Quantifying the cellular NAD+ metabolome using a tandem liquid chromatography mass spectrometry approach.

Nicotinamide adenine dinucleotide (NAD+) is an essential pyridine nucleotide that serves as a key hydride transfer coenzyme for several oxidoreductases. It is also the substrate for intracellular secondary messenger signalling by CD38 glycohydrolases, DNA repair by poly(adenosine diphosphate ribose) polymerase, and epigenetic regulation of gene expression by a class of histone deacetylase enzymes known as sirtuins. The measurement of NAD+ and its related metabolites (hereafter, the NAD+ metabolome) represents an important indicator of cellular function. A study was performed to develop a sensitive, selective, robust, reproducible, and rapid method for the concurrent quantitative determination of intracellular levels of the NAD+ metabolome in glial and oocyte cell extracts using liquid chromatography coupled to mass spectrometry (LC/MS/MS). The metabolites were separated on a versatile amino column using a dual HILIC-RP gradient with heated electrospray (HESI) tandem mass spectrometry detection in mixed polarity multiple reaction monitoring mode. Quantification of 17 metabolites in the NAD+ metabolome in U251 human astroglioma cells could be achieved. Changes in NAD+ metabolism in U251 cell line, and murine oocytes under different culture conditions were also investigated. This method can be used as a sensitive profiling tool, tailoring chromatography for metabolites that express significant pathophysiological changes in several disease conditions and is indispensable for targeted analysis.