| Background:Fentanyl is an intravenous narcotic analgesics with high efficiency and most commonly used in liver transplantation. As an opiate receptor agonist, it takes effect very rapidly, lasts a very short time in blood, does not release histamine, and has little effect on cardiovascular function. It is metabolized mainly in the liver, and its metabolites and about 10%original drug are discharged by the kidneys. In liver transplant operation, there is no liver metabolism during anhepatic phase. How is fentanyl metabolized? And what will influence this proscess?These questions remain unclear. Cytochrome P450 (CYP), a member of the hemoprotein gene superfamily, encodes a series of metabolic enzymes, participates biological transformation of lipophilc compounds in various structures, and enhances the water-solubility of the metabolic products which will be prone to be discharged so as to reduce the toxicity of these exogenous compounds. The liver has the highest contents of CYP3A1 among other tissues and organs. Its expression is modulated in an immediate, tissue-specific and environment-related manner. In the anhepatic phase of liver transplantation, internal milieu changes vigorously, and the levels of some hormones may affect extrahepatic expression of CYP3A1, thus influencing extrahepatic metabolism of fentanyl. In small intestine, CYP3A4 is the main form and distributed in the epithelial cells.Objective:In this study, fentanyl metabolism in anhepatic rats was studied, and the changes in CYP3A1 enzymatic activity, gene expression and protein expression in small intestine before and after the anhepatic phase were also investigated. This study was aimed to preliminarily elucidate how the anhepatic state influences the CYP3A1 expression and what fentanyl metabolism is in the anhepatic phase.Materials and Methods: This study is composed of three parts:1. Fentanyl plasma cocentration changes in the anhepatic rats.Five SPF male SD rats were used to plot the standard curve of blood fentanyl concentration, and another 20 were equally divided into control and treatment group (anhepatic group). After all rats were etherized, a 24# vein detaining-pin was inserted into their right deep cervical vein to collect blood sample, and another detaining-pin was set to the right femoral vein to add fentanyl. In treatment group, fentanyl (20μg/kg) was infused after hepatic hilum blocking, and blood sample (0.5 ml) was colleted in 1, 2, 3, 5, 10, 15, 30, 45, 60, 70 and 90 min after the infusion. Rats of control group did not receive hepatic hilum blocking. The fentanyl concentration was measured with LC-MS/MS and analyzed with DAS2.0 Pharmacokinetics Program. The standard curve of rat blood fentanyl concentration was plotted by high performance liquid chromatograph-mass spectrogram using standard fentanyl (1μg/ml, purity 99.9%), and the method for measuring fentanyl concentration with small volume of blood sample was established.2. Extrahepatic expression of fentanyl metabolism related CYP3A1 in the anhepatic phase.Totally 30 SPF male SD rats were equally and randomly divided into the control groups (group B1, as before the anhepatic phase), rats with hepatic portal blocking for 30 min (group B2, as 30 min anhepatic phase), and rats with 60 min blocking (groupB3, as 60 min anhepatic phase). All rats were treated as done in part 1 for drug injection and infusion. The intestinal specimen 4 cm from the stomachus pyloricus was taken out, and the activity of CYP3A1 in the specimens was detected by colorimetry.3. Extrahepatic expression of CYP3A1 gene and protein in the anhepatic phase. The rats were randomly divided into (n=10 in each group): control group (group C1, before the anhepatic phase), group C2 (30 min anhepatic phase) and group C3 (60 min anhepatic phase).After etherization, small intestine were harvested, and CYP3A1 mRNA and protein expression was detected by RT-PCR and Western blotting respectively.Results:1. Under the selected chromatographic conditions, the samples and the internal standard of fentanyl showed satisfactory and completely-separated peaks and had no interference of impurity peak, and maintained in a sound time period. The standard curve equation of fentanyl was y = 0.0156 x+0.0072 (r=0.9997, P<0.05). The minimal detectable concentration of fentanyl was 0.5 ng/ml, indicating the detection method is highly precise.2. Before and after the anhepatic phase, single dose of fentanyl did not change significantly with CL and Vd (P>0.05), AUC and T1/2βchanged obviously.(P<0.05).3. CYP3A1 activity in small intestine was significantly higher after 30 min or 60 min anhepatic phase than before the anhepatic phase (P<0.05), but no significant difference was seen between 30 min and 60 min of the anhepatic phase (P>0.05).4. The expression of CYP3A1 gene and protein in rat small intestine was significantly higher after 30 min or 60 min anhepatic phase than before the anhepatic phase (P<0.05), but there is no significant difference between 30 min and 60 min anhepatic phase(P>0.05).Conclusions:1. The method of measuring blood fentanyl concentration in small volume of blood sample is specific, simple and accurate, with little time variation and wide linear range. It can be used as a conventional assay measuring blood fentanyl concentration.2. Melabolism of fentanyl during the anhepatic phase is characterized with no significantly changed CL and Vd, prolonged T1/2βand obviously increased AUC.3. Liver is the main organ to metabolize fentanyl, but there maybe other ways for fentanyl metabolization.4. CYP3A1 activity in small intestine is significantly higher in the anhepatic phase, indicating it maybe one of the mechanism for frntanyl metabolized in small intestine.5. The expression of CYP3A1 gene and protein in rat small intestine is significantly higher in the anhepatic phase, which maybe one of the mechanism about the elevated enzymic activity and extrahepatic metabolism of fentanyl. Simultaneously, it maybe one of the reasons that generate the metabolic characters in the rat small intestine about fentanyl. |
