Mechanism of Microbial Transformation of Cholesterol into Coprostanol

The conversion of cholesterol into coprostanol by cecal contents from rats under anaerobic conditions was studied using deuterated water, [4β‐³H,4‐¹⁴C]‐ and [3α‐³H,4‐¹⁴C]cholesterol. On the basis of the following experimental findings, it was concluded that the conversion of cholesterol into coprostanol proceeds to at least 50% by means of the intermediate formation of Δ⁴‐cholestenone. Conversion of [3α‐³H,4‐¹⁴C]cholesterol into coprostanol occurred with loss of 50% of the tritium. The tritium retained in coprostanol was located in the C‐3α position. However, part of the tritium retained might have been introduced in a reductive step following an initial oxidative step. Evidence for this possibility was provided by the finding that coprostanol formed from [4‐¹⁴C]cholesterol in the presence of [3α‐³H]β‐sitosterol contained significant amounts of tritium in the C‐3α position. After conversion of part of [3α‐³H,4‐¹⁴C]cholesterol into coprostanol, unchanged cholesterol had a higher ³H/¹⁴C ratio than the cholesterol added to the incubation mixture, indicating that oxidation of the 3β‐hydroxyl group is at least partly rate limiting in the over‐all conversion of cholesterol into coprostanol. After incubation of cholesterol in the presence of deuterated water, the coprostanol isolated had a deuterium content of about 1.4 atoms at C‐2, C‐3 and C‐4, about 0.5 atoms at C‐5 and about 0.1 atoms at C‐6, indicating extensive oxido‐reduction at C‐3 during or after conversion of cholesterol into coprostanol. Coprostanol isolated after incubation of [4β‐³H,4‐¹⁴C]cholesterol retained 60% of the tritium. The main part of this tritium had been transferred to the C‐6 position, showing that the conversion of cholesterol into coprostanol involves isomerization of the Δ⁵ double bond to a Δ⁴ double bond. Incubation of Δ⁴‐[4‐¹⁴C]cholestenone resulted in efficient formation of coprostanol.