7a,12a-Dihydroxy-4-cholesten-3-one

During the 1960 s, the above sequence of reactions was confirmed by different in vitro studies. Mendelsohn and Staple showed that labelled cholesterol could be converted into 5j8-cholestane-3a,7a,12 -triol by 20000 X g supernatant fluid of rat liver homogenates [23]. The enzymatic conversion of cholesterol into 7a-hydroxy-cholesterol was first shown by Danielsson and Einarsson using the microsomal fraction fortified with NADPH [24]. The conversion of 7 -hydroxycholesterol into 7 -hydroxy-4-cholesten-3-one was found to be catalysed by the microsomal fraction fortified with NAD [25]. The latter steroid was converted into 7a,12a-dihydroxy-4-cholesten-3-one by the microsomal fraction and NADPH [26]. The conversion of 7 -hydroxy-4-cholesten-3-one and 7a,12a-dihydroxy-4-cholesten-3-one into the corresponding 3a-hydroxy-5/8-saturated steroids was catalysed by soluble NADPH-de-pendent enzymes [25,27,28]. Since Hutton and Boyd found that 4-cholestene-3 ,7 -diol was a product of 7a-hydroxy-4-cholesten-3-one in vitro [25], it was first... 

In the presence of 100000 X g supernatant fraction of liver homogenate, 7 -hy-droxy-4-cholesten-3-one as well as 7a,12a-dihydroxy-4-cholesten-3-one are efficiently converted into the corresponding 3a-hydroxy-5 8-steroids via the 3-oxo-5/8-steriods [27,28] (cf. Fig. 3). The NADPH-dependent A -3-oxosteroid 5/S-reductase active on 7a-hydroxy-4-cholesten-3-one and 7a,12 -dihydroxy-4-cholesten-3-one has been partially purified by Bersdus [112,113]. The preparation was also active towards 3-oxo-A -steroids of the C19, Cj, and C24 series. The results of some inhibition experiments indicated that there were different A -3-oxosteroid 5j8-reductases with different substrate specificities in the preparation. 

The microsomal 26-hydroxylase in rat liver has a higher substrate specificity than the mitochondrial. Of a number of C27-steroids, only 5j8-cholestane-3a,7a,12a-triol, 5)8-cholestane-3a,7a-diol, 7a-hydroxy-4-cholesten-3-one and 7a,12a-dihydroxy-4-cholesten-3-one were 26-hydroxylated to a significant extent [126]. In addition to hydroxylation in the 26 position, 5)8-cholestane-3a,7a,12a-triol was hydroxylated by the microsomal fraction of rat liver in the 23, 24 , 24/8 and 25 positions [40]. The hydroxylation in the 25 position was about as efficient as that in the 26 position. 

Bjorkhem and Einarsson showed that rat liver microsomes in the presence of NADPH are able to convert 7a-hydroxy-4-cholesten-3-one and 7a,12a-dihydroxy-4-cholesten-3-one into the corresponding 3-oxo-5a-steroids (Fig. 11) [168], Liver microsomes from female rats were 3-4 times more active than those from male rats. A similar sex difference was not found in human hver microsomes. The 3-oxo-5a-steroids were efficiently converted into alio bile acids in bile fistula rats. In the presence of both NADPH and NADH, the 3-oxo-5a-steroids were converted into the corresponding 3/3-hydroxy-5a-steroids. As mentioned above, the 3-oxo-5a-steroids are efficiently converted into the corresponding 3a-hydroxy-5a-steroids in the cytosol. 

The quantitative importance of the pathway involving 7a-hydroxy-4-cholesten-3-one and 7a,12a-dihydroxy-4-cholesten-3-one as intermediates in formation of alio bile acids is not known. At least in the rat, the capacity of the microsomal A" -3-oxosteroid 5a-reductase is high, and thus it is surprising that such small amounts of alio bile acids are formed under normal conditions in mammals. It seems probable that this enzyme is inhibited under in vivo conditions. Hoshita et al. found an efficient conversion of 7a,12a-dihydroxy-4-cholesten-3-one into 7a,12a-dihy-droxy-5a-cholestan-3-one in the microsomal fraction of liver from Iguana iguana [170], Alio bile acids are predominant in this species of iguana and it was suggested that the microsomal A -3-oxosteroid 5a-reductase is of major importance for their formation. 

