In animals, NAD+-consuming activities and cell division necessitate ongoing NAD+ synthesis, either through a de novo pathway that originates with tryptophan or via salvage pathways from three NAD+ precursor vitamins, Nam, nicotinic acid (NA) and nicotinamide riboside (NR)2. Dietary NAD+ precursors, which include tryptophan and the three vitamins, prevent pellagra. Though NR is present in milk6,7, the cellular concentrations of NAD+, NADH, NADP+ and NADPH are much higher than those of other NAD+ metabolites8,9, such that dietary NAD+ precursor vitamins are largely derived from enzymatic breakdown of NAD+. Thus, although milk is a source of NR6,7, the more abundant sources of NR, Nam and NA are unprocessed foods, in which plant and animal cellular NAD+ metabolites are broken down to these compounds. Human digestion and the microbiome10 play roles in the provision of these vitamins in ways that are not fully characterized. In addition, the conventional NAD+ precursor vitamins, NA and Nam, have long been supplemented into human and animal diets to prevent pellagra and promote growth11,12. Though NR has been available as a GMP-produced supplement since 2013 and animal safety assessment indicates that it is as nontoxic as Nam13, no human testing has been reported.
Different tissues maintain NAD+ levels through reliance on different biosynthetic routes and precursors14,15 (Fig. 1). Because NAD+-consuming activities frequently occur as a function of cellular stresses3 and produce Nam, the ability of a cell to salvage Nam into productive NAD+ synthesis through Nam phosphoribosyltransferase (NAMPT) activity versus methylation of Nam to N-methylnicotinamide (MeNam) regulates the efficiency of NAD+-dependent processes16. NAD+ biosynthetic genes are also under circadian control17,18. Both NAMPT expression and NAD+ levels decline in a number of tissues as a function of aging and overnutrition19,20,21,22.
High-dose NA but not high-dose Nam is prescribed to treat and prevent dyslipidemias, although its use is limited by painful flushing23,24. Whereas it takes only ∼15 mg per day of NA or Nam to prevent pellagra, pharmacological doses of NA can be as high as 2–4 g. Despite the >100-fold difference in effective dose between pellagra prevention and dyslipidemia treatment, we proposed that the beneficial effects of NA on plasma lipids might simply depend on function of NA as an NAD+ boosting compound1. According to this view, sirtuin activation would likely be part of the mechanism because Nam is an NAD+ precursor in most cells14,15 but inhibits sirtuins at high doses25.
On the basis of the ability of NR to elevate NAD+ synthesis, increase sirtuin activity and extend lifespan in yeast6,26, NR has been employed in mice to elevate NAD+ metabolism and improve health in models of metabolic stress. Notably, NR allows mice to resist weight gain on high-fat diet27, prevent noise-induced hearing loss28 and maintain the regenerative potential of stem cells in aging mice, providing a longevity advantage29. In addition, the hepatic NAD+ metabolome has been interrogated as a function of prediabetic and type 2 diabetic mouse models. The data indicate that levels of liver NADP+ and NADPH, which are required for resistance to ROS, are severely challenged by diet-induced obesity, and that diabetes and the NAD+ metabolome can be partially controlled while diabetic neuropathy can be blocked by oral NR30.
Data indicate that NR is a mitochondrially favoured NAD+ precursor31 and in vivo activities of NR have been interpreted as depending on mitochondrial sirtuin activities27,28, though not to the exclusion of nucleocytosolic targets32,33. Similarly, nicotinamide mononucleotide (NMN), the phosphorylated form of NR, has been used to treat declining NAD+ in mouse models of overnutrition and aging19,20. Beneficial effects of NMN have been shown to depend on SIRT120. However, because of the abundance of NAD+-dependent processes, the effects of NR and NMN may depend on multiple targets including sirtuins, PARP family members, cADPribose synthetases, NAD+-dependent oxidoreductases and NADPH-dependent ROS detoxification enzymes30.
To translate NR technologies to people, it is necessary to determine NR oral availability and utilization in different tissues. Here we began with targeted quantitative NAD+ metabolomics of blood and urine in a pilot experiment in which a healthy 52-year-old man took 1,000 mg of NR daily for 7 days. These data indicate that blood cellular NAD+ rose 2.7-fold after one dose of NR and that NA adenine dinucleotide (NAAD) unexpectedly increased 45-fold. We then performed a detailed analysis of 128 mice comparing oral NR, Nam and NA in a manner that eliminated the possibility of circadian artefacts. These data indicate that NR boosts hepatic NAD+ and NAD+- consuming activities to a greater degree than Nam or NA. Further experiments clarified that NR is a direct precursor of NAAD and that NAAD sensitively reports on increased NAD+ metabolism in mouse liver and heart. Finally, we performed a clinical study with 12 healthy human subjects at three single doses of NR. We demonstrated that NR supplementation safely increases NAD+ metabolism at all doses and validated elevated NAAD as an unexpected, sensitive biomarker of boosting NAD+. The unique oral bioavailability of NR in mice and people and methods established herein enable clinical translation of NR to improve wellness and treat human diseases.
Results
Oral NR increases human blood NAD+ with elevation of NAAD
GMP-synthesized NR showed no activity as a mutagen or toxin13. Despite use as an over-the-counter supplement, no data addressing human availability were available. A healthy 52-year-old male (65 kg) contributed blood and urine before seven days of orally self-administered NR (1,000 mg per morning). Blood was taken an additional nine times during the first day and at 24 h after the first and last dose. Blood was separated into peripheral blood mononuclear cells (PBMC) and plasma before quantitative NAD+ metabolomics by liquid chromatography (LC)-mass spectrometry (MS)9, which was expanded to quantify methylated and oxidized metabolites of Nam30. As shown in Supplementary Table 1 and Fig. 2, the PBMC NAD+ metabolome was unaffected by NR for the first 2.7 h. In six measurements from time zero through 2.7 h, NAD+ had a mean concentration of 18.5 μM; while Nam had a mean concentration of 4.1 μM and the methylated and oxidized Nam metabolite, N-methyl-2-pyridone-5-carboxamide (Me2PY), had a mean concentration of 2.6 μM. However, at 4.1 h post ingestion, PBMC NAD+ and Me2PY increased by factors of 2.3 and 4.2, respectively.