Isotope-assisted metabolic flux analysis (isotope MFA) is a methodology for the measurement of intracellular metabolic fluxes through a metabolic network. Isotope MFA proceeds by sequentially performing isotope labeling experiments, measuring the ensuing isotopomer patterns (usually by mass spectrometry) and using a mathematical model to interpret these patterns. Because isotope MFA directly measures how carbon flows and partitions in metabolic pathways, it complements and is a more direct gauge of metabolic activity than other ’omics methods such as transcriptomics or proteomics. Isotope MFA is a complex procedure due to the significant number of isotope measurements needed and the development of a mathematical model that relates isotopomers to fluxes. Accurate identifiability of fluxes in isotope MFA depends on the specific types of isotopically labeled substrates supplied to the cell culture under study. Previous studies have predominantly focused on carbon (13C) labels. This is natural because most metabolites have carbon backbones. However, there is potential for employing isotopes of other elements including 18O and 15N in isotope MFA. Here, we will present a computational and experimental evaluation of the potential of two types of 18O-labeled glucose (2-18O and 5-18O glucose) in isotope MFA. Our computational studies have revealed that these 18O-labeled varieties of glucose complement metabolic information available from 13C-labeled varieties including 1-13C and 6-13C glucose. We will present results demonstrating the merits of the 18O labels as well as the complementation between 18O and 13C labels. Furthermore, we will present experimental results on a microbial cell culture that evaluate whether the use of these labels can lead to quantification of metabolic fluxes with enhanced precision. Finally, we will also present the experimental and computational enhancements necessary for simultaneously measuring and processing isotopomers of multiple elements.

Multi-element metabolic flux analysis toward improved quantification of intracellular metabolism

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.