Lipidomic profiling and liver tissue autofluorescence of fatty livers correlated with the organ susceptibility to cold storage preservation

Introduction: Although fatty livers are more prone to primary dysfunction and nonfunction after transplantation, they represent a possibility to reduce the discarded organs (1). Animal models of hepatic steatosis are essential to understand the mechanisms underlying preservation injury of graft under hypothermic conditions. (2). Liver tissue autofluorescence (AF) is an innovative resource to monitor in situ fluorescing fatty acids (3) . Aim: In this study, we investigated how the lipidomic profile correlates with the liver susceptibility to cold storage preservation using two rat models of NAFLD: the methionine and choline deficient (MCD) diet and the Obese Zucker fa/fa rats. Method: Experimental models: NAFLD was induced in male Wistar rats by 2-week MCD diet (n=7); 12-week male old obese (fa/fa) Zucker rats were also used (n=7). Isolated livers were preserved in UW solution at 4°C for 6 hours (Cold Storage, CS) followed by reperfusion (2 hours). Assays: Perfusate and biliary enzyme release were measured by Hitachi747. Bile production and portal pressure were evaluated, tissue glutathione (GSH) and lipid peroxides were evaluated. Analysis of fatty acid (FA): before preservation, liver FA profiling was performed on liver lipid extracts by Gas Chromatography-Mass Spectrometry (GS-MS) and parallel estimation of fluorescing free FA (FFA) by fitting analysis of autofluorescence (AF) spectra recorded under 366 nm excitation (see below). Results: According to the FA analysis by GS-MS, in Obese Zucker and MCD rats the total saturated (SFA)/polyunsaturated fatty acid (PUFA) ratio was 1.5 and 0.71, respectively, When compared with Obese Zucker Zats, MCD rats showed a three-time decrease in saturated stearic acid (%: 6,7±0.4 versus 20.5±1.5), an eight-time increase in polyunsaturated linoleic acid (%: 31,7±0.9 versus 5.1±1.3) and a three-time decrease in polyunsaturated arachidonic acid (AA). Arachidonic acid levels measured by AF fully reflected the changes observed by GC-MS analysis. After 6 hours CS and 2 hours reperfusion, a three-time increase in AST and two-time increase in LDH were found comparing Zucker obese rats versus MCD group. Bile production was lower and biliary enzymes were higher in Obese Zucker rats when compared with MCD group (Figure 4). A marked rise of portal pressure were found in Zucker obese rats versus MCD group (mmHg: 12.9±1.6 versus 8.2±1.3, <0.05 ). Conclusions: Our results suggest that cellular injury is strongly associated with the liver composition in FAs, which could represent an effective prognostic target; fatty livers from Zucker obese donors characterized by higher levels of saturated FA, are more prone to CS injury as compared with MCD livers with high PUFA content. On the contrary, livers from MCD appear markedly less susceptible to CS as demonstrated by lower necrosis as well as by decreased biliary damage and increased bile flow. The present study suggests that the preservation technique should be chosen on the basis of the liver lipidomic profiling which might be proposed as a prescreening tool. Supported by GC-MS analysis, the ability of AF to promptly reveal the liver lipidomic profiling can be proposed for the prescreening donor livers.