The nanomolar sensing of nicotinamide adenine dinucleotide in human plasma using a cycling assay in albumin modified simulated body fluids

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.