Effect of Oxidative Stress in Fertile and Non Fertile Women

Effect of Oxidative Stress in Fertile and Non Fertile Women3. MATERIAL AND METHODSThe materials and systems employ in the study entitled Comparative study of effect of oxidative var. in fertile and non fertile women was carried out in the Faculty of wellness and Medical Sciences, SHIATS, Allahabad.The detail of experimental techniques employed is as followsMATERIALSSTUDY AREAThe beginning have of infertile and fertile selected married young-bearing(prenomoal)s having child bearing epoch (25-35yrs) without any metabolic disorder from different gynecologist clinical hospitals and infertility centers of Allahabad.COLLECTION OF SAMPLE AND SITE OF EXPERIMENTThe present study was carried out by collecting venous blood taste (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 gro up 25-35 yrs.3.2 Glassw areAll the applesauceware used were washed properly with detersive and rinsed with amend water and autoclaved prior to use.Fig.3.1 Flow chart for fertile and infertile femalesInstrumentation The following instruments were used during the course of studyAutoclave cartridge remover equilibrium (Remi)Cooling centrifuge (remi C-28)Hot air oven (tempo)HomogenizerIncubatorMicropipette tips (100 and guanine l)pH meterSpectrophotometerWeighing balanceCentrifugeColorimeter terrene InvestigationThe issue 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 for3.5.1 Evaluation of Routine biochemical Parmeters-Hb By Sahli ( window glass hematin) method.Blood Sugar By deity/POD methodGlycosylated Hb By Cation method blood serum ProteinBiuret methodSerum lipid profileSerum add C holesterol By Autopack kit methodSerum Triglyceride By Autopack Kit MethodSerum HDL Cholesterol By Autopack Kit MethodSerum LDL cholesterol Friedwald methodSerum VLDL cholesterol Friedwald method3.5.2 Thyroid Profile-Serum T3 enzyme-linked-immunosorbent serologic assay MethodSerum T4 ELISA MethodSerum TSH ELISA Method3.5.3 Female Reproductive hormonesSerum Estrogen ELISA MethodSerum Progesteron ELISA MethodSerum follicle stimulating hormone (FSH) ELISA Method3.5.4 Oxidative Stress marker-Melondialdehyde (MDA) By the santos (1978)method3.5.5 Antoioxidant level-Catalase Brannan (1981) methodCeruloplasmin By Spectrophotometric methodSuperoxide dimutase (SOD) By Mishra and Fridovich (1972)Method bringing close together communications protocol of routine biochemical protocol The body weight and height was calculated manually with the help of weight balance and length scale respectively.Body chew index (BMI) The Body mass index was calculated when body weight is divided by the squ are of height.3.5.1 Estimation of HemoglobinHemoglobin reacts with0.1N hydrochloric acid and physical bodys a brown colour multiplex called hematin.The resulting color after dilution is compa vehement with expectard brown glass reference blocks of a sahli hemoglobinometer.ReagentN hydrochloric acid.Distilled water. performance-By using pasture pipette add 0.1N hydrochloric acid in the tube up to the mark 20Add 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. usher the reading.Normal valueIn female 12-14mg/dlIn males 14-16 mg/dl3.5.2 Estimation of Blood GlucoseEstimation of blood glucose was carried out by using commercial available GOD-POD glucose reagent kit (Autospan, Span symptomatic limited, Surat, India).Glucose oxidase (GOD) oxidizes glucose to gluconic acid and hydrogen peroxide. In presence of enzyme peroxidase, released urine2 is coupled with phenol and 4-aminoanrttipyrine (4-AAP) to form coloured quinoneimine dye. The absorbance of dye is directed proportional to glucose assiduity in the sample (Kaplan, 1984)Glucose + O2 + H2O Gluconic acid+ H2 O2H2O2 + phenol + 4-AAP Qinoneimine Dye + H2OReagents1) Glucose reagentPhosphate bufferGlucose OxidasePeroxidase4-amino antipyrine.2) Glucose diluents3) Glucose holdardProcedure- provision of working Solution All the reagent are ready -to-use. pipette into turn up tube markedBlankStandardTestSerum/plasma20 