On the Properties of the 12alpha-Hydroxylase in Cholic Acid Biosynthesis. Bile Acids and Steroids 198

Abstract

The 12a-hydroxylation of the steroid nucleus in cholic acid biosynthesis was studied in the microsomal fraction of rat liver homogenate. The reaction was shown to require NADPH. A participation of flavins in the 12a-hydroxylase system or a requirement for metal ions could not be shown. Carbon monoxide inhibited partly the 12a-hydroxylase system. The effect of some inhibitors of steroid hydroxylations was tested and the influence of sex and age was studied. The relative rates of 12a-hydroxylation of a number of C,, steroids were measured and were found to be : 7a-hydroxycholest-4-en-3-one, 1 .O ; 5~-cholestane-3a,7o-diol, 0.4 ; 7a-hydroxy-5/3-cholestan%one, 0.2 ; cholest-5-ene-3~,7a-diol, 0.1 ; cholesterol and cholest-4-en-3-one, < 0.1.The main primary bile acids formed from cholesterol in the rat are cholic acid and chenodeoxycholic acid. The major pathways for the formation of these bile acids have now been elucidated (for a review see [l]). The quantitative importance of the different pathways has, however, not been finally established. The first step in the conversion of cholesterol into cholic acid is hydroxylation in the 7a-position to yield cholest-5-ene-3/3,7a-diol. Subsequently, cholest-5-ene-3/?,7a-diol is converted either into cholest-5-ene-3B,7a,lZa-triol or into 7a-hydroxycholest-4-en-3-oneBoth cholest-5-ene-3/3,7a,l2a-triol and 7a-hydroxycholest-4-en-3-one are transformed into 7a,-12a-dihydroxycholest-4-en-3-0ne, which in turn is converted into 5j3-cholestane-3~,7a,l2a-triol [2,3,5]. An alternative pathway for the formation of 58-cholestane-3~,7a,l2a-triol involves the transformation of 7a-hydroxycholest-4-en-3-one into 5B-cholestane-3~,7a-diol, which is then hydroxylated in the 12cc-position [4]. Thus, 12a-hydroxylation can occur with a t least three different substrates, cholest-5-ene-38,7a-diol, 7a-hydroxycholest-4-en-3-one, and 58-cholestane-3cc,7a-diol. I n previous investigations from this laboratory evidence has been presented to indicate that the main substrate for the 12a-hydroxylase in cholic acid formation is 7a-hydroxycholest-4-en-3-one [Z, 31. Present information indicates that the first steps in the major pathway for the biosynthesis of chenodeoxycholic acid are the same as those in the biosynthesis of cholic acid, the branching point being the 12a-hydroxylation [6]. Hydroxylation in the 12a-position is, thus, a key reaction in cholic Nomenclature. The following systematic names are given to bile acids referred to by trivial names: cholic acid, 3c(,7c(, 12cr-trihydroxy-5~-cholanoic acid; chenodeoxycholic acid, 3cr,7a-dihydroxy-5~-cholanoic acid.

Mechanisms of Bile Acid Biosynthesis

“…The reaction is catalyzed by the microsomal fraction fortified with NADPH (15,37). The conversion of 5-cholestene-3{1,7a-diol into 5-cholestene-3{1, 7a, l2a-triol, which is a reaction in another pathway for the formation of 5{1-cholestane-3a,7a,12a-triol, is also catalyzed by the microsomal fraction fortified with NADPH (30,37), as is the 12a-hydroxylation of 5{1-cholestane-3a,7a-diol and 7a-hydroxy-5{1-cholestan-3-one (37). The rates of 12a-hydroxylation of these C27-steroids differ considerably: the rate with 5-cholestene-3{1,7a-diol is about one-tenth and with 5{1-cholestane-3a,7a-diol about half of that with 7a-hydroxy-4-cholesten-3-one (37).…”

Formation and Metabolism of Cholest‐5‐ene‐3β,12α‐diol

“…Rabbit liver lacks such a 7a-hydroxylating system and since 12a-hydroxy-5/3-cholestan-3-one and 5/3-cholestane-3a,l2a-diol were not metabolized to cholic acid in the bile fistula rabbit, it can be concluded that rat liver as well as rabbit liver are not able to 7a-hydroxylate 12a-hydroxy-5/3-cholestan-3-one and 5~-cholestane-3a,l2a-diol to any significant extent. When 12a-hydroxycholest-4-en-3-one and cholest-5-ene-3~,12a-diol [5] were administered to bile fistula rabbits about 3 and loo/,, respectively, of the metabolites formed could be accounted for as cholic acid. These data agree with the results of the experiments with rat liver homogenates.…”

“…Another pathway for the formation of 7a,l2a-dihydroxycholest-4-en-3-one has been described involving the 12a-hydroxylation of 7 whydroxycholest-4-en-3-one catalyzed by the microsomal fraction fortified with NADPH [ 2 ] . Recently, some properties of the 12a-hydroxylase system were reported including measurements of the rate of 12a-hydroxylation of a number of C,,-steroids [3]. The rate of 12a-hydroxylation of 7a-hydroxycholest-4-en-3-one was found to be about ten times faster than Note.…”

Hydroxylations in Biosynthesis of Bile Acids. Cytochrome P-450 LM4 and 12alpha-Hydroxylation of 5beta-Cholestane-3alpha,7alpha-diol

“…Low sensitivity of the 12~-hydroxylase system towards carbon monoxide has been observed with microsomal fraction and crude reconstituted systems [2,[4][5][6]101. However, the sensitivity was higher -up to 80 inhibition-with purified cytochroine P-450 LM4 from starved rabbits.…”