Tac assay kit

Total and nonprotein antioxidant defenses in serum were analyzed by using a commercial colorimetric biochemical kit, according to the manufacturer's instructions (TAC Assay Kit, Sigma Aldrich, Milwaukee, USA). The method is based on Cu²⁺ conversion to Cu⁺ by both small molecules and proteins. Small antioxidant molecules were isolated from protein antioxidants by using a chemical mask, which prevents Cu²⁺ reduction by proteins. The antioxidant capacity (mM) was analyzed in a spectrophotometer at 570 nm and estimated from a standard curve using trolox as the antioxidant reference.

The activity of antioxidant enzymes glutathione-S-transferase (GST), catalase (CAT), and superoxide dismutase (SOD) was analyzed in heart samples homogenized in ice-cold phosphate buffer (pH = 7.0) by being centrifuged for 15 min at 5°C and 3500×g. The kinetic method of hydrogen peroxide (H₂O₂) decomposition was used to evaluate CAT activity [28 (link)]. The xanthine oxidase method, which is based on the reduction of nitroblue tetrazolium and the production of H₂O₂, was used to estimate SOD activity [29 (link)]. GST activity was estimated from the rate of NADPH oxidation, which was analyzed in the spectrophotometer at 340 nm [30 (link)]. All results were normalized by protein levels, which were measured in the supernatant using the Bradford method [31 (link)].
The nonenzymatic defenses in the heart homogenate were analyzed by using a total antioxidant capacity assay kit, according to the manufacturer's instructions (TAC Assay Kit, Sigma Aldrich, Milwaukee, USA). The method was based on the inhibition of antioxidant enzymes and the prevention of Cu²⁺ oxidation by small antioxidant molecules, which is analyzed in a spectrophotometer at 570 nm. The antioxidant capacity was estimated from a standard curve, using trolox as the antioxidant reference.

Total antioxidant capacity (TAC) and lipid peroxidation were estimated in cell lysates for all groups. The end product of lipid peroxidation, malondialdehyde (MDA), was detected spectrophotometrically at 534 nm using a commercial kit (Biodiagnostic Co., Cairo, Egypt). TAC was estimated in Trolox equivalents at 570 nm according to the manufacturer’s instructions using the TAC assay kit (sigma-Aldrich, USA).