Stereospecific Dehydrogenation of (25R)- and (25S)-3α,7α,12α-Trihydroxy 5β-Cholestanoic Acids by Acyl-Coa Oxidase in Rat Liver Light Mitochondrial Fraction

From a stereochemical point of view, the dehydrogenation mechanism of the biotransformation of 3α,7α,12α-trihydroxy-5β-cholestanoic acid (THCA) into (24E)-3α,7α,12α-trihydroxy-5β-cholest-24-enoic acid (Δ²⁴-THCA) has been studied with capillary gas chromatography (GC)/negative ion chemical ionization (NICI)-mass spectrometry. After incubation of (24R,25R)-or (24S,25S)-[24,25-²H₂] THCA, synthesized from (24E)-Δ²⁴-THCA by a deuterated diimide reduction, with a rat liver light mitochondrial fraction, 5β-cholestanoic acids were extracted and derivatized into a pentafluorobenzyl (PFB) ester-dimethylethylsilyl (DMES) ether. Subsequent resolution into THCA and Δ²⁴-THCA was attained by GC on a cross-linked 5% phenylmethyl silicone fused-silica capillary column monitored with a corresponding characteristic carboxylate anion [M-PFB]^- in the NICI mode. The stereospecific elimination of a pro-R hydrogen at C-24 in both (25R)-and (25S)-THCA indicated syn-elimination for the former, whereas anti-elimination for the latter was observed.