Quantification of XXXX metabolism using transient metabolic flux analysis.

@entity1501 @entity3042 @entity8959 @entity17262 @entity0 @entity5 @entity2041 @entity8582 @entity563 @entity505 @entity1499 @entity855
BACKGROUND: Systematic quantitative methodologies are needed to understand the heterogeneity of cell metabolism across cell types in normal physiology, disease, and treatment. Metabolic flux analysis (MFA) can be used to infer steady state fluxes, but it does not apply for transient dynamics. Kinetic flux profiling (KFP) can be used in the context of transient dynamics, and it is the current gold standard. However, KFP requires measurements at several time points, limiting its use in high-throughput applications. RESULTS: Here we propose transient MFA (tMFA) as a cost-effective methodology to quantify metabolic fluxes using metabolomics and isotope tracing. tMFA exploits the time scale separation between the dynamics of different metabolites to obtain mathematical equations relating metabolic fluxes to metabolite concentrations and isotope fractions. We show that the isotope fractions of @entity563 and @entity8959 are at steady state 8 h after addition of a tracer, while those of @entity3042 and @entity2041 are following a transient dynamics with an approximately constant turnover rate per unit of metabolite, supporting the application of tMFA to the analysis of @entity1501 metabolism. Using tMFA, we investigate the heterogeneity of @entity1501 metabolism and the response to the antifolate @entity1499 in @entity0 cells. Our analysis indicates that @entity1499 not only inhibits @entity17262 synthesis but also induces an increase in the @entity8582 / @entity855 ratio, activation of @entity8582 kinase (AMPK), and the inhibition of protein and @entity2041 synthesis. We also find that in some @entity5 cells, the generation of one- @entity505 units from @entity563 exceeds the biosynthetic demand. CONCLUSIONS: This work validates tMFA as a cost-effective methodology to investigate cell metabolism. Using tMFA, we have shown that the effects of treatment with the antifolate @entity1499 extend beyond inhibition of @entity17262 synthesis and propagate to other pathways in central metabolism.
