以滨蒿内酯为底物的细胞色素P-450活性分化。快速一步高效液相色谱分析。

Differentiation of cytochrome P-450 activities with scoparone as substrate. A rapid one-step HPLC-analysis.

作者信息

Müller-Enoch D, Greischel A

机构信息

Department of Physiological Chemistry, University of Ulm/Donau, Fed. Rep. of Germany.

出版信息

Arzneimittelforschung. 1988 Oct;38(10):1520-2.

PMID:3196394

Abstract

A direct and highly sensitive high performance liquid chromatography (HPLC) method for measuring cytochrome P-450 activities in biological probes, with the substrate scoparone, is described. Using 10-100 microliter of a supernatant of an incubation mixture of scoparone (0.1 mmol/l), microsomal fractions (1 nmol P-450/ml), MgCl2 (2 mmol/l), and NADPH (4 mmol/l), the products scopoletin and isoscopoletin as well as the substrate scoparone were separated by HPLC on an ODS-Hypersil RP-18 column. The compounds were detected by a UV-spectrophotometer (345 nm) and quantified by the aid of an external calibration curve. The limits of detection were 10 pmol for the products scopoletin and isoscopoletin. This method allows the direct quantification of P-450 activities and the simultaneous differentiation between "3-methylcholanthren-like" or "phenobarbital-like" induction states by estimation of the scopoletin: isoscopoletin ratios.

摘要

描述了一种直接且高灵敏度的高效液相色谱（HPLC）方法，用于测定生物样品中细胞色素P - 450的活性，该方法以滨蒿内酯为底物。使用10 - 100微升由滨蒿内酯（0.1 mmol/l）、微粒体组分（1 nmol P - 450/ml）、MgCl2（2 mmol/l）和NADPH（4 mmol/l）组成的孵育混合物的上清液，在ODS - Hypersil RP - 18柱上通过HPLC分离产物东莨菪素和异东莨菪素以及底物滨蒿内酯。通过紫外分光光度计（345 nm）检测这些化合物，并借助外标曲线进行定量。产物东莨菪素和异东莨菪素的检测限为10 pmol。该方法可直接定量P - 450活性，并通过估算东莨菪素：异东莨菪素的比率，同时区分“3 - 甲基胆蒽样”或“苯巴比妥样”诱导状态。