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首页> 外文期刊>Journal of Lipid Research >Regulation of 25- and 27-hydroxylation side chain cleavage pathways for cholic acid biosynthesis in humans, rabbits, and mice. Assay of enzyme activities by high-resolution gas chromatography;-mass spectrometry.
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Regulation of 25- and 27-hydroxylation side chain cleavage pathways for cholic acid biosynthesis in humans, rabbits, and mice. Assay of enzyme activities by high-resolution gas chromatography;-mass spectrometry.

机译：调节人，兔子和小鼠中胆酸生物合成的25和27羟基化侧链裂解途径。通过高分辨率气相色谱法;质谱法测定酶活性。

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In classic cholic acid biosynthesis, a series of ring modifications of cholesterol precede side chain cleavage and yield 5beta-cholestane-3alpha, 7alpha, 12alpha-triol. Side chain reactions of the triol then proceed either by the mitochondrial 27-hydroxylation pathway or by the microsomal 25-hydroxylation pathway. We have developed specific and precise assay methods to measure the activities of key enzymes in both pathways, 5beta-cholestane-3alpha, 7alpha, 12alpha-triol 25- and 27-hydroxylases and 5beta-cholestane-3alpha, 7alpha, 12alpha, 25-tetrol 23R-, 24R-, 24S- and 27-hydroxylases. The extracts from either the mitochondrial or microsomal incubation mixtures were purified by means of a disposable silica cartridge column, derivatized into trimethylsilyl ethers, and quantified by gas chromatography;-mass spectrometry with selected-ion monitoring in a high resolution mode. Compared with the addition of substrates in acetone, those in 2-hydroxypropyl-beta-cyclodextrin increased mitochondrial triol 27-hydroxylase activity 132% but decreased activities of the enzymes in microsomal 25-hydroxylation pathway (triol 25-hydroxylase and 5beta-cholestane-3alpha, 7alpha, 12alpha, 25-tetrol 23R-, 24R-, 24S- and 27-hydroxylases) 13;-60% in human liver. The enzyme activities in both pathways were generally 2- to 4-times higher in mouse and rabbit livers compared with human liver. In all species, microsomal triol 25-hydroxylase activities were 4- to 11-times larger than mitochondrial triol 27-hydroxylase activities but the activities of tetrol 24S-hydroxylase were similar to triol 27-hydroxylase activities in our assay conditions. The regulation of both pathways in rabbit liver was studied after bile acid synthesis was perturbed. Cholesterol feeding up-regulated enzyme activities involved in both 25- (64;-142%) and 27- (77%) hydroxylation pathways, while bile drainage up-regulated only the enzymes in the 25-hydroxylation pathway (178;-371%). Using these new assays, we demonstrated that the 25- and 27-hydroxylation pathways for cholic acid biosynthesis are more active in mouse and rabbit than human livers and are separately regulated in rabbit liver.
机译：在经典的胆酸生物合成中，胆固醇的一系列环修饰在侧链裂解之前发生，并产生5β-胆甾烷3α，7α，12α-三醇。然后，三醇的侧链反应通过线粒体27-羟基化途径或微粒体25-羟基化途径进行。我们已经开发出了特定而精确的测定方法来测量两种途径中的关键酶的活性，即5beta-胆甾烷-3alpha，7alpha，12alpha-三醇25-和27-羟基酶和5beta-胆固醇3alpha，7alpha，12alpha，25-四醇23R-，24R-，24S-和27-羟基酶。线粒体或微粒体温育混合物的提取物通过一次性硅胶柱纯化，衍生化为三甲基甲硅烷基醚，并通过气相色谱法进行定量;质谱分析，以选定模式监控高分辨率。与在丙酮中添加底物相比，在2-羟丙基-β-环糊精中的底物将线粒体三醇27-羟化酶活性提高132％，但在微粒体25-羟化途径中降低了酶的活性（三醇25-羟化酶和5beta-胆甾烷-3alpha （7α，12α，25-四醇23R-，24R-，24S-和27-羟基酶）13; -60％在人肝脏中。与人肝相比，在小鼠和兔肝中，这两种途径的酶活性通常高2-4倍。在所有物种中，微粒体三醇25-羟化酶活性比线粒体三醇27-羟化酶活性大4至11倍，但在我们的测定条件下，四萜24S-羟化酶的活性类似于三醇27-羟化酶的活性。在胆汁酸合成受到干扰后，研究了兔肝中这两种途径的调节。胆固醇喂养的酶活性上调涉及25-（64; -142％）和27-（77％）的羟化途径，而胆汁排泄仅上调25-羟基化途径中的酶（178; -371％ ）。使用这些新的检测方法，我们证明了胆酸生物合成的25-和27-羟基化途径在小鼠和兔子中比人的肝脏更活跃，并且在兔子肝脏中受到单独调节。

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