transport systems that pass phospholipids across the membrane. The
flipase actively transports amino-phospholipids from the outer to the inner monolayer,
whereas the scramblase, when activated, moves all phospholipids in both directions, thereby
scrambling phospholipid distribution. Loss of normal phospholipid asymmetry with the
appearance of anionic phosphatidylserine on the erythrocyte surface happens in various
haemolytic anaemias. Phosphatidyl serine exposure is heterogeneous, occurring on mature
erythrocytes and transferring - positive stress reticulocytes. Causes for this exposure are
inactivation of ATP-dependent amino-phospholipid translocase (which transports
phosphatidylserine from the outer to inner membrane surface), premature activation of
apoptosis in marrow cells, and sickling-induced membrane damage (Frenette, 2002)20. High
HbF concentrations protect sickle erythrocytes from phosphatidyl serine externalisation
(Grigg, 2001, Setty, 2002) 21, 22. Consequences of such exposure are exacerbation of anaemia
because of enhanced phagocytic recognition and removal, increased adhesion to the
endothelium and extracellular matrix components and development of a pro-coagulant
erythrocyte phenotype, since phosphatidyl serine promotes assembly of clotting factors on
cell surfaces. The dysfunctional lipid bilayer thus contributes greatly to sickle-cell
pathophysiology (figure 4) (Kaul et al, 2000) 23.
 Figure 4: Perturbations in erythrocyte membrane lipids in sickle-cell disease
SM=sphingomyelin. PC=phosphatidylcholine. PE=phosphatidylethanolamine. PS=phosphatidylserine.
AV=annexin V. Ca++=calcium ions. Phosphatidylserine exposed on the cell surface forms a docking site for
haemostatic factors, such as the prothrombinase complex (factor Xa, Va, and II). Additionally,
Phosphatidylserine is recognised by macrophages and interacts with proteins such as annexin V, allowing its
measurement by flow cytometry. Cell-surface phosphatidylserine also aids erythrocyte adhesion to the vascular
endothelium and thrombospondin, an extracellular matrix component. Figure taken from Stuart et al, 20044.

Extra-erythrocyte-related pathophysiological changes

Natural history studies and animal data implicate the leucocyte as of major
importance in the pathophysiology of sickle-cell disease. Raised white cell counts predict
disease severity and mortality, whereas an increased baseline white cell count is an
independent risk factor for acute chest syndrome and cerebral infarction. The syndromes of
acute chest and multiple organ failure have also occurred after administration of myeloid
colony stimulating factors (Ingram, 1959)24. Qualitative abnormalities in circulating
leucocytes indicative of an activated phenotype include evidence for degranulation, down-
regulation of L-selectin (a surface membrane glycoprotein that initiates leucocyte-endothelial

Sickle
cell
disease
attachment), activation of the respiratory burst, and raised leukotriene B4 concentrations
(Ferrone, 2004) 25. Leucocyte size, rigidity, and adhesive characteristics are relevant to
micro-vascular blood flow, with transgenic mouse models providing evidence of vascular
inflammation, and leucocyte involvement in the vaso-occlusive event (Li, Trush, 1994)26.
Models of hypoxia-reoxygenation also lend support to the hypothesis that micro vessel
occlusion is a form of reperfusion injury, in which oxidant stress and inflammation leads to
chronic end organ damage. Quantitative and qualitative reductions in leucocytes during
hydroxyurea treatment correlate with amelioration of disease severity. The platelet does not
seem to be critical to the pathophysiology of acute microvascular occlusion, although its role
in large-vessel disease (e.g. cerebral vasculopathy) has yet to be determined. Qualitative
platelet abnormalities are secondary to activation (presumably, because of in-vivo thrombin
generation). Quantitative changes are a result of functional asplenia. Activation-related
changes occur during steady state, with further modulations during vaso-occlusive crises.
However, available data do not forge a primary link between platelets and either haemostatic
perturbation related to sickle-cell disease or vaso-occlusive changes. These studies
corroborate trials of antiplatelet drugs, in which there was no beneficial effect. A recently
postulated correlation between platelet activation and pain episode frequency is difficult to
interpret since platelet and pain assessments were not concurrent. Evidence for perturbation
of the endothelium include histological studies of vascular changes circulating endothelial
cells during painful crises and increased levels of circulating adhesion molecules, such as
soluble VCAM-1 (Nose, 2000)27. Solovey and colleagues have conclusively proven that the
endothelium in sickle-cell disease is activated by showing increased numbers of
microvascular circulating endothelial cells that express tissue factor, VCAM-1, intercellular
adhesion molecule-1 (ICAM-1), E-selectin, and P-selectin (signifying their procoagulant,
proadhesive, and proinflammatory phenotype). Data from animal studies also support the
notion that the phenotype of circulating endothelial cells reflects in-situ microvasculature.
Activation frequently takes place via nuclear factor (NF) B, a transcription factor that up
regulates many pro-inflammatory, pro-adhesive, and pro-coagulant endothelial molecules in
response to inflammatory stimuli and cytokines. Other transcription factors might include
early growth response (EGR)-1 and activator protein (AP)-1. Thus, the endothelium is under
a constant barrage of stimuli, resulting in a state of chronic activation and providing a
dysfunctional template on which micro vessel occlusion and large-vessel vasculopathy
occurs. Fluid-phase coagulation in sickle-cell disease is a process of perturbed and activated
haemostasis with in-vivo thrombin generation and thromboses. In general, patients with the
HbSC disease have milder abnormalities than their homozygous HbSS counterparts (Finkel,
 2000) 28. Key and colleagues recorded both a rise in whole-blood tissue factor in sickle-cell
disease and the presence of circulating endothelial cells expressing a tissue factor phenotype.
These findings suggest that the activated endothelium is one pathophysiological source for
coagulation activation. An additional trigger for the thrombophilic state is the
phosphatidylserine-positive sickle erythrocyte. Thrombin mimics many cytokine-associated
vascular effects, and could provide a crucial link between coagulation activation and
adhesion. Thrombin mediates endothelial cell retraction with exposure of pro-adhesive matrix
elements, and causes endothelial expression of P-selectin (that modulates leucocyte rolling,
red-cell-endothelial interactions, and platelet endothelial interactions.) Although initial trials
of anticoagulation treatment were not beneficial in vaso-occlusive crises, they were not well
controlled. Notably, in-vitro studies have shown that heparin inhibits erythrocyte-endothelial
adhesion via inhibition of P-selectin and that N-3 fatty-acid dietary supplementation reduces
pain episode rates, with concomitant evidence for a reduction in thrombin activity.
Haemostatic activation could be implicated in the genesis of macrovasculopathy. A
preliminary study 80 reported increases in pro-thrombin fragment F1.2 and erythrocyte
phosphatidyl serine in a paediatric population — these biomarkers correlated with increased
transcranial doppler-flow velocities. The time is ripe for studies investigating the prevalence
of thrombosis and for clinical trials highlighting the anti-adhesive properties of heparin or
other anti-thrombins in the modulation of vaso-occlusion (Li, Trush, 1994) 26.

