Formation of Chenodeoxycholic Acid

The structural changes involved in the conversion of cholesterol into chenodeoxycholic acid are the same as those in the formation of cholic acid with the exception that no 12a-hydroxyl group is introduced. It has been shown that the mechanisms of conversion of the zl -3i5-hydroxy configuration of cholesterol into the 3a,7a-dihydroxy-5/5 configuration of chenodeoxycholic acid are the same as those in the formation of cholic acid. Similarly, the mechanisms of oxidation of the side chain are the same for chenodeoxycholic acid and cholic acid. Whereas it is now possible to formulate a few probable sequences for these events in cholic acid formation, available information 

In the early studies of the metabolism of cholesterol and other C27-steroids in the presence of mitochondrial preparations from rat and mouse liver, several metabolites were isolated that could be intermediates in the biosynthesis of chenodeoxycholic acid (11). Cholesterol, 5-cholestene-3j, 7a-diol, 7a-hydroxy-4 Cholesten-3-one, and 5/5-cholestane-3a,7a-diol were found to be converted into the corresponding 26-hydroxy derivatives by mitochondrial preparations. In addition, these preparations were shown to catalyze the 7a-hydroxylation of 5-cholestene-3/9,26-diol and the oxidation of 5-cholestene-3i, 7a-diol into 7a-hydroxy-4-cholesten-3-one. All the 26-hydroxy compounds were found to be transformed predominantly into chenodeoxy- 

With respect to the oxidation of the side chain in chenodeoxycholic acid formation, it may be inferred from the early studies with mitochondrial preparations that it involves an co-oxidation followed by a / -oxidation (cf. Section IIB). More direct evidence has been presented by Dean and White-house (87,91), who showed that mitochondrial preparations from rat liver catalyze the oxidation of 5-cholestene-3/ ,26-diol into 3/ -hydroxy-5-choles-tenoic acid and the formation of propionic acid from 3/5-hydroxy-5-choles-tenoic acid. Mitropoulos and Myant (97) have shown that mitochondrial preparations from rat liver catalyze the conversion of cholesterol into 5-cholestene-3/ ,26-diol, 3/ -hydroxy-5-cholestenoic acid, 3/5-hydroxy-5-chole-noic acid, lithocholic acid, and chenodeoxycholic acid (Fig. 5). Additional evidence for a pathway to chenodeoxycholic acid involving the successive, intermediary formation of above-mentioned compounds is provided by the finding that 3/ -hydroxy-5-cholenoic acid is converted into lithocholic acid and chenodeoxycholic acid by mitochondrial preparations (98). 

The importance, quantitatively, of the different pathways for chenodeoxycholic acid formation has not been fully established. Several lines of evi- 

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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. 

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... 

Yamasaki et al. have studied a pathway for formation of chenodeoxycholic acid in the rat involving intermediate formation of 7a-hydroxycholesterol, 3 -hydroxy-5-cholenoic acid and 7a-hydroxy-3-oxo-4-cholenoic acid [176-179]. In this pathway, changes in the side chain occur after the rate-limiting step. From the data available, it is not possible to evaluate the quantitative importance of this pathway. 

Yamasaki and his co-workers have proposed an alternative pathway to chenodeoxycholic acid via the side chain degradation of cholest-5-ene-3/S,7a-diol to form 3jS,7a-dihydroxychol-5-en-24-oic acid [119]. The formation of chenodeoxycholic acid from 3jS,7a-dihydroxychol-5-en-24-oic acid was demonstrated in rats [146] and hamsters [147]. 

Fig. 3. Formation of chenodeoxycholic acid some reactions catalyzed by mitochondrial preparations. I, Cholesterol II, 5-cholestene-3/9,7a-diol III,7a-hydroxy-4-cholesten-3-one VIII, 5i -cholestane-3a,7a-diol XVI, 5-cholestene-3i3,26-dioI XVII, 5-cholestene-3)3,7a,26-trioI XVIII, 7a,26-dihydroxy-4-choIesten-3-one XIX, 5 -cholestane-3a,7a,26-triol XX, chenodeoxycholic acid.

Section III In agreement with the suggested major pathway for formation of chenodeoxycholic acid, Hanson (192) has found that in man cholesterol is converted into 3a,7a-dihydroxy-5)5-cholestanoic acid, which in turn is metabolized predominantly into chenodeoxycholic acid. Yamasaki and collaborators (193-196) have provided evidence for the presence in rat of a pathway to chenodeoxycholic acid involving the conversion of 5-cholestene-3)5,7a-diol into 3/5,7a-dihydroxy-5-cholenoic acid. 

Support for the concept of an unsaturated intermediate in the formation of allo-acids is provided by recent experiments of Yamasaki et at. (98, 89). After administration of 3-ketochol-4-enoic-24- - C acid to rats and examination of the biliary metabolites, all four isomers of 3-hydroxycholanoic acid were identified other di- and trihydroxy acids were not investigated. Of the four possible 3-hydroxy-isomers about twice as much lithocholate was present as each of the other isomers. Similar results were obtained following administration of 3/3-acetoxychol-5-enoic-24-i- C acid in addition, 3f,6 -dihydroxy-5a-cholanoic acids were obtained. Yamasaki et al. (89) propose that a 3/3-dehydrogenase converts the 3/3-hydroxy-J -cholenoic acid to the a,/3-unsaturated ketone from which both 5 and 5 acids are derived, whereas hydroxylation of the above acid provides the diol from which only 5 acids are produced, somewhat analogous to the scheme of metabolism proposed by Mitropoulos and Myant (132) for the formation of chenodeoxycholic acid and the muricholic acids. 

F7. Fromm, H., Sarva, R. P., and Bazzoli, F., Formation of ursodeoxycholic acid from chenodeoxycholic acid in the human colon studies of the role of 7-ketolithocholic acid as an intermediate. /. Lipid Res. 24, 841-853 (1983). 

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... 

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. 

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. 

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]. 

Use A low conversion rate of cholesterol to B. in the liver leads to an oversaturation of bile with cholesterol and can result in the formation of cholesterol gallstones. Oral administration of a large amount of chenodeoxycholic acid can dissolve small gallstones. However, the 7a-dehydroxylation of the administered exogenous chenodeoxycholic acid by intestinal bacteria can result in the formation of high concentrations of toxic lithocholic acid (risk of development of liver and bile cirrhosis). 

Fig. 5. Conversion of cholesterol into chenodeoxycholic acid by means of the intermediary formation of lithocholic acid. I, Cholesterol XVI, 5-cholestene-3ft26-diol XX, chenodeoxycholic acid XXI, 3j5-hydroxy-5-cholestenoic acid XXII, 3/ -hydroxy-5-cholenoic acid XXIII, lithocholic acid.

Chenodeoxycholic acid is converted into a-muricholic acid (3a,6i8,7a-trihydroxy-5jS-cholanoic acid) and j8-muricholic acid (3a,6)J,7/J-trihydroxy-5j8-cholanoic acid) in the mouse and the rat and probably also in man (68, 102, Chapter 11 in this volume). a-Muricholic acid is a precursor of jS-muri-cholic acid in a reaction involving the intermediary formation of the 7-oxo compound (Chapter 11 in this volume). In the rat, /8-muricholic acid has been shown to be formed also from 3a,7j8-dihydroxy-5i8-cholanoic acid, which is a minor metabolite of chenodeoxycholic acid, and from 3a,6)9-dihydroxy-5)5-cholanoic acid, which is a metabolite of lithocholic acid (Chapter 11 in this volume). The microsomal 6i8-hydroxylase system in rat liver catalyzing the conversion of (tauro)chenodeoxycholic ac d into (tauro)a-muricholic acid has been studied by Hsia and collaborators (103-105), who... 

Most bile salts excreted in the feces are of the secondary type. Their formation is discussed in Section VII. The daily fecal excretion of bile salts in healthy subjects is highly variable and easily influenced by dietary alterations. Values from several studies are given in Table VIII. Bile salts virtually disappear from the stools during prolonged fasting, and turnover nearly ceases (19). Primary bile salts appear in the stools of patients with diarrhea (1). Patients taking cholestyramine excrete the usual pattern of secondary bile salts (57), so that apparently bacterial dehydroxylation of bile salts can occur in the presence of this resin. Patients with total external bile fistulas have no bile salts in the feces (2) this does not exclude transintestinal excretion of bile salts but makes it unlikely. As mentioned earlier, the predominance of chenodeoxycholic acid in blood and bile is often reflected in a predominance of lithocholate over deoxycholate in the feces (27). 

It seems to be a general observation that the proportion of chenodeoxy-cholic acid is increased in liver cirrhosis. Thus the ratio cholic acid/cheno-deoxycholic acid has been found to be decreased in the bile (23), serum (52,134,193,195-198), and urine (88,199) of cirrhotic subjects. Since the ratios of cholic acid, chenodeoxycholic acid, and deoxycholic acid appear to be approximately the same in bile and serum (200,201), and perhaps also in urine, it seems quite obvious that the bile acid pattern in any of these three sources is similar to that produced by the liver. Simultaneous determinations of bile acids from bile, serum, and urine have not been made, however. The relative increase of chenodeoxycholic acid has been interpreted to indicate a hindrance of 12a-hydroxylation in liver injury when the formation of cholic acid is decreased in favor of chenodeoxycholic acid (202). This, on the other hand, changes the pattern of secondary bile acids so that relatively more lithocholic acid is formed in the colon (191,200,202), the amount of deoxycholic acid being reduced (23,52,134,193,195-198), particularly because quantitatively only a small portion of the bile acids escapes daily from the ileum to the colon (23). 

Lithocholic acid has been associated with the development and progression of human liver cirrhosis (188,202). This acid is found in human serum, particularly in cirrhotic patients (202,210), in whom chenodeoxycholic acid is the predominant bile acid. Serum lithocholic acid is decreased by cholestyramine and neomycin in cirrhosis, and it has been suggested that the treatment of cirrhotic patients with these drugs warrants consideration (202). Usually, however, the correlation between the levels of lithocholic acid and its precursor chenodeoxycholic acid is poor (188,193). Long-term treatment of patients with lithogenic bile with chenodeoxycholic acid led to an almost complete predominance of this bile acid in bile, yet the amount of lithocholic acid was not increased significantly (96). Predominance of chenodeoxycholic acid appears to be related to the parenchymal cell function (195) the poorer it is the more predominant is chenodeoxycholic acid among the bile acids. However, a simultaneous decrease of hepatic secretory function possibly associated with intrahepatic biliary obstruction reduces the quantitative flow of chenodeoxycholate to the colon, so that bacterial formation and... 

Early in vitro studies showed that mitochondria from livers of hyperthyroid rats did not oxidize cholesterol-26- C to C02 at a faster rate than similar preparations from normal animals (12). A more recent study (13) led to the conclusion that the effects of thyroid hormones on bile acid metabolism must take place at a biosynthetic step preceding side-chain oxidation, perhaps involving hydroxylation of the steroid nucleus. However, it must be realized that the normal substrate for side-chain oxidation leading to the formation of cholic acid from cholesterol is not cholesterol itself but presumably 3a,7afl2a-trihydroxy-5/5-cholestane (14,15), and the substrate for the side-chain oxidation leading to chenodeoxycholate is, presumably, 3a,7a-dihydroxy-5/5-cholestane (16). Thus results of in vitro experiments in which cholesterol is employed as the substrate must be interpreted with caution, since mitochondria do not have the enzyme system required for formation of the triol and diol from cholesterol. 

FIGURE 14.7 Derivatization for ESI-MS/MS. (A) Preparation of the dimethylaminoethylester of dihydroxycholestanoic acid bis acetate. (B) Preparation of aminoethanesulfonate derivative of chenodeoxycholic acid. (C) Formation of sulfate ester of cholesterol. (D) Preparation of oxime of testosterone. 

Comparative formation of lithocholic acid from chenodeoxycholic and ursodeoxycholic acids in the colon. Gastroenterology, 83 753 (1982). 

Salon, G., Tint, G. S., I liav, B., Deering, N. and Mosbach, E. H. (1974) Increased formation of ursodeoxycholic acid in patients treated with chenodeoxycholic acid. J. din. Invest., 53, 612. 

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