Effect of Oxidative Stress in Fertile and Non Fertile Women

Effect of aerophilous Stress in Fertile and Non Fertile Women3. MATERIAL AND METHODSThe materials and methods used in the study entitled comparative study of effect of oxidative stress in fertile and non fertile women was carried break in the aptitude of Health and Medical Sciences, SHIATS, Allahabad.The detail of experimental techniques employed is as followsMATERIALSSTUDY AREAThe countercurrent specimen of infertile and fertile selected married females having child bearing age (25-35yrs) without any metabolic disorder from various gynecologist clinical hospitals and infertility centers of Allahabad.COLLECTION OF SAMPLE AND SITE OF EXPERIMENTThe present study was carried out by collecting venous blood test (5ml) of fertile and non fertile selected married females in Allahabad.Group-I 250 trite healthy fertile women without any metabolic disorder,Group-II 250 infertile female without any metabolic disorder.All the suit of the two crowds were between the age group 25-35 y rs.3.2 GlasswargonAll the glassw atomic number 18 used were washed correctly with detergent and rinsed with distill pee system and autoclaved prior to use.Fig.3.1 Flow chart for fertile and infertile femalesInstrumentation The adjacent instruments were used during the course of studyAutoclaveCentrifugeBalance (Remi)Cooling centrifuge (remi C-28)Hot transmission line oven (tempo)HomogenizerIncubatorMicropipette tips (100 and grand l)pH meterSpectrophotometerWeighing balanceCentrifugeColorimeterRoutine investigationThe routine investigation of the subject include BMI and weight and history which was taken by asking the subjects to surfeit 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 ( mordant hematin) method.Blood Sugar By GOD/POD methodGlycosylated Hb By Cation method blood blood blood serum ProteinBiuret methodSerum lipide profileSerum append Cholesterol By Au billack fit out up ruleSerum Triglyceride By Autopack Kit MethodSerum HDL Cholesterol By Autopack Kit MethodSerum low-density lipoprotein cholesterin Friedwald methodSerum very low density lipoprotein cholesterin Friedwald method3.5.2 Thyroid Profile-Serum T3 enzyme-linked-immunosorbent serologic bridle 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) methodCeruloplas arc morsel By Spectrophotometric methodsuperoxide anion dimutase (SOD) By Mishra and Fridovich (1972)Methodapproximation protocol of routine biochemical protocol The body weight and height was figure manually with the help of weight balance and length scale respectively. luggage compartment mass index ( BMI) The Body mass index was calculated when body weight is divided by the square of height.3.5.1 Estimation of hemoglobinHemoglobin reacts with0.1N hydrochloric blistery and forms a embrown colour complex called hematin.The resulting color after dilution is compared with standard brown glass reference blocks of a sahli hemoglobinometer.ReagentN hydrochloric acid.Distilled water. mathematical operation-By utilise pasture pipette make up 0.1N hydrochloric acid in the tube up to the mark 20Add 20ul blood to the tube.Leave the ancestor for 10 mins.Dilute the solution by adding few drops of distill water at a term till the color matches with the glass plate in the comparator. sound out the reading. sane 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 diagnostic limited, Surat, India).Glucose oxidase (GOD) oxidizes glucose to gluconic a cid and hydrogen peroxide. In charge of enzyme peroxidase, released H2O2 is coupled with oxybenzene and 4-aminoanrttipyrine (4-AAP) to form coloured quinoneimine dye. The absorbance of dye is directed proportionate to glucose dumbness 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 standard subprogram- zeal of working Solution All the reagent are ready -to-use.Pipette into test tube marked mindlessStandardTestSerum/plasm20 lCholesterol Standard20 lMix intumesce and incubate at 37 C for 10 trans go throughs at manner tempDistilled water1500 l1500 l1500 lThe absorbance of the test 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 divagateFasting 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 FractionBlood separation ( HbA1a,HbA1c,HbA1c)ReagentsGlycohaemoglobin Ion Exchange rosin ReagentCation-Exchange Resin (8mg/ml)Glycohaemoglobin Lysing ReagentLysing Agent (10 m M)Glycohaemoglobin CalibratorCalibrator (10%)PROCEDUREThe reaction mixture contained 500L Lysing Reagent and 100 L whole blood and another tube 500 L Lysing Reagent and 100 L Calibrator mix and allow in it to stand for 5 minutes till lysis is complete. Add 0.1 ml of t he hemolysate from step-1 into the approximately marked Ion-Exchange Resin tubes. limiting 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 intemperately 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 place 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 violet color complex, (biuret reaction). Tartarate is added as a stabiliser 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)ReagentsTotal reagent bulls eye II sulphatePotassium Sodium TartaratePotassium IodideSodium HydroxideProtein standardProcedure- prep of working Solution All the reagents are ready -to-use.Pipette into test tube marked cleanStandardTestSerum/plasma20 lProtein Standard20 lTotal protein reagent megabyte l1000 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 free Cholesterol and fatty acids. In subsequent reaction , cholesterol oxidase (CHOD) oxidize s 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 heart 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. handle 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 density Lipoprotiens (LDL) Cholesterol, Very Low Density Lipoprotiens (VLDL) cholestero l and Chylomicron fractions are precipitated by addition of polyethylene Glycol 6000 (PEG) .After Centrifugation, the High Density Lipoprotien (HDL) Fraction in the supernatant is unconquerable with CHOD-PAP method.ProcedurePreparation of working Solution All the reagent are ready -to-use.STEP-I HDL-Cholesterol separation recognise 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. stool the clear supernatant for HDL-Cholesterol.STEP-II HDL-Cholesterol Estimation.Pipette into test tube markedBlankStandardTest supported form step-I__10 ulHDL-Cholesterol Standard_10 ul_Cholesterol Reagent1000 ul1000 ul1000 ulMix Well. plow 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= Total 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 adenosine tri inorganic phosphate 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 t he 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 check to the content 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/plasm aReagents requiredTricholoro acetic acid TCASulfuric Acid HCLSodium sulfateN-Butanol5-1,1,1,3,3 Tetra Ethoxypro-pane (Standard)ProcedureMalondialdehyde (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 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 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.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 bodily process in serum/plasmaReagents required change buffer (0.2M)Kcl (0.015 M)Epinephrine (0.025M)Preparation of the sampleCollect blood withou t using an anticoagulant such as heparin, citrate or EDTA.Allow blood to clot for 30 minutes at 25CCentrifuge 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 constitute 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 last-place 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 25C.(UV-1800 SHIMADZU)Suitable control wanting(p) 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 oxid ation of PPD to yield a colored product, which is believed to correspond either to Bandrowskis base or to Wuersters 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 ethanoate 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 likewise 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 open 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 to the lowest degree 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 from lysed RBC suspension, further dilute by phosphate buffer(pH-7.0) here(predicate) 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