MATERIAL AND METHODS

MATERIAL AND METHODS 3.1 SOURCE OF DATA The present data which is case controlled study was done during the period from 1 st Oct 2010 to 1 st Jan 2012. All the participants were recruited from outpatient departments of the Hospital of National Institute of Unani medicine, Magadi Main Road, Kottege Palya. Bangalore. Located near industrial area of Bangalore. The study was done on a total of 302 subjects of which 198 were diabetic and 104 were controls, written consent was obtained from each subjects after explaining the reasons, objective and methodology of the research study. The copy of the consent form attached as annexure 1. 3.2 SPECIMEN COLLECTION AND PRESERVATION The participants of the study were required to fast for at least 10 hours before the sample collection; 5ml of the blood was collected in plain dry vacutainer tube. The patients were asked to have breakfast and to come again after 1-1/2 hours for post prandial sample collection. The vacutainer tube of the fasting sample was made to stand for 25-30 minutes for separation of serum from the cells. Immediately after, the tubes were centrifuged and serum was separated for the analysis. From the fasting serum sample, glucose estimation, lipid profile, liver function tests, kidney function tests, Gamma Glutamyltransferase, hs-crp and Lipoprotein (a) was analyzed. Again the samples were collected for post prandial blood sugar for which only 2ml of the blood was drawn in vacutainer tube and serum separated and analysis was carried out for the post prandial blood sugar levels, 32

3.3 Inclusion criteria A comprehensive literature survey was done from various research journals to design the study format and proforma. The copy of which is attached in Annexure 2. In building up of the proforma utmost care is taken to make it broad based so that most of the desirable aspects could be included or incorporated in the study. Information of the patient about sex, diagnosis and disease history, lifestyle of the patients and family history was collected from each subject. A criterion for inclusion of the subjects was taken as fasting blood sugar more than 110 mg/ at more than two occasions. All the diagnosis was done by physicians of the hospital. Normal subjects were selected from healthy random sampling from different age groups attending the outpatient departments of the National Institute of Unani Medicine and the staffs have also participated in the study. a. Clinically investigated and diagnosed patients with Type-2 diabetes mellitus. b. Patients Investigated and Diagnosed for Hyper / Dyslipidemea. c. Patients of either sex between ages 25 to 60 years. 3.4 Exclusion Criteria a. Patients suffering from other serious illness / diseases. b. Patients with Hypothyroidism c. Patients with Pregnancy. d. Patients on Hemodialysis. e. Patients with complications of Diabetes. f. Patients with Diabetic Nephropathy / Neuropathy. g. Patients who are Alcoholics. 3.5 Laboratory Investigations The diagnostic tests of the patients was carried out at the Biochemistry Laboratory. Hospital Block, National Institute of Unani 33

Medicine. (Govt of India.), Magadi Main Road. KottegePalya. Bangalore 560091 STUDY DESIGN Subjective Parameters 1. Type-2 diabetes Mellitus 2. Hyperlipidimea / Dyslipidimea. 3. Obesity 4. History of the patient Objective Parameters 1. Blood sugar (FBS, PPBS). 2. Serum Cholesterol. 3. Serum Triglyceride. 4. High Density Lipoprotein (HDL). 5. Low Density Lipoprotein (LDL). 6. LDL: HDL ratio. 7. Lipoprotein (a). 8. Gamma Glutamyltransferase (GGT). 9. Hs-C Reactive Protein (hs-crp). 34

3.5.1 Estimation of Blood Glucose The estimation of fasting plasma glucose (FBG) level was done by GOD-POD method of Trinder (1969). (Aspen Diagnostics) 3.5.5.1.1 Principle Glucose determination after enzymatic oxidation by glucose oxidase, The quinoneimine is a colorimetric indicator, which is formed from 4 aminotipyrine and phenol by hydrogen peroxide by the catalytic action of peroxidase. β - D glucose +O 2 GOD Gluconic acid +H 2 O 2 H 2 O 2 + 4-aminoantipyrine + phenol POD Red Dye + 4-H 2 O 3.5.5.1.2 Reagents composition Reagent: Phosphate buffer PH 7.5 Phenol 5mmol/l 4-Aminoantipyrine 0.5mmol/l Glucose oxidase (GOD) 10kU/l Peroxidase (POD) 1 ku/l Standard 100mg/dl 3.5.5.1.3 System General parameters. Method End point Slope of reaction Increasing Wavelength 505 nm (49-530) 35