Oxidative stress

Cells growing aerobically are exposed to reactive oxygen species (ROS) generated
during metabolism. These include hydrogen peroxide (H2O2), the hydroxyl radical (OH-), and
the superoxide anion (O2-), which can damage proteins, lipids, carbohydrates, and DNA.
Oxidative stress occurs when cellular defense mechanisms are unable to cope with existing
ROS, caspase independent mechanism activating apoptosis. In respiring cells, the primary
source of ROS is leakage of electrons from the mitochondrial respiratory chain.
Saccharomyces cerevisiae cells that lack functional mitochondria or an intact electron
transport chain or that have been treated with mitochondrial inhibitors are viable, but
sensitive to ROS. ROS can lead to cell death; however, cells possess a variety of defenses

Cells including cell-cycle delay, the induction of enzymes such as catalases, peroxidases, and
have superoxide dismutases, and the synthesis of antioxidants such as glutathione, vitamins C and
distinct E, and ubiquinol (Bandhopadhyay et al, 1999)29. Human and yeast cells can mount an
mechan
ism
adaptive response in which exposure to a low dose of an oxidant induces resistance to a
higher dose. In S. cerevisiae, there is overlap in the stress systems induced by treating cells
with various oxidants. However, differences have been noted in sensitivity, in adaptive and
cell-cycle responses to different oxidants or toxic products of ROS damage, and in activation
of the transcription factor Yap1p by H2O2 or diamide. Most studies of oxidative stress at the
molecular level have used a range of external oxidants or free radical generating compounds.
Genome wide surveys of changes in transcripts and proteins have identified many genes that
are activated or repressed in response to a few oxidants. These studies have provided insight
on regulatory responses, but they have not revealed the relevance of these genes to survival,
repair of damage, or cellular recovery. A number of antioxidant and repair processes have
been characterized by isolating mutants affected by antioxidants or resistant to oxidants, or
genes that confer resistance when over expressed. Despite these studies, many mechanisms
whereby cells are damaged by particular oxidants or protect themselves from damage remain
to be identified. The availability of a genome wide set of deletion strains have enabled
comprehensive screening of cellular functions involved in stress responses (Halliwell,
1995)30. Some genes that play a role may, when deleted, confer no specific phenotype
because of gene redundancy or compensatory parallel pathways. However, this approach
relies on the sheer number of genes under study and the fact that mutations affecting some
components of an important function or pathway will show a phenotype of sensitivity or
resistance. A preliminary screen has been performed for three types of oxidative stress on 600
strains of the EUROFAN collection, because this collection consists mainly of strains that
were individually deleted for unknown function, the results of that survey were more relevant
to identifying gene function than to identifying cell functions important in stress resistance.
These results also indicated that many more genes involved in maintaining cellular resistance
remained to be identified. This study aimed at obtaining a comprehensive view of the cellular
functions needed to maintain cellular viability to ROS by screening the S. cerevisiae deletion
mutant collection by using five different ROS: H2O2, linoleic acid 13-hydroperoxide
(LoaOOH, a product of lipid peroxidation), menadione (superoxide- generating agent),
cumene hydroperoxide (CHP, an aromatic hydroperoxide), and the thiol-oxidant diamide
(Herrick, 1910) 31.

Oxidative stress represents an imbalance between the production and manifestation of

reactive oxygen species and a biological system's ability to readily detoxify the reactive
intermediates or to repair the resulting damage. Disturbances in the normal redox state of
tissues can cause toxic effects through the production of peroxides and free radicals that
damage all components of the cell, including proteins, lipids, and DNA (Hanh et al, 1927) 32.
Chemically, oxidative stress is associated with increased production of oxidizing
species or a significant decrease in the capability of antioxidant defenses, such as glutathione.
The effects of oxidative stress depend upon the size of these changes, with a cell being able to
overcome small perturbations and regain its original state. However, more severe oxidative
stress can cause cell death and even moderate oxidation can trigger apoptosis, while more
intense stresses may cause necrosis (Pauling, 1964) 33.
Free radicals are natural by-products of our own metabolism. These are electrically
charged molecules that attack our cells, tearing through cellular membranes to react and
create havoc with the nucleic acids, proteins, and enzymes present in the body. These attacks
by free radicals, collectively known as oxidative stress, are capable of causing cells to lose
their structure, function and can eventually destroy their structure, function and can
eventually destroy them. They are continuously produced by our body’s use of oxygen such
as in respiration and some cell – mediated immune function. They are also generated through
environmental pollutants, cigarette smoke, automobile exhaust, radiation, air-pollution,
pesticide. Radicals generated in the body and the antioxidant defence systems that scavenge
these free radicals prevent them from causing deleterious effects in the body. The antioxidant
defence systems in the body can only protect the body when the amount of the free radical is
within the normal physiological level. But when this balance is shifted towards more of free
radicals, increasing their burden in the body either due to environmental conditions or
produced within the body, it leads to oxidative stress, which may result in tissue injury and
subsequent diseases. Since free radicals play such an important role in the disease scenario of
an individual, a thorough understanding of the various physiologically significant free
radicals is of paramount importance (Pauling et al, 1949) 34.
Free radical tends to rob electrons from the molecules in the immediate surroundings
in order to replace their own losses. Reactive oxygen species (ROS) is a collective term,
which includes not only the oxygen radicals (O2- and OH) but also some non-radical
derivatives of oxygen. These include hydrogen peroxide (H2O2), hypochlorous acid (HOCl)
and ozone (O3). The superoxide anion radical (O2-) appears to play the central role and other
reactive intermediates are formed from it. ROS are also produced in the organism as a part of
the primary immune defense. Phagocytic cells such as neutrophils or macrophages defend
against foreign organism by generating O2- and nitric oxide as a part of the killing
mechanism. Despite the existence of endogenous defense mechanism against ROS, it has
been observed that whenever either the level of the cellular antioxidant system goes down or
when the ROS reach abnormally high levels, oxidative damage to the cells occurs leading to
several pathological conditions. Over about 100 disorders like rheumatoid arthritis,
hemorrhagic shock, CVS disorders, cystic fibrosis, metabolic disorders, neurodegenerative
diseases, gastrointestinal ulcerogenesis, cancer, cardiovascular disease, Down’s syndrome,
Friedreich’s ataxia, aging, age related diseases and AIDS have been reported as ROS
mediated. Some specific examples of ROS mediated diseases include Alzheimer’s disease,
Parkinson’s disease, atherosclerosis, cancer, Down’s syndrome and ischemic reperfusion
injury in different tissues including heart, liver, brain kidney and GIT (Neel, 1949) 35.

These ROS are primarily generated following release of electrons from complex I and
complex III of the electron transport chain at the mitochondria, and to a lesser extent by
peroxisomes and by the activity of NADPH oxidases (Nox) at the plasma membrane (BOX
2). They have important functions in cell signalling in general but their role in regulating cell
cycle progression is poorly understood. ROS levels increase significantly as cells pass from
G1 into S phase of cell cycle and ROS are required for S phase entry, as demonstrated by the
cell cycle arrest induced by quenching ROS. However, cell cycle checkpoints are also
activated by increased ROS, indicating that cellular proliferation relies on maintaining ROS
levels within a functional range. ROS have been proposed to modulate the cell cycle in a
variety of ways, including through effects on the activity of enzymes with redox-sensitive
amino acids at their catalytic sites that are required for cell cycle progression. These include
ribonucleotide reductase, which is crucial for nucleotide biosynthesis. ROS have also been
proposed to inhibit the turnover of key cell cycle progression factors by as yet unknown
mechanisms. For example, ROS stabilize levels of F-box only protein 5 (FBXO5, also known
as EMI1), an E2f target gene that inhibits the anaphase-promoting complex (APC) in S and
G2 phases of cell cycle by acting as a pseudo-substrate, thereby preventing premature entry
into mitosis (Watson et al, 1948) 43.

