Determination of the total glutathione content, reduced & oxidized glutathione ratio, and activities of glutathione peroxidase and glutathione reductase

The total glutathione (GSH) content was determined as described previously⁴². Briefly, 50 µL of diluted serum (in PBS 10 mM, pH 7.2) or total GSH standard was combined with 80 µL of a DTNB/NADPH mixture (10 μL of 4 mM DTNB and 70 μL of 0.3 mM NADPH) in a 96-well microplate. Next, 20 μL (0.06 U) of a GSH-reductase (GSH-Rd) solution was added to each well. The serum levels of reduced and oxidized GSH were determined by using the GSH & GSSG assay kit (DoGen Bio Co., Ltd., Seoul, Republic of Korea) according to the manufacturer’s instructions, and GSH/GSSG ratio was calculated based on their concentration. Absorbance was measured using a plate reader at 412 nm (Molecular Devices).

The GSH-peroxidase (GSH-Px) activity was determined according to the method of Paglia⁴³. Briefly, 50 μL of NADPH reagent (5 mM NADPH, 42 mM GSH and 10 units/mL of GSH-Rd in 1.25 mL of distilled water) was added to 890 μL of GSH-Px buffer (50 mM Tris HCl, pH 8.0, with 0.5 mM EDTA). Then, 50 μL of serum and 10 μL of 30 mM tert-butyl hydroperoxide solution were added to the mixture. The final absorbance was measured at 340 nm using a UV-visible spectrophotometer (Molecular Devices).

GSH-Rd activity was determined according to the method of Worthington with slight modifications⁴⁴. Briefly, 150 μL of GSSG with 30 µL of GSH-Rd assay buffer (100 mM potassium phosphate buffer, pH 7.5, with 1 mM EDTA) was added to 30 µL of the serum sample and diluted with GSH-Rd dilution buffer (100 mM potassium phosphate buffer, pH 7.5, with 1 mM EDTA and 1 mg/mL bovine serum albumin). Then, 75 μL of DTNB and 2 mM NADPH were added, and the absorbance was read at 412 nm.