Dataset Information

High-Throughput Indirect Quantitation of 13C Enriched Metabolites Using 1H NMR.

ABSTRACT: Nuclear magnetic resonance (NMR) spectroscopy is widely used in metabolomics to perform quantitative profiling of low-molecular weight compounds from biological specimens. The measurement of endogenous metabolites using NMR has proven to be a powerful tool to identify new metabolic biomarkers in physiological and pathological conditions, and to study and evaluate treatment efficiency. In this study we present a rapid approach to indirectly quantify 13C enriched molecules using one-dimensional (1D) 1H NMR. We demonstrate this approach using isotopically labeled [1,6-13C]glucose and in four different cell lines. We confirm the applicability of this approach for treatment follow-up, utilizing a renal cancer cell line with rapamycin as a tool compound to study changes in metabolic profiles. Finally, we validate the applicability of this method to study metabolic biomarkers from ex vivo tumor extracts, after infusion, using isotopically enriched glucose. Given the high throughput and increased sensitivity of direct-detect 1H NMR, this analytical approach provides an avenue for simple and rapid metabolic analysis of biological samples including blood, urine, and biopsies.

SUBMITTER: Di Gialleonardo V

PROVIDER: S-EPMC5148664 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature

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High-Throughput Indirect Quantitation of 13C Enriched Metabolites Using 1H NMR.

Di Gialleonardo Valentina V Tee Sui Seng SS Aldeborgh Hannah N HN Miloushev Vesselin Z VZ Cunha Lidia S LS Sukenick George D GD Keshari Kayvan R KR

Analytical chemistry 20161026 22

Nuclear magnetic resonance (NMR) spectroscopy is widely used in metabolomics to perform quantitative profiling of low-molecular weight compounds from biological specimens. The measurement of endogenous metabolites using NMR has proven to be a powerful tool to identify new metabolic biomarkers in physiological and pathological conditions, and to study and evaluate treatment efficiency. In this study we present a rapid approach to indirectly quantify 13C enriched molecules using one-dim ...[more]

PMID: 27749041

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Project description:PurposeTo perform in vitro high-resolution 900 MHz magnetic resonance spectroscopy (NMR) analysis of human brain tumor tissue extracts and analyze for the oncometabolite 2-hydroxyglutarate (2HG) and other brain metabolites, not only for 1H but also for 13C with indirect detection by heteronuclear single quantum correlation (HSQC).Material and methodsFour surgically removed human brain tumor tissue samples were used for extraction and preparation of NMR samples. These tissue samples were extracted with 4% perchloric acid and chloroform, freeze-dried, then dissolved into 0.28 mL of deuterium oxide (D2O, 99.9 atom % deuterium) containing 0.025 wt % sodium 3-(trimethylsilyl)propionate-2,2,3,3-d4 (TSP). All samples were adjusted to pH range of 6.9-7.1 before finally transferred to 5 mm Shigemi™ NMR microtube. NMR experiments were performed on Bruker DRX 900 MHz spectrometer with 1H/13C/15N Cryo-probe™ with Z-gradient, without further temperature control for the samples. All chemical shift values were presented relative to TSP at 0.00 ppm for both 1H and 13C. 1H 1D, 1H-13C HSQC, 1H-1H correlation spectroscopy (COSY) and 1H-13C heteronuclear multiple bond correlation (HMBC) spectra were acquired and analyzed.Results2-hydroxyglutarate, an oncometabolite associated with gliomas with IDH mutations, was successfully detected and assigned by both 1H-13C HSQC and 1H-1H COSY experiments as well as 1H 1D experiments in two of the tissue samples. In particular, to our knowledge this work shows the first example of detecting 900 MHz 13C-NMR spectral lines of 2-hydroxyglutarate in human brain tumor tissue samples. In addition to the oncometabolite 2-hydroxyglutarate, at least 42 more metabolites were identified from our series of NMR experiment.ConclusionThe detection of 2-hydroxyglutarate and other metabolites can be facilitated by homonuclear and heteronuclear two-dimensional 900 MHz NMR spectroscopy even in case of real tumor tissue sample extracts without physical separation of metabolites.

Dataset Information

High-Throughput Indirect Quantitation of 13C Enriched Metabolites Using 1H NMR.

Publications

High-Throughput Indirect Quantitation of <sup>13</sup>C Enriched Metabolites Using <sup>1</sup>H NMR.

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