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The metabolism of 3 alpha,7 alpha-dihydroxy-25,26-bishomo-5 beta-cholane-26-sulfonate (bishomoCDC-sul), the sulfonate analogue of bishomochenodeoxycholic acid, and its effect on biliary bile acid composition were studied during chronic administration in the hamster. After oral administration of radiolabeled bishomoCDC-sul, more than 80% of the radioactivity was excreted into the feces within 7 days, both as the unchanged sulfonate (38.5%) and two more polar metabolites (50.0% and 11.5%). The half time of the fecal excretion was 1.6 days. In gallbladder bile, the unchanged sulfonate and its major metabolite accounted for 19.1% and 19.8% of total bile acids, respectively. In another experiment, hamsters were fed bishomoCDC-sul with antibiotics to evaluate the site of biotransformation. Even when the number of intestinal microorganisms was greatly reduced, the same three metabolites were found in the feces: bishomoCDC-sul (44.0%) and the two polar metabolites (30.8% and 25.1%). The major metabolite was isolated from feces of the hamsters fed bishomoCDC-sul without antibiotics. Its chemical structure was identified by mass spectrometry and nuclear magnetic resonance spectroscopy as the 15 alpha-hydroxylated derivative, namely sodium 3 alpha,7 alpha,15 alpha-trihydroxy-25,26-bishomo-5 beta-cholane-26-sulfonate. These results indicate that after oral administration, the sulfonate analogue of bishomochenodeoxycholic acid underwent enterohepatic circulation like a natural bile acid and was transformed, in part, into the 15 alpha-hydroxylated derivative and another more polar metabolite in the liver of hamsters. There was no evidence that bishomoCDC-sul was dehydroxylated to a lithocholic acid analogue during enterohepatic cycling.

The effects of beta-muricholic acid and hyocholic acid on cholesterol cholelithiasis were examined in two animal models. The following experiments were carried out: A) In a gallstone prevention study, prairie dogs were fed the lithogenic diet with or without 0.1% beta-muricholic or 0.1% hyocholic acid for eight weeks. B) In a second prevention study, hamsters were fed the lithogenic diet with or without 0.1% beta-muricholic acid or 0.1% hyocholic acid for six weeks. C) In a gallstone dissolution study, hamsters were fed the lithogenic diet for six weeks to induce stones; stone dissolution was examined during administration of a cholesterol-free purified diet with or without 0.1% beta-muricholic acid or 0.1% hyocholic acid. In the prevention study in prairie dogs (A), both bile acids failed to prevent stone formation, the cholesterol saturation index of bile was 0.89 in the lithogenic controls, remained unchanged with hyocholic acid and increased to 1.52 in the beta-muricholic acid group. In the prevention study in hamsters (B), beta-muricholic acid completely inhibited the cholesterol cholelithiasis (0% stone incidence); the cholesterol saturation index of bile was 1.78 (compared to lithogenic controls, 1.37). Hyocholic acid reduced stone incidence to 16% with a cholesterol saturation index of 0.98. In the dissolution study in hamsters (C), preexisting cholesterol gallstones were not dissolved by either hydrophilic bile acid after feeding these bile acids for an additional six weeks; at the end of the experiment, the cholesterol saturation indices were below unity.(ABSTRACT TRUNCATED AT 250 WORDS)

