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We examined the effect of diet on gallstone incidence and the composition of biliary phosphatidylcholines in methyltestosterone-treated female hamsters. These hamsters were fed a nutritionally adequate purified lithogenic diet containing 2% corn oil, 4% butterfat, 0.3% cholesterol, and 0.05% methyltestosterone, resulting in a cholesterol gallstone incidence of 86%. This incidence was lowered when mono- and polyunsaturated fats or fatty acids were added to the diet: 2.5% oleic acid resulted in total prevention of cholesterol cholelithiasis, 2.5% linoleic acid, and 4% safflower oil (78% linoleic acid content) reduced gallstone incidence to 26 and 8%, respectively. An additional 4% butterfat (29% oleic acid content) produced gallstones in 50% of the animals. At the end of the 6-wk feeding period, the bile of all hamsters was supersaturated with cholesterol. The major biliary phosphatidylcholine species in all groups were (sn-1-sn-2): 16:0-18:2, 16:0-18:1, 18:0-18:2, 16:0-20:4, and 18:2-18:2. The safflower oil- and linoleic acid-fed hamsters exhibited an enrichment of 16:0-18:2 (16-18%); added butterfat or oleic acid increased the proportion of 16:0-18:1 (9 and 25%, respectively). We conclude that the phosphatidylcholine molecular species in female hamster bile can be altered by dietary fats/fatty acids and that mono- and polyunsaturated fatty acids play a role in suppressing the induced cholelithiasis.

The effect of the sulfonate analogues of ursodeoxycholic acid, namely sodium 3 alpha,7 beta-dihydroxy-24-nor-5 beta-cholane-23-sulfonate (norUDC-SO3Na) and sodium 3 alpha, 7 beta-dihydroxy-5 beta-cholane-24-sulfonate (UDC-SO3Na), on biliary lipid secretion was studied in bile fistula rats. During intravenous infusion of the two sulfonate analogues, bile flow and biliary lipid secretion were stimulated in a dose-dependent manner. This suggests that the analogues exert an effect on biliary lipid secretion comparable to that of the naturally occurring bile acid, ursodeoxycholyltaurine (UDC-tau). The effects of norUDC-SO3Na and UDC-SO3Na on bile flow were similar but slightly smaller than that of UDC-tau. The output of bile salts was similar with both sulfonates but greater than that with UDC-tau. The infusion of norUDC-SO3Na or UDC-SO3Na induced cholesterol secretion and phospholipid secretion more significantly than UDC-tau infusion. The increase in phospholipid secretion was particularly pronounced during high-dose administration of norUDC-SO3Na. Although norUDC-SO3Na stimulated cholesterol secretion more intensely than the other two bile salts, it also facilitated phospholipid output, perhaps as a compensatory mechanism, and the biliary cholesterol/phospholipid ratio was decreased to a greater extent by the sulfonates than by UDC-tau. Consequently, the administration of norUDC-SO3Na or UDC-SO3Na produces a more "stable" bile than UDC-tau, suggesting that these sulfonates possess potential cholelitholytic activity.

The type of dietary fat strongly affects the incidence of gallstones in the hamster model of cholesterol cholelithiasis. The present study was designed to determine whether dietary fats could affect gallstone formation by altering the microstructure (vesicular/micellar ratio) of cholesterol in bile. Golden Syrian hamsters from Sasco (Omaha, NE) or Charles River (Wilmington, MA) were fed nutritionally adequate semipurified diets to which were added: (i) 4.0% butterfat without added cholesterol; (ii) 1.2% palmitic acid plus 0.3% cholesterol; or (iii) 4.0% safflower oil plus 0.3% cholesterol. Gallstone incidence and the percentage of cholesterol in vesicles and micelles were determined after two- or six-week feeding periods. Three out of ten Sasco hamsters fed the 1.2% palmitic acid diet for two weeks had cholesterol stones, while none of the eight Charles River animals had stones. In the Sasco hamsters, a significant proportion of the biliary cholesterol was found in void volume vesicles (28.8%) and small vesicles (17.1%); Charles River hamsters had negligible proportions (1.1%) of cholesterol in void volume vesicles and 15.4% in small vesicles. Cholesterol gallstones were most abundant in Sasco hamsters fed 1.2% palmitic acid for six weeks (nine out of ten animals); the mean cholesterol saturation index of the bile was 1.27. A significant proportion of the biliary cholesterol was eluted in the void volume vesicles (21.4%) and in small vesicles (15.0%). Five of the eight identically treated Charles River hamsters had cholesterol stones; the cholesterol saturation index averaged 1.36, and the biliary cholesterol was present in void volume vesicles (31.3%) and small vesicles (14.3%). Vesicles were not detected in the bile of hamsters fed cholesterol-free diets, and none of these animals developed cholesterol gallstones. Safflower oil diets inhibited stone formation even though the cholesterol saturation index was above unity. After six weeks, biliary cholesterol transported in void volume vesicles was highest for Sasco hamsters (13.3%) as compared to Charles River animals (6.9%), but total cholesterol transported in void volume vesicles plus small vesicles was similar in both groups (33.5% vs. 26.2%), respectively. These results suggest that in both strains of hamsters dietary fat influences gallstone formation by modulating the vesicular/micellar distribution of biliary cholesterol. Apparently, the presence of cholesterol/phospholipid vesicles in bile is associated with cholesterol gallstone formation.

