Central metabolism is comprised of a network of pathways that interconnect.  Eukaryotic cells have evolved multiple routes to feed substrates into the major energy pathways that produce ATP and other compounds essential for cell survival.  For example, the TCA cycle is a major oxidative pathway for generating ATP, but can be accessed via various routes that include glycolysis, fatty acid oxidation, and amino acid catabolism.  When cells are low on glucose, they may switch to fatty acid oxidation or amino acid catabolism to keep the TCA cycle replenished.  In a typical metabolomics experiment, it is unclear from what pathway these downstream intermediates derived from, and even when major shifts in metabolism have occurred, their levels may not be differentiated.  By using 13C tracers (such as 13C glucose or 13C palmitate), one can measure the rate of 13C incorporation into various downstream metabolites to decipher differences in metabolic states of cells.  This can be done in both cell culture as well as animal models. 

We have developed LC-MS/MS methods to determine 13C enrichment in glycolysis, pentose phosphate pathway, TCA cycle, amino acids, and acylcarnitines.  We measure multiple isotopologue forms of critical compounds such as TCA intermediates, since the labeling composition will vary through multiple rounds of the cycle.  We report the enrichment ratios (13C/total C) as well as absolute concentrations of each metabolite.