The prevention of cholesterol gallstone formation by three bile acid analogs, sodium 3 alpha,7 alpha-dihydroxy-5 beta-cholane-24- sulfonate, sodium 3 alpha,7 beta-dihydroxy-5 beta-cholane-24-sulfonate and sodium 3 alpha,6 alpha-dihydroxy-5 beta-cholane-24-sulfonate, was examined in a hamster model of cholesterol cholelithiasis. Sodium taurochenodeoxycholate, sodium tauroursodeoxycholate and sodium taurohyodeoxycholate were studied simultaneously for comparison. Gallstones and cholesterol crystals were induced in 14 of 15 hamsters fed a bile acid-free, semipurified lithogenic diet containing 0.3% cholesterol and 4% butterfat for 6 wk. The addition of 0.1% sodium taurochenodeoxycholate and sodium tauroursodeoxycholate to the lithogenic diet had little effect on the formation of gallstones or biliary cholesterol crystals. In contrast, sodium 3 alpha,7 alpha-hydroxy-5 beta-cholane-24- sulfonate and sodium 3 alpha,7 beta-dihydroxy-5 beta-cholane-24-sulfonate, when fed at the same dose, prevented cholesterol gallstone formation significantly. Sodium taurohyodeoxycholate and sodium 3 alpha,6 alpha-dihydroxy-5 beta-cholane- 24-sulfonate inhibited cholesterol gallstone formation effectively. The cholesterol saturation index of bile was greater than 1.00 in all groups, with the exception of the group fed sodium 3 alpha,7 alpha-dihydroxy-5 beta-cholane-24-sulfonate. Liver and serum cholesterol levels tended to be lower in most of the groups that were fed bile acids. This effect was most pronounced in the animals receiving sodium taurohyodeoxycholate. At the end of the experiment, the administered sulfonate analogs were detected in gallbladder bile.(ABSTRACT TRUNCATED AT 250 WORDS)

We tested bile acid oxazoline derivatives of chenodeoxycholic (CDC-OX), 7-ketolithocholic (7-KLC-OX), ursodeoxycholic (UDC-OX), and deoxycholic (DC-OX) as inhibitors of the 7-epimerization of the primary bile acids cholic acid (CA) and CDC in cultures of four species of bacteria and the human fecal flora. The organisms tested elaborate a 7 alpha- and/or 7 beta-hydroxysteroid dehydrogenase (HSDH); they were Escherichia coli (7 alpha-HSDH), Bacteroides fragilis (7 alpha-HSDH), Clostridium absonum (7 alpha- and 7 beta-HSDH) and Eubacterium aerofaciens (7 beta-HSDH). None of the oxazolines affected 7 alpha-OH oxidation of CA or CDC by E. coli or the growth of the organism. All the oxazolines (except UDC-OX) inhibited the growth of B. fragilis and its 7 alpha-HSDH. In contrast, only DC-OX blocked 7 alpha-OH epimerization of CA by C. absonum. Surprisingly, the other three oxazolines enhanced 7 alpha-OH epimerization of CA, but not that of CDC, which was inhibited (CDC-OX greater than 7-KLC-OX much greater than UDC-OX). Enzymic data suggest that CDC-OX in the presence of CA can induce a greater level of both 7 alpha- and 7 beta-HSDH than CA or CDC-OX alone, CDC-OX being more toxic in the presence of CDC. Formation of urso-bile acid from 7-keto substrates by E. aerofaciens is totally blocked by the oxazolines (except UDC-OX). Similarly, suppression of urso-bile acid formation from primary bile acids by the human fecal flora was evident with DC-OX greater than 7-KLC-OX greater than CDC-OX much greater than UDC-OX, the last being ineffective. The inhibitory activity of the oxazolines on the 7-dehydroxylation of primary bile acids by human fecal flora followed the same order.

The effect of 7-methyl substituted bile acid and bile alcohol analogues on cholesterol metabolism was studied in the hamster. Animals were fed chow plus 0.1% cholesterol supplemented with 0.1% of one of the following steroids: chenodeoxycholic acid, 7-methyl-chenodeoxycholic acid, 7 beta-methyl-24-nor-5 beta-cholestane-3 alpha, 7 alpha, 25-triol, cholic acid, 7-methyl-cholic acid, or 7 beta-methyl-24-nor-5 beta-cholestane-3 alpha, 7 alpha, 12 alpha,25-tetrol. Cholesterol absorption was determined from fecal analysis after feeding of radiolabeled cholesterol and beta-sitosterol. Of the six compounds studied, chenodeoxycholic acid and 7-methyl-chenodeoxycholic acid decreased intestinal cholesterol absorption (17% and 31% decrease, respectively). Only 7-methyl-chenodeoxycholic acid decreased serum cholesterol concentration (29% decrease), but there were no analogous changes of liver and biliary cholesterol concentration and cholesterol saturation of bile. Total fecal neutral sterol excretion was increased in the groups fed chenodeoxycholic acid and 7-methyl-chenodeoxycholic acid. In addition, the production of coprostanol was increased in both groups. These data suggest that 7-methyl-chenodeoxycholic acid resembles chenodeoxycholic acid in its effect on cholesterol metabolism and may be a potential candidate for further studies of its gallstone-dissolving properties.

