| Biliary atresia (BA) is the most common neonatal cholestatic disorder characterized by complete fibrotic obliteration of the lumen of all or part of the biliary tree within 3 months of life. Kasai procedure (portoenterostomy) is an optimal surgical intervention for patients with biliary atresia before 12 weeks.Continuous intravenous infusion of morphine is frequently practiced for postoperative pain relief in infants after Kasai procedure. In addition to pain relief, morphine induces respiratory depression which in turn would cause life threatening hypoxemia crisis.Children with biliary atresia have a serious degree of hepatic dysfunction resulting in impaired hepatic elimination of drugs in general is to be expected.The capacity of the liver to metabolize morphine depends on uridine-diphosphate glucuronosyl-transferases (UGT) enzyme activity as well as hepatic blood flow. At present, most data of morphine metabolism in liver disease subjects are from animal studies or researches in adult cirrhotic patients. Nevertheless, the available data can not explain each other. The efficiency of morphine removal by the liver of children with biliary atresia is unknown.There is growing evidence suggesting that overabundance of reactive oxygen species (ROS) may play an important role in cholestatic liver injury. The mechanism of ROS mediated hepatocyte death involves lipid peroxidation, apotosis as well as oncotic necrosis. Melatonin has been proven to have the greatest impact not only on oxidative stress, but also on defense systems that removes free radicals, restoring the oxidative balance in treated experimental animals.Since ROS are the principal toxic mediators by which cholestasis damages hepatocytes, it could also be contributed to both UGT enzyme and hepatic blood flow impairments in children with biliary atresia. Consequently, it would decrease the morphine metabolism in children with biliary atresia.The aims of this study were to investigate the status of morphine metabolism in very young children undergoing Kasai procedure, as well as the effects of ROS on morphine metabolism in these bile obstructed subjects. PartⅠReactive Oxygen Species and Morphine Metabolism in Biliary Atresia InfantsPurpose:To investigate both the morphine metabolism and the correlation between morphine metabolism and ROS in biliary atresia infants.Methods:Patients study:Fifty infants scheduled for Kasai procedure and 20 infants without liver disease(age≤3 Months) scheduled for major abdomen/pelvic surgery received a continuous intravenous infusion of 8μg/kg/min morphine during 48 hours. Blood was sampled 24 hours after infusion initiation and plasma morphine concentration (steady-state concentration) was measured by high performance liquid chromatography (HPLC). Clearance of morphine was calculated as infusion rate divided by the steady-state concentration. ROS was determined by Fenton action from liver tissue samples of subjects undergoing Kasai procedure. Regression analysis of ROS vs. steady-state morphine concentration was performed by Curve Estimation. Animal study:Thirty Wistar rats were randomized into three groups:sham operation, bile duct ligation (BDL) and bile duct ligation plus melatonin (BDL+melatonin). Melatonin was injected intraperitoneally 500μg/kg/day for 6 days. On the 6th day of ligation (or sham), blood was sampled 3 hours after morphine 5 mg/kg intrperitoneally and plasma morphine concentration was measured by HPLC. ROS was determined by Fenton action from liver tissue samples.Results:Patients results:Blood samples were obtained from 34 subjects in biliary atresia group and 16 subjects in control group. Steady-state morphine concentration of biliary atresia patients and controls were 5.8(4.5～16.4)ng/ml and 6.1 (3.2～10.9)ng/ml respectively (P>0.05). Clearance was 22.6 (8.1～29.4)ml/kg/min and 21.8 (12.6～42.1)ml/kg/min respectively (P>0.05).47 liver tissue samples were obtained in biliary atresia group. Negative Logarithmic regression between steady-state morphine concentration and ROS was found in subjects younger than 60 days(Y=79.5-15.11nX, n=14, R2=0.577, P=0.001)。