Metabolic flux analysis (MFA) using stable isotope labeling is an important tool for explaining the flux of certain elements through the metabolic pathways and reactions within a cell. An isotopic label is fed to the cell, then the cell is allowed to grow utilizing the labeled feed. For stationary metabolic flux analysis the cell must reach a steady state (the isotopes entering and leaving the cell remain constant with time) or a quasi-steady state (steady state is reached for a given period of time). The isotope pattern of the output metabolite is determined. The output isotope pattern provides valuable information, which can be used to find the magnitude of flux, rate of conversion from reactants to products, through each reaction.
The figure demonstrates the ability to use different labels to determine the flux through a certain reaction. Assume the original metabolite, a three carbon compound, has the ability to either split into a two carbon metabolite and one carbon metabolite in one reaction then recombine or remain a three carbon metabolite. If the reaction is provided with two isotopes of the metabolite in equal proportion, one completely labeled (blue circles), commonly known as uniformly labeled, and one completely unlabeled (white circles). The pathway down the left side of the diagram does not display any change in the metabolites, while the right side shows the split and recombination. As shown, if the metabolite only takes the pathway down the left side, it remains in a 50–50 ratio of uniformly labeled to unlabeled metabolite. If the metabolite only takes the right side new labeling patterns can occur, all in equal proportion. Other proportions 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.