Flow cell temp 37º C Sample volume 10 µl Reagent volume 1000 µl Incubation 10 minutes at 37º C Standard Conc. 100mg/dl Unit mg/dl Linearity upto 500mg/dl Zero setting Reagent Blank. 3.5.1.4. Test Procedure- Dispense into tubes Pipette into Tubes Blank Standard Test Standard - 10 µl - Sample - - 10 µl Reagent 1000 µl 1000 µl 1000 µl Incubate for 10 min at 37º C. Mix and read absorbance at 505nm against Blank. 3.5.1.5 Reference Range: Fasting 70-110mg/dl Post Prandial Up to 140mg/dl 36

3.5.2. ESTIMATION OF CHOLESTEROL The estimation of serum cholesterol was done by end point method of Allainet al. (1974) (Siemens Kit). 3.5.2.1 Principle Cholesterol Esters +H 2 O Chol Esterase Cholesterol +Fatty Acids Cholesterol +O 2 Chol Oxidase Cholest-4-en-3-one + H 2 O 2 2H 2 O 2 +Phenol+4-Aminoantipyrine Peroxidase Red quinine +4-H 2 O 3.5.2.2 Reagents composition Reagent 1 (Enzyme/Chromogen) Cholesterol Esterase 30IU/L Cholesterol Oxidase 250IU/L Peroxidase 1000IU/L 4-Aminoantipyrine 0.5 mmol/l Reagent 1A Buffer: Pipes buffer, ph6.95 50 mmol/l Phenol 24 mmol/l Sodium Cholate 0.5mmol/L Standard (Cholesterol 200mg/dl) 37

3.5.2.3 General parameters. Method End point Slope of reaction Increasing Wavelength 500 nm (490-550) Flow cell temp 30º C Sample volume 10 µl Reagent volume 1000 µl Incubation 5 minutes at 37º C Standard Conc. 200mg/dl Unit mg/dl Linearity up to 500mg/dl Zero setting Reagent Blank. 3.5.2.4 Test Procedure Dispense into tubes Pipette into Tubes Blank Standard Test Reconstituted 1000 µl 1000 µl 1000 µl reagent Standard - 10 µl - Sample - - 10 µl Incubate for 5 min at 37º C. Mix and read absorbance at 500nm 3.5.2.5 Reference Range: Serum: Up to 200 mg/dl 38

3.5.3. Triglyceride The estimation of triglycerides was done by end point method of McGowan et al., (1983)(Siemens) 3.5.3.1 Principle Triglyceride +H 2 O Lipoprotein Lipase Glycerol + Fatty Acid Glycerol +ATP Glycerol Kinase, Mg2 + Glycerol-3-Phosphate +ADP Glycerol-3-Phosphate + O 2 GPO DihydroxyacetonePhophate +H 2 O 2 2H 2 O 2 + 4-Aminoantipyrine + ADPS Peroxidase Red quinone GPO = Glycerol-3-phosphate Oxidase ADPS = N-Ethyl-N-(3-sulfopropyl)-m-anidisin The intensity of the purple colored complex is measured. 3.5.3.2 Reagents Lipoprotein Lipase 1100U/L Glycerol Kinase 450U/L Glycerol-3-phosphate Oxidase 5000U/L Peroxidase 350U/L 4-Aminoantipyrine 0.7mmol/L ATP 0.3mmol/L Reagent buffer, ph 7.0 50 mmol/l ADPS 0.9mmol/L Magnesium salt 17.8 mmol/l Standard (Triglyceride 200mg/) 39

3.5.3.3 General system parameters. Method End point Slope of reaction Increasing Wavelength 546 nm (520-570) Flow cell temp 30º C Sample volume 10 µl Reagent volume 1000 µl Incubation 5 minutes at 37º C Standard Conc. 200mg/dl Unit mg/dl Linearity up to 1000mg/dl Zero setting Reagent Blank. 3.5.3.4 Test Procedure Dispense into tubes Pipette into Tubes Blank Standard Test Reconstituted reagent 1000 µl 1000 µl 1000 µl Standard - 10 µl - Sample - - 10 µl Incubate for 5 min at 37º C. Mix and read absorbance at 546(520-570nm) 3.5.3.5 ReferenceRange: up to 150 mg/dl. 40