Erythropoiesis and oxidative stress

Analysis of the mouse models discussed above has revealed how the interplay
between oxidative stress sensing pathways and the cell cycle machinery in HSCs is important
for the long-term survival of the animal. However, a cursory glance at the phenotypes of mice
with defects in ROS management or of humans with mutations in ROS regulatory enzymes,
such as glutathione S-transferase-θ or acute stresses such as blood loss or occupational
exposure to chemicals such as benzene is a function of red blood cell performance. The red
blood cell is important for mammalian viability, not just in terms of its capacity to transport
oxygen in the form of oxy-haemoglobin to peripheral tissues, but in its antioxidant capacity.
Peroxynitrite and superoxide taken up from the environment through red blood cell-specific
membrane anion channels are reduced by glutathione and other antioxidants, which are
expressed at particularly high levels in red blood cells. In addition to handling extracellular
ROS, the red blood cell is equipped to reduce the endogenous ROS that is generated at high
levels owing to routine tasks including oxygen and iron uptake, haem biosynthesis and globin
chain imbalance management. In particular, the oxidation of ferrous (Fe2+) haemoglobin to
ferric (Fe3+) haemoglobin produces superoxide and hydroxyl radicals that need to be
quenched on a scale unparalleled in other mammalian cell types. Failure to manage ROS
production owing to inherited genetic defects, such as mutations in glucose-6-phosphate
dehydrogenase, which converts NADP to NADPH (required for glutathione reduction),
results in reduced red blood cell lifespan and life-threatening anaemia under stressed
conditions. Red blood cells also express high levels of antioxidants, including catalase,
peroxyredoxins and SODs. Interestingly, FOXO3A becomes increasingly nuclear in
localization and transcriptionally active during erythroid maturation. The significance of
FOXO3A regulation of SOD2 and catalase for red blood cell production is manifested by
increased ROS in Foxo3 a-null erythroblasts and the reduced numbers and lifespan of mature
red blood cells in Foxo3 a-null mice. Given the tight regulation of antioxidant expression
during erythroid maturation, it is also tempting to speculate that ROS has an instructive role
in erythroid differentiation, perhaps by promoting cell cycle arrest (Ingram, 1958) 36.

Classical Peroxidation Reactions:

The reactions of oxygen free radicals with polyunsaturated lipids have been
extensively researched because of their involvement in rancidity and the development of
undesirable odours and flavours in foods. Historically these reactions are the most frequently
cited consequence of oxygen radical production in plant cells. The lipid bilayer membrane is
composed of a mixture of phospholipids and glycolipids that have fatty acid chains attached
to carbon 1 and 2 of the glycerol backbone by an ester linkage. The peroxidation reactions
differ among these fatty acids depending on the number and position of the double bonds on
the acyl chain.

The peroxidation of lipids involves three distinct steps: initiation, propagation and
termination. The initiation reaction between an unsaturated fatty acid (e.g. linoleate) and the
hydroxyl radical involves the abstraction of an H atom from the methylvinyl group on the
fatty acid (reaction 1); in the case of linoleate this occurs at carbon-11. The remaining carbon
centred radical, forms a resonance structure sharing this unpaired electron among carbons 9
to 13. In the propagation reactions, this resonance structure reacts with triplet oxygen, which
is a biradical having two unpaired electrons and therefore reacts readily with other radicals.
This reaction forms a peroxy radical (reaction 2). In the case of linoleate, addition occurs at
either carbon - 9 or 13. The peroxy radical then abstracts an H atom from a second fatty acid
forming a lipid hydroperoxide and leaving another carbon centred free radical (reaction 3)
that can participate in a second H abstraction (reaction 2). Therefore, once one hydroxyl
radical initiates the peroxidation reaction by abstracting a single H atom, it creates a carbon
radical product (R) that is capable of reacting with ground state oxygen in a chain reaction.
The role of the hydroxyl radical is analogous to a "spark" that starts a fire. The basis for the
hydroxyl radical's extreme reactivity in lipid systems is that at very low concentrations it
initiates a chain reaction involving triplet oxygen, the most abundant form of oxygen in the
cell. The lipid hydroperoxide (ROOH) is unstable in the presence of Fe or other metal
catalysts because ROOH will participate in a Fenton reaction leading to the formation of
reactive alkoxy radicals:

(1)

(2)

(3)

(4)

Therefore, in the presence of Fe, the chain reactions are not only propagated but amplified.
Note that two radicals are produced by the summation of reactions 1 to 4. Among the
degradation products of ROOH are aldehydes, such as malondialdehyde, and hydrocarbons,
such as ethane and ethylene that are commonly measured end products of lipid
peroxidation.Malondialdehyde(MDA) is the end-product of lipid peroxidation, which is a
process where reactive oxygen species degrade polyunsaturated lipids (Pryor WA et al,
1975)37. This compound is a reactive aldehyde and is one of the many reactive electrophile
species that cause toxic stress in cells and form advanced glycation endproducts (Farmer EE,
Davoine et al)38.The production of this aldehyde is used as a biomarker to measure the level
of oxidative stress in an organism (Del Rio D, Moore K, et al)39.
There have been several reports demonstrating the important role of lipid peroxide in
atherosclerotic disorders and aging process. Although lipid peroxide has been measured by
various methods, the Thiobarbituric acid (TBA) method seems to be most suitable for the
determination of serum lipid peroxide because of its high sensitivity. Measurements of serum
lipid peroxide using TBA reaction have been performed by several workers. However, it has
been pointed out that sialic acids have a great influence on lipid peroxide determination in
these methods. Fluorometric TBA method has also been described but its reproducibility
seems to be less satisfactory (Suke et al, 2008) 40.

Ferric reducing ability of plasma (FRAP): At low ph, when ferric – tripyridyl triazine
(FeIII – TPTZ) complex is reduced to the ferrous (FeII) form, an intense blue colour with an
absorption maximum at 593 nm develops. The reaction is nonspecific, and any half - reaction
which has a less – positive redox potential, under reaction conditions, than the FeIII/FeII –
TPTZ half reaction will drive FeIII – TPTZ reduction. Test conditions favor reduction of the
complex and, thereby, colour development, provided that a reductant (antioxidant) is present.
In FRAP assay, excess FeIII is used, and the rate – limiting factor of FeII – TPTZ, and hence
colour, formation is the reducing ability of the sample (Manodori et al, 1998) 10.

Total Thiols(T-SH): These include extremely important substances such as glutathione,

which are among the body’s most important free radical scavengers. Reduced thiols (-SH) are
the major antioxidants in the cell and the source for such reduced thiols are GSH and protein
bound thiols. Both GSH and protein bound thiols contributes maximum to the total thiols
pool in the cell. Low levels of total thiols pool have been shown to be associated with various
disorders with increased generation of free radicals (Chan AC, 1999)41 (Halliwell B,
2007)42.
Hundekar et al studied on antioxidant status and lipid peroxidation in sickle cell
anemia. They found significantly elevated plasma MDA level and activity of erythrocytic
superoxide dismutase.Again plasma total antioxidant capacity and activities of erythrocytic
catalase were reduced significantly in SCA patients. These observations provide the evidence
of imbalance between oxidant and antioxidant status leading to chronic oxidative stress.
 Arinola et al studied antioxidant levels and trace elements status in Nigerian sickle
cell disease patients with plasmodium parasitaemia. They have noted a significant reduction
in total antioxidant status in patients with sickle cell anemia. Similar changes were also noted
by Foluke et al in sickle cell anemia patients in steady state.

