Metabolic flux analysis and fluxomics-driven determination of reaction free energy using multiple isotopes.

•The use of multiple isotopes facilitates a comprehensive and quantitative analysis of metabolic pathways.•Benefits and drawbacks of 13C, 2H, 18O, and 15N tracing in metabolism.•Isotope tracing delineates the relative contribution of alternative pathways to the production of key metabolites and cofactors.•Isotope tracing reveals the different fates of a metabolite.•Improved metabolic flux quantitation leads to thermodynamic quantitation in central carbon metabolism.