Effect of Oxidative Stress in Fertile and Non Fertile Women

Effect of Oxidative Stress in Fertile and Non Fertile Women 3. MATERIAL AND METHODS The materials and methods used in the study entitled â€Å"Comparative study of effect of oxidative stress in fertile and non fertile women† was carried out in the Faculty of Health and Medical Sciences, SHIATS, Allahabad. The detail of experimental techniques employed is as follows: MATERIALS STUDY AREA The blood sample of infertile and fertile selected married females having child bearing age (25-35yrs) without any metabolic disorder from different gynecologist clinical hospitals and infertility centers of Allahabad. COLLECTION OF SAMPLE AND SITE OF EXPERIMENT: The present study was carried out by collecting venous blood sample (5ml) of fertile and non fertile selected married females in Allahabad. Group-I 250 normal healthy fertile women without any metabolic disorder, Group-II 250 infertile female without any metabolic disorder. All the subject of the two groups were between the age group 25-35 yrs. 3.2 Glassware: All the glassware used were washed properly with detergent and rinsed with distill water and autoclaved prior to use. Fig.3.1: Flow chart for fertile and infertile females Instrumentation: The following instruments were used during the course of study Autoclave Centrifuge Balance (Remi) Cooling centrifuge (remi C-28) Hot air oven (tempo) Homogenizer Incubator Micropipette tips (100 and 1000  µl) pH meter Spectrophotometer Weighing balance Centrifuge Colorimeter Routine Investigation The routine investigation of the subject include BMI and weight and history which was taken by asking the subjects to fill a from including 9their approval to be a part of the study. 3.5 Routine biochemical analysis:- All of the blood sample were analyzed for 3.5.1 Evaluation of Routine biochemical Parmeters:- Hb : By Sahli (acid hematin) method. Blood Sugar :By GOD/POD method Glycosylated Hb : By Cation method Serum Protein:Biuret method Serum lipid profile Serum Total Cholesterol : By Autopack Kit Method Serum Triglyceride : By Autopack Kit Method Serum HDL Cholesterol : By Autopack Kit Method Serum LDL cholesterol : Friedwald method Serum VLDL cholesterol : Friedwald method 3.5.2 Thyroid Profile:- Serum T3 :ELISA Method Serum T4 :ELISA Method Serum TSH :ELISA Method 3.5.3 Female Reproductive hormones: Serum Estrogen: :ELISA Method Serum Progesteron: :ELISA Method Serum follicle stimulating hormone (FSH) :ELISA Method 3.5.4 Oxidative Stress marker:- Melondialdehyde (MDA): By the santos (1978)method 3.5.5 Antoioxidant level:- Catalase: Brannan (1981) method Ceruloplasmin: By Spectrophotometric method Superoxide dimutase (SOD): By Mishra and Fridovich (1972)Method Estimation protocol of routine biochemical protocol : The body weight and height was calculated manually with the help of weight balance and length scale respectively. Body mass index (BMI): The Body mass index was calculated when body weight is divided by the square of height. 3.5.1 Estimation of Hemoglobin: Hemoglobin reacts with0.1N hydrochloric acid and forms a brown colour complex called hematin.The resulting color after dilution is compared with standard brown glass reference blocks of a sahli hemoglobinometer. Reagent: N hydrochloric acid. Distilled water. Procedure:- By using pasture pipette add 0.1N hydrochloric acid in the tube up to the mark 20 Add 20ul blood to the tube. Leave the solution for 10 mins. Dilute the solution by adding few drops of distill water at a time till the color matches with the glass plate in the comparator. Read the reading. Normal value: In female: 12-14mg/dl In males: 14-16 mg/dl 3.5.2 Estimation of Blood Glucose: Estimation of blood glucose was carried out by using commercial available GOD-POD glucose reagent kit (Autospan, Span diagnostic limited, Surat, India). Glucose oxidase (GOD) oxidizes glucose to gluconic acid and hydrogen peroxide. In presence of enzyme peroxidase, released H2O2 is coupled with phenol and 4-aminoanrttipyrine (4-AAP) to form coloured quinoneimine dye. The absorbance of dye is directed proportional to glucose concentration in the sample (Kaplan, 1984) Glucose + O2 + H2O Gluconic acid+ H2 O2 H2O2 + phenol + 4-AAP Qinoneimine Dye + H2O Reagents: 1) Glucose reagent Phosphate buffer Glucose Oxidase Peroxidase 4-amino antipyrine. 