A method to monitor the NAD+ metabolome—from mechanistic to clinical applications

Maria Pilar Giner, Stefan Christen, Simona Bartova, Mikhail V. Makarov, Marie E. Migaud, Carles Canto, Sofia Moco*

*Corresponding author for this work

Research output: Contribution to JournalArticleAcademicpeer-review


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 ge-netic‐ 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 chroma-tography‐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 sep-arate a total of 18 metabolites belonging to pathways leading to NAD+ biosynthesis, including pre-cursors, 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.

Original languageEnglish
Article number10598
Pages (from-to)1-16
Number of pages16
JournalInternational Journal of Molecular Sciences
Issue number19
Early online date30 Sept 2021
Publication statusPublished - 1 Oct 2021

Bibliographical note

Funding Information:
M.P.G., S.C., S.B., C.C. and S.M. received no external funding. M.V.M and M.E.M. were supported by R21AT009908 from National Institutes of Health, National Center for Complementary and Integrative Health (http://www.nccih.nih.gov/). The authors thank all the volunteers who donated their blood for implementing this method and acknowledge the assistance of Fabienne Praplan and Ondine Walter (Nestle Research) in the collection and biobanking of human blood samples, as well as Jim Kaput who helped in proofreading the manuscript.

Publisher Copyright:
© 2021 by the authors. Licensee MDPI, Basel, Switzerland.

Copyright 2021 Elsevier B.V., All rights reserved.


  • Mass spectrometry
  • Metabolomics
  • NAD


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