Content And Activity Of 10 Cytochrome P450 Isoform In Human Normal Liver

Cytochrome P450（CYP）,mainly exists in liver microsomes.It plays a very important role in the biotransformation of exogenous compounds and contriubutes to metabolism of about 80%of therapeutically usual drugs.CYP1,CYP2 and CYP3 are three major members of P450 enzyme system.There are ten kinds of CYP450isozymes,which are CYP1A2、CYP2A6、CYP2B6、CYP2C8、CYP2C9、CYP2C19、CYP2D6、CYP2E1、CYP3A and CYP3A5,respectively.The reaction catalyzed by P450 enzyme is a critical step in the metabolism in vivo.And the foundamental reason for the pharmacokinetics of drugs in vivo is due to the large individual difference in the contents and activities of the CYP450 enzymes involved in metabolism.With the rapid development of protein content quantification technology,stable isotope dilution internal standard-multiple reaction monitoring-mass spectrometry（SID-MRM-MS）has become an ideal method for protein quantification because of its unique reproducibility and accuracy.With the research and understanding of CYP,finding that it plays an important role in the metabolism of human body.At present,the study on the content and activity of CYP450 is limited to its content and activity in liver microsomes.Although liver microsome as one of the traditional research of CYP enzyme system is widely used,but in order to objectively reflect the real state of the liver,to accurately predict the actual situation of the body,It is necessary to analyze and discuss the physiological parameters of liver from microscopic to macroscopic level.Therefore,determining the content of the ten kinds of CYP in microsomes,liver tissue and liver,and at the enzyme level,microsome level,liver tissue level,and liver level.The correlations between the content and activity of 10 CYP isoforms at the same level were studied,and the correlations between the content and activities of the same CYP isoforms at different levels were also studied,which can provide important reference data of physiology for individualized rational use of drugs in clinical practice.Methods1 Human liver samplesThe samples were collected from 90 human normal liver tissue,including 34males and 56 females,aged from 20 to 75 years old.There were 12 cases of drinkers and 12 smokers,respectively.2 Determination of human liver microsome protein concentrationHuman liver microsomes were prepared by low temperature differential centrifugation.The Bradford method was used to determine the concentration of human liver microsome protein.3 Determination of CYP content and activity in microsomesSID-MRM-MS method was used to determine the content of ten CYPs in human liver microsomes.Ten kinds of CYP probes were selected to determine the corresponding enzyme activity.4 Determination of CYP activity at enzyme levelCYP activity at the enzyme level is equal to CYP activity at microsome level divided by CYP isoform content in microsomes5 Determination of CYP content and activity at liver tissue levelCYP content in liver tissue is equal to CYP content in microsomes multiplied by the content of microsome protein in liver tissue（MPPGL）.CYP activity at liver tissue level is equal to CYP activity at microsome level multiplied by MPPGL.6 Prediction of CYP content and activity at liver levelCYP content in liver is equal to CYP content in liver tissue multiplied by liver weight ratio（LW/BW）.CYP activity at liver level is equal to CYP activity at liver tissue level multiplied by LW/BW.7 Statistical analysesAll data were statistically analyzed using SPSS 21.0 software and plotted using GraphPad Prism 5.0 mapping software.Most of the data obtained from this experiment did not conform to normal distribution,non-parametric test analysis was used.The test levelαwas 0.05.Results1 Content of 10 CYP protein in human normal liverIn the liver samples of 90 normal individuals,the content of 10 CYP protein were in non-normal distribution.The highest content were CYP2C9 and CYP2E1,and the lowest content was CYP2B6.In addition,in microsomes,CYP2E1 had the highest content,with a median of 101.84 pmol/mg protein and a range of 11.49-166.20pmol/mg protein.The lowest content was CYP2B6,the median was 4.54 pmol/mg protein and the range was 1.68-46.19 pmol/mg protein.In liver tissue,CYP2C9 was the highest,with a median of 3.67 nmol/g liver and a range of 0.41-21.72 nmol/g liver.The lowest content was CYP2B6,the median was 0.21 nmol/g liver and the range was0.04-2.89 nmol/g