2) Glucose diluents 3) Glucose standard Procedure:- Preparation of working Solution: All the reagent are ready -to-use. Pipette into test tube marked Blank Standard Test Serum/plasma 20  µl Cholesterol Standard 20  µl Mix well and incubate at 37 C for 10 minutes at room temp Distilled water 1500  µl 1500  µl 1500  µl The absorbance of the test was taken after standard at 490-550 nm. Calculation: Serum/plasma glucose concentration (mg/dl) = Absorbance of test x 100(Conc. of Std) Absorbance of Std Normal Range: Fasting glucose: 65-110mg/dlPost Prandial: Upto140 mg/dl. 3.5.3 Estimation of Glycosylated hemoglobin (HbA1C) The Glycosylated hemoglobin was estimated by (ion exchange resin method) commercially available kit (ERBA Diagnostic Mannheim, Transasisa Bio-Medicals limited, Solan India).A hemolysed preparation of the whole blood is mixed continuously for 5 min with a weak binding cation resin. During this time, HbAo binds to the resin. After the mixing period, a filter is used to separate the supernatant containing the Glycohaemoglobin from resin (Trivelli et al 1971) Hemolysed whole+ Cation exchange resin Fast Fraction Blood separation ( HbA1a,HbA1c,HbA1c) Reagents: Glycohaemoglobin Ion Exchange Resin Reagent Cation-Exchange Resin (8mg/ml) Glycohaemoglobin Lysing Reagent Lysing Agent (10 m M) Glycohaemoglobin Calibrator Calibrator (10%) PROCEDURE: The reaction mixture contained 500 µL Lysing Reagent and 100  µL whole blood and another tube 500  µL Lysing Reagent and 100  µL Calibrator mix and allow it to stand for 5 minutes till lysis is complete. Add 0.1 ml of the hemolysate from step-1 into the approximately marked Ion-Exchange Resin tubes. Close the cap and allow continuous gentle mixing for 5 minutes. Allow the resin to settle to assay temperature for 5 minutes. Position the resin separator in the tube and push down the separators until the resin is firmly packed. Read the absorbance of each tube at 415 nm against deionised water bank. For the fraction of hemoglobin add 20  µL sample hemolysate in 5.0 ml deionised water in calibrator 20  µL Calibrator Hemolysate in 5.0 ml deionised water, mix well and read the absorbance of calibrator and sample at 415 nm against deionised water. Normal Range: 6- 8.3 % Hb 3.5.4 Estimation of Serum Protein: The protein was estimated (Biuret method, End method) by commercially available kit (ERBA diagnostic Mannheim, Transasia Bio-Medicals Limited, Solan, India). The peptide bonds of protein react with copper II ion in alkaline solution to form blue violet color complex, (biuret reaction). Tartarate is added as a stabilizer whilist iodide is used to prevent auto-reduction of the alkaline cooper complex. The absorbance of color complex is proportional to protein concentration (Tietz 1986) Reagents: Total reagent Copper II sulphate Potassium Sodium Tartarate Potassium Iodide Sodium Hydroxide Protein standard Procedure:- Preparation of working Solution: All the reagents are ready -to-use. Pipette into test tube marked Blank Standard Test Serum/plasma 20  µl Protein Standard 20  µl Total protein reagent 1000  µl 1000  µl 1000  µl The absorbance of the test was taken after standard at 546 nm. Calculation: Serum/plasma total protein concentration (g/dl) = Absorbance of test x 6.5 Absorbance of Std Normal Range: Serum Total protein : 6.4-7.8 g/dl 3.5.5 Estimation of lipid profile: Determination of total cholesterol in serum/plasma: Method Name: CHOD-PAP method Principle: Cholesterol esters are hydrolyzed by Cholesterol Esterase (CE) to give free Cholesterol and fatty acids. In subsequent reaction , cholesterol oxidase (CHOD) oxidizes the 3-OH group of free Cholesterol to liberate cholest-4-en-3-one and Hydrogen Peroxide. In presence of Peroxidase (POD), Hydrogen Peroxide couple with 4-Amonoantipyrine (4-AAP) and phenol to produce red Quinoneimine dye . Absorbance of colored dye is measured at 505 nm and is proportional to amount of total cholesterol concentration in the sample. Procedure: Preparation of working Solution: All the reagent are ready -to-use. Pipette into test tube marked Blank Standard Test Serum/plasma 10 µl Cholesterol Standard 10  µl