ON TESTIS AND EPlDlDYMlS OF RATS - Pondicherry University ...

3.19.2 Determination of nitric oxide
Production of nitric oxide was assayed by measuring the accumulation of
nitrite/ nitrate in tissue using Griess reagent as described by Green el a/. (1982). The
assay mixture contained I mL of tissue supernatant and I mL of Griess reagent (see
Appcndix 1.15). After a I0 min incubation at room temperature absorbance was read
at 550 nm in Systronics Spectropbotometer. A standard curve was prepared by using
known concentrations of sodium nitrite.
3.19.3 Hydrogen peroxide generation assay
Hydrogen peroxide generation was assayed by the method of Pick and Keisari
(198 1). The reaction mixture contained 1.641 mL phosphate buffer (50 mM, pH 7.6),
54 pL of horseradish peroxidase (8.5 units/ mL), 30 pL of 0.28 nM phenol red, 5.5
nM of 165 LLL of dextrose (see Appendix 1.16) and 600 pL of enzyme source. Blank
was prepared simultaneously by adding all the reagents except the tissue extract. The
reaction mixture was ~ncubated at 35OC for 30 rnin. The reaction was terminated by
addition of 60 pL of I0 N sodium hydroxide. Absorbance was read at 610 m against
a reagent blank on a Systronics Spectrophotometer. For preparation of standard
curve, known amounts of hydrogen peroxide and all the above reagents except
enzyme source were incubated for 30 min at 35°C and then 60 pL of 10 N sodium
hydroxide was added and optical density was read at 610 nm.
3.20 Determination of antioxidants in tissues
Estimations of antioxidants such as reduced glutathione, a-tocopherol and
ascorbic acid were done by the methods as described below.
3.20.1 Estimation of reduced glutathione
Total reduced glutathione content was measured by the method of Moron ef
a/. (1979). To 0.5 mL of tissue homogenate 2 mL of 5% trichloro acetic acid waa
added and mixed well. It was centrifuged at 800 g for 10 min and 2 mL of the