NAD Metabolome Analysis in Human Cells Using 1H NMR Spectroscopy

Abstract

Nicotinamide adenine dinucleotide (NAD) and its phosphorylated form, NADP, are the major coenzymes of redox reactions in central metabolic pathways. Nicotinamide adenine dinucleotide 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 by sirtuins. The regulation of these metabolic and signaling processes depends on NAD availability. Generally, human cells accomplish their NAD supply through biosynthesis using different forms of vitamin B3: 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 adenylylated to the dinucleotides NAD and NAAD, respectively. Here, we have developed an NMR-based experimental approach to detect and quantify NAD(P) and its biosynthetic intermediates in human cell extracts. Using this method, we have determined 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 additional presence of both NAR and NR, intracellular pools of deamidated NAD intermediates (NAR, NAMN and NAAD) were also detectable. We have also tested this method to quantify NAD+ in human platelets and erythrocytes. Our results demonstrate that 1H NMR spectroscopy provides a powerful method for the assessment of the cellular NAD metabolome.

Equilibrative Nucleoside Transporters Mediate the Import of Nicotinamide Riboside and Nicotinic Acid Riboside into Human Cells

“…Culture media from HEK293 cells were collected and stored at −80 • C. To precipitate proteins, the samples were incubated on ice with two volumes of acetonitrile for 30 min and then centrifuged at 15,000× g for 30 min at 4 • C. Supernatants were then treated in the same way as the cell extracts. All NMR experiments were performed using a Varian DirectDrive NMR System 700-MHz spectrometer equipped with a 5-mm z-gradient salt-tolerant as described in [46]. Briefly, the one-pulse sequence with the suppression of solvent signal by presaturation was used for acquisition of 1 H spectra.…”

Degradation of Extracellular NAD+ Intermediates in Cultures of Human HEK293 Cells

“…All NMR experiments were performed using a Varian DirectDrive NMR System 700-MHz spectrometer equipped with a 5-mm z-gradient salt-tolerant as described in [27]. Briefly, the one-pulse sequence with the suppression of solvent signal by presaturation was used for acquisition of 1 H spectra.…”

“…In the present study, we applied a previously developed 1 H-NMR-based metabolomics approach [27] to conduct a systematic analysis of the metabolism of exogenous NAD + intermediates in cultures of human HEK293 cells and reveal how they are utilized to maintain intracellular NAD + levels.…”

NADomics: Measuring NAD+ and Related Metabolites Using Liquid Chromatography Mass Spectrometry

“…NMR-based experimental approaches have been developed to quantify the NADome in human cells. 1 H NMR spectroscopy has been previously used to quantify the NADome in human platelets and erythrocytes [43]. While the relative concentration can be quantified directly from the NMR spectrum for each metabolite, irrespective of the complexity of the sample, this approach may require up to 2 h of data acquisition to achieve a signal-to-noise ratio of at least 5 for a sample containing approximately 2 µM of metabolite [43].…”

“…1 H NMR spectroscopy has been previously used to quantify the NADome in human platelets and erythrocytes [43]. While the relative concentration can be quantified directly from the NMR spectrum for each metabolite, irrespective of the complexity of the sample, this approach may require up to 2 h of data acquisition to achieve a signal-to-noise ratio of at least 5 for a sample containing approximately 2 µM of metabolite [43]. This can limit the application of NMR for the quantification of the NADome in some biological specimens, where some metabolites may be present in very low nanomolar levels [15].…”