7a-Hydroxy-4-cholesten-3-one

In 3-oxosteroid A4-steroid 5)3-reductase deficiency, key intermediates for cholic and chenodeoxycholic synthesis, 7a-hydroxy-4-cholesten-3-one and 7a,12a-dihy-droxy-4-cholesten-3-one undergo side-chain oxidation and conjugation to produce... 

In work published in 1989, Hylemon et al. used cholesterol oxidase to convert 7a-hydroxycholesterol to 7a-hydroxy-4-cholesten-3-one. Cholesterol that remained was converted to 4-cholesten-3-one. 7/3-Cholesterol, which was added as an internal steroid recovery standard, was oxidized to 7/3-hydroxy-4-cholesten-3-one. These steroid products were analyzed by Qg reversed-phase chromatography on an Altex Ultrasil-ODS column (4.6 mm x 25 cm) using 70 30 (v/v) mixture of acetonitrile and methanol (Fig. 9.83). The eluate was monitored at 240 nm, and the amount of product determined from a calibration curve. 

Figure 9.83 Separation of 7a-hydroxy-4-cholesten-3-one (A), 7/3-hydroxy-4-cholesten-3-one (B), and 4-cholesten-3-one (C) by C-18 reveised-phase HPLC.

The second step in the synthesis of bile acids, according to Hylemon et al. (1991), is the conversion of 7a-hydroxycholesterol to 7a-hydroxy-4-cholesten-3-one by NAD+-dependent 3/3-hydroxy-A5-C27-steroid oxidoreductase. This enzyme is located in the endoplasmic reticulum of liver, and its catalysis of the 3/3-hydroxy group also results in isomerization of the double bond from A5 to A4. 

Axelson M, Bjorkhem I, Relhner E, Einarsson K. The plasma level of 7a-hydroxy-4-cholesten-3-one reflects the activity of hepatic cholesterol 7a-hydroxylase in man. Febs Lett 1991 284 216-8. 

Brydon WG, Nyhiin H, Eastwood MA, Merrick MV. Serum 7a-hydroxy-4-cholesten-3-one and selenoho-mocholyltaurine (SeHCAT) whole body retention in the assessment of bile acid induced diarrhoea. Eur J Gastroenterol Hepatol i996 8 117-23. 

Serum 7a-hydroxy-4-cholesten 3-one concentrations in the evaluation of bile acid malabsorption in patients with diarrhoea correlation to the SeHCAT test. Gut 1993 34 698-701. 

Sauter GH, Munzing W, von Ritter C, Pamngartner G. Bile acid malabsorption as a cause of chronic diarrhea diagnostic value of 7a-hydroxy-4-cholesten-3-one in serum. Dig Dis Sci 1999 44 14-9. 

Cholic acid differs from chenodeoxycholic acid in having an extra hydroxyl group at C-12. The enzyme responsible for producing this difference, 7a-hydroxy-4-cholesten-3-one 12a-hydroxylase, thus acts at a key branch point in the biosynthesis of bile acids and might be expected to be regulated in order to control the relative amounts of cholic acid and chenodeoxycholic acid produced. Like other hydroxylation steps in bile acid biosynthesis, 12a-hydroxylation requires a specific form of cytochrome P-450, which is present in the cytochrome P-45OLM4 fraction of rabbit liver microsomes (H6). The activity of I2a-hydroxylase has been postulated to be decreased in patients with liver cirrhosis to explain the low proportion of cholic add relative to chenodeoxycholic add in the bile of these patients (V9). Conversely, the activity of this enzyme may be high in patients with cerebrotendinous xanthomatosis, as the bile of these individuals contains mostly cholic acid... 

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... 

The above investigations led to the formulation of the sequence of reactions shown in Fig. 3 for the nuclear changes in the conversion of cholesterol into cholic acid and chenodeoxycholic acid. In this scheme, the 12a-hydroxyl group is introduced at the stage of 7a-hydroxy-4-cholesten-3-one. It was shown that also 7a-hy-droxycholesterol could be 12a-hydroxylated by the microsomal fraction of a liver homogenate [31]. Since that hydroxylation occurred at a much lower rate, it was believed to represent a minor pathway [32]. The conversions shown in Fig. 3 were later also demonstrated in human Uver [33]. 

The idea that only one enzyme is involved in the conversion of 7a-hydroxy-cholesterol into 7a-hydroxy-4-cholesten-3-one was recently supported by an investigation by Wikvall [97]. He solubilized the enzyme activity from rabbit liver micro-somes by treatment with a mixture of sodium cholate and the non-ionic detergent Renex 690. The enzyme was purified about 200-fold by different chromatographic steps. The purified enzyme showed only one protein band with an apparent molecular weight of 46 000. Whereas the microsomal fraction had a broad substrate... 

In most studies on 12 -hydroxylation, labelled 7a-hydroxy-4-cholesten-3-one has been used as the substrate [32], and it is believed to be the most important substrate also in vivo. It cannot be excluded that 5j8-cholestane-3a,7 -diol as well as 7a-hy-droxycholesterol are substrates for the 12a-hydroxylase in minor pathways in vivo. 

