NR is the superior hepatic NAD+ precursor vitamin

On the basis of known NAD+ biosynthetic pathways31, it was difficult to understand how levels of NAAD rose in human PBMCs after an oral dose of NR. Though NR did not elevate Nam in blood samples at any time during the n=1 experiment, it remained possible that NR was partially converted to Nam before salvage synthesis to NAD+. Such conversion to Nam might allow bacterial hydrolysis of Nam to NA by pncA gene products—potentially in the gut10—and subsequent conversion to NAD+ through an NAAD intermediate. NAAD was reported in mouse liver when 500 mg kg−1 of radioactive Nam was injected intraperitoneally (IP) into the body cavity of mice37. However, NAAD was observed in kidneys, ovaries, lung, heart and brain in addition to liver in mice IP-injected with 500 mg kg−1 of NA but not Nam38. Moreover, careful analysis of mouse liver perfused with radioactive NA and Nam indicated that NAAD is produced from NA but not Nam at physiological concentrations39. To our knowledge, formation of blood or tissue NAAD from oral administration of Nam or NR has never been observed.
Although some mouse experiments have been done with IP administered NR at dosages of 1,000 mg kg−1 twice per day28, NR is active as an oral agent at a daily dose of 400 mg kg−1 by supplementation into food27,29,30 and demonstrated potent NAD+ boosting activity in the n=1 human experiment at 15 mg kg−1 (Fig. 2). On the basis of weight/surface area, the conversion between human adult dose and mouse dose is a factor of 12.3 (ref. 40), suggesting that mice should be administered 185 mg kg−1 to achieve comparable levels of supplementation with the human pilot experiment. We therefore designed a reverse translational experiment in which mice were administered 185 mg kg−1 of NR or the mole equivalent doses of Nam and NA by oral gavage. To ascertain the timecourse by which these vitamins boost the hepatic NAD+ metabolome without the complication of circadian oscillation of NAD+ metabolism17,18, we euthanized all mice at ∼2 pm. Thus, gavage was performed at 0.25, 1, 2, 4, 6, 8 and 12 h before tissue harvest. To stop metabolism synchronously, mouse livers were harvested by freeze-clamping. As shown in Fig. 5, we additionally performed saline gavages at all time points and euthanized mice for quantitative NAD+ metabolomic analysis to ensure that animal handling does not alter levels of NAD+ metabolites. The flat timecourses of saline gavages established methodological soundness. Baseline levels of hepatic NAD+ metabolites (pmol mg−1) at 2 pm were 1,000±35 for NAD+, 230±29 for Nam, 210±20 for NADP+, 66±13 for ADPR and <15 for all other NAD+ metabolites. Hepatic levels of NA, NAR, NAMN, NAAD have baselines of <4. As a point of orientation to quantitative metabolomics in tissue samples, 1,000 pmol mg−1 is ∼1 mM, 200 is ∼200 μM and 10 is ∼10 μM.