The effect of chenodeoxycholic acid, ursodeoxycholic acid and hyodeoxycholic acid on gallstone dissolution was studied in the prairie dog. Cholesterol gallstones were found in all animals after feeding a semipurified diet plus 1.2% cholesterol for six wk. Gallstone regression was examined by feeding a chow diet containing the bile acids (chenodeoxycholic acid, ursodeoxycholic acid or hyodeoxycholic acid) alone (30 mg/kg/day) or in combination (chenodeoxycholic acid plus ursodeoxycholic acid) for an additional six wk. Chenodeoxycholic acid was effective in dissolving established cholesterol gallstones (two out of 16 animals still had stones) and cholesterol crystals (six out of 16 animals had crystals); the hydrophilic bile acids, ursodeoxycholic acid and hyodeoxycholic acid, were ineffective in the six-wk regression study. The lithogenic indices averaged 1.09 at the end of the induction period; all biles became unsaturated with respect to cholesterol after the six-wk regression period (group 1, 0.82; group 2, 0.66; group 3, 0.81; group 4, 0.84; group 5, 0.66). Cholesterol levels in liver, plasma and bile were elevated after the six-wk induction phase (4.59 mg/g, 610 mg/dl and 0.36 mg/ml, respectively) but returned to near normal levels after the six-wk regression period. Biliary bile acids contained increased levels of the dietary bile acid administered to each group. This experiment shows that relatively hydrophobic bile acids may be more effective than hydrophilic bile acids for gallstone dissolution during the period studied.

Taurolithocholic acid and deoxycholic acid were perfused into isolated prairie dog livers. Taurolithocholic acid was 7 alpha-hydroxylated to form taurochenodeoxycholic acid, whereas deoxycholic acid was conjugated and 7 alpha-hydroxylated to form taurocholic acid. The low concentrations of secondary bile acids observed in prairie dog bile are due, at least in part, to active bile acid 7 alpha-hydroxylase(s) in the liver of these animals.

A microcomputer program to calculate the cholesterol saturation of bile is described. The program is designed to accept most of the conventional concentration units for bile salts, phospholipid, and cholesterol. The calculated cholesterol saturation can be corrected for the presence of ursodeoxycholic acid conjugates in bile. The program is designed to make appropriate statements when the input data produce results that are out of range of the solubility data available in the published literature. The program makes possible not only a very rapid calculation of the cholesterol saturation of bile, but eliminates arithmetical errors that are occasionally encountered during conventional calculations.

The effect of various dietary additions such as cholesterol, beta-sitosterol, bile acids, and bile acid analogs on gallstone formation was studied in the hamster. Gallstones were formed in 50% of the animals fed a high glucose, fat-free diet. Administration of 0.2% cholesterol or 1% beta-sitosterol had no effect on the incidence of gallstones. Ursodeoxycholic acid (0.5%) and its analog ursodeoxy-oxazoline [2-(3 alpha, 7 beta-dihydroxy-24-nor-5 beta-cholanyl)-4,4-dimethyl-2- oxazoline] were ineffective in preventing gallstones. Hyodeoxycholic acid and hyodeoxy-oxazoline [2-(3 alpha,6 alpha-dihydroxy-24-nor-5 beta-cholanyl)-4,4-dimethyl-2- oxazoline] at the same dosage effectively prevented gallstones, while the trihydroxy bile acid, hyocholic acid, was not effective. Of all the dietary regimens tested, only hyodeoxycholic acid significantly lowered serum cholesterol. The lithogenic diet produced a five-fold increase in hepatic HMG-CoA reductase activity; this activity was not affected by dietary cholesterol or beta-sitosterol. Hyodeoxycholic acid and hyocholic acid feeding increased the reductase activity by an additional 50% while the other bile acids had no effect. beta-Sitosterol doubled the cholesterol 7 alpha-hydroxylase activity whereas hyodeoxy-oxazoline lowered it. Hyodeoxycholic acid-fed animals had significantly lower cholesterol absorption than the animals on the lithogenic diet alone. Biliary cholesterol content increased dramatically in the animals fed the lithogenic diet and was increased still further by ursodeoxycholic acid, hyodeoxycholic acid, and hyodeoxy-oxazoline. These data show that hyodeoxycholic acid and hyodeoxy-oxazoline do not prevent gallstones by inhibiting hepatic cholesterol synthesis or biliary cholesterol secretion.