3.5.4. ESTIMATION OF HDL-CHOLESTEROL The estimation of serum HDL was done by precipitation method of Lopez- Virellaet.al. (1977) (Siemens Kit) 3.5.4.1 Principle Chylomicrons VL and L fractions in serum or plasma are separated from H by precipitating with Phosphotungstic Acid and Magnesium Chloride. After centrifugation, the Cholesterol in the H fraction, which remains in the supernatant is assayed with enzymatic cholesterol method, using cholesterol Esterase, Cholesterol Oxidase, Peroxidase and the chromogen 4- Aminoantipyrine/Phenol. Phosphotungstate Serum/ Plasma HDL Fraction+ (LDL+VLDL+Chylomicrons) Mg 2+ (Supernatant) (precipitate) 3.5.4.2 Reagents Reagent 1 (Enzyme/Chromogen) : Cholesterol Esterase 30IU/L Cholesterol Oxidase 250IU/L Peroxidase 1000IU/L 4-Aminoantipyrine 0.5mmol/L Reagent 1A Buffer: Pipes buffer, ph6.95 50mmol/L Phenol 24mmol/L Sodium Cholate 0.5mmol/L 41

3.5.4.3 Precipitation step. Dispense into Centrifuge tubes Test Sample 200µl Precipitating Reagent 2 200µl Mix well, centrifuge separate the supernatant immediately and test for HDL- Cholesterol. 3.5.4.4 General parameters. Method End point Slope of reaction Increasing Wavelength 500 nm (492-550) Flow cell temp 30º C Sample volume 20 µl Reagent volume 1000 µl Incubation 5 minutes at 37º C Standard Conc. 200mg/dl Unit mg/dl Zero setting Reagent Blank. 42

3.5.4.5 Test Procedure Dispense into tubes Blank Standard Test Reconstituted Reagent 1mL 1mL 1mL Standard - 20 µl - Supernatant - - 20 µl Incubate for 5 min at 37º C Mix and read absorbance at 500nm 3.5.4.6 Reference Range: 30-70mg/dl 3.5.4.7 CALCULATION OF LDL-CHOLESTEROL L CHOLESTEROL mg/ = TOTAL CHOL-Triglyceride H CHOL 5 3.5.4.8 Reference Range: Up to 100mg/dl 3.5.5. ESTIMATION OF SGOT (AST Aspartate Aminotransferase): The estimation of SGOT was done by IFCC methods of Bergmeyeret al., (a) (1986) (Siemens kit) 3.5.5.1 Principle L-Aspartate + α-ketoglutarategotoxaloacetate + L Glutamate Oxaloacetate + NADH + H + MDH L-Malate + NAD + AST = Aspartate Aminotransferase MDH = Malate Dehydrogenase 43

3.5.5.2 Reagents Reagent 1 (Enzyme) MDH 800U/L LDH 4000U/L NADH > 0.2.0mmol/L Α-Ketoglutarate> 13 mmol/l Reagent 1A (Buffer) : Tris buffer, ph 7.8 L-Aspartate 264mmol/L 3.5.5.3 General system parameters. Method Kinetic Slope of reaction Decreasing Wavelength 340 nm Flow cell temp 37º C Delay Time 60 secs No of Readings 4 Interval 60 secs Sample volume 100 µl Reagent volume 1000 µl Path length 1cm Factor 1746 Unit IU/L Zero setting Distilled Water. 44

Linearity Up to 300IU/L 3.5.5.4Test Procedure Dispense into tubes TEST Reconstituted Reagent 1ml Sample 100 µl Mix and read immediately at 340nm 5.5.5.5 Reference Range: Up to 50 IU/L 3.5.6. ESTIMATION OF SGPT (ALT- Alanine Aminotransferase ): The estimation of SGPT was determined by IFCC method of Berg Meyeret al., (b) (1986). (Siemens kit) 3.5.6.1 Principle L-Alanine + α-ketoglutarategpt Pyruvate + L Glutamate Pyruvate + NADH + H + LDH L-Lactate + NAD+ ALT= Alanine Aminotransferase LDH= Lactate dehydrogenase 3.5.6.2 Reagents Reagent 1 (Enzyme) LDH 1200U/L NADH > 0.2.0mmol/L Α-Ketoglutarate> 16 mmol/l 45

Reagent 1A (Buffer) : Tris buffer, ph 7.5 110mmol/L L-Alanine 550mmol/L 3.5.6.3 General system parameters. Method Kinetic Slope of reaction Decreasing Wavelength 340 nm Flow cell temp 37º C Delay Time 60 sec. No of Readings 4 Interval 60 sec. Sample volume 100 µl Reagent volume 1000 µl Path length 1cm Factor 1746 Unit IU/L Zero setting Distilled Water. Linearity Up to 250IU/L 46

