Glutathione (GSH) assay

Glutathione content was determined according to the procedure of Beutler [46] with some modifications. Briefly, 0.20 ml of tissue supernatant was mixed with 1.5 ml precipitating solution containing 1.67 % glacial metaphosphoric acid, 0.20 % Na-EDTA and 30 % NaCl. The mixture was allowed to stand for 5 min at room temperature and centrifuged at 1000g for 5 min. One ml clear supernatant was mixed with 4 ml 0.30 M Na₂HPO₄ and 0.50 ml DTNB reagent (40 mg 5, 5′ dithiobis-(2-nitrobenzoic acid dissolved in 1 % sodium citrate). A blank was similarly prepared in which 0.20 ml water was used instead of the brain supernatant. The absorbance of the color was measured at 412 nm in a spectrophotometer.