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

Abstract

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.

Keywords: NAD+; ageing; biomarker; nicotinamide; plasma.

Figure 1

NAD⁺ metabolism in eukaryotic cells. NAD⁺ anabolism from tryptophan occurs by the de novo kynurenine pathway (KP). NAD⁺ precursors via the salvage pathway include nicotinamide (NAM), nicotinic acid (NA), nicotinamide riboside (NR), and nicotinic acid riboside (NAR). The enzyme nicotinamide phosphoribosyltransferase (NAMPT) converts NAM to nicotinamide mononucleotide (NMN). Nicotinamide mononucleotide adenylyltransferase (NMNAT1-3) converts NMN to NAD⁺. NAM can be methylated to N-methyl-nicotinamide (MeNAM) by the action of nicotinamide N-methyltransferase (NNMT). NADH represents the reduced form of NAD⁺. NADP⁺ is the phosphorylated form of NAD⁺. NADP⁺ can be reduced to NADPH by NAD kinases (NADK1,2). PARPs, Sirtuins, and CD38 NAD⁺ glycohydrolases are known as NAD⁺ consumers, leading to the generation of NAM. Nicotinic acid phosphoribosyltransferase (NAPRT) converts NA to nicotinic acid mononucleotide (NAMN), which is then converted to NAD⁺ by NMNAT1-3. NAR needs to be converted to NAMN to yield NAD⁺ synthesis via nicotinamide riboside kinases (NRK1,2). NRK1,2 also convert NR to NMN. NAR can form NA via purine nucleoside phosphorylase (PNP). PNP are also capable of converting NR to NAM.

Figure 2

Schematic representation of the role of NAD⁺ in purinergic signalling. Extracellular NAD⁺ and ATP are released from damaged cells. ATP binds to the ATP-sensitive P2X7 receptor of monocytic cells. Activation of inflammasomes and caspase-1 induces cleavage of pro-IL-1β and release of bioactive IL-1β. NAD⁺ binds to P2Y receptors and activates iPLA2β, leading to the production and release of bioactive mediators which serve as nicotinic agonists.

Figure 3

NADomics workflow. The NADomics workflow involves profiling the NADome with statistically significant variations in biological samples, e.g., blood, urine, and CSF. The specific NAD⁺ metabolite ID including chemical structure and concentrations can be elucidated using LC-MS/MS. Analysis is the final step to elucidate associations between the identified metabolite and its role in physiology and disease.