Materials and Methods

Transcription

1 Materials and Methods

2 The study was conducted at School of Health Sciences, Kannur University Thalassery Campus, Palayad, and Pushpagiri Institute of Medical Sciences and Research Center, Thiruvalla, Kerala, India. One hundred and twelve clinically proved patients below the age of 65 years formed the test group. Fifty age and sex matched control subjects from among the siblings of the patients and staffs of the institute formed the control groups. None of the subjects were suffering from autoimmune disease, and had no liver and renal involvement. Detailed clinical, epidemiological and anthropometric characteristics were recorded using proforma (Appendix). Ten ml of fasting venous blood was collected from all the subjects after getting the informed consent. Three ml of blood was immediately transferred aseptically to heparinized vacuutainers and used for invitro mutagen sensitivity analysis. The remaining 7ml of blood was allowed to clot and serum separated and the following investigations were carried out in the serum sample. 1. Total cholesterol 2. High density lipoprotein cholesterol (HDL) 3. Low density lipoprotein cholesterol (LDL) 4. Triglycerides 5. Malondialdehyde (MDA) 103

3 6. Ascorbic acid (Vitamin C) 7. Glutathione (GSH) 1. Total Cholesterol (Rifai et al, 1999) Cholesterol is estimated in serum by enzymatic direct quantitative determination on Roche automated clinical chemistry analyzers. Cholesterol esters are enzymatically hydrolyzed by cholesterol esterase to cholesterol and free fatty acids. Free cholesterol, including that originally present, is then oxidized by cholesterol oxidase to cholest-4-en-3-one and hydrogen peroxide. The hydrogen peroxide combines with 4-amino phenazone phenol to form a chromophore (quinoneimine dye) which may be quantitated at nm. Cholesterol esters CHOL: ESTERASE Cholesterol+ Fatty acids + H 2 O CHOL: OXIDASE Cholesterol + O 2 Cholest-4-en-3-one + H 2 O 2 2H 2 O AminoPhenazone +4-Chlorophenol PEROXIDASE 4(p-benzoquinone-monoimino) -phenazone +HCl+ 4 H 2 O 2. HDL Cholesterol (Sugiuchi et al, 1995) HDL Cholesterol is estimated in serum by homogenous enzymatic colorimetric assay on Roche automated clinical chemistry analyzers. In the presence of magnesium sulfate, dextran sulfate selectively forms watersoluble complexes with LDL, VLDL and chylomicrons which are resistant to PEG-modified enzymes. The cholesterol concentration of HDL-cholesterol is 104

4 determined enzymatically by cholesterol esterase and cholesterol oxidase coupled with PEG to the amino groups (approx. 40%). Cholesterol esters are broken down quantitatively into free cholesterol and fatty acids by cholesterol esterase. In the presence of oxygen, cholesterol is oxidized by cholesterol oxidase to Δ 4 -cholestenone and hydrogen peroxide. In the presence of peroxidase, the hydrogen peroxide generated reacts with 4- amino-antipyrine and HSDA to form a purple-blue dye. The color intensity of this dye is directly proportional to the cholesterol concentration and is measured photometrically. Homogenous Enzymatic colorimetric assay. HDL Cholesterol esters + H 2 O PEG - CHOL ESTERASE HDL Cholesterol + Fatty acids PEG - CHOL: OXIDASE HDL Cholesterol + O 2 Δ 4 -Cholestenone + H 2 O 2 H 2 O AAP +HSDA + H 2 O+ H + PEROXIDASE Purple blue pigment (585 nm) + 5 H 2 O HSDA - Sodium N-(2-hydroxy-3-sulfoprpyl)-3,5-dimethoxyaniline 4-4- AAP -4- Amino Anti Pyrine 3. LDL Cholesterol (Armstrong and Seidel, 1985) LDL Cholesterol is estimated in serum by homogenous enzymatic colorimetric assay on Roche automated clinical chemistry analyzers. Direct determination of LDL-cholesterol takes advantage of the selective micellary solubilization of LDL-cholesterol by nonionic detergent. Cholesterol esters are broken down quantitatively into free cholesterol and fatty acids by 105

