Isotopically nonstationary 13C metabolic flux analysis.

Jazmin LJ, Young JD
Methods Mol Biol. 2013 985: 367-90

PMID: 23417813 · DOI:10.1007/978-1-62703-299-5_18

(13)C metabolic flux analysis (MFA) is a powerful approach for quantifying cell physiology based upon a combination of extracellular flux measurements and intracellular isotope labeling measurements. In this chapter, we present the method of isotopically nonstationary (13)C MFA (INST-MFA), which is applicable to systems that are at metabolic steady state, but are sampled during the transient period prior to achieving isotopic steady state following the introduction of a (13)C tracer. We describe protocols for performing the necessary isotope labeling experiments, for quenching and extraction of intracellular metabolites, for mass spectrometry (MS) analysis of metabolite labeling, and for computational flux estimation using INST-MFA. By combining several recently developed experimental and computational techniques, INST-MFA provides an important new platform for mapping carbon fluxes that is especially applicable to animal cell cultures, autotrophic organisms, industrial bioprocesses, high-throughput experiments, and other systems that are not amenable to steady-state (13)C MFA experiments.

MeSH Terms (10)

Algorithms Animals Carbohydrate Metabolism Carbon Isotopes Cells, Cultured Humans Isotope Labeling Kinetics Mass Spectrometry Models, Biological

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