Extracellular NAD+ response to post-hepatectomy liver failure: bridging preclinical and clinical findings.

Liver fibrosis progressing to cirrhosis is a major risk factor for liver cancer, impacting surgical treatment and survival. Our study focuses on the role of extracellular nicotinamide adenine dinucleotide (eNAD+) in liver fibrosis, analyzing liver disease patients undergoing surgery. Additionally, we explore NAD+'s therapeutic potential in a mouse model of extended liver resection and in vitro using 3D hepatocyte spheroids. eNAD+ correlated with aspartate transaminase (AST) and bilirubin after liver resection (AST: r = 0.2828, p = 0.0087; Bilirubin: r = 0.2584, p = 0.0176). Concordantly, post-hepatectomy liver failure (PHLF) was associated with higher eNAD+ peaks (n = 10; p = 0.0063). Post-operative eNAD+ levels decreased significantly (p < 0.05), but in advanced stages of liver fibrosis or cirrhosis, this decline not only diminished but actually showed a trend towards an increase. The expression of NAD+ biosynthesis rate-limiting enzymes, nicotinamide phosphoribosyltransferase (NAMPT) and nicotinamide mononucleotide adenylyltransferase 3 (NMNAT3), were upregulated significantly in the liver tissue of patients with higher liver fibrosis stages (p < 0.0001). Finally, the administration of NAD+ in a 3D hepatocyte spheroid model rescued hepatocytes from TNFalpha-induced cell death and improved viability (p < 0.0001). In a mouse model of extended liver resection, NAD+ treatment significantly improved survival (p = 0.0158) and liver regeneration (p = 0.0186). Our findings reveal that eNAD+ was upregulated in PHLF, and rate-limiting enzymes of NAD+ biosynthesis demonstrated higher expressions under liver fibrosis. Further, eNAD+ administration improved survival after extended liver resection in mice and enhanced hepatocyte viability in vitro. These insights may offer a potential target for future therapies.

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

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.