2163-LB: Metabolic Fluxomics with Heavy Water Labeling

Introduction and Objective: We introduce a heavy water (2H2 O) labeling technique to measure fluxes across a metabolome. The principle is that metabolic pathways comprise enzymatic reactions that exchange hydrogen between solvent water and covalent C-H bonds. Methods: The number (n) of exchangeable C-H sites is calculated from combinatorial probabilities (isotope ratios) after 2H2 O labeling, in a single LC-MS/MS analysis. This reveals contributions from different pathways traversed. Results: We first validated targeted fluxes in vivo in mice - e.g., hepatic glycolysis and glyceroneogenesis; plasma gluconeogenesis. We then measured untargeted fluxomics in central metabolism (Figure), including effects of inborn errors (Citrin deficiency) and T2D. Global flux ratios include sources of UDP-glucose, glycolytic and TCA cycle intermediates, acetyl-CoA, pentose phosphates, amino acids, purines and pyrimidines. The list can be expanded. Fig. Central metabolic fluxes in mouse liver. Numbers represent n (exchangeable C-H bonds), revealing pathway contributions. Conclusion: Labeling with 2H2 O has several benefits over 13C- methods. 13C- tracers enter locally whereas 2H2 O is distributed globally, is immune to non-linear error propagation and label recycling, and experimental logistics are simpler. This method has implications for diabetes research. Metabolic flux signatures, changes during diabetogenesis and effects of therapeutics can be explored. This fluxomics technique is translatable to humans. Disclosure N. Ziari: None. M.K. Hellerstein: Research Support; Lilly USA LLC. Consultant; Lilly USA LLC. Funding University of California CRCC predoctoral fellowship