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

3.21.4 Assay of glutathione peroxidase
Glutathione peroxidase (EC. 1.1 1.1.9) was assayed by the method of
Mohandas rt al. (1984). The assay mixture contained 1.59 mL of phosphate buffer
(100 mM, pH 7.6). 100 pL of 10 mM EDTA, 100 pL of sodium azidc, 50 pL of
glutathione reductase, 100 pL of glutathione reduced, I00 pL of 200 mM NADPH, I0
pL of hydrogen peroxide (see Appendix 1.23) and 10 pL enzyme source. Blank was
prepared simultanwusly by adding all the reagents except the enzyme extract. The
extinction coefficient of NADPH was measured at 340 nm against blank containing
all the components except the enzyme at 10 sec intervals for 3 min on a Systronics
Spectrophotometer.
3.22 Estimation of lipid peroxidation
A break-down product of lipid peroxidation thiobarbituric acid reactive
substance (TBARS) was measured by the method of Buege and Aust (1976). Briefly,
the stock solution contained equal volumes of trichloroacetic acid 15% (w/ v) in 0.25
N hydrochloric acid and 2-thiobarbituric acid 0.37% (wl v) in 0.25 N hydrochloric
acid (see Appendix 1.24). 1 mL of the tissue test samples and 2 mL of stock reagent
were mixed in a screw-capped centrifuge tube, vortexed and heated for 15 niin in a
boiling water bath. Blank was prepared simultaneously by adding all the reagents
except the enzyme exrract. After cooling on ice the precipitate was removed by
centrifugation at 1000 g for 15 min and absorbance of the supernatant was measured
at 532 nm against blank. A standard curve was constructed extrapolating the amount
of malondialdchyde to the measured absorbance
3.23 Statistical analysis
The data in the experimental groups of rats were obtained from the individual
rat, which received identical treatments. All other biochemical estimations were
carried out in duplicate. The significance of the results has been analyzed using one-