Model-based metabolic flux analysis (MFA) using isotope-labeled substrates has provided great insight into intracellular metabolic activities across a host of organisms. One challenge with applying MFA in mammalian systems, however, is the need for absolute quantification of nutrient uptake, biomass composition, and byproduct release fluxes. Such measurements are often not feasible in complex culture systems or in vivo. One way to address this issue is to estimate flux ratios, the fractional contribution of a flux to a metabolite pool, which are independent of absolute measurements and yet informative for cellular metabolism. Prior work has focused on "local" estimation of a handful of flux ratios for specific metabolites and reactions. Here, we perform systematic, model-based estimation of all flux ratios in a metabolic network using isotope labeling data, in the absence of uptake/release data. In a series of examples, we investigate what flux ratios can be well estimated with reasonably tight confidence intervals, and contrast this with confidence intervals on normalized fluxes. We find that flux ratios can provide useful information on the metabolic state, and is complementary to normalized fluxes: for certain metabolic reactions, only flux ratios can be well estimated, while for others normalized fluxes can be obtained. Simulation studies of a large human metabolic network model suggest that estimation of flux ratios is technically feasible for complex networks, but additional studies on data from actual isotopomer labeling experiments are needed to validate these results. Finally, we experimentally study serine and methionine metabolism in cancer cells using flux ratios. We find that, in these cells, the methionine cycle is truncated with little remethylation from homocysteine, and polyamine synthesis in the absence of methionine salvage leads to loss of 5-methylthioadenosine, suggesting a new mode of overflow metabolism in cancer cells. This work highlights the potential for flux ratio analysis in the absence of absolute quantification, which we anticipate will be important for both in vitro and in vivo studies of cancer metabolism.

Biological samples such as tissues, blood, or tumors are often complex and harbor heterogeneous populations of cells. Separating out specific cell types or subpopulations from such complex mixtures to study their metabolic phenotypes is challenging because experimental procedures for separation may disturb the metabolic state of cells. To address this issue, we developed a method for analysis of cell subpopulations using stable isotope tracing and fluorescence-activated cell sorting followed by liquid chromatography-high-resolution mass spectrometry. To ensure a faithful representation of cellular metabolism after cell sorting, we benchmarked sorted extraction against direct extraction. While peak areas differed markedly with lower signal for amino acids but higher signal for nucleotides, mass isotopomer distributions from sorted cells were generally in good agreement with those obtained from direct extractions, indicating that they reflect the true metabolic state of cells prior to sorting. In proof-of-principle studies, our method revealed metabolic phenotypes specific to T cell subtypes, and also metabolic features of cells in the committed phase of the cell division cycle. Our approach enables studies of a wide range of adherent and suspension cell subpopulations, which we anticipate will be of broad importance in cell biology and biomedicine.

Mammalian cell types exhibit specialized metabolism, and there is ample evidence that various co-existing cell types engage in metabolic cooperation. Moreover, even cultures of a single cell type may contain cells in distinct metabolic states, such as resting or cycling cells. Methods for measuring metabolic activities of such subpopulations are valuable tools for understanding cellular metabolism. Complex cell populations are most commonly separated using a cell sorter, and subpopulations isolated by this method can be analyzed by metabolomics methods. However, a problem with this approach is that the cell sorting procedure subjects cells to stresses that may distort their metabolism. To overcome these issues, we reasoned that the mass isotopomer distributions (MIDs) of metabolites from cells cultured with stable isotope-labeled nutrients are likely to be more stable than absolute metabolite concentrations, because MIDs are formed over longer time scales and should be less affected by short-term exposure to cell sorting conditions. Here, we describe a method based on this principle, combining cell sorting with liquid chromatography-high resolution mass spectrometry (LC-HRMS). The procedure involves analyzing three types of samples: (1) metabolite extracts obtained directly from the complex population; (2) extracts of "mock sorted" cells passed through the cell sorter instrument without gating any specific population; and (3) extracts of the actual sorted populations. The mock sorted cells are compared against direct extraction to verify that MIDs are indeed not altered by the cell sorting procedure itself, prior to analyzing the actual sorted populations. We show example results from HeLa cells sorted according to cell cycle phase, revealing changes in nucleotide metabolism.