Figure 2. Metabolic effect of diamide challenge in RBCs from WT and G6PD_Med- mice.

RBCs from WT and G6PD_Med- mice were incubated with 1 mM diamide (A). RBCs were tested at 0 minutes (no diamide), 30 minutes, 1 hour, 2 hours, 6 hours, and 12 hours from incubation with diamide prior to metabolomics analysis. Multivariate analyses including PLS-DA (B) and hierarchical clustering analysis (C) clearly indicate a time-dependent effect of the treatment on RBCs (PC1 explaining 45.5% of the total variance) and highlight the impact of G6PD activity (PC3 explaining 10.7% of the total variance). Significant metabolites by repeated measures 1-way ANOVA are shown in the heatmap in C. (D) The experiment was repeated by incubating RBCs in the presence of [1,2,3-¹³C₃]-glucose. By quantifying isotopologs M+2 and M+3 of lactate (and the relative ratio), fluxes through PPP versus glycolysis can be determined (E — median ± range) and confirm a significantly lower activation of this pathway in RBCs from G6PD_Med- mice upon diamide challenge. n = 4 for both groups. **P < 0.01, ***P < 0.001. EMP, Embden-Meyerhof-Parnas.