Metabolic Flux Analysis

Metabolic Flux Analysis and Tracer Experiments

• Metabolic fluxes can be described by mass balances of internal metabolites using the vector of metabolite concentrations (X), flux distribution vector (v), and stoichiometric matrix (S).
• The stoichiometric matrix for Metabolite Balancing in MFA is constructed according to material balance equations.
• A metabolic network reconstruction consists of stoichiometrically balanced biochemical reactions, represented in a matrix labeled S, with rows for metabolites and columns for reactions.
• Growth is incorporated into the reconstruction with a biomass reaction, and exchange reactions are used to represent the flow of metabolites in and out of the cell.
• At steady state, the flux through each reaction is given by Sv = 0, defining a system of linear equations with multiple possible solutions.
• Linear programming is used to identify a flux distribution that maximizes or minimizes an objective function within the space of allowable fluxes defined by mass balance equations and reaction bounds.
• Tracer experiments, MS, and NMR can be used to infer metabolic flux through the use of isotopic tracers, with 13C-MFA considered a gold standard for accurate and precise flux information.
• The workflow for 13C-MFA involves designing an optimal tracer experiment, measuring metabolite labeling, synthesizing tracer data using computational models, and reaching the best global fit for flux estimation.
• Classic MFA, Flux Balance Analysis (FBA), and constraint-based reconstruction and analysis (COBRA) methods are other techniques for quantifying metabolic fluxes in biological systems.
• These techniques require no 13C-labeling experiments and involve fewer experimental data, but to constrain metabolic fluxes, the stoichiometric matrix for the given reaction networks must be written.
• Computational programs help reach the best global fit for flux estimation, followed by statistical analysis to evaluate the fit, and further labeling experiments could be proposed if the solution is not optimal.
• Key references for metabolic flux analysis include works by de Falco et al. (2022), Emwas et al. (2022), Kwilas et al. (2015), and Nielsen (2014), as well as the study by Orth et al. (2010) on flux balance analysis.