Cholesterol Reagent 1000  µl 1000  µl 1000  µl Mix well. Incubate at 37’c for 10 minutes or at room temperature (15-30’c) for 30 minutes. Read the absorbance of the sample Standard against blank. Calculation: Cholesterol concentration (mg/dl) = Absorbance of test x 200(Conc. of Std) Aborbance of Std Normal Range: 150-250 mg/dl. 3.5.6 Determination of HDL Cholesterol in serum/plasma: Method Name: CHOD-PAP Principle: Low density Lipoprotiens (LDL) Cholesterol, Very Low Density Lipoprotiens (VLDL) cholesterol and Chylomicron fractions are precipitated by addition of polyethylene Glycol 6000 (PEG) .After Centrifugation, the High Density Lipoprotien (HDL) Fraction in the supernatant is determined with CHOD-PAP method. Procedure: Preparation of working Solution: All the reagent are ready -to-use. STEP-I: HDL-Cholesterol separation Take 0.5 ml of serum /plasma in to a glass tube. Add 50ul precipitating reagent. Mix well; leave it for 10 min at room temperature. Centrifuge at 3000 rpm for 10 min. Take the clear supernatant for HDL-Cholesterol. STEP-II: HDL-Cholesterol Estimation. Pipette into test tube marked Blank Standard Test Supernatant form step-I _ _ 10 ul HDL-Cholesterol Standard _ 10 ul _ Cholesterol Reagent 1000 ul 1000 ul 1000 ul Mix Well. Incubate at 37’c for 5 minutes or at Room temperature (15-30 ºC) for 30 minutes.. Read the absorbance of the sample Standard against blank at 510 nm. Calculation: HDL-Cholesterol concentration (mg%)= Absorbance of test x 200(Conc. of Std) Absorbance of Std Normal Range: Men=30-60 mg%, Women= 40-70 mg%. 3.5.7 Estimation of Low Density Lipoprotein (LDL) LDL= Total Triglyceride HDL 5-HDL LDL cholesterol were obtained by calculation using the empirical relationships of (Friedwald et.al.1995) 3.5.8 Estimation of Very Low Density Lipoprotein (VLDL) VLDL =Total triglycerides/5 VLDL cholesterol were obtained by calculations using the empirical relationships of (Freidwald et.al 1995) 3.5.9 Determination of Triglyceride in serum/plasma: Method Name: GPO-TRINDER Principle: Lipoprotein lipase hydrolyses triglycerides to glycerol and free fatty acid. The glycerol formed with ATP in the presence of glycerol Kinase forms Glycerol 3 Phosphate which is oxidized by the enzyme glycerol phosphate oxidase to form hydrogen peroxide. The hydrogen peroxide further reacts with phenolic compound and 4-aminoantioyrine by the catalytic action of peroxidase to form a red coloured quinoneimine dye complex. Intensity of the colour formed is directly proportional to the amount of triglycerides present in the sample. The intensity of chromogen (Quinoneimine) formed is proportional to the Triglyceride in the sample when measured at 505nm (500-540nm). Preparation of working Solution: Allow the reagent bottle and AQUA-4 to attain room temperature .Add the amount of AQUA-4 indicated on the label to the contents of each vial. Swirl to dissolve, allow to stand for 10 min at room temperature. Procedure: STEP-II: HDL-Cholesterol Estimation. Pipette into test tube marked Blank Standard Test Working reagent 1000 ul 1000 ul 1000 ul Distill Water 10 ul _ _ Standard 10 ul Sample 10 ul Mix Well. Incubate at 37’c for 10 minutes. Read the absorbance of the sample Standard against blank at 505 nm (500-540nm) or 505/670nm on bichromic analysers against reagent blank. Calculation: Triglyceride (mg/dl) = Absorbance of test x Conc. of Std (mg/dl) Absorbance of Std Normal Range: Normal fasting levels: 25-160mg/dl. Oxidative stress marker : 3.6.1. Determination of Melon di aldehyde (MDA) in serum/plasma: Reagents required: Tricholoro acetic acid TCA Sulfuric Acid HCL Sodium sulfate N-Butanol 5-1,1,1,3,3 Tetra Ethoxypro-pane (Standard) Procedure: Malondialdehyde (MDA) Assay: Lipid peroxidation in the plasma is evaluated by the spectrophotometric method based on the reaction between MDA and Thiobituric acid (TBARS). Briefly, to 0.5 ml plasma, 2.5 of 20% tricholoro acetic acid (TCA) in 2M sodium sulfate is added. After precipitating the protein with TCA and washing with 0.05sulfuric acid. It was incubated in a boiling water bath for 30 min. After cooling, the samples are exactracted with n-butaneol and centrifuged at 3500rpm. The absorbance of samples is determined at 530nm. Calculation: TBARS (A) =10 