Abstract
The relationship between anemia, SOD (superoxide dismutase) and G6PD (glucose-6-phosphate) in SCD (sickle cell disease) patients were determined for the better understanding of their pathophysiology. Hemolytic anemia is one of the most common complications of sickle cell. It can be influenced by different factors, malaria infections, oxidative stress, dehydration, environmental stress and much more. SOD are enzymes that help with the regulation of oxidative stress and G6PD deficiency together with SCA are at high frequencies in malaria epidemic regions. Both SCA and G6PD deficiency patients suffer from anemia and can be said to coexist in some individuals. This study aims to find out the relationship between anemia, SOD and G6PD in SCD patients, if they coexist together or influence each other’s manifestations. A total of 70 patients were used including AA and AS genotypes as control. Anemia indices including hemoglobin (Hb) concentration, hematocrit were tested for. The presen
CHAPTER ONE
INTRODUCTION
1.0 BACKGROUND
Sickle cell disease (SCD) is a group of genetic disorder that is inherited in an autosomal recessive
manner due to the homozygous or heterozygous state of the mutation. It is caused by a single base
mutation in the β-globin gene of hemoglobin, where GAT is replaced by GTT in the 6th codon of
exon 1 resulting to valine instead of glutamic acid on the sixth position in chromosome 11. In the
normal adult hemoglobin (HbA), there are 2 α-globin chains and 2 β-globin chains that form a
globin tetramer. They are stabilized by intramolecular points of contact, without any interaction
between them. When they bind or release oxygen they retain their normal shape but in the mutated
β-globin there is a hydrophobic interaction between the adjacent valine amino acids which align
into polymers and distort the shape of the red blood cells. These polymers, which are poorly
soluble, distort the normal shape of the red blood cells, changing it to a sickle or crescent shape
which prevents the normal flow of blood in the blood vessels (microcirculation) and increasing its
adhesion to the endothelium of the vessels. This leads to vaso-occlusive crisis and hemolytic
anemia which are the hallmark of the disease. SCD is a systemic pleiotropic disease that affects
almost all the organs of the body or causes tissue infarction and a good number of other clinical
manifestations throughout the affected individual’s life as a result of the polymerization of the beta
hemoglobin under deoxygenated, acidic or dehydrated conditions and hypoxia. Sickled RBCs are
more readily destroyed or are broken down prematurely by the reticulo-endothelial system due to
their rigidity makes them filtered by the spleen. Most of the clinical manifestations are protean in
nature and vary in frequency and severity among patients. SCD is a hemoglobinopathy in which
the single base substitution mutation in the β-globin chain can result to either hemoglobin S, C, β+
or β æ thalassemia, D, E or OArab and are all known as hemoglobin variants but when they are
2combined with HbS they are known as SCD variants. Individuals, who are affected with sickle cell
anemia which is one of the variants of SCD, have two copies of the mutated gene (HbSS). Other
heterozygote individuals have one copy of the Hb S and other variant which could be Hb C, Hb β+
or β æ thalassemia. The mutation in HbSS is Glu6Val, in HbSC is Glu6Lys, the mutation in
hemoglobin D glutamine replaces glutamic acid at position 121 of the gene and the mutations that
cause the Sβ+ or the Sβº are deletions or additions of a single base substitute or more in the HBB
gene (Serjeant 2013; Ashley-Koch et al., 2000; Heiman and Greist, 2010; Bunn, 1997; Booth et
al., 2010; Al-Jafar et al., 2016; Kaur et al., 2013; Ballas, 2002; Ballas et al 2010; Wild and Bain,
2006 and Emecheba et al., 2017).
