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: Adenine; Aging; Animal models; Animals; Biological properties; Biosynthesis; Blood; Blood Donors; Catabolites; Cell culture; Chromatography; Chromatography, Liquid - methods; Coenzymes; Cofactors; Enzymes; Hep G2 Cells; Humans; Hydrophobic and Hydrophilic Interactions; Intermediates; Kinases; Liquid chromatography; Mass spectrometry; Mass spectroscopy; Metabolism; Metabolites; Metabolome; metabolomics; Metabolomics - methods; Methods; Mice; Mice, Inbred C57BL; Monitoring, Physiologic - methods; NAD; NAD - biosynthesis; NADH; Nicotinamide; NMR; Nuclear magnetic resonance; Oxidation; Oxidation-Reduction; Pilot Projects; Plasma - chemistry; Plasma - metabolism; Poly(ADP-ribose); Poly(ADP-ribose) polymerase; Retention; Ribose; Serum - chemistry; Serum - metabolism; Sirtuins; Tandem Mass Spectrometry - methods; Therapeutic applications; Urine - chemistry; Urine - physiology; Workflow; 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.