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 have developed NMR-based experimental approach to identify NAD and its intermediates in culture of human cells. Using this method we have detected and quantified NAD, NADP, NMN and Nam pools in HEK293 cells cultivated in the standard culture medium containing Nam as the only NAD precursor. When cells were grown in the presence of NR and NAR, we have additionally identified intracellular pools of deamidated NAD intermediates (NAR, NAMN and NAAD). Also, we have characterized the potential of different extracellular NAD precursors to maintain the synthesis of intracellular NAD.


NAD metabolism in human cells
4 Nicotinamide adenine dinucleotide (NAD) and its phosphorylated form NADP are the major coenzymes of redox reactions in central metabolic pathways. Besides its central role in cellular metabolism, NAD also serves as substrate of several families of regulatory proteins: protein deacetylases (Sirtuins), ADP-ribosyltransferases (ARTs) and Poly-ADP-ribosyl polymerases (PARPs), which govern vital processes including gene expression, DNA repair, apoptosis, aging, cell cycle progression and many others. NAD is also used by ADP-ribosyl cyclases (CD38, CD157) to produce calcium mobilizing messengers, including ADP-ribose (ADPR) and cyclic ADPribose (cADPR). The regulation of these metabolic and signaling processes depends on NAD availability. Generally, human cells regulate their NAD supply through biosynthesis using various precursors: tryptophan (Trp), the pyridine bases nicotinamide (Nam) and nicotinic acid (NA) or the nucleosides Nam riboside (NR) and NA riboside (NAR). Nam and NA are converted to the corresponding mononucleotides (NMN and NAMN) by nicotinamide phosphoribosyltransferase (NamPRT) and nicotinic acid phosphoribosyltransferase (NAPRT), respectively. NMN and NAMN are also generated through phosphorylation of NR and NAR, respectively, by nicotinamide riboside kinases (NRK). NAMN and NMN are converted to the corresponding dinucleotide (NAAD or NAD+) by NMN adenylyltransferases (NMNAT). NAD synthetase (NADS) amidates NAAD to NAD+ (1). Here we developed NMR-based experimental approach for quantitative analysis of NAD(P) and its major intermediates in cell extracts and culture medium.

Experimental procedure
Cell Culture HEK293 cells were cultivated in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) fetal bovine serum. NA, NR, or/and NAR were added to the cell culture medium at a concentration of 100 µM. Cells were also treated with FK866 (inhibitor of NamPRT) at a concentration of 1 µM.
NMR sample preparation 100 µM standard solutions of Nam, NA, NR, NAR, NMN, NAMN, NAD, NAAD, NADP, NADH, and NADPH were prepared using DBP buffer: D 2 O-based buffer containing 50 mM NaPi (pH 6.5) and 1 mM sucrose as a chemical shift reference (δ( 1 H), 5.42 ppm) and internal standard for quantification. Samples were stored at -80 °C until NMR analysis. Culture medium from HEK293 cells was collected and stored at -80 °C. To precipitate proteins, the samples were incubated on ice with 2 volumes of acetonitrile for 30 min and then centrifuged at 15,000 g for 30 min at 4 °C. Supernatants were lyophilized and resuspended in DBP buffer. 10 7 HEK293 cells grown on 100 mm culture dish were washed twice with ice cold PBS, extracted with 67% acetonitrile for 30 min at 4 °C, scraped and centrifuged at 15,000 g for 30 min at 4 °C. The supernatant fraction (extract) was then lyophilized and resuspended in DBP buffer.

NMR Analysis
All NMR experiments were performed using a Varian DirectDrive NMR System 700-MHz spectrometer equipped with a 5-mm z-gradient salt-tolerant. VNMRJ 4.2, Mestrelab Mnova 10.0 was used for data analysis.
To optimize parameters of NMR spectra acquisition: 1) Pulse sequence with the suppression of solvent signal was used. This pulse sequence allowed recording the spectra with maximal sensitivity. 2) Relaxation time for each metabolite signal was measured and used to optimize the relaxation delay and other parameters of spectra recording. 3) 1H-spectrum for each metabolite of interest was obtained and several characteristic peaks for the identification and quantification of metabolites were chosen. 4) Number of scans was determined to obtain signal/noise ratio more than 5 for quantification of metabolites. 5) Concentration of metabolite was calculated as a ratio of weighted average intensity of metabolite signals to sucrose signal intensity, where weight equals (1-exp{-(Repetition time)/(Relaxation time)}), Repetition time = Acquisition time + Relaxation delay. 6) Signal intensity was calculated as an integral of multiplet fitted to spectrum.  We have developed NMR-based experimental approach for the NAD metabolome analysis in various biological samples. This method can be used for the identification and quantification of NAD(P) and its major intermediates at minimal concentration of 0.5 µM with an error no more than 10%. This method is optimized for the quantitative analysis of NAD metabolome in cell extracts and culture medium.