3045-LB: Unbiased Global Metabolic Fluxomics (UGMF) in Monogenetic Disorder Citrin Deficiency (CD): Unexpected Alterations in Metabolic Source of Urea Cycle Intermediates

Introduction and Objective: A goal of UGMF is to identify unexpected flux alterations in disease or genetic variation. Current UGMF methods introduce tracers at a specific metabolic location. This biases global results and introduces complications. The exception is 2H2O, which distributes universally and enters covalent C-H bonds through well described reactions, revealing metabolic history. Methods: We present a UGMF technique with 2H2O and combinatorial analysis and apply this to CD, a monogenic disorder of aspartate:glutamate mitochondrial transporter. CD and WT mice were injected with 2H2O for 24 hr. Liver extracts were analyzed by LC-MS/MS. Inaccuracy of isotope ratios was corrected by machine learning. Pathway contributions were inferred from number of C-H bonds exchanging with 2H2O. Results: Significant differences were found for many metabolites, including lower gluconeogenic input in CD. An unexpected result was highly significant (p < 10-5) differences in source of urea cycle (UC) intermediates ornithine (Orn) and citrulline (Ctln): 40 - 50% from de novo synthesis in CD versus 15% in WT (Fig) Conclusion: 2H2O UGMF revealed a novel change in metabolic source of UC intermediates in CD. Greater input from de novo synthesis may represent compensatory increase in extrahepatic Orn/Ctln synthesis or slowing of Arginase flux, and may be a biomarker of ammonia toxicity Disclosure N. Ziari: None. Z. Yu: None. R. Ramamurthy: None. H.G. Van Blarcom: None. J. Baur: Research Support; Current; Eli Lilly and Company. Research Support; Ended; Calico. Advisory Panel; Current; Cytokinetics Inc. Other - Donated reagents (no financial support); Current; Elysium Health. Research Support; Current; Metro Biotech. M.K. Hellerstein: Research Support; Current; Lilly, Merck & Co., Inc.