RED BLOOD CELL MORPHOLOGY AS A MARKER OF OXIDATIVE STRESS IN EARLY TYPE 2 DIABETES PATIENTS AND EFFICACY OF ANTIOXIDANTS AS AN ADD ON THERAPY TO STANDARD TREATMENT - A RANDOMIZED, OPEN LABEL, COMPARATIVE PILOT STUDY Dissertation submitted to THE TAMILNADU DR. M.G.R. MEDICAL UNIVERSITY In partial fulfillment for the award of the degree of DOCTOR OF MEDICINE IN PHARMACOLOGY INSTITUTE OF PHARMACOLOGY MADRAS MEDICAL COLLEGE CHENNAI - 600 003 OCTOBER 2016 CERTIFICATE This is to certify that the dissertation entitled, “RED BLOOD CELL MORPHOLOGY AS A MARKER OF OXIDATIVE STRESS IN EARLY TYPE 2 DIABETES PATIENTS AND EFFICACY OF ANTIOXIDANTS AS AN ADD ON THERAPY TO STANDARD TREATMENT - A RANDOMIZED, OPEN LABEL, COMPARATIVE PILOT STUDY” submitted by DR. ROHINI ANN MATHEW, in partial fulfilment for the award of the degree of Doctor of Medicine in Pharmacology by The Tamil Nadu Dr.M.G.R.Medical University, Chennai is a bonafide record of the work done by her in the Institute of Pharmacology, Madras Medical College during the academic year 2013-16. DEAN DIRECTOR AND PROFESSOR, Madras Medical College & Institute of Pharmacology, Rajiv Gandhi Govt. General Hospital Madras Medical College, Chennai – 600 003. Chennai – 600 003. CERTIFICATE OF THE GUIDE This is to certify that the dissertation entitled, “RED BLOOD CELL MORPHOLOGY AS A MARKER OF OXIDATIVE STRESS IN EARLY TYPE 2 DIABETES PATIENTS AND EFFICACY OF ANTIOXIDANTS AS AN ADD ON THERAPY TO STANDARD TREATMENT - A RANDOMIZED, OPEN LABEL, COMPARATIVE PILOT STUDY” submitted by DR. ROHINI ANN MATHEW, in partial fulfillment for the award of the degree of Doctor of Medicine in Pharmacology by The Tamil Nadu Dr.M.G.R.Medical University, Chennai is a record of original work done by her under my guidance and supervision in the Institute of Pharmacology, Madras Medical College during the academic year 2013-16. Place: Dr. B.VASANTHI, M.D., Date: Director and Professor, Institute of Pharmacology, Madras Medical College, Chennai- 600 003. DECLARATION I, Dr. ROHINI ANN MATHEW solemnly declare that the dissertation titled “RED BLOOD CELL MORPHOLOGY AS A MARKER OF OXIDATIVE STRESS IN EARLY TYPE 2 DIABETES PATIENTS AND EFFICACY OF ANTIOXIDANTS AS AN ADD ON THERAPY TO STANDARD TREATMENT - A RANDOMIZED, OPEN LABEL, COMPARATIVE PILOT STUDY” has been prepared by me and submitted to TN Dr.MGR Medical University, Chennai in partial fulfillment of the rules and regulations for the M.D degree examination in Pharmacology. Date: DR. ROHINI ANN MATHEW Place: TURNITIN ANTI-PLAGIARISM SOFTWARE – CERTIFICATE ACKNOWLEDGEMENT I am grateful to the Dean, Dr. Isaac Christian Moses, M.D, FICP, FACP., Madras Medical College and Rajiv Gandhi Government General Hospital, Chennai for allowing me to avail the facilities needed for my dissertation work. I am very thankful to Dr.Sudha Seshayyan, M.S., Vice Principal and Professor of Anatomy, Madras Medical College for her encouragement that helped me to accomplish my goal. I would like to express my special thanks and deepest gratitude to Dr. B.Vasanthi, M.D., Director, Professor and Guide, Institute of Pharmacology, Madras Medical College, Chennai for her remarkable guidance, valuable suggestions and continuous encouragement. I am grateful to her for enforcing strict validation of my work and her constant and untiring support that made me to complete my dissertation successfully. I record my sincere thanks to Dr.P.Dharmarajan M.D, DIP.DIAB; Professor and Head of the Institute of Diabetology for granting me permission and complete support to do this study in the Department of Diabetology. I wish to express my sincere thanks to Dr.K.M.Sudha, M.D., Professor of Pharmacology, Madras Medical College for her enduring encouragement and support. I am grateful to Assistant Professors of the Department Dr.S.Deepa M.D, Dr.G.Chenthamarai M.D, Dr.S.Suganeshwari M.D, Dr.Ramesh Kannan M.D, Dr. R.Vishnupriya M.D, and Tutor in Pharmacology, Dr.A.C. Yegneshwaran M.D, who supported me throughout and provided the necessary information needed during the study. I also extend my sincere thanks to all other staff members and colleagues of the Institute of Pharmacology for their wholehearted support and valuable suggestions throughout the study. Last but not the least, I sincerely thank my family and friends for their continuous encouragement, patience, valuable support and sincere prayers, without which I could not have completed this work successfully. I also wish to thank the patients who voluntarily participated in this study. ABBREVIATIONS DM - Diabetes Mellitus WHO - World Health Organization ROS - Reactive oxygen species RBC - Red blood cell PPARα - peroxisome proliferator activated receptor alpha FFA - free fatty acid BMI - Body mass index ECG - Electro cardiography GLP1 - Glucagon