Determination of metabolite concentrations in human brain tumour biopsy samples using HR-MAS and ERETIC measurements.

Accurate determination of the concentration of the metabolites contained in intact human biopsies of 10 glioblastoma multiforme samples was achieved using one-dimensional (1)H high-resolution magic angle spinning (HR-MAS) NMR combined with ERETIC (electronic reference to in vivo concentrations) measurements. The amount of sample used ranged from 6.8 to 12.9 mg. Metabolite concentrations were measured in each sample using two methods: with DSS (2,2-dimethyl-2-silapentane-5-sulfonate sodium salt) as an internal reference and with ERETIC as an external electronically generated reference. The ERETIC signal was shown to be highly reproducible and did not affect the spectral quality. The concentrations calculated by the ERETIC method in model solutions were shown to be independent of the salt concentration in the range typically found in biological samples (0-250 mM). The ERETIC method proved to be straightforward to use in tissues and much more robust than the internal standard method. The concentrations calculated using the internal DSS concentration were systematically found to be higher than those determined using the ERETIC technique. These results indicate a possible interaction of the DSS molecules with the biopsy sample. Moreover, variations in the sample preparation process, with possible loss of DSS solution, may hamper the quantification process, as happens in one of the ten samples analysed. In this study, the ERETIC method was validated on model solutions and used in brain tumour tissues. Calculated metabolite concentrations obtained with the ERETIC procedure matched the values determined in the same type of tumours by in vivo, ex vivo and in vitro methodologies.

1H and 13C HR-MAS spectroscopy of intact biopsy samples ex vivo and in vivo 1H MRS study of human high grade gliomas.

High-resolution magic angle spinning (HR-MAS) one- and two-dimensional 1H and 13C nuclear magnetic resonance (NMR) spectroscopy has been used to study intact glioblastoma (GBM) brain tumour tissue. The results were compared with in vitro chemical extract and in vivo spectra. The resolution of 1H one-dimensional, 1H TOCSY and 13C HSQC HR-MAS spectra is comparable to that obtained on perchloric extracts. 13C HSQC HR-MAS spectra have been particularly useful for the identification of 37 different metabolites in intact biopsy tumours, excluding water and DSS components. To our knowledge, this is the most detailed assignment of biochemical compounds obtained in intact human tissue, in particular in brain tumour tissue. Tissue degradation during the recording of the NMR experiment was avoided by keeping the sample at a temperature of 4 degrees C. Detailed metabolical compositions of 10 GBM (six primary, two secondary and two unclassified) were obtained. A good correlation between ex vivo and in vivo MRS has been found.