3.5.6.4 Test Procedure Dispense into tubes TEST Reconstituted Reagent 1ml Sample 100 µl Mix and read immediately at 340nm 5.5.6.5 Reference Range: Up to 40 IU/L 3.5.7. ESTIMATION OF ALKALINE PHOSPHATASE The estimation of ALKP was determined by PNPP methods of Z. Klin. Chem. U. Klin). 3.5.7.1 Principle Alkaline Phosphatase hydrolyses p-nitrophenyl phosphate (PNPP) into p- Nitrophenol and Phosphate. At the alkaline ph of the buffered medium.p- Nitrophenol is yellow. The color developed by hydrolysis is measured at 405 nm and is proportional to alkaline phosphatase activity. p-nitrophenyl phosphate + H 2 O ALKP p- Nitrophenol + Phosphate 3.5.7.2 Reagents Reagent 1 (Substrate): p-nitrophenol Phosphate 10 mol/l Reagent 1A(Buffer) : Diethanolamine 1 mol/l Magnesium Chloride 0.5mmol/L 47

3.5.7.3 General system parameters Method Kinetic Slope of reaction Increasing Wavelength 405 nm Flow cell temp 25º C Delay Time 60 sec. No of Readings 4 Interval 30 sec. Sample volume 100 µl Reagent volume 1000 µl Path length 1cm Factor 1826 Unit IU/L Zero setting Distilled Water. Linearity Up to 700 IU/L 3.5.7.4 Test Procedure Dispense into tubes TEST Reconstituted Reagent 1ml Sample 30 µl Mix and read immediately at 405nm 3.5.7.5 Reference Range: 60-170 IU/L 48

3.5.8. ESTIMATION OF BLOOD UREA The estimation of blood urea was done by kinetic method of Fawcett and Scott,(1960)(Siemens). 3.5.8.1 Principle Urea + 2H 2 O Urease + -2 2NH 4 + CO 3 + α-ketoglutarate + NH 4 + NADH GDPH L- Glutamate + NAD + +H 2 O GLDH: Glutamate dehydrogenase 3.5.8.2 Reagents R 1 TRIS Buffer ph 7.8 120mmol/L α-ketoglutarate 7mmol/L ADP 0.6mmol/L Urease 6kU/L GLDH 1kU/L R2: NADH 0.25mmol/L Standard: 50mg/dl 3.5.8.3 General System parameters Method Fixed Time Slope of reaction Decreasing Wavelength 340 nm (49-530) Delay Time 30 sec. Delta Time 60 sec. 49

Flow cell temp 37º C Sample volume 10 µl Reagent volume 1000 µl Standard Conc 50mg/dl Unit mg/dl Linearity up to 300mg/dl Zero setting Reagent Blank 3.5.7.4 Test Procedure Dispense into tubes Pipette into Tubes Standard Test Standard 10 µl - Sample - 10 µl Reagent 1000 µl 1000 µl Mix well, Incubate for app 30 sec at 37º C. Then read absorbance A1. After exactly 60sec further read A2 at 340nm. 3.5.7.5 Reference Range: 10-50mg/dl 50

3.5.8. ESTIMATION OF CREATININE: The estimation of serum Creatinine was done by picrate method of Henry et.al. (1974). 3.5.8.1 Principle Creatinine in alkaline solution reacts with picrate to form a red-orange compound. Under the specific condition of the assay, the rate of development of the color is proportional to the concentration of creatinine in the sample when measured at 500nm Creatinine + Picrate 3.5.8.2 Reagents Alkaline medium Orange color Reagent 1 (Picrate): Picric Acid 34.9 mmol/l Sodium Hydroxide 45mmol/L Reagent 2 (Sodium Hydroxide) Sodium Hydroxide 0.26mol/L S. Creatinine 0.020g/L 3.5.8.3 General System parameters. Method Fixed Time Slope of reaction Increasing Wavelength 500 nm (49-530) Delay Time 30 sec. No of Readings 2 51

Flow cell temp 25º C, 30ºC, 37ºC. Sample volume 100 µl Reagent volume 1000 µl Pathlength 1cm Standard Conc. 2mg/dl Unit mg/dl Linearity Up to 10mg/dl Zero setting Distilled Water 3.5.7.4 Test Procedure Dispense into tubes TEST Working solution 1ml Standard 100 µl Mix and read immediately at 500nm. 3.5.7.5 Reference Range: Males 0.6-1.1mg/dl and Females 0.5-0.9mg/dl 3.5.8 ESTIMATION OF GAMMA-GLUTAMYLTRANSFERASE (GGT/GGTP). The estimation of GGT was done by Tris method of Bablock W. et al (1998). 52

