Glutathione reductase activity kit

The activity of GR was assayed using a Glutathione Reductase Activity Kit (Enzo Life Sciences®, Butler Pike Plymouth Meeting, PA, USA). GR, together with its cofactor NADPH, catalyzes the reduction of oxidized glutathione (GSSG) to glutathione (GSH). In this assay, the oxidation of NADPH to NADP+, during the GR reaction, is spectrophotometrically monitored by the decrease in absorbance (range of 1.7 to 0.2). The activity of GR is expressed in terms of NADPH oxidation. In brief, 50 μL of plasma, blank or GR standard; 100 μL of Master Mix (10X GR buffer, GSSG reagent, and distilled water); and 100 μL of NADP solution were added to initiate the reaction per well. The plate was immediately transferred to an Epoch ELISA reader (BioTek, Winooski, VT, USA), and the absorbance at 340 nm was measured every minute for 10 minutes at room temperature. For the activity calculation, one unit of GR was considered to oxidize 1 μmol of NADPH per minute at 25°C, pH 7.5, and the molar extinction coefficient (E) for NADPH was considered 3.732 × 10⁻³ mL/nmol. The data are expressed in U/mL.

The activity of GPx and the calculation of its determination were carried out as described by [21 (link)] using a glutathione reductase activity kit (Enzo Life Sciences^®, Butler Pike Plymouth Meeting, PA, USA). In brief, 50 μL of hippocampal supernatant diluted 1:5, blank or GR standard; 100 μL of Master Mix (10X GR buffer, GSSG reagent, and distilled water); and 100 μL of NADP solution were added to each well to initiate the reaction. The absorbance was measured at 340 nm every minute for 10 min at room temperature. The data were expressed in U/mg of protein [21 (link)].