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Originally published In Press as doi:10.1194/jlr.D200039-JLR200 on December 1, 2002

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Journal of Lipid Research, Vol. 44, 640-644, March 2003
Copyright © 2003 by Lipid Research, Inc.

Methods

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{dagger}, Ronald J. A. Wanders* and Sacha Ferdinandusse1,*

* University of Amsterdam, Academic Medical Center, Departments of Clinical Chemistry and Pediatrics, Laboratory for Genetic Metabolic Diseases (F0-224), P.O. Box 22700, 1100 DE Amsterdam, The Netherlands
{dagger} Department of Pediatrics, University of Ghent, Ghent, Belgium

1 To whom correspondence should be addressed. e-mail: s.ferdinandusse{at}amc.uva.nl

D-bifunctional protein (D-BP) plays an indispensable role in peroxisomal ß-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.

Abbreviations: D-BP, D-bifunctional protein; D-BP DH, D-BP 3-hydroxyacyl-CoA dehydrogenase; D-BP HY, D-BP enoyl-CoA hydratase; THCA, trihydroxycholestanoic acid; PTS, peroxisomal targeting signal

Supplementary key words peroxisomal fatty acid oxidation disorders • very long chain fatty acids • trihydroxycholestanoic acid


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