SUMMARY AND CONCLUSION 7. SUMMARY AND CONCLUSION Oxidative stress has been repetitively hallmarks of many diseases linked with metabolic or vascular disorders. Diabetes mellitus is the most rapidly growing chronic disease in the world. To understand the essence of aetiopathogenic mechanisms, which are at the root of diabetic complications development is an essential challenge to modern medical science and practice. Recently diabetic micro- and macroangiopathy are considered to be polyaetiological multifactorial diseases where persistent hyperglycaemia plays the leading part. On the other hand it contributes to the origin of oxidative stress. Oxidative stress is the fast becoming medical and nutritional buzzword for 21 st century. It is thought to be increased in a system where the rate of free radical production is increased and/or the antioxidant mechanisms are impaired. A Study Of Oxidative Stress And Antioxidant Status In The Pathogenesis Of Diabetic Complications 2
Overwhelming evidence has accumulated showing that antioxidants may prevent oxidative damage and thus protect against the adverse effects of oxidants. According various studies in diabetes increased oxidative stress and decreased antioxidative capacity was observed. Based on the findings the role of nutritional and pharmacological antioxidants turned into a subject of intense research. In point of view the aim of present project was to study the role oxidative stress and antioxidants in pathogenesis of diabetic complications and evaluation of efficacy of antioxidant supplementations which may help in minimizing late complications of diabetes mellitus. The present study was conducted in the Department of Biochemistry of Dr. V. M. Medical College, Solapur and Pad. Dr. D. Y. Patil Medical College; Pimpri, Pune, during the period of August 2004 to May 2009. In all 380 subjects were enrolled in the study. Forty age and sex matched healthy subjects were included in each group of two different control groups ( A & B) to compare with respect of type 1 and type 2 DM group. A Study Of Oxidative Stress And Antioxidant Status In The Pathogenesis Of Diabetic Complications 3
The test group included total 300 freshly diagnosed cases of diabetes mellitus i.e. type 1 (n=150) and type 2 (n=150). All the patients were selected from Shri Chhatrapati Shivaji Maharaj Sarvopchar Rugnalaya; Solapur and Pad. Dr. D.Y. Patil Medical College, Hospital and Research Centre, Pune. World Health Organization Criteria i.e. fasting plasma glucose 126 mg/dl along with classic symptoms of diabetes mellitus or postprandial plasma glucose 200 mg/dl at more than one occasion was applied for a diagnosis. No patients had ketoacidosis and had taken any hypolipidemic, antidiabetic, or antioxidant containing drugs. All the patients underwent details history, biochemical investigations which include hemogram, BUL, serum creatinine, lipid profile, electrolytes, Urinary protein etc. For exclusion from the study, retinopathy was confirmed by fundal examination by observing presence of microaneurysm, soft exudates, and intraretinal hemorrhages. Nephropathy was considered to be present, if there was proteinurea 1 gm/l in urine sample. The A Study Of Oxidative Stress And Antioxidant Status In The Pathogenesis Of Diabetic Complications 4
cardiovasculopathy was diagnosed by ECG findings and history of myocardial infarction. All the subjects included in the study were volunteered after proper consent and reported for follow-up at right time. The study approved by local ethical committee of the institute. The test group was categorized into different group as follows and each group includes 30 patients. Depending upon supplementation of different antioxidants in type 1 DM was categorized into Group I to V. Group I (only insulin), Group II (Insulin + Vitamin C), Group III (Insulin + Vitamin E), Group IV (Insulin + Vitamin(E+C)) and Group V (Insulin + (A-Z) tablets). Type 2 DM was categorized into Group VI to X. Group VI (only Drugs (metformin & sulphonylurea) Group VII (Drugs + Vitamin C), Group VIII (Drugs + Vitamin E), Group IX (Drugs Vitamin(E+C) and Group X (Drugs + (A-Z) tablets). Vitamin C (Celin 500 mg/day), Vitamin E - (Evinal 400 mg/day) and (A-Z) tablets - Multivitamin-Multimineral Antioxidants tablets. A Study Of Oxidative Stress And Antioxidant Status In The Pathogenesis Of Diabetic Complications 5
