Journal of Chromatography B, 900 (2012) 24–31 Contents lists available at SciVerse ScienceDirect Journal of Chromatography B jo u r n al hom epage: www.elsevier.com/locate/chromb Detection of  4 -3-oxo-steroid 5-reductase deficiency by LC–ESI-MS/MS measurement of urinary bile acids Akina Muto a , Hajime Takei a , Atsushi Unno a , Tsuyoshi Murai b , Takao Kurosawa b , Shoujiro Ogawa c , Takashi Iida c , Shigeo Ikegawa d , Jun Mori e , Akira Ohtake f , Takayuki Hoshina g , Tatsuki Mizuochi h , Akihiko Kimura h , Alan F. Hofmann i , Lee R. Hagey i , Hiroshi Nittono a,∗ a Junshin Clinic Bile Acid Institute, Haramachi, Meguro-ku, Tokyo 152-0011, Japan b Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido, Kanazawa, Ishikari-Tobetsu, Hokkaido 061-0293, Japan c Department of Chemistry, College of Humanities & Sciences, Nihon University, Sakurajousui, Setagaya-ku, Tokyo 156-8550, Japan d Faculty of Pharmaceutical Sciences, Kinki University, Kowakae, Higashi, Osaka 577-8502, Japan e Department of Pediatrics, Kyoto Prefectural University of Medicine Graduate School of Medical Science, Kajii-cho, Kawaramachi Hirokoji, Kamigyo-ku, Kyoto 602-8566, Japan f Department of Pediatrics, School of Medicine, Saitama Medical University, Moroyama, Iruma-gun, Saitama 350-0495, Japan g Department of Pediatrics, Graduate School of Medical Sciences, Kyushu University, Maidashi, Higashi-ku, Fukuoka 812-8582, Japan h Department of Pediatrics and Child Health, Kurume University School of Medicine, Asahi-machi, Kurume-shi, Fukuoka 830-0011, Japan i Department of Medicine, University of California, San Diego, La Jolla, CA 92093-0063, USA a r t i c l e i n f o Article history: Received 5 January 2012 Accepted 18 May 2012 Available online 24 May 2012 Keywords: Abnormal bile acid  4 -3-Oxo-steroid 5-reductase deficiency Inborn errors of bile acid metabolism Profile LC–ESI-MS/MS GC–MS a b s t r a c t 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 fol- lowed 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, sim- ple and sensitive analytical method for the identification and characterization of 39 conjugated and unconjugated bile acids, including  4 -3-oxo- and  4,6 -3-oxo-bile acids (markers for  4 -3-oxo-steroid 5-reductase deficiency), using liquid chromatography-electrospray ionization tandem mass spectrom- etry (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; quan- tification 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  4 -3-oxo-steroid 5-reductase deficiency as well as a third patient with an elevated concentration of abnormal conjugated and unconjugated  4 -3-oxo-bile acids. The  4 -3-oxo- bile acids concentration recovered in three patients with 5-reductase deficiency were 48.8, 58.9, and 49.4 mol/mmol creatinine, respectively by LC–ESI-MS/MS. © 2012 Elsevier B.V. All rights reserved. 1. Introduction Bile acids are biosynthesized from cholesterol in a process that utilizes multiple partially overlapping enzymatic pathways to make substantial changes to the steroid ring nucleus and side- chain, a process that utilizes a minimum of 16 enzymes [1]. Some of the first inborn errors in these pathways were detected nearly 30 years ago [2,3] by the detection of intermediates in the bile ∗ Corresponding author at: Junshin Clinic Bile Acid Institute, 2-1-22, Haramachi, Meguro-ku, Tokyo 152-0011, Japan. E-mail address: bile-res@eco.ocn.ne.jp (H. Nittono). acid biosynthetic pathway. Since that time, inborn errors of bile acid formation involving isolated defects in most of the enzymes in the biosynthetic pathway have been reported [4]. However, such enzymatic defects are extremely rare. The most common genetic defect in bile acid biosynthesis appears to be  4 -3-oxo-steroid 5- reductase deficiency, first described by Setchell et al. [5]. In children with defects in the gene or promoter region of  4 -3-oxo-steroid 5-reductase (gene AKR1D1), the urinary bile acid profile contains 7-hydroxy-3-oxo-4-cholenoic acid (as its glycine m/z 444 and tau- rine m/z 494 conjugates), as well as its 12-dihydroxy-metabolite (again as glycine m/z 460 and taurine m/z 510 conjugates), as shown in Fig. 1. Chenodeoxycholic acid and cholic acid, the nor- mal primary bile acids in man are nearly undetectable. However, 1570-0232/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jchromb.2012.05.023