900MHz (1)H-/(13)C-NMR analysis of 2-hydroxyglutarate and other brain metabolites in human brain tumor tissue extracts

PURPOSE: To 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 (1)H but also for (13)C with indirect detection by heteronuclear single quantum correlation (HSQC). MATERIAL AND METHODS: Four 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 (D(2)O, 99.9 atom % deuterium) containing 0.025 wt % sodium 3-(trimethylsilyl)propionate-2,2,3,3-d(4) (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 (1)H/(13)C/(15)N 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 (1)H and (13)C. (1)H 1D, (1)H-(13)C HSQC, (1)H-(1)H correlation spectroscopy (COSY) and (1)H-(13)C heteronuclear multiple bond correlation (HMBC) spectra were acquired and analyzed. RESULTS: 2-hydroxyglutarate, an oncometabolite associated with gliomas with IDH mutations, was successfully detected and assigned by both (1)H-(13)C HSQC and (1)H-(1)H COSY experiments as well as (1)H 1D experiments in two of the tissue samples. In particular, to our knowledge this work shows the first example of detecting 900 MHz (13)C-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. CONCLUSION: The 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.