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

Nicotinamide adenine dinucleotide (NAD⁺) and its metabolome (NADome) play important roles in preserving cellular homeostasis. Altered levels of the NADome may represent a likely indicator of poor metabolic function. Accurate measurement of the NADome is crucial for biochemical research and developing interventions for ageing and neurodegenerative diseases. In this mini review, traditional methods used to quantify various metabolites in the NADome are discussed. Owing to the auto-oxidation properties of most pyridine nucleotides and their differential chemical stability in various biological matrices, accurate assessment of the concentrations of the NADome is an analytical challenge. Recent liquid chromatography mass spectrometry (LC-MS) techniques which overcome some of these technical challenges for quantitative assessment of the NADome in the blood, CSF, and urine are described. Specialised HPLC-UV, NMR, capillary zone electrophoresis, or colorimetric enzymatic assays are inexpensive and readily available in most laboratories but lack the required specificity and sensitivity for quantification of human biological samples. LC-MS represents an alternative means of quantifying the concentrations of the NADome in clinically relevant biological specimens after careful consideration of analyte extraction procedures, selection of internal standards, analyte stability, and LC assays. LC-MS represents a rapid, robust, simple, and reliable assay for the measurement of the NADome between control and test samples, and for identifying biological correlations between the NADome and various biochemical processes and testing the efficacy of strategies aimed at raising NAD⁺ levels during physiological ageing and disease states.