lCholesterol Standard20 l meld well and incubate at 37 C for 10 minutes at room tempDistilled water1500 l1500 l1500 lThe absorbance of the running play was taken after standard at 490-550 nm.CalculationSerum/plasma glucose concentration (mg/dl) =Absorbance of test x 100(Conc. of Std)Absorbance of StdNormal RangeFasting glucose 65-110mg/dlPost Prandial Upto140 mg/dl.3.5.3 Estimation of Glycosylated hemoglobin (HbA1C)The Glycosylated hemoglobin was estimated by (ion e xchange resin method) commercially available kit (ERBA Diagnostic Mannheim, Transasisa Bio-Medicals limited, Solan India).A hemolysed preparation of the whole blood is flux 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 FractionBlood separation ( HbA1a,HbA1c,HbA1c)ReagentsGlycohaemoglobin Ion Exchange Resin ReagentCation-Exchange Resin (8mg/ml)Glycohaemoglobin Lysing ReagentLysing Agent (10 m M)Glycohaemoglobin CalibratorCalibrator (10%)PROCEDUREThe reaction florilegium contained 500L Lysing Reagent and 100 L whole blood and another tube 500 L Lysing Reagent and 100 L 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 L sample hemolysate in 5.0 ml deionised water in calibrator 20 L 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 % Hb3.5.4 Estimation of Serum ProteinThe 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 majestic color complex, (biuret reaction). Tartarate is added as a stabilizer whilist iodide is used to prevent auto-reduction of the alkaline cooper complex. The a bsorbance of color complex is proportional to protein concentration (Tietz 1986)ReagentsTotal reagentCopper II sulphatePotassium Sodium TartaratePotassium IodideSodium HydroxideProtein standardProcedure- conceptualisation of working Solution All the reagents are ready -to-use.pipet into test tube markedBlankStandardTestSerum/plasma20 lProtein Standard20 lTotal protein reagent railway yard l super acid l1000 lThe absorbance of the test was taken after standard at 546 nm.CalculationSerum/plasma total protein concentration (g/dl) =Absorbance of test x 6.5Absorbance of StdNormal RangeSerum Total protein 6.4-7.8 g/dl3.5.5 Estimation of lipid profileDetermination of total cholesterol in serum/plasmaMethod Name CHOD-PAP methodPrinciple Cholesterol esters are hydrolyzed by Cholesterol Esterase (CE) to give quit 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. I n presence of Peroxidase (POD), Hydrogen Peroxide couple with 4-Amonoantipyrine (4-AAP) and phenol to establish red Quinoneimine dye . Absorbance of colored dye is measured at 505 nm and is proportional to amount of total cholesterol concentration in the sample.ProcedurePreparation of working Solution All the reagent are ready -to-use.Pipette into test tube markedBlankStandardTestSerum/plasma10lCholesterol Standard10 lCholesterol Reagent1000 l1000 l1000 lMix well. Incubate at 37c for 10 minutes or at room temperature (15-30c) for 30 minutes. Read the absorbance of the sample Standard against blank.CalculationCholesterol concentration (mg/dl) =Absorbance of test x 200(Conc. of Std)Aborbance of StdNormal Range 150-250 mg/dl.3.5.6 Determination of HDL Cholesterol in serum/plasmaMethod Name CHOD-PAPPrinciple Low tightfistedness Lipoprotiens (LDL) Cholesterol, Very Low density Lipoprotiens (VLDL) cholesterol and Chylomicron fractions are precipitated by addition of polyethylene Glyco l 6000 (PEG) .After Centrifugation, the High Density Lipoprotien (HDL) Fraction in the supernatant is heady with CHOD-PAP method.ProcedurePreparation of working Solution All the reagent are ready -to-use.STEP-I HDL-Cholesterol separationTake 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 effloresce supernatant for HDL-Cholesterol.STEP-II HDL-Cholesterol Estimation.Pipette into test tube markedBlankStandardTestSupernatant form step-I__10 ulHDL-Cholesterol Standard_10 ul_Cholesterol Reagent1000 ul1000 ul1000 ulMix Well. Incubate at 37c for 5 minutes or at Room temperature (15-30C) for 30 minutes.. Read the absorbance of the sample Standard against