Nicotinamide Adenine Dinucleotide as a Potential Biomarker for Graft Viability During Normothermic Machine Perfusion—An Ex Vivo Rodent Study

ABSTRACT Background The shortage of donor organs has increased the use of extended criteria donor (ECD) grafts in liver transplantation. Normothermic machine perfusion (NMP) could be used as a platform for real‐time quality assessment, which is crucial since ECD grafts are more susceptible to ischemia–reperfusion injury (IRI). As nicotinamide adenine dinucleotide (NAD + ) is a central mediator of cellular antioxidativedefense, reduced intracellular NAD (iNAD + ) levels in grafts sustaining greater injury may reflect diminished antioxidative capacity and could serve as a marker to assess graft quality. This study aimed to investigate iNAD + in comparison to existing damage‐associated parameters for liver graft quality in a small animal NMP model simulating ECD conditions through advanced donor age, prolonged cold ischemia time (CIT), and donation after circulatory death (DCD). Methods Fifty‐nine male Sprague–Dawley rat livers were analyzed across six experimental groups defined by donor age (3 months [young] or 12 months [elderly]), procurement modality (standard procurement or donation after circulatory death [DCD]), and cold ischemia time (CIT; 6 h or 12 h), followed by 6 h of NMP. No livers were excluded from analysis. iNAD + levels were quantified using a colorimetric enzymatic assay. Results Perfusate iNAD + levels continuously rose over time during NMP and differed significantly between intervention groups ( p = 0.003). iNAD+ correlated negatively with ALT levels (R = −0.36, p = 0.024) and liver tissue necrosis in grafts (perfusate R = −0.24, p = 0.17; tissue R = −0.19, p = 0.2), although these associations did not reach statistical significance. Long CIT, higher donor age, and DCD procurement were associated with lower iNAD+ levels. iNAD+ did not correlate with lactate clearance or bile production (both p > 0.05). A non‐significant trend toward a positive association between iNAD+ and glucose metabolism was observed ( R = 0.29, p = 0.077). Discussion Higher perfusate iNAD+ levels were associated with better organ performance, consistent with iNAD+ reflecting the graft's antioxidative metabolic capacity. Lower iNAD+ levels in elderly and DCD grafts aligned with established damage markers. Perfusate iNAD+ may therefore offer a complementary tool for evaluating metabolic activity and organ performance during NMP, pending prospective validation.