Figure 4.

Tracer compositions should be scrutinized to optimize information for pathways of interest. The example displays a simple glycolytic pathway in which we assume that reactions only flow in the forward direction. Uniformly labeled glucose results in identical isotopomers from the PPP (purple box) and glycolysis. Using a combination of 50% fully labeled glucose with 50% unlabeled glucose can better inform PPP fluxes, as the complex atom rearrangement in the PPP leads to M+1, M+2, M+4, and M+5 isotopomers (shown in red box). The percent composition of these isotopomers can be used to determine flux activity at the PPP branchpoints. A more direct option is to use [1,2-¹³C₂] glucose, which generates an M+1 isotopomer after entering the PPP by being metabolizing into R5P that is further metabolized into M+1 F6P and G3P. In contrast, the glucose that is directly metabolized into F6P generates M+2 isotopomers. Therefore, the ratio of M+1 and M+2 isotopomers in metabolites such as F6P and G3P, as well as derivatives such as lactate and pyruvate, can be compared to determine relative activity at the PPP branchpoint. Red = carbons that pass through the PPP; black = carbons that pass through the main glycolytic pathway. The red, dashed boxed encompasses isotopomers that can only be formed after passing through the PPP. Abbreviations: glucose-6-phosphate (G6P), ribose-5-phosphate (R5P), fructose-6-phosphate (F6P), glyceraldehyde-3-phosphate (G3P), pentose phosphate pathway (PPP).