Hydroxyurea in sickle-cell anaemia

In vivo evidence of mild disease in individuals with sickle cell disease with high
concentrations of HbF, paved the way for a search for compounds that would increase HbF in
vivo. Hydroxyurea, an S-phase-cytotoxic drug, is the only one that has been widely used to
increase HbF concentrations in sickle-cell disease. Its effect on HbF synthesis is ascribed to
the premature commitment of erythroid precursors during the marrow regeneration that
follows drug-related cytoreduction. Thus, effectiveness of increasing HbF concentrations is
tied to the cytoreductive property of hydroxyurea, and is response dependent on the capacity
of the marrow to withstand moderate drug dosages. In responders, the number of F cells (red
cells containing HbF) and the amount of HbF per F cell increase, and the number of dense
cells and reticulocytes decrease, with improved erythrocyte survival. Other beneficial effects
of hydroxyurea include modulation of sickle erythrocyte adhesive properties and enhanced
NO production. In a landmark study in adults with homozygous HbSS disease, hydroxyurea
therapy reduced the frequency of painful crises, acute chest syndrome, admission rates, and
the need for blood transfusions. A 9-year follow-up of the study has shown reduced mortality
in the hydroxyurea - treated cohort. In children with HbSS disease, data are similar to those
for adults. Hydroxyurea related toxic effects are mild and reversible with no evidence of
growth failure. Notably, a beneficial effect on preservation of splenic function was seen in
very young children given hydroxyurea. A placebo-controlled pilot trial of hydroxyurea
treatment in young children (aged 12–18 months at enrolment) has begun in the USA, in an
attempt to prevent chronic organ dysfunction. Until results are available, the drug is not
recommended for this age group. However, hydroxyurea can be cautiously considered in
infants at high risk, as identified by Miller and co-workers. Three early life predictors of
adverse outcomes included hand-foot syndrome, severe anaemia (Hb concentration < 70 g/L),
and leucocytosis (Ingram, 1958) 36. An additional indication is the child with stroke, who for
various reasons might need to discontinue prophylactic transfusion therapy. In this situation
hydroxyurea could decrease the rate of stroke recurrence. The drug also seemed to relieve
pain in children with HbSC disease, although its use in children and adults with HbSC
disease is fairly limited. Hydroxyurea should be given in a structured environment of medical
care, attention to compliance, and initial escalating therapy with maximum tolerated doses
between 20–30 mg/kg. Optimum doses provide a balance between haematological toxic
effects and increased concentrations of HbF, with the highest tolerated dose yielding the
greatest response. Disadvantages include a large number of non-responders, and potential
long-term carcinogenic or leukaemogenic effects. Additionally, women and men on
hydroxyurea should use contraception, discontinuing use of the drug before conception. The
effects of the drug in human foetuses are unknown, although teratogenic effects are seen in
animals. As well as hydroxyurea, other inducers of HbF synthesis include butyric acid (a
short chain fatty acid given either alone or in combination with hydroxyurea). Decitabine, a
DNA hypomethylating drug that greatly increases HbF concentrations at non-cytotoxic doses,
also is worthy of further investigation (Watson et al, 1948)43.

Other promising treatments

Ion-channel blockers
As discussed earlier, cation homoeostasis is important in sickle pathogenesis. Combination
therapy with additional agents (acting synergistically) merits further consideration.

Anti-adhesion and anti-inflammatory treatment: future prospects

Anti-adhesion and anti-inflammatory treatments also have potential prospects to ameliorate
sickle-cell disease. Since the endothelium is the template on which adhesion occurs,
inhibition strategies providing maximum benefit might include the targeting of single
molecules (such as P-selectin) that mediates heterotypic cell-endothelial interactions. Anti-P-
selectin antibodies and heparin have shown promising results that lend support to this theory.
Transcription factor activation can lead to inflammatory, pro-adhesive, and pro-coagulant
gene expression. Since these effectors, such as NF-B, are increased in sickle transgenic mice
models, another targeted approach is the inhibition of transcription (Mutlu-Türkoglu U,
1997)53 regulation of endothelial expression of pro-inflammatory and pro-adhesive molecules
(Suke et al, 2008)40. Glucocorticoids inhibit NF-B and reduce cytokine production. Although
such anti-inflammatory effects have proven beneficial in the acute management of vaso-
occlusion and acute chest syndrome, the rebound of symptoms after discontinuation of
steroid use, and the potentially harmful effect of a further increase of leucocyte counts must
be considered. A vascular lubricant (poloxamer 188; a non-ionic surfactant copolymer) has
been assessed, with a small reduction in the duration of acute vaso-occlusion. Antiadhesive
treatment might be targeted at specific erythrocyte or leucocyte-endothelial interactions. High
fetal Hb levels that occur in infancy, or can be induced by drugs such as hydroxyurea,
decrease sickle red cell adhesion molecule expression, protect against the adhesion process,
and are associated with a decrease in vaso-occlusive crises and chronic organ damage.
(Prakash M, et al)54. Monoclonal antibodies against endothelial integrin-v-3 prevented
vascular obstruction in an animal model of vaso-occlusion. Abciximab (antibody against
platelet integrin – Iib-3) used in the treatment of acute coronary syndromes, cross-reacts with
v-3 integrin. Thus, future studies on transgenic animal models assessing its effectiveness (or
other specific v-3 inhibitors) in the modulation of vaso-occlusion might be desirable.
Antagonists to leucocyte integrins of the 2 or CD18 subfamily, and antioxidants such as the
xanthine oxidase inhibitor allopurinol, deserve further investigation as modulators of both
reperfusion injury and chronic inflammation (Gaetani GF et al, 1996)55. Thus while
preliminary studies on animals show that pharmacological inhibition of endothelial cell
activation or heterocellular adhesion (or both) is feasible, the route of administration, and
haemostatic-related effects of some compounds (monoclonal antibodies against v -3 and
heparin), might make oral antioxidants, and NF-B antagonists better suited for initial clinical
trials (Hu Mi et al,1994)44.

Nitric oxide
Reference has been made to a role for NO in the acute and chronic complications of sickle-
cell disease. This signalling molecule, whose precursor is L-arginine, is continuously
produced in the endothelium by a constitutive NO synthase, which is functionally calcium
dependent. NO increases cyclic guanosine monophosphate production, leading to
dephosphorylation of myosin light chains. Thus, in the vessel wall, NO induces relaxation of
smooth muscle and vasodilation. Other mechanisms related to sickle-cell disease include the
role of NO as a cytoprotective mediator, inhibiting gene transcription of pro-adhesive and
pro-inflammatory molecules such as endothelial VCAM-1 and P-selectin. Effects on
circulating cellular elements include inhibition of platelet aggregation, leucocyte adhesion
and migration, quenching of superoxide, and inhibition of erythrocyte-endothelial adhesion.
NO bio-availability is maintained by a balance between endothelial production and
consumption, this balance is disrupted in sickle-cell disease. Although NO synthase is up
regulated by anaemia, shear stress, and tissue hypoxia, NO bio-availability is impaired,
especially in male individuals with the disease. This unavailability is due to rapid scavenging
of NO by cell-free haemoglobin and free-oxygen radicals, together with low concentrations
of substrate L-arginine. Additionally, cell-free haemoglobin amounts are also higher in men
with sickle-cell disease than in a woman, which partly explains the recorded differences in
NO bio-availability between the sexes. The lung is most affected by perturbations in NO, in
which a reduction in NO seems to be the mechanism underlying hypoxic pulmonary
vasoconstriction, and a predisposition to acute chest syndrome (Pauling et al, 1949)34.
Reduced NO with raised concentrations of endothelin-1 and chronic haemolysis have been
implicated in pulmonary hypertension associated with sickle-cell disease. Furthermore,
dysregulation of microcirculatory vascular tone, partly due to reduced NO bio-availability, is
thought to have a role in the pathophysiology of vaso-occlusion. Transgenic models and case
reports suggest a beneficial effect of NO inhalation in acute chest syndrome. A pilot study of
children with vaso-occlusion also showed a trend towards lower opioid use and pain scores
than controls. A larger study is needed for definitive results. Clinical trials of NO in
pulmonary hypertension are also in progress. Although NO inhalation is cumbersome, use of
oral L-arginine improves endothelial function, and seems to modify pulmonary hypertension
related to sickle - cell disease. Concentrations of this amino-acid are reduced during vaso-
occlusive crises, with increases in NO after L-arginine is given. Gardos-channel inhibition
has been shown in transgenic mice fed with arginine with concomitant beneficial effects on
haematological indices (Herrick, 1910) 31.

Haemopoietic cell transplantation

Haemopoietic cell transplantation is the only available potentially curative therapy for sickle-
cell disease. Estimated risk of death from HLA identical-stem-cell transplantation in the
disease is 5%. Rapid development of procedures such as non-myeloablative conditioning
regimens that lead to stable mixed chimerism, cord-blood transplantation, and transplantation
from unrelated stem cell donors holds promise. Although initial transplantations were
restricted by eligibility criteria that included evidence for substantial single-organ
dysfunction, less restrictive criteria will apply as mortality rates improve. The goal is to
successfully replace the host’s marrow with normal genotype cells before development of
organ dysfunction. Choice of appropriate candidates will be helped by identification of
epistatic genes that improve the definition of risk (Frenette, 2002)20.

