Chenodeoxycholic acid 6/3-hydroxylation

Hydroxylation, shortening of the hydrocarbon chain, and addition of a carboxyl group convert cholesterol in a complex series of reactions to the bile acids, cholic acid, and chenodeoxycholic acid. 

Primary BAs, cholic acid (CA), and chenodeoxycholic acid (CDCA), are synthesised via the 5/3-saturation of the cholesterol double bond by enzymes of the hepa-tocyte microsomal fraction, epimerisation of the 3/j-hydroxyl group to the 3a-con-figuration, and further insertion of a 7 -hydroxyl group, with or without a further 12a-hydroxyl group. After shortening of the side chain by three carbons, oxidation of the terminal carbon of the side chain occurs to form the carboxylic group [3]. Alternative metabolic sequences add to the complexity of this metabolic pathway (Fig. 5.4.2). 

Bacteria in the intestine can remove glycine and taurine from bile salts, regenerating bile acids. They can also convert some of the primary bile acids into "secondary" bile acids by removing a hydroxyl group, producing deoxycholic acid from cholic acid and lithocholic acid from chenodeoxycholic acid (Figure 18.11). 

The C>4 bile acids arise from cholesterol in the liver after saturation of the steroid nucleus and reduction in length of the side chain to a 5-carbon add they may differ in the number of hydroxyl groups on the sterol nucleus. The four acids isolated from human bile include cholic acid (3,7,12-tiihydroxy), as shown in Fig. 1 deoxycholic acid (2,12-dihydroxy) chenodeoxycholic acid (3,7-dihydroxy) and lithocholic acid (3-hydroxy). The bile acids are not excreted into the bile as such, but are conjugated through the C24 carboxylic add with glycine or... 

Scalia and Games developed a packed column SFC method for the analysis of free bile acids cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholic acid (LCA), and ursodeoxycholic acid (UDCA) [32]. The baseline separation of all five bile acids was achieved on a packed phenyl column with a methanol-modified carbon dioxide in less than 4 min. The elution order showed a normal-phase mechanism because the solutes eluted in the order of increasing polarity following the number of hydroxyl groups on the steroid nucleus. The method was also applied to the assay of UDCA and CDCA in capsule and tablet formulations. The method was found to be linear in the range 1.5-7.5 ng/ml (r > 0.99, n = 6). The average recoveries (n= 10) for UDCA and CDCA were 100.2% with a RSD of 1.7% and 101.5% with a RSD of 2.2%, respectively. The reproducibility of the method was less than 1.5% (n = 10) for both UDCA and CDCA. 

The bile acids are produced in the liver by the metabolism of cholesterol. They are di- and trihydroxylated steroids with 24 C atoms. The structure of cholic acid was seen earlier (Sec. 6.6). Deoxycholic acid and chenodeoxycholic acid are two other bile acids. In the bile acids, all the hydroxyl groups have an a orientation, while the two methyl groups are /3. Thus, one side of the molecule is more polar than the other. However, the molecules are not planar but bent because of the cis conformation of the A and B rings. 

The conversion of cholesterol to bile salts begins when hydroxyl groups are introduced into the phenanthrene ring of cholesterol by the action of cholesterol 7-a-hydroxylase, followed by modification of the side chain. Cholic acid and chenodeoxycholic acid are produced, as shown in Fig. 13-24. 

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. 

Bile acids contain hydroxyl groups, which are usually substituted at positions, C-3, C-7, or C-12 of the steroid nucleus. The three major bile acids found in man are 3a,7a,12a-trihydroxy-5P-cholan-24-oic acid 3a,7a-dihydroxy-5p-cholan-24-oic add and 3a,12a-dihydroxy-5p-cholan-24-oic acid. Because of the complexities of steroid nomenclature, bile acids are nearly always referred to by trivial names. 11108, the three major human bile acids are named cholic acid, chenodeoxycholic acid, and deoxycholic acid, respectively, and their chemical structures are shown in Fig. 1. Human bile does, however, contain small amounts of other bile acids, such as lithocholic acid (3a-hydroxy-5P-cholan-24-oic add) and ursodeoxycholic add (3a,7p-dihydroxy-5p-cholan-24-oic acid) (see Fig. 1). 

The bile acids cholic acid and chenodeoxycholic acid are synthesized from cholesterol in the liver (Dl, S3). Several structural modifications are necessary to convert cholesterol, with its 27 carbon atoms, C-5,6 double bond and 3p-hydroxyl group, to a 24-carbon atom, saturated, 3,7 and 12a-hydroxyl-ated bile acid. The major reactions in this transformation are shown in Figs. 3 and 4. The reactions are catalyzed by mitochondrial, microsomal, soluble, and possibly peroxisomal enzymes. 