x OD of sample/OD of control (Blank) x mg/ml protein. ) Normal Range: 0.5-2.0 nmol/ml 3.7. Estimation of enzymatic antioxidants: 3.7.1 Estimation of SOD activity in serum/plasma: Reagents required: Carbonate buffer (0.2M) Kcl (0.015 M) Epinephrine (0.025M) Preparation of the sample: Collect blood without using an anticoagulant such as heparin, citrate or EDTA. Allow blood to clot for 30 minutes at 25à ¡Ã‚ ´Ã‚ ¼C Centrifuge the blood at 2000 rpm for 15 minutes at 4à ¡Ã‚ ´Ã‚ ¼c.Pipette off the top yellow serum layer without disturbing the white Buffy layer. Procedure: 1 .The reaction mixture composed of 0.1 ml of carbonate buffer (0.2M, pH 10.2), 0.8ml KCl (0.015 M) 0.1 ml of diluted blood and water to make the final volume to 3.0 ml. 2. The reaction was started by adding 0.2 ml of epinephrine (0.025 M). 3. Change in absorbance was recorded at 480 nm at 15 sec interval for 1 min at 25à ¡Ã‚ ´Ã‚ ¼C.(UV-1800 SHIMADZU)Suitable control lacking enzyme preparation was run simultaneously.( Mishra and Fridivicl;1972). Calculation: one unit of enzyme activity is defined as the amount of enzyme causing 50% inhibition of auto oxidant of epinephrine under experimental condition. SOD Activity= Normal range: 12-16 unit/mg protein 3.7.2 Estimation of Ceruloplasmin activity in serum/plasma: At pH 5.4, ceruloplasmin catalyzes the oxidation of PPD to yield a colored product, which is believed to correspond either to Bandrowski’s base or to Wuerster’s red . The rate of formation of the colored oxidation product is proportional to the concentration of serum ceruloplasmin if a correction is made for nonenzymatic oxidation of PPD. Therefore, simultaneous assays are carried pH 5.45, which has been warmed to 37 ºC.The contents of the flask are adjusted to pH 5.45 at 37 ºC by dropwise addition of sodium hydroxide solution (1 mol/liter), and diluted to the mark with acetate buffer solution. The solution is stable for3h. Procedure (1) Into two test tubes (12 X 75 mm), labeled R (reaction) and B (blank), 2 ml of acetate buffer solution was pipetted. (2) Serum, 0.1 ml, is added to each tube. (3) Tubes R and B are placed in a water bath at 37 ºC to reach thermal equilibrium. A flask  containing buffered PPD solution is also placed in the water bath. (4) Warmed, buffered PPD solution (1 ml) is added to both tubes. The contents of the tubes are mixed, and the tubes are kept unstoppered in the water bath. The water bath is covered, to avoid exposure of the tubes to light. (5) After 5 min, 50  µl of sodium azide solutionis pipetted into tube B, and the contents are mixed. The tube is replaced in the water bath. (6) Exactly 30 min later, 50  µl of sodium azide solution is added to tube R, and the contents are mixed. (7) Samples R and B are transferred to spectrophotometer cuvette (light path, 1 cm), and absorbance is measured at 530 nm with a spectrophotometer. The color of the samples remains stable for at least 6 hrs. Calculations Ceruloplasmin (g/liter) = 0.752 (A AB), where AR is the absorbance of sample R, and AB is the absorbance of sample B. Normal range: 20-37mg/dl 3.7.3 Estimation of Catalase (CAT) activity in serum/plasma: Reagents: H2O2(1.2mM) Phosphate Buffer (pH-7.0)(0.05M) Peroxidase /potassium dichromate Procedure: The catalase activity of the hemolysate is determined by adopting the method of Brannan et.al. The assay is based on the disappearance of H2O2 in the presence of the enzyme source at 26 à ¡Ã‚ ´Ã‚ ¼C. In brief the hemolysate is prepared from lysed RBC suspension, further dilute by phosphate buffer(pH-7.0) Here the reaction mixture containing 0.05M phosphate buffer (pH-7.0), 1.2mM H2O2 and 0.2ml of diluted hemolysate is allowed to stand for 25 min. At the end of which reaction is stopped by the addition of 2.5 ml peroxidase reagent containing peroxidase and the red coloured compound chromogen system. Peroxidase reduced the H2O2 to give a compound and absorbance measure at 505 nm. Calculation: Activity= Std Conc.= 20 µ mol Std.OD =0.02 Unit=  µ mol/minute/mg protein Normal range: 3-5 unit/mg protein STATISTICAL ANALYSIS OF THE DATA: The results were analyzed using Duncan multiple range test. All the data are expressed as mean. Differences between the groups were considered significant at pË‚0.05