Gene-therapy advances
Gene therapy has been successful in the sickle transgenic mouse. A lentivirus construct
containing an A–T87Q globin gene variant to resemble HbF was generated. This vector was
made optimum for transfer to haemopoietic stem cells and gene expression in the erythroid
lineage. Transduced haemopoietic stem cells were transplanted into mice in two mouse
models with sickle-cell disease by marrow ablation. Long-term expression was achieved,
without pre-selection, in all transplanted mice. Erythroid-specific accumulation of the anti-
sickling protein was noted for up to 52% of total haemoglobin and in virtually all circulating
erythrocytes. The mouse models showed inhibition of red-cell dehydration and sickling, in
addition to correction of haematological indices, splenomegaly, and hyposthenuria. Clinical
trials are being considered, although unexpected difficulties could arise before gene therapy
becomes a reality. Nevertheless, there are good reasons to be optimistic (Ingram, 1958) 36.

Future areas of research

With respect to sickle-cell research, natural history studies and clinical trials have provided
incremental increases in our knowledge about this monogenic event that results in a systemic
disorder of monumental complexity. Although some issues have been successfully tackled,
and gene therapy holds promise, many questions remain. What are the genetic and
environmental modifiers of clinical phenotype? Will genetic determinants of the
inflammatory response, adhesion, haemostatic activation or related surrogate biomarker
values be able to determine future treatment, or identify fetuses at risk for severe disease?
Can we unravel CNS vasculopathy; or improve pregnancy related complication rates? Can
we do anything about acute chest syndrome and chronic pulmonary disease? Is the fully
functional spleen or kidney a realistic therapeutic goal? In countries where stem-cell
transplantation or gene therapy will be initially impractical, can we develop safe, easily
administered, and effective treatments that will revolutionise care without straining
resources? The challenges remain for this archetype of the single-gene disease in the genomic
era of medical research.
 Materia
ls & Methods
 MATERIALS AND METHODS

Subjects: - Total sixty-six (66) subjects were selected (children 5-15 age, male and female
mixed population) and divided into groups according to the haemoglobin profile: AA
(normal), AS (SCD trait subject) and SS (SCD patient). They were classified into the groups
by haemoglobin electrophoresis and high-performance liquid chromatography (HPLC)
methods and by family history.

Inclusion criteria: The AA and AS subjects were healthy and not anemic, they had no
underlying medical problems and did not take vitamin or mineral supplements etc.

Exclusion criteria: Patients who had been transfused or had hemolytic crisis for at least 3
months prior to the study and who is in treatment with desferral (iron chelator) were not
enrolled in the study.

Sample: Taking all aseptic and antiseptic precautions 5ml of blood is drawn from the ante
cubital vein. The needle is removed from the syringe and the blood is immediately
transferred carefully into EDTA vials. The vials were stoppered and centrifuged at 3000rpm
for 5 minutes. The separated plasma is transferred into fresh vials and stored in -200C.The
plasma is used for the further oxidative stress investigations.

• Total thiol (T-SH): - The T-Sh level is calculated using Hu and Dillard, 199443
method.

Reagents required: -
 1. Tris – base (0.25M) – EDTA (20mM) buffer; ph maintained is 5.2.
(Sigma Aldrich)
2. 10mM DTNB in absolute methanol (HiMedia).
3. Absolute methanol.

Reagents are stable for upto 2 weeks which are stored at 4˚C.

Procedure: -

1. An aliquot of plasma i.e. 0.200ml is mixed in a 10ml test tube with 0.6ml of the Tris –
EDTA buffer followed by addition of 40µl of 10mM DTNB and 3.16ml of absolute
methanol.
2. The test tube is capped and the colour is developed after 15 – 20 min.
3. This is followed by centrifugation at 3000rpm for 10 minute at ambient (370C)
temperature.
4. The absorbance of the supernatant is measured at 412nm which is abbreviated as ‘A’
and subtracted from a DTNB blank i.e. ‘B’.

Calculation: -

Total thiol group is calculated using an absorptivity of 13600 cm-1 m-1 as follows: -

[(A – B – 0.03) × 4.00.2] [13.6 - (A – B – 0.03) × 1.47mM]

• Ferric reducing ability of plasma (FRAP): The FRAP level is calculated using
Benzie and strain 199644 method.

Reagents required:

1. 300mMol/liter Acetate buffer, pH – 3.6.

2. TPTZ (2, 4, 6 – tripyridyl – s – triazine).
3. 20mMol/liter FeCl3.6H2O.

Procedure:
 1. Prepare 300mMol Acetate buffer of pH - 3.6 by mixing 3.1gram sodium Acetate
trihydrate, 16ml Glacial Acetic Acid and the volume is made up to 1 liter by adding
distilled water .
2. 10mMol TPTZ is prepared by mixing 33milligram of TPTZ in 40mMol HCl. The
40mMol HCl is prepared by adding 332µl in 100milliliter of distilled water.
3. Thirdly, 20mMol FeCl3.6H2O is prepared by dissolving 324milligram of FeCl3.6H2O

in 100milliliter of distilled water.

4. 1mMol FeSO4 is prepared for standard calculation by mixing 332milligram of FeSO4

in 500milliliter of distilled water.

5. Working reagent is prepared as Acetate buffer: TPTZ: FeCl3 in 10:1:1 ratio.
6. The absorbance is calculated using spectrophotometer. Standard is made by mixing
3ml of working reagent and 100µl FeSO4 and the absorbance was obtained at 593nm.
7. Now, the test sample is placed in spectrophotometer. The working reagent is taken as
3ml and plasma is100µl.
8. The FRAP value of sample is calculated using under given formula.

Calculation:

0-4min. ∆A593 test0-4min. ∆A593standard ×FRAP value of standard

Solution.

• Malondialdehyde (MDA)

The MDA level is calculated using method by Satoh et al, 197845.

Reagents required:

1. TCA (Sigma-Aldrich).
2. 0.05M H2SO4.
3. TBA (Sigma-Aldrich).
4. n – butanol.
5. 1,1,3,3 – tetramethoxy propane (HiMedia).

Procedure:
 1. Prepare 20%TCA by mixing 20gm of TCA in 100ml of distilled water.
2. Prepare 0.05M H2SO4 by adding 273ml of H2SO4 in 100ml of distilled water.
3. TBA is prepared of required concentration by mixing 220mg of TBA in 100ml of
Na2SO4.
4. Standard MDA is prepared by mixing 1,1,3,3 tetramethoxy propane in 0.05M H2SO4
to prepare 10µM solution.
5. 0.5ml of plasma is taken and 2.5ml of TCA is added in it and kept for 10 minute in
room temperature.
6. It is then centrifuged at 3500rpm for 10 minute. Supernatant is decanted.
7. The precipitate is washed once with 2ml of 0.05M H2SO4.
8. To this precipitate 2ml of H2SO4 and 3ml of TBA is added and then incubated in
boiling water bath for 30 minute and then cooled under tap water.
9. To this chromogen, add 4ml of n – butanol by continuously shaking it vigorously.
10. It is then centrifuged at 3000 rpm for 10 minute. Separate the organic layer and then

see the absorbance in spectrophotometer at 530 nm.

Statistical analysis
A statistical analysis is performed using unpaired student t-test and ANOVA. The results are
expressed as mean ± SD.
 Result
and Observation

RESULTS AND OBSERVATION

 A total of 66 subjects were taken for the study which is divided in to three groups: AS
comprising of 23 subjects, SS comprising of 23 subjects and AA comprising of 20 subjects.
The cases as well as the controls were taken from the school children of Chhattisgarh after
taking consent from their parents/guardian. They were classified in to three groups after
doing electrophoresis and HPLC and family history. Blood samples were collected in EDTA
vials and later analysed for oxidative stress markers and other tests. Statistical analysis was
done among the three groups in relation to age, sex and oxidative stress markers viz: MDA,T-
SH and FRAP.