Certain bile acid oxazoline derivatives (100 microM), but not corresponding unconjugated bile acids (100 microM), were found to inhibit the growth of Eubacterium sp. V.P.I. 12708. The growth inhibition was correlated with the polarity of the steroid portion of the bile acid oxazoline. Primary cultures of adult rat hepatocyte monolayer cultures converted [7 epsilon-14C]methylchenooxazoline3 into MeOH-H2O soluble derivatives. Certain intestinal bacteria were capable of metabolizing [17 epsilon-14C]methylchenooxazoline as well as the MeOH-soluble hepatocyte derivative(s). These results suggest that bile acid oxazoline derivatives may undergo hepatic, as well as bacterial metabolism during enterohepatic circulation.

Sterol balance studies using isotopic and chromatographic techniques were performed in rats fed diets supplemented with ursodeoxycholic acid. Compared to controls, ursodeoxycholic acid dramatically altered sterol metabolism. Ursodeoxycholic acid was absorbed and circulated in the enterohepatic circulation. The biliary bile acid composition was significantly altered with ursodeoxycholic acid the predominant biliary bile acid (67%). Cholesterol absorption was depressed by 34%; bile acid synthesis was depressed by 30%; however, cholesterol balance was significantly increased. It is apparent that the effects of ursodeoxycholic acid on sterol metabolism are different in several respects from chenodeoxycholic acid.

Bile acid amides and oxazolines were synthesized by a sequence of steps involving the reaction of the free bile acid with formic acid to yield the formyloxy derivative, preparation of the formyloxy acid chloride, condensation of the acid chloride with 2-amino-2-methyl-1-propanol to give the amide and, finally, cyclization of the amide with thionyl chloride to give the oxazoline. The oxazolines were characterized by physical constants, thin layer and gas-liquid chromatography and identified by elemental analysis and gas-liquid chromatography-mass spectrometry. Some of the bile acid oxazoline derivatives alter the activity of bacterial 7-dehydroxylases in vitro, and inhibit the growth of certain anaerobic bacteria in pure culture.

Confluent cultures of Hep G2 cells were found to synthesize chenodeoxycholic and cholic acids continually. Chenodeoxycholic acid was synthesized at the rate of 58 +/- 8.6 micrograms/96 h, a rate more than 7-fold greater than that for cholic acid. Addition of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol but not the -3 alpha, 7 alpha-diol was followed by an increase in cholic acid synthesis, thus indicating a relatively low 12 alpha-hydroxylase activity. Endogenous synthesis of monohydroxy bile acid ester sulfates was found, with maximum rates of 135 and 74 micrograms/96 h for lithocholic and 3 alpha-hydroxy-5-cholenoic acids, respectively. Incubation of Hep G2 cells in medium containing 25% D2O permitted a comparison of the precursor/product relationship of cholesterol with 3 beta-hydroxy-5-cholenoic acid. The pattern of incorporation of deuterium was in accordance with that expected, thus allowing the conclusion that this monohydroxy bile acid is derived from cholesterol and should be considered together with chenodeoxycholic and cholic acids as a primary bile acid.

The acute effects of cholic acid ingestion on methylazoxymethanol acetate [(MAM) CAS: 592-62-1]-treated conventional and germfree rats were investigated. Male SD rats were divided into 4 treatment groups. The first group received control chow; the second group, control chow plus 0.5% cholic acid; the third group, control chow plus MAM; and the fourth group, control chow plus 0.5% cholic acid plus MAM. Fecal bile acids, cholesterol, cholesteral degradation products, and neutral sterols, as well as labeling indices and numbers of epithelial cells per crypt column, were measured after 6 weeks of treatment. The administration of MAM to germfree groups diminished both fecal bulk and the amount of fecal water. MAM did not affect the fecal bile acid composition. Analysis of the fecal bile acids in conventional rats fed cholic acid demonstrated that half of the bile acids were in a form of deoxycholic acid. In the germfree groups fed cholic acid, 90% of the bile acids appeared unaltered in the feces. Neither in the germfree nor in the conventional groups was an effect seen of MAM on the output of fecal neutral sterols. The addition of cholic acid to the food decreased the output of neutral sterols both in the conventional (P less than .001) and in the germfree (P less than .02) animals. The germfree animals showed a reduced amount of neutral steroid excretion (P less than .01) when compared to the findings for the conventional groups. MAM had no influence on the fecal cholesterol or coprostanol output. The consumption of 0.5% cholic acid decreased the total output of cholesterol (P less than .05). The excretion of coprostanol was significantly diminished in the conventional rats fed cholic acid (P less than .001). No difference in labeling indices was observed between conventional and germfree rats, whether treated with cholic acid, MAM, or cholic acid plus MAM. However, all germfree groups showed less epithelial cells per crypt column (P less than .001) than did conventional animals.

