Effect Of CYP3A4*1G Genetic Polymorphism On The Human Liver Microsomal Fentanyl Metabolism

Background and ObjectiveFentanyl is a potent opioid narcotic analgesic,which has been widely used in thefield of clinical anesthesia, patient controlled analgesia, painless labor. The obviousindividual differences of adverse reaction and analgesic effect was found in theclinical study of fentanyl. Fentanyl turn into norfentanyl， under the participation ofthe cytochrome P4503A4(CYP3A4) in the liver. The enzyme activity of cytochromeP4503A4has the obvious individual differences. The metabolism of fentanyl wasobviously influenced by the transcription of CYP3A4and the enzyme activity. Theresult of our previous study indicated that the CYP3A4*1G genetic polymorphismcan influence the fentanyl metabolism and decrease fentanyl consumption in theproject of patient-congtrolled intravenous. As this point, there are two kinds ofcompletely different results in the past. During the existing roughly40SNPs ofCYP3A4, CYP3A4*1G has the top frequency. The mass gene sequencing found themutation frequency of CYP3A4*1G in Japanese and Chinese is24.9%and26%. Theinfluence of CYP3A4*1G on the metabolism in vitro of fentanyl has few reports. Aswe know, due to the key role of CYP3A4participate in the metabolism of fentanyl,our team conducted this study to discuss the influence of CYP3A4*1G polymorphism on the human liver microsomal fentanyl metabolism. Study thegenetic factors to individual differences of fentanyl metabolism without interferencefactors. We think with the present study, it will provide an important and freshtheoretical evidence and foundation, in the respect of individual therapy of fentanylclinical administration.Materials and Methods1. SubjectsThe study design was approved and written informed consent was obtainedfrom the sufferer, under the approval of the Institutional Ethics Committee of thefirst affiliated hospital of Zhengzhou University. Total eighty-eight liver tissuesamples were taken during surgical removal in the first affiliated hospital ofZhengzhou University. The patients aged20-65yr, without diabetes, kidney disease,abnormal liver function, acute and chronic hepatitis, liver cirrhosis, long history ofalcoholism, didn’t use any drug or food that induce or suppress CYP3A4one monthbefore operation. Excise normal tissue on the outside edge of the surgical resection,which was confirmed by pathology, and preserve in liquid nitrogen.2. Genotyping assaysVenous blood samples were very improtant, in this study we collected from allsufferers. The DNA was extracted from leukocytes of the blood we collected, themethod was standard phenol/chloroform procedure. Polymerase chainreaction-restriction fragment length polymorphism (PCR-RFLP) was used toconduct genotype of CYP3A4*1G allele. Reconfirm the results of the genotypes ofwe appraised,we selected individuals with the direct sequence analysis randomly.3. Preparation of liver microsomesLiver microsomes was prepared by calcium precipitation. Make livermicrosomes are suspended in the0.25M sucrose solution, dispensed and saved in-80℃.Use the BCA protein concentration determination kit determinate the proteinconcentration.4. Metabolism of fentanyl in vitroFentanyl as the metabolic substrate in the incubation reaction, incubating timeis0,5,10,15,20min. Then terminate the reaction and extract. Inject sample afterredissolve with mobile phase. Use HPLC-UV to detect the concentration of fentanyl. 5. The determination of CYP3A4mRNA levelTizol was be used to extract total RNA, and ultraviolet spectrophotometer todetect the sample and ratio of A260nm/A280nm represent the concentration of RNA.cDNA was synthesized and use Real-Time PCR to quantitate the template.Results1. Frequency of CYP3A4*1G alleleThe rate of CYP3A4*1G allele in the patients of hepatobiliary surgery was0.188. We detected the allelic frequency of CYP3A4*1G allele and found it was inHardy-Weinberg equilibrium (P>0.05).2. Metabolism of fentanyl in vitroIn the former work of our laboratory, we select30cases of the samplesrandomly to metabolize fentanyl in vitro. The rate of fentanyl in chinese livermicrosome is1.64±0.77nmol min-1per mg of protein. The lowest and highest rate is0.31nmol min-1and2.84nmol min-1per mg of protein. There were obviousdifferences in the individual susceptibility.3. Association of CYP3A4*1G gene polymorphism with the metabolism offentanyl in vitroThe rate of fentanyl metabolism in chinese liver microsome of wild type,heterozygous and homozygous are1.89±0.58nmol min-1mg-1protein,1.82±0.65nmol min-1mg-1protein,0.85±0.37nmol min-1mg-1protein. The rate offentanyl metabolism in homozygous genotype group, compare with in heterozygousgenotype and wild genotype group is significantly decreased (P<0.05), but thedifference was not be found by us between heterozygous genotype group and wildgenotype group.4. Effect of different gender on metabolic rate of fentanyl in vitro49cases of liver microsomes grouped according to gender, female group of22patients and male group of27cases. Female group aged51.73±9.13years old,male group aged50.41±11.20years old, age difference was not statisticallysignificant(P>0.05). Using two independent sample t test to compare two groups ofpatents, there was no statistically significant difference in vitro metabolic rate offentanyl(P>0.05).5. Effect of different age on metabolic rate of fentanyl in vitro 49cases of determination of fentanyl metabolism in vitro aged50.98±10.24years old. Use Spearman rank correlation to analysis and discover the different agesand fentanyl metabolic rate in vitro have no correlation(P>0.05).6. Effect of different hepatobiliary disease on metabolic rate of fentanyl in vitro49cases of liver microsomes grouped according to different hepatobiliarydisease, hepatic hemangioma group of20patients, liver cancer group of12cases,gallbladder tissue lesions group of17cases. Use multiple sets of quantitative datacomparison to analysis and discover there was no statistically significant betweenthe three groups of patients(P>0.05).7. Real time PCR determination of mRNA levels of CYP3A4Successful determination of mRNA in36cases with liver specimens,CYP3A4*1/*1group of23cases, CYP3A4*1/*1G group of11cases,CYP3A4*1G/*1G group of2cases. The result showed that there was significantindividual differences of mRNA level of liver tissue, the highest level of19.11,minimum of0.11. Average of CYP3A4*1/*1，CYP3A4*1/*1G and CYP3A4*1G/*1Gwere6.49,3.28and1.20, there was statistically significant between the three groupsof patients(P<0.05).8. The correlation of CYP3A4mRNA level and metabolism of fentanyl in vitroThere are17cases of the samples were determined the CYP3A4mRNA leveland metabolism of fentanyl in vitro. The CYP3A4mRNA level and metabolism offentanyl in vitro have positive correlation.Conclutions1. CYP3A4*1G polymorphism has a significant effect on the hepatic microsomalcytochrome P4503A4enzyme in the metabolism of fentanyl, homozygous mutantreduces the metabolic rate of fentanyl. This may cause individual differences in theclinical pharmacodynamics of fentanyl;2. Sex, age and various hepatobiliary diseases has no significant effect on thehepatic microsomal cytochrome P4503A4enzyme in the metabolism of fentanyl.These may not cause individual differences in the clinical pharmacodynamics offentanyl;3. The CYP3A4mRNA level and metabolism of fentanyl in vitro have positive correlation.