After 12 hours fast, venous blood sample were collected in different bulbs under aseptic conditions. 1. Fluoride bulb was used for fasting blood glucose estimation by Glucose Oxidase Peroxidase method 2. EDTA bulb was used for glycosylated hemoglobin (HbA1c) estimation by resin binding method. 3. Plain bulb was used for estimations of serum superoxide dismutase (SOD) by Marklund and Marklund, nitric oxide (NO) by Cortas and Wakid, malondialdehyde (MDA) by Satosh K. Total Antioxidant Status by FRAP method. 4. Acid citrate bulb was used for erythrocyte reduced glutathione (GSH) measurement by method of Beutler et al. 5. Heparin bulb used for estimation of platelet aggregation by ADP induced aggregation method. 6. Erythrocyte ghost prepared by Dodge method. The estimation of membrane protein by lowery s, membrane cholesterol by Zak et al and membrane phospholipids by Fiske- Subarrow method was done. A Study Of Oxidative Stress And Antioxidant Status In The Pathogenesis Of Diabetic Complications 6
Baseline level of all of the above biochemical parameters were measured at the time of enrollment in the study for all subjects. But Group I to X was again reassessed for the same parameters after follow up of 90 days of antioxidant supplementation along with antidiabetic drugs. The glycated hemoglobin (HbA1c) levels were used as an index of metabolic control. Statistical Analysis: The sample size was decided in consultation with statistician. The results were expressed as mean ± S.D. Comparison of control and test groups was done by unpaired t test. The change in parameters before and after antioxidant supplementation was studied by paired t test. Comparison of efficacy of antioxidant supplementation was tested by ANOVA. The present study shows no significant change in FPG levels after 90 days as compared to baseline levels in diabetes mellitus. Concentration of HbA1c was significantly decreased (p<0.001) in all group at 90 days as compared to baseline concentration. The patients were stabilized with insulin injections (long acting and short acting dosage) and A Study Of Oxidative Stress And Antioxidant Status In The Pathogenesis Of Diabetic Complications 7
antidiabetic drugs, no effect of antioxidant supplementations on FPG levels but it was observed that glycated hemoglobin (HbA1c) was better controlled by antioxidant supplementation along with antidiabetic drugs. Significant rise (p<0.001) in concentration of serum MDA and NO in DM group as compared to controls. Significant fall in antioxidants (p<0.001) like serum SOD, erythrocyte GSH and total antioxidant binding capacity i.e. FRAP was observed in DM as compared with controls. Lower glutathione and elevated lipid peroxidation are risk factors for the development of pathological states such as retinopathy, neuropathy, cataracts and atherosclerosis. The membrane proteins and phospholipids concentrations were significantly decreased (p<0.001) in DM group as compared to controls. In contrast membrane cholesterol concentration was significantly increased (p<0.001) in DM as compared controls. Aggregations of platelets were also significantly increased (p<0.001) in DM as compared to controls. The oxidative stress and antioxidant status was of test group 90 days as compared to control, concentration A Study Of Oxidative Stress And Antioxidant Status In The Pathogenesis Of Diabetic Complications 8
of serum MDA was significantly increased (p<0.001) in that group which on only antidiabetic treatment. The significant reduction(p<0.001) was observed in all type antioxidant supplementation but more reduction was by vitamin vitamin( E+C) and (A-Z) tablets along with antidiabetic drugs. Vitamin E exerts it s antioxidant property by preventing chain propagation. Vitamin C acts as water soluble antioxidant by inhibiting initiation of lipid peroxidation and also required for regeneration of vitamin E. The net result is reduction in lipid peroxidation resulting in reduced serum MDA levels. The concentration of serum nitric oxide showed significant reduction (p<0.001) after 90 days as compared to baseline in all treatment groups. Nitric oxide reacts with superoxide anion and generates peroxynitrite, further this peroxynitrite get decompose to highly toxic hydroxyl radical. This could probably attribute to increased oxidative stress which may further cause complications of type 2 DM. A Study Of Oxidative Stress And Antioxidant Status In The Pathogenesis Of Diabetic Complications 9
