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

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. 

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

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.

Figure 9.84 Separation of 7a-hydroxy-4-cholesten-3-one (A) from 4-cholesten-3-one (B) by Ci8 reversed-phase HPLC. Microsomes (1 mg) prepared from cholestyramine-fed rats. (From Hylemon et al., 1991.)

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. 

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 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. 

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. 

There was a significant isotope effect in the conversion of [3a- H]7a-hydroxy-cholesterol into 7 -hydroxy-4-cholesten-3-one, indicating that abstraction of a hydride ion from the 3a position may be rate limiting in the reaction. 

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.

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. 

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. 

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 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 

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. 

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. 

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. 

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 

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]. 

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 

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 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. 

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