In a study by Ali and Elliott it was shown that 5a-cholestane-3 ,7a-diol was an even better substrate for the 12a-hydroxylase in rabbit liver microsomes than 7a-hydroxy-4-cholesten-3-one (156%) [104]. This reaction is probably of importance in the formation of allocholic add. The high specificity of the 12 -hydroxylase towards the coplanar 5a-sterol nucleus is also evident from the finding that allochenodeoxycholic acid can be converted into allocholic acid in rats, both in vivo and in vitro [105,106, Chapter 11]. Based on the known structural requirements of the 12a-hydroxylase, Shaw and Elliott prepared competitive inhibitors with different substitutions in the C,2 position [107]. The best inhibitor of those tested was found to be 5a-cholest-ll-ene-3a,7 ,26-triol. Theoretically, such inhibitors may be used to increase the endogenous formation of chenodeoxycholic acid in connection with dissolution of gallstones. 

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. 

The mitochondrial enzyme has a broad substrate specificity and catalyses 26-hydroxylation of a number of C27-steroids. The most important substrates in vivo are believed to be 5)8-cholestane-3a,7a-diol, 7a-hydroxy-4-cholesten-3-one and 5j8-cholestane-3a,7a,12a-triol. Bjorkhem and Gustafsson found that 5j8-cholestane-3a,7a,12a-triol and 7a-hydroxy-4-cholesten-3-one were the best substrates in rat liver mitochondria and that the least efficient 26-hydroxylation occurred with cholesterol as substrate [126,130]. There was also a small extent of 25-hydroxylation of cholesterol in the mitochondrial fraction [130]. The major part of the 26-hydroxylase is bound to the inner mitochondrial membranes [130,131]. Thus the hydroxylase activity is low with intact mitochondria and NADPH as cofactor. Under such conditions citric acid and isodtric acid, which are able to penetrate the inner mitochondrial membrane, stimulate 26-hydroxylation much more efficiently than NADPH [130,131]. It is evident that citric acid and isocitric acid generate NADPH inside the mitochondrial membrane. When using leaking mitochondria, NADPH stimulates the reaction about as efficiently as isocitrate [130,131]. 

The mitochondrial 26-hydroxylase is inhibited by biliary drainage and is not influenced by starvation or treatment with phenobarbital [126]. Gustafsson reported that the mitochondrial 26-hydroxylation of cholesterol, 7a-hydroxycholesterol and 7a-hydroxy-4-cholesten-3-one was stimulated whereas 26-hydroxylation of 5)8-cholestane-3a,7a,12a-triol was inhibited by biliary obstruction [127]. Whether the... 

Conversion of 7a-hydroxy-4-cholesten-3-one into alio bile acids... 

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. 

In the microsomal 5 a saturation of the double bond in 7a-hydroxy-4-cholesten-3-one there is a stereospecific transfer of a hydride ion from the 4B position of NADPH to the 5 a position of the steroid [169]. From the results of experiments on the stereochemistry of the addition of the proton to the 4 position, it was concluded that the reduction of the double bond is likely to involve a non-stereospecific addition of hydrogens or a cis addition rather than a trans addition. 

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.

Studies on patients with the rare inborn disease cerebrotendinous xanthomatosis provide unique possibilities to evaluate the relative importance of different intermediates as substrates for the mitochondrial 26-hydroxylase. These patients appear to have a complete lack of mitochondrial 26-hydroxylase activity and may thus be expected to accumulate the normal substrates for the 26-hydroxylase in their livers. According to an investigation by Bjbrkhem et al., in which the level of different intermediates in a hver biopsy was determined by isotope dilution-mass spectrometry, 7a-hydroxy-4-cholesten-3-one and 5 3-cholestane-3a,7a,12a-triol seem to be the most important normal substrates for the 26-hydroxylase also in man [183]. 

The microsomal 12a-hydroxylase seems to be influenced by the flux of bile acids through the liver in a similar way as cholesterol 7a-hydroxylase. Biliary drainage in rats leads to a 2-fold stimulation of 12a-hydroxylase [44] perhaps due to reduced intake of food [252]. However, it was shown later that 12a-hydroxylation of 7a-hydroxy-4-cholesten-3-one was inhibited by feeding rats different taurine-conjugated bile acids at the 1% level [110]. Ahlberg et al. showed that the microsomal 12a-hydroxylase in human liver was inhibited by about 50% after treatment for 8 weeks with chenodeoxycholic acid, 15 mg/kg body weight [HI]. The increased ratio between cholic acid and chenodeoxycholic acid observed after treatment with cholestyramine is also consistent with an inhibitory effect of reabsorbed bile acids on the 12a-hydroxylase [219]. 

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.

Conversion of 5-Cholestene-S, 7a-diol into 7a-Hydroxy 4 cholesten-3-one... 

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... 

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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