The Relationship Between ATP Contents Of Graft Liver And Early Function Of Donor Liver After Transplantation

BACKGROUND:Liver transplantation has become a standard treatment for end-stage liver disease,but it has several outstanding problems.The graft primary dysfuction is one of them,contributing significantly to a more than 20% per year mortality rate for patiens in liver transplantation centers in the United States.To predict the graft function precisely in time and regulate corresponding cure for cutting down mortality of patients,it is very important to establish a series of detection methods and sensitive factors used for evaluating the donor liver quality early.So several units are subject to find independent reliable simple methods and parameters abroad.Bile flow is a reliable factor to predict the early graft function proved by most researches.To study the relationship between adine nucleotides or their catabolites and early graft function is one of the hot,but few researches are made in our nation.PART I:Objective :To establish an animal model of modified cuff technique orthotopic of liver transplantation in rat.Methods: 150 cases of rat orthotopic liver transplantation were performed by using the two-cuffed technique with some modification in graft harvesting, recipients anesthesia, the anastomoses of suprahepatic vena cava and the perioperative treatment.Results: Of the last 30 cases , the mean time of anhepatic period was 13min, and the mean operation time was 45min. The 24 hours and 2 week survival rates were 97% and90% respectively.Conclusions: The modified two-cuffed methods is convenient and easy to duplicated. It can enhance the stability and survival rate of rat orthotopic liver transplantation models.PART II:Objective: To set up an rapid - simple, accurate RP-HPLC method for the ditermination of ATP contents in tissues of rat donor liver and to observe the change of ATP levels of donor liver preserved in 4°C Ringer's lactate solution.Methods: ATP contents of rat donor liver with defferent CIT were detected by RP-HPLC. chromatographic conditions were as follows: Kromasil? CigODS column (4.6mm X 250mm, 5um), 0.15mol/L potassium dihydrogen phosphate buffer solution (pH=6.00) as mobile phase, the flow rate 1.0 mL/min and UV detector at 254 nm.Results: The calibration curve of ATP showed a good linearity in 3.125-200ug/ml. Its average recovery was (99.03±1.78)% and RSD was 1.80%,interday RSD ^1.08%, intraday RSD ^4.94%.Conclusions: HPLC is a simple rapid reliable low-costed method for determination of ATP content. ATP levels of rat donor liver were decreased sharply followed CIT prolonging and becoame a good correlation with CIT.PART III:Objective: To study relationship between ATP levels of rat graft liver and its early graft function..Methods: . The liver grafts were preserved in 4°C Ringer's lactate solution and transplanted orthotopically,and its tissue ATP content was analyzed with high-performance liquid chromatography. The groups for detecting paranmeters,whose cold ischemic time(CIT) were 3 and 5 hours,were to analyze ATP levels at the time of reperfusion and 2 hours after it, to measure bile output and serum ALT> AST at 2 hours after reperfusion,then to cut liver for histological evaluation with HE staining and apoptosis of the hepatocytes and SEC analyzed by TUNEL. Another rats taken for the survial study underwent transplantation within correspondent CIT.For the relationship study between tissue ATP content prior to portal-venous declamping and bile output at 30 min after reperfusion, 16 rats were taken for orthotopic liver transplantation within defferent CIT by random.Results: The tissue ATP levels in the grafts decreased followed cold preservation time.There were distinct differences in ATP contents,bile flow,enzyme levels,survival rates and morphology within groops and was a good correlation between tissue ATP contentprior to portal-venous declamping and bile output at 30 min after reperfusion.Conclusion: The levels of cellular ATP are related to the graft early function and are a reliable indicator for assessing the extent of ischemic damage to the liver and for early graft early function.