IMPORTANCE OF THE RESEARCH PROPOSAL. separately on the effect of vitamin E, C and O.S in the diabetes mellitus and

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1 IMPORTANCE OF THE RESEARCH PROPOSAL Diabetes mellitus and hypertension are the most common occurring noncommunicable disorders in every country (developed or developing). Review of literature indicates that so far many studies have been done, separately on the effect of vitamin E, C and O.S in the diabetes mellitus and hypertensive patients and shows positive/beneficial effects. But none of the studies indicate the combined effect of antioxidants and Tulsi therapy on serum lipids and serum glucose levels in diabetic and hypertensive patients. Therefore, this study was undertaken to investigate the role of vitamin E, C and Tulsi supplements on the levels of lipids and blood glucose in diabetic and hypertensive patients. All these three things have no adverse effects on our body. So this study may be very helpful in treating diabetes mellitus and hypertension. 33

2 MATERIAL AND METHODS The present study was conducted in the department of Physiology, Subharti Medical College, Meerut. Approval of the project plan was taken from the research and ethical committee of the Institute vide letter no. SMC/EC/2010/88; dated: 27/11/2010. Study was also approved and registered by Clinical Trials RegistryIndia, NIMS (ICMR), vide registration no. CTRI/2012/06/002728; dated:14/06/2012 and reference no. REF/2012/05/ MATERIAL The present study was conducted on 320 patients aged 25 years and above and suffering from type 2 diabetes and hypertension visiting the OPD of department of Medicine, Subharti Medical College & Hospital, Meerut and also visiting the clinic & research centre (diabetology and endocrinology) at SB6, Shastri Nagar, Ghaziabad. Patients were selected by simple random sampling method. American Diabetes Association (ADA) criteria were used for diagnosis of diabetes (81). Before conducting the study, informed written consent were taken from each subjects. (Annexure1) 34

3 INCLUSION CRITERIA Patients whose fasting blood sugar (FBS), triglycerides (TG) and cholesterol levels were in the ranges that did not need to change their treatment plan, were included in this study. EXCLUSION CRITERIA Patients with complications like nephropathy, retinopathy, cardiovascular and other endocrinal disorders and patients already on antioxidant supplementation or on antiresorptive therapy were excluded from the study. METHOD After taking full history and relevant physical & general examination, the study group (320 patients) was further sub divided into 4 groups, each consisting of 80 patients. 35

4 S. No. GROUPS NUMBER 1 Control 80 2 Patient with Vitamin E & C therapy 80 3 Patient with Tulsi therapy 80 4 Patient with Vitamin E, C and Tulsi therapy 80 Group 1 The control group were not given any kind of supplementation. Group 2 The experimental group, patients were given supplements in tablet form viz: vitamin C (CELIN 500 mg of Glaxosmithkline Drug company), vitamin E (EVINAL 400mg or 200 mg tab of Alembic Limited Drug company). Vitamin E&C were given to the patient, one tablet each per day (01 O.D.) just after the breakfast. Group 3 i.e. the experimental group were given Tulsi (as Tulasi tab of Himalaya Herbal Healthcare Pharmaceutical company). Tulsi tablets were given one tablet twice a day before meals (01 B.D.). Group 4 i.e. the experimental group were given supplements of Vitamin C & E and Tulsi in tablet form as given to group 2 and group 3 collectively. All the above supplements were given for 45 days along with the prescribed treatment by physician. 36

5 Tools and Techniques : Various tools and techniques were employed to elicit information on the general profile, clinical parameters and bio chemical parameters. A detailed information (General Profile, Clinical parameters) of each subject were recorded. 1. Questionnaires to collect general information and clinical parameters 2 Anthropometric measurements 3. Bio Chemical Analysis 1 Questionnaires (Annexure II) For collecting necessary information, questionnaires were developed. These were designed to collect demographic information on age, sex, family profile, educational qualification, occupation, marital status working hours, type of diet followed, consumption of alcohol and habit of smoking of the patient. And also the information regarding, any family history of coronary heart disease (CHD), diabetes and hypertension. Questions were designed to gather information relating to the number of years since they were detected diabetic (hyperglycemic) and hypertensive (hypercholesterolemics) and the type of treatment followed by them. 37

