Oxidative modulation of glucose metabolism during RBC storage. (A) Metabolic analysis of glycolytic intermediates in AS-3 RBCs from storage day 1 to storage day 42. Despite ongoing glycolysis, as illustrated by lactate accumulation, glyceraldehyde 3-phosphate and diphosphoglycerate reach steady-state levels by storage days 14-21. (B) Metabolic flux analysis was performed by spiking 11 mM ¹³C_1,2,3-glucose (in addition to the 55 mM dextrose in AS-3 formula) into RBC concentrates at day 0. The isotopologues +3 or +2 of lactate accumulate proportionally to glucose catabolic fluxes through the Embden-Meyerhof glycolytic pathway or through the PPP, respectively. The first carbon atom of glucose is lost in the form of CO₂ during metabolism through the oxidative phase of the PPP. Metabolic fluxes were determined in lactate, and absolute quantitation of total lactate and isotopologues was performed directly against spiked in ¹³C₁-lactate (upon correction for 3.3% natural abundance) and indirectly by determining the percentage of labeled lactate in total lactate, quantified through classic spectrometric approaches. PPP to glycolysis ratios were determined by dividing lactate isotopologues (+2/+3) at each tested time point (2-42, on a weekly basis). (C) An overview of the GAPDH monomer structure (pdb ID: 3GPD) is shown. Structural models of the enzyme active site pocket, highlighting Cys152 and His179 are shown in presence of the substrate glyceraldehyde 3-phosphate or the N-term cytosolic domain of band 3 (PDB ID:3BTB), in agreement with Eisenmesser and Post.²³