Sterol balance studies, using both isotopic and chromatographic techniques, were carried out in hamsters fed semipurified diets to detect changes in sterol metabolism during the early period of the lithogenic stimulus. The balance studies examined animals in the first two weeks on the experimental lithogenic diets. The variables were as follows: dose of cholesterol (group 1, 0.05% vs. group 2, 0.2%); dietary fat (fatty acid) (group 2, butterfat vs. group 4, palmitic acid); source of hamster [group 2, Sasco (Omaha, NE) vs. group 3, Charles River (Wilmington, MA)]; average weight of animals (group 4, 60 g vs. group 5, 119 g). Animals in groups 1, 2, 3 and 5 maintained almost constant weight throughout the two-week balance study. Liver and plasma cholesterol levels increased in groups 2-5 with increasing dose of dietary cholesterol. The highest levels were found in group 4 (liver cholesterol, 32.7 mg/g; plasma cholesterol, 367 mg/dL). Sterol balance measurements showed that bile acid synthesis remained low (range 0.55-1.01 mg/d) for all groups regardless of the intake of dietary cholesterol (range, 3.27-20.90 mg/d). The dietary cholesterol absorbed from the intestine (range, 2.91-18.91 mg/d) was stored in the liver; this storage was reflected in the negative values for cholesterol balance for all groups (range, -0.70 to -14.97 mg/d). These studies did not reveal any correlations between parameters of sterol balance and cholelithiasis.

In the present study, we examined the effect of the following factors on a hamster model of cholesterol cholelithiasis: (i) the source of the golden Syrian hamsters (Sasco, Omaha, NE or Charles River, Wilmington, MA), (ii) the sex of the experimental animals and (iii) their age (4 wkvs. 8 wk of age). All hamsters were fed a semipurified diet which contained cholesterol (0.3%) and palmitic acid (1.2%). No cholesterol gallstones formed in any of the female hamsters regardless of age or source. The 4-week-old male hamsters from Sasco had the greatest incidence of gallstones (93%). The 8-week-old male hamsters tended to have a lower incidence of cholesterol gallstones than the younger ones, regardless of the commercial supplier (67vs. 93% for Sasco and 27vs. 40% for Charles River). Female hamsters has higher liver and serum cholesterol levels than the male hamsters; Charles River hamsters had lower serum cholesterol concentrations than the Sasco animals. Total biliary lipid concentrations were highest in Sasco male hamsters, but biliary cholesterol (mol%) was lower in the males than in the females (4.2–4.5%vs. 6.1–7.1%) regardless of age. The cholesterol saturation indices were higher in the Sasco females than the corresponding males; these values were lower in the Sasco hamsters than the Charles River animals, regardless of age or sex. The male Sasco hamsters had a higher total biliary bile acid concentration (98.9 mg/mL) than the Sasco females (58.9 mg/mL) and the Charles River animals (24.6% mg/mL for males and 38.2 mg/mL for females). The percentage of chenodeoxycholic acid in bile was significantly lower, and the percentage of cholic acid was higher in all females as compared to males. We conclude that there is a sex, age and “strain” difference in cholesterol cholelithiasis in hamsters; it is important to consider these factors when working with the hamster model of gallstone disease. All female hamsters were markedly resistant to the induction of cholesterol gallstone disease.

The metabolism of sodium 3 alpha,7 beta-dihydroxy-5 beta-cholane-24-sulfonate and sodium 3 alpha,7 beta-dihydroxy-24-nor-5 beta-cholane-23-sulfonate was studied in hamsters. In bile fistula animals these sulfonate analogs of ursodeoxycholic acid were absorbed mainly from the terminal ileum and secreted rapidly into the bile without biotransformation or conjugation. After oral administration, the sulfonate analogs were excreted in the feces at the same rate as chenodeoxycholic acid and its metabolic products. The intestinal microorganisms transformed chenodeoxycholic acid largely into lithocholic acid; the sulfonate analogs were completely resistant to biotransformation. After a 2-week feeding period, the sulfonate analogs of ursodeoxycholic acid accounted for 24.0% and 16.9% of total biliary bile acids. These sulfonates did not affect the proportions of the natural bile acids in the bile, and the ratio of glycine-conjugated bile acids to taurine-conjugated bile acids was not altered by feeding the sulfonates. In contrast, when ursodeoxycholic acid was fed, the proportions of the natural bile acids and the glycine/taurine ratio were changed. These results suggest that the sulfonate analogs had no profound effect on endogenous bile acid metabolism and did not cause a depletion of the hepatic taurine pool during enterohepatic circulation. The sulfonates had no effect on intestinal cholesterol absorption and serum cholesterol levels.

