Quantitative Metabolic Flux Analysis

Cells were grown in Dulbecco's modified eagle media (DMEM) without pyruvate (CELLGRO) with 10% dialyzed fetal bovine serum (Invitrogen) in 5% CO₂ at 37°C and harvested at ∼80% confluency. Stable knockdown cell lines were generated by shRNA-expressing lentivirus with puromycin selection. IDH1, IDH2 and ALDH1L2 knockdown was generated by transfecting cells with siRNA. For confirmation of knockdown, see Extended Figure 10. For metabolite measurements, metabolism was quenched and metabolites extracted by aspirating media and immediately adding -80°C 80:20 methanol:water. Supernatants from two rounds of extraction were combined, dried under N₂, resuspended in water, placed in 4°C autosampler, and analyzed within 6 h by reversed-phase ion-pairing chromatography negative-mode electrospray-ionization high-resolution MS on a stand-alone orbitrap (Thermo)^{6 (link)}. Fluxes from ¹⁴C-labeled substrates to CO₂ were measured by adding trace ¹⁴C-labeled nutrient to normal culture media, quantifying radioactive CO₂ release^{14 (link)}, and correcting for intracellular substrate labeling according to percentage of radioactive tracer in the media and fraction of particular intracellular metabolite deriving from media uptake, as measured using ¹³C-tracer. To assess the potential contribution of various metabolic pathways to NADPH production, we analyzed feasible steady-state fluxes of a genome-scale human metabolic network model^{12 (link)} constrained by experimentally measured uptake and excretion fluxes and growth rate of the iBMK cell line. The flux balance equations were solved in MATLAB with the objective function formulated to minimize the total sum of fluxes^{14 (link)}. NADPH consumption by reductive biosynthesis was determined based on reaction stoichiometries, experimentally measured cellular biomass composition, growth rate, fractional de novo synthesis of fatty acids (by ¹³C-labeling from U-¹³C-glucose and U-¹³C-glutamine), and fractional synthesis of proline from glutamate versus arginine (by ¹³C-labeling from U-¹³C-glutamine). Correction for the deuterium kinetic isotope effect was based on the assumption that total metabolic fluxes are not impacted. Let x be the fractional labeling of the relevant substrate hydrogen, F_U be the NADPH production flux from unlabeled substrate and F_L be the NADPH production flux from the labeled substrate.
F_L/x is the flux in cases without a discernible kinetic isotope effect (e.g., for ¹³C). The remaining term is the correction factor for the kinetic isotope effect: