Assessment of NAD+metabolism in human cell cultures, erythrocytes, cerebrospinal fluid and primate skeletal muscle

The reduction-oxidation state of NAD+/NADH is critical for cellular health with NAD+ and its metabolites playing critical roles in aging and pathologies. Given the inherent autooxidation of reduced dinucleotides (i.e. NADH/NADPH), and the well-established differential stability, the accurate measurement of NAD+ and its metabolites is technically challenging. Moreover, sample processing, normalization and measurement strategies can profoundly alter results. Here we developed a rapid and sensitive liquid chromatography mass spectrometry-based method to quantify the NAD+ metabolome with careful consideration of these intrinsic chemical instabilities. Utilizing this method we assess NAD+ metabolite stabilities and determine the presence and concentrations of NAD+ metabolites in clinically relevant human samples including cerebrospinal fluid, erythrocytes, and primate skeletal muscle. •A rapid and sensitive LC-MS/MS-based method to quantify the NAD+ metabolome was developed.•NAD+ metabolite stabilities were assessed in different matrices.•Sample processing results in significant changes in NAD+ and its metabolites levels.•Multiple NAD + metabolites identified in cerebrospinal fluid for the first time.