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)

In an established hamster model of cholesterol cholelithiasis, a semipurified lithogenic diet containing 4% butterfat and 0.3% cholesterol leads to the production of cholesterol gallstones in only 50-60% of animals after a 6-wk feeding period. The purpose of this study was to investigate whether gallstone incidence could be increased while feeding a nutritionally adequate diet of moderate cholesterol content. The semipurified lithogenic diet was modified as follows: (i) substitution of 1.2% palmitic acid for 4% butterfat, and (ii) varying the amount of dietary cholesterol from 0.0 to 0.3% with either butterfat or palmitic acid as the lipid component of the diet. Substitution of palmitic acid for butterfat produced a significantly higher incidence of cholesterol gallstones (94% vs. 53%). Palmitic acid also raised the incidence of gallstones when added to the 0.1% and 0.2% cholesterol diets as compared to butterfat: 0% vs. 44% and 50% vs. 81%, respectively. Gallstone incidence increased from 0% to nearly 100% when the cholesterol content of the palmitic acid diets was raised from 0.0% to 0.3%, indicating a dose response effect with respect to dietary cholesterol. Hamsters fed cholesterol-free diets did not form gallstones. Increased dietary cholesterol led to increased liver weight associated with a significant increase in liver cholesterol concentration. However, the palmitic acid groups had significantly lower liver cholesterol values than the corresponding butterfat groups. Serum and biliary cholesterol concentrations increased with increasing dietary cholesterol intake, but there were no differences between the butterfat and palmitic acid groups.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of aspirin on cholesterol cholelithiasis was examined in the hamster and the prairie dog. In the prairie dog, diets were composed of semipurified components of chow, plus cholesterol (1.2%), with and without aspirin. Animals were studied for either 2 weeks or 4 weeks. Cholesterol gallstones were present in all groups at the end of each period; aspirin did not alter the incidence of cholelithiasis. All animals studied had cholesterol crystals in the bile when they were killed. Liver cholesterol levels in prairie dogs with and without aspirin tended to be lower in animals fed chow than in animals fed semipurified diets. There were no significant differences in cholesterol levels in the plasma or bile. The cholesterol saturation index of all biles approached unity when animals were fed chow with aspirin; animals fed the semipurified diets had cholesterol saturation indices of less than 1.0. The prairie dogs fed aspirin plus cholesterol in the semipurified diet showed increased levels of biliary chenodeoxycholic acid amidates and concomitant decreased levels of cholic acid amidates compared with animals fed the same diet without aspirin. Hamsters fed aspirin plus cholesterol in a semipurified diet tended to have a greater incidence of gallstones than animals given no aspirin (80% vs. 55%). Liver and bile cholesterol levels were similar with and without aspirin; plasma cholesterol levels increased significantly with aspirin [14.20 vs. 7.80 mmol/L (549 vs. 301 mg/dL)]. Lithogenic indices in all hamsters were above unity; biliary lipids, total lipid concentration, and biliary bile acid composition were similar. These results show that the addition of aspirin to a lithogenic diet does not reduce the incidence of cholelithiasis.

The metabolism of 7-ethyl- and 7-propyl-chenodeoxycholic acids was studied in hamsters. Both bile acid analogs were absorbed efficiently by the intestine and secreted into the bile at rates similar to those of chenodeoxycholic acid. After intraduodenal administration into bile fistula hamsters, the 7-alkyl analogs were present in bile as the glycine and taurine conjugates. The glycine/taurine ratios were: chenodeoxycholic acid, 1.9; 7-ethyl analog, 0.3; and 7-propyl analog, 0.2. After oral administration, during a 21-day feeding experiment, the 14C-labeled analogs were recovered quantitatively in the feces. Chenodeoxycholic acid was largely 7-dehydroxylated to lithocholic acid in the intestinal tract. In contrast, the 7 alpha-hydroxy group of the 7-alkyl bile acids was completely resistant to bacterial action. 7-Ethyl-chenodeoxycholic acid was transformed in part to a compound tentatively identified as 7 alpha-hydroxy-3-oxo- 7 beta-ethyl-5 beta-cholanoic acid while 7-propyl-chenodeoxycholic acid was excreted unchanged. In the hamsters used, the 7-alkyl bile acid analogs did not inhibit the bacterial dehydroxylation of chenodeoxycholic acid. At the end of the 21-day feeding period, analysis of the gallbladder bile showed that 7-methyl-, 7-ethyl-, and 7-propyl-chenodeoxycholic acids accounted for 38, 31, and 12% of total bile acids, respectively. The 7-alkyl bile acids decreased cholesterol absorption; the 7-propyl analog caused significant decrease in serum and liver cholesterol concentration. These experiments demonstrate that the 7-ethyl- and 7-propyl chenodeoxycholic acids, just like the 7-methyl-analog, are absorbed by the intestine and participate in the enterohepatic circulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolism of sodium 3 alpha,7 alpha-dihydroxy-5 beta-cholane-24-sulfonate, the sulfonate derivative of chenodeoxycholic acid, was studied in hamsters. In bile fistula hamsters, the sulfonate analogue was efficiently absorbed from the ileum and secreted rapidly into the bile without any modification such as conjugation. However, absorption from the jejunum was smaller than that observed for the ileum. After oral administration, the sulfonate analogue of chenodeoxycholic acid was recovered quantitatively in the feces as the unchanged form in contrast to simultaneously administered chenodeoxycholic acid, which was entirely converted to lithocholic acid during its passage through the intestinal tract. These results demonstrate that the sulfonate analogue is absorbed mainly from the ileum by active transport, enters the enterohepatic circulation like the endogenous conjugated bile acids, and completely resists bacterial degradation.

