Subjects for the study:
Four groups of human male volunteers, each consisting of twenty members aged between 35-60, residing in Anantapur town, Andhra Pradesh, India, were the subjects for the present study. The subjects were selected for the study based on information through a specially designed questionnaire. These volunteers were categorized into four groups viz., controls (who were non-alcoholics and non-diabetic), alcoholics (who consumed 70-120 g alcoholic beverage/day for the past 7-10 years), Diabetics (NIDDM patients who are on metformin, glycomate medication as prescribed by the physician), Alcoholic diabetics (NIDDM patients who consume 70-120 g alcoholic beverage/day for the past 7-10 years and also are on metformin, glycomate medication as prescribed by the physician). The beverages consumed by the chosen alcoholics include 80 proof hard liquors such as whisky, rum, Gin and brandy of various brands containing up to 40% alcohol. All the volunteers were well explained about the experimentation, a written consent was obtained and were asked to continue with their normal regular local diet throughout the period of study. Enough care was taken to prevent the effects of diet, water or sampling time, and daily activities of the subjects. The chosen subjects were not on medication for any other known chronic diseases or illnesses and were free from use of any other drugs and anaesthetics. The study was approved by our institutional ethical committee. Base line characteristics of the selected subjects are presented in Table-1.
Blood collection and Experimentation:
Venous blood samples were collected from volunteers into heparin test tubes after overnight fasting and were used for analysis immediately. Biochemical studies using plasma, erythrocyte lysate and erythrocyte membrane were carried out.
Plasma glucose and HbA1C:
Plasma glucose was estimated by GOD-POD enzymatic method using Monozyme diagnostic kit . Plasma HbA1C was estimated by using ERBA diagnostic kit.
Determination of plasma and erythrocyte total nitrate and nitrite levels
Nitrite and nitrate levels have been determined in plasma and erythrocyte as mentioned above . Plasma and red cell lysate samples were treated with 30% zinc sulphate to deproteinize samples followed by centrifugation at 4000 g for 5 minutes. Nitrite is measured using Griess reagent from 1.0 ml plasma aliquots and erythrocyte lysate (1 percent sulfanilamide, 2.5 percent phosphoric acid, and 0.1 percent 1-naphthylethylene diamine). One millilitre of supernatant aliquots was spun with cadmium granules separately for 90 minutes for nitrite conversion, and Griess was then added to the nitrate. The amounts of nitrite have been calculated using a typical sodium nitrite curve.
Estimation of plasma Enzymes and Lipid Profile
Activities of plasma aspartate transaminase (AST; EC 188.8.131.52), alanine transaminase (ALT; EC 184.108.40.206), alkaline phosphatase (ALP; EC 220.127.116.11) and gamma glutamyl transferase (gGT; EC 18.104.22.168) were measured. Total cholesterol (TC), phospholipids, HDL-C and triglycerides (TG) were determined using commercially available kits (Erba Mannheim, Germany) as described earlier . LDL-C and VLDL-C were calculated using the formula described previously .
Erythrocyte membrane preparation
Erythrocyte membranes were prepared as described previously . The red blood cells were lysed with 5mM phosphate buffer (PH 8.0) and spun at 15000 x g for 30 minutes after being rinsed with phosphate buffered saline (PH 7.2). For analysis, we selected membrane ghosts that were devoid of haemoglobin after another wash with 5 mM phosphate buffer.
Determination of erythrocyte membrane TBARS
The produced malondialdehyde was used to determine the amount of lipid peroxidation (LPO) by treating the samples with 2ml of thiobarbituric acid reagent, as reported before [17, 18].
Erythrocyte membrane total cholesterol, phospholipids and C/P ratio
Erythrocyte membrane lipids were extracted as described previously . Methanol (5 ml) was added to the lysed membrane preparations, followed by the addition of chloroform (10 ml). The filtrate was removed from the mixture after 30 minutes, and the residue was utilised for another extraction. The pooled filtrates were used for the estimation of cholesterol content  and phospholipids .
Data were subjected to statistical analyses, values are means S.D. of 20 subjects in each group. Two-sided paired Student’s t-test was performed for finding significant difference between the groups. A p < 0.05 was considered statistically significant.