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Determination of Non-Enzymatic Antioxidants Estimation of Ascorbic Acid (Vitamin C) The level of ascorbic acid was estimated by the method of Omaye et al. (1979). The ascorbic acid was oxidized to copper to form dehydro ascorbic acid and diketoglutaric acid. These products when treated with DNPH form the derivative, bis-2, 4-dinitrophenyl hydrazone that undergoes rearrangement to form a product with absorption maxima at 520nm. Thiourea provides a mild reducing medium, which helps to prevent the interference from ascorbic acid chromogens. Reagents 1.	Tris-HCl buffer - 0.025 M, pH 7.5 2.	DNPH reagent - 2g of DNPH was dissolved in 100 ml of 9 N H2SO4. To this 4 g of thiourea was added and mixed. 3.	TCA - 6% 4.	TCA - 4% 5.	H2SO4 - 85% 6.	Standard ascorbic acid - 10 mg of L-ascorbic acid was dissolved in 100 ml of 4% TCA. This was diluted to prepare a working standard of concentration 100 g/ml. 7.	Activated charcoal. Procedure 0.5 ml of plasma/tissue homogenate was mixed thoroughly with 1.5 ml of 6% TCA and centrifuged for 20 min at 3500  g. To 0.5 ml of the supernatant, 0.5 ml of DNPH reagent was added and mixed well. The tubes were allowed to stand at room temperature for an additional 3 h. Removed, placed in ice-cold water and added 2.5 ml of 85% H2SO4 and allowed to stand for 30 min. A set of standards containing 10-50 g of ascorbic acid was taken and processed similarly along with a blank, containing 0.5 ml of 4% TCA. The colour developed was read at 530nm. Ascorbic acid values were expressed as M/mg - tissue mg/dl - plasma Estimation of -Tocopherol (Vitamin E) -tocopherol was estimated by the method of Desai (1984). This method involves the reduction of ferric ion to ferrous ion by -tocopherol and the formation of a red coloured complex with 2, 2′-dipyridyl. Absorbance of the chromophore was measured at 520nm. Reagents 1.	Petroleum ether 60-80C 2.	Double distilled ethanol 3.	2, 2′-dipyridyl in ethanol - 0.2% 4.	Ferric chloride in ethanol - 0.5% 5.	Stock standard - 100 mg of -tocopherol in 100 ml of distilled ethanol. 6.	Working standard - Stock solution was diluted to a concentration of 10 g/ml in distilled ethanol. Procedure To 0.1 ml of plasma/lipid extract, 1.5 ml of ethanol and 2 ml of petroleum ether were added, mixed and centrifuged. The supernatant was evaporated to dryness at 80C. To this 0.2 ml of 2, 2′- dipyridyl solution and 0.2 ml of ferric chloride solution was added. Mixed well and kept in dark for 5 min and added 2 ml of butanol. The intense red colour developed was read at 520nm. Standard -tocopherol in the range of 10-100 g were taken and treated similarly along with blank containing only the reagent. The values were expressed as M/mg - tissue mg/dl - plasma Estimation of Reduced Glutathione The level of reduced glutathione (GSH) was determined by the method of Moron et al. (1979). Reagents 1.	TCA - 10% 2.	0.6 mM of DTNB in 0.2 M sodium phosphate 3.	Phosphate buffer - 0.3 M, pH 8.4 4.	Standard glutathione - 10 mg of reduced glutathione in 100 ml of distilled water. Procedure 0.5 ml of plasma/homogenate was mixed with 10% TCA in a 1:1 ratio and then centrifuged for 10 min at 5000 rpm. The clear supernatant was then mixed with 2 ml of phosphate buffer and 0.5 ml of DTNB. After the incubation for 10 min, the absorbance was measured at 412nm. A reagent blank with 1 ml of distilled water and standards containing 20-100 g glutathione were processed similarly along with test samples. The level of GSH was expressed as g/mg protein - tissue mg/dl - plasma Estimation of Total Sulphydrl Groups The levels of total sulphydrl groups (TSH) were estimated by the method of Ellman (1959). This method was based on the development of yellow colour when DTNB was added to the compounds containing sulphydryl groups. Reagents 1.	Phosphate buffer - 0.2 M, pH 8.0 2.	TCA - 5% 3.	Ellman’s reagent - 19.8 mg of DTNB in 100 ml of 1% sodium citrate solution 4.	Disodium hydrogen phosphate - 0.3 M 5.	Standard glutathione solution - 10 mg of reduced glutathione was dissolved in 100 ml of distilled water. Procedure A known weight of tissue was homogenized in phosphate buffer. From this 0.5 ml was pipetted out and precipitated with 2 ml of 5% TCA. 1 ml of the supernatant was taken after centrifugation and added to it 0.5 ml of Ellman’s reagent and 3 ml of phosphate buffer. The yellow colour developed was read at 412nm. A series of standards were treated in a similar manner along with a blank containing 3.5 ml of buffer. The values were expressed as mg/g of tissue Assay of Enzymatic Antioxidants Assay of Superoxide Dismutase (E.C.1.15.1.1) The activity of superoxide dismutase (SOD) was determined by the method of Kakkar et al. (1984). Superoxide radicals react with NBT in the presence of NADH and produce formazan blue. SOD removes the superoxide radicals and inhibits the formation of formazan blue. The intensity of colour is inversely proportional to the activity of the enzyme. Reagents 1.	Sodium pyrophosphate buffer - 0.025 M, pH 8.3 2.	PMS - 186 M 3.	NBT - 300 M 4.	