This study was designed to elucidate the effect of the synthetic androgen, methyltestosterone, on bile flow and biliary lipid secretion in female hamsters. Animals were divided into four groups and fed the following diets: group 1, lithogenic diet for three weeks; group 2, lithogenic diet + 0.05% methyltestosterone for three weeks; group 3, lithogenic diet for six weeks; group 4, lithogenic diet + 0.05% methyltestosterone for six weeks. At the end of each experimental period, the hamsters were operated on to establish external biliary fistulas. During the depletion of the endogenous bile acid pool (for two hours), the basal bile flow of group 4 was significantly smaller than that of group 3. Basal bile acid output was significantly lower in the methyltestosterone-fed groups 2 and 4 than in control groups 1 and 3. In contrast, groups 2 and 4 secreted more cholesterol than groups 1 and 3. Group 4 had a higher ratio of cholesterol output to phospholipid output than group 3. Increasing doses of taurocholate were infused after the bile acid depletion period, and it was found that methyltestosterone did not change the bile acid independent bile flow. The increments in cholesterol or phospholipid output induced per increment of bile acid output (linkage coefficients) were analyzed by linear regression. The methyltestosterone-fed groups (groups 2 and 4) had a higher linkage coefficient of cholesterol output to bile acid output than the control groups (groups 1 and 3). The linkage coefficients of phospholipid output to bile acid output of groups 2 and 4 were also higher compared to groups 1 and 3. The linkage coefficient of cholesterol output to phospholipid output of group 2 was higher than that of group 1. These results suggest that methyltestosterone stimulated the cosecretion mechanism of cholesterol and phospholipid in bile associated with an increasing ratio of cholesterol to phospholipid. In conclusion, the synthetic androgen, methyltestosterone, caused a decrease in basal bile flow and bile acid secretion, and an increase in basal cholesterol secretion and the biliary cholesterol-to-phospholipid ratio. These findings explain, in part, how methyltestosterone intensifies the formation of cholesterol gallstones in female hamsters.

Vesicles and micelles, the major carriers of cholesterol in bile, play a role in the formation of cholesterol gallstones. A simple and rapid ultracentrifugation method was developed to isolate these biliary cholesterol carriers when only microliter amounts of bile were available. The proposed method employs a 46 to 0% sucrose density gradient, a NVT90 near-vertical rotor, and a centrifugation time of one hour. As little as 25 microL of bile can be used with no disruption of the carriers. The method was validated by comparison with gel filtration column chromatography using 6 mM taurocholate in the elution buffer. The sucrose linear density gradient ultracentrifugation procedure described here is simple, fast, and compares favorably with the gel filtration chromatography method for the separation of cholesterol carriers from bile.

The effects of β-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% β-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% β-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% β-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 β-muricholic acid group. In the prevention study in hamsters (B), β-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. These studies suggest that, in the hamster animal model, hydrophilic bile acids may be useful for the prevention of gallstones but not dissolution of preestablished cholesterol gallstones.

This paper reports the chemical synthesis of a new bile acid analogue, namely sodium 3 alpha,7 alpha-dihydroxy-25,26-bishomo-5 beta-cholane-26-sulfonate (bishomoCDC-sul) from chenodeoxycholic acid and describes its metabolism in the hamster. The structure of the new compound was confirmed by proton and carbon-13 nuclear magnetic resonance spectroscopy. After intravenous infusion of [3H]-labeled sulfonate into bile fistula hamsters, it was extracted by the liver and secreted into the bile; more than 65% of the radioactivity was recovered in the bile within 1 h. Following intraduodenal administration of the [3H]sulfonate and [14C]chenodeoxycholyltaurine, both compounds were excreted into the bile more slowly; only 41 and 43% of the radioactivity, respectively, were recovered in the bile during the four-hour experimental period. In contrast, when the labeled compounds were injected into the terminal ileum, both the sulfonate and chenodeoxycholyltaurine were rapidly absorbed and secreted into the bile; 84 and 97%, respectively, of the radioactivity were recovered during a four-hour period. Chromatographic analysis demonstrated that in these short-term experiments most (95%) of the sulfonate was secreted into the bile without biotransformation regardless of the route of administration. When infused intravenously at increasing doses, bishomoCDC-sul induced cholestasis at an infusion rate of 1 mumol/min/kg. These results suggest that sodium 3 alpha,7 alpha-dihydroxy-25,26-bishomo-5 beta-cholane-26-sulfonate was absorbed from the terminal ileum by active transport, extracted by the liver, and secreted into the bile in a manner similar to that of the natural bile acids.

We tested two hypotheses, i) whether the type and the amount of fat in the diet will affect the formation of cholesterol gallstones in the hamsters, and ii) whether palmitic acid, a major fatty acid component of butterfat, can act as a potentiator of cholesterol cholelithiasis in the hamster. Young, male golden Syrian hamsters (Sasco) were fed a semipurified diet containing casein, corn starch, cellulose and cholesterol (0.3%) to which various types and amounts of fat (butterfat, olive oil, menhaden oil, corn oil) were added. All diets contained 2% corn oil to supply essential fatty acids to the growing hamsters. No deaths or illness occurred during the experiment. Animals fed the semipurified diet plus 4% butterfat (group 1) had a gallstone incidence of 63%. Replacement of butterfat with either olive oil, corn oil or menhaden oil prevented the formation of cholesterol gallstones entirely (groups 2–4). When total butterfat was increased from 4% to 8% (group 8), the incidence of cholesterol gallstones increased to 80%. Substitution of 4% olive oil (group 5), corn oil (group 6), or menhaden oil (group 7) for the additional 4% butterfat significantly reduced gallstones to 35%, 45% and 30%, respectively. The replacement of 4% butterfat with 1.2% palmitic acid gave the highest incidence of cholesterol gallstones (95%). These results suggest that butterfat (and one of its components, palmitic acid) intensifies gallstone formation in this model whereas mono- and polyunsaturated fats act as inhibitors of cholesterol cholelithiasis. A fatty acid, possibly palmitic acid, appears to act as lithogen in our model.

