7a-Hydroxylation

Tetrahydropyranyl ethers have been prepared from the quasi-axial 7a-hydroxyl in a 3)5-acetoxy-A -7a-ol, but in this case enhanced reactivity is due to the adjacent double bond. °... 

CYP7A1 catalyzes the 7a-hydroxylation of cholesterol, the first and rate limiting step of bile acid synthesis. This is also the principal way to eliminate cholesterol. CYP7B1 is primarily expressed in brain and catalyzes the synthesis of various neurosteroids and also the 7a-hydroxylation of oxysterols. 

Bile acids are C24 acids that are biosynthetically derived from cholesterol. The 7a-hydroxylation of cho-... 

The second method of preparation (shown in Scheme 2) depends on treating dehydroepiandrosterone (prepared from cholestrol or sitosterol) with acetylene to form the 17a-ethnyl-17p-hydroxy derivative, which is carbonated to the 17a-propionic acid. Reduction of the unsaturated acid in alkaline solution yields the saturated acid, which cyclizes to the lactone on acidification. Bromination to the 5,6-dibromo-compound, followed by oxidation of the hydroxyl group to the ketone, and then dehydro-bromination to the 7a-hydroxyl derivative, produces spironolactone when esterified with thiolacetic acid. 

Other microorganisms which are known to 7a-hydroxylate various steroidal substrates are presented in Table 7. 

Angyal and Young report second-order kinetics for the oxidation of the camphane-2,3-diols. The cis- isomers are oxidised much more rapidly than the trans- isomers, and a temperature of 80 °C had to be used for kinetic measurements on the latter. It seems likely that the rigidity of the camphane skeleton prevents the formation of a cyclic diol-periodate ester from the trans- isomers. Possibly the reaction at 80° is completely different in nature from the normal oxidation of 1,2-diols by periodate. The same workers report that cholestane-3j8,6j8,7a-triol, in which the and 7a hydroxyl groups are axial-axial, is inert towards periodate. 

Storage of frozen rat liver microsomes for 20 or 48 days at - 15°C resulted in a progressive decrease in enzyme activity for the 6/3- and 16a-hydroxylation of testosterone, but la -hydroxylation activity was stable (36). The in vitro addition of 10-4 M chlorthion almost completely inhibited the 16a-hydroxylation of testosterone by rat liver microsomes but only inhibited the 6/3- and 7a-hydroxylation reaction by 31 and 14 percent, respectively (36,37). 

Treatment of immature male rats with phenobarbital for three days increased the 6 ft-, la-, and 16a-hydroxyl ati on of testosterone by liver microsomes to different degrees (36). The 16a-hydroxyl ati on reaction was stimulated several-fold, whereas the 6ft- and 7a-hydroxylations were stimulated to a smaller extent. In contrast to these results, the administration of 3-methylcholanthrene had little or no stimulatory effect on the 6ft- or 16a-hydroxylation of testosterone by liver microsomes but caused a significant increase in the 7 a-hydroxy 1 ati on reaction. 

Patients also develop cholesterol gallstones from a defect in bile acid synthesis. The defect is in the mitochondrial C27-steroid 27-hydroxylase. In these patients, the reduced formation of normal bile acids, particularly chenodeoxycholic acid, leads to the up-regulation of the rate limiting enzyme Tct-hydroxylase of the bile acid synthetic pathway (discussed later). This leads to accumulation of 7a-hydroxylated bile acid intermediates that are not normally utilized. 

Regulation of bile acid formation from cholesterol occurs at the 7a-hydroxylation step and is mediated by the concentration of bile acids in the enterohepatic circulation. 7a-Hydroxylase is modulated by a phosphorylation-dephosphorylation cascade similar to that of HMG-CoA reductase (Figure 19-11) except that the phosphorylated form of 7a-hydroxylase is more active. 

