ASSESSMENT OF THE LEVELS OF NITRIC OXIDE, MALONDIALDEHYDE AND TOTAL ANTIOXIDANT ACTIVITY AS INDICATORS OF OXIDATIVE STRESS IN NIGERIAN SICKLE CELL ANAEMIC PATIENTS

TABLE OF CONTENTS

TITLE PAGE

CERTIFICATION

DEDICATION

ACKNOWLEDGEMENT

TABLE OF CONTENT

LIST OF FIGURES

ABSTRACT

CHAPTER ONE

1.0       Introduction

1.1       The Hemoglobin Molecule and Sickle Cell Disease

CHAPTER TWO

2.0       Literature Review

2.1       Sickle Cell Disease

2.2       Complications of Sickle Cell Anemia

2.3       Fundamental Genetics of Sickle Cell Disease

2.4       Types of Sickle Cell Disease

2.5       Blood Cell Auto-oxidation

2.6       Sickling and Hemolysis

2.7       RBC Adhesion, Vaso-occlusion and Sickle Cell Crises

2.8       Natural History of the Vaso-occlusive Crisis

2.9       Hypoxia/Reperfusion Injury

2.10     Oxidative Stress

2.11     Reactive Oxygen Species

2.12     Generation of Reactive Oxygen Species in Sickle Cell Disease

2.13     Antioxidants

2.14     Nitric oxide and Vascular Function

2.15     Fate of Nitric Oxide in Sickle Cell Anaemia

2.16     Lipid Peroxidation

2.17     Objectives of the Study

CHAPTER THREE

3.0       Patients and Methods

3.1       Sample Collection and Methodology

3.2       Antioxidant Supplementation

3.3       Reagents and Sources

3.4       Dosage Administered

3.5       Biochemical Analysis

3.6       Statistical Analysis

CHAPTER FOUR

4.0       Results

4.1       Nitric Oxide Levels

4.2       Malondialdehyde Levels

4.3       Antioxidant Activity Levels

4.4       Hemoglobin Level

CHAPTER FIVE

5.0       Discussion

5.1       Conclusion

References

Appendix

ABSTRACT

Sickle cell anaemia (SCA) is a hereditary disorder associated with severe haemolyticanaemia, periodical vaso-occlusive pain and premature death. Oxidative stress plays important role in the pathophysiology of sickle cell anaemia. The present study was undertaken to evaluate the levels of plasma lipid peroxidation product, malondialdehyde (MDA), serum nitric oxide (NO) and total antioxidant activity (TAOA), and the effect of antioxidant supplementation (Vitamins A,C and E) on oxidative stress. A total of 52 subjects participated in the study, including 32 homozygous (HbSS) sickle cell patients and 20 age and sex matched healthy controls. The above parameters were measured before and after six weeks of antioxidant supplementation. Results obtained showed a statistically significant decrease and increase in baseline level of NO and MDA respectively in HbSS group when compared with the control (p<0.05). A slight decrease in the total antioxidant activity level was observed although statistically non-significant with (p>0.05). Patients mean blood hemoglobin was significantly (p<0.05) decreased in the HbSS group being 7.09±1.02 g/dL as compared to that of the control that was 13.65±1.39 g/dL. A significant (p<0.05) positive correlation was found between hemoglobin level and nitrite(r=0.7). Also there was a significant (p<0.05) negative correlation between hemoglobin and malondialdehyde levels (r=-0.7). No significant correlation was seen between hemoglobin and total antioxidant activity levels. After the supplementation with the antioxidants, a statistically significant increase (p<0.05) in the level of NO was observed whereas a slight decrease and increase in the levels of MDA and TAOA respectively were observed although statistically not significant (p>0.05). These observations provide evidence of imbalance between oxidant and antioxidant status leading to oxidative stress in SCA. Therefore antioxidant supplementation is essential in sickle cell individuals in the steady state as well as in crisis to prevent further oxidative damage to the erythrocytes.

CHAPTER ONE

1.0             INTRODUCTION

1.1             THE HEMOGLOBIN MOLECULE AND SICKLE CELL DISEASE

Found in red blood cells, hemoglobin is a globular protein that carries oxygen molecules (O₂)

and carbon dioxide molecules (CO₂₎ throughout the body. Each hemoglobin protein structure

consists of four polypeptide subunits, which are held together by ionic bonds, hydrogen bonds, hydrophobic interactions, and van der Waals forces, as well as four heme pigments, one in each of the subunits (Sadava et al., 2008).

The hemoglobin molecule is made up of four polypeptide chains: two alpha chains of 141 amino acid residues each, and two beta chains of 146 amino acid residues each. The alpha and beta chains have different sequences of amino acids, but fold up to form similar three-dimensional structures (Perutz, 1990).

Sickle cell disease (SCD) is a class of hemoglobinopathy, which results from a single mutation in the gene for β-globin chain of hemoglobin inducing the substitution of valine for glutamic acid at the sixth amino acid position (Halliwell, 2007). This mutation leads to the production of abnormal hemoglobin (hemoglobin S or HbS). In addition to homozygous sickle cell disease (HbSS), other forms such as HbSC (compound heterozygosity for sickle cell trait and hemoglobin C) and HbSβ-thalassemia (compound heterozygosity for sickle cell trait and β thalassemia trait) also exist. The pathogenesis of SCD occurs due to the aggregation of deoxygenated HbS. This aggregation alters the normal biconcave disc shape into a rigid, irregularly shaped, unstable cell (Halliwell, 2007), causing intravascular hemolysis, chronic haemolytic anaemia and vasoocclusive crisis. Sickle cell disease patients are susceptible to increased oxidative stress (Kings and Farber 2001), because mutant red blood cell which contains haemoglobin is one of the enhancers of peroxidative reaction......

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