The effect of two hydrophilic bile acids, murideoxycholic acid (3α,6β-dihydroxy-5β-cholanoic acid) and ursodeoxycholic acid, on cholesterol and bile acid metabolism and hepatic pathology and gallstone composition was studied in the prairie dog. Cholesterol gallstones were induced by feeding a diet containing 1.2% cholesterol for 75 days. The animals were divided into six groups, and gallstone regression was studied as follows: groups 2 and 5, chow plus 0.2% cholesterol; groups 3 and 6, chow plus 0.2% cholesterol plus 0.15% ursodeoxycholic acid; groups 4 and 7, chow plus 0.2% cholesterol plus 0.15% murideoxycholic acid. Animals in groups 2 to 4 were killed after an additional 6 wk; animals in groups 5 to 7 were killed after an additional 12 wk. Gallstone dissolution did not occur in any group. The gallstones in groups 2, 3, 5 and 6 were typical cholesterol aggregates, as determined by polarized light microscopy and Fourier transform infrared spectrometry. The gallstones of the murideoxycholic acid group were large, solitary, dark stones that appeared radiopaque under 22 kVp x-ray examination. Scanning electron microscopy showed that in these stones the cholesterol crystals had been replaced by an amorphous material, both within the stone and on the stone surface. Chemical analysis indicated that at the end of 12 wk the calcium/sodium salt of the taurine conjugate of murideoxycholic acid (murideoxycholyl taurine) comprised 70% of the stones; protein, cholesterol and small amounts of other bile salts were also present. In vitro studies confirmed the insolubility of the sodium and calcium salts of murideoxycholyl taurine. These studies indicate that the hydrophilic bile acids, murideoxycholic acid and ursodeoxycholic acid, did not achieve gallstone dissolution under the conditions used. In the animals fed murideoxycholic acid, an insoluble calcium salt of murideoxycholyl taurine replaced cholesterol as the major constituent of gallbladder stones. This is the first example of an insoluble dihydroxy taurine-conjugated bile acid; administration of the unconjugated bile acid induced precipitation of a kind of gallstone not previously reported. The final result was transformation of cholesterol stones to bile salt stones. (HEPATOLOGY 1991;14:158–168.)

Cholesterol gallstones were present in prairie dogs fed alfalfa plus corn with and without exogenous cholesterol (0.4%). The diets fed to the animals for eight weeks contained alfalfa plus corn in fixed proportions of 50∶50, 85∶15 and 15∶85 (w/w). At sacrifice, all animals were healthy but had not gained weight; no deaths occurred during the experiment. Cholesterol gallstones were present in all groups. In the absence of exogenous cholesterol, the highest stone incidence was found in the animals which received the lowest fiber (highest corn) diets (alfalfa plus corn, 50∶50, 67%; alfalfa plus corn, 15∶85, 83%). Cholesterol gallstone incidence was 100% when exogenous cholesterol was added to the alfalfa plus corn diets (50∶50 and 15∶85). No pigment gallstones were detected in any animal. Liver and plasma cholesterol concentrations were highest in the animals receiving alfalfa plus corn (15∶85) plus 0.4% cholesterol (4.29 mg/g, and 356 mg/dl, respectively). These values were lowest in animals receiving 85% alfalfa plus 15% corn without cholesterol (2.19 mg/g and 88 mg/dl, respectively). Lithogenic indices were below 1.00 in all groups. Biliary bile acids were mainly amidates of cholic acid and chenodeoxycholic acid, with the former predominating. Thus, gallstones can be formed in prairie dogs in the absence of exogenous cholesterol; gallstone incidence is reduced by dietary fiber.

