**3.5 Glutathione peroxidase activity**

The activity of glutathione peroxidase (GPx) is derived from the oxidation of reduced β-NADPH in a conjugated glutathione reductase (GR) system using H2O2 (12 mM) as a substrate. Glutathione peroxidase reacts with H2O2 oxidizing reduced glutathione (GSH) to oxidized glutathione (GSSG). In brief, the GPx assay was performed in duplicate in a 96-well UV Costar plate. Each well contained 215 μL assay buffer (AB: 50 mM potassium phosphate, 1 mM EDTA, pH 7.0), 5 μL GSH (30.7 mg/mL in water), 5 μL GR (0.1 U/mL in AB), and 20 μL of sample, and 5 μL NAD(P)H was added to the mixture. Two readings were recorded [38]. The first was the background of oxidation at 340 nm for 3 min in 30 s intervals for samples (A1) and blank (A1b). The second reading was performed after adding 50 μL H2O2. This reading monitored the decrease of H2O2 due to NAD(P)H oxidation at 340 nm for 2 min. The GPX activity was expressed in μmol/mg of protein.
