4.5.4. Glutathione Levels

GSH levels were evaluated in the erythrocytes of peripheral blood. Fifty μL samples were centrifuged at 3000× *g* for 15 min at 0 ◦C. The erythrocytes were suspended in 5% cold (*w*/*v*) metaphosphoric acid, mixed and stored at 0 ◦C for 15 min. Subsequently, the suspension was centrifuged at 14,000× *g* for 10 min at 4 ◦C. The clarified supernatant was collected, and GSH levels were analyzed according to the instructions using Trevigen's HT Glutathione Assay Kit (Item No. 7511-100-K; Gaithersburg, MD, USA). The absorbance was read at 405 nm in a Multiskan™ FC microplate reader (Thermo Scientific™, Vantaa, Finland). The levels of GSH were determined in duplicate (per sample) according to the GSH standard curve.
