Deoxycholic acid 6/3-hydroxylation

A and B are in cis position relative to each other (see p. 54). One to three hydroxyl groups (in a position) are found in the steroid core at positions 3, 7, and 12. Bile acids keep bile cholesterol in a soluble state as micelles and promote the digestion of lipids in the intestine (see p.270). Cholic add and cheno-deoxychoMc acid are primary bile acids that are formed by the liver. Their dehydroxylation at C-7 by microorganisms from the intestinal flora gives rise to the secondary bile acids lithocholic acid and deoxycholic acid. 

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. 

Bile-acid formation in rats involves hydroxylation to give 7a- and 6j8-hydroxy-derivatives. In many cases, no isotope effect was observed on hydroxylation of the appropriate labelled sterol. These examples involve cytochrome P-450 in the oxidation. However, oxidation of [7a- H,24- C]deoxycholic acid or tauro-deoxycholic acid to the corresponding cholic acid showed an isotope effect of 3.8 on examination of recovered starting material. 

Rat liver microsomes hydroxylate 5/8-cholestane-3a ,7a,12Q -triol at C-25 and C-26 both activities are dependent on cytochrome P450 and there is some evidence that different types of the latter are involved. A mitochondrial steroid 24-hydroxylase that accepts 3a,7a,12a-trihydroxy-5/3-cholestanoic acid has been extracted from rat liver apparently this is not a mixed-function oxidase although the presence of oxygen was obligatory for its action. Bile acids hydroxylated at C-23 have been formed from sodium cholate and deoxycholate in preparations from Viperinae species and a steroid-12ct-hydroxylase from liver microsomes has been studied.Sitosterol has been confirmed to be a precursor of C24 and C29 bile acids in mammalian liver, and here hydroxylation at C-26 precedes that at C-7. ° "... 

Free-radical attack on tertiary C—H bonds has been used for the direct hydroxyla-tion of steroids,but with almost random attack at the available tertiary centres. In a novel regio- and stereo-specific version of this procedure, the solid inclusion complex of deoxycholic acid and di-t-butyl diperoxycarbonate (4 1) gave the 5/8-hydroxy-derivative of deoxycholic acid as the only hydroxylated product, on heating at 90 °C or by photolysis. An X-ray study of the inclusion complex showed a normal arrangement of deoxycholic acid molecules to form a channel, but the guest peroxy-compound is apparently disordered within the channel, as it could not be located. 

In 1957, we reported on the use of the detergent deoxycholate for the solubilization of aminoazo dye N-demethylase, and we also reported an inhibitory effect of CO on aminoazo dye N-demethylase activity (49). In a key study in 1968, Lu Coon described the deoxycholate-dependent solubilization and resolution of a liver microsomal fatty acid >-hydroxylation system into three components by column chromatography, and they were able to reconstitute catalytic activity by combining the three fractions (50). The three fractions were identified as cytochrome P450,... 

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). 

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. ... 

Bremmelgaard, A. and Sjovall, 1. (1980) Hydroxylation of cholic, chenodeoxycholic, and deoxycholic acids in patients with intrahepatic cholestasis. J. Lipid Res. 21, 1072-1081. 

In a similar study of the metabolism of [24- C]12a-hydroxy-5)8-cholanic acid given intraperitoneally to male rats with bile fistulas [83], the identified biliary metabolites were 7a,12a-dihydroxy-5)8-cholanic acid (26%), deoxycholic acid (18%), cholic acid (15%), 6)8,12a-hydroxy-5)8-cholanic acid (0.8%), and 12% of unchanged 12a-hydroxy-5)8-cholanic acid. Thus, 5)8-cholanic acid and 12a-hydroxy-5)8-cholanic acids are hydroxylated in vivo preferentially in the order 7a, 3a, and 6)8. The preference for 7a-hydroxylation may be related to the concentration and properties of the active enzyme. Although no in vitro studies have been carried out, these studies infer the ability of hepatic tissue to provide characteristic 3a-hydroxy bile acids from derivatives devoid of a C-3 oxygen. How often this activity is required is questionable, because of the abundance of 3-hydroxylated sterol derivatives provided to and by the liver. 

