Abstract
Nicotinamide adenine dinucleotide (NAD), a prominent member of the pyridine nucleotide family, plays a pivotal role in cell-oxidation protection, DNA repair, cell signalling and central metabolic pathways, such as beta oxidation, glycolysis and the citric acid cycle. In particular, extracellular NAD+ has recently been demonstrated to moderate pathogenesis of multiple systemic diseases as well as aging. Herein we present an assaying method, that serves to quantify extracellular NAD+ in human heparinised plasma and exhibits a sensitivity ranging from the low micromolar into the low nanomolar domain. The assay achieves the quantification of extracellular NAD+ by means of a two-step enzymatic cycling reaction, based on alcohol dehydrogenase. An albumin modified revised simulated body fluid was employed as standard matrix in order to optimise enzymatic activity and enhance the linear behaviour and sensitivity of the method. In addition, we evaluated assay linearity, reproducibility and confirmed long-term storage stability of extracellular NAD+ in frozen human heparinised plasma. In summary, our findings pose a novel standardised method suitable for high throughput screenings of extracellular NAD+ levels in human heparinised plasma, paving the way for new clinical discovery studies.
Highlights
Nicotinamide adenine dinucleotide (NAD), a prominent member of the pyridine nucleotide family, plays a pivotal role in cell-oxidation protection, DNA repair, cell signalling and central metabolic pathways, such as beta oxidation, glycolysis and the citric acid cycle
High performance liquid chromatography (HPLC) methods followed by mass spectrometry (HPLC-MS), and especially tandem mass spectrometry (HPLC-MS/MS), have managed to achieve remarkable sensitivity and specificity for iNAD+ thanks to their ability to separate the respective metabolites through elution with highly specialised columns and successively combining the relaxation time of specific metabolites with their mass spectrum[15]
In order to emulate the enzyme kinetics of ADH in human heparinised plasma and to gain assay sensitivity, several standard matrices were compared against human plasma, namely DEPC water, revised simulated body fluid adjusted with albumin (r-SBFA) matrices featuring g/L (r-simulated body fluids (SBF)) and r-SBFA, using β-NAD Standards
Summary
Nicotinamide adenine dinucleotide (NAD), a prominent member of the pyridine nucleotide family, plays a pivotal role in cell-oxidation protection, DNA repair, cell signalling and central metabolic pathways, such as beta oxidation, glycolysis and the citric acid cycle. We present an assaying method, that serves to quantify extracellular NAD+ in human heparinised plasma and exhibits a sensitivity ranging from the low micromolar into the low nanomolar domain. The assay achieves the quantification of extracellular NAD+ by means of a two-step enzymatic cycling reaction, based on alcohol dehydrogenase. We evaluated assay linearity, reproducibility and confirmed long-term storage stability of extracellular NAD+ in frozen human heparinised plasma. Our findings pose a novel standardised method suitable for high throughput screenings of extracellular NAD+ levels in human heparinised plasma, paving the way for new clinical discovery studies. Intracellular NAD+ (iNAD+) was determined to be in the range of 10–40 M, whilst eNAD+ in pig plasma was found at a fraction of this concentrations, namely 240–290 nM11–13. Each singular measurement of LC-based assays consumes significant amounts of time, from ten minutes up to one hour, making their application to high throughput screenings impractical
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