Estimation of tissue malondialdehyde (MDA) and antioxidant enzymes

For homogenate preparation, the desired tissues were quickly removed, cleaned and washed in ice-cold saline. They were finely minced and homogenized in 0.1 M phosphate buffer pH 7.4 using glass Teflon homogenizer. The homogenate was centrifuged at 6,000 rpm for 30 min at 40 °C then the supernatant was used for biochemical estimations.

MDA levels and superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) enzyme activities were assayed according to the manufacturer’s protocol (MDA; CAT No.: MD 2528, SOD; CAT. No.: SA 2520, CAT; CAT. No.: CA 2516; GPx; CAT. No.: GP 2524; Biodiagnostic, Dokki, Egypt). Principally, the colorimetric assay of MDA involves the reaction of MDA with thiobarbituric acid (TBA) in acidic medium at 95 °C to form thiobarbituric acid reactive product, and the absorbance of the resultant pink product can be measured at 534 nm. SOD assay relies on the ability of the SOD to inhibit the phenazine methosulphate-mediated reduction of nitro-blue tetrazolium dye. CAT assay colorimetric method depends on the measurement of the hydrogen peroxide (H₂O₂) substrate remaining after the action of CAT present in the sample. The rest of H₂O₂ reacts with 3,5-dichloro-2-hydroxybenzene sulfonic acid and 4-aminophenazone to yield a colored chromophore with a color intensity that is inversely proportional to the amount of CAT in the sample. The activity of GPx was measured indirectly, based on the principle that oxidized glutathione (GSSG), produced upon reduction of an organic peroxide by GPx, is immediately recycled to its reduced form (GSH) by glutathione reductase (GR). This is accompanied by oxidation of NADPH (GR coenzyme) to NADP+, which was monitored by the decrease in absorbance at 340 nm. The protein contents of the tissue samples (i.e., liver, kidney and testis) were determined by the method of Lowry et al. (1951) using bovine serum albumin as a standard.