NAD metabolome analysis in cultured human cells using ¹H NMR spectroscopy

Nicotinamide adenine dinucleotide (NAD) and its phosphorylated form NADP are the major coenzymes of redox reactions in central metabolic pathways. NAD is also used to generate second messengers (such as cyclic ADP-ribose) and serves as substrate for protein modifications (including ADP-ribosylation and protein deacetylation). The regulation of these metabolic and signaling processes depends on NAD availability. Generally, human cells regulate their NAD supply through biosynthesis using various precursors: nicotinamide (Nam) and nicotinic acid as well as nicotinamide riboside (NR) and nicotinic acid riboside (NAR). These precursors are converted to the corresponding mononucleotides NMN and NAMN,which are then adenylylated to the dinucleotides NAD or NAAD, respectively. Here, we developed NMR-based experimental approach to identify NAD and its intermediates in cultures of human cells. Using this method we detected and quantified NAD, NADP, NMN and Nam pools in HEK293 cells cultivated in standard culture medium containing Nam as the only NAD precursor. When cells were grown in the presence of NR and NAR, we additionally identified intracellular pools of deamidated NAD intermediates (NAR, NAMN and NAAD). We also characterized the potential of different extracellular NAD precursors to maintain the synthesis of intracellular NAD. This work was supported by RSF grant 16-14-10240.