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

supcrnutant was taken. Hlunk was prcpurod simultaneously by addilly ull tho rcuycnls
except the tissue extract. To this 1 mL of Ellman's reagent and 4 mL of 0.3 M
disodium hydrogen phosphate (see Appendix 1.17) were added. l'hc yellow colour
developed was read at 412 nm on a Systronics Spectrophotometer against blank. A
series of standards, reduced glutathione were treated in a similar manner along with a
blank.
3.20.2 Estimation of a-tocopherol
Tissue a-tocopherol was estimated by the method of Desai (1984):The assay
mixture contained 0.5 mL of tissue homogenate, 2 rnL of petroleum ether and 1.6 mL
of ethanol. Blank was prepared simultaneously by adding all the reagents except the
enzyme extract. The assay mixture was mixed and centrifuged at 800 g for 10 min.
To the supernatant, 0.2 mL of 2.2'-dipyridyl solution (see Appendix 1.18) was added,
mixed well and kept in the dark for 5 min. An intense red colour was developed and
read at 520 nm on a Systronics Spectrophotometer against blank. Various
concentrations of a-tocopherol were taken and treated similarly along with a blank.
The colour in the aqueous layer was read at 520 nrn and used for standard.
3.20.3 Estimation of ascorbic acid
Ascorbic acid was assayed by the method of Fisher (1962). Approximately,
100 mg of testis was taken in 10 mL of cold 2.5% metaphosphoric acid and
homogenized with the help of glass teflon homogenizr. The homogenate was
centrifuged at 800 g for 20 min at 4OC. The reaction mixture contained 1 mL of
supernatant (diluted with 2.5% metaphosphoric acid) and I mL of 2.6-dichlorophenol
indophenol acetate solution (see Appendix 1.19). Blank was prepared sin~ultaneously
by adding all the reagents except the tissue extract. The optical density was measured
in a within 30 xc at 520 nm in a Systronics Spectrophotometer. A standard
calibration curve was prepared using different concentrations of standard ascorbic
acid.