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目的研究中国人群CYP2D6基因多态性对阿托伐他汀(A)在体外人肝微粒体代谢的影响,探讨CYP2D6基因在阿托伐他汀代谢过程的作用。方法收集肝胆手术患者手术旁正常肝组织样本,并提取人肝微粒体(HLM)及肝组织DNA。使用Taqman基因分型技术测定CYP2D6*3、*4、*5、*6、*9、*10和*41这7个位点多态性。采用HLM孵育A使其代谢,并用LC-MS/MS方法测定A及其代谢产物2-羟基阿托伐他汀(2A)和4-羟基阿托伐他汀(4A)的浓度,分析CYP2D6基因多态性对A体外代谢的影响。按照相关文献及分型结果将样本的CYP2D6代谢表型分为强代谢型(EMs)和中等代谢型(IMs)表现型。结果 A在人肝微粒体中生成2A和4A的酶反应速率常数分别为53.7及48.5μmol/L;最大反应速率分别为140.2及181.7 pmol/(mg·min)。A在55例人肝微粒体中的代谢呈现多态性,最小和最大速率分别为109.9和9.3 pmol/(mg·min)。本研究中CYP2D6*3、*4、*5、*6、*9、*10、*41等位基因突变频率分别为0%、1.82%、2.73%、0%、0%、69.09%、2.73%。CYP2D6*4、*5、*10、*41等位基因突变对阿托伐他汀在人肝微粒体反应体系中的代谢无明显影响。A的减少速率在EMs组比在IMs组高,但差异无统计学意义(P>0.05)。2A的生成速率在EMs和IMs组分别为(14.68±7.62)vs.(8.70±3.50)pmol/(mg·min),P=0.06。4A的生成速率在EMs和IMs组分别为(20.32±13.10)vs.(11.04±4.88)pmol/(mg·min),P=0.07。结论 CYP2D6*4、*5、*10、*41等位基因突变对阿托伐他汀代谢影响较小,而CYP2D6*4、*5、*10、*41多等位基因突变引起的代谢表型的差异对阿托伐他汀体外代谢生成2-羟基阿托伐他汀、4-羟基阿托伐他汀有一定的影响。
Objective To investigate the effect of CYP2D6 gene polymorphism on the metabolism of human liver microsomes in vitro by CYP2D6 gene and to explore the role of CYP2D6 gene in atorvastatin metabolism. Methods Normal liver tissue specimens of patients with hepatobiliary surgery were collected and the human liver microsomes (HLM) and liver tissue DNA were extracted. The polymorphisms of CYP2D6 * 3, * 4, * 5, * 6, * 9, * 10 and * 41 were detected by Taqman genotyping. A was incubated with HLM to metabolize it, and the concentrations of A and its metabolites, 2-hydroxyatoform (2A) and 4-hydroxyatulinvastatin (4A), were determined by LC- MS / MS method to analyze the CYP2D6 polymorphism Effects of Sex on A in Vitro Metabolism. The CYP2D6 metabolic phenotypes of the samples were divided into two groups according to the relevant literature and typing results: strong metabolizable (EMs) and intermediate metabolizable (IMs) phenotypes. Results The enzyme reaction rate constants of 2A and 4A in human liver microsomes were 53.7 and 48.5μmol / L, respectively. The maximum response rates were 140.2 and 181.7 pmol / (mg · min), respectively. A showed polymorphism in 55 human liver microsomes with the minimum and maximum rates of 109.9 and 9.3 pmol / (mg · min), respectively. The frequencies of CYP2D6 * 3, * 4, * 5, * 6, * 9, * 10 and * 41 alleles in this study were 0%, 1.82%, 2.73%, 0%, 0%, 69.09% and 2.73 %. CYP2D6 * 4, * 5, * 10, * 41 allelic mutations atorvastatin in human liver microsomal response in the metabolism had no significant effect. The reduction rate of A in the EMs group was higher than that in the IMs group, but the difference was not statistically significant (P> 0.05). The formation rate of 2A in EMs and IMs groups was (14.68 ± 7.62) vs. (8.70 ± 3.50) pmol / (mg · min), and the rate of P = 0.06.4A in EMs and IMs groups was (20.32 ± 13.10 ) vs. (11.04 ± 4.88) pmol / (mg · min), P = 0.07. Conclusion The CYP2D6 * 4, * 5, * 10, * 41 alleles have little effect on the metabolism of atorvastatin, while the metabolic phenotypes caused by CYP2D6 * 4, * 5, * 10, * 41 multiple alleles The differences in atorvastatin in vitro metabolism of 2-hydroxy atorvastatin, 4-hydroxy atorvastatin have a certain impact.