Oxidative stress drives GAPDH irreversible modification and pentose phosphate pathway activation. (A) An overview of the metabolic flux analysis experiment, where four units were spiked with 11 mM ¹³C_1,2,3-glucose and then stored either under normoxic (blue), hyperoxic (>95% SO₂, magenta) or hypoxic (5% SO₂, light green) conditions (n = 4 per group). (B) Hemoglobin oxygen saturation in normoxic red blood cells reached ∼95% by storage week 3, when 2,3-DPG reservoirs were totally consumed (supplemental Figure 2). (C) MetHb accumulation was higher in hyperoxic red blood cells during the first 3 storage weeks, before SO₂ became comparable in both groups, whereas it remained significantly lower throughout storage duration in hypoxic red blood cells in comparison with normoxic and hyperoxic treatments. (D) PPP to glycolysis ratios were determined by dividing lactate isotopologues (+2/+3) at each tested time point (2-42, on a weekly basis). Continuous lines: median (blue, normoxic control; magenta, hyperoxic red blood cells); dashed lines: interquartile ranges. Although PPP remains active in all 3 groups, hyperoxic red blood cells are characterized by the highest activation rate. (E) Targeted SRM-based relative quantitation was performed for intracellular irreversibly oxidized Cys152 of GAPDH (normalized vs the relative abundance of the unmodified IISNASCTTNCLAPLAK peptide containing the active site residue) in normoxic, hyperoxic, and hypoxic red blood cells at storage day 2, 21, and 42. Significantly higher levels of irreversibly oxidized Cys152 of GAPDH were determined at storage day 2 but not at later time points, when SO₂, methemoglobin levels and PPP:glycolysis ratios were comparable in either groups. * and # indicate significance (P < .05; < 0.01; < 0.001) vs normoxic controls or hyperoxic units, respectively.