Figure 2. Isotope-assisted metabolic flux helps quantify multiple TCA cycle turns.

Cyclic metabolic networks, such as the TCA cycle, generate a different set of isotopomers at each turn. This simplified TCA cycle example assumes that uniformly labeled glucose and an unlabeled oxaloacetate (OAA) are the only carbon contributors. Each color represents a different TCA cycle turn. The initial unlabeled OAA reacts with glucose-derived, fully labeled acetyl-CoA to form [M+2] citrate via citrate synthase (CS). This citrate is then metabolized into α-ketoglutarate (AKG) via isocitrate dehydrogenase (IDH). As the AKG progresses through the TCA cycle (metabolites not shown), “flipped” isotopomers of OAA with either the first and second or the third and fourth carbons labeled can occur. When these two OAA isopomers react with fully labeled acetyl-CoA to form [M+4] citrate in the second TCA cycle turn, two unique citrate and AKG isotopomers are created. With each turn of the TCA cycle, more isotopomers are created in different concentrations until, with infinite TCA cycle turns, the metabolites reach metabolic steady state.