Detection of Delta(4)-3-oxo-steroid 5 beta-reductase deficiency by LC-ESI-MS/MS measurement of urinary bile acids

作者：Muto Akina; Takei Hajime; Unno Atsushi; Murai Tsuyoshi; Kurosawa Takao; Ogawa Shoujiro; Lida Takashi; Ikegawa Shigeo; Mori Jun; Ohtake Akira; Hoshina Takayuki; Mizuochi Tatsuki; Kimura Akihiko; Hofmann Alan F; Hagey Lee R; Nittono Hiroshi^*

来源：Journal of Chromatography B-Analytical Technologies in the Biomedical and Life Sciences, 2012, 900: 24-31.

DOI：10.1016/j.jchromb.2012.05.023

• Abnormal bile acid
• Delta(4)-3-Oxo-steroid 5 beta-reductase deficiency
• Inborn errors of bile acid metabolism
• Profile
• LC-ESI-MS/MS
• GC-MS

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 Delta(4)-3-oxo- and Delta(4).6-3-oxo-bile acids (markers for Delta(4)-3-oxo-steroid 5 beta-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 Delta(4)-3-oxo-steroid 5 beta-reductase deficiency as well as a third patient with an elevated concentration of abnormal conjugated and unconjugated Delta(4)-3-oxo-bile acids. The Delta(4)-3-oxo-bile acids concentration recovered in three patients with 5 beta-reductase deficiency were 48.8, 58.9, and 49.4 mu mol/mmol creatinine, respectively by LC-ESI-MS/MS.

• 出版日期2012-7-1