Direct Estimation of Metabolic Flux by Heavy Isotope Labeling Simultaneous with Pathway Inhibition: Metabolic Flux Inhibition Assay
Heavy isotope labeled metabolites are readily detected by mass spectrometry and are commonly used to analyze the rates of metabolic reactions in cultured cells. The ability to detect labeled metabolites—and infer fluxes—is influenced by a number of factors that can confound simplistic comparative assays. The accumulation of labeled metabolites is strongly influenced by the pool size of the metabolite of interest and also by changes in downstream reactions, which are not always fully perceived. Here, we describe a method that overcomes some of these limitations and allows simple calculation of reaction rates under low nutrient, rapid reaction rate conditions. Acutely increasing the pool of the metabolite of interest (by adding a pulse of excess unlabeled nutrient to the cells) rapidly increases accumulation of labeled metabolite, facilitating a more accurate assessment of reaction rate.
Key wordsMetabolic flux inhibition assay Carbon-13 Heavy isotope metabolic tracer Pathway activity Pulse-stop-flux
ODKM and ACN are funded by Cancer Research UK Career Development Fellowship C53309/A19702. KHV and CFL are funded by CRUK Grant C596/A10419.
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