Content And Activity Of 10 CYPs And Microsomal Protein In Human Livers

The human cytochrome P450(CYPs) are major players in the metabolism of a diverse array of endogenous and exogenous compounds, including 70–80% of all drugs currently in clinical use. Wide individual variation in enzyme activity of CYP leads to unpredictable drug responses and drug toxicity. Content and polymorphism of CYP play major roles in individual variation.CYP gene polymorphisms have been intensely investigated, but no report to date has characterized the absolute content of 10 CYPs over a large sampling of normal human livers due to the limit of liver samples and reliable quantified method. Recently, mass spectrometry(MS)-based proteomics for absolute quantification of proteins has been developed, which can simultaneously distinguish and quantify highly homologous proteins with high accuracy and precision in the same sample. Due to the lack of information regarding the absolute contents of CYP isoforms in normal liver samples, the individual variation, transcriptional regulation, genetic and epigenetic regulatory mechanism of CYP content can’t be developed systematically.The amount of microsomal protein per gram of liver(MPPGL) is a critical scaling factor used in physiologically-based pharmacokinetics(PBPK) models to extrapolate in vitro rates of metabolism to drug clearance in vivo. In order to assure the accuracy of predicted values for in vivo clearance, the MPPGL level should be determined precisely and individual variations in MPPGL should be considered. Unfortunately, to date there are only a limited number of studies concerning MPPGL amounts in human samples.The in vitro-in vivo extrapolation(IVIVE) method affords researchers the opportunity to produce quantitative data on drug metabolism prior to studying pharmacokinetics in vivo. However, generally there was a significantly predictive bias for in vivo clearance from the in vitro metabolic data because existing variation was not considered and instead mean parameter value reconstructed from very small data sets were used. The accuracy of the predictions will be increased if the individual values for the important liver sample parameters are introduced into IVIVE.In this study, the absolute protein content of 10 CYP isoforms was detected by multiple reaction monitoring coupled with stable isotope dilution mass spectrometry(SID-MRM-MS) and the m RNA levels of 10 CYPs were measured in 100 human liver samples. The transcription factors and mi RNA were detected in 107 human livers. 10 CYP activities at isoform level were characterized for the first time in human liver microsomes(HLM). Gene polymorphism with a frequency of >1% were used to analyze their effects on protein, m RNA and activities of CYPs, respectively. Furthermore, the relationships between and among CYP protein contents, CYP m RNA levels, and CYP activities had been assessed.Next, 128 liver samples were collected to characterize individual variations in the contents of MPPGL and the metabolic activities of 10 CYPs based on microsomes(VM) and liver tissues(VL). For the first time, the distributions and activities of MPPGL and CYP were assessed, the differences between VM and VL were compared and the correlations among VM or VL were analyzed. Furthermore, individual variations in the in vivo hepatic clearance of tolbutamide, used as a CYP2C9 probe substrate, were predicted using five important individual parameter values in large number of liver samples.Methods 1.1. Human Liver samples Human liver samples were obtained from 128 Chinese patients. There were 40 men and 88 women with the mean age 47.2 years. There were 13 smokers or drinkers, respectively.1.2. Preparation of liver microsomes HLMs were prepared by differential centrifugation. Total HLM protein concentrations were measured by Bradford method.1.3. Genotypes of 10 CYPs Genetic polymorphisms of 10 CYPs with frequencies of > 1% in Han Chinese population were genotyped by SNP Mass ARRAY.1.4 Quantification of CYP protein by SID-MRM-MS in HLMs A Qcon CAT protein consists of 23 stable isotope-labeled peptides from 10 CYPs in which two or three peptides were selected for each targeted protein was employed to quantify 10 CYPs in human liver microsomes. After prokaryotic expression, the Qcon CAT protein and HLM were digested and analyzed by SID-MRM-MS.1.5 Measurement of CYP m RNA and mi RNA levels by real-time quantitative PCR The c DNA for real-time reverse transcription PCR was