使用[2-¹⁴C]氯唑沙宗通过薄层色谱法测定微粒体中细胞色素P450 2E1的活性。

Determination of cytochrome P450 2E1 activity in microsomes by thin-layer chromatography using [2-14C]chlorzoxazone.

作者信息

Zerilli A, Lucas D, Berthou F, Bardou L G, Ménez J F

机构信息

UFR Odontologie, Brest, France.

出版信息

J Chromatogr B Biomed Appl. 1996 Feb 23;677(1):156-60. doi: 10.1016/0378-4347(95)00483-1.

DOI:10.1016/0378-4347(95)00483-1

PMID:8925089

Abstract

A thin-layer chromatographic assay was developed as an alternative method for the determination of cytochrome P450 2E1 (CYP2E1) in microsomes using [2-14C]chlorzoxazone. After incubation of microsomes with 0.125 microCi/mmol chlorzoxazone, chlorzoxazone and its single metabolite, 6-hydroxychlorzoxazone, were extracted using chloroform-2-propanol (85:15, v/v) and chromatographed on silica gel 60 F254 plates with acetone-hexane (45:55, v/v) as solvent . The plates were then exposed to X-ray film for 2 days to localize the radiolabelled chlorzoxazone and 6-hydroxychlorzoxazone. The metabolite and substrate regions were scraped and counted in a liquid scintillation analyzer. This method is sensitive enough to determine constitutive and induced CYP2E1 activities in liver or kidney microsomes. The precision of the method was similar to that of the HPLC method. The correlation coefficient between both methods was found to be 0.97 (n = 21). Therefore, the TLC method constitutes a valuable tool for the determination of chlorzoxazone metabolism in microsomes.

摘要

开发了一种薄层色谱测定法，作为使用[2-¹⁴C]氯唑沙宗测定微粒体中细胞色素P450 2E1（CYP2E1）的替代方法。微粒体与0.125微居里/毫摩尔氯唑沙宗孵育后，使用氯仿-异丙醇（85:15，v/v）提取氯唑沙宗及其单一代谢物6-羟基氯唑沙宗，并在硅胶60 F254板上以丙酮-己烷（45:55，v/v）作为溶剂进行色谱分析。然后将板暴露于X射线胶片2天，以定位放射性标记的氯唑沙宗和6-羟基氯唑沙宗。刮下代谢物和底物区域，并在液体闪烁分析仪中计数。该方法灵敏度足以测定肝脏或肾脏微粒体中组成型和诱导型CYP2E1活性。该方法的精密度与HPLC方法相似。发现两种方法之间的相关系数为0.97（n = 21）。因此，TLC方法是测定微粒体中氯唑沙宗代谢的有价值工具。