12a-Hydroxylase

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... 

S2). More recent studies have shown that patients with cirrhosis are able to efficiently convert 7a-hydroxy-cholesterol into cholic acid (G8, P8), suggesting that 12a-hydroxylase activity is near normal. Other evidence from in vivo studies in man with labeled preciusors suggests that 12a-hydroxylase activity is not important in the regulation of the ratio between cholic acid and chenodeoxycholic add in human bile (B21). The possibility that different pools of cholesterol are utilized for the biosynthesis of cholic acid and chenodeoxycholic acid is now being investigated. 

If the cholesterol 7a-hydroxylating system is of regulatory importance, a short half-life of the enzyme can be expected. Already in 1968 Einarsson and Johansson obtained evidence that this is the case [87]. It was calculated that the half-life for the breakdown of the 7a-hydroxylase was only 2-3 h. Other enzyme activities involved in the biosynthesis of bile acids from cholesterol, such as the 12a-hydroxylase, had a considerably longer half-life time. It seems likely that a cytochrome P-450 species with short half-life time is the component of the cholesterol 7a-hydroxylase. This... 

Also the microsomal 12a-hydroxylase is dependent upon cytochrome P-450. In early work, the involvement of cytochrome P-450 was questioned, due to the relatively low sensitivity towards carbon monoxide of this hydroxylation [32,99]. Conclusive evidence that the 12 -hydroxylation involves participation of cytochrome P-450 was obtained by Bemhardsson et al., who showed that the hydroxylation could occur in a reconstituted system consisting of a crude fraction of cytochrome P-450 from rat Uver, NADPH-cytochrome P-450 reductase and a phosphohpid [100]. The specificity depended upon the cytochrome P-450 component. Starvation is known to stimulate 12a-hydroxylation, and use of cytochrome P-450 from starved rats led to increased rate of 12a-hydroxylation. 

The cytochrome P-450 component in the 12a-hydroxylase is more sensitive to high ionic strength than most other species of cytochrome P-450 [77]. There is no isotope effect in the 12 -hydroxylation of a 12a- H-labelled substrate [102], showing that cleavage of the C-H bond in the ternary complex is not rate limiting. 

Recently, Danielsson et al. reported that the activity of a purified 12a-hydroxylat-ing system from rabbit liver microsomes could be modulated by protein fractions from rabbit hver microsomes and cytosol [103]. The microsomal protein fraction had a stimulatory effect whereas the cytosolic protein was inhibitory. Addition of ATP and MgCl2 or NaF had no effect on the activities of the two protein fractions, indicating that phosphorylation-dephosphorylation was of little or no importance. The microsomal 12a-hydroxylase stimulator also stimulated cholesterol 7a-hydroxyl-ase activity. 

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. 

The activity of the 12a-hydroxylase in rat liver is decreased by treatment with thyroid hormone and increased after thyroidectomy [108,109]. The activity also seems to be inhibited by feeding bile acids [110,111]. 

Oftebro et al. reported that the mitochondrial fraction of a liver homogenate from a biopsy of a CTX patient was completely devoid of 26-hydroxylase activity [193]. The possibility that there had been a general inactivation of the mitochondrial fraction seems excluded since there was a significant 25-hydroxylase activity towards vitamin D,. There was a substantial accumulation of 5 -cholestane-3a,7a,12a-triol, the immediate substrate for the 26-hydroxylase in cholic acid biosynthesis. It was suggested that the accumulation of 5)3-cholestane-3a,7a,12a-triol would lead to increased exposure to the action of the microsomal 23-, 24- and 25-hydroxylases. The alternative 25-hydroxylase pathway would then be of importance for the formation of cholic acid in patients with CTX (Fig. 13). If the 25-hydroxylase pathway has an insufficient capacity, this would explain the accumulation of the different 25-hydroxylated intermediates in patients with CTX. A lack of the mitochondrial 26-hydroxylase would also lead to accumulation of intermediates in chenodeoxycholic acid biosynthesis such as 5)8-cholestane-3a,7a-diol and 7a-hy-droxy-4-cholesten-3-one. Such accumulation would lead to increased exposure to the microsomal 12a-hydroxylase which would yield a relatively higher biosynthesis of cholic acid. This would explain the marked reduction in the biosynthesis of chenodeoxycholic acid in patients with CTX. 

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]. 

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. 2. Composite of GA metabolic pathways in higher plants. Reactions arc catalysed by i, 7-oxidase ii, 13-hydroxylase iii, 20-oxidase iv, 33-hydroxylase v, 23-hydroxylase vi, GA,/GA7-forming enzyme vii, 23, 33-dihydroxylase (in pumpkin endosperm) viii, 12a-hydroxylase. Bold arrows depict major pathways in shoots.

Structure and chromosomal assignment of the sterol 12a-hydroxylase gene (CYP8B1) in... 

Zhang, M. and J.Y. Chiang (2001). Transcriptional regulation of the human sterol 12a-hydroxylase gene (CYP8BI) Roles of hepatocyte nuclear factor 4a in mediating bile acid repression. J. Biol. Chem. 276, 41690 1699. 

Yang, Y, M. Zhang, G. Eggertsen, and JY. Chiang (2002). On the mechanism of bile acid inhibition of rat sterol 12a-hydroxylase gene CYP8BI) transcription Roles of a-fetoprotein transcription factor and hepatocyte nuclear factor 4a. Biochim. Biophys. Acta 1583, 63-73. 

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 observations upon which the pathways in Fig. 2 are based are consistent with the view (14) that oxidation of the cholesterol side chain inhibits 12a-hydroxylation. The 12a-hydroxylase enzyme (or enzymes) ap-... 

Li Y, Mezei O, Shay NF (2007) Human and murine hepatic sterol-12a-hydroxylase and other xe-nobiotic metabolism mRNA are upiegulated by soy isoflavones. JNutr 137 1705-1712... 

Jahan A, Chiang JY (2005) Cytokine regulation of human sterol 12a-hydroxylase (CYP8B1) gene. Am J Physiol Gastrointest Liver Physiol 288 G685-695... 

Abrahamsson A, Gafvels M, Reihner E, Bjorkhem I, Einarsson C, Eggertsen G (2005) Polymorphism in the coding part of the sterol 12a-hydroxylase gene does not explain the marked differences in the ratio of eholic aeid and ehenodeoxycholic acid in human bile. Scand J Clin Lab Invest 65 595-600... 

Einarsson K, Akerlund JE, Reihner E, Bjorkhem I (1992) 12a-hydroxylase activity in human liver and its relation to cholesterol 7a-hydroxylase activity. J Lipid Res 33 1591-1595... 

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. 

The size of the bile acid pool in the rat is about 14—20 mg, but may vary with the diet, being lower when semi-synthetic diets are fed (Eriksson, 1960 PoRTMAN and Murphy, 1958). The half life of cholic and chenodeoxycholic acids is 2—3 days in normal rats (Lindstedt and Norman, 1956), but is much higher in the absence of intestinal microorganisms (Lindstedt and Norman, 1956 Gustafsson et al., 1957). The intestinal flora hydrolyze the circulating bile salts and, normally, only free bile acids are found in the feces. Thyroid hormone exerts a marked effect on bile acid metabolism. In the euthyroid rat the cholic chenodeoxy cholic ratio is 4 1, but this is almost reversed in the hyperthyroid rat (Eriksson, 1957). The thyroid effect appears to be an inhibition of the 12a-hydroxylase, since the extent of side chain oxidation of cholesterol-26-is the same in eu-, hyper-, or hypothyroid rats (Kritchevsky et al., 1962). 

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