Estimation of Antioxidant Enzyme Activities

The activity of GR in hippocampal tissue was investigated based on the protocol illustrated by Factor et al.³² Briefly, 20 µL of hippocampal homogenate was added to oxidized glutathione (0.44 mM), EDTA (0.30 M), in phosphate buffer (0.1 M) at pH 7.0. The reaction was developed by pipetting NADPH (0.036 M). The oxidation rate of NADPH was followed by the reduction in absorbance at 340nm with duration. One unit of the enzyme was known as the quantity of enzyme required to oxidize 1 µmol of NADPH per minute.

Based on the procedures described by Paglia and Valentine,³³ the hippocampal activity of GPx was assayed. In brief, hippocampal supernatant (200 µL) was added to 1 mL phosphate buffer (75 mM) at pH 7.0, 10 mL glutathione (150 mM), 10 mL glutathione reductase (340 U/mL), 30 mL (25 mM EDTA), 30 mL NADPH (5 mM), 10 mL Triton X-100 (20%), and 50 µL H₂O₂ (7.5mM). The oxidation of NADPH to NADP⁺ was examined at 340 nm for 3 minutes. One unit of GPx activity was presented as the amount of GSH (nanomoles) oxidized/minute/milligram of protein (U/mg protein).

CAT activity in the hippocampal supernatant was estimated according to the method illustrated by Aebi.³⁴ In order to evaluate the CAT activity, the enzymatic reaction mixture (total volume 1 mL) contained 50 mM potassium phosphate (pH 7.0), 19 mM H₂O₂, and 50 µL of homogenate supernatant was employed. The molar attenuation coefficient of H₂O₂ was examined by a UV-VIS spectrophotometer at 240 nm. One unit of catalase activity was determined as the level of enzyme decomposing H₂O₂ (1 µmol)/minute/milligram of hippocampal tissue protein (U/mg protein).

The determination of Hippocampal SOD activity was performed following Misra and Fridovich’s technique.³⁵ This method depended on the capacity of epinephrine oxidation at pH 10.2 resulting in the formation of adrenochrome and superoxide radicals (O₂.⁻). The suppression of SOD activity was investigated based on reducing the absorbance at 480 nm.