like peptide 1 DPP 4 - Dipeptidyl peptidase 4 SGLT 2 - Sodium glucose co-transporter 2 DNA - Deoxy ribonucleic acid GIP - Gastric inhibiting polypeptide. ATP - Adenosine triphosphate AMPK - AMP dependant protein kinase DKA - Diabetic ketoacidosis PEP - Phospho enol pyruvate TAG -Tri acyl glycerol TNF α -Tumor necrosis factor alpha IL-6 - Interleukin 6 IL-1 - Interleukin 1 iNOS - inducible Nitric oxide synthase IRS - insulin receptor substrate MAP - Mitogen activated protein kinase GLUT - Glucose transporter Lep-Rb - Leptin receptor b RNS - Reactive nitrogen species NADPH - Nicotinamide adenine dinucleotide phosphate AGE - Advanced glycation end products PKC - Protein kinase C DAG - Di acyl glycerol NF-kB - Nuclear factor kappa B NO - Nitric oxide CFU –E - Colony forming unit – erythrocyte HMP - Hexose monophosphate G6PD - Glucose 6-phosphate dehydrogenase GSH - Glutathione LDL - Low density lipoprotein IgG - Immunoglobulin G IgM - Immunoglobulin M CYP - Cytochrome P PUFA - Poly unsaturated fatty acids ANOVA - Analysis of variance CONTENTS S.NO TOPICS PAGE NO 1. INTRODUCTION 1 2. REVIEW OF LITERATURE 4 3. AIM AND OBJECTIVE 72 4. METHODOLOGY 73 5. RESULTS 85 6. DISCUSSION 109 7. CONCLUSION 114 8. BIBLIOGRAPHY 9. APPENDICES INTRODUCTION INTRODUCTION Diabetes mellitus is a heterogeneous group of metabolic disorders characterized by chronic hyperglycemia resulting from defects in insulin secretion, insulin action or both 1. It is classified into 2 major types: insulin dependent (Type 1) and non-insulin dependent (Type 2) 2. Type 1 diabetes mellitus is characterized by a specific destruction of the pancreatic beta cells mostly associated with immune mediated damage 3. Type 2 diabetes mellitus is characterized by a gradual change in glucose homeostasis due to insulin resistance and/or reduced insulin secretion 4. Type 2 diabetes mellitus is now taking its place as one of the main threats to human health in the 21st century5. The World Health Organization (WHO) has estimated that by 2025 there will be about 300 million people living with diabetes worldwide, with India alone contributing a massive 57.2 million 6. Though the metabolic derangements in Type 1 diabetes can be easily explained due to lack of insulin, for Type 2 diabetes it is multi-factorial influenced by genetic and environmental factors. Oxidative stress plays an important role in the pathogenesis of Type 2 diabetes and its complications 7. In diabetic patients there is a significant decrease in the activity of enzymatic antioxidant defense system like Superoxide dismutase, glutathione reductase, glutathione peroxidase and catalase. 8 Reactive oxygen species (ROS) generated from chronic hyperglycemia in these patients disrupts the critical balance between oxidants and antioxidants 9 and is implicated in the development of long-term microvascular and macrovascular complications associated with high morbidity and mortality in these patients 10. Free radicals are highly reactive molecular species with an unpaired electron that can cause damage to nucleic acids, proteins and lipids in the cell membrane and plasma lipoproteins. Tissue damage caused by these free radicals is often termed as “Oxidative damage”11. Red blood cells (RBCs) are highly susceptible to oxidative damage as they are the first cells to be exposed to oxidative stress. Presence of high cellular concentration of oxygen and hemoglobin, lack of nucleus and mitochondria, inability to synthesize enzymes and protein make RBCs vulnerable to oxidative stress 12. Therefore, structural damage like crenated edges and Heinz bodies in RBCs caused by ROS can be used as a marker of oxidative stress in Diabetes mellitus. The level of antioxidants is reduced in diabetic patients and supplementation of non enzymatic antioxidants like Vitamin E and C can reduce this oxidative damage. Therefore in this study RBC morphology is used as a marker of oxidative stress in Type 2 Diabetes and the effect of antioxidants like Vitamin E and C is studied in reversing this oxidative damage. REVIEW OF LITERATURE LITERATURE REVIEW DIABETES MELLITUS DEFINIITON Diabetes mellitus is characterized by chronic hyperglycemia with disturbances of carbohydrate, fat, and protein metabolism resulting from defects in insulin secretion, insulin action, or both. 13 ETIOLOGICAL CLASSIFICATION 1 1. Type 1 (beta cell destruction leading to absolute insulin deficiency) Autoimmune Idiopathic 2. Type 2 (hyperglycemia, insulin resistance and relative insulin deficiency) 3. Other specific types: - Genetic defects of β-cell function/insulin action - Endocrinopathies - Drug- or- chemical-induced - Infections 4. Gestational diabetes. EPIDEMIOLOGY 6,7 The worldwide prevalence of Diabetes Mellitus has risen dramatically over the past two decades. The