The aim of the present study is to examine the efficacy of 6-hydroxy substituted bile acids on the prevention of cholesterol gallstones in a new hamster model of cholesterol cholelithiasis. Male golden Syrian hamsters were fed a nutritionally adequate semipurified lithogenic diet consisting of casein, cornstarch, soluble starch, butterfat, corn oil, and cellulose plus 0.3% cholesterol. Six different bile acids were added to this diet at the 0.05% level: chenodeoxycholic acid, ursodeoxycholic acid, hyodeoxycholic acid, murideoxycholic acid, 6 beta-methyl-hyodeoxycholic acid, and 6 alpha-methyl-murideoxycholic acid. At the end of the 6-wk feeding period, the control group receiving the lithogenic diet had a 55% incidence of gallstones. It was found that all bile acids had inhibited the formation of cholesterol gallstones; complete prevention of gallstones was observed with all 4 3,6-dihydroxy bile acids, whereas chenodeoxycholic acid and ursodeoxycholic acid were somewhat less effective (80% and 75% prevention, respectively). The accumulation of cholesterol in serum and liver induced by the lithogenic diet was inhibited to some extent by all of the bile acids; hyodeoxycholic acid, murideoxycholic acid, and 6 beta-methyl hyodeoxycholic acid were most effective in this respect. The administered bile acids tended to predominate in bile in the case of chenodeoxycholic acid, hyodeoxycholic acid, and 6 beta-methyl-hyodeoxycholic acid. In contrast, ursodeoxycholic acid seemed to be converted to chenodeoxycholic acid and murideoxycholic acid to hyodeoxycholic acid. Only 4% of the 6-methyl analogue of murideoxycholic acid, 6 alpha-methyl-murideoxycholic acid, was recovered in gallbladder bile. These experiments show that the new hamster model of cholesterol cholelithiasis is suitable for gallstone-prevention studies. It was not possible to draw definite conclusions concerning the mechanism of action of the administered bile acids on the basis of cholesterol saturation or the presence of liquid crystals. The detailed mechanism of gallstone prevention by hydrophilic bile acids in this model remains to be elucidated.

Hyodeoxycholic acid and its isomer, 6 beta-hyodeoxycholic acid, when added to a lithogenic diet prevented the formation of cholesterol gallstones and crystals in prairie dogs. This beneficial effect occurred in the presence of bile supersaturated with cholesterol. Hyodeoxycholic acid abolished the feedback inhibition of hepatic hydroxymethylglutaryl coenzyme A reductase activity, the rate-limiting enzyme of cholesterol synthesis, and prevented elevations in serum and liver cholesterol observed in animals fed a 0.4 percent cholesterol diet. The gallbladder bile of the animals fed hyodeoxycholic acid and 6 beta-hyodeoxycholic acid contained abundant liquid crystals. This suggests that these bile acids prevented the transition of cholesterol from its liquid crystalline phase to solid crystals and stones.

This paper describes the conversion of 5beta-cholestane-3alpha,7alpha,12alpha,25-tetrol into 5beta-cholestane-3alpha,7alpha,12alpha,24beta,25-pentol by liver microsomes. A sensitive radioactive assay for measuring the formation of 5beta-cholestane-3alpha,7alpha,12alpha,24beta,25-pentol was developed. Optimal assay conditions for human and rat microsomal systems were established. A higher 24beta-hydroxylation activity was detected in rat than in human liver under the conditions employed. The hydroxylation of 5beta-cholestane-3alpha,7alpha,12alpha,25-tetrol by the rat liver microsomal fraction fortified with NADPH was stimulated about two-fold by administration of phenobarbital. Phenobarbital treatment also stimulated hydroxylations at C-23, C-24alpha, and C-26. Carbon monoxide markedly inhibited all side-chain hydroxylations. In contrast, side-chain hydroxylase activities were not affected in animals deprived of food for 48 hr. These results are consistent with a previously postulated cholic acid biosynthetic pathway involving 5beta-cholestane-3alpha,7alpha,12alpha,24beta,25-pentol as a key intermediate in man and in the rat.

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.

This paper describes studies dealing with the nature of the C27 pentahydroxy bile alcohols present in the bile and feces of two patients with cerebrotendinous xanthomatosis (CTX). The presence of a bile alcohol having the structure 5β-cholestane-3α,7α,12α,24α,25-pentol was confirmed by separation of the two 24-hydroxy epimers of 5β-cholestane-3α,7α,12α,24,25-pentol and characterization of the dpimers by gas-liquid chromatography and infrared and mass spectrometry. Tentative assignment of the 24α and 24β configuration was made on the basis of molecular rotation differences. A second major bile alcohol excreted by the CTX subjects was 5β-cholestane-3α,7α,12α,23xi,25-pentol. Its structure was determined by infrared spectrometry, proton magnetic resonance spectrometry, and mass spectrometry because a reference compound was not available.

Feces from normal subjects and patients with cerebrotendinous xanthomatosis were incubated anaerobically with labeled chenodeoxycholic acid and ursodeoxycholine acid for known periods, and the bile acids formed were analyzed by TLC and scintillation counting. In the normal subjects, 80% of the chenodeoxycholic acid and 41% of the ursodeoxycholic acid were 7-dehydroxylated to lithocholic acid during 2 hr of incubation. In contrast, the fecal flora of the CTX patients transformed only 5% of chenodeoxycholic acid and less than 1% of ursodeoxycholic acid to lithocholic acid during the same time period. In several subjects (normals and CTX), the intestinal flora converted chenodeoxycholic acid to ursodeoxycholic acid without the accumulation of the hypothetical intermediate 7-ketolithocholic acid (3 alpha-hydroxy-7-keto-5 beta-cholanoic acid). These results indicate that the fecal bacterial flora is capable of 7-dehydroxylating chenodeoxycholic acid and ursodeoxycholic acid to yield lithocholic acid. Apparently the enzymes involved are relatively stereospecific since the 7 beta-hydroxy group of ursodeoxycholic acid was removed more slowly than the 7 alpha-hydroxy group of chenodeoxycholic acid.

