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

Abstract

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-hydroxyglu-tarate (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-hydro-xyglutarate 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 homonu-clear and heteronuclear two-dimensional 900 MHz NMR spectroscopy even in case of real tumor tissue sample extracts without physical separation of metabolites.