liver.Similar to liver tissue,CYP2C9 was the highest in the liver,with a median of 77.85 nmol/kg and a range of 9.37-440.88 nmol/kg.The lowest content was CYP2B6,the median was 4.75 nmol/kg and the range was 0.75-60.49nmol/kg.2 Activities of 10 CYP protein in human normal liverThe K_m,V_maxax and CL_intnt of 10 CYPs in the liver samples of 90 normal livers were all in non-normal distribution.The K_m of the 10 CYPs among individual differences are small,all below 40 fold.The Vmax of 10 CYPs at enzyme level,microsome level,liver tissue level and liver level are various.The degree of individual differences of the CYP1A2,CYP2A6,and CYP2C8 showed an upward trend with the expansion of the research system.Other CYPs first decreased from the enzyme level to the microsome level,then increased again.The results of the liver tissue level and the liver level were close to each other.Similar to V_max,the CL_int of 10 CYPs at the enzyme level,microsome level,liver tissue level,and total liver level were also different from each other.With the expansion of the research system,individual differences in CYP1A2,CYP2B6,CYP2C9,CYP2E1,CYP3A4 and CYP3A5 firstly decreased and then increased.The individual differences of CYP2A6 and CYP2D6 increased firstly and then decreased;The degree of individual differences between CYP2C8 and CYP2C19kept increasing and the levels of liver tissue and liver were close.3 Correlation of 10 CYP content and activityThe correlations among the levels of 10 CYP and their corresponding activities were analyzed statistically at microsome level,liver tissue level and liver level.3.1 Microsome levelThere was no correlation between content of CYP1A2 and Km（P>0.05）,but there was a significant correlation between CYP1A2 and V_max and CL_int（P<0.05）.The protein content of CYP2D6 was negatively correlated with Km（P<0.05）,weakly correlated with its corresponding CLint（P<0.05）,but had no correlation with its corresponding V_max（P>0.05）.There was no correlation between the protein content of CYP2B6,CYP2C8,CYP2C9,CYP2C19,and CYP3A4 and their corresponding microsome activities（P>0.05）.The remaining CYP content and its activity were correlated to different degrees.3.2 Liver tissue levelThere was a significant correlation between the protein content of enzyme isoform and their corresponding activities（P<0.05）except CYP2B6.And there was a strong correlation between the content of CYP1A2 and CYP2E1 and its corresponding V_maxax and CLint（r>0.6,P<0.05）.The contents of CYP2C19,CYP3A4,and CYP3A5 were weakly correlated with their corresponding Vmax and CLint（0.5<r<0.6,P<0.05）.The protein content of CYP2D6 was moderately correlated with V_max and CL_int of CYP2D6（0.4<r<0.6,P<0.05）.3.3 Liver levelThe protein content of enzyme isoforms and their corresponding V_max and CL_intnt were significantly correlated（P<0.05）except CYP2B6.The protein contents of CYP1A2 and CYP2E1 and their corresponding activities were highly correlated（r>0.6,P<0.05）.The correlation between the protein content of CYP2C19,CYP3A4 and CYP3A5 and their corresponding activities were weak（0.5<r<0.6,P<0.05）.4 Correlation of 10 CYP protein content4.1 Microsome levelThere was no correlation between the protein content of CYP1A2 and the protein content of CYP2B6 and CYP2C19（P>0.05）but there was a significant correlation with the content of other protein（P<0.01）.There was no correlation between the protein content of CYP2B6 and any other enzyme protein content（P>0.05）.The protein content of CYP2D6 was not significantly correlated with the protein content of CYP2B6,but was significantly correlated with other enzyme contents（P<0.01）.The protein content of CYP3A5 had no correlation with the protein content of CYP2B6 and CYP2C19（P>0.05）,but had significant correlation with other enzyme content.4.2 Liver tissue levelThe protein content of CYP1A2 was significantly correlated with the protein content of other enzymes and strongly correlated with the protein content of CYP2C8,CYP2C9 and CYP2E1（P<0.01）.The protein content of CYP2C9 was significantly correlated with the protein content of other enzymes,and strongly correlated with the protein content of CYP1A2,CYP2C8 and CYP2E1（P<0.01）.Except that there was no correlation between CYP2C19 protein content and protein content of CYP3A5,there was significant correlation between protein content of CYP2E1,CYP3A5 and other CYPs.4.3 Liver levelThere was no significant correlation in protein content among CYP2B6,CYP2C19,CYP3A4,CYP3A5,and