Reduced glutathione (GSH) is considered one of the most important scavengers of reactive oxygen species (ROS), and its ratio with oxidized glutathione (GSSG) may be used as a marker of oxidative stress [41 (link)]. Total glutathione (GSH) was determined in vivo by an enzymatic recycling procedure: the sulphydryl group of the molecule reacts with 5,5′-dithiobis-2-nitrobenzoic acid (DTNB, Ellman’s reagent) (Sigma-Aldrich, St. Louis, MO, USA) producing a yellow colored 5-thio-2-nitrobenzoic acid (TNB), and the disulfide is reduced by NADPH in the presence of glutathione reductase.
Hepatic tissue was homogenized on ice in percloric acid solution 5%. The homogenate was centrifuged at 10,000× g, 4 °C for 10 min and the acid-soluble fraction was collected and neutralized with 2 M KHCO3. After a centrifugation to remove the formed potassium perchlorate, the supernatant was divided to quantify both GSH and GSSG. For GSH detection, the sample was mixed with 6 mM DTNB and 0.3 mM NADPH. After incubation at 37 °C for 3 min, 200 U/mL glutathione reductase was added and immediately the rate of produced TNB was read at 415 nm using the EnSpire® Multimode Plate Reader (Perkin Elmer, Waltham, MA, USA). Glutathione concentrations were calculated using appropriate standards and normalized by µg of proteins.
Hepatic tissue was homogenized on ice in percloric acid solution 5%. The homogenate was centrifuged at 10,000× g, 4 °C for 10 min and the acid-soluble fraction was collected and neutralized with 2 M KHCO3. After a centrifugation to remove the formed potassium perchlorate, the supernatant was divided to quantify both GSH and GSSG. For GSH detection, the sample was mixed with 6 mM DTNB and 0.3 mM NADPH. After incubation at 37 °C for 3 min, 200 U/mL glutathione reductase was added and immediately the rate of produced TNB was read at 415 nm using the EnSpire® Multimode Plate Reader (Perkin Elmer, Waltham, MA, USA). Glutathione concentrations were calculated using appropriate standards and normalized by µg of proteins.