Sphk1 and hypoxia impact metabolic fluxes through glycolysis and the pentose phosphate pathway in stored murine RBCs. Tracing experiments with 1,2,3-¹³C₃-glucose in stored RBCs from WT and Sphk1 mice (A) afford discrimination of metabolic fluxes through glycolysis and the PPP (B) via determination of the ratio of +3 and +2 lactate isotopologues (C). Increases in glycolysis were observed in hypoxia in WT mice but not in Sphk1 mice. (D) However, glycolysis/PPP ratios were basally lower in Sphk1 mice than in controls and were increased by storage in hypoxia in both groups (despite Sphk1 preserving higher PPP activation compared with WT) (median ± ranges). (E) A summary overview of the main findings of this study is provided.