CYP2C19（P>0.05）.However,there was a significant correlation among other enzyme content（P<0.01）.4.4 Microsome,liver tissue and liver levelSpearman correlation analysis was performed to analyze the correlation of 10CYPs among the microsome level,liver tissue level and liver level in 90 human normal liver.5 Correlation of 10 CYP protein activities5.1 Enzyme levelThere was no correlation between the V_max of CYP1A2 and the V_max of CYP2B6,CYP2C19,CYP3A4（P>0.05）and there was a correlation with the Vmax of other CYPs（P<0.05）.There was no significant correlation between the V_max of CYP2C8 and the V_max of CYP2C19,but there were correlations with the V_maxax of other CYPs.The V_maxax of CYP2C19 is only related to CYP2A6.The correlation between the Vmax of CYP3A5and the V_maxax of CYP3A4 is the strongest.The CL_int of CYP2C19 and the CL_int of CYP2C9 were significantly correlated（P<0.05）,but had no correlation with other isoforms（P>0.05）.The CLint of CYP2D6and CYP2E1 had no correlation with the CL_int of CYP2C19,but had significant correlation with CLint of other isoforms.5.2 Microsome levelThe Vmax of CYP2A6 was only correlated with the Vmax of CYP2C19（P<0.01）and there was no correlation with the Vmax of other CYPs（P>0.05）.There was no correlation between the V_max of CYP2C8 and the V_max of CYP2A6 and CYP2E1.there was a strong correlation with the Vmax of other CYPs（P<0.01）.The Vmax of CYP2E1 is significantly correlated with the V_max of CYP2C9 and CYP2C19.There was no correlation between the CL_int of CYP2A6 and other CYPs（P>0.05）.The CLint of CYP2D6 was only correlated with the CLint of CYP2B6（P<0.05）.The CLint of CYP2C19 was only significantly correlated with that of CYP3A4（P<0.01）.The CL_int of CYP2E1 was only correlated with the CL_int of CYP1A2 and CYP2C19（P<0.01）.5.3 Liver tissue levelThe V_max of CYP2B6 was not correlated with the V_max of CYP2A6 but it was significantly correlated with the Vmax of other CYPs（P<0.05）.There was no correlation between the Vmax of CYP2D6 and the Vmax of CYP2A6（P>0.05）,but weakly related with other CYPs（P<0.05）.The CL_intnt of 10 CYPs were significantly correlated（P<0.05）.5.4 Liver levelThere was no correlation between the V_max of CYP2A6 and the V_max of CYP2B6and CYP2D6（P>0.05）.There was a significant correlation between the Vmax of other isoforms（P<0.05）.There was a strong correlation between the Vmax of CYP2C9 and the V_max of CYP2E1（P<0.01）and there was a significant correlation between the CL_intnt of 10 CYPs at the liver level（P<0.05）.5.5 Enzyme,microsome,liver tissue and liver levelSpearman correlation was used to analyze the correlation of 10 CYP activities at enzyme level,microsome level,liver tissue level and liver level.Between the enzyme and the microsome level,except that the Vmax of CYP2C19 and CYP3A5 had no correlation between the two levels（P>0.05）,the V_max of other CYPs was correlated between the two levels（P<0.05）.Except that the CLint of CYP2A6 and CYP2C19 had no correlation between the two levels,other CYPs have correlations between the two levels.Between the enzyme and liver tissue level,except that the Vmax of CYP2C9,CYP2C19,and CYP3A5 were not correlated between the two levels（P>0.05）,the V_maxax of other CYPs was correlated between the two levels（P<0.05）.The CLint of CYP2A6,CYP2C19 and CYP3A5 had no correlation between the two levels.But the CL_int of other CYPs have correlations between the two levels.Between the enzyme and liver level,except that the Vmax of CYP1A2,CYP2C9,CYP2C19 and CYP3A5 were not correlated between the two levels（P>0.05）,the Vmax of other CYPs was correlated between the two levels（P<0.05）.Except that the CL_int of CYP2A6 and CYP2C19 had no correlation between the two levels,the CL_int of other CYPs had correlations between the two levels.Between microsome and liver tissue level and between the microsome and liver level,the V_max and CL_int of all CYPs were significantly correlated between the two levels（P<0.05）.Between the liver tissue and liver level,the Vmax and CLint of all CYPs were strongly correlated between the two levels（r>0.90,P<0.05）.Conclusions1.First time provided normal liver values of the 10 CYP isoforms protein content in human at microsome level,liver tissue level and liver level.There are obvious individual differences at each level.2.At the microsome level,the protein content of CYP2A6 had a significant effect on its activity,but at any level,the protein content of CYP2B6 had no effect on its activity.