*2.6. Glutathione Redox State Measurement*

To 0.5 mg/mL of muscle homogenate, 5% (*v*/*v*) sulfosalicylic acid was added, and it underwent two cycles of freezing and thawing. Subsequently, it was centrifuged at 10,000 rpm for 5 min and the supernatant was extracted.

Total glutathione (GSH + GSSG) and oxidized glutathione (GSSG) were determined by an enzymatic method. GSH + GSSG levels were determined using 90 μL of the supernatant, resuspended in phosphate buffer (K2HPO4, 0.1 M, pH 7.5), and mixed with 3 mM 5,5 -dithiobis 2-nitrobenzoic acid (DTNB) and 0.115 μ/mL glutathione reductase in a final volume of 1 m. After 5 min incubation at room temperature, 2 mM NADPH was added and the reaction kinetics were determined for 15 min. The increase in absorbance at 412 nm measured on a Shimadzu UV-2550 spectrophotometer was converted to GSH concentration using a standard curve with known GSH values [28,29].

For GSSG determination, the same DNTB recycling assay was applied after incubating for 1 h at room temperature with 4% vinylpyridine (*v*/*v*) to derivatize the reduced GSH [30]. Reduced glutathione (GSH) was determined by subtracting the concentration of GSH + GSSG minus that of GSSG. Data were expressed as μMoles/mg protein.
