Assays for NAD⁺-Dependent Reactions and NAD⁺ Metabolites

Nicotinamide adenine dinucleotide (NAD⁺) is an essential redox cofactor and signaling molecule that controls the activity of enzymes involved in metabolism, DNA repair, and cellular survival, such as the PARPs, CD38, and the sirtuins. Here, we describe three methods for measuring the activity of these enzymes: the etheno-NAD⁺ assay measures NAD⁺ hydrolase activity using an NAD⁺ analog to produce a fluorescent product that is measured in real time; the PNC1 assay converts a native product of NAD⁺ hydrolysis, nicotinamide, into a quantitative fluorescent readout; and liquid chromatography tandem mass spectrometry (LC-MS/MS) is used to characterize the entire NAD⁺ metabolome in a sample. These methods will enable new insights into the roles that NAD⁺ and the enzymes that utilize it play in health and disease.