The metabolism of ursodeoxycholic acid was investigated in 7 subjects during treatment with ursodeoxycholic acid, 1 g per day and chenodeoxycholic acid, 1 g per day for 2 weeks. The molar proportions of biliary cholesterol, lecithin, and bile salts were determined before and during bile acid treatment and the pool sizes and daily turnover of ursodeoxycholic acid were measured by isotope dilution techniques after administration of [24-14C]ursodeoxycholic acid. Ursodeoxycholic acid became the major biliary bile acid (56%) during ursodeoxycholic acid treatment while the proportions of chenodeoxycholic acid declined from 32 to 18%, and cholic acid from 38 to 17%. Deoxycholic acid and lithocholic acid were virtually absent from the bile. During chenodeoxycholic acid administration, chenodeoxycholic acid became the major biliary bile acid (59%), cholic acid declined to 22% of the biliary bile acids, while the proportion of lithocholic acid rose from 1.3 to 3.4%, and an additional 4% were sulfated. Ursodeoxycholic acid increased to 8% of the total bile acids. The molar percent of biliary cholesterol decreased from 8 to 5% during both ursodeoxycholic acid and chenodeoxycholic acid administration. After [24- 14C]ursodeoxycholic acid was administered intravenously the biliary specific activity- time curves of ursodeoxycholic acid decayed exponentially during both bile acid treatment periods. In the ursodeoxycholic acid-treated subjects, the ursodeoxycholic acid decay curve intersected with the rising specific activity curve of chenodeoxycholic acid and revealed a precursor-product relationship. The average ursodeoxycholic acid pool measured 938 ± 458 mg with a daily production rate of 900 ± 438 mg per day (absorption). The average ursodeoxycholic acid pool during chenodeoxycholic acid treatment measured 123 ± 82 mg and the production rate was 91 ± 35 mg per day (synthesis). These studies demonstrate that substantial quantities of ursodeoxycholic acid are absorbed when this bile acid is fed or are synthesized when chenodeoxycholic acid is given. The reduction in bile lithogenicity during ursodeoxycholic acid administration implies that this bile acid like chenodeoxycholic acid may be effective in dissolving cholesterol gallstones in man.

The effect of dietary 7 beta-methyl-cholic acid [0.075% in rodent chow (6.4 mg/animal per day)] on cholesterol and bile acid metabolism was studied and compared with that of cholic acid in the hamster. Following oral administration of 7 beta-methyl-cholic acid for 3 weeks, the glycine-conjugated bile acid analog became a major constituent of gallbladder bile. Biliary cholic acid concentration decreased significantly, while that of chenodeoxycholic acid remained unchanged. Serum and liver cholesterol levels were increased by dietary 7 beta-methyl-cholic acid and by cholic acid. Hepatic microsomal HMG-CoA reductase activity was inhibited (30% of the control value) by both bile acids; cholesterol 7 alpha-hydroxylase activity was not affected. In chow controls and cholic acid-fed animals, bacterial 7-dehydroxylation of [14C]chenodeoxycholic acid and [14C]cholic acid was nearly complete. In contrast, dietary 7 beta-methyl-cholic acid effectively prevented the 7-dehydroxylation of the two primary bile acids. These results show that dietary 7 beta-methyl-cholic acid is preserved in the enterohepatic circulation and has an effect on serum and liver cholesterol concentrations similar to those produced by the naturally occurring cholic acid. 7 beta-Methyl-cholic acid is an efficient inhibitor of the bacterial 7-dehydroxylation of the primary bile acids in the hamster.

Sterol metabolism studies using a combination of thin-layer chromatography, gas-liquid chromatography and isotopic methods were carried out in rats fed diets containing: (a) control chow (group 1); (b) 0.8% β-sitosterol (group 2); (c) 0.8% β-sitosterol plus 0.5% sodium taurochenodeoxycholate (group 3); and (d) 0.8% β-sitosterol plus 0.5% sodium taurocholate (group 4). Feeding β-sitosterol led to an inhibition of cholesterol absorption, increased cholesterol output (cholesterol balance, 28.8 mg/day vs. 19.5 mg/day in controls) and no significant increase in bile acid synthesis. Rats receiving sodium taurochenodeoxycholate or sodium taurocholate in addition to β-sitosterol showed: (a) decreased levels of bile acid synthesis; (b) low cholesterol absorption; and (c) increased output of fecal and endogenous neutral sterols. Levels of cholesterol in plasma were not significantly different from those in the control animals. Liver cholesterol levels were significantly lower in the taurochenodeoxycholate plus β-sitosterol (group 3) vs. the taurocholate plus β-sitosterol animals (group 4). However, biliary cholesterol concentrations were elevated in both bile acid-plant sterol groups (0.38 mg/ml in group 3; 0.44 mg/ml in group 4 vs. 0.19 mg/ml in group 1). There were no significant differences in sterol metabolism between groups of rats fed sodium taurochenodeoxycholate plus β-sitosterol compared to rats fed sodium taurocholate plus β-sitosterol.

