Abstract
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.
Highlights
Material and methodsFour surgically removed human brain tumor tissue samples were used for extraction and preparation of NMR samples
Gliomas make up approximately 30% of all brain and central nervous system tumors and 80% of all malignant brain tumors [1]
2-hydroxyglutarate, an oncometabolite associated with gliomas with isocitrate dehydrogenases (IDH) mutations, was successfully detected and assigned by both 1H-13C heteronuclear single quantum correlation (HSQC) and 1H-1H correlation spectroscopy (COSY) experiments as well as 1H 1D experiments in two of the tissue samples
Summary
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, 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 transferred to 5 mm ShigemiTM NMR microtube. NMR experiments were performed on Bruker DRX 900 MHz spectrometer with 1H/13C/15N Cryo-probeTM with Z-gradient, without further temperature control for the samples.
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