Secondary bile adds Products of deconjugated and reduced primary acids. Bacteria in the intestine remove the 7a-hydroxyl group, leaving the secondary bile acids. 

Deoxycholic acid, oxygenated in the presence of Fe sulphate and ascorbic acid in a phosphate buffer, afforded the 15a-hydroxy-derivative in low yield. 7a-Hydroxylation under similar conditions has been reported previously. ... 

From the above investigations, summarized in Fig. 2, it was concluded that 7a-hydroxylation of cholesterol may be the first step in the conversion of cholesterol into bile acids, and that 5/S-cholestane-3a,7a,12a-triol probably is an intermediate in cholic acid formation. Since 5)S-cholestane-3a,7a-diol was rapidly converted into chenodeoxycholic acid and only to a small part into cholic acid [19], it was concluded that 5i8-cholestane-3a,7a-diol is a corresponding intermediate in the formation of chenodeoxychohc acid. Samuelsson showed that the conversion of cholesterol into bile acids most probably involves a ketonic intermediate, since [3a- H]cholesterol lost its tritium when converted into chohc acid [1,20]. Since... 

Assay of the enzyme activity is complicated by the fact that 7a-hydroxylation of cholesterol also may occur due to auto-oxidation or secondary to lipid preoxidation. In addition it is difficult to equilibrate exogenous cholesterol with endogenous microsomal cholesterol. 

In the first studies on 7a-hydroxylation, the microsomal fraction together with the... 

Most studies on substrate specificity of cholesterol 7a-hydroxylase have been performed with intact microsomes. Results of such studies may be difficult to interpret since the enzyme system is embedded in a lipoprotein membrane, and may not be directly accessible to potential substrates [59]. Thus, differences in the rate of 7a-hydroxylation of various steroids could be due to differences in the rate at which the substrate reaches the active site of. the enzyme rather than to differences in the intrinsic ability of the enzyme to interact catalytically with the substrate [59], Further, occurrence of 7a-hydroxylation of a certain steroid may not reflect the substrate specificity of cholesterol 7a-hydroxylase activity since different species of cytochrome P-450 are present in the microsomes. 

Esters of cholesterol cannot be directly attacked by the enzyme [60,61]. Rat liver microsomes catalyse 7a-hydroxylation of cholestanol at a rate comparable to that of cholesterol [62]. Since 7a-hydroxylation of cholestanol is stimulated after treatment with cholestyramine, it appears likely that the same enzyme is involved as in hydroxylation of cholesterol. Shght changes in the side chain lead to marked loss of activity [63,64]. Loss of a terminal methyl group reduces the rate to about 50% and addition of an ethyl group at C-24 (sitosterol) leads to almost complete loss of the activity. From a study with a great number of structurally closely related steroids, Aringer concluded that cholesterol 7a-hydroxylase requires a rather flat steroid (A", A or 5a) and an equatorial or quasi-equatorial hydroxyl group at C-3 [65]. 

Mechanism of 7a-hydroxylation of cholesterol and experiments with purified enzyme components... 

Bjorkhem et al. were the first to demonstrate 7a-hydroxylation of cholesterol in a reconstituted system consisting of partially purified cytochrome P-450, NADPH-cytochrome P-450 reductase, NADPH and a synthetic phosphatidylcholine [81]. The latter component is known to facilitate transfer of electrons from NADPH-cytochrome P-450 reductase. In later experiments with highly purified components there was no need for addition of lipids [82]. The specificity of the hydroxylation was found to be dependent mainly upon the cytochrome P-450 fraction [81]. It was further concluded that the cytochrome P-450 component must be different from the bulk of cytochrome P-450 in the liver. This is in accord with the finding that biliary drainage, which increases the rate of 7a-hydroxylation of cholesterol severalfold, has no effect on other hydroxylations [44] and results in no change or a sHght reduction in the total cytochrome P-450 content. Treatment with phenobarbital which increases the rate of hydroxylation of a number of drugs and also increases the content... 

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