Sensitive analysis of serum 3 alpha, 7 alpha, 12 alpha,24-tetrahydroxy-5 beta-cholestan-26-oic acid diastereomers using gas chromatography-mass spectrometry and its application in peroxisomal D-bifunctional protein deficiency

The final steps in bile acid biosynthesis take place in peroxisomes and inv olve oxidative cleavage of the side chain of C-27-5 beta-cholestanoic acids leading to the formation of the primary bile acids cholic acid and chenode oxycholic acid. The enoyl-CoA hydratase and beta-hydroxy acyl-CoA dehydroge nase reactions involved in the chain shortening of C-27-5 beta-cholestanoic acids are catalyzed by the recently identified peroxisomal D-bifunctional protein. Deficiencies of D-bifunctional protein lead, among others, to an a ccumulation of 3 alpha,7 alpha,12 alpha,24-tetrahydroxy-5 beta-cholest-26-o ic acid (varanic acid). The ability to resolve the four C-24, C-25 diastere omers of varanic acid has, so far, only been carried out on biliary bile ac ids using p-bromophenacyl derivatives. Here, we describe a sensitive gas ch romatography-mass spectrometry (GC/MS) method that enables good separation of the four varanic acid diastereomers by use of ZR-butylester-trimethylsil ylether derivatives, This method showed the specific accumulation of (24R,2 5R)-varanic acid in the serum of a patient with isolated deficiency of the D-3-hydroxy acyl-CoA dehydrogenase part of peroxisomal D-bifunctional prote in, whereas this diastereomer was absent in a serum sample from a patient s uffering from complete D-bifunctional protein deficiency. Zn samples from b oth patients an accumulation of (24S,25S)-varanic acid was observed, most l ikely due to the action of L-bifunctional protein on Delta 24E-THCA-CoA. Th is GC/MS method is applicable to serum samples, obviating the use of bile f luid, and is a helpful tool in the subclassification of patients with perox isomal D-bifunctional protein deficiency.