Conditions for 13C NMR detection of 2-hydroxyglutarate in tissue extracts from isocitrate dehydrogenase-mutated gliomas

Abstract

13C NMR spectroscopy of extracts from patient tumor samples provides rich information about metabolism. However, in IDH-mutant gliomas 13C labeling is obscured in glutamate and glutamine by the oncometabolite, 2-hydroxyglutaric acid (2HG), prompting development of a simple method to resolve the metabolites. J-coupled multiplets in 2HG were similar to glutamate and glutamine and could be clearly resolved at pH 6. A cryogenically-cooled 13C probe but not J-resolved heteronuclear single quantum coherence spectroscopy significantly improved detection of 2HG. These methods enable the monitoring of 13C-13C spin-spin couplings in 2HG expressing IDH mutant gliomas.

Lactate Dehydrogenase C Produces S-2-Hydroxyglutarate in Mouse Testis

“…Specifically, high levels of R-2HG are produced by mutant isocitrate dehydrogenase 1 and 2 (IDH1/2) in brain cancer and acute myeloid leukemia. 4–7 S-2HG has recently been found to accumulate in renal cell carcinoma. 8 The evolutionarily conserved R-2-hydroxyglutarate (D2HGDH) and S-2-hydroxyglutarate dehydrogenases (L2HGDH) normally prevent accumulation of R-2HG and S-2HG, respectively, by catalyzing their conversion back to α -ketoglutarate.…”

Metabolomic signature of brain cancer

“…SIRM typically uses carbon ( 13 C), nitrogen ( 15 N), deuterium ( 2 H), and oxygen ( 18 O) metabolic datasets generated using stable isotope‐assisted metabolomics to calculate intracellular and extracellular metabolic fluxes. The stable isotopes most widely utilized in brain cancer studies include 13 C‐glucose, 13 C‐glutamine, and 13 C‐acetate …”

“…Alternatively, two dimensional NMR spectroscopy acquired using a large number of increments on the indirect dimension can provide 13 C‐ 13 C scalar coupling information and thus valuable information to determine the exact position of the labelled atom . 13 C NMR has been successfully applied to study tumor metabolism of primary human GBM and IDH‐mutated gliomas by infusion of 13 C‐labeled nutrients (glucose, acetate and glutamine) …”

“…The stable isotopes most widely utilized in brain cancer studies include 13 C-glucose, 13 C-glutamine, and 13 C-acetate. [59][60][61] FIGURE 1 Major metabolomic pathways involved in brain tumor metabolism. The highlighted boxes represent the potential metabolic pathways involved in brain tumor: glycolytic intermediates, glutamine, lipids, and TCA cycle metabolites significantly alter in malignant brain tumors.…”

“…75,76 13 C NMR has been successfully applied to study tumor metabolism of primary human GBM and IDH-mutated gliomas by infusion of 13 C-labeled nutrients (glucose, acetate and glutamine). [59][60][61] FIGURE 2 Workflow for brain tumor metabolomics. Metabolites are extracted from biological samples (cell, tissue, and biofluids) with the addition of internal standards using liquid-liquid extraction; polar and non-polar aliquots are dried and subsequently analyzed by liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance spectroscopy (NMR).…”