Submitted on October 21, 2002
Revised on December 1, 2002
Accepted on November 19, 2002
A novel HPLC-based method to diagnose peroxisomal D-bifunctional protein enoyl-CoA hydratase deficiency
Jolein Gloerich, Simone Denis, Elisabeth G. van Grunsven, Georges Dacremont, Ronald J.A. Wanders, and Sacha Ferdinandusse
Laboratory Genetic Metabolic Diseases, Academic Medical Center, Amsterdam 1100 DE
Corresponding Author: s.ferdinandusse{at}amc.uva.nl
D-bifunctional protein (D-BP) plays an indispensable role in peroxisomal 
-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 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 co-incubation of yeast homogenate expressing D-BP hydratase 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 hydratase activity to the D-BP deficiency subgroups type I or type II.
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