5 mm nmr tube

The methods of NMR-based metabolomics from Xiao et al.^{76 (link)} and Zhang et al.^{24 (link)} were employed with minimal modification as described below^{24 (link),76 (link)}. Urine was further centrifuged at 9,700 × g for 10 minutes at 4 °C and the supernatant was collected. The supernatant (500 μL) was added to 50 μL of phosphate buffer in D₂O (1.5 M potassium sodium phosphate buffer, pH 7.4), which contained 0.05% TSP (w/v) and 0.55% NaN₃ (w/v), and the mixture was centrifuged at 9,700 × g for 10 min at 4 °C. The final supernatant (520 μL) was placed into a 5-mm NMR tube (Wilmad-Labglass, Vineland, NJ, USA) for 1D ¹H-NMR, 2D ¹H-¹H TOCSY and ¹H-¹³C HSQC. All NMR spectra were measured at 298 K on a 500 MHz Bruker Avance DRX 500 (Billerica, MA), employing a 5-mm inverse triple-resonance probe. Water suppression was executed using a standard NOESYGPPR1D pulse sequence (RD-G1-90°-t1-90°-tm-G2-90°-acq) with 16 ppm spectra width, 32 k data points, 128 transients and 1.5 s relaxation delay. 2D ¹H-¹H TOCSY was conducted with the ‘mlevgpph19’ pulse sequence with 2048 × 256 data points, 32 transients and 2.0 s relaxation time. ¹H-¹³C HSQC was carried out with the ‘hsqcetgoorsiso2.2’ pulse sequence with 1024 × 128 data points, 128 transients and 1.5 s relaxation time.

Before analysis, the spirit drinks were filtered or centrifuged (especially required for turbid samples). For the sample preparation, 500 µL of spirit drinks was mixed with 100 µL buffer solution, and 400 µL water–ethanol-mixture (190 mL and 50 mL) resulting in a dilution factor of 50%, compared to the spirit drink’s original concentrations. For direct measurement, 600 µL of the sample preparation was transferred into a 5 mm NMR tube (with <1% volume variation, e.g., Wilmad Labglass Inc., Vineland, NJ, USA).

Amicon Ultra 0.5-mL 3 kDa (Millipore, Billerica, MA, USA) was used to filter the thawed plasma samples at 10,000× g at 4 °C for 3 h to remove lipid and proteins. For NMR measurement, 300 µL plasma sample, 250 µL D₂O and 50 µL D₂O containing 0.05% sodium trimethylsilyl-[2,2,3,3-2H4]-1-propionate (TSP; Sigma-Aldrich, St. Louis, MO, USA) as an internal chemical shift reference was added to a 5 mm NMR tube (Wilmad, Vineland, NJ, USA). For the urine samples, 100 µL phosphate buffer in D₂O [38 (link)] (pH = 7.4) containing 0.05% TSP was transferred to 500 µL of thawed urine in a 5 mm NMR tube.

Lyophilized samples were reconstituted in 500 μl D₂O (D₂O, Aldrich, 99.9%); 50 μl of 3 mM 2,2-dimethyl-2- silapentane-5-sulfonate (DSS, Cambridge Labs) were added to serve as a lock signal and chemical shift references (δ = 0.0). Solution pH was adjusted with DCl and NaOD to 7.4. Final volume was brought to 600 μL with D₂O making the solution 25 mM in DSS and the sample was transferred to a 5mm NMR tube (Wilmad, Vineland, NJ).
¹H NMR data acquisition was performed at 600 MHz using a Varian spectrometer with a 5mm HCN triple resonance probe at 25°C. Muscle spectra were generated from 128 scans with a basic ¹H acquisition protocol consisting of a 45° tip angle, a relaxation delay of 12.8 sec and an acquisition time of 7 sec. Liver spectra were generated from 1028 scans with a basic ¹H acquisition protocol consisting of a 45° tip angle, a relaxation delay of 2 sec and an acquisition time of 1.9 sec. Phase and baselines were corrected manually before integration. Chemical shifts were assigned relative to the internal standard signal at 0 ppm.

Three hundred microliters of thawed serum samples were aliquoted into a microcentrifuge tube and followed by 300 µL of a solution containing 0.75 M potassium phosphate buffer (pH 7.4), 5.81 mM of trimethylsilyl-2,2,3,3-tetradeuteropropionic acid (TSP; Sigma-Aldrich, St. Louis, MO, USA) and a trace amount of sodium azide dissolved in deuterium oxide. Samples were vortexed to ensure complete homogeneity and a final volume of 540 µL of each sample was transferred to a 5 mm NMR tube (Wilmad Lab Glass, Vineland, NJ, USA) and analysed.

A 400 MHz Bruker Avance II NMR spectrometer (Bruker Biospin, Rheinstetten, Germany) was used to perform 19F NMR with a 5 mm broadband probe, which can operate at 376.5 MHz for ex vivo experiments. Excised organs (liver, lungs, and spleens and 4T1 subcutaneous tumors) were harvested and flash frozen by liquid nitrogen. The prepared sample (0.4 ml, mixed with D2O) was transferred to a 5 mm NMR tube (Wilmad, Vineland, NJ, USA). As a reference compound, 5-fluorocytosine (0.1 ml, 5 mM 19F concentration) was added to determine the chemical shift and 19F concentration for each sample. The pH value of the sample was confirmed to be around 7 when preparing the sample using Bromothymol blue indicator and the temperature was maintained at 37 C during the experiment. The acquisition parameters were as follows: frequency = 376.5 MHz, spectral width = 350 ppm, relaxation delay = 5 s, data points = 64 k. After phase and baseline correction of the acquired ¹⁹F NMR spectra using the Topspin software (Bruker Biospin, Rheinstetten, Germany), the ¹⁹F NMR signals were quantified relative to the 5-fluorocytosine signals (reference) by peak integration. The total ¹⁹F content of the excised organs (4T1 tumor, liver, spleen and lungs) was determined and the results were normalized to the tissue weight generating a signal expressed as a number of fluorine atoms per gram of tissue.