Carrier individuals have one copy of the mutated hemoglobin and normal hemoglobin (Hb AS)
and are said to have sickle cell trait. They are also said to be protected from malaria infection,
resulting to the high frequency of the Hb S variants in malaria epidemic regions. Not all the variants
of the mutated hemoglobin are detrimental, a concept known as genetic polymorphism. Millions
of people worldwide are affected with this disease with the highest population in Africa but it can
also be seen in Sub-Saharan Africa, Eastern Saudi Arabia, Central India Mediterranean, Afro
Caribbean, South and Central American, Arab and East Indian. Some of these variants are frequent
in some of these populations than others. The two commonest hemoglobin variants in Nigeria are
HbS and HbC. HbS is distributed fairly well in Nigeria but HbC is commonly seen among the
westerns (Yoruba) and decreases from the west eastwards. It was estimated in Nigeria around 1982
that 30,000 infants are born each year with SCD as it is seen as the country with the highest affected
individuals with the trait ranging from 20-30%. As of recent it is estimated that >40 million
individuals are carriers, >150,000 infants are born each year with the disease and about 1 million
survive past childhood (Galadanci et al., 2013; Emecheba et al., 2017 and Grosse et al., 2011).
3According to Robbins, (2014) the major cause of the symptoms in patients with SCD is the sickling
of the red blood cells. The clinical or phenotypic manifestations of SCD are grouped into three,
which include hemolytic anemia, pain episodes or crisis and severe organ damage. The sickled
cells are unable to deliver oxygen to tissues in the body and this leads to tissue or organ damage.
They also die faster than normal cells which lead to anemia, a blood condition that the red blood
cells are lower than normal and it is a major symptom in patients with SCD. Due to their
inflexibility they are unable to pass through small capillaries, causing blockage in the blood vessels
leading to severe vaso-occlusive crisis. Other signs and symptoms of sickle cell disease which vary
from person to person and can change over time include; acute pain (sickle cell or vaso-occlusive
crisis), frequent infections, pulmonary complications, leg ulcers, priapism, brain complications
(clinical stroke and silent stroke), eye problem, retarded growth and puberty, kidney problem
(nocturnal enuresis), gallstones, liver complications (intrahepatic cholestasis), joint complications
(avasualar or aseptic necrosis) and metal health. Lack of a large, readily accessible population for
clinical studies has contributed to the absence of standard definitions and diagnostic criteria for
the numerous complications of SCD and inadequate understanding of SCD pathophysiology
(Ballas et al., 2010).
Most of these complications found in SCD patients can be triggered by a lot of factors such as
malaria infections, stress, temperature change (favorably warmth) and dehydration. As discoveries
are been made, new body of evidence has shown that oxidative stress is a significant pathway
sickle cell complications and morbidity as it enhances the sickling phenomenon of the cells. These
could all contribute to the heterogeneous phenotypic expression of the disease. Oxidative stress is
an imbalanced redox status caused by over production of oxidants (reactive oxygen specie) and
depletion of antioxidants. This excess oxidant state leads to release of heme, auto-oxidation of
4hemoglobin, uncoupling of nitric oxide synthase activity leading to a decrease in NO. It has been
observed that the antioxidant defense systems in SCD are ineffective in neutralizing the excess
oxidant specie. Normal erythrocytes counter oxidative stress using self-sustaining activities of
antioxidant defense enzymes such as superoxide dismutase (SOD) which is a key enzyme in
dismuting super radicals into hydrogen peroxide. The activity of this enzyme is seen to be higher
in SCA patients.
Anemia is a medical condition where the red blood cells or hemoglobin level in the body is lower
than the normal level. Sickle cell disorders are associated with variable degrees of anemia
depending on genotype, with the most severe decrease in hemoglobin level seen in sickle cell
anemia and the least severe in hemoglobin S-β+ thalassemia. Normal red blood cells live for 120
days while the sickle cells live for 10-30 days as a result of continuous breakage of the cells. When
the body is short of red blood cells, the tissues do not receive adequate amount of oxygen and this
leads to fatigue or weakness. Severe anemia episodes may result from a variety of causes, including
hyperhemolysis, acute splenic sequestration, and aplastic crises (Ballas et al., 2010). Although
chronic hemolytic anemia is a major feature of sickle cell disorders, a marked drop in hemoglobin
with an increased hemolytic rate is referred to as hyperhemolysis. Hemolytic anemia varies
intensively among the genotypes of sickle cell disease and it may be the driving force behind some
complications of sickle cell disease because of its effects on Nitric oxide (NO) bioavailability
which its decrease is associated with pulmonary hypertension, priapism, leg ulceration, and
possibly with non-hemorrhagic stroke (Kato et al., 2007).