The following results are obtained:-

Age & Sex:

The age of both the control and the test groups are shown in table 2. The statistical
parameters of age distribution of control and test subjects are given in table 3.

From the table 2, it is seen that the difference in age distribution between the three
groups is not significant (p > 0.05).

The control group comprised 20 individuals age ranging from 5 to 13yrs with a mean
value of 10 & a median of 10.

The test group was divided in to 2 subgroups according to age comprising of 5 – 15

years. The mean age of the two groups was 12.5 and 11.5 and the median were 12.5 and 11.5
respectively.

Table 2: Table showing age and sex distribution in the study.

AA AS SS
Age Age Age
Sl. no. sex Sl. no. sex Sl. no. Sex
(yrs.) (yrs.) (yrs.)
1 6 M 1 12 F 1 6 M
2 9 M 2 11 M 2 15 F
3 5 M 3 5 M 3 14 M
4 7 F 4 6 M 4 12 F
5 6 F 5 10 F 5 9 F
 6 13 M 6 11 M 6 10 F
7 11 M 7 8 F 7 9 M
8 11 F 8 14 M 8 8 F
9 10 M 9 8 M 9 12 F
10 12 F 10 8 M 10 14 M
11 7 M 11 7 M 11 14 F
12 8 F 12 5 M 12 15 M
13 6 F 13 12 F 13 7 F
14 13 M 14 11 F 14 8 F
15 12 M 15 12 F 15 11 F
16 11 F 16 9 M 16 12 M
17 10 M 17 10 F 17 15 M
18 10 F 18 11 F 18 8 F
19 13 M 19 12 M 19 6 F
20 9 M 20 7 M 20 8 F
21 8 M 21 10 M
22 10 F 22 13 M
23 14 M 23 11 M
24 13 F
25 14 M

Table 3: Statistical parameters of age distribution.

Description Control AA AS SS
No of obs. (n) 20 25 23
Range (years) 5 – 13 5 – 14 5 – 15
Mean 10 12.5 11.5
SEM 0.316 0.707 0.737
Median 10 12.5 11.5
SD 1.4142 3.5356 3.5356

Statistical parameters of age of control and tests subjects

 Table 4: Table showing ANOVA result of age.
DF (↓) = F
DF(→) = AA ↔ AS ↔ SS ??

Showing the significance in the difference of age between the three

groups

Sex: The sex of both the control and test group is shown in table 2. The sex distribution of
both the control and experimental groups are shown in table 5.

In the control group there are 20 individuals comprising of 12 males and 8 females i.e. 60%
and 40% respectively as shown in table 5.

In the experimental group AS there are 25 individuals comprising of 15 males and 10 females
i.e. 60% and 40% respectively, as shown in table 5.

Table 5: Table showing Sex Distribution in population.

AA AS SS
Sex
No. % No. % No. %
Male 12 60 15 60 10 43.48
Female 8 40 10 40 13 56.52
Total 20 100 25 100 23 100

Graph 1: Sex distribution of subjects

MDA

The values of MDA level in the control, AS and SS are shown in the following table:

Table 6: Statistical parameters of MDA

 Description Control AA AS SS
No of obs.(n) 20 23 23
Range 0.9 – 2.4 1.0 – 2.8 1.8 – 5.2
Mean 1.6775 1.9304 3.3087
SEM 0.099 0.124 0.187
Median 1.7 1.8 3
SD 0.4462 0.5927 0.8959

In the normal control group, the MDA level ranges from 0.9 to 2.4 with a mean + SD value
of 1.6775 + 0.4462 and a median of 1.7. The standard error of mean is 0.099 and coefficient
of variation is 26.58.In the AS patients, the MDA level ranges from 1.0 to 2.8 with a mean +
SD value of 1.9304 + 0.5927 and a median of 1.8. The standard error of mean is 0.124 and
coefficient of variation is 30.72.
In the SS patients, the range of MDA varies from 1.8 to 5.2 with a mean + SD value of
3.3087 + 0.8959 and a median of 3. The standard error of mean is 0.187 and coefficient of
variation is 27.08.
The difference of mean value of MDA level between the three groups is shown in table which
is found to be significant with a F-ratio of 36.9 for 2df across(→) and 63df vertically(↓) at
5% level of significance.

DF (↓) = 63 F
Df(→) = 2 AA ↔ AS ↔ SS 36.9

The difference in the mean value of the MDA between the control and the AS is shown in
table and it is found that it has a t value of 1.5723 with a p value of > 0.10 which is not much
significant but the t value between the control group and the SS are 7.5161 with a p value of
<0.001 which is highly significant.

FRAP
The values of FRAP level in the control, AS and SS are shown in the following table:

Description Control AA AS SS
No of obs. (n) 20 23 23
 Range 490 – 1340 385 – 1160 230 – 700
Mean 883.2 719.348 414.478
SEM 47.26 41.02 24.26
Median 872 715 406
SD 211.25 196.73 116.35

In the normal control group, the FRAP level ranges from 490 to 1340 with a mean + SD
value of 883.2 + 211.25 and a median of 872. The standard error of mean is 47.26 and
coefficient of variation is 23.92. In the AS patients, the FRAP level ranges from 385 to 1160
with a mean + SD value of 719.348 + 196.73 and a median of 715. The standard error of
mean is 41.02 and coefficient of variation is 27.35.
In the SS patients, the range of FRAP varies from 230 to 700 with a mean + S.E.M value of
414.478 + 116.35 and a median of 406. The standard error of mean is 24.26 and coefficient of
variation is 28.07.
The difference of mean value of FRAP level between the three groups is shown in table
which is found to be significant with an F-ratio of 38.9 for 2df across (→) and 63df vertically
(↓) at 5% level of significance.

DF (↓) = 63 F
Df(→) = 2 AA ↔ AS ↔ SS 38.9

The difference in the mean value of the FRAP between the control and the AS is shown in
table and it is found that it has a t value of 2.6827 with a p value of < 0.02 which is
significant but the t value between the control group and the SS are 9.3464 with a p value of
<0.001 which is highly significant.
T-Sh
The values of T-Sh level in the control, AS and SS are shown in the following table:

Description Control AA AS SS
No of obs.(n) 20 23 23
Range 310 – 456 274 – 432 190 – 342
Mean 375.55 336.43 249.61
SEM 9.55 9.501 8.28
 Median 373 320 250
SD 42.6669 45.5739 39.6974

In the normal control group, the T-Sh level ranges from 310 to 456 with a mean + SD value
of 375.55 + 42.6669 and a median of 373. The standard error of mean is 9.55 and coefficient
of variation is 11.36. In the AS patients, the T-Sh level ranges from 274 to 432 with a mean +
SD value of 336.43 + 45.5739 and a median of 320. The standard error of mean is 9.501 and
coefficient of variation is 13.54.
In the SS patients, the range of T-Sh varies from 190 to 342 with a mean + SD value of
249.61 + 39.6974 and a median of 250. The standard error of mean is 8.28 and coefficient of
variation is 15.9.
The difference of mean value of T-Sh level between the three groups is shown in table which
is found to be significant with a F-ratio of 49.8 for 2df across(→) and 63df vertically(↓) at
5% level of significance.

DF (↓) = 63 F
Df(→)=2 AA ↔ AS ↔ SS 49.8

The difference in the mean value of the T-Sh between the control and the AS is shown in
table and it is found that it has a t value of 2.9454 with a p value of < 0.01 which is very
significant. The t value between the control group and the SS are 10.2141 with a p value of
<0.001 which is highly significant.
Graph 2: Values are expressed mean ± SD; n = 20 subjects for AA, 23 for
AS and 23 for SS. * Significantly different from control AA
(p > 0.10). # significantly different from AA (p < 0.001).

Graph 3: Values are expressed mean ± SD; n = 20 subjects for AA, 23

for AS and 23 for SS. * Significantly different from control
AA (p < 0.01). # significantly different from AA (p < 0.001).