Methods are described for the chemical synthesis of three new bile acid analogs, namely, 3 alpha, 7 alpha-dihydroxy-7 beta-methyl-5 beta-cholanoic acid, 3 alpha, 7 beta-dihydroxy-7 alpha-methyl-5 beta-cholanoic acid and 3 alpha-hydroxyl-7 xi-methyl-5 beta-cholanoic acid. The starting material, 2-(3 alpha, 7 xi-dihydroxy-7 xi-methyl-24-nor-5 beta- cholanyl )-4, 4-dimethyl-2-oxazoline upon mild hydrolysis in aqueous HCl yields the two epimeric 3 alpha, 7-dihydroxy-7-methyl-5 beta-cholanoic acids which can be separated as the methyl esters by silica gel column chromatography. More rigorous hydrolysis in boiling methanolic HCl yields a mixture of unsaturated compounds which can be separated as their methyl esters into three fractions by silica gel-AgNO3 column chromatography. The fractions containing 3 alpha-hydroxy-7-methyl-5 beta-chol-6-enoic acid and 3 alpha-hydroxy-7-methylene-5 beta-cholanoic acid when subjected to catalytic hydrogenation yield 3 alpha-hydroxy-7 xi-methyl-5 beta-cholanoic acid.

Rats of the Wistar and Sprague-Dawley strains were injected with sodium phenobarbital (100 mg/kg body wt/day) for 8 days. Fecal bile acid excretion was measured on days 6 and 8 of the experiment, and biliary bile acid composition, hepatic microsomal cholesterol, 7alpha-hydroxylase, and 7alpha-hydroxy-4-cholesten-3-one 12alpha-hydroxylase were determined at the end of the study. In the Wistar rat, injection of phenobarbital produced a doubling of fecal bile acid output (controls, 5.3 mg/rat/day; treated rats, 10.6 mg/rat/day) and a two-threefold increase in cholesterol 7alpha-hydroxylase. The fecal bile acid output of Sprague-Dawley rats increased 20% in response to phenobarbital (controls, 9,5 mg/rat/day; treated rats, 11.6 mg/rat/day). The activity of cholesterol 7alpha-hydroxylase remained unchanged. In both strains, phenobarbital treatment produced a decrease in the proportion of cholic acid in total bilary bile acids (controls, 85%; treated groups, 65%). This was associated with a decrease of 7alpha-hydroxy-4-cholesten-3-one 12alpha-hydroxylase activity by ca. 50%. Biliary cholesterol concentrations were reduced in phenobarbital treated rats of both strains, but liver cholesterol concentrations remained unchanged. The drug produced a 25% increase in liver wt, on the average.

