Detection of Δ4-3-oxo-steroid 5β-reductase deficiency by LC-ESI-MS/MS measurement of urinary bile acids.
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(4)-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 49.4 μmol/mmol creatinine, respectively by LC-ESI-MS/MS.
Junshin Clinic Bile Acid Institute, Haramachi, Meguro-ku, Tokyo, Japan.
Akina
A
Muto
Hajime
H
Takei
Atsushi
A
Unno
Tsuyoshi
T
Murai
Takao
T
Kurosawa
Shoujiro
S
Ogawa
Takashi
T
Iida
Shigeo
S
Ikegawa
Jun
J
Mori
Akira
A
Ohtake
Takayuki
T
Hoshina
Tatsuki
T
Mizuochi
Akihiko
A
Kimura
Alan F
AF
Hofmann
Lee R
LR
Hagey
Hiroshi
H
Nittono
D001647:Bile Acids and Salts / Q000652:urine*; D002675:Child, Preschool; D002853:Chromatography, Liquid / Q000379:methods*; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D007223:Infant; D010088:Oxidoreductases / Q000172:deficiency*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry / Q000379:methods*
D001647:Bile Acids and Salts; D010088:Oxidoreductases; C051860:cholestenone 5 beta-reductase