NADH - 780 M 5.	Glacial acetic acid 6.	n-butanol 7.	Chloroform (CHCl3) 8.	Ethanol Procedure 0.5 ml of tissue homogenate was diluted to 1 ml with water. Then added 2.5 ml of ethanol and 1.5 ml of CHCl3 (all the reagents were chilled). This mixture was shaken for 1 min at 4C and then centrifuged. The enzyme activity in the supernatant was determined. The assay mixture contained 1.2 ml of sodium pyrophosphate buffer (0.025 M, pH 8.3), 0.1 ml of 186 M PMS, 0.3 ml of 30 M NBT, 0.2 ml of 780 M NADH, appropriately diluted enzyme preparation and water in a total volume of 3 ml. The reaction was started by the addition of NADH. After incubation at 30C for 90 sec the reaction was stopped by the addition of 1 ml glacial acetic acid. The reaction mixture was stirred vigorously and shaken with 4 ml of n-butanol. The intensity of the chromogen in the butanol layer was measured at 560nm against butanol blank. A system devoid of enzyme served as control. One unit of the enzyme activity is defined as the enzyme reaction which gave 50% inhibition of NBT reduction in one minute under the assay conditions and expressed as specific activity in units/mg protein. Assay of Catalase (E.C.1.11.1.6) The activity of catalase (CAT) was determined by the method of Sinha (1972). Dichromate in acetic acid was converted to perchromic acid and then to chromic acetate, when heated in the presence of H2O2. The chromic acetate formed was measured at 620nm. Reagents 1.	Phosphate buffer - 0.01M, pH 7.0 2.	H2O2 - 0.2 M 3.	Potassium dichromate - 5% 4.	Dichromate-acetic acid reagent - 1:3 ratio of potassium dichromate was mixed with glacial acetic acid. From this 1 ml was diluted again with 4 ml acetic acid 5.	Standard H2O2 - 0.1 ml of 0.2 M H2O2 was diluted to 100 ml using distilled water. Procedure To 0.9 ml of phosphate buffer, 0.1 ml of tissue homogenate and          0.4 ml of H2O2 were added. After 60 sec, 2 ml of dichromate acetic acid reagent was added. The tubes were kept in boiling water bath for 10 min and the colour developed was read at 620nm. Standards in the range of 2-10 mol were taken and proceeded as test with blank containing reagent alone. The activities were expressed as moles of H2O2    consumed/min/mg protein. Assay of Glutathione Peroxidase (E.C.1.11.1.9) The activity of glutathione peroxidase (GPx) was estimated by the method of Rotruck et al. (1973). A known amount of enzyme preparation was allowed to react with H2O2 in the presence of GSH for a specified time period. Then the remaining GSH was measured by the method of Ellman. 2GSH + H2O2                   GSSG + 2H2O Reagents 1.	Tris-HCl buffer - 0.4 M, pH 7.0 2.	Sodium azide - 10 mM 3.	TCA - 10% 4.	Ethylenediaminetetraacetic acid (EDTA) - 0.4 mM 5.	H2O2 - 20 mM 6.	Ellman’s reagent - 19.8 mg of DTNB in 100 ml of 1% sodium citrate solution. 7.	Reduced glutathione - 2 mM Procedure To 0.2 ml of Tris buffer, 0.2 ml of EDTA, 0.1 ml of sodium azide and 0.5 ml of tissue homogenate were added. To the mixture, 0.2 ml of glutathione followed by 0.1 ml of H2O2 was added. The contents were mixed well and incubated at 37C for 10 min along with a tube containing all the reagents except sample. After 10 min the reaction was arrested by the addition of 0.5 ml of 10% TCA, centrifuged and the supernatant was assayed for glutathione by the method of Ellman (1959). The activities were expressed as g of GSH consumed/min/mg protein. Assay of Glutathione-S-Transferase (E.C.2.5.1.18) Activity of glutathione-S-transferase (GST) was measured in tissue homogenate by following the increase in absorbance at 340nm using CDNB as substrate by the method of Habig et al. (1974). 2GSH + H2O2		           GSSG + 2H2O Reagents 1.	Phosphate buffer - 0.3 M, pH 6.5 2.	Reduced glutathione - 30 mM 3.	CDNB - 30 mM was prepared in 95% ethanol. Procedure The reaction mixture contained 1 ml of phosphate buffer, 0.1 ml of CDNB, 0.1 ml of tissue homogenate and 0.7 ml of distilled water. The reaction mixture was incubated at 37C for 5 min and then the reaction was started by the addition of 0.1 ml of 30 mM glutathione. The absorbance change was read at 340nm for 5 min. Reaction mixture without the enzyme was used as the blank. Calculation Activity  = 9.6 was the difference in the micromolar extinction co-efficient between CDNB-GSH conjugate formed/min/mg protein. The activity of GST was expressed as moles of CDNB-GSH conjugate formed/min/mg protein. Assay of Glutathione Reductase (E.C.1.6.4.2) Glutathione reductase (GR) that utilizes NADPH to convert GSSG to the reduced form was assayed by the method of Staal et al. (1969). Reagents 1.	Phosphate buffer - 0.3 M, pH 6.8 2.	EDTA - 0.25 M 3.	GSSG - 12.5 mM 4.	NADPH - 3 mM Procedure The reaction mixture containing 1 ml of phosphate buffer, 0.5 ml of EDTA, 0.5 ml of GSSG and 0.2 ml of NADPH was made up to 3 ml with water. After the addition of 0.1 ml of tissue homogenate, the change in optical density at 340nm was monitored for 2 min at 30 sec intervals. The activity of GR was expressed as n moles of NADPH oxidized/min/mg protein.

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