Fig. 11. Sequence of reactions in the conversion of cholesterol into alio bile acids with 7a-hydroxy-4-cholesten-3-one and 7a,12a-dihydroxy-4-cholesten-3-one as intermediates.

The final ratio between cholic acid and chenodeoxycholic acid in bile is influenced also by factors other than the activity of the hepatic 12a-hydroxylase. Thus, the differential rates of enterohepatic cycling, intestinal absorption and degradation are of importance. Ahlberg et al. did not find a correlation between microsomal 12a-hydroxylase activity and the ratio between cholic acid and chenodeoxycholic acid in the bile of some normo- and hyperlipidaemic patients [253]. In a recent in vivo study, Bjorkhem et al. failed to show a correlation between the apparent 12a-hydroxylase activity and the ratio between biliary cholic and chenodeoxycholic acid in healthy subjects and a patient with liver cirrhosis [254]. In this study, a mixture of [ H]7a,12a-dihydroxy-4-cholesten-3-one and [ C]7a-hydroxy-4-choles-ten-3-one was administered intravenously and the relative 12a-hydroxylase activity was calculated from the ratio between and in cholic acid. 

Fig. 1. Conversion of cholesterol into 5i -cholestane-3a,7a,12a-triol. I, Cholesterol II, 5-cholestene-3i5,7a-diol III, 7a-hydroxy-4-cholesten-3-one IV, 5-cholestene-3/5,7a,12a-triol V, 7a,12a-dihydroxy-4-cholesten-3-one VI, 7a-hydroxy-5/5-cholestan-3-one VII, 7a,12a-dihydroxy-5/5-cholestan-3-one VIII, 5 -cholestane-3a,7a-dio IX, 5i -choIes-tane-3a7a, 12a-triol.

In the apparently major pathway for the conversion of cholesterol into 5 -cholestane-3a,7a,12a-triol, the step following the formation of 7a-hydroxy-4-cholesten-3-one is a 12a-hydroxylation yielding 7a,12a-dihydroxy-4-cholesten-3-one (Fig. 1). The reaction is catalyzed by the microsomal fraction fortified with NADPH (15,37). The conversion of 5-cholestene-3, 7a-diol into 5-cholestene-3/5,7a,12a-triol, which is a reaction in another pathway for the formation of 5/5-cholestane-3a,7a,12a-triol, is also catalyzed by the microsomal fraction fortified with NADPH (30,37), as is the 12a-hydroxylation of 5/5-cholestane-3a,7a-diol and 7a-hydroxy-5)5-cholestan-3-one (37). The rates of 12a-hydroxylation of these C27-steroids differ considerably the rate with 5-cholestene-3/5,7a-diol is about one-tenth and with 5 -cholestane-3a,7a-diol about half of that with 7a-hydroxy-4-cholesten-3-one (37). Einarsson (37) and Suzuki et al. (38) have studied some properties of the 12a-hydroxylase system with special reference to the possible participation of electron carriers such as NADPH-cytochrome c reductase and cytochrome P-450. The 12a-hydroxylation of 7a-hydroxy-4-cholesten-3-one was inhibited by cytochrome c, indicating that NADPH-cytochrome c reductase might be involved. However, no direct evidence for the participation of flavins was obtained. If NADPH-cytochrome c reductase participates, it is not rate-limiting, since the activity of this enzyme increases upon treatment with thyroxine whereas the activity of the 12a-hydroxylase decreases (39). Suzuki et al. (38) found no inhibition of 12a-hydroxylation by carbon monoxide, whereas Einarsson (37) obtained some inhibition. The 12a-hydroxylase activity was unaffected by methylcholanthrene treatment (40) and lowered by phenobarbital treatment (37,38). These observations indicate that the cytochrome(s) P-450 induced by methylcholanthrene and... 

Conversion of 7a,12a-Dihydroxy-4 cholesten-3-one into 5p-Cholestane-3a,7a,12a-triol... 

