A Method to Monitor the NAD+ Metabolome—From Mechanistic to Clinical Applications

• Published in: International journal of molecular sciences Vol. 22; no. 19; p. 10598
• Main Authors: Giner, Maria Pilar, Christen, Stefan, Bartova, Simona, Makarov, Mikhail V., Migaud, Marie E., Canto, Carles, Moco, Sofia
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 30.09.2021; MDPI
• Subjects: Animals; Biosynthesis; Blood Donors; Chromatography; Chromatography, Liquid - methods; Enzymes; Hep G2 Cells; Humans; Hydrophobic and Hydrophilic Interactions; Kinases; Metabolism; Metabolites; Metabolome; Metabolomics - methods; Methods; Mice; Mice, Inbred C57BL; Monitoring, Physiologic - methods; NAD - biosynthesis; NMR; Nuclear magnetic resonance; Oxidation; Oxidation-Reduction; Pilot Projects; Plasma - chemistry; Plasma - metabolism; Retention; Serum - chemistry; Serum - metabolism; Tandem Mass Spectrometry - methods; Urine - chemistry; Urine - physiology; NAD; metabolomics; mass spectrometry
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms221910598

Nicotinamide adenine dinucleotide (NAD+) and its reduced form (NADH) are coenzymes employed in hundreds of metabolic reactions. NAD+ also serves as a substrate for enzymes such as sirtuins, poly(ADP-ribose) polymerases (PARPs) and ADP-ribosyl cyclases. Given the pivotal role of NAD(H) in health and disease, studying NAD+ metabolism has become essential to monitor genetic- and/or drug-induced perturbations related to metabolic status and diseases (such as ageing, cancer or obesity), and its possible therapies. Here, we present a strategy based on liquid chromatography-tandem mass spectrometry (LC-MS/MS), for the analysis of the NAD+ metabolome in biological samples. In this method, hydrophilic interaction chromatography (HILIC) was used to separate a total of 18 metabolites belonging to pathways leading to NAD+ biosynthesis, including precursors, intermediates and catabolites. As redox cofactors are known for their instability, a sample preparation procedure was developed to handle a variety of biological matrices: cell models, rodent tissues and biofluids, as well as human biofluids (urine, plasma, serum, whole blood). For clinical applications, quantitative LC-MS/MS for a subset of metabolites was demonstrated for the analysis of the human whole blood of nine volunteers. Using this developed workflow, our methodology allows studying NAD+ biology from mechanistic to clinical applications.