6 Pretesting of the questionnaire The prepared questionnaires were pretested on ten type II diabetic and hypertensive patients from the same OPD and according to the responses obtained necessary modifications were made to elicit the desired information. These subjects were not included in the study. 2. Anthropometric Measurements Nutritional anthropometry is defined as measurement of the variations of the physical dimensions and the gross composition of the human body at different age levels and degree of nutrition (82). In the present study, the following anthropometric measurements were taken to assess the nutritional status of the subject: Weight A bathroom scale was used to record the weight of all the subjects. The weighing scale had a least count of 0.5 Kg. The accuracy of the balance was checked by standardizing it with known weight from time to time. It was made sure that the patients are light at the time of measurement. Height The height scale was made on the wall. The patient was made to stand barefoot on the floor, with feet together, knees straight and heels, buttocks 38

7 and shoulder blade in contact with the vertical surface of the wall. To measure height, a ruler was placed over the head crushing the hair and making contact with the top of the head. The measurement was taken at the examiner s eye level. The above mentioned measurements were used to calculate the Body Mass Index (BMI), which accounts for differences in body composition by defining the level of adiposity according to the relationship of weight and height. It is expressed as: BMI = Weight (in Kilograms) Height² (in meters) This index is regarded as a sensitive indicator of obesity. The following classification has been used to grade the subjects: Table Grade BMI (Kg/m 2 ) underweight Normal Obese Grade I Grade II Grade III < Source: Garrow, 1994(83). 39

8 Blood pressure Blood Pressure measurement of all subjects, were recorded using a mercury sphygmomanometer both before and after the period of supplementation. The following criteria (JNC7) has been used for the classification of patients according to the blood pressure measured in them respectively (84). CLASSIFICATION OF BLOOD PRESSURE (for adults >18 yrs old) JNC6 Category SBP/DBP JNC7 Category Optimal <120/80 Normal Normal /8084 PreHypertension Borderline /8589 Hypertension >140/90 Hypertension Stage /9099 Stage1 Hypertension Stage / Stage3 >180/110 Stage2 Hypertension Pulse rate (beats per minute) was also counted by placing the fingers over the radial artery at the wrist for 60 seconds. 40

9 III. Biochemical Analysis All the subjects were made to get their serum lipid profile & blood glucose done before and after 45 days of the study. Blood samples (5 ml) were collected by vein puncture after an overnight fasting in two different vials.grey cap Fluoride vial for sugar estimation and Red cap Plain vial for lipid profile estimation. After centrifugation of vials for 10 minutes at rpm speed serum was separated and stored at a temperature of 28ºC. It was brought to room temperature just prior to lipid estimation. With the collected blood, estimation of following parameters were done: (a) Total serum cholesterol (TC) (b) Serum high density cholesterol (HDL) (c ) Serum low density lipoprotein Cholesterol (LDL) (d) Serum very low density lipoprotein Cholesterol (VLDL) (e) Serum triglycerides (TG) (f) LDL/HDL ratio (g) TC/HDL ratio (h) Blood Sugar fasting and postprandial. 41

10 SERUM LIPID ESTIMATION The serum lipid profile was analysed using fully enzymatic diagnostic kit (Excel Diagnostics). Serum Cholesterol (85) Reagents: Cholesterol esterase, cholesterol oxidase, peroxidase, 4 aminoantipyrine, phenol. Principle: The Cholesterol esters are hydrolysed to free cholesterol and fatty acids by Cholesterol esterase (CE) enzyme and free Cholesterol to cholesten 4en3 one with the simultaneous production of hydrogen peroxide (H 2 O 2 ) in the presence of Cholesterol Oxidase (CO) enzyme. The H 2 O 2 reacts with 4amino antipyrine and phenol in the presence of peroxidase enzyme to yield a red coloured complex (Quinoneimine) which is read at 505 nm (500540nm). a. Cholesterol esters +H 2 O CE Cholesterol + fatty acids b. Cholesterol + O 2 CO Cholesterol 4en3 one + H 2 O 2 c. 2H 2 O 2 + 4Aminoantipyrine + Phenol Peroxidase Quinone imine dye + 4H 2 O 42

