Extraction of NAD
Participants fasted and visited the Clinical Research Support Center Phase 1 Unit of the University of Tokyo Hospital in the morning. The blood samples were collected from forearms of the participants. The samples in heparinized tubes were frozen at -80 °C and analyzed at the University of Toyama. The extraction and analysis of NAD + related metabolites (NAD + , NA, nicotinic acid riboside (NAR), NANM, NMN, NR, and NAM) were performed as previously described [50 µL of whole blood and 450 µL of 50% MeOH were mixed and vortexed for 10 s, followed by the addition of an equal volume of chloroform (500 μL). The mixture was centrifuged at 13,000 × g at 4 °C for 10 min, and then aqueous phase and organic phase were separated. The upper aqueous phase was transferred to a new tube, and this liquid–liquid extraction was repeated. After this procedure, the samples were dried. The dried aqueous phase was reconstituted in LC/MS-grade water. The metabolites were analyzed by using an Agilent 6460 Triple Quad mass spectrometer (Agilent Technologies Inc., Santa Clara, CA, USA) and Agilent 1290 HPLC system (Agilent Technologies Inc.). Analytes were separated on an Atlantis T3 column (2.1 × 150 mm, particle size 3 µm, Waters) using mobile phase A (5 mM ammonium formate) and mobile phase B (methanol) at a flow rate of 150 µL/min and column temperature of 40 °C. In addition, the programmed mobile phase gradient was following: 0–10 min, 0–70% B; 10–15 min, 70% B; 15–20 min, 0% B. Analysis of chromatograms were performed by using MassHunter Quantitative Analysis software (Agilent Technologies Inc.). Various concentrations of the standard compounds were measured to make standard curves for quantification.