Appearance of a peak of NAAD after NR administration could either be due to inhibition of de novo synthesis of NAD+ or from a deamidating activity that occurs at high NAD+. If NAAD is not derived from ingested NR, then it should not incorporate heavy atoms. However, if NAAD is derived from ingested NR, then it should incorporate heavy atoms that reflect the rate at which the putative deamidating activity occurs with respect to NAD+-consuming activities and the degree of heavy atom incorporation into NAD+. As shown in Fig. 7e, at the 2 h time point, NAAD contained roughly the same heavy atom composition as NAD+ (Fig. 7a), that is, 45% contained at least one heavy atom and 8% incorporated both heavy atoms. Thus, NR is the biosynthetic precursor of NAD+, NADP+ and NAAD. The data suggest that the activity that converts NAD+ to NAAD occurs at high NAD+ concentrations at a rate comparable to the rate of NAD+ turnover to Nam. Given the lack of formation of NA from either Nam or NR in the mouse liver (Fig. 5), the only other reasonable possibility is that NMN is deamidated to NAMN when NAD+ metabolism increases. Incorporation of the Nam and ribosyl moieties of NR into NAAD establishes this metabolite as both a biomarker of increased NAD+ metabolism and a direct product of NR utilization.
NR safely increases PBMC NAD+ metabolism and NAAD in people
The n=1 human experiment illustrated the potential of 1,000 mg NR to boost human NAD+ metabolism. We therefore conducted a controlled experiment with 12 consented healthy men and women to determine the effect of three single doses of NR on blood and urine NAD+ metabolites with monitoring of subjects for potential adverse events. Considering that the recommended daily allowance of vitamin B3 as Nam or NA is ∼15 mg per adult, we tested three doses of the higher molecular weight compound NR Cl (100, 300 and 1,000 mg) that correspond to 2.8, 8.4 and 28 times recommended daily allowance. Body weights of the subjects varied. However, we had already observed the timecourse of changes in a human NAD+ metabolome with daily doses of 1,000 mg in a healthy 65 kg male. Participants were randomized to receive doses of NR in different sequences with 7-day washout periods between data collection. Participants and investigators were blinded to doses. Blood and urine collections were performed over 24 h following each dose. Participants were asked to self-report perceived discomforts.
At 500 mg of niacin, 33 of 33 participants experienced flushing compared with one out of 35 participants on placebo46. In this study, two individuals self-reported flushing at the 300 mg dose but not at the 100 mg or 1,000 mg dose, and two individuals self-reported feeling hot at the 1,000 mg dose but not at lower doses. Over the total of 36 days of observation of study participants, there were no serious adverse events and no events that were dose-dependent. To assess whether NR might be associated with authentic and dose-dependent episodes of flushing, future experiments will incorporate a validated flushing symptom questionnaire47.
As shown in Fig. 8 and Supplementary Data Files 1 and 2, the NAD+ metabolome was quantified in the PBMC and plasma fractions at pre-dose and at 1, 2, 4, 8 and 24 h after receiving oral NR. Urinary NAD+ metabolites (Supplementary Data File 3) were quantified in pre-dose, 0–6 h, 6–12 h and 12–24 h collections.