Optimized protocol for quantification of extracellular nicotinamide adenine dinucleotide: evaluating clinical parameters and pre-analytical factors for translational research

Abstract

Nicotinamide adenine dinucleotide (NAD⁺), a coenzyme for more than 500 enzymes, plays a central role in energy production, metabolism, cellular signaling, and DNA repair. Until recently, NAD⁺ was primarily considered to be an intracellular molecule (iNAD⁺), however, its extracellular species (eNAD⁺) has recently been discovered and has since been associated with a multitude of pathological conditions. Therefore, accurate quantification of eNAD⁺ in bodily fluids such as plasma is paramount to answer important research questions. In order to create a clinically meaningful and reliable quantitation method, we analyzed the relationship of cell lysis, routine clinical laboratory parameters, blood collection techniques, and pre-analytical processing steps with measured plasma eNAD⁺ concentrations. Initially, NAD⁺ levels were assessed both intracellularly and extracellularly. Intriguingly, the concentration of eNAD⁺ in plasma was found to be approximately 500 times lower than iNAD⁺ in peripheral blood mononuclear cells (0.253 ± 0.02 μM vs. 131.8 ± 27.4 μM, p = 0.007, respectively). This stark contrast suggests that cellular damage or cell lysis could potentially affect the levels of eNAD⁺ in plasma. However, systemic lactate dehydrogenase in patient plasma, a marker of cell damage, did not significantly correlate with eNAD⁺ (n = 33; r = -0.397; p = 0.102). Furthermore, eNAD⁺ was negatively correlated with increasing c-reactive protein (CRP, n = 33; r = -0.451; p = 0.020), while eNAD⁺ was positively correlated with increasing hemoglobin (n = 33; r = 0.482; p = 0.005). Next, variations in blood drawing, sample handling and pre-analytical processes were examined. Sample storage durations at 4°C (0-120 min), temperature (0° to 25°C), cannula sizes for blood collection and tourniquet times (0 - 120 s) had no statistically significant effect on eNAD⁺ (p > 0.05). On the other hand, prolonged centrifugation (> 5 min) and a faster braking mode of the centrifuge rotor (< 4 min) resulted in a significant decrease in eNAD⁺ levels (p < 0.05). Taken together, CRP and hemoglobin appeared to be mildly correlated with eNAD⁺ levels whereas cell damage was not correlated significantly to eNAD⁺ levels. The blood drawing trial did not show any influence on eNAD⁺, in contrast, the preanalytical steps need to be standardized for accurate eNAD⁺ measurement. This work paves the way towards robust eNAD⁺ measurements, for use in future clinical and translational research, and provides an optimized hands-on protocol for reliable eNAD⁺ quantification in plasma.

Keywords: clinical parameters; enzymatic assay; extracellular; intracellular; nicotinamide adenine dinucleotide (NAD+); plasma; pre-analytical factors; quantification.