blank at 510 nm.CalculationHDL-Cholesterol concentration (mg%)=Absorbance of test x 200(Conc. of Std)Absorbance of StdNormal Range Men=30-60 mg%, Women= 40-70 mg%.3.5.7 Estimation of Low Density Lipoprotein (LDL)LDL= Tota l Triglyceride HDL5-HDLLDL 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/5VLDL cholesterol were obtained by calculations using the empirical relationships of (Freidwald et.al 1995)3.5.9 Determination of Triglyceride in serum/plasmaMethod Name GPO-TRINDERPrinciple 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 obscure and 4-aminoantioyrine by the catalytic action of peroxidase to form a red coloured quinoneimine dye complex. forcefulness of the colour formed is directly proportional to the amount of triglycerides present in the sample.The intensity of chromogen (Quinoneimine) formed is proportio nal 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 confine of each vial. Swirl to dissolve, allow to stand for 10 min at room temperature.ProcedureSTEP-II HDL-Cholesterol Estimation.Pipette into test tube markedBlankStandardTestWorking reagent1000 ul1000 ul1000 ulDistill Water10 ul__Standard10 ulSample10 ulMix Well. Incubate at 37c 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.CalculationTriglyceride (mg/dl) =Absorbance of test x Conc. of Std (mg/dl)Absorbance of StdNormal Range Normal fasting levels 25-160mg/dl.Oxidative stress marker 3.6.1. Determination of Melon di aldehyde (MDA) in serum/plasmaReagents requiredTricholoro acetic acid TCASulfuric Acid HCLSodium sulfateN-Butanol5-1,1,1,3,3 Tetra Ethoxypr o-pane (Standard)ProcedureMalondialdehyde (MDA) adjudicate 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 atomic number 11 sulfate is added.After precipitating the protein with TCA and washing with 0.05sulfuric acid.It was incubated in a boiling water lavatory for 30 min.After cooling, the samples are exactracted with n-butaneol and centrifuged at 3500rpm.The absorbance of samples is determine at 530nm.CalculationTBARS (A) =10 x OD of sample/OD of control (Blank) x mg/ml protein. )Normal Range 0.5-2.0 nmol/ml3.7. Estimation of enzymatic antioxidants3.7.1 Estimation of SOD activity in serum/plasmaReagents requiredCarbonate buffer (0.2M)Kcl (0.015 M)Epinephrine (0.025M)Preparation of the sampleCollect blood without using an anticoagulant such as heparin, citrate or EDTA.Allow blood to clot for 30 minutes at 25CCentr ifuge the blood at 2000 rpm for 15 minutes at 4c.Pipette off the top yellow serum layer without disturbing the white Buffy layer.Procedure1 .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 playscript 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 25C.(UV-1800 SHIMADZU)Suitable control lacking enzyme preparation was run simultaneously.( Mishra and Fridivicl1972).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 protein3.7.2 Estimation of Ceruloplasmin activity in serum/plasmaAt pH 5.4, ceruloplasmin catalyzes the oxidation of PPD to yield a colored product, which is believed to correspond either to Bandrowskis base or to Wuerst ers 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 37C.The contents of the flask are adjusted to pH 5.45 at 37C 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 37C to reach thermal equilibrium. A flaskcontaining 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 l 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 l 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.CalculationsCeruloplasmin (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/dl3.7.3 Estimation of Catalase (CAT) activity in serum/plasmaReagentsH2O2(1.2mM)Phosphate Buffer (pH-7.0)(0.05M)Peroxidase / potassium dichromateProcedureThe 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 C.In brief the hemolysate is prepared fr om 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.CalculationActivity= Std Conc.= 20 molStd.OD =0.02Unit= mol/minute/mg proteinNormal range 3-5 unit/mg proteinstatistical 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 p0.05