11 Procedure: Reaction Parameters Reaction Type : End point with standard Wavelength : 500 nm (Green Filter) Flowell temperature : 30 0 C Incubation : 5 minutes at 37 0 C Sample Volume : 0.01 ml Reagent volume : ml The working reagent was made by adding the given reagents in specified amounts. To this standard solution or sample was added. This was mixed well and incubated for 5 minutes at 37 0 C. Absorbance of test and standard was measured at 500 nm (Green filter) against reagent blank. The color of the final reaction mixture was stable for 1 hour. 43

12 Protocol : Pipette into Test Tubes Blank Std. Test Working reagent (ml) Deionized water (ml) 0.01 Standard (ml) 0.01 Sample (ml) 0.01 Mix and read the optical density (OD) at 500 nm against blank after 5 minute incubation (37 0 C). The final color is stable for at least 1 hour. Calculation: Cholesterol Conc.(mg%) = Serum HDL Cholesterol (86) Reagents: Phosphotungstic acid, magnesium chloride. Principle: HDL is separated from other protein fractions by treating serum with phosphotungstic acid and mangnesium chloride (mgcl 2 ). HDL remains in solution while all other lipoprotein fractions are precipitated. 44

13 Centrifugation separates HDL fraction as a clear supernatant; cholesterol content of which is estimated by enzymatic method. Serum + Phosphotungstic acid Supernatant + Precipitate Procedure Separation of HDL Cholesterol Pipette Into Centrifuge Tube Sample (ml) HDL Precipitating reagent (ml) Test The reagent and the sample were mixed well and stand at R. T for 10 minutes. Centrifuge at 3000 rpm for 10 minutes. The clear supernatant was separated immediately and cholesterol content was determined as given below: Protocol Pipette into Test Tubes Blank Std. Test Working reagent (ml) Deionized water (ml) Standard (ml) 0.01 Sample (ml)

14 The contents were mixed well and incubate for 5 minutes at 37 0 C and read the optical Density at 500 nm against blank. Calculation: HDL Conc (mg/dl) =. Serum Triglycerides (87) Reagents: P Cholorphenol, Lipoprotein Lipase (LPL), Glycerokinase (G.K), Glycerole 3Oxidase (GPO), Peroxidase (POD), 4Aminophenazone (4AP) and ATP. Principle of the Method Sample triglycerides incubated with liporoteinlipase enzyme (LPL), liberate glycerol and free fatty acids. Glycerol is converted to glycerol3phosphate (G3P) and adenosine5diphosphate (ADP) in the presence of glycerokinase enzyme and ATP. Glycerol3Phosphate (G3P) is then converted by glycerol phosphate dehydrogenase enzyme (GPD) to dihydroxyacetone phosphate (DAP). Hydrogen peroxide (H 2 O 2 ) reacts with 4aminophenazone (4AP) and Pchlorophenol in the presence of peroxidase (POD) enzyme to give a red colored dye Quinone. 46

15 Tnglycerides + H 2 O LPL Glycerol + free fatty acids Glycerol + ATP Glycerokinase G3P+ADP G3P+O 2 GPO DAP + H 2 O 2 H 2 O 2 + 4AP + PChlorophenol peroxidase Quinone + H 2 O The intensity of the color formed is proportional to the triglycerides concentration in the sample. Procedure: Reaction type : End Point Wavelength : 505 nm (490550) Flowcell temperature : 37 0 C Incubation : 15 minutes at 37 0 C Sample Volume : 0.01 ml. Reagent volume : ml Pipette into Test Tubes Blank Std. Test Working reagent (ml) Deionized water (ml) 0.01 Standard (ml) 0.01 Sample (ml)