Graph 4: Values are expressed mean ± SD; n = 20 subjects for AA, 23

for AS and 23 for SS. * Significantly different from control
AA (p < 0.02). # significantly different from AS (p < 0.001).
Discussion

Discussion

The generation of reactive oxygen species is a steady-state cellular event in respiring

cells. Their production can be grossly amplified in response to a variety of pathophysiological
conditions such as inflammation,immunologic disorders, hypoxia, hyperoxia, metabolism
of drugs or alcohol, exposure to UV or therapeutic radiation, and deficiency in antioxidants
enzymes. Uncontrolled production of reactive oxygen species often leads to damage of
cellular macromolecules (DNA, protein and lipids). A number of major cellular defense
mechanisms exist to neutralize and combat the damaging effects of these reactive substances.
Enzymatic systems function to directly or sequentially remove of reactive oxygen species
(catalase, glutathione peroxidase and superoxide dismutase), thereby terminating their
activities (Gaetani GF et al, 1996, Prakash M et al, 2008)53, 54. SCA is a hereditary disorder
with higher potential for oxidative damage due to chronic redox imbalance in red cells that
often results in clinical manifestation of mild-to severe hemolysis in patients with this genetic
disorder (Mutlu-Türkoglu U et al, 1997)53.
Congenital haemoglobin mutations may alter the delicate balance of free-radical
generation and antioxidant defense systems in the red cell. Oxidative stress may thus play a
role in the pathophysiology of the clinical manifestations of the disease. SCA is primarily a
disorder of red blood cells (RBCs), which are a significant source of free radicals in
biological systems. RBCs have a rich oxygen supply and are densely packed with redox-
active haemoglobin residues. The bonding interaction between heme-iron and oxygen in
oxygenated haemoglobin is associated with an electron transfer (Capo C et al, 1979)50. In
response, there is an integrated network of the antioxidant system, consisting of both
enzymes and low-molecular-weight compounds, which help to mitigate oxidative stress and
injury to the red cell and tissues in general. Congenital haemoglobin mutations may alter this
balance and create a pro-oxidant reactive milieu. This may be contributory to the
pathophysiology of the abnormalities that underlie the clinical course of SCA. The present
‘study of oxidative stress in sickle cell trait subjects and sickle cell anemia patients of School
children of Chattisgarh’ was undertaken in an effort to find out the relative level of oxidative
stress in sickle cell anemia patients and sickle cell trait as compared to normal which in the
long run may aid the clinician for better preventive and curative measures for these patients.

In the current study, MDA, FRAP and total- thiol were used as oxidative stress
marker, their levels were measured in the school children of Chhattisgarh and the values were
statistically analyzed with the age and sex matched control group. The study was carried out
from July 2010 to December 2010 at Pt. J. N. M. Medical College, Raipur. The blood
samples were collected from 25 AS and 23 SS patients were included in the present study. In
the present study, out of 25 AS patients, two samples were haemolysed, so 23 samples were
available for studying the oxidative stress markers.
All the participants were included in the study after sickle screen test (solubility test)
and HPLC analysis of blood. Subjects were excluded having past 3 month history of vaso-
occlussive crisis, blood transfusion and serious illness.

From the present study, it is seen that the majority of the patients are in the age group of 5-12
years.
The age and sex distribution of the normal control and the sickle cell trait and disease
patients group are depicted in table 2 , and that the age and sex of the normal control and
sickle cell trait and sickle cell disease patients are evenly distributed and there is no
significant difference (p > 0.05) among the three groups. Thus, the study group was evenly
matched and excludes the possibility of interference due to these factors

In the present study, it is found that sickle cell trait is higher in the Sahu community
(26.08%) followed by Kurmi and Dhiwar community (17.39%) each with lowest number in
the Muslim community(4.34%).Similarly in the SS group, Highest number of cases were
found from Sahu community(34.78%) followed by Kurmi community(21.74%).

Existence of oxidative stress in sickle cell disease is well proved by numerous studies.
Previous studies of Hebbel and colleagues47 have shown that, under ambient oxygen tensions,
sickle cells spontaneously generate O2•¯, H2O2 and OH• approximately two times more, when
compared to normal RBCs. Furthermore, these workers have also demonstrated that
hemichrome may facilitate OH• production in the presence of O2•¯ and H2O2.In the present
study,it is seen that the mean MDA level among the three groups is significant with a F ratio
of 36.9. This study agrees with previous study on MDA (De Jong K et al, 2001)17 (Miller ST
et al)17 where MDA level is significantly elevated. Accumulation of malondialdehyde disturbs
the organization of phospholipids in the human erythrocyte membrane bi-layer. Membrane
damage considered as an important factor contributing towards pathophysiology due to the
formation of irreversible sickle cells (ISC). Peroxidative reactions have long been recognized
as potential factors that contribute to degenerative cellular processes. RBCs are particularly
susceptible to peroxidative damage because they contain haemoglobin, one of the most
powerful catalysts for initiation of peroxidative reaction (Arinola OG et al, 2008)51. It is also
stated that excess quantity of malondialdehyde can promote erythrophagocytosis (Foluke F et
al)52.

We have used FRAP as a marker for total antioxidant status in the present study.It is
found that the FRAP level in both the sickle cell heterozygous(AS)and homozygous(SS)
patients is significantly decreased as compared to the control with F-ratio of ------ .The AS
and the SS group has p < 0.001 when compared with the control group. These results
correlates well with previous studies (Pryor WA et al, 1975 Mutlu-Türkoglu U et al, 1997)37,
53
.

Low levels of total thiols pool have been shown to be associated with various
disorders with increased generation of free radicals (Farmer EE et al, 2007)38. The total thiol
levels amongst the three groups viz AA, AS and SS is estimated. It is found that the mean T-
Sh level within the three groups significantly varies with a F ratio of ----.The level of total
thiol in the AS is very low compared to AA with p < 0.001.Similarly the total thiol level in
the SS is also significantly decreased compared to control with p < 0.001.Our study shows
significant decrease in total thiol level in sickle cell patients both AS and SS but not much
study is available on total thiol level in sickle cell anemia. It is therefore hoped that more
studies on this parameter may help in establishing the importance of this parameter in sickle
cell disease.

In the present study, two SS cases were found with cardiac problem, two SS cases
presented with renal disease, while one SS case presented with both cardiac and renal disease.
The blood Hb level is significantly decreased in SS with mean Hb ----while in AS and AA
with mean Hb level is ---- and ----- respectively.
 Summary
and Conclusion

Summary and Conclusion

Our study agrees with current evidence that oxidative stress has an important role to
play in the pathophysiology of the deleterious vaso-occlusive crises observed in SCA
patients. It thus strengthens the case for the use of agents that increase the total antioxidant
capacity of these patients with a view to improving their clinical course.
 This study was carried out so as to assess the effect of oxidative stress in sickle cell
trait and sickle cell anemia patients and this was done taking three parameters into
consideration i.e. Malondialdehyde (MDA), Total thiol (T-Sh) and Ferric Reducing ability of
Plasma (FRAP). The AA and AS subjects were healthy and not anemic, they had no
underlying medical problems and did not take vitamin or mineral supplements, etc. 5ml blood
was drawn from the patients antecubital vein and through centrifugation plasma is separated
and stored in -20◦C.

The present study was carried out at Pt. J. N. M. Medical College. In the study, we
have taken 23 AS and SS patients each. Twenty age and sex matched normal control groups
in the age group of 5 – 13 years were included. Under all aseptic measures patients as well as
control groups blood sample were taken from median cubital vein and blood samples were
analysed for MDA, T-Sh and FRAP and comparative study was done within the three groups.

The mean age of AS is 12.5 with range of 5 – 13 years. The mean age of SS is 11.5
with range of 5- 15 years.

In the present study the median MDA level within the three groups were found to be
significant with an F – ratio of 36.9. The MDA level between the control (AA) and the AS
is......... with the p – value of > 0.10. The MDA level between the patient and the AA is
highly significant with p < 0.001.

