Antioxidant Enzyme Activity Evaluation

The superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione S-transferase (GST) activity was evaluated as already described previously [47 ,48 (link)]. SOD activity was evaluated by quantitative sandwich enzyme immunoassay technique using a commercial kit (Cusabio, Houston, Texas). The results were expressed as U/mg of protein. For GPx activity, the absorbance was monitored at 340 nm at 37 °C for 10 min, and the results were expressed as μmol of reduced glutathione (GSH)/min/mg of protein. The GR activity was evaluated by measuring the consumption of nicotinamide adenine dinucleotide phosphate (NADPH) as a cofactor in the reduction of oxidized glutathione to reduce GSH. The results were expressed as U of GR/mg of protein. One U of enzyme activity was defined as the amount of GR that oxidizes 1 μmol of NADPH per min. The GST activity was measured using 1-chloro-2,4-dinitrobenzene (CDNB) as a substrate. The results were expressed as U of GST/mg of protein. One U of enzyme activity was defined as the amount of GST that produces 1 μmol of the conjugate of GSH with CDNB per min. The total protein concentration in the homogenate was measured using the method of Bradford.