Oxidative Stress Markers Quantification

The concentration of malondialdehyde (MDA) as a marker of oxidative stress and the levels of total antioxidant capacity (TAC), catalase (CAT) and superoxide dismutase (SOD) activity were measured using commercial kits and according to the manufacturer’s protocol (Kiazist, Iran). Briefly, kidney tissues were homogenized in lysis buffer containing protease inhibitors (Sigma–Aldrich, USA). After centrifugation by a 3-18KS Sigma centrifuge (Sigma, Germany), supernatants were collected for next analysis. MDA level was quantified by measuring thiobarbituric acid reactive substances produced in the reaction of MDA with thiobarbituric acid. TAC level was measured based on the capacity to convert Cu²⁺ to Cu⁺ ion. The activity of catalase was determined according to the reaction of the enzyme with methanol in the presence of hydrogen peroxide and measurement of generated formaldehyde. SOD activity was assayed by measuring the dismutation of superoxide radicals generated by the xanthine/xanthine oxidase system. Protein concentration of lysates was measured using Bradford method. Then the levels of oxidative stress markers were normalized to protein content [20 (link), 21 (link)].