In the previous sections, the conversion of cholesterol into 5j -cholestane-3a,7a,12a-triol by means of the intermediary formation of 5-cholestene-3/5,7a-diol, 7a-hydroxy-4-cholesten-3-one, 7a,12a-dihydroxy-4-cholesten-3-one, and 7a,12a-dihydroxy-5i -cholestan-3-one has been discussed. Mention was made also of another pathway for the formation of 5i -cholestane-3a,7a,-12a-triol in which 5-cholestene-3i, 7a-diol is 12a-hydroxylated prior to oxidation of the 3 5-hydroxyl group. The importance of a pathway involving 5-cholestene-3i, 7a,12a-triol as an intermediate has not been conclusively established. It appears, however, that this pathway is less important, quantitatively, than the one involving 7a-hydroxy-4-cholesten-3-one as an intermediate. The main finding to support this contention is the difference in the rate of 12a-hydroxylation by the microsomal fraction fortified with NADPH between 7a-hydroxy-4-cholesten-3-one and 5-cholestene-3i, 7a-diol. As mentioned in section II A3, the rate of 12a-hydroxylation of 7a-hydroxy-4-cholesten-3-one is about ten times faster than that of 5-cholestene-3, 7a-diol (37). On the other hand, upon incubation of cholesterol with the 20,000g supernatant fluid, about equal amounts of 5-cholestene-3i, 7a,12a-triol and 7a,12a-dihydroxy-4-cholesten-3-one are formed (30). Since 5-cholestene-3j, 7a,12a-triol is converted efficiently into 7a,12a-dihydroxy-4-cholesten-3-one (30), part or all of the 7a,12a-dihydroxy-4-cholesten-3-one may have been formed by means of the intermediary formation of 5-cholestene-3), 7a,12a-triol. An interpretation of these contrasting findings could be that the 12a-hydroxylation of 5-cholestene-3i, 7a-diol requires not only the microsomal... 

In the next step, the conversion of 7a-hydroxy-4-cholestene-3-one into 5j8-cholestene-3a, 7a, 12a-triol involves the 12 hydroxylation of the substrate by a NADPH microsomal enzyme. The product of the reaction, the 7a, 12a-dihydroxy-4-cholestene-3-one, is converted into the 7a, 12a-dihydroxy-5j8-cholestene-3-one by a soluble A, 3-oxysteroid-5j8-reductase, and 3a-hydroxysteroid dehydrogenase yields the final intermediate 5j -cholestene-3a, 7a, 12a-triol. 

Although the major pathway for the formation of the cholestenetriol involved the 7a-hydroxy-4-choles-tene-3-one as an intermediate, a pathway in which a third hydroxylation precedes the oxidation of the 3j -hydroxyl group through the conversion of 5-cho-lestene-3j8, 7a-diol into 5-cholestene-3j8, 7a, 12a-triol has also been described. The reaction involves the same 12a-hydroxylase that is involved in the conversion of 7a-hydroxy-4-cholestene-3-one into 7a, 12a-dihydroxy-4-cholestene-3-one, but the hydroxylation is much more rapid with the latter substrate. 

Introduction of a 26-hydroxyl group almost completely prevents introduction of a 12 -hydroxyl group in rat liver. In contrast, there is an efficient conversion of 5)8-cholestane-3a,7a,26-triol and 7a,26-dihydroxy-4-cholesten-3-one into both cholic and chenodeoxychohc acid in human hver [180-182], From these findings it is possible to formulate a number of different pathways in the biosynthesis of cholic acid in human hver, with introduction of the 26- and the 12a-hydroxyl groups at different stages. 

The 3a-hydroxysteroid dehydrogenase preparation described by Berseus (42) has been found to catalyze the oxidation of 4-cholestene-3a,7a-diol into 7a-hydroxy-4-cholesten-3-one (52). It is interesting that the reverse reaction proceeds at a rate which is at least ten times slower. Similarly, the rate of oxidation of 5j -cholestane-3a,7a,12a-triol into 7a,12a-dihydroxy-5j -choles-tan-3-one, catalyzed by 3a-hydroxysteroid dehydrogenase, is about ten times slower than the rate of reduction (42). 

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