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- butylestertrimethylsilylether 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 n-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.


Chemistry and Biochemistry

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This is an Open Access article under the Creative Commons CC-BY license.


Bile acids, D-bifunctional protein, GC/MS, Peroxisome

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Journal of Lipid Research