Noesygppr1d

Before MRS analysis, samples were dissolved in deuterium oxide (D₂O, Sigma-Aldrich Corporation, USA). The pH of all samples was adjusted to the same level (pH ~ 7) by perchloric acid and potassium hydroxide. MR spectroscopy was performed using a Bruker Avance III Ultrashielded Plus 600 MHz spectrometer (Bruker Biospin GmbH, Germany) equipped with a 5 mm QCI Cryoprobe with integrated, cooled preamplifiers for ¹H, ²H, and ¹³C. This MR system provided a fully automated experiment in combination with Icon-NMR on TopSpin v3.1 software (Bruker Biospin). The MR spectra were obtained at 28.05 °C using a standard protocol [29 (link)], for proton one-dimensional nuclear Overhauser effect spectroscopy (1D-NOESY) (noesygppr1d; Bruker) with the following acquisition parameters: 128 scans, acquisition time of 2.73s, relaxation delay of 4s, free induction decay (FID) size of 65536, mixing time of 10ms, spectral width of 20.0243 ppm, and a total scan time of 349s.

Tumor samples from both ¹³C glutamine-labeled (n = 5 to six per group, experiment 3) and unlabelled (N.A.: natural abundance, n = 3 per group, experiment 1) tumors (39.9 ± 1.1 mg) were cut to fit into a 50-μl zirconium HR MAS rotor (4-mm diameter). Lock reference containing D₂O with formate (25 mM) was added to the rotor (~ 16 μl). The HR MAS MR spectra were recorded using a Bruker Advance DRX600 spectrometer (14.1 T) (Bruker Biospin GmbH, Germany) containing a ¹H/¹³C MAS probe. Samples were spun at 5 kHz at magic angle, and the temperature was kept at 4 °C during the whole experiment. NMR spectra were acquired using the following NMR sequences and acquisition parameters: One-dimensional ¹H NOESY pulse sequence with water presaturation (Bruker; noesygppr1d). Acquisition time was 2.7 s, repetition time 6.7 s, sweep width was 30 ppm, and 128 scans were acquired. The ¹³C MR spectra were acquired using a single pulse experiment, with ¹H decoupling applied during recycle delay and acquisition (Bruker; zgpg30). The flip angle was 30°, acquisition time 0.9 s, repetition time 1.9 s, sweep width 250 ppm, and 16 k scans were obtained. Total acquisition time per sample was 9 h.

All samples were analyzed on an AVANCE III HD 600 MHz NMR Bruker spectrometer equipped with a cryo-probe CPQCI 1H-31P/13C/15N and a SampleJET^TM autosampler set to 5 mm shuttle mode with the tubes refrigerated at 6 °C. All analyses were run under automation by IconNMR. In order to suppress the water signal and avoid any baseline distortion following acquisition, the spectra were acquired using the AU program, Bruker pulse program: noesygppr1d (relaxation delay-90°-t1-90°-tm-90°-acquisition, Bruker Biospin GmbH, Ettlingen, Germany) at 300 K. The optimal 90° pulse was calculated for each sample using the automated routine. Tuning and matching was adjusted and the sample was locked to the solvent optimized for H₂O/D₂O samples and shimmed using the automated routine of IconNMR. The relaxation delay (RD) and mixing time (tm) were set at 2 s and 10 ms, respectively. A total of 32k complex data points were accumulated for 32 scans over a spectral width of 16 ppm using a 1.70 s acquisition delay. All spectra were then automatically phased, baseline corrected, and referenced to the internal TSP signal at 0 ppm.

