Quantification of Glutathione Redox Status

Glutathione was measured as previously published (18 (link), 20 (link)). Briefly, GSH and GSSG were measured using the Shimadzu Nexera X2 UHPLC system (Mandel Scientific). Quadriceps and diaphragm were homogenized in 50 mM Tris buffer with 10 mM boric acid, 2 mM l-serine, 20 μM acivicin, and 5 mM N-ethylamide and acidified using TCA (for GSH) and PCA (for GSSG). Samples were separated with a Zorbax C18 column (Agilent Technologies). GSH was assessed by UV-HPLC (265 nm wavelength) monitoring of NEM-GSH using a 0.25% glacial acetic acid mobile phase with 6% acetonitrile at 1.05 mL/min flow rate detected at 265 nm. GSSG was assessed by fluorescent HPLC (excited at 350 nm and detected at 420 nm emission) using HPLC/UHPLC fluorescence detector (Mandel Scientific). GSSG samples were diluted in 0.5 M NaOH and run using 25 mM Na₂HPO₄ in HPLC-grade water with 15% methanol mobile phase at a 0.5 mL/min flow rate by tracking o-pthalymide–tagged GSH.