Associating metabolomics and mass isotopomer analysis reveals unexpected metabolite transfers between pathways. This can be achieved by (i) regional or specific labeling of the metabolome, and (ii) measuring the concentration and mass isotopomer distribution of metabolites, including phosphoesters and CoA esters.

Regional labeling. Rat livers were perfused with 40% NaH¹³CO₃, lactate and various inhibitors of gluconeogenesis. The labeling of citric acid cycle (CAC) and gluconeogenic intermediates reveals that labeling of phosphoenolpyruvate is less than (i) triose phosphates and (ii) half of glucose (especially in the presence of inhibitors of PEP production). The data are compatible with unknown reactions linking a CAC intermediate and glyceraldehyde-3-P.

Specific labeling. Rat livers were perfused with M9 [U-¹³C₉]azelate (C₉ dicarboxylate believed to be partially oxidized in peroxisomes). We demonstrated that (i) M9 azelate undergoes complete oxidation to mitochondrial M3 malonyl-CoA + M2 acetyl-CoA, (ii) M3 malonyl-CoA is converted to M3 methylmalonyl-CoA, an anaplerotic substrate of the CAC, (iii) M9 azelate is a major contributor of mitochondrial acetyl-CoA, and (iv) the MID of CAC intermediates reveals entry of label via both methylmalonyl-CoA and acetyl-CoA.

These metabolite transfers between pathways could not have been identified by simple metabolomics without isotopomer analysis. Supported by NIH Roadmap grant R33DK070291.