For each sample, one-dimensional ¹H-NMR spectra were acquired on a Bruker 600 MHz Avance III HD spectrometer (Bruker BioSpin with TopSpin 3.5pl7) operating at 600.13 MHz proton Larmor frequency and equipped with a 5 mm PA TXI 1H-13C-15N and 2H-decoupling probe including a z-axis gradient coil, an automatic tuning-matching and an automatic sample changer (SampleJet). The temperature was kept stable within 0.1 K using a BCU I. Before starting measurements manually, samples were kept inside the NMR probe head for at least 5 min to equilibrate temperature at 300 K. The standard Nuclear Overhauser Effect SpectroscopY (NOESY) presat pulse sequence (noesygppr1d; Bruker BioSpin) was used to detect both signals of small metabolites and high molecular weight macromolecules. The parameters of the experiment were: 512 scans, 32,768 data points, a spectral width of 12.0166 ppm, an acquisition time of 2.27 s, a relaxation delay of 2 s, and a mixing time of 0.01 s. Fourier-transformed spectra were automatically corrected for phase and baseline distortions using Topspin 3.2 (Bruker BioSpin) and then automatically calibrated to the proton signal of TSP-d4 at 0.00 ppm.

Blood serum lipoproteins were measured on a Bruker 600 MHz Avance Neo NMR spectrometer using the Bruker IVDr lipoprotein subclass analysis protocol. Serum samples were thawed, and 330 µL of each sample mixed with 330 µL of Bruker serum buffer (Bruker, Rheinstetten, Germany). The samples were mixed gently, and 600 µL of the mixed sample were transferred into a 5 mm SampleJet rack tube (Bruker). Proton spectra were obtained at a constant temperature of 310 K using a standard nuclear Overhauser effect spectroscopy (NOESY) pulse sequence (Bruker: noesygppr1d), a Carr–Purcedll–Meiboom–Gill (CPMG) pulse sequence with presaturation during the relaxation delay (Bruker: cpmgpr1d) to achieve water suppression, and a standard 2D J-resolved (JRES) pulse sequence (Bruker: jresgpprqf). Data analysis was carried out using the Bruker IVDr LIpoprotein Subclass Analysis (B.I.LISA^TM, Bruker, Rheinstetten, Germany) method.

Serum levels of total HDL-C, HDL-TG, HDL-PL, HDL-apoA-I, and HDL-apoAII, as well as of their 4 size/density subclasses (HDL1: 1.063–1.100 kg/L; HDL2: 1.100–1.112 kg/L; HDL3: 1.112–1.125 kg/L; HDL4: 1.125–1.210 kg/L), were measured on a Bruker 600 MHz Avance Neo NMR spectrometer using the Bruker IVDr lipoprotein subclass analysis protocol, as described [17 (link),60 (link)]. Briefly, serum samples were thawed, and 330 µL of each sample was mixed with 330 µL of Bruker serum buffer (Bruker, Rheinstetten, Germany). The samples were mixed gently and 600 µL of the mixed sample was transferred into a 5 mm SampleJet rack tube (Bruker). Proton spectra were obtained at a constant temperature of 310 K using a standard Nuclear Overhauser Effect Spectroscopy (NOESY) pulse sequence (Bruker: noesygppr1d), a Carr–Purcell–Meiboom–Gill (CPMG) pulse sequence with presaturation during the relaxation delay (Bruker: cpmgpr1d) to achieve water suppression, and a standard 2D J-resolved (JRES) pulse sequence (Bruker: jresgpprqf). Data analysis was carried out using the Bruker IVDr LIpoprotein Subclass Analysis (B.I.LISA^TM, Bruker Biospin, Rheinstetten, Germany).

NMR spectroscopy was performed at the MR Core Facility, Norwegian University of Science and Technology (NTNU), and ¹H NMR spectra were acquired on a 600 Mhz Bruker Avance III NMR spectrometer (Bruker Biospin GmbH, Rheinstetten, Germany) equipped with an autosampler (Sample Jet). Temperature during acquisition was 300 K and a 5 mm CPQCI cryoprobe was used to sample 1 D proton spectra with a preprogrammed water presaturation pulse sequence (noesygppr1d, Bruker library); the recycle delay was 3 s, and the mixing time 10 ms. Spectra were collected into 65 K data (SW 12,019 Hz) and the FID transformed with line broadening 0.3 Hz and zero filling 1.0. Phasing, baseline correction and chemical shift calibration (using the TSP signal as reference, δ 0.0 ppm) of the frequency domain spectra was done using Bruker TopSpin v. 3.0.