Another clinical symptom that can be associated with SCD is G6PD deficiency. Glucose-6
phosphate dehydrogenase deficiency is a genetic disorder that results to an inadequate production
of G6PD enzyme. This enzyme helps to regulate many biochemical processes in the body
5including the proper and normal functioning of the red blood cells. This deficiency causes the red
blood cells to break prematurely called hemolysis leading to a common medical problem called
hemolytic anemia. This anemia could lead to paleness, jaundice, fatigue, rapid heart rate and so
on. In individuals with this deficiency, hemolytic anemia can be triggered by bacterial or viral
infections, antibiotics or antimalaria drugs, favism which is caused by eating fava beans. This
deficiency occurs exclusively in males. This deficiency results from mutations in the G6PD gene.
This gene provides the instruction for producing the enzyme which is involved with the chemical
reactions that prevent reactive oxygen species from accumulating to toxic levels in the body. With
these mutations occurring, the production or structure of the enzyme is altered leading to an
accumulation of the reactive oxygen species and would be harmful to the red blood cells. This
gene is found on the X-chromosome and since males have only one copy of this chromosome they
are more affected then females that have 2 copies of the chromosome and it is very rare for the
mutation to occur on both genes. G6PD deficiency just like SCD is prevalent where malaria is
epidemic and very common among the black population with a protective role against malaria.
The presence of the G6PD deficiency can lead to an increase in the severity of crisis in SCD
patients. Studies have also shown that this deficiency is prevalent in SCD patients more than the
general population but this could be otherwise in some other population. The coexistence of this
relationship can lead to hemolytic anemia, acute splenic sequestration and vaso-occlusive crisis.
Patients usually are asymptomatic, these disorders do not alter the hemoglobin (Hb) levels and
RBC count in stable conditions (Genetic home reference, 2018; Benkerrou, et al., 2013; Memon,
et al., 2016, Firempong, et al., 2016 and Al-Nood, 2011).
Over the years, measures like prenatal screening, parent education, better medical care,
immunization and the use of penicillin prophylaxis have been used to increase the life expectancy
6of affected individual, having the three basic therapeutics modalities as hydroxyurea, blood
transfusion and bone marrow transplant. World Health Organization (WHO) has promoted several
national screening programs with the goal of informing reproductive choice in other to reduce
severely affected infants (Kaur et al., 2013).
This study is designed to access the relationship between sickle cell disease and the factors that
trigger their complications. Hemolytic anemia is the most common clinical manifestation found in
each of the single genotype of this disorder. Oxidative stress is known to increase the anemia rate
in SCD patients and leads to vaso-occlusive crisis and any other clinical complications and how it
affects G6PD patients is quite unclear but antimalaria drugs can trigger hemolytic crisis.
Individuals that have only G6PD deficiency tend to have hemolytic anemia as the main clinical
symptom, and the relationship between SCD and G6PD is not definite for all population but they
are common in black population and have a protective role against malaria. Thus, it will be of
interest to evaluate the influence of oxidative stress, anemia and G6PD deficiency on SCD patients.
1.1 STATEMENT OF PROBLEM
Nigeria is said to have the highest number of SCD cases, having the two most common variants
as SS and SC. Despite the high burden of SCD in Nigeria, it has been difficult to improve the care
and management of diseases. Most of the new treatments, therapies, and creation of awareness is
lacking or is not widely available especially in the rural regions. The pathophysiology of the diseases,
to an extent is not really understood resulting from lack of assessable data which also leads to the inability
of providing a permanent cure for the disease. Studying the various factors that triggers their crisis and
the degree of their phenotypic manifestations would give a better understanding of the disease
7pathophysiology and more data would be available in order to provide better improved treatment
of the disease.
1.2 AIMS AND OBJECTIVES
This study intends to assess the relationship between anemia, SOD and G6PD deficiency and how
they increases the vaso-occlusive crisis in SCD patients visiting ESUTH and UNTH in Enugu
State, Nigeria
The objectives of this study are to determine:
ï‚· Screen patients for sickle cell anemia based on their genotype status-using questionnaire.
ï‚· Assess hemoglobin concentration and hematocrit level in patients.
ï‚· Quantify the level of superoxide dismutase (SOD) activity in patients.
ï‚· To determine the absence or presence of G6PD deficiency in SCD patients.