Nor-ursodeoxycholate, the C23 analogue of ursodeoxycholate, is a potent choleretic agent in rodents when given acutely but, to be used in humans, chronic toxicity studies are required. In the rabbit, ingestion of ursodeoxycholate or chenodeoxycholate leads to accumulation of lithocholate, its major bacterial metabolite, in biliary bile acids, which causes inflammation in portal tracts of the liver and bile duct proliferation. To test whether chronic administration of nor-ursodeoxycholate would cause an analogous accumulation of nor-lithocholate and hepatotoxicity, rabbits were fed a Chow diet containing nor-ursodeoxycholate (5 or 50 mg/day): control groups received Chow alone, and "disease control" groups received Chow plus ursodeoxycholate or Chow plus chenodeoxycholate. After 3 wk, animals were killed, liver sections were interpreted by a pathologist, and the steroid moiety of the glycine (and taurine) conjugates of gallbladder bile acids were analyzed by high-pressure liquid chromatography. Ingestion of nor-ursodeoxycholate did not cause hepatotoxicity, and neither it nor its presumed metabolite, nor-lithocholate, accumulated in biliary bile acids. To explain this unexpected finding, the hepatic metabolism of nor-ursodeoxycholate was investigated in biliary fistula rabbits. Nor-ursodeoxycholate was well absorbed from the intestine and secreted in the bile as a glucuronide as well as the unchanged compound, but conjugation with glycine and taurine was not observed. As glucuronides are poorly absorbed from the gut, it is proposed that the hepatic biotransformation of nor-ursodeoxycholate to a glucuronide rather than to a glycine amidate in the liver prevented its accumulation in the bile acid pool. Thus, shortening the side chain of ursodeoxycholate by a single carbon atom resulted in a bile acid with novel metabolism, which when administered chronically, does not accumulate in the enterohepatic circulation and does not cause hepatotoxicity.

7 beta-Methyl-chenodeoxycholic acid (7-MeCDC, 3 alpha, 7 alpha-dihydroxy-7 beta-methyl-5 beta-cholan-24-oic acid), 7 alpha-methyl-ursodeoxycholic acid (7-MeUDC, 3 alpha, 7 beta-dihydroxy-7 alpha-methyl-5 beta-cholan-24-oic acid), 7 xi-methyl-lithocholic acid (7-MeLC, 3 alpha-hydroxy-7 xi-methyl-5 beta-cholan-24-oic acid) and ursodeoxycholylsarcosine (UDCS) were tested as inhibitors of bacterial bile acid 7 alpha-dehydroxylase activity. At a concentration of 50 microM, 7-MeCDC and 7-MeUDC inhibited enzyme activity by 66% and 12%, respectively. 7 alpha-Dehydroxylase activity was not inhibited in the presence of 7-MeLC and UDCS. None of the four bile acid analogs tested inhibited the growth of Eubacterium sp. V.P.I. 12708 at concentrations up to 100 microM.

The in vivo conversion of several 5 beta-cholestane intermediates to primary bile acids was investigated in three patients with total biliary diversion. The following compounds were administered intravenously: 5 beta-[G-3H]-cholestane-3 alpha, 7 alpha-diol, 5 beta-[G-3H]cholestane-3 alpha, 7alpha, 26-triol, and 5 beta-[24-14C]cholestane-3 alpha, 7 alpha-25-triol. Bile was then collected quantitatively at frequent intervals for the next 21 to 28 h. The administered 5 beta-[G-3H]cholestane-3alpha, 7alpha, 26-triol was found to be efficiently converted to cholic and chenodeoxycholic acids in two patients; 61 and 75% of the administered label was found in primary bile acids. The proportion of labeled cholic to chenodeoxycholic acid was 1.20 and 1.02 in the bile of these patients, indicating that the C-26 triol was efficiently converted to cholic acid. The ratio of cholic to chenodeoxycholic acid (mass) in the bile of these patients was 1.23 and 2.32. The 5 beta-cholestane-3alpha, 7alpha-diol intermediate was also efficiently converted (71%) to both primary bile acids. The cholic to chenodeoxycholic acid ratios by mass and label were similar (2.97 versus 2.23). By contrast, the 5beta-cholestane-3alpha, 7alpha, 25-triol was poorly converted to bile acids in three patients. Following the administration of this compound almost all of the administered radioactivity found in the bile acid fraction was in cholic acid (5 to 19%) and very little (less than 5%) was found in chenodeoxycholic acid. These findings indicate that ring hydroxylation at position 12 is not materially hindered by the presence of a hydroxyl group on the side chain at C-26 in patients with biliary diversion. The labeled C-26-triol which was efficiently converted to both primary bile acids in a proportion similar to that which was observed for the bile acids synthesized by the liver suggests that this 5beta-cholestane derivative may be a major intermediate in the synthesis of both cholic and chenodeoxycholic acids.

