[1]张海朋),赵杰)#,张莉蓉),等.人肝脏组织CYP3A4酶活性的高效液相色谱法测定[J].郑州大学学报(医学版),2013,(04):544.
 ZHANG Haipeng,ZHAO Jie,ZHANG Lirong,et al.Establishment of a HPLC method to detect CYP3A4 activity in human hepatic tissue[J].JOURNAL OF ZHENGZHOU UNIVERSITY(MEDICAL SCIENCES),2013,(04):544.
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人肝脏组织CYP3A4酶活性的高效液相色谱法测定
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《郑州大学学报(医学版)》[ISSN:1671-6825/CN:41-1340/R]

卷:
期数:
2013年04期
页码:
544
栏目:
应用研究
出版日期:
2013-07-20

文章信息/Info

Title:
Establishment of a HPLC method to detect CYP3A4 activity in human hepatic tissue
作者:
张海朋1赵杰1#张莉蓉2王晓飞2薛文华1
1)郑州大学第一附属医院药学部 郑州 4500522)郑州大学基础医学院药理学教研室 郑州 450001
Author(s):
ZHANG Haipeng1)ZHAO Jie1)ZHANG Lirong2)WANG Xiaofei2)XUE Wenhua1)
1)Department of Pharmacy, the First Affiliated Hospital, Zhengzhou University, Zhengzhou 4500522)Department of Pharmacology, College of Basic Medical Sciences,Zhengzhou University, Zhengzhou 450001
关键词:
高效液相色谱 肝脏CYP3A4
Keywords:
highperformance liquid chromatographyliverCYP3A4
分类号:
R979.1
摘要:
目的:建立测定人体肝脏组织中CYP3A4酶活性的HPLC方法。方法:取21例肝癌、16例肝血管瘤、13例胆组织病变患者的肝组织,制作肝微粒体,体外孵育后以咪达唑仑(MDZ)为探针药物,用高效液相色谱法测定CYP3A4的Km值。HPLC色谱条件:色谱柱为Angel C18柱(4.6 mm×250 mm,5 μm),流动相为甲醇:乙腈:水(体积比531334),流速1 mL/min,检测波长220 nm,柱温25 ℃,进样量40 μL。结果:1OH MDZ与MDZ的出峰时间分别为10.05与15.02 min,分离充分(R>1.5),1OH MDZ质量浓度的范围为200~6 000 μg/L, 高、中、低浓度回收率均>90%,日内、日间精密度RSD均<5%,稳定性RSD均<5%。50例患者Km值1.75~30.17 μmol/L;不同性别、年龄及肝胆病变患者的Km值差异无统计学意义(P>0.05)。 结论:该法稳定、快速、重复性好,可用于人体肝组织中CYP3A4酶活性的测定;性别、年龄及肝胆病变情况对CYP3A4酶活性无影响。
Abstract:
Aim: To establish a HPLC method for determination of CYP3A4 activity in human hepatic tissue.Methods: Human liver microsome of 21 cases of liver cancer,16 cases of hepatic hemangioma,and 13 cases of gallbladder lesion was prepared for HPLC analysis. The analytical column was Angel C18 column (4.6 mm×250 mm, 5 μm), mobile phase consisted of methanolacetonitrilewater(volume ratio=531334)with flow rate at 1 mL/min, detective wavelength at 220 nm, and column temperature at 25 ℃,and the injection volume was 40 μL. Results: 1OH MDZ and MDZ retention time was 10.05 and 15.02 min with symmetrical peak and full separation (R>1.5) . The linear range of 1OH MDZ was 200~6 000 μg/L. Extraction recoveries at low, medium and high concentrations were all higher than 90%, respectively. The intraday and interday precision RSD was less than 5%, and the stability RSD was less than 5%. The statistical analysis showed that the liver diseases, gender or age had no significant effect on CYP3A4 activity(P>0.05). Conclusion: The HPLC method is rapid, stable with high repeatability. It is suitable for the determination of CYP3A4 activity in human liver. The liver diseases, gender and age have no significant effects on the Km.

参考文献/References:

[1]李晓宇,刘皋林. CYP450酶特性及其应用研究进展 [J].中国临床药理学与治疗学, 2008, 13(8):942
[2]薛正楷.稳定表达人CYP3A4基因与Bama小型猪CYP3A基因的HepG2细胞株的建立及探针药物代谢表征的比较研究
[D].重庆:重庆医科大学,2009.
[3]Thummel KE, Wilkinson GR .In vitro and in vivo drug interactions involving human CYP3A [J]. Annu Rev Pharmacol Toxicol,1998,38:389
[4]Guengerich FP.Cytochrome P4503A4: regulation and role in drug metabolism [J].Annu Rev Pharmacol Toxicol,1999,39:1
[5]Wandel C,Brocker R,Bohrer H,et al.Midazolam is metabolized by at least three different cytochrome P450 enzymes [J]. Br J Anaesth,1994,73(5):658
[6]Thummel KE, Shen DD, Podoll TD, et al. Use of midazolam as a human cytochrome P450 3A probe: in vivo in vitro correlation in liver transplant patients [J]. J Pharmacol Exp Ther, 1994,27(1):7549
[7]郭勇, 郑穗平. 酶学
[M]. 广州: 华南理工大学出版社, 2000:219
[8]YasuiFurukori N,Inoue Y,Tateishi T.Sensitive determination of midazolam and 1hydroxymidazolam in plasma by liquidliquid extraction and column switching liquid chromatography with ultraviolet absorbance detection and its application for measuring CYP3A activity [J].J Chromatogr B Analyt Technol Biomed Life Sci,2004,811(2):153
[9]Kumar A, Mann HJ, Remmel RP. Simultaneous analysis of cytochrome P450 probesdextromethorphan, flurbiprofen and midazolam and their major metabolites by HPLCmassspectrometry/flu orescence after single step extraction from plasma [J]. J Chromatogr B Analyt Technol Biomed Life Sci, 2007, 853(1/2):287
[10]Zhu B, Liu ZQ, Chen GL, et al. The distribution and gender difference of CYP3A activity in Chinese subjects [J].Br J Clin Pharmacol, 2003, 55(3):264
[11]Wo1bold R,Klein K,Burk O,et al. Sex is a major determinant of CYP3A4 in human liver [J].Hepatology,2003,38(4):978

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备注/Memo

备注/Memo:
#通讯作者,男, 1969年5月生, 博士, 副主任药师, 研究方向: 临床药理学,Email:Zhaojie@zzu.edu.cn
更新日期/Last Update: 2013-07-25