title: A novel HPLC-based method to diagnose peroxisomal D-bifunctional protein enoyl-CoA hydratase deficiency.
abstract: D-bifunctional protein (D-BP) plays an indispensable role in peroxisomal beta-oxidation, and its inherited deficiency in humans is associated with severe clinical abnormalities. Three different subtypes of D-BP deficiency can be distinguished: 1) a complete deficiency of D-BP (type I), 2) an isolated D-BP enoyl-CoA hydratase deficiency (type II), and 3) an isolated D-BP 3-hydroxyacyl-CoA dehydrogenase deficiency (type III). In this study, we developed a method to measure D-BP dehydrogenase activity independent of D-BP hydratase (D-BP HY) activity to distinguish between D-BP deficiency type I and type II, which until now was only possible by mutation analysis. For this assay, the hydratase domain of D-BP was expressed in the yeast Saccharomyces cerevisiae. After a coincubation of yeast homogenate expressing D-BP HY with fibroblast homogenate of patients using the enoyl-CoA ester of the bile acid intermediate trihydroxycholestanoic acid as substrate, D-BP dehydrogenase activity was measured. Fibroblasts of patients with a D-BP deficiency type II displayed D-BP dehydrogenase activity, whereas type I and type III patients did not. This newly developed assay to measure D-BP dehydrogenase activity in fibroblast homogenates provides a quick and reliable method to assign patients with deficient D-BP HY activity to the D-BP deficiency subgroups type I or type II.
authors.affiliation: University of Amsterdam, Academic Medical Center, Department of Clinical Chemistry, Laboratory for Genetic Metabolic Diseases (F0-224), P.O. Box 22700, 1100 DE Amsterdam, The Netherlands.
authors.forename: Jolein
authors.initials: J
authors.lastname: Gloerich
authors.forename: Simone
authors.initials: S
authors.lastname: Denis
authors.forename: Elisabeth G
authors.initials: EG
authors.lastname: van Grunsven
authors.forename: Georges
authors.initials: G
authors.lastname: Dacremont
authors.forename: Ronald J A
authors.initials: RJ
authors.lastname: Wanders
authors.forename: Sacha
authors.initials: S
authors.lastname: Ferdinandusse
mesh_terms: D015067:17-Hydroxysteroid Dehydrogenases; D015094:3-Hydroxyacyl CoA Dehydrogenases / Q000032:analysis* / Q000172:deficiency* / Q000276:immunology; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004746:Enoyl-CoA Hydratase / Q000032:analysis* / Q000172:deficiency* / Q000276:immunology; D005347:Fibroblasts; D006801:Humans; D006836:Hydro-Lyases / Q000032:analysis* / Q000172:deficiency* / Q000276:immunology; D015151:Immunoblotting; D009097:Multienzyme Complexes / Q000032:analysis* / Q000172:deficiency* / Q000276:immunology; D018901:Peroxisomal Disorders / Q000175:diagnosis* / Q000201:enzymology*; D064029:Peroxisomal Multifunctional Protein-2; D020675:Peroxisomes / Q000201:enzymology*; D015003:Yeasts
chemical_list: D009097:Multienzyme Complexes; 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