| Cytochrome b5(Cyt b5) is an small heme-containing protein in the endoplasmic reticulum, which plays an important role in bile acid desaturation, hemin protein reduction, steroid production and drug metabolism process by transferring electron to a variety of enzymes. Cytochrome P450(CYP) is distributed mainly in the human liver, and participates in more than 75% of drug metabolism. The main isoforms of CYPs include CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2E1, CYP2D6, CYP3A4 and CYP3A5. The main reason of clinical drug individual differences is the difference of CYP activities. The lastest research showed that Cyt b5 could transfer electrons to CYPs during the catalytic reaction, increasing the catalytic reaction efficiency of CYPs. The expression of Cyt b5 in a large number of normal human livers is still unknown, and the effect of Cyt b5 content in human microsomes on the activity of CYP is rarely reported. The relationships between Cyt b5 and polymorphic activities of CYP haven’t been reported.It is more accurate to reflect the natural state influence of Cyt b5 on CYP metabolism in human liver microsomes, but little is known about the effects of Cyt b5 on CYPs in human liver microsomes. Therefore, for the first time, we studied the impact of Cyt b5 content on CYP activity in normal human liver microsomes. We intend to use a large sample of normal human livers to perform related experiments. Firstly, we used the q PCR method to detect the m RNA of Cyt b5 in 107 human liversamples. Secondly we detected the content of Cyt b5 in 123 samples human liver microsomes by spectroscopy, and then analyzed the effects of Cyt b5 on the enzyme kinetics parameters of CYPs to find out the cause of individual differences in CYP activity.Method 1.1 Collection of human liver samples We collected 123 normal human liver samples from the First Affiliated Hospital of Zhengzhou University, People Hospital of Henan province and Henan Tumor Hospital. There were 40 men and 80 women aged from 20 to 75. All the subjects didn’t use the medicines recently which had effects on the CYP activity. This experimental protocol was approved by the Ethics Committee of Zhengzhou University and each subject signed the informed consent. 1.2 Preparation of the human liver microsomes Human liver microsomes were determined by differential centrifugation. 1.3 Determination of the content of Cyt b5 The content of Cyt b5 was determined by spectroscopy. 1.4 Determination of the level of Cyt b5 m RNA Total RNA from human liver was isolated by Trizol And then 1μg of total RNA was reversely transcripted into c DNA. Two-step process was used for PCR amplification in the 7500 Fast Real-Time PCR System. GAPDH was used as internal gene and 2-△ CT was used to calculate the relative expression levels. 1.5 Determination of the CYP activity The substrates for CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2E1, CYP2D6, CYP3A4, and CYP3A5 are phenacetin,, coumarin, bupropion, paclitaxel, tolbutamide, omeprazole, dextromethorphan, chlorine chlorzoxazine, and midazolam, respectively. Metabolite concentrations were analyzed by HPLC. 1.6 Genotyping for CYP gene Genomic DNA was isolated from human liver by DNA extraction kit and genotypes were analyzed by SNP Mass ARRAY. 1.7 Statistical Methods The data were analyze by SPSS17.0 and the pictures were performed by Prime 5.0. All data were tested by the normality and most of data were the non-normal distribution. The non-parametric test was used to analyze the data. P<0.05. is considered significant.Result 2.1 Cyt b5 content 2.1.1 Cyt b5 m RNA The relative levels of Cyt b5 in the 107 normal human livers ranged from 0.61-7.44 with a median of 1.59. The difference fold is 12. 2.1.2 Cyt b5 protein content The content of Cyt b5 in the 124 normal human livers ranged from 33.75-641.27 with a median of 270.01. The difference fold is 19. 2.2 Effect of demographic factors on Cyt b5 m RNA and protein content. 2.2.1 Effect of Demographic factors on Cyt b5 m RNA Gender, age, smoking and drinking had no effects on the the Cyt b5 m RNA. 2.2.2 Effect of Demographic factors on Cyt b5 protein The content of Cyt b5 in male is significantly higher than the female(P=0.002) and smoking group is significantly higher than no-smoking(P=0.006). Other demographic factors had no significant effects on the Cyt b5 content. 2.3 Effect of Cyt b5 on the overall activity of 10 CYP 2.3.1 Effect of Cyt b5 m RNA level on the overall activity of 10 CYP Significant correlation between Cyt b5 m RNA and Vmax of CYP2B6(r=0.619), CYP2D6(r=0.269) and CYP3A4/5(r=0.439), Km of CYP2C8(r=0.236) and CYP3A4/5(r=0.223), Clint of CYP2B6(r=0.452) and CYP2D6(r=0.438) were observed. 2.3.2 Efect of Cyt b5 content on the overall activity of 10 CYP Significant correlation between the Cyt b5 content and Vmax of CYP1A2(r=0.377), CYP2B6(r=0.416), CYP2C19(r=0.282), CYP2D6(r=0.467), CYP2E1(r=0.316) and CYP3A4/5(r=0.322), Clint of CYP1A2(r=0.343), CYP2B6(r=0.391), CYP2C9(r=0.240), CYP2C19(r=0.283), CYP2D6(r=0.375), CYP2E1(r=0.352) and CYP3A4(r=0.197) were observed. There is no correlation between the Cyt b5 content and the Km of the CYP. 2.4 Effect of Cyt b5 on the polymorphic activities of 10 CYP 2.4.1 Effect of the Cyt b5 m RNA on the polymorphic activities of 10 CYP Cyt b5 m RNA had significant effects on the activities of CYP1A2 5347C>T,-3113A>G, CYP2A6 *4, CYP2B6 516G>T, 785A>G, CYP2C8*1B, *1C, CYP2D6 100C>T, 1661G>C, 2850C>T, CYP3A4 20070T>C, 20230G>A, and CYP3A5*3. 2.4.2 Effect of the Cyt b5 content on the polymorphic activities of 10 CYP Cyt b5protein content had significant effects on the activities of CYP1A2-3860G>A,-163C>A, 5347C>T,-3113A>G, 2159G>A, CYP2A6*1B, CYP2B6 516G>T, 785A>G, CYP2C9 *3, CYP2C19 *2*3, CYP2D6100C>T, 1661G>C, 2850C>T, CYP2E1-1293G>C, 7632T>A,-333T>A,-352A>G, CYP3A4 20070T>C, 20230G>A,and CYP3A5*3.Conclusion 1. Cyt b5 expression level has a large individual difference in normal human livers. 2. Sex and smoking have significant effects on the Cyt b5 protein. 3. Cyt b5 m RNA and protein have a significant effect on the overall activity of 10 CYP in human liver microsomes. 4. The effects of Cyt b5 m RNA and protein on the polymorphic activities of 10 CYP are SNPs dependent. |
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