Assessment of NADmetabolism 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.