Figure 2.

(A) Schematic flow chart of ¹³C-based metabolic flux analysis. Exemplary results for flux ratios and absolute fluxes are given in the bottom boxes. (B) Example of inferring relative fluxes through the three initial pathways of glucose catabolism in E. coli from mass spectrometry data. A positional label is introduced by feeding [1-¹³C]glucose, and ¹³C-pattern are detected in alanine, which derives its carbon backbone directly from pyruvate. Although unique isotope pattern occurs in intact alanine molecules, the lack of positional information in the detected mass distribution cannot discriminate between glycolysis and the ED pathway. For discrimination of these two pathways, additionally the C2–C3 moiety of alanine must be analyzed, which occurs by fragmentation in some MS instruments. For flux ratios, the relative contribution of these pathways to the formation of alanine (pyruvate) is calculated directly from the detected abundance of the different mass isotope isomers by probabilistic equations. For absolute fluxes, a best-fit flux solution is obtained by extensive computations that seek to minimize the error between fitted intracellular fluxes not only to the six shown, but also to all other detected mass spectra and physiological uptake and production rates.