Dissecting mammalian cell metabolism through 13C- and 2H-isotope tracing: Interpretations at the molecular and systems levels

Isotopic tracers have been widely used to probe specific pathways within metabolic networks. Isotopically labeled tracers are metabolized through various metabolic routes, generating differential labeling patterns. Correct interpretations of the labeling data require understanding of atomic transitions of isotopes along biochemical reactions. Here, we review common 13C- and 2H-isotopic tracers used to investigate mammalian cell metabolism. Labeling patterns obtained from those tracers are presented and explained through stepwise tracking in the tricarboxylic acid (TCA) cycle, glycolysis, pentose phosphate pathway (PPP) and anaplerotic reactions. Metabolic insights regarding relative pathway activities and substrate utilization patterns are obtained by analyzing distinct enrichment profiles of metabolites. Finally, we introduce and demonstrate the use of 13C-metabolic flux analysis (13C-MFA) for deciphering complex metabolic networks at the systems level.