Chemical and enzymic studies on the characterization of intermediates during the removal of the 14α-methyl group in cholesterol biosynthesis. The use of 32-functionalized lanostane derivatives

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Abstract
By using cell-free preparations of rat liver it was shown that the removal of the 14.alpha.-methyl group (C-32) of steroids containing either a .DELTA.7(8) or a .DELTA.8(9) double bond is attended exclusively by the formation of the corresponding 7,14- and 8,14-dienes, respectively. Cumulative evidence from a variety of experimental approaches suggests that .DELTA.8(14)-steroids are not involved as intermediates on the major pathway of cholesterol biosynthesis. The metabolism of [32-3H]lanost-7-ene-3.beta.,32-diol (I) results in the formation of radioactive formic acid, no labeled formaldehyde being formed. By using appropriately labeled species of the compound (I) it was found that the release of formic acid and the formation of 4,4-dimethylcholesta-7,14-dien-3.beta.-ol (III) were closely linked processes, and that in the conversion of (I) into (III), 3.beta.-hydroxylanost-7-en-32-al (II) is an obligatory intermediate. Both the conversion of [I) into (II) and the further metabolism of (II) to (III) exhibited a requirement for NADPH and O2. This suggests that the oxidation of the 32-hydroxy group of (I) to the aldehyde group of (II) does not occur by the conventional alcohol dehydrogenase type of reaction, but may proceed by a novel mechanism involving the intermediacy of a gem-diol. A detailed overall pathway for the 14.alpha.-demethylation in cholesterol biosynthesis is considered and proposals about the mechanism of individual steps in the pathway are made.