| Summary: | The final steps in bile acid biosynthesis take place in peroxisomes and involve oxidative cleavage of the side chain of C27-5β-cholestanoic acids leading to the formation of the primary bile acids cholic acid and chenodeoxycholic acid. The enoyl-CoA hydratase and β-hydroxy acyl-CoA dehydrogenase reactions involved in the chain shortening of C27-5β-cholestanoic acids are catalyzed by the recently identified peroxisomal d-bifunctional protein. Deficiencies of d-bifunctional protein lead, among others, to an accumulation of 3α,7α,12α,24-tetrahydroxy-5β-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 Δ24E-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.—Vreken, P., A. van Rooij, S. Denis, E. G. van Grunsven, D. A. Cuebas, and R. J. A. Wanders. Sensitive analysis of serum 3α,7α,12α,24-tetrahydroxy-5β-cholestan-26-oic acid diastereomers using gas chromatography–mass spectrometry and its application in peroxisomal d-bifunctional protein deficiency. J. Lipid Res. 1998. 39: 2452–2458.
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