The total thiol level in among the three groups is significant with the variance ratio of
(F ratio) 49.8. The total thiol level within the AS and control group is highly significant with
p < 0.001. It is found that the total thiol level in SS patients is significantly decreased
compared to AA with p < 0.001.

The ANOVA study within the three groups for FRAP was significant with an F –
ratio 38.9. The unpaired t – test between AA and AS and AA and SS were carried out which
is highly significant with p < 0.001.

The oxidative stress parameters taken are biochemical test whose result seen is
deduced through spectrophotometer.

Malondialdehyde (MDA)
The MDA level in SS patients was more significantly as compared to AS and AA . mean +
S.E.M value of 1.6775 + 0.099

Total-thiol (T-Sh)

The T-Sh level in AA patients were more as compared to AS and AS showed greater increase
in T-Sh as compared to SS (see graph 2).

Ferric reducing ability of Plasma (FRAP)

The FRAP level in SS patients minimum as compared to AA and AS. AS patients have lower
FRAP level than AA (see graph 3).

Conclusion

Chhattisgarh is a state in the central India which has very high prevalence of sickle
cell disease. The present study was carried out to understand the level of oxidative stress
amongst the sickle cell anemia patients and carrier. Since oxidative stress is very high among
these patients, it is emphasised that more thrust should be given in sickle cell research and
development of agents to increase the total antioxidant capacity of these patients with a view
to improve their clinical course.
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disposal of hydrogen-peroxide within human erythrocytes. Blood ; 47:1595 – 1599.
 PROFORMA

Name:-
……………………………………………………………………………………………....

Father’s Name: -
……………………………………………………………………………………...

Age…………… Sex……………… Caste…………….

Religion………………………………....

Permanent Address:- …..….

………………………………………………………………….

…...
……………………………………………………………………..

……………………………………………………………...
…………..

Correspondence Address:-
………………………………………………………………………….

……………………………………………………………………
……

………………………………………………………………………
….

Telephone No: - Mobile …………………………………………………………………..

Residence ...………………………………………………………………..

Diagnosis:-
SS AA AS

Medical History:-
 1. Weight: 4. Height:
2. Fever: 5. Chest Pain:
3. Haemoglobin: 6. Joint Pain:

• History of blood transfusion:-

• Blood Group:-
• Penicillin prophylaxis: - Done /not done
• Antibiotic prophylaxis: - Taken / not taken

Master Chart
AA
Name Age Sex Address MDA T-Sh FRAP
Ranju Sen 12 F 2 0.98 0.96
Krit DhritBhare 11 M 0.98 0.95 0.96
Guhan Singh 10 M 1 0.94 1
Mamta Sager 12 F 2 0.92 0.94
Baldev Kaushal 14 M 1.5 1 0.94
Jyotsana Mandal 15 F 2 1.05 1.25
Ramnath 8 M 1 0.98 1.5
Ghanshyam
Tandi 7 M 3 0.96 0.92
Durga Sawra 10 F 2.5 0.9 0.9
Hiteshwari
Kaiwarth 6 M 2.5 0.98 0.94
Mohini Sahu 11 F 3 0.92 0.94
Manbai Banjare 12 F 2 0.9 0.94
Aayan lunkad 10 M 1.5 0.82 0.96
Dheeraj Pratab
Deo 8 M 1.5 0.94 0.96
Deepali Sahu 9 F 1.75 1 0.98
Pulkit Pruthi 7 M 1.25 0.96 1
Rahul Singh 12 M 2 0.82 1.25
Anil Agarwal 13 M 2 0.96 0.94
Vikash Singh 15 M 2.5 0.94 1.25
Jayant Bahadur 15 M 3 0.91 0.96
Savant Sahu 14 M 2.8 0.92 0.98
Rashi Baghel 14 F 1.2 0.86 0.94
Anamika Tiwari 10 F 1.8 0.9 1.05
Govind Ram 8 M 2.2 1 1.25
Saurabh Tilak 9 M 1 0.86 0.9
Bunty Rao 13 M 2.6 1 0.9
Nidhi Telkar 8 F 1.5 1 0.98
Salojna Bisen 9 F 1.2 0.88 0.92
Saroj Sonwane 7 F 1.2 0.84 0.88
 AS
Name Age Sex Address MDA T-Sh FRAP
Rekha Sahu 9 F 2.5 0.86 0.86
Anju Agarwal 8 F 3 0.86 0.84
Laxmi Kaushal 11 F 2 0.82 0.95
Nemchand Sahu 9 M 2 0.9 0.83
Jagdish
Chandrakar 11 M 2.75 0.92 0.87
Aditya
Gadhewal 12 M 2.75 0.86 0.85
Ananya
Srivastav 15 F 1.5 0.88 0.8
Rahul Agarwal 14 M 1.25 0.9 0.86
Vimla Bai 8.5 F 3.75 0.88 0.88
Ved Sahu 7 M 2.75 0.86 0.83
Parul Vishkarma 10 F 2.5 0.84 0.82
Tanmay Aiyer 10 M 2.5 0.84 0.88
Vikas Sonwane 11 M 3.5 0.9 0.86
Vishwaroop
Satnami 13 M 2.25 0.82 0.85
Pankaj
Suryavanshi 12 M 2 0.88 0.84
Vibha Desai 11 F 2.75 0.88 0.85
Ankit Talreja 10 M 1.5 0.86 0.85
Anil Kumar 14 M 2.8 0.9 0.8
Surya Sonkar 15 M 3.5 0.84 0.88
Vansh Sonkar 15 M 2.5 0.86 0.8
Abeer Lunkad 10 M 2.8 0.9 0.83
Shivi Sahu 16 F 2.4 0.84 0.78
Debashish Anant 15 M 2.8 0.88 0.9
Drigraj Khodiyar 14 M 3 0.98 0.78
Sunil Patra 8 M 3.05 0.9 0.8
Avinash Tripathi 7 M 2.6 0.84 0.8
Gokul Soni 8 M 3.2 0.86 0.85
Mona Deshlahre 13 F 2.8 0.98 0.88
Anju
Khandelwal 12 F 3.5 0.86 0.8
 SS
Name Age Sex Address MDA T-Sh FRAP
Rani Besra 12 F 3 0.69 0.76
Kirti Yadav 15 F 3 0.68 0.7
Laxmi Dhruv 15 F 2.75 0.7 0.65
Satrupa Bairagi 12 F 2.5 0.72 0.7
Kavita Verrma 11 F 4 0.64 0.7
Manish Verma 12 M 4.5 0.65 0.8
Latish Verma 13 M 1 0.7 0.76
Gopi Verma 8 M 1.5 0.69 0.74
Namita Sahu 14 F 2 0.7 0.74
Aashish Sahu 11 M 3.5 0.69 0.72
Dolly Soni 8 F 3.5 0.68 0.76
Durga Nayak 15 F 3 0.67 0.78
Aarti Jal 14 F 3 0.71 0.78
Ghanshyam Sahu 14 M 3.25 0.72 0.8
Sanjay Sharma 13 M 4 0.7 0.75
Tulsi Patel 11 F 3.5 0.69 0.74
Laxman Bagh 10 M 1.5 0.7 0.75
Kishore Ku. Sahu 12 M 1.5 0.69 0.78
Kamesh Verma 11 M 3.5 0.69 0.76
Tumar Prasad
Verma 9 M 3 0.69 0.72
Gayetram Netam 14 M 1.5 0.7 0.8
Deepak Sahu 15 M 2 0.68 0.64
Sanjay Deshlahre 12 M 3.05 0.98 0.7
Anupam Toppo 15 M 1.2 0.8 0.8
Radhe lal 10 M 1.5 0.84 0.74
Dinesh Gaikwad 8 M 2.5 0.76 0.72
Pramod Telkar 14 M 2 1.5 0.72
Rahul sinha 13 M 2.5 0.7 0.68
Lileshwar Khare 14 M 1.5 0.86 0.8
Mohit Pathak 11 M 1.8 0.9 0.78