16 The contents were mixed and absorbance of test and standard was measured against the reagent blank at 505 nm (490550). The color is stable for at least 30 minutes. Test Results: Triglycerides (mg/dl) = Calculations VLDL = 1/5 TG LDL = TC (HDL + VLDL ) [LDL in mg% = Total cholesterol (HDL + 1/5 TG)] Glucose Estimation in human Serum/Plasma (88). Reagents Phosphate Buffer, Glucose Oxidase, peroxidase, 4Aminoantipyrine (4 AAP), Phenol Preservative, Dextrose Preservative. Principle Glucose Oxidase (GOD) oxidizes Glucose to Gluconic acid and Hydrogen Peroxide. In the presence of enzyme peroxidase, released Hydrogen Peroxide is coupled with Phenol and 4 Aminoantipyrine (4AAP) to coloured 48

17 Quinoneimine dye. Absorbance of coloured dye is measured at 505 nm and is directly proportional to glucose concentration in the Sample. Glucose + O 2 + H 2 O Glucose Oxidase Gluconic Acid + H 2 O 2 H 2 O 2 + Phenol + 4AAP Peroxidase Quinoneimine dye + H 2 O Collection of Sample For fasting Glucose estimation: Patient should fast for 1012 hours. For Post Prandial (PP) Sample: Patient should report to the laboratory 2 hours after meal. Procedure: Reaction Parameters Reaction type : End Point Wavelength : 505 nm (490550) Flowcell temperature : 37 0 C Incubation : 10 minutes at 37 0 C Sample Volume : 0.01 ml Reagent volume : ml 49

18 Pipette into Test Tubes Blank Std. Test Working reagent (ml) Deionized water (ml) 0.01 Standard (ml) 0.01 Sample (ml) 0.01 Mix well. Incubate at 37 0 C for 10 minutes. Blank the analyzer with Reagent Blank. Measure absorbance of Standard followed by the Test. Calculate results as per given calculation formula. Calculation : Serum/Plasma Glucose (mg/dl) = 50

19 LIPID PROFILE : Total cholesterol (TC), high density lipoprotein cholesterol (HDLC) and triglycerides (TG) were estimated by enzymatic methods, employing kits using in Biochemistry lab of Subharti Medical College and Hospital, Meerut. It is VITROS 250 fully automatic analyzer; made by Johnson and Johnson. It works on the principle of Reflectance Spectrophotometry, Dry chemistry enzymatic. VLDL and LDL cholesterol were calculated by the standard formula as described. LDL Cholesterol will be estimated by using Friedewald WT (89) formula as follows: VLDL = 1/5 TG LDL = TC (HDL + VLDL ) [LDL in mg% = Total cholesterol (HDL + 1/5 TG)] DATA COLLECTION The data was gathered in two phases i.e. presupplementation and postsupplementation. In presupplementation phase, after a preliminary discussion regarding the study and establishing a rapport with the patients, their informed consent (Annexure I) was taken for including them in the study. Information on the general and clinical profile were gathered using the questionnaire 51

20 (AnnexureII) and their anthropometric measurements viz : height, weight, blood pressure and pulse rate were also recorded. After gathering all the necessary data, supplementation were recommended to each patient in experimental group in the presence of physician along with the prescribed treatment. Dose and method of consuming the Supplementation of antioxidants ( Vitamin C and E) and Tulsi were explained briefly to the patients. The patients were asked to revisit the clinic/opd after 45 days/ six weeks. Telephonic contacts during the supplementation phase were made to ensure the regular intake of supplementation in prescribed format. After 45 days all the patients were asked to revisit the OPD/Clinic and repeat their biochemical (lipid and sugar) estimation and some cardiovascular parameters (BP and pulse rate). ANALYSIS OF DATA Statistical analysis was done using Graph Pad Instat 3.10,32 bit for windows created in July 10, Publisher is Graph Pad Software Inc. Mean and standard deviations (± S.D.) of all the observations were calculated. 52

21 Comparison were done between the pre supplementation and post supplementation values of all four groups applying student t test (paired) for intra group comparisons. ANOVA (analysis of variance) for intergroup and student t test (unpaired) for inter group comparisons were applied. P 0.05 was considered as statistically significant. 53