Hydroxylation. Urinary acids hydroxylated at position 6, especially 6a, include hyodeoxycholate, hyocholate, 3a,6 /, 12a-trihydroxy- and 3a,6a,7a,12 -tetrahy-droxy-5 -cholanates. Hyocholate, the major bile acid in urine, serum and duodenal fluid of a child with intrahepatic cholestasis [109], is a major urinary metabolite of chenodeoxycholate in patients with intrahepatic cholestasis, while 3a,6a,12a-trihy-droxy-5j3-cholanate is only a minor metabolite of deoxycholate [123]. The 3a,6a,7a,12a-tetrahydroxy acid, obtained from urine of patients with liver diseases [194] and from gastric contents of neonates with duodenal atresia [203] is another metabolite of cholic acid [123] several unidentified tetrols have yet to be char-... 

The intestinal microflora of man and animals can biotransform bile acids into a number of different metabolites. Normal human feces may contain more than 20 different bile acids which have been formed from the primary bile acids, cholic acid and chenodeoxycholic acid [1-5], Known microbial biotransformations of these bile acids include the hydrolysis of bile acid conjugates yielding free bile acids, oxidation of hydroxyl groups at C-3, C-6, C-7 and C-12 and reduction of oxo groups to give epimeric hydroxy bile acids. In addition, certain members of the intestinal microflora la- and 7j8-dehydroxylate primary bile acids yielding deoxycholic acid and lithocholic acid (Fig. 1). Moreover, 3-sulfated bile acids are converted into a variety of different metabolites by the intestinal microflora [6,7]. 

FIGURE 18-6 Chemical structures of major sterols and cholesterol derivatives. The major sterols in animals (cholesterol), fungi (ergosterol), and plants (stigmasterol) differ slightly in structure, but all serve as key components of cellular membranes. Cholesterol is stored as cholesteryl esters in which a fatty acyl chain (R = hydrocarbon portion of fatty acid) is esterified to the hydroxyl group. Excess cholesterol is converted by liver cells into bile acids (e.g., deoxycholic acid), which are secreted into the bile. Specialized endocrine cells synthesize steroid hormones (e.g., testosterone) from cholesterol, and photochemical and enzymatic reactions in the skin and kidneys produce vitamin D. 

The trogerophane based on the deoxycholic acid chiral template in 25 [and in its (/ ,/ )-epimer] was used for an asymmetric synthesis of the enantiopure Troger s base analogues after hydrolysis.As the two hydroxyl groups of deoxycholic acid have different reactivities for esterification, unsymmetric Troger s bases could be asymmetrically synthesized as well. 

The changes of the cholesterol molecule that occur in its conversion into bile acids include epimerization of the 3 -hydroxyl group, reduction of the double bond, introduction of hydroxyl groups in positions C-7 (cheno-deoxycholic acid), C-7 and C-12 (cholic acid), or C-6 and C-7 (a- and P-muricholic acids, hyocholic acid), and transformation of the C27 side chain into a C24-carboxylic acid. 

Allocholanoic acids (5a-cholanoic acids) are found mainly in lower animals (68,76), but small amounts of allocholic acid (3a,7a,12a-trihydroxy-5a-cholanoic acid), allodeoxycholic acid (3a,12a-dihydroxy-5a-cholanoic acid), and probably also allochenodeoxycholic acid (3a,7a-dihydroxy-5a-cholanoic acid) may be present in bile and feces of mammals (68,76,102). Karavolas et al. (Ill) and Ziller et al. (112) have shown that cholestanol is converted into allocholic acid and allochenodeoxycholic acid in rats with a biliary fistula. The conversion of cholestanol into allocholic acid has also been shown in the rabbit (113). Allodeoxycholic acid is a secondary bile acid, formed from allocholic acid (113,114) and deoxycholic acid (115,116). The early steps in the sequence of reactions from cholestanol to allocholic acid (Fig. 6) have been the subject of two recent investigations. Shefer et al. (17) have shown that the microsomal fraction of rat liver homogenate fortified with NADPH catalyzes 7a-hydroxylation of cholestanol. Bjorkhem and Gustafsson (117) have compared the rates of 7a-hydroxylation of cholestanol,... 

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). 

The demonstration by Bergstrom et al. (4,5) in 1953 of the conversion of deoxycholic acid to taurocholic acid in the rat in vivo and by rat liver slices paved the way for studies on 7a-hydroxylation and conjugation of bile acids. The early work on the synthesis of bile acid conjugates in vitro utilized slices or homogenates of rat and human liver, and the enzymatic reaction was followed by the incorporation of radioactivity from carboxyl- C-labeled bile acids into the corresponding taurine and glycine conjugates (6,7). The... 

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