The bile salts present in gallbladder bile of the West Indian manatee, Trichechus manatus latirostris, an herbivorous marine mammal of the tropical and subtropical margins of the Atlantic Ocean, were found to consist of a mixture of bile alcohol sulfates. Bile acids, previously believed to be present in all mammals, were not detected. Using chromatography, mass spectrometry, and 1H- and 13C-nuclear magnetic resonance spectroscopy, the major bile alcohol was identified as 5 beta-cholestane-3 alpha,6 beta,7 alpha-25,26-pentol; that is, it had the nuclear structure of alpha-muricholic acid and the side chain structure of bufol. This compound has not been described previously and the trivial name "alpha-trichechol" is proposed. The second most abundant compound was 5 beta-cholestane-3 alpha,7 alpha,25,26-tetrol. Other bile alcohols were tentatively identified as 5 beta-cholestane-3 alpha,6 beta,7 beta,25,26-pentol (named beta-trichechol), 3 alpha,6 alpha,7 beta, 25-26-pentol (named omega-trichechol) and 5 beta-cholestane-3 alpha,6 beta,7 alpha,26-tetrol. The 1H and 13C NMR spectra of the four 6,7 epimers of 3,6,7 trihydroxy bile acids are described and discussed. All bile alcohols were present as ester sulfates, the sulfate group being tentatively assigned to the 26-hydroxy group. 12-Hydroxy compounds were not detected. The manatee is the first mammal found to lack bile acids, presumably because it lacks the enzymes required for oxidation of the 26-hydroxy group to a carboxylic acid. Trichechols, like other bile salts, are water-soluble end products of cholesterol metabolism; whether they also function as biological surfactants in promoting biliary cholesterol secretion or lipid digestion is unknown.

The purpose of this study was to compare the effects of the naturally occurring bile acids (chenodeoxycholic acid and cholic acid) with their 7-methyl analogues (3 alpha,7 alpha-dihydroxy-7 beta-methyl-5 beta-cholanoic acid and 3 alpha,7 alpha,12 alpha-trihydroxy-7 beta-methyl-5 beta-cholanoic acid) on gallstone formation and prevention and cholesterol metabolism in the prairie dog. Sixty animals were fed a semipurified diet, containing 0.4% cholesterol, with one of the following acids (0.1%): chenodeoxycholic, cholic, 3 alpha,7 alpha-dihydroxy-7 beta-methyl-5 beta-cholanoic, or 3 alpha,7 alpha,12 alpha-trihydroxy-7 beta-methyl-5 beta-cholanoic acid. This concentration of dietary bile acids amounts to a dose of 27-30 mg/kg.day. After 8 wk, 89% of control animals had gallstones and 94% had cholesterol crystals. Chenodeoxycholic and 3 alpha,7 alpha-dihydroxy-7 beta-methyl-5 beta-cholanoic acids decreased the incidence of gallstones to 50%. Cholic acid and 3 alpha,7 alpha,12 alpha-tri-hydroxy-7 beta-methyl-5 beta-cholanoic acid did not prevent gallstone formation. The liver cholesterol level was decreased by chenodeoxycholic acid, whereas cholic and 3 alpha,7 alpha,12 alpha-trihydroxy-7 beta-methyl-5 beta-cholanoic acids increased serum and liver cholesterol. Each administered bile acid became the predominant biliary bile acid and 7-methyl analogues did not increase secondary bile acids. Fecal analysis of radioactive metabolites using 14C-labeled 7-methyl analogues showed that these compounds are resistant to bacterial 7-dehydroxylation. It was concluded that 3 alpha,7 alpha-dihydroxy-7 beta-methyl-5 beta-cholanoic acid inhibited gallstone formation as effectively as chenodeoxycholic acid, whereas both cholic and 3 alpha,7 alpha,12 alpha-trihydroxy-7 beta-methyl-5 beta-cholanoic acids were not effective. The effects of 7-methyl analogues on the parameters of cholesterol metabolism that we studied were similar to those of their parent compounds, chenodeoxycholic and cholic acids. Thus, 3 alpha,7 alpha-dihydroxy-7 beta-methyl-5 beta-cholanoic acid but not 3 alpha,7 alpha,12 alpha-trihydroxy-7 beta-methyl-5 beta-cholanoic acid offers promise in cholelitholytic therapy for the prevention and possibly dissolution of cholesterol gallstones.