Simultaneous 2H and 13C Metabolic Flux Analysis of Liver Metabolism Using NMR and GC-MS—Methods Validation and New Applications

Hepatic metabolism is critically altered by the pathology of many diseases, including obesity, diabetes and NAFLD. Quantitative estimation of gluconeogenic flux and TCA cycle turnover are clinically useful parameters as they represent major biosynthetic and energetic pathways that are perturbed by these diseases. NMR spectroscopy is an important technique used to study hepatic fluxes since it can precisely distinguish between 2H and 13C metabolic tracers, and provide position-specific enrichment. However, the throughput of NMR for flux analysis is hindered by its low sensitivity, making microscale experiments that have limited biological mass (e.g., primary hepatocytes, tissue biopsies or mouse blood analysis) often infeasible. Thus, we investigate an alternative approach based on GC-MS measurements of glucose mass isotopomers and mathematical modeling of metabolic fluxes. In contrast with NMR, MS approaches are very sensitive, but cannot directly distinguish nuclei or positional enrichment information. Hence, we validated a GC-MS method in isolated perfused mouse liver and in vivo infusions in rats against NMR measurements. A medium-scale metabolic network containing atom transitions for both hydrogen and carbon atoms in the gluconeogenic pathway and TCA cycle was able to deconvolute 2H and 13C labeling information and estimate metabolic fluxes. Although some fluxes were significantly different between the NMR and GC-MS, there was excellent correlation between the two approaches. Finally, we applied GC-MS based simultaneous 2H and 13C approach in primary mouse hepatocytes cultured in 60 mm dishes, an experiment not feasible using standard NMR equipment. This opens future possibilities for drug screening and translational flux studies. Disclosure S. Deja: None. J.A. Fletcher: None. B. Kucejova: None. X. Fu: None. J. Young: None. S.C. Burgess: None.