A novel HPLC-based method to diagnose peroxisomal D-bifunctional protein enoyl-CoA hydratase deficiency.
J Lipid Res
; 44(3): 640-4, 2003 Mar.
Article
em En
| MEDLINE
| ID: mdl-12562856
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.
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Base de dados:
MEDLINE
Assunto principal:
Cromatografia Líquida de Alta Pressão
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Transtornos Peroxissômicos
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Peroxissomos
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Enoil-CoA Hidratase
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Hidroliases
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3-Hidroxiacil-CoA Desidrogenases
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17-Hidroxiesteroide Desidrogenases
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Complexos Multienzimáticos
Idioma:
En
Ano de publicação:
2003
Tipo de documento:
Article