title: Sensitive analysis of serum 3alpha, 7alpha, 12alpha,24-tetrahydroxy- 5beta-cholestan-26-oic acid diastereomers using gas chromatography-mass spectrometry and its application in peroxisomal D-bifunctional protein deficiency.
abstract: The final steps in bile acid biosynthesis take place in peroxisomes and involve oxidative cleavage of the side chain of C27-5beta-cholestanoic acids leading to the formation of the primary bile acids cholic acid and chenodeoxycholic acid. The enoyl-CoA hydratase and beta-hydroxy acyl-CoA dehydrogenase reactions involved in the chain shortening of C27-5beta-cholestanoic acids are catalyzed by the recently identified peroxisomal d-bifunctional protein. Deficiencies of d-bifunctional protein lead, among others, to an accumulation of 3alpha,7alpha,12alpha, 24-tetrahydroxy-5beta-cholest-26-oic acid (varanic acid). The ability to resolve the four C24, C25 diastereomers of varanic acid has, so far, only been carried out on biliary bile acids using p -bromophenacyl derivatives. Here, we describe a sensitive gas chromatography-mass spectrometry (GC/MS) method that enables good separation of the four varanic acid diastereomers by use of 2R-butylester-trimethylsilylether derivatives. This method showed the specific accumulation of (24R,25R)-varanic acid in the serum of a patient with isolated deficiency of the d-3-hydroxy acyl-CoA dehydrogenase part of peroxisomal d-bifunctional protein, whereas this diastereomer was absent in a serum sample from a patient suffering from complete d-bifunctional protein deficiency. In samples from both patients an accumulation of (24S,25S)-varanic acid was observed, most likely due to the action of l-bifunctional protein on Delta24E-THCA-CoA. This GC/MS method is applicable to serum samples, obviating the use of bile fluid, and is a helpful tool in the subclassification of patients with peroxisomal d-bifunctional protein deficiency.
authors.affiliation: Emma Children's Hospital and Department of Chemistry, Academic Medical Center, University of Amsterdam, P. O. Box 22700, 1100 DE Amsterdam, The Netherlands.
authors.forename: P
authors.initials: P
authors.lastname: Vreken
authors.forename: A
authors.initials: A
authors.lastname: van Rooij
authors.forename: S
authors.initials: S
authors.lastname: Denis
authors.forename: E G
authors.initials: EG
authors.lastname: van Grunsven
authors.forename: D A
authors.initials: DA
authors.lastname: Cuebas
authors.forename: R J
authors.initials: RJ
authors.lastname: Wanders
mesh_terms: D015067:17-Hydroxysteroid Dehydrogenases; D015094:3-Hydroxyacyl CoA Dehydrogenases / Q000097:blood*; D002777:Cholestanols / Q000097:blood*; D004746:Enoyl-CoA Hydratase / Q000097:blood; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D006836:Hydro-Lyases / Q000097:blood*; D008830:Microbodies / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000097:blood* / Q000172:deficiency*; D018901:Peroxisomal Disorders / Q000097:blood*; D064029:Peroxisomal Multifunctional Protein-2; D011237:Predictive Value of Tests; D012680:Sensitivity and Specificity; D013237:Stereoisomerism
chemical_list: D002777:Cholestanols; D009097:Multienzyme Complexes; C024759:3,7,12,24-tetrahydroxycholestanoic acid; D015067:17-Hydroxysteroid Dehydrogenases; D015094:3-Hydroxyacyl CoA Dehydrogenases; D006836:Hydro-Lyases; D064029:Peroxisomal Multifunctional Protein-2; C104587:HSD17B4 protein, human; D004746:Enoyl-CoA Hydratase