Deo Anand K, Bandiera Stelvio M
Faculty of Pharmaceutical Sciences, University of British Columbia, 2146 East Mall, Vancouver, BC, Canada.
Drug Metab Dispos. 2009 Sep;37(9):1938-47. doi: 10.1124/dmd.109.027763. Epub 2009 Jun 1.
3alpha-Hydroxy-5 beta-cholan-24-oic (lithocholic) acid is a relatively minor component of hepatic bile acids in humans but is highly cytotoxic. Hepatic microsomal oxidation offers a potential mechanism for effective detoxification and elimination of bile acids. The aim of the present study was to investigate the biotransformation of lithocholic acid by human hepatic microsomes and to assess the contribution of cytochrome P450 (P450) enzymes in human hepatic microsomes using human recombinant P450 enzymes and chemical inhibitors. Metabolites were identified, and metabolite formation was quantified using a liquid chromatography/mass spectrometry-based assay. Incubation of lithocholic acid with human liver microsomes resulted in the formation of five metabolites, which are listed in order of their rates of formation: 3-oxo-5 beta-cholan-24-oic (3-ketocholanoic) acid, 3 alpha,6 alpha-dihydroxy-5 beta-cholan-24-oic (hyodeoxycholic) acid, 3 alpha,7 beta-dihydroxy-5 beta-cholan-24-oic (ursodeoxycholic) acid, 3 alpha,6 beta-dihydroxy-5 beta-cholan-24-oic (murideoxycholic) acid, and 3 alpha-hydroxy-6-oxo-5 beta-cholan-24-oic (6-ketolithocholic) acid. 3-Ketocholanoic acid was the major metabolite, exhibiting apparent K(m) and V(max) values of 22 muM and 336 pmol/min/mg protein, respectively. Incubation of lithocholic acid with a of human recombinant P450 enzymes revealed that all five metabolites were formed by recombinant CYP3A4. Chemical inhibition studies with human liver microsomes and recombinant P450 enzymes confirmed that CYP3A4 was the predominant enzyme involved in hepatic microsomal biotransformation of lithocholic acid. In summary, the results indicate that oxidation of the third carbon of the cholestane ring is the preferred position of oxidation by P450 enzymes for lithocholic acid biotransformation in humans and suggest that formation of lithocholic acid metabolites leads to enhanced hepatic detoxification and elimination.
3α-羟基-5β-胆烷-24-酸（石胆酸）是人体肝胆汁酸中含量相对较少的成分，但具有高度细胞毒性。肝微粒体氧化为胆汁酸的有效解毒和消除提供了一种潜在机制。本研究的目的是研究人肝微粒体对石胆酸的生物转化，并使用人重组细胞色素P450（P450）酶和化学抑制剂评估人肝微粒体中P450酶的作用。鉴定了代谢产物，并使用基于液相色谱/质谱的分析方法对代谢产物的形成进行了定量。石胆酸与人肝微粒体孵育产生了五种代谢产物，按其形成速率排序如下：3-氧代-5β-胆烷-24-酸（3-酮胆烷酸）、3α,6α-二羟基-5β-胆烷-24-酸（猪去氧胆酸）、3α,7β-二羟基-5β-胆烷-24-酸（熊去氧胆酸）、3α,6β-二羟基-5β-胆烷-24-酸（鼠去氧胆酸）和3α-羟基-6-氧代-5β-胆烷-24-酸（6-酮石胆酸）。3-酮胆烷酸是主要代谢产物，其表观K(m)和V(max)值分别为22 μM和336 pmol/min/mg蛋白。石胆酸与人重组P450酶孵育表明，所有五种代谢产物均由重组CYP3A4形成。用人肝微粒体和重组P450酶进行的化学抑制研究证实，CYP3A4是参与肝微粒体石胆酸生物转化的主要酶。总之，结果表明，在人体内，胆甾烷环的第三个碳原子氧化是P450酶对石胆酸进行生物转化的首选氧化位置，提示石胆酸代谢产物的形成导致肝脏解毒和消除能力增强。
