In yeast, deletion of NR kinase 1 (NRK1) does not eliminate utilization of NR26. As shown in Fig. 1, NR can be phosphorylyzed to Nam by purine nucleoside phosphorylase and still contribute to NAD+ synthesis through Nam salvage26,34. However, as shown in Fig. 2, Nam concentration in the human subject's PBMCs merely fluctuated in a range of 2.6 to 7.1 μM throughout all 11 observations. The 4.2-fold increase in Me2PY concentration at the 4.1 h time point suggests that increased cellular NAD+ accumulation is accompanied by increased NAD+-consuming activities linked to increased methylation and oxidation of the Nam product.
In the subject's PBMCs at 7.7 and 8.1 h post ingestion, NAD+ and Me2PY peaked, increasing above baseline concentrations by 2.7-fold and 8.4-fold, respectively. At these times, unexpectedly, NAAD, the substrate of glutamine-dependent NAD+ synthetase35,36, which is only expected to be produced in biosynthesis of NAD+ from tryptophan and NA2, was elevated from less than 20 nM to as high as 0.91 μM. Whereas NAAD lagged the rise in PBMC NAD+ by one time point, the relative rise in PBMC NAD+ was not as pronounced as the spike in NAAD, which was at least 45-fold above the baseline level. Although contrary to expectations, these data suggested that NR might be incorporated into NAAD after formation of NAD+ and chased back to the NAD+ peak as NAD+ declines.
Complete NAD+ metabolomic data from the human subject's PBMCs, plasma and urine are provided in Figs 2, ,3,3, ,44 and Supplementary Tables 1–3. These data show that all of the phosphorylated compounds—NAMN, NAAD, NAD+, NADP+, NMN and ADPR—are found exclusively in blood cells and are not found in plasma or urine. Notably, the peak of NADP+, which represents cellular NADP+ plus NADPH oxidized in extraction, and the peak of ADPR, which signals an increase in NAD+-consuming activities, co-occur with peak NAD+. Using methods optimized for recovery of nucleotides, NR was not recovered. As shown in Figs 3 and and4,4, the major time-dependent waste metabolite in plasma and urine was Me2PY, which rose ∼10-fold from pre-dose to time points after NAD+ peaked in PBMCs.