Identification and specific makers of abnormal bile acids excreted in inborn errors of bile acid biosynthesis

从胆固醇合成胆盐是一个复杂的生化途径，涉及至少16种酶。胆汁酸生物合成的大多数先天性错误导致中间体和/或其代谢物的过度形成，其在血液中积累并部分通过尿液排泄。早期发现是重要的，因为口服胆汁酸治疗可以改善病情。在过去，胆汁酸生物合成中的这些中间体已经通过用FAB-MS筛选然后用GC-MS详细表征在新生儿血液或尿液中检测到。这两种方法已经证明难以自动化，并且目前大多数实验室使用LC-MS/MS筛选候选样品。用于鉴定和表征39种结合和非结合胆汁酸的简单和灵敏的分析方法，包括Δ^-3-氧代-和Δ^<4，6>-3-氧代-胆汁酸（Δ 4>-3-氧代-类固醇5β-还原酶缺乏的标志物），使用液相色谱-电喷雾电离串联质谱法（LC/ESI-MS/MS）。在该程序中，将浓缩的脱盐尿液样品（用乙醇稀释）直接进样至LC-ESI-MS/MS中，采用ESI和负离子模式操作;通过选择反应监测（SRM）进行定量。为了评估我们的新方法的性能，我们将其与使用GC-MS的经验证的方法进行了比较，分析了两名遗传学证实的Δ^4-3-氧代-类固醇-5 β-还原酶缺乏症患者以及第三名异常结合和未结合Δ^4-3-氧代-胆汁酸浓度升高的患者的尿液。通过LC/ESI-MS/MS，在3名5β-还原酶缺乏症患者中恢复的Δ^4-3-氧代胆汁酸浓度分别为48.8、58.9和49.4μmol/mmol肌酐。

The synthesis of bile salts from cholesterol is a complex biochemical pathway involving at least 16 enzymes. Most inborn errors of bile acid biosynthesis result in excessive formation of intermediates and/or their metabolites that accumulate in blood and are excreted in part in urine. Early detection is important as oral therapy with bile acids results in improvement. In the past, these intermediates in bile acid biosynthesis have been detected in neonatal blood or urine by screening with FAB-MS followed by detailed characterization using GC-MS. Both methods have proved difficult to automate, and currently most laboratories screen candidate samples using LC-MS/MS. Here, we describe a new, simple and sensitive analytical method for the identification and characterization of 39 conjugated and unconjugated bile acids, including Δ^-3-oxo- and Δ^<4, 6>-3-oxo-bile acids(markers for Δ^4-3-oxo-steroid 5β-reductase deficiency), using liquid chromatography-electrospray ionization tandem mass spectrometry(LC/ESI-MS/MS). In this procedure, a concentrated, desalted urinary sample(diluted with ethanol) is injected directly into the LC-ESI-MS/MS, operated with ESI and in the negative ion mode ; quantification is obtained by selected reaction monitoring(SRM). To evaluate the performance of our new method, we compared it to a validated method using GC-MS, in the analysis of urine from two patients with genetically confirmed Δ^4-3-oxo-steroid-5β-reductase deficiency as well as a third patient with an elevated concentration of abnormal conjugated and unconjugated Δ^4-3-oxo-bile acids. The Δ^4-3-oxo-bile acids concentration recovered in three patients with 5β-reductase deficiency were 48.8, 58.9, and 49.4μmol/mmol Creatinine, respectively by LC/ESI-MS/MS.