13C Metabolic flux analysis (13C MFA)

Our in-house developed software, Isodyn [https://github.com/seliv55/isodyn], was used to simulate the transfer of the tracers from [1,2-¹³C₂]-glucose or [U-¹³C₅]-glutamine medium into intracellular metabolites. Isodyn is a program written in C++ and designed to simulate the dynamics of metabolite labeling by stable isotopic tracers^18–22. This program automatically constructs and solves a large system of ordinary differential equations which describe the evolution of isotopologue concentrations of metabolites produced in glycolysis, TCA cycle and PPP. Initially, all the metabolites except for introduced labeled substrates with known isotopologue composition in the medium are considered to be non-labeled and initial total concentrations of intracellular metabolites are calculated as a function of model parameters assuming a steady state at the initial moment. There is a function designed specifically for each type of reaction (i.e. carboxylation, decarboxylation) and these functions simulate transformation of carbon skeleton (specific transition of labeled carbon) and consumption and production rates of each isotopologue in the considered system. These transformations redistribute ¹³C isotopes in all metabolites, so that, individual rates which determine the values of the derivatives for the isotopologues are calculated for each isotopologue. To solve this system, a method of numerical integration is chosen arbitrarily (Runge-Kutta, BDF, Dassl). Isodyn simulates a real-time course of label propagation starting from the initial values of experimental conditions of incubation. As it compares the experimental and computed data for corresponding time points, reaching an isotopic steady state is not necessary.