synthesized from 1 μg total RNA using the Prime Script RT reagent kit with g DNA Eraser or Gene Copoeia All-in-One? mi RNA q RT-PCR Detection Kit(for mi RNA). CYPs m RNA expression was detected by two-step or three-step(for mi RNA) real-time PCR using ABI 7500 Fast Real-Time PCR system.1.6. Determination of CYP activity The incubation mixtures contained probe, phosphate buffer, Mg CL2, NADPH. Incubation conditions were ensured linear metabolite formation with respect to reaction time and protein concentration. Incubations were initiated by the addition of NADPH and were terminated by adding ice-cold acetonitrile or ethyl acetate or perchloric acid. Substrate metabolites were determined by HPLC-UV or HPLC-FLD.1.7 Determination MPPGL The activity of POR as measured in homogenates and microsomes produced from the same liver tissue sample was used to estimate the amount of MPPGL. MPPGL ={rate of reductionhomogenate(n M min-1g liver-1)}/{rate of reductionmicrosome /(n M min-1mg microsomal protein-1).1.8. Prediction of tolbutamide in vivo hepatic clearance The whole liver intrinsic clearance(CLint, liver) was estimated and the hepatic clearance(CLH) of tolbutamide hydroxylation was then predicted using the well-stirred model. According to the different combinations of mean and individual values of the five parameters, seven methods were employed. The accuracy of the predictions was assessed from the average fold-error(AFE).1.9 Statistical analyses Because most data sets were not normally distributed, nonparametric methods were generally used for statistical analyses. Non-parametric Spearman rank correlation analysis was performed to calculate the correlation coefficient(r). A P value < 0.05 was considered statistically significant(two-tailed). SPSS statistics 21 software was used for statistical analyses. Graphs were generated using Graph Pad Prism software 5.04.2 Results 2.1 Expression levels of CYP m RNA and protein in human livers2.1.1 Protein contents of CYPs Protein contents of 10 CYP were not normally distributed in 100 HLMs. The median content of 10 CYPs were 4.62-102.04pmol/mg. CYP3A4 exhibited the biggest individual variations(129-fold) and those for the remaining enzymes were 11- to 100-fold. Protein contents of almost all the CYP isoforms were significantly correlated with each other(P<0.05).2.1.2 m RNA Expression Levels of CYP m RNA expression levels of 10 CYPs were also not normally distributed in 107 human livers. The median expression levels of 10 CYPs were 0.19-5.94. CYP2A6 exhibited the biggest individual variations(168-fold) and those for the remaining enzymes were 8- to 122- fold. m RNA levels of almost all the CYPs were also significantly correlated with each other(P<0.05).2.1.3 Relationship between individual CYP m RNA and corresponding protein Except for CYP1A2(r=0.244, P<0.05) and CYP2A6(r=0.371, P<0.01), no significant correlation was observed between protein and the corresponding m RNA expression levels of the investigated CYPs.2.2 Effects of factors on CYP expression 2.2.1 Transcription factors m RNA expression levels of PXR, HNF4α, CAR, and AHR were not normally distributed in 107 human livers. Compared with CYP, the m RNA expression levels of 4 TFs were low, but individual variations were large(12-to 85-fold). The m RNA levels of almost all the TFs were significantly correlated with each other except PXR and AHR(P<0.05).There were significant correlations between all the TFs and the CYP m RNA expression except AHR and CYP2C9、CYP2D6(P<0.05). The correlation between TFs and CYP protein were poor.2.2.2 mi RNA Expression levels of 7 mi RNA were not normally distributed in 106 human livers. Among them, the abundance of mi RNA-27 b was the highest and mi RNA-34 a was relatively low. mi RNA-27 b exhibited the biggest individual variations(618-fold) and the remaining mi RNA had 89- to 312-fold variation.mi RNA were negatively correlated with m RNA level of CYPs(P<0.05). There were poor correlations between mi RNA and the CYP protein content except for mi RNA-27 and 4 CYP(P < 0.05). There were no correlations between mi RNA and the translation efficiency of CYP protein(P > 0.05).2.2.3. Effects of polymorphisms on CYP m RNA and proteinEffects on m RNA CYP1A2 3113A>G, CYP2A6*4, CYP2A6*9, CYP2D6 1661G>C, CYP2D6*10 and CYP3A5*3 had significant effects on m RNA levels. Effects on protein CYP1A2 5347C>T, CYP1A2 2159G>A, CYP2A6*4, CYP2A6*9, CYP2C9*3, CYP2D6 *10, CYP2D6 1661G>C, CYP2E1-1293G>C, CYP2E1 7632T>A, CYP2E1-333T>A and