Fatty liver preservation and autofluorescence real time monitoring of oxidative damage

Introduction: The risk of disease progression or organ dysfunction during surgery and transplantation of fatty livers is increased by oxidative stress. A rat liver model of NASH (induced with methionine/choline-deficient diet-MCD) exhibited a general subversion of metabolism reflecting in alterations of autofluorescence (AF) emission properties. These consisted in NAD(P)H and flavin AF signal changes, ascribable to redox metabolism alterations, and rising in vitamin A, protein, and lipopigment AF contribution. Aims: Since lipopigments derive from the oxidation of unsaturated lipids, fluorescing fatty acids and their oxidized products were investigated in isolated MCD livers under different preservation conditions as early AF biomarkers of oxidative alterations. Material and Methods: Control and 2 week MCD diet livers were isolated and submitted to 6-h Cold Storage (CS) or subnormotermic Machine Perfusion (MP) preservation, followed by reperfusion at 37°C with oxygenated medium. In vivo AF analysis -exc 366nmwas performed via fiber optic probe. Each endogenous fluorophore contribution to the overall AF emission was estimated through spectral fitting procedure, similarly to in situ biochemical analysis. Tissue oxidative stress (TBARS, and GSH) and mitochondrial dysfunction (ATP/ADP) were assayed with conventional methods. Results: Liver AF emission amplitude was influenced to different degree by CS and MP preservation and subsequent reperfusion, consistently with a MCD liver mitochondrial dysfunction affecting the response to oxygen availability and temperature, in terms of NAD(P)H/flavin redox changes. Both controls and MCD diet livers showed spectral shape alterations indicating a rising in oxidized lipids after preservation, in a good correlation with TBARS. The phenomenon more marked in MCD than in control livers indicated a strong influence of oxidative stress, independently from CS or MP preservation. To note that fluorescing fatty acids were not depleted, consistently with dynamic equilibria in liver lipid pool composition. Conclusions: The in vivo, real time AF analysis was validated to evidence the rising of oxidative effects induced by external stimuli even in a model of mild metabolic alterations. These results provide further support in AF experimental applications to assess drug response and toxicity, and in the set-up of innovative organ preservation strategies. (Supported by Fondazione Cariplo, grant n° 2011-0439).