Hafez, T; (2015) The effect of liver warm ischaemia reperfusion injury and modulation on bile composition evaluated by magnetic resonance spectroscopy. Doctoral thesis , UCL (University College London).

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Abstract

Orthotopic liver transplantation has become the preferred treatment for a variety of end-stage liver disease. As competency and survival rates increase, so does increasing demand, which puts greater strain on a static donor pool. This increases pressure to accept more marginal grafts, e.g. non-heart-beating and steatotic donors. However, graft dysfunction post-transplantation contributes considerably to postoperative morbidity and mortality. Proton nuclear magnetic resonance (1HNMR) spectroscopy is a powerful technique to explore the biochemical composition of biological fluids; it is rapid, non-invasive, nondestructive and it can detect metabolites at millimolar concentrations. In this study, 1HNMR assessed the changes in bile composition during liver ischaemia/reperfusion. The primary hypothesis was that bile composition changes during liver ischaemia reperfusion injury (IRI). The aims were to document these changes, identify biliary markers for liver IRI using 1HNMR, validate these markers using known modulators, determine if the same was true for steatotic livers and attempt to understand the mechanisms by which these changes happen in relation to the redox state of the liver. Materials and methods: A rabbit model of hepatic lobar IRI was used. In most experiments 3 groups were used (n=6): Sham group (laparotomy alone), I/R group (1hr ischaemia and 6hrs reperfusion), and a modulation group similar to I/R with the addition of N-acetylcysteine (I/R+NAC: 150mg/kg of NAC) and glycine (I/R+glycine: 5mg/kg glycine). Steatosis was induced by feeding with a 2% cholesterol diet for 8 weeks. Experiments were repeated on steatotic animals with Sham, I/R, I/R+NAC and IPC+I/R (5min ischaemia followed by 10min of reperfusion before prolonged ischaemia was induced) groups. The following parameters were measured: portal blood flow, bile flow (BF) and bile 1HNMR spectroscopy, hepatic microcirculation, intracellular tissue oxygenation, serum ALT, AST and ICG clearance were measured at 1, 2, 5 and 7 hours following reperfusion. Results: Bile spectroscopy demonstrated significant changes in bile composition following I/R and alterations with NAC, glycine and IPC. These changes are evident despite a constant post-reperfusion rate of BF. They were also present in steatotic livers, and were modulated by NAC and IPC. In experiment 1: BF, COX and biliary acetate decreased following I/R while AST, ALT, biliary PC and lactate increased along with PMN accumulation in sinusoids, KC hypertrophy, necrosis and apoptosis in normal livers. In experiment 2: BF, COX and biliary acetate decreased following I/R while ALT, PC, conjugated bile acids and lactate increased in the I/R group in normal livers. NAC administration attenuated the increase in ALT and lactate following I/R in the NAC+I/R group compared to the I/R group. Changes in conjugated bile acids seem to reflect changes in BF. In experiment 3: I/R+glycine was associated with increased BF, bile acid, acetate, pyruvate, glucose, acetoacetate, and decreased bile lactate and PC levels in normal livers. In experiment 4: NAC administration in steatotic livers reduced the extent of IRI, increased portal blood flow and liver parenchymal perfusion. NAC increased BF, biliary acetate and pyruvate and reduced acute liver injury, ALT and biliary PC. In experiment 5: IPC protected the steatotic liver from IRI and maintained hepatic oxygenation, tissue perfusion and mitochondrial redox state. COX activity was decreased by IRI in the fatty liver, but can was protected by IPC. Conclusions: This thesis has demonstrated changes in bile composition during warm normal liver I/R and its modulation with NAC and glycine. It has also demonstrated changes in bile composition during warm steatotic liver I/R and its modulation with NAC and IPC. It has noted several metabolites that are consistently changed, as well as the bile redox ratio of metabolites that provides a clearer indication of liver redox state than individual metabolites.

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