CYP3A5 A>G had significant effects on protein content.2.2.3. Effects of polymorphisms on CYP m RNA and proteinEffects on m RNA CYP1A2 3113A>G, CYP2A6*4, CYP2A6*9, CYP2D6 1661G>C, CYP2D6*10 and CYP3A5*3 had significant effects on m RNA levels.Effects on protein CYP1A2 5347C>T, CYP1A2 2159G>A, CYP2A6*4, CYP2A6*9, CYP2C9*3, CYP2D6 *10, CYP2D6 1661G>C, CYP2E1-1293G>C, CYP2E1 7632T>A, CYP2E1-333T>A and CYP3A5 A>G had significant effects on protein content.2.2.4 Effects of demographic factors Comparing with age 1 group(20-45 years old), CYP3A5 protein contents were significantly higher in age 2(46-60 years old) and age 3(61-75 years old) groups(P<0.05). Statistically significant differences in CYP1A2 m RNA between male and female donors were seen(P=0.014)). Donor age had noticeable effect on all the detected CYP m RNA levels except CYP1A2, CYP2D6 and CYP2E1. The total trend was m RNA expression levels decreased with the increasing of age.2.3 CYP activities at isoform level The median of Km, Vmax and CLint for 10 CYP were 1.9-230.4μm/L,1.2-21 pmol/min/pmol CYP,and 0.012-10.8μl /min/pmol CYP, respectively. There were large individual variations of CYP activities at isoform level. Vmax of CYP3A4 exhibited the largest individual variation(1755-fold). The remaining enzymes had relative smaller individual variation(8.6- to 575-fold).Vmax of almost all the CYP isoforms were significantly correlated with each other except CYP2C19(P<0.05). CLint of almost all the CYP isoforms were significantly correlated with each other except CYP2C19(P<0.05). There was no correlation among Km of 10 CYPs except 7 pairs.2.4 Effects of polymorphisms on CYP activities at isoform level CYP1A2 5347C>T, CYP1A2 2159G>A, CYP2A*4, CYP2A6*9, CYP2D6*10, CYP2D6 1661G>C and CYP3A5*3 had significant effects on CYP activities at isoform level(P<0.05).2.5 MPPGL content The values for MPPGL levels in 128 samples indicated a not-normal distribution, with minimal and maximal values of 6.71 and 127.95 mg microsomal protein per gram liver, representing a 19-fold variation. The average overall MPPGL content was about 39.46 mg/g liver. Age, gender, smoking, alcohol consumption and tissue resources had no effects on MPPGL values.2.6 CYP activity based on microsomes(VM) and on liver tissues(VL) The median VM of 10 CYPs were 31.50-677.94 pmol/min/mg protein. The two biggest individual variations in VM took place in the activity of CYP2C19 and CYP2A6, reaching to 232 and 109-fold, and those for other CYPs were 6- to 72-fold. The median VL of 10 CYPs were 0.96-29.05nmol/min/g liver. The individual variations of CYP activities were even more pronounced. VL of CYP2A6 exhibited the largest individual variation(210-fold), followed by that of CYP2C19, showing 144-fold individual variation. The individual variations for the remaining enzymes were 23- to 86-fold.Inter-individual variations in the VL of CYPs were much higher than those of corresponding VM except CYP2C19. The fold-changes in VL of CYPs exceeded those of corresponding CYP VM by 18%-278%, respectively. Rank change of individual VL compared with corresponding VM in 10 CYPs was obvious. For CYP1A2, CYP2C8,CYP2C9 and CYP2E1, the place change exceeding 20% accounted for 47%, 41%, 40% and 41% of the total samples, respectively.2.7 Prediction of tolbutamide hepatic clearance The mean values for the predicted CLH of tolbutamide obtained by seven methods were all 0.113 ml/min/kg, but the predicted ranges showed obvious variations. Method G gave merely the mean value, whereas method A predicted the largest individual variation(51-fold between the highest and lowest values).Conclusions: 1. Physiological data of 10 CYP isoform contents and MPPGL have been determined in a large number of human normal livers microsomes. Both of them have large individual variations.2. CYP isoform activity and activity based on liver are two new methods to express CYP activity. The former stands for the real activity of CYP and the latter is closer to the in vivo situation.3. A new method to predict the individual variations in hepatic clearance is established. The variations in the in vivo clearance rates of tolbutamide are successfully predicted using the individual values of five different parameters.4. Transcription factors, mi RNA, CYP gene polymorphisms, and demographic factors have significant effects on individual variations in CYP protein content and m RNA levels, in which the effects of transcription factors and gene polymorphisms are more extensive.