The synthesis of 12α-hydroxylated bile acids (12HBAs) and non-12α-hydroxylated bile acids (non-12HBAs) occurs via classical and alternative pathways, respectively. The composition of these BAs is a crucial index for pathophysiologic assessment. However, accurately differentiating 12HBAs and non-12HBAs is highly challenging due to the limited standard substances. Here, we innovatively introduce 12α-hydroxysteroid dehydrogenase (12α-HSDH) as an enzymatic probe synthesized by heterologous expression in Escherichia coli, which can specifically and efficiently convert 12HBAs in vitro under mild conditions. Coupled to the conversion rate determined by liquid chromatography-high resolution mass spectrometry (LC–HRMS), this enzymatic probe allows for the straightforward distinguishing of 210 12HBAs and 312 non-12HBAs from complex biological matrices, resulting in a BAs profile with a well-defined hydroxyl feature at the C12 site. Notably, this enzyme-driven LC-HRMS approach can be extended to any molecule with explicit knowledge of enzymatic transformation. We demonstrate the practicality of this BAs profile in terms of both revealing cross-species BAs heterogeneity and monitoring the alterations of 12HBAs and non-12HBAs under asthma disease. We envisage that this work will provide a novel pattern to recognize the shift of BA metabolism from classical to alternative synthesis pathways in different pathophysiological states, thereby offering valuable insights into the management of related diseases.

中文翻译：

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用于特异性识别 12α-羟基胆汁酸的酶驱动 LC-HRMS 方法


12α-羟基化胆汁酸 (12HBA) 和非 12α-羟基化胆汁酸 (non-12HBA) 的合成分别通过经典途径和替代途径进行。这些 BA 的组成是病理生理学评估的重要指标。然而，由于标准物质有限，准确区分 12HBA 和非 12HBA 极具挑战性。在这里，我们创新性地引入了12α-羟基类固醇脱氢酶（12α-HSDH）作为在大肠杆菌中异源表达合成的酶探针，它可以在温和条件下在体外特异性、高效地转化12HBA。结合液相色谱-高分辨率质谱 (LC-HRMS) 测定的转化率，该酶探针可以从复杂的生物基质中直接区分 210 个 12HBA 和 312 个非 12HBA，从而获得具有良好的 BA 谱。在 C12 位点定义羟基特征。值得注意的是，这种酶驱动的 LC-HRMS 方法可以扩展到任何具有明确的酶转化知识的分子。我们证明了该 BA 谱的实用性，既可以揭示跨物种 BA 异质性，又可以监测哮喘疾病下 12HBA 和非 12HBA 的变化。我们预计这项工作将提供一种新的模式来识别不同病理生理状态下 BA 代谢从经典合成途径向替代合成途径的转变，从而为相关疾病的管理提供有价值的见解。