胆汁酸生合成における炭素-炭素結合切断反応に関する研究

胆固醇生物合成胆酸涉及C-24/C-25键的断裂，这种碳-碳键断裂反应的前体是3-α，7-α，12-α-三羟基辅酶-26-酸，这种酸被认为以类似于脂肪酸β氧化的机制代谢成胆酸。在中间体中，已经积累了THCA-CoA、24-烯-THCA-CoA和24-羟基-THCA-CoA的中介作用的实验证据。相比之下，对24-oxo-THCA-CoA的形成和代谢知之甚少。我们在这方面所做的努力被描述在这里。包括上述三个THCA-CoA衍生物的潜在底物是化学合成的，并与大鼠肝脏轻线粒体部分或Hep G2孵育。24-氧代-THCA-辅酶A如果是在孵育过程中产生的，应通过随后的碱处理使中间体α-酮-羧酸容易地脱羧化而转化为27-NOR-3-α，7-α，12-三羟基-24-辅基-24-酮(“乙基酮”)。THCA-CoA、24-烯-THCA-CoA和24-羟基-THCA-CoA的三甲基硅醚衍生物的GC-MS分析清楚地检测到了“乙酮”。这些结果表明这三种辅酯在酶作用下转化为24-氧代-THCA-CoA。同样，24-氧代-THCA-CoA与24-氧代-THCA-CoA反应生成了胆酸(或由24-氧代-THCA-CoA合成的胆酰-CoA)。

Cholic acid biosynthesis from cholesterol involves C-24/C-25 bond cleavage.A precursor of this carbon-carbon bond cleavage reaction is 3α, 7α, 12α-trihydroxycoprostan-26-oic acid(THCA) and this acid is thought to be metabolized into cholic acid by a mechanism similar to that of the β-oxidation of fatty acids. Among the intermediates, experimental evidence for the intermediary role of THCA-CoA, 24-ene-THCA-CoA and 24-hydroxy-THCA-CoA has been accumulated. In contrast, little has been known on the formation and metabolism of 24-oxo-THCA-CoA. Our efforts on this line are described here.The potential substrates including the three THCA-CoA derivatives mentioned above were chemically synthesized, and incubated with rat liver light mitochondria fraction or Hep G2. 24-Oxo-THCA-CoA, if produced during incubation, should be transformed into 27-nor-3α, 7α, 12α-trihydroxy-24-coprostan-24-on ("ethyl ketone") by the subsequent alkaline treatment inducing facile decarboxylation of the intermediate β-keto-carboxylic acid. GC-MS analysis of the trimethylsilyl ether derivatives coming from THCA-CoA, 24-ene-THCA-CoA and 24- hydroxy-THCA CoA clearly detected the "ethyl ketone". These results indicated the enzymic transformation of these three CoA-esters into 24-oxo-THCA-CoA.Similarly 24-oxo-THCA-CoA was incubated and the subsequent HPLC analysis of the p-bromophenacyl ester derivative indicated the enzymic formation of cholic acid(or cholyl-CoA from 24-oxo-THCA-CoATaken together, it is concluded that 24-oxo-THCA-CoA is the final intermediate of cholic acid biosynthesis.