The activity of serum SOD was significantly decreased (p<0.001) after 90 days as compared to baseline activity in group I and VI but it was significantly improved (p<0.001) by antioxidant supplementation. In diabetic patients, the autoxidation of glucose results in the formation of hydrogen peroxide which inactivates SOD and this accumulated hydrogen peroxide may be one of the explanations for decreased activity of SOD in type 2 diabetic patients. No effects of antioxidants were seen on erythrocyte GSH concentration. Reduced glutathione functions as a direct free radical scavenger as a cosubstrate for glutathione peroxide (GPx) it was improved by antidiabetic agents but get more benefit by antioxidant supplementation. FRAP was significantly increased (p<0.001) after 90 days treatment of antioxidants along with antihyperglycemic agents. Decreased activity of antioxidant enzymes and depletion of total antioxidant capacity may increase the susceptibility of diabetic patients to oxidative injury. A Study Of Oxidative Stress And Antioxidant Status In The Pathogenesis Of Diabetic Complications 10
Rise in mean difference of membrane protein concentration was non significant in both type of diabetes. Membrane cholesterol was significantly decreased (p<0.001) and membrane phospholipids were significantly increased (p<0.001) after 90 days of antioxidant supplementation in all groups of DM except I and VI. Alteration in the membrane cholesterolphospholipids ratio results in morphologically abnormal erythrocytes with decreased life span. Platelet aggregation significantly reduced (p<0.001) in both type of DM after 90 days of treatment when compared with baseline levels. Vit E modulates prostaglandin synthesis and hence platelet adhesiveness and thrombosis. In present study different antioxidants were evaluated statistically by ANOVA method. It was observed that supplementation of vitamin (E+C) and tablets of (A-Z) are more efficient than individual supplementation of vitamin C or E in both types of diabetes mellitus. The oxidative stress was drastically reduced and antioxidants status was improved by supplementation (E+C) and A-Z along with antidiabetic treatment. As A Study Of Oxidative Stress And Antioxidant Status In The Pathogenesis Of Diabetic Complications 11
oxidative stress was decreased, membrane cholesterol and phospholipids ratio was maintained and deformability of membrane was minimized. Also platelet aggregation was reduced because of reduced oxidative stress. All these achieved only by vitamin (E+C) or (A-Z) tablets along with regular treatment of diabetes and helps in minimizing late complications of diabetes. There was no change in membrane protein concentration after 90 days but antioxidants may prevent future loss of protein by nonenzymatic glycosylation. Effect of antioxidants supplementation was not seen on nitric oxide it can improve by antidiabetic drugs especially insulin. There is controversy regarding vitamin E supplementation. Some study shows detrimental instead of beneficial effect because of proxidant effect of vitamin E. But proxidant action is depending on doses of vitamin E, duration of supplementation and also duration of diabetes. But if there is co-supplementation it may show beneficial effect. The antioxidants are working synergistically, vitamin C is required for regeneration vitamin E, trace elements A Study Of Oxidative Stress And Antioxidant Status In The Pathogenesis Of Diabetic Complications 12
required for activation of antioxidant enzymes. If one of them is defected whole get disturbed. In diabetes deficiency of trace elements and vitamin are observed which is responsible for increased oxidative stress which further causes late complications. High doses of single antioxidant supplements may perturb the antioxidant-prooxidant balance of cell systems. Therefore mixtures of antioxidants therapies possibly in combination with trace elements and vitamins to diminish oxidative stress and improve glycemic control may provide better therapeutic options for diabetics. Deficiency of trace elements and antioxidant vitamins and increased oxidative stress was observed in diabetes mellitus which is responsible late complications. Hence the present study suggests supplementation of antioxidants individually or for better benefits conjointly vitamin(e+c) or multiantioxidants (A-Z) tablets along with antidiabetic drugs to minimize late micro as well as macrovasular complications of diabetes mellitus. A Study Of Oxidative Stress And Antioxidant Status In The Pathogenesis Of Diabetic Complications 13