can occur depending on how much of the original metabolite follows the left side of the pathway versus the right side of the pathway. Here the proportions are shown for a situation in which half of the metabolites take the left side and half the right, but other proportions can occur. These patterns of labeled atoms and unlabeled atoms in one compound represent isotopomers. By measuring the isotopomer distribution of the differently labeled metabolites, the flux through each reaction can be determined. MFA combines the data harvested from isotope labeling with the stoichiometry of each reaction, constraints, and an optimization procedure resolve a flux map. The irreversible reactions provide the thermodynamic constraints needed to find the fluxes. A matrix is constructed that contains the stoichiometry of the reactions. The intracellular fluxes are estimated by using an iterative method in which simulated fluxes are plugged into the stoichiometric model. The simulated fluxes are displayed in a flux map, which shows the rate of reactants being converted to products for each reaction. In most flux maps, the thicker the arrow, the larger the flux value of the reaction. Isotope labeling measuring techniques Any technique in measuring the difference between isotopomers can be used. The two primary methods, nuclear magnetic resonance (NMR) and mass spectrometry (MS), have been developed for measuring mass isotopomers in stable isotope labeling. Proton NMR was the first technique used for 13C-labeling experiments. Using this method, each single protonated carbon position inside a particular metabolite pool can be observed separately from the other positions. This allows the percentage of isotopomers labeled at that specific position to be known. The limit to proton NMR is that if there are n carbon atoms in a metabolite, there can only be at most n different positional enrichment values, which is only a small fraction of the total isotopomer information. Although the use of proton NMR labeling is limiting, pure proton NMR experiments are much easier to evaluate than experiments with more isotopomer information. In addition to Proton NMR, using 13C NMR techniques will allow a more detailed view of the distribution of the isotopomers. A labeled carbon atom will produce different hyperfine splitting signals depending on the labeling state of its direct neighbors in the molecule. A singlet peak emerges if the neighboring carbon atoms are not labeled. A doublet peak emerges if only one neighboring carbon atom is labeled. The size