4.3. HRMAS NMR Technical Features

All HRMAS NMR spectra were obtained on a Bruker Advance III 500 spectrometer (Bruker GmbH, Germany) installed at Hautepierre University Hospital, Strasbourg, operating at a proton frequency of 500.13 MHz. A one-dimensional (1D) proton spectrum using a Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence was acquired with an interpulse delay of 285 μs and an acquisition time of 10 min for each tissue sample. The number of loops was set to 328, giving the CPMG pulse train a total length of 93 ms. To confirm resonance assignments in a few representative samples, two-dimensional (2D) heteronuclear experiments (1H–13C) were also recorded immediately after ending the 1D spectra acquisition. Metabolites were assigned using standard metabolite chemical shift tables available in the literature [14].

HRMAS NMR spectra were recorded on a Bruker Advance III 500 spectrometer operating at a proton frequency of 500.13 MHz and equipped with a 4-mm double resonance (1H, 13C) gradient HRMAS probe. A Bruker Cooling Unit was used to regulate the temperature by cooling down the bearing air flowing into the probe. To minimize the effects of tissue degradation, all ex vivo spectra were acquired at a temperature of 4 °C. This value was calibrated exactly using a 100% methanol sample. To keep the rotation sidebands out of the spectral region of interest and to minimize sample degradation, all NMR experiments were conducted on samples spinning at 3502 Hz. For each sample, a one-dimensional proton spectrum using a Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence with presaturation of the water signal was acquired. To eliminate signal losses due to B1 inhomogeneity, the inter-pulse delay between the 180° pulses of the CPMG pulse train was synchronized with the sample and set to 285 µs.

The number of loops was set to 328, thus giving the CPMG pulse train a total length of 93 ms. The parameters for the CPMG experiment were set as follows: sweep width, 14.2 ppm; number of points, 32k; relaxation delay, 2 s; and acquisition time, 2.3 s. A total of 128 FIDs (free induction decay) were acquired resulting in an acquisition time of 10 min. Spectra were referenced by setting the lactate doublet chemical shift to 1.33 ppm. Peaks of interest were automatically defined by an in-house program using MATLAB 7.0 (MathWorks, Natick, MA, USA). In order to confirm resonance assignments, two-dimensional (2D) heteronuclear experiments (1H–13C) were also recorded immediately after the end of 1D spectra acquisition. Because the duration of these experiments was long, they were acquired on only a few samples representative of each class of tissues and exclusively for signal assignment. Significant tissue degradation occurs during this long measurement time; therefore, 1H CPMG experiments were completed before 2D signal assignment experiments. Metabolites were assigned using standard metabolite chemical shift tables available in the literature [14]. Data were zero-filled to a 2k × 1k matrix and weighted with a shifted square sine-bell function before Fourier transformation (FTIR). HRMAS NMR signals were bucketed into integral regions 0.01 ppm wide (ppm range, 8.65–1) using AMIX 3.8 software (Bruker GmbH, Germany) and exported into SIMCA P (version 11.0, Umetrics AB, Umeå, Sweden). To accommodate the influence of metabolite present at both high and low concentrations, without emphasizing spectral noise, unit variance scaling was employed for all analyses. Spectra were referenced for chemical shift according to the lactate peak.