Animal results: ROS concentration of rats from sham operation, BDL and BDL+melatonin group was 5.9 (4.4～12.9)U/mg protein,12.5 (10.1～17.1)U/mg protein,8.1 (5.6～9.2) U/mg protein respectively (P=0.004). Morphine concentration was 4.6 (2.4～17.2)ng/ml,1.4 (1.0～2.8)ng/ml,8.6 (1.0～20.8) ng/ml respectively (P>0.05).Conclusion:Capacity of morphine metabolism was not impaired in biliary atresia infants. Morphine clearance had an upward tend when ROS was increasing. ROS augmented morphine metabolism in bile obstructed rats, which implied that did not necessarily decrease morphine metabolism in infants with biliary atresia.PartⅡEffects of Reactive Oxygen Species on UGT mRNA/protein Level in Infants with Biliary AtresiaPurpose:To identify the effects of reactive oxygen species (ROS) on both UGT mRNA and UGT protein level in infants with biliary atresia.Methods:Patient study:UGT mRNA and protein expression level was determined by Real time PCR and Western blot respectively from the 47 liver tissue samples of subjects undergoing Kasai procedure recruited in PartⅠRegression analysis of both ROS vs. UGT mRNA and ROS vs. UGT protein was performed by Curve Estimation. Animal study:UGT mRNA and protein expression level was also determined by Real time PCR and Western blot respectively from the liver tissue samples of the 30 rats in PartⅠ.Results:Patient results:Relative UGT mRNA level of 47 samples from biliary atresia patients was 7.88(4.59～13.49). Relative UGT protein level was 0.49(0.25～0.79). Negative Logarithmic regression between UGT mRNA vs. ROS(Y= 27.2-4.31nX, R2= 0.2, p= 0.002), UGT protein vs. ROS(Y =1.136-0.1581nX, R2= 0.128, P= 0.014) were both found. Animal results: Relative UGT mRNA level of liver tissue samples from sham operation, BDL and BDL+melatonin group was 0.81(0.70-0.86),0.28(0.22～0.29) and 0.43(0.35～0.68) respectively(P=0.001). Relative UGT protein level was 0.39(0.16～0.59),0.12(0.12～0.15) and 0.33(0.28～0.42) respectively (P=0.001).Conclusion:Both UGT mRNA and UGT protein level was determined by ROS downwardly in infants with biliary atresia. ROS induced downregulation of both UGT mRNA and protein in bile obstructed rats, which implied ROS was contributed to UGT downregulation in infants with biliary atresia.PartⅢEffects of Reactive Oxygen Species on Hepatic Blood Flow in Infants with Biliary AtresiaPurpose:To investigate the effects of reactive oxygen species (ROS) on hepatic blood flow in infants wit h biliary atresia.Methods:Patient study:Both hepatic blood flow and hepatic arterial perfusion index (HPI) was evaluated by Dynamic Contrast-Enhanced MRI (DCE MRI) prior to surgery in 12 of the 47 biliary atresia patients involved in PartⅠ. DCE MRI evaluation was also performed in 6 hydronephrosis patients who were part of the controls in PartⅠ. Regression analysis of ROS vs. HPI and HPI vs. steady-state morphine concentration was performed by Curve Estimation. Animal study:Hepatic blood flow was accessed by DCE MRI in 15 rats selected randomly from the 30 rats prepared in PartⅠ(5 each group) before morphine administration on the 6th day of ligation or sham operation.Results:Hepatic blood flow of patients from each group were similar, which is 3.51(2.72～3.88) ml/min/100ml for biliary atresia and 3.15(2.66～4.42) ml/min/100ml for hydronephrosis (P>0.05). HPI was higher in infants with biliary atresia [0.65(0.58～0.70) vs.0.54(0.47～0.65), P=0.04]. Logarithmic regression between HPI and ROS was found (Y= 0.245+0.1021nx, R2= 0.58, P=0.004). Inverse regression between steady-state morphine concentration and ROS was found (Y=4.559/X-2.446, R2=0.519, P=0.001). Animal results:Relative hepatic blood flow(% renal blood flow) of subjects from sham operation, BDL and BDL+melatonin group was 0.37±0.13,2.00±0.70 and 0.61±0.14 respectively,P=0.002.Conclusion:Infants with biliary atresia had unchanged hepatic blood flow and increased hepatic arterial blood flow. Morphine concentration is dependant on hepatic arterial flow. The latter depended on ROS downwardly. ROS induced hepatic blood flow increasing in bile obstructed rats, which implied stable hepatic blood flow was maintained by ROS in biliary atresia infants. |
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