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NRH, NAR and NARH were custom synthesized as described previously [29 (link)]. Twelve individual solutions of NR, NAR, NRH, and NARH were prepared in water, DMEM, and blood plasma. The concentration of NR, NAR, and NRH stock solutions in water and DMEM was 55 mM, while the concentration of the NARH stock solution in water and DMEM was 30 mM. In plasma, the stock solution was ten times diluted (5.5 and 3.0 mM, respectively) as compared to water and DMEM media. A 450 mL volume of solution was mixed with 50 μL of D2O, and the resulting mixture was vortexed three times. NMR spectral acquisition (ns = 8) was then performed in water suppression mode using a Bruker Avance III HD NMR spectrometer equipped with a 400 MHz magnet Ultrashield Plus, with temperature fixed to 300 K for all NMR measurements. TopSpin 3.2 (Bruker BioSpin (Billerica, MA, USA)) was used for all NMR spectral acquisition and preprocessing, and the automation of sample submission was performed using ICON-NMR (Bruker BioSpin). All samples were automatically shimmed. The FID was processed automatically using ICON-NMR (Bruker BioSpin), and phasing was refined manually. The solutions were examined at 1, 12, and 24 h by ¹H NMR analysis. Chemical shift allocations for the individual compounds are shown in Figure S5.

The supernatants were examined at 0, 1, and 24 h by ¹H NMR analysis for the presence of any remaining NRH after supplementation at 100 μM in HEK293T and HepG3. Samples were prepared as follows: 450 μL of cell culture supernatant was mixed with 50 μL of D₂O, and the resulting mixture was vortexed three times. NMR spectral acquisition (ns = 2048) was then performed using a Bruker Avance III HD NMR spectrometer equipped with 400 MHz magnet Ultrashield Plus, with temperature fixed to 300 K for all NMR measurements. TopSpin 3.2 (Bruker BioSpin) was used for all NMR spectral acquisition and preprocessing, and the automation of sample submission was performed using ICON-NMR (Bruker BioSpin). All samples were automatically shimmed. The FID were processed automatically using ICON-NMR (Bruker BioSpin), and phasing was refined manually. NRH, NR, and nicotinamide were all detected (S1 Fig). NRH is oxidized to NR in aqueous solution over time, while nicotinamide is present at ~16 μM in DMEM. Nicotinamide accumulates in the media over time as NRH is consumed by the cells.

All measurements were performed on a Bruker Avance III 600 Ascend NMR spectrometer (Bruker, Ettlingen, Germany), operating at 600.13 MHz for ¹H observation, equipped with a TCI cryoprobe incorporating a z axis gradient coil and automatic tuning-matching (ATM). Experiments were acquired at 300 K in automation mode after loading individual samples by a Bruker Automatic Sample Changer, interfaced with the software IconNMR (Bruker). For each sample a 1D sequence with pre-saturation and composite pulse for selection (zgcppr Bruker standard pulse sequence) was acquired, with 16 transients, 16 dummy scans, 5 s relaxation delay, size of fid of 64K data points, a spectral width of 12019.230 Hz (20.0276 ppm) and an acquisition time of 2.73 s. The resulting FIDs were multiplied by an exponential weighting function corresponding to a line broadening of 0.3 Hz before Fourier transformation, automated phasing and baseline correction. Metabolite identification were based on ¹H and ¹³C assignment by 1D and 2D omo and eteronuclear experiments and by comparison with literature data [19 (link),26 (link),47 (link),48 (link),49 (link)]. NMR data processing was performed by using TopSpin 3.5 (Bruker). All spectra were referenced to the TSP signal (δ = 0.00 ppm).