3.5.8.1 Principle L-Ɣ- Glutamyl-3-carboxy-4-nitronolide +Glycylglucine L-Ɣ- GlutamyGlycylglucine + 5-amine-2-nitrobenzoate. 3.5.8.2 Reagents TRIS 100mmol/l Glucylglycine 100mmol/l L-Ɣ- Glutamyl-3-carboxy-4-nitronolide 4mmol/l REAGENTS STANDERDIZED TO IFCC Reaction type: KINETIC METHOD 3.5.8.3 ASSAY PROCEDURE. Dispense into tubes Pipette in Tubes Volumes Working Reagent 1000 µl Test 100 µl Mix and read at 405nm. Read absorbance initially after 60seconds and again after 1 & 2minutes. 3.5.8.4 Reference Range: 60-170 IU/L 53

3.5.9 LIPOPROTEIN (A) TEST The estimation of Lp(a) was done by Gaubalz JW, et al. 1983 3.5.9.1 PRINCIPLE Anti-humanLp(a) coated latex particles are agglutinated when mixed with samples containing Lp(a). This agglutination causes an absorbance change depending upon the Lp(a) contents of the patient samples, which can be interpolated in a calibration curve. 3.5.9.2 Reagents Lipoprotein (a) R1 Buffer Solution ph 8.3 Lipoprotein (a) R2 Lipoprotein latex Lipoprotein (a) calibrator 3.5.9.3 ASSAY PROCEDURE (AGAPPE DIAGNOSTICS) CALIBRATOR SAMPLE Calibrator Sample R1 Buffer R2 Latex 15 µl - 800µl 200 µl - 15 µl 800 µl 200 µl Mix and read the absorbance against blank after 10sec and after 4minutes of the latex addition 54

3.5.9.4 Reference Range: Desirable 20mg/dl Borderline risk: 20-30mg/dl High risk: 31-50mg/dl Very high risk: 50mg/dl 3.5.10 hs-crp TEST (Turbilatex) The estimation of hs-crp was done by Thomas A et al. 2000. 3.5.10.1 Principle: This is a quantitative turbidometric test for the measurement of low levels of C- Reactive Protein in human serum/plasma. 3.5.10.2 Reagents Diluent Ultra1 Tris buffer 20mmol/L,pH 8.2, Sodium azide 0.95 g/l Latex-ultra (R2). Latex particles coated with goat IgG anti- human CRP, ph 7.3 Sodium azide 0.95g/L U-CRP CAL Liquid Calibrator, 3.5.10.3 ASSAY PROCEDURE Pipette in Tubes Volumes Working Reagent 1000 µl Test/Sample 10 µl 55

Mix well and read the absorbance immediately (A1) and after 4 minutes (A2) of the sample addition. 3.5.10.4 Reference range: Up to 3mg/dl. 3.6 Blood pressure Blood pressure was measured by the nursing staffs of the department with accurate care and proper management of the patients in RELAXED STATE. SYSTOLIC above 130mm Hg DYSTOLIC above 90mm Hg Normal 120/80mm Hg. 3.7 Body Mass Index (BMI) = (Weight in Kg / Hieght in meters 2 ) Up to 25 perfect Normal Overweight 25.0-29.9 Obese 30 or more. 56

3.8 Statistical Analysis SPSS (Statistical Package for Social Science) Software (SPSS Version 17.0: SPSS Inc. Chicago, IL) was used for the statistical calculations of the study. The prevalence was carried out using Microsoft Excel program. Results for continuous variables were presented as mean ± SD. The difference between the studied groups was also tested for their significance using Students t-test. The difference between the studied variables was analyzed using Pearson s correlation or Spearman s0. rho correlation test. Differences and correlations were considered as significant at p< 0.05. The differences and the correlations were considered highly significant at p< 0.01. 3.8.1 Statistical Methods: Descriptive and inferential statistical analysis has been carried out in the present study. ROC curve analysis is performed to find the diagnostic role of GGT, Lp(a) and Hs-CRP 11. Significant figures + Suggestive significance (P value: 0.05<P<0.10) * Moderately significant (P value: 0.01<P 0.05) ** Strongly significant (P value: P 0.01) 57