Evaluation of oxidative/antioxidative markers in rats' brain homogenate

For the detection of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and malondialdehyde (MDA), immediately after dissection, the brain was rinsed in ice-cold phosphate buffer saline (PBS) solution pH 7.4, blotted with filter paper, weighed, and homogenized utilizing a tissue homogenizer in cold buffer (5 ml per gram tissue). The activity of GPx was evaluated in the brain tissues as previously described by Paglia and Valentine³⁹ utilizing available kits (Cat. GP 2524, Biodiagnostic Co.), the assay based on the recycling of oxidized glutathione reduced state by the enzyme glutathione reductase and the oxidation of NADPH to NADP+ which is associated by a reduction in absorbance at 340 nm. The levels of SOD were evaluated in tissues using the available kits (Cat. SD 25 21 Biodiagnostic Co.) according to Nishikimi, et al.⁴⁰. The assay relied on the inhibition of nitroblue tetrazolium dye reduction through phenazine methosulphate enzyme at 560 nm for 5 min for control and sample at 25 °C. Moreover, the level of CAT was estimated via the laboratory-supplied kits (Cat. CA 2517, Biodiagnostic Co.) based on the method detailed by Aebi⁴¹ by interaction with H₂O₂, and then inhibition of this reaction by CAT inhibitor at 510 nm. Estimation of MDA levels was performed using the commercially available kits (Cat. MD 2529, Biodiagnostic Co.) as formerly performed by Mihara and Uchiyama⁴². The assay was based on the reaction between thiobarbituric acid and MDA at the absorbance of 534 nm, resulting in a pink-colored complex.