Substrate specificity of THCA-CoA oxidases from rat liver light mitochondrial fractions on dehydrogenation of 3α,7α,12α-trihydroxy-5β- cholestanoic acid CoA thioester

The substrate specificity of rat liver peroxisomal 3α, 7α, 12α- trihydroxy-5β-cholestanoyl-CoA (THCA-CoA) oxidases, which catalyze the dehydrogenation of 3α,7α,12α-trihydroxy-5β-cholestanoic acid (THCA) CoA thioester, having an asymmetric center at C-25, to form (24E)-3α,7α,12α- trihydroxy-5β-cholest-24-enoic acid (Δ²⁴-THCA) CoA thioester, was studied. The stable isotope labeled substrates, [3,7,12-¹⁸O₃]-(25R)- and (25S)-THCA CoA thioesters were synthesized by an exchange reaction of carbonyl oxygens on asteroid nucleus of 3,7,12-trioxo-5β-cholestanoic acid, followed by metal hydride reduction and condensation reaction with CoA. After incubation of a mixture of unlabeled (25R)- and ¹⁸O-labeled (25S)-THCA CoA thioester, or vice versa, with hepatic peroxisomal THCA-CoA oxidases, biotransformed Δ²⁴-THCA was determined by liquid chromatography/atmospheric pressure chemical ionization mass spectrometry. The Δ²⁴-THCA was derived only from (25S)-THCA CoA thioester, indicating that the 25S epimer of THCA is a preferential substrate on dehydrogenation by THCA-CoA oxidases.