Intra-individual Variation And Correlation Of Cytochrome P450 Activities In Human Liver Microsomes

Most exogenous and endogenous chemicals undergo biotransformation and metabolism in liver. Liver plays an important role in metabolism usually through CYPs.Individual variation in the activities of CYPs, traditionally inter-individual variation of CYPs activities, was the main reason for individual variation in metabolism, response and toxicity of drugs, which could be used to guide personalized medicines known as an important part of precision medicine, were reaserched by lots of authors.Actually, intra-individual variation in the activities of CYPs, which means different liver-localized CYPs present different activities in a given individual under same conditions also contributes to personalized medicines. However, intra-individual variation of CYPs activities that was the hidden but very apparent key to realize personalized medicines was never explored by researchers. To achieve the goal of personalized medicine, it is imperative that completely understand the extraordinary intra-individual variation and real correlation in the activities of CYPs.Here we performed an in vitro study of human liver microsomes from 105 normal human liver samples that focused on the intra-individual variation and correlation in the activities RMAs of 10 CYPs?CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1 and 3A4/5?. Meanwhile, we investigated the correlation in the relative activities and the effects of CYP polymorphism on intra-individual differences,which might provide theoretical basis for personalized medicine.Methods 1 Collection of human liver samplesHuman liver samples with normal liver function were obtained from 105 humans. The age ranged from 20 to 75 years. All donors with normal liver function checked by pathological examination and imaging were free of infectious disease?HBV or HCV?. This study was approved by the ethics committee of Zhengzhou University and every voluntter wrote informed consent.2 Preparation of human liver microsomes and concentration determinationHuman liver microsomes were prepared by differential centrifugation and microsome protein level was determined by Bradford method.3 Enzymatic activity of 10 CYPs in human liver microsomesSubstrate of CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1 and 3A4/5 were phenacetin, coumarin, amphetamines, paclitaxel, tolbutamide, omeprazole, dextromethorphan, chlorzoxazone and midazolam. Explore the optimum protein concentrate and incubating time for each CYPs' incubation system. Measured 10 CYPs 'enzyme kinetic parameter(K_m, V_max, CL_int) from 105 normal Chinese human liver microsomes.4 Genotypes of 10 CYPsGenomic DNA was isolated from human liver tissue using a genomic DNA purification kit. Polymorphisms in the 10 CYPs with frequencies more than 1% in Chinese subjects were detected.5 Statistical analysisCalculation of enzyme kinetic parameters was performed by GraphPad Prism 5.0 software. The relative metabolic activity(K_m, V_max and CL_int) of each CYP enzyme in each individual was calculated by dividing the absolute value of K_m, V_max and CL_int of each individual CYP enzyme by the median of the corresponding parameters of 105 individuals variations of RMAs in 10 CYPs for one person were expressed as intra-individual percentage coefficient of variation?ICV?, which was calculated as standard deviation of relative K_m, V_max and CL_int in one case divided by the corresponding average value for that case.Hierarchical cluster analysis was performed using SPSS 17.0?SPSS Inc., Chicago, IL, USA? to detect correlation and similarity of 10 CYPs activities.Statistical analysis was performed using SPSS 17.0 software. One-way ANOVA and Bonferroni test were used for RMAs potency of 10 CYPs comparisons, P<0.05 was considered statistically significant.Results 1 Concentration determination of human liver microsomesThe standard curve and regression equation were established?C=1.5898A-0.0512, R2=0.9939?. Median protein concentrations of 105 normal Chinese human liver microsomes were 8.39?1.31¹1.66? mg·ml^-1.2 Method validation of 10 CYPsNeither the substrate of 10 CYPs nor other impurities interfered the chromatographic peak for metabolites, which indicated high specificity. Intra-day and inter-day variation less than 10%, with recovery rate range from 90% to 110%, which indicated high precision and accuracy. The established HPLC methods are in compliance with the requirements for the test.The optimum protein concentration of incubation system for CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1 and 3A4/5 were 0.3, 0.3, 0.5, 0.5, 0.5, 0.5, 0.3, 0.3 and 0.2 mg·ml^-1, respectively. The optimum incubation time for CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1 and 3A4/5 were 30, 30, 60, 120, 60, 90, 20, 30 and 5min, respectively.3 Intra-individual variation of 10 CYPs 3.1 Intra-individual variation of 10 CYPsThere was variation in the activities of 10 CYPs from 105 normal Chinese human liver microsomes, as well as the activities of 10 CYPs in a case. The ICVs of relative K_m, V_max and CL_int for 10 CYPs were 45.88%?18.31%?140.14%?, 44.03%?16.79%?127.42%? and 52.00%?24.20%?134.81%? for 105 cases.3.2 Relative metabolic activities of 10 CYPsRank the relative V_max, K_m and CL_int of 10 CYPs. The first three of relative V_max were CYP3A4, CYP2C19 and CYP2D6. The first three of relative K_m were CYP2D6, CYP1A2 and CYP2C19. The first three of relative CL_int were CYP2D6, CYP2B6 and CYP3A4.4 Correlation between 10 CYPsThis paper employs the method of Pearson correlation analysis to investigate Correlation between 10 CYPs. According to V_max, the strongest correlativity were between CYP2C8 and CYP2C9?r =0.537, P=3.48E-09?. However, There exists weak correlation among K_m of 10 CYPs, except for CYP1A2 and CYP2C9?r = 0.475, P=3.04E-07?. In addition, the correlation among CL_int of 10 CYPs was also lower.5 Hierarchical cluster analysis of 10 CYPsFor relative V_max, hierarchical cluster analysis divided 10 CYPs into two groups according to correlation and similarity and group I included CYP2C8, CYP2C9, CYP2D6, CYP2B6 and CYP3A4/5, group II included the rest. The highest correlation and similarity held in CYP2C8 and CYP2C9.Adopting hierarchical cluster analysis for the values of relative K_m, the 10 CYPs could be divided into two groups according to correlation and similarity, the first group included CYP1A2, CYP2C9, CYP2B6, CYP2C19, CYP2A6, CYP2C8 and CYP2D6, the second included CYP2E1 and CYP3A4/5. Among the 10 CYPs, the highest correlation and similarity was present in CYP1A2 and CYP2C9.Adopting hierarchical cluster analysis for the values of relative CL_int, the 10 CYPs were divided into two groups, the first group included CYP2C8, CYP2C9, CYP1A2 and 2B6, the second included CYP2A6, CYP2D6, CYP2C19, CYP2E1, and CYP3A4/5 the third include the rest. There was the highest correlation and similarity between CYP2C8 and CYP2C9.6 Genetic polymorphisms affected intra-individual variation of 10 CYPsPolymorphisms in the 10 CYPs with frequencies more than 1% in Chinese subjects were detected. We have found that most of genetic polymorphisms do not affect intra-individual of CYPs. However, CYP2A6*4, CYP2B6 785A>G and CYP2D6 100C>T had a significant impact on there relative activities.Conclusions1. There were large intra-individual variation in the activities of 10 CYPs, as well as intra-individual variable coefficient of relative K_m, V_max and CL_int. 2. The correction between 10 CYPs in a case were complicated. According to V_max and CL_int, The highest correlation and similarity held in CYP2C8 and 2C9. Among the K_m of 10 CYPs, the highest correlation and similarity was present in CYP1A2 and 2C9. 3. CYP2A6*4, CYP2B6 785A>G and CYP2D6 100C>T can affect the intra-individual of 10 CYPs.