5 cholesterol esterase. In the presence of oxygen, LDL cholesterol is oxidized by cholesterol oxidase to Δ 4 -cholestenone and hydrogen peroxide. In the presence of peroxidase, the hydrogen peroxide generated reacts with 4- aminoantipyrine and HSDA to form a purple-blue dye. The color intensity of this dye is directly proportional to the cholesterol concentration and is measured photometrically. Homogenous Enzymatic colorimetric assay. LDL Cholesterol esters Fatty acids + H 2 O DETERGENT CHOL: ESTERASE CHOL: OXIDASE Cholesterol + Free LDL Cholesterol + O 2 Δ 4 Cholestenone + H 2 O 2 PEROXIDASE 2 H 2 O AAP Purple blue pigment (585 nm) +HSDA + H 2 O+ H H 2 O HSDA -Sodium N-(2-hydroxy-3-sulfoprpyl)-3,5-dimethoxyaniline 4-AAP - 4- Amino Anti Pyrine 4. Triglycerides (Mc Gowan et al, 1983) Triglycerides in the serum were estimated by enzymatic GPO-PAP method on Roche automated clinical chemistry analyzers. The triglycerides in the sample are hydrolysed by a combination of microbial lipases to give glycerol and fatty acids. The glycerol is phosphorylated by ATP in the presence of glycerol kinase (GK) to produce glycerol-3-phosphate. The glycerol-3-phosphate is oxidised by molecular oxygen in the presence of GPO (glycerol phosphate oxidase) to produce hydrogen peroxide and 106

6 Dihydroxyacetone phosphate (DHAP). The hydrogen peroxide produced then reacts with 4-aminophenazone and 4-chlorophenol, catalysed by peroxidase (POD) to give a red dye with an absorbance maximum at 500 nm. The increase in absorbance is proportional to the triglyceride content of the sample. Triglycerides + 3 H2O Glycerol + ATP LIPASE GK, Mg2+ Glycerol + 3 Fatty acids Glycerol-3-phosphate + ADP GPD Glycerol-3-phosphate Dihydroxyacetone phosphate + O2 + H 2 O 2 2H 2 O AminoPhenazone POD +4-Chlorophenol 4(p-benzoquinone-monoimino) -phenazone +HCl+ 4 H 2 O ll the reagents, calibrators, controls used for Serum Triglyceride, Serum Total Cholesterol, Serum HDL Cholesterol and Serum LDL Cholesterol were from Roch Diagnostics and the analysis was carried out in fully automated analyzer, Hitachi 912. Quality control was done with Precinom Universal, Precipath Universal, Precinorm lipids and Precipath lipids of Roch Diagnostics. 5. Malondialdehyde [MDA] The assay is based on the reaction of malondialdehyde (MDA) with thiobarbituric acid (TBA); forming a MDA-TBA 2 adduct according to a modified version of Satoh s (1978) and Yagi s (1984) methods. 107

7 In brief, 0.5 ml serum was mixed with 4 ml 1/12 N H 2 SO 4 in a centrifuge tube and shaken gently. After 0.5 ml 10% phosphotungstic acid was added to the tube and it was left to stand at room temperature for 5 minutes, the mixture was centrifuged at 3000 rpm for 10 minutes. The supernatant was discarded and the sediment was mixed with 2.5 ml 1/12 N H 2 SO 4. The centrifugation was repeated and the supernatant was discarded again. The sediment was resuspended in 2.5 ml 1/12 N H 2 SO 4 and in fresh 3 ml 0.2% thiobarbituric acid reagent, mixed thoroughly and heated in a bath of boiling water for one hour. After cooling in cold water, the resulting chromogen was extracted with 3.0 ml n-butyl alcohol by vigorous shaking. The organic phase was separated by centrifugation at 3000 rpm for 10 minutes, and its absorbance was recorded at a wavelength of 532 nm using UV-VIS Spectrophotometer. The level of absorbance was converted into nmol/ml MDA from a standard curve generated with 1, 1, 3, 3- tetraethoxypropane (SIGMA). 6. Ascorbic Acid [Vitamin C] (Varley, 2002) Protein free supernatant was prepared by adding 2.0 ml of serum and 6.0ml of 5% TCA. To 0.5 ml protein free supernatant 1.5 ml DTC reagent (2, 4- dinitrophenyl hydrazine-thiourea-cuso reagent) was added. 1.5 ml DTC reagent and 0.5ml distilled water was taken as blank. All the tubes are incubated at room temperature for1 hour. Then 2 ml ice cold sulphuric acid was added to the tubes. The intensity of the yellow colour developed was read at 520 nm in UV VIS Spectrophotometer. A standard graph was plotted 108

