2.2. Glutathione Assays

To measure GSH and GSSG levels from the same sample, snap-frozen liver pieces (~0.3 g) were homogenized in 0.8 mL of 10 mM HCl and proteins were removed by adding 5-sulfosalicylic acid to 1% (w/v) followed by centrifugation. Reaction mixes contained 120 mM NaH₂PO₄, pH 7.4, 5.3 mM EDTA, 0.75 mM DTNB (5,5′-dithiobis-2-nitrobenzoic acid; Sigma-Aldrich D8130, St. Louis, MO, USA), 0.24 mM NADPH and 1.2 IU/mL yeast Gsr (Sigma-Aldrich G9297), and 5 μL of a 1/20 dilution of clarified lysate, and were assayed at room temperature by absorbance at 412 nm in a Versamax plate reader (Molecular Devices, San Jose, CA, USA) [23,43]. Standard curves contained dilutions ranging from 0 to 1600 pmole GSH. To measure GSSG, 25 uL of the deproteinized lysate was added to 465 µL 120 mM NaH₂PO₄, pH 7.4, 5.3 mM EDTA and 10 µL 1 M 2-vinylpyridine in ethanol was immediately added. Samples were incubated at room temperature for 1–3 h in darkness to block free thiols. Assays used 20 µL of the blocked lysate in 120 mM NaH₂PO₄, pH 7.4, 5.3 mM EDTA, 0.75 mM DTNB, 0.24 mM NADPH and 1.2 IU/mL recombinant yeast Gsr (Sigma-Aldrich G9297). Standard curves contained 0–500 pmole GSSG and 20 mM 2-vinylpyridine. GSH concentrations were calculated by subtraction of GSSG concentration from total glutathione concentrations. Protein content was determined by the bicinchoninic acid (BCA) method following the manufacturer’s protocols (Sigma-Aldrich BCA1). In vivo redox probe imaging technologies have revealed that total glutathione pools in live cell cytosol are exceptionally reduced (GSH:GSSG ratios in cytosol of living cells ~10⁴) [44]. However, in biochemical assays, homogenization releases abundant GSSG from the endoplasmic reticulum (ER) [45], releases ROS from compartments including ER and peroxisomes, and exposes samples to environmental oxidants. As such, measured GSSG levels in biochemical analyses are typically orders of magnitude higher than the actual cytosolic level that was present in the pre-homogenized living cells or tissue (GSH:GSSG ratios typically 10¹–10²). To assess how much GSSG in our samples arose from post-homogenization oxidation of GSH as opposed to release of compartmentalized GSSG, a separate GSSG assay was carried out wherein fresh-harvested liquid nitrogen-snap-frozen liver pieces and frozen buffer (containing 50 mM Tris pH 7.5, 150 mM NaCl, 1% NP-40, 50 mM N-ethylmaleimide, 30 mM iodoacetimide, 0.6% sulfosalicylic acid and 5% metaphosphoric acid) were co-pulverized into a fine homogeneous powder at −80 °C using a custom-fabricated ultra-low temperature Teflon/tungsten-carbide bead homogenizer (Imperium Engineering, Butte, MT, USA) driven by a B. Braun Melsungen Mikro-Dismembrator-II powerhead (Melsungen, Germany). The frozen powder was then thawed and incubated at room temperature to alkylate the GSH, followed by deproteination and dilution into the GSSG assay as described above. This procedure is not compatible with GSH measurements, so it was only used for GSSG validation. The results, shown in Supplementary Figure S3 had 2- to 3-fold less GSSG than did the non-alkylated samples (Figures 3–5 and Figure S1), yet also confirmed that total glutathione dynamics measured in this study were associated with loss of GSH and not substantial changes in the redox status of the glutathione pool.