Apply High Performance Liquid Chromatography To Study The Effect Of Cold Preservation Reperfusion Injury On Bile Acid Metabolism Of Rat Liver After Liver Transplantation

Objective:1. A method for the determination of individual free and conjugated bile acids in rat bile by reverse phase high performance liquid chromatography (RP-HPLC) with pre-column derivatization and fluorescence detection was developed.2. The Bile acids in rat bile within 14 d posttransplantation of liver transplantation group and control group were analyzed by RP-HPLC, so as to discuss the change tendency of bile acids in rat liver after cold preservation reperfusion injury, and to propose some new strategies for the prevention and treatment of posttransplant biliary complications.Methods:1. Free and glycine-conjugated bile acids are derived by 4-bromomethyl-7-methoxycoumarin with dicyclohexano-18-crown-6 as the catalyst. Taurine-conjungated bile acids was hydrolysed by cholylglycine hydrolase before derivatization.2. Chromatographic separation was performed with gradient elution on a Waters Nova C18 column (3.9×150 mm,5μm) at a flow rate of 1.0 mL/min. Mobile phases A is methanol-acetonitrile (1:2, v/v) and B is ultrapure water. The column was kept at 20℃. The elutions were monitored by a fluorophotometer at 330 nm (λex) and 400 nm (λem), and quantitated by internal standard curves with aurid acid (LA) as an internal standard. The analysis time was 20 min.3. Bile acids were extracted from bile samples using Chromabond C18 ec cartridges.4. The Bile acids in rat bile within 14d posttransplantation of liver transplantation group (with 1 hour and 12 hour graft preservation) and control group were analyzed by RP-HPLC.Results:1. The assay was linear in the range 5-2400μmol/L. The linear regression coefficients ranged from 0.9989 to 0.9997. The detection limits ranged from 1.2 to 2.5μmol/L for different bile acids. The within-day precisions were below 3.67%, the between-day precision were below 6.83%. The average recoveries were 87.80﹪-106.71﹪.2. Contrast to the control group, the concentration of hydrophobic bile acids (TCA and TCDCA) increased significantly and the concentration of hydrophilic bile acids (TUDCA and THDCA) decreased continuously after a short-time increase. The hydrophobicity index (HI) elevated obviously. furthermore, the HI elevated with the prolong of graft preservation time.Conclusion:1. This method was competent for determining of individual free and conjugated bile acids in rat bile. With wide linear range, it is sensitive, reproducible, rapid, and suitable for the determination of bile acids in rat bile in scientific research.2. the proportion of hydrophobic bile acids increased after cold preservation/reperfusion injury (CPRI), which is probably one of the major factors leading to the increase of bile toxicity after liver transplantation and may correlate to the pathogenesis of bile duct injury of liver allografts.