8 with standard concentration adjusted to 0.25 mg%, 0.5 mg%, 1 mg%, 1.5 mg%, and 2 mg%. 7. Glutathione [GSH] (Beutler and Kelley, 1963) 5, 5 -Dithiobis (2-nitrobenzoic acid) (DTNB) and glutathione (GSH) react to generate a yellow coloured product 2-nitro-5-thiobenzoic acid and glutathione disulfide (GSSG). Protein free supernatant was prepared by adding 3.8 ml precipitating reagent and added 200 µl of serum sample and 1ml distilled water in a test tube. Mixed well and filtered through a coarse filter paper in to a centrifuge tube. Centrifuged and collected the supernatant. To 1 ml filtrate added 0.5 ml DTNB reagent and 4 ml phosphate buffer as test. A blank was taken with 0.5 ml DTNB reagent, 4 ml phosphate buffer, 0.6 ml distilled water and 0.4 ml precipitating reagent. A standard tube was taken by adding 1 ml GSH standard (20 mg %), 0.5 ml DTNB reagent and 4 ml phosphate buffer. All the tubes were capped and mixed by inversion and read the OD at 412 nm within four minutes in UV VIS Spectrophotometer. 8. Invitro Mutagen Sensitivity Analysis Peripheral blood was collected aseptically by venipuncture. Peripheral blood lymphocyte micro culture was performed as described by Moorhead et al (1960) for determining the mutagen sensitivity. The culture medium was RPMI 1640, supplemented with 20% fetal calf serum, 0.5ml phytohaemagglutinin, 100-units/ml penicillin and 100 µg/ml streptomycin. Bleomycin was added to induce chromosomal breakage according to the 109

9 method of Hsu et al (1985) units/ml of bleomycin treatment was given 5hrs (late S-G2 phase) before harvesting (66 th hour). The cultures were incubated for 72 hrs at 37 0 C. At the end of 70 th hr all the cultures were treated with colcemid (0.04µgm/ml) to accumulate mitoses before harvesting. The cells centrifuged at 1500 rpm for 10 minutes, removed the supernatant. Re-suspended in 10 ml warm KCl hypotonic solution (0.075M) after this added 2 drops of freshly prepared fixative (Methanol: Acetic acid) in 3:1 ratio. The tube was centrifuged again at 1500 rpm for 10 minutes. This step was repeated many times until the supernatant was clear and the cell button gets white. Dropped the suspension drop by drop (7-8 drops) on pre cleaned chilled slides with a fine bore pipette, flame the slide on spirit lamp, blow on the material gently, dried and stored it in a slide box for ageing. The slides were stained with Giemsa. For mutagen induced chromosome sensitivity analysis the mean number of breaks/cell (b/c) was calculated by the following formula. Only frank chromatid breaks were scored. Calculation Number of breaks counted X 46 Mean number of breaks/cell (b/c) = Total number of metaphase score 100 The frequency of breaks was expressed as b/c for comparison. Any individual expression 0.8 b/c was considered sensitive and that 1.0 b/c was 110

10 Geimsa Stained Metaphases

11 Metaphases showing chromatid breaks

12 considered hypersensitive. A minimum of 100 metaphases per culture was scored and data were analyzed. The frequency of chromatid breaks was considered as a measure of an individual s DNA repair capacity. All the chemicals and standards for MDA, ascorbic acid, GSH and invitro mutagen sensitivity analysis were procured from Sigma. Statistical Analysis Results were expressed as mean ± SD. Independent sample t test was performed using SPSS (windows version 17.0) for comparing various risk/lifestyle factors, biochemical and genetic characteristics such as total cholesterol, LDL cholesterol, HDL cholesterol, MDA, ascorbic acid, GSH and mean b/c value of the study subjects and the control subjects. Analysis of variance (ANOVA) was done to compare the mean b/c value with and without various risk/ life-style factors among subgroups. Probability values of p < 0.05 were considered to be statistically significant. Association between various risk/lifestyle factors, biochemical and genetic characteristics and socio-economic variables and CAD is analyzed using chi-square test. The contribution of various risk/lifestyle factors and biochemical and genetic characteristics for coronary artery disease and the contribution of various risk/lifestyle factors and biochemical characteristics for high mean b/c value (sensitive) was studied by Logistic regression analysis. 111