Cholic acid is biosynthesized from cholesterol in liver via β-oxidation of 3α,7α,12α-trihydroxy-5β-cholestan-26-oic acid (THCA). Stereochemical aspects of this β-oxidation have now been investigated by preparation of the potential biosynthetic intermediate carboxylic acids including their ^2H- or ^<13>C-labelled forms, followed by their incubation with rat liver homogenate in the presence of CoA and ATP. Analysis of the incubation products by HPLC, NMR and MS revealed the followings: (1) (25R)-THCA, (25S)-THCA and Δ^<24E>-THCA were converted to cholic acid and four C-24/C-25 diastereoisomers of 24-hydroxy-THCA. Incubation of the latters produced cholic acid and Δ^<24E>-THCA. Thus all these compounds in the form of CoA esters, including the recently found 24-oxo-THCA-CoA seem to be intermediates of cholic acid biosynthesis. (2) Stereochemically defined four deuteriated (at C-24) THCA were chemically prepared. Their incubation and the FAB-MS spectra of the resulting Δ^<24E>-THCA indicated that deuterium was retained in the Δ^<24E>-THCAs derived from [24-pro-S-^2H]-substrates, while deuterium was lost during conversion from [24-pro-R-^2H]-substrates. We proposed that the dehydrogenation occurs in syn-mode, wherin (25R)-THCA-CoA, not (25S)-epimer, is a substrate for the oxidase; the (25S)-isomer would isomerize into (25R)-epimer prior to the dehydrogenation. (3) Hydration of Δ^<24E>-THCA-CoA appears to be non-stereospecific because the four 24-hydroxy-THCAs were produced from Δ^<24E>-THCA. Although we have no definite explanation of this phenomenon, the possibility of interconversion of the four alcohols via 24-oxo compound was excluded by the observation that deuterium of [24-^2H]-Δ^<24E>-THCA were retained in all the four 24-hydroxy-THCAs.