The exact contributions of these alternate pathways to total hepatic bile acid synthesis in normal subjects is not certain, although 26-hydroxylation is usually regarded as the major pathway. In addition, it should be pointed out that current views of hepatic cholic acid and chenodeoxycholic acid synthesis are based primarily oh studies carried out in the rat. More recent studies, which have involved the administration of labeled bile acid intermediates to patients, have suggested that bile acid biosynthesis is more complex than previously thought and that multiple pathways exist to convert cholesterol to bile acids (Vll). 

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... 

Recent investigations into the mechanism of action of these bile acids indicate that ursodeoxycholic acid has certain advantages over chenodeoxycholic acid in the context of the overall homeostasis of cholesterol metabolism (F6). In contrast to chenodeoxycholic acid, ursodeoxycholic acid does not suppress bile acid synthesis (H7), possibly because the a-orientation of the 7-hydroxyl group of chenodeoxycholic acid is required to inhibit cholesterol 7a-hydroxylase activity. Thus, cholesterol breakdown into bile acids is not reduced by ursodeoxycholic acid. Other favorable factors are that ursodeoxycholic acid has a reduced capacity to solubilize cholesterol into micellar solution compared to chenodeoxycholic acid and intestinal cholesterol absorption is decreased by this bile acid (F6, H7). However, in gallbladder bile the relative limitation of ursodeoxycholic acid for micellar solubilization of cholesterol is compensated for by an ability to transport... 

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... 

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 properties of the ihicrosomal 26-hydroxylase seem to differ from those of the other microsomal side-chain hydroxylations. Treatment with phenobarbital had little or no influence on 26-hydroxylation of 5yS-cholestane-3a,7a,12a-triol but stimulated the other hydroxylations up to 8-fold [126]. Thyroid hormone stimulated the microsomal 26-hydroxylase, which might be of importance for the regulation of the ratio between cholic and chenodeoxycholic acid in the rat (cf. below). Biliary obstruction inhibited [127] whereas biliary drainage and starvation have little or no effect [126]. 

From in vitro experiments it may be concluded that there are several possible pathways for formation of chenodeoxycholic acid in rat liver. A number of different 26-hydroxylated steroids are converted into chenodeoxycholic acid in bile fistula... 

Salen et al. reported that liver microsomes from 2 patients with CTX had a decreased capacity to 24/8-hydroxylate 5)8-cholestane-3a,7a,12a,25-tetrol [185]. It was suggested that the basic metabolic defect is a relative deficiency of the 24j8-hy-droxylase. To explain the severe metabolic consequences of such a defect, it must be assumed that the 25-hydroxylase pathway is the major pathway in the biosynthesis of cholic acid. This hypothesis does not explain the marked reduction in the biosynthesis of chenodeoxycholic acid. In view of the very low activity of the microsomal 25-hydroxylase towards 5)3-cholestane-3a,7a-diol in human hver [41] it is evident that a 25-hydroxylase pathway cannot be of importance in the normal... 

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 regulation of the overall biosynthesis of bile acids has been studied intensively during the last decade, and only a small fraction of all the pubhcations can be reviewed here. Cholesterol 7a-hydroxylase is the rate-limiting enzyme in the biosynthesis of both chohc acid and chenodeoxycholic acid. The publications in which a correlation has been demonstrated between bile acid biosynthesis and 7a-hydroxyl-ation of cholesterol have been reviewed by Myant and Mitropoulos [59]. In the present review, emphasis will be put on the feedback regulation of the cholesterol 7a-hydroxylase by the bile-acid flux through the hver, the relation between HMG-CoA reductase and cholesterol 7 -hydroxylase and possible mechanisms for the regulation. 

Microsomal 12a-hydroxylation is the only unique step in the formation of chohc acid and is likely to be of regulatory importance for the ratio between newly synthesized cholic and chenodeoxycholic acid. Introduction of a 26-hydroxyl group seems to prevent subsequent 12a-hydroxylation in rat liver and the 26-hydroxylase could thus also have a regulatory role. It is possible that there are different precursor pools for the synthesis of cholic acid and chenodeoxycholic acid in rats. If so, the relative size of the two pools could be of importance for the relative rate of formation of the two bile acids. 

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]. 

Mitropoulos et al. have measured the rate of excretion and the specific activities of cholic acid and chenodeoxycholic acid in bile fistula rats fed [ H]cholesterol and infused with [ " C]mevalonate or [ C]7a-hydroxycholesterol [255]. It was concluded that newly synthesized hepatic cholesterol was the preferred substrate for the formation of cholic acid. It could not be excluded, however, that part of the chenodeoxycholic acid had been formed from a pool of cholesterol different from that utilized in cholic acid biosynthesis. The mitochondrial pathway, starting with a 26-hydroxylation, could have accounted for a significant fraction of the chenodeo-... 

The major pathway for the formation of chenodeoxycholic add is thought to be the same as that for cholic acid, with the exception that no 12a-hydroxylation occurs. Thus, 3a,7a-dihydroxy-5i8-cholestan-26-oic acid is a probable intermediate, and Hanson has shown that this acid can be made from cholesterol and is effidently converted to chenodeoxycholic acid but only to a very limited extent to cholic acid [131]. 

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