Background: Two-hydroxyglutarate (2HG) is present at low concentrations in healthy mammalian cells as both an L and D enantiomer. Both the L and D enantiomers have been implicated in regulating cellular physiology by mechanisms that are only partially characterized. In multiple human cancers, the D enantiomer accumulates due to gain-of-function mutations in the enzyme isocitrate dehydrogenase (IDH) and has been hypothesized to drive malignancy through mechanisms that remain incompletely understood. While much attention has been dedicated to identifying the route of 2HG synthesis, the metabolic fate of 2HG has not been studied in detail. Yet the metabolism of 2HG may have important mechanistic consequences influencing cell function and cancer pathogenesis, such as modulating redox potential or producing unknown products with unique modes of action.
Results: By applying our isotope-based metabolomic platform, we unbiasedly and comprehensively screened for products of L- and D-2HG in HCT116 colorectal carcinoma cells harboring a mutation in IDH1. After incubating HCT116 cells in uniformly (13)C-labeled 2HG for 24 h, we used liquid chromatography/mass spectrometry to track the labeled carbons in small molecules. Strikingly, we did not identify any products of 2HG metabolism from the thousands of metabolomic features that we screened. Consistent with these results, we did not detect any significant changes in the labeling patterns of tricarboxylic acid cycle metabolites from wild type or IDH1 mutant cells cultured in (13)C-labeled glucose upon the addition of L, D, or racemic mixtures of 2HG. A more sensitive, targeted analysis revealed trace levels of isotopic enrichment (<1 %) in some central carbon metabolites from (13)C-labeled 2HG. However, we found that cells do not deplete 2HG from the media at levels above our detection limit over a 48 h time period.
Conclusions: Taken together, we conclude that 2HG carbon is not readily transformed in the HCT116 cell line. These data indicate that the phenotypic alterations induced by 2HG are not a result of its metabolic products.
Keywords: 13C-tracing; 2-Hydroxyglutarate; Cancer metabolism; Isotope-based metabolomics; Mass spectrometry; Metabolomics.