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

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

doi:10.3390/ijms221910598

Abstract

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.

Highlights

Many metabolic pathways depend on the redox couples, nicotinamide adenine dinucleotide NAD+ and reduced form NADH, and respective phosphorylated forms, nicotinamide adenine dinucleotide phosphate NADP+ and reduced form NADPH
All metabolites vary in hydrophilicity despite being pyridine derivatives with molecular masses ranging between 122 (Nam) and 745 (NADPH) Da
Whole blood is ~45% red blood cells (RBCs) and ~1% white blood cells with the remaining volume as plasma. These results suggest that our workflow is amenable to detect the NAD+ metabolome in the blood of free-living populations and that whole blood is the best matrix for clinical samples

Summary

Introduction

Many metabolic pathways depend on the redox couples, nicotinamide adenine dinucleotide NAD+ and reduced form NADH, and respective phosphorylated forms, nicotinamide adenine dinucleotide phosphate NADP+ and reduced form NADPH. NAD(H) and NADP(H) both serve as hydride carriers in the cell (donors and acceptors), but they act differently. NAD(H) plays a crucial role in catabolism, energy metabolism (glycolysis, fatty acid oxidation, tricarboxylic acid cycle and electron transport chain), while NADP(H) is involved in anabolic reactions (biosynthesis of nucleic acids, fatty acids, cholesterol and steroid hormones) as well as in antioxidant response and detoxification processes [1]. Beyond their established biochemical roles in metabolism, these molecules have important cell signaling functions.

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