Lithocholic acid 3-formate

Steroidal amides. Herz and Mantecon have prepared amides of lithocholic acid 3-formate in satisfactory yields by the EEDQ method. 

They developed a continuum elastic-free energy model that suggests these observations can be explained as a first-order mechanical phase transition. In other recent work on steroids, Terech and co-workers reported the formation of nanotubes in single-component solutions of the elementary bile steroid derivative lithocholic acid, at alkaline pH,164 although these tubules do not show any chiral markings indicating helical aggregation. 

Deconjugation and dehydroxylation reactions occur in the colon, leading to the formation of dozens of new distinct BAs, by the action of the colonic bacteria. The final products enter the enterohepatic circulation and reach the liver where they are reconjugated mostly to either glycine or taurine. Some lithocholic acid, the most toxic substance produced in the body and a known carcinogen, enters the liver where it is sulfated or esterified to glucuronic acid and excreted. 

Bile acids within the enterohepatic circulation that undergo absorption in the terminal ileum encounter a relatively low number of species and population of bacteria and return to the liver in portal blood relatively unchanged. However, the approximately 5% of the bile-acid pool that enters the colon provides substrate for the extensive microbial population that deconjugate and oxidise hydroxyl groups leading to formation of the secondary bile acids deoxycholic and lithocholic acids that are the major bile acids in faeces. 

The interest in bile acids as potential carcinogens was subject to investigation as early as 1940 when Cook et al. reported in Nature that repeated injection of deoxycholic acid into the flanks of mice could induce tumour formation in mice." Furthermore, Kelsey and Pienta showed that treatment of hamster embryo cells with lithocholic acid could cause cell transformation. ... 

When chenodeoxycholic acid therapy was first introduced, there was some anxiety that this bile acid, or its bacterial metabolite lithcholic acid, might cause liver damage in man. This possible complication has not eventuated. Lithocholic acid is toxic to the liver in many animal species but in man, it is converted to sulfolithocholate and excreted (A5). Nevertheless, up to one-third of patients undergoing chenodeoxycholic acid treatment do show transient rises in serum levels of aspartate aminotransferase activity. The mechanism of this hypertransaminasemia is obscure, although it could possibly be related to lithocholate formation (D8). In any case, hepatotoxicity very rarely occurs at a clinically significant level (SI4). 

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). 

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). 

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... 

Neomycin is a polybasic, poorly absorbed antibiotic which forms insoluble precipitates with bile salts (99). It lowers serum cholesterol concentrations in man (100-102) and chickens (99) and increases fecal bile acid excretion. It inhibits the hepatotoxic effects of lithocholic acid ingestion in chickens (99) and prevents bacterial conversion of cholate to deoxycho-late (103). Neomycin, 6-12 g/day, induces a malabsorption syndrome, with mucosal changes similar to those of sprue (104). Bile salt metabolism is thus affected in at least three ways by neomycin (1) a binding effect similar to that of cholestyramine, (2) suppression of deconjugation and secondary bile formation caused by antimicrobial properties, and (3) possible impairment of absorption of bile salts by intestinal mucosa. The first probably accounts for most of the increased fecal excretion of bile salts. 

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... 

Norman and Palmer found that metabolites of lithocholic acid in human bile were not hydrolyzed by 1 N NaOH, 6 hr, 110°C, or by 4.5 N NaOH, 6 hr, 130°C. Complete hydrolysis was achieved with 5 n NaOH, 48 hr, 135 °C. Such conditions resulted in formation of several products from 3-keto-5p-cholanoic acid (60). 

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

The liver, and also bacteria in the small and large intestine, can cause other structural modifications to bile acids as they undergo their entero-hepatic cycle. The formation of sulfate esters, already mentioned with respect to lithocholate in Section 4.2.1, is carried out primarily in the liver in man by a sulfotransferase (Lll). Other bile acids can also be sulfoconjugated to a small extent, mainly at the 3a-hydroxyl position. Bacteria, which have been isolated anaerobically from human feces, are known to possess bile acid sulfatase activity, which removes the 3a-sul te group of chenodeoxycholic and cholic acids (H24). The action of this bacterial enzyme probably explains why only trace amounts of sul ted bile acids, which are poorly absorbed in the intestine, are detected in the feces (12). Another type of bile acid conjugate, which has been identified in the urine of healthy subjects and patients with hepatobiliary disease, is the glucuronide (A7, S41). Both the liver and extrahepatic tissues, such as the kidney and small intestinal mucosa, are capable of glucuronidation of bile acids in man (M14). 

Although cholesterol is the major source of 5)9-bile acids, an unsaturated acid, 3)8-hydroxy-5-cholenic acid [174] has been found in meconium, mainly as the sulfate [175], in bile of a boy with a deficiency of 3)8-hydroxysteroid dehydrogenase [176], and in urine of healthy persons and individuals with liver disease [164]. The details of metabolism of 3)8-hydroxy-5-cholenic acid to lithocholate have not been entirely elucidated, but the mechanism for conversion of the 3/8-hydroxy-A to the 3-oxo-A derivative has been formulated in the C27 series (cf. Chapter 9). Briefly, the 3)8-ol is dehydrogenated by a microsomal enzyme fortified with NAD to provide the 3-oxo-A system [177,178]. Whether a A - A" isomerase is essential is not known, since there is no direct evidence for the formation of the intermediary 3-oxo-A system the rate-limiting step is the dehydrogenation of the 3)8-ol which may prevent accumulation of the 3-oxo-A system [177]. The reduction of the double bond at 4-5 to the 5)8- or 5a-bile acid is catalyzed by the respective A -3-oxosteroid 5)8- or 5 -reductase obtained from liver cytosol [170], and has been purified about 10-fold [178]. The formation of the 3-oxo-5/9 derivative requires the enzyme and NADPH the proton from the A side (4A-NADPH) appeared in the product as the 5)8-H, whereas the proton at C-4 is derived from the aqueous medium. Formation of the 5a derivative requires (4B-NADPH) in a similar mechanism (Fig. 4) [179], Reduction of the 3-0X0 product is then catalyzed by 3a-hydroxysteroid dehydrogenase as discussed above. 

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). 

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. 

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