Sterol balance measurements using isotopic and chromatographic techniques were carried out in rats fed diets containing beta-sitosterol (0.8%) and cholesterol (1.2%). The activities of the rate-limiting enzymes of cholesterol synthesis (beta-hydroxy-beta-methylglutaryl-CoA reductase, EC 1.1.1.34) and bile acid synthesis (cholesterol 7 alpha-hydroxylase) were determined in the same animals. Cholesterol feeding increased cholesterol absorption from 1.2 to 70 mg/day. The increased absorption was compensated for by inhibition of hepatic cholesterol synthesis, enhanced conversion of cholesterol to bile acids (from 13.7 to 27.3 mg/day) and a slight increase in the excretion of endogenous neutral steroids (from 7.7 to 11.2 mg/day). Despite the adaptation there was accumulation of cholesterol in the liver (from 2.2 to 9.2 mg/g). Beta-Sitosterol feeding inhibited cholesterol absorption (calculated absorption was zero). In these rats there was enhanced cholesterol synthesis (from 20.0 to 28.8 mg/day, but no change in the rates of bile acid formation. Measurements of the activities of the rate-limiting enzymes showed fair correlation with cholesterol-bile acid balance. In cholesterol fed animals, beta-hydroxy-beta-methylglutaryl-CoA reductase was inhibited 80% and cholesterol 7 alpha-hydroxylase was enhanced 61%. In beta-sitosterol-fed animals, the reductase was increased 2-fold and cholesterol 7 alpha-hydroxylase was not significantly different from controls.

Sterol metabolism studies using a combination of isotopic and chromatographic procedures were carried out in rats fed either a low-cholesterol stock diet or a stock diet containing 0.1% cholesterol. The primary bile acids, sodium taurochenodeoxycholate and sodium taurocholate, were added to the stock diets at a level of 0.5%, as required. Feeding sodium taurochenodeoxycholate and sodium taurocholate led to a decrease in acidic steroid synthesis, cholesterol turnover, and cholesterol balance, compared to controls. Sodium taurochenodeoxycholate feeding did not influence cholesterol absorption, but rats fed sodium taurocholate showed a twofold increase in cholesterol absorption as well as an accumulation of cholesterol in the liver. Rats receiving diets containing sodium taurochenodeoxycholate or sodium taurocholate plus cholesterol (0.1%) showed decreased acidic steroid synthesis, cholesterol turnover, and cholesterol balance, compared to the corresponding controls (Group 2, high cholesterol diet). Significantly larger amounts of cholesterol were absorbed in the taurocholate group (34.1 mg/day); these animals increased their concentrations of cholesterol in liver and plasma. The rats fed taurocholate plus 0.1% cholesterol differed from those fed taurochenodeoxycholate plus 0.1% cholesterol in the following respects: a) increased cholesterol absorption (35%), and b) accumulation of cholesterol in liver and plasma.

The metabolic fate of the bile add analogs, 3α,7α-dihydroxy-7β-methyl-5β-cholanoic acid and 3α,7β-dihydroxy-7α-methyl-5β-cholanoic acid, was investigated and compared with that of chenodeoxycholic acid in hamsters. Both bile acid analogs were absorbed rapidly from the intestine and excreted into bile at similar to that of chenodeoxycholic acid. In the strain of hamster studied, the biliary bile were conjugated with both glycine and taurine. After continuous intravenous infusion, chenodeoxycholic acid the analogs became the major bile acid constituents in bile. After oral administration of a single dose of these compounds, fecal analysis revealed the existence of unchanged material (25–35%) as well as considerable amounts of metabolites (65–75%). The major metabolites excreted into feces were more polar than the starting material and were tentatively identified as trifaydroxy-7-methyl compounds by radioactive thin-layer chromatography. However, monohydroxy compounds were also found in the fecal extracts. These results show that chenodeoxycholic acid and ursodeoxycholic acid with a methyl group at the 7-position are resistant to bacterial 7-dehydroxylation than the normally occurring bile acids and that a certain proportion of these analogs is hydroxylated to give the corespondiag trihydroxy compound(s), In a control experiment, about 5% of administered chenodeoxychoulic acid was metabolized to a trihydroxy feile acid, but most of the compound (95%) was transformed into lithocholic acid.

This report describes the gas-liquid chromatography-mass spectrometry (GLC-MS) of the trimethylsilyl ethers of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol with mono- or dihydroxy substitution in the side chain. Compounds with 24- and 25-unsaturation in the side chain were also studied. The gas-liquid chromatographic separation of the different bile alcohols was carried out using 3% QF-1 and 1% HI-EFF 8BP as column packings. Both columns were useful in that the retention times of the trimethyl-silyl ethers of the various 5 beta-cholestanetetrols varied linearly with the position of the side-chain trimethylsiloxyl substituent. The major fragmentations in the GLC-MS of all bile alcohols tested were due to the trimethylsiloxyl side-chain substituent(s). A trimethylsiloxyl group at C-22 was the most effective in promoting side-chain fragmentation, followed, in order of decreasing effectiveness, by substituents at carbons 25, 23, 24, and 26. The side-chain fragments generally gave rise to one or two intense mass peaks and a series of weaker peaks, 90 mass units apart, owing to the loss of successive molecules of trimethylsilanol. Most 5 beta-cholestane-triols, -tetrols, and -pentols can be unequivocally identified by their strong side-chain fragmentation peaks, except for 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha, 26-tetrol, the trimethylsilyl ether of which exhibited very low intensity side-chain fragments.

Oxidation products of cholesterol have been shown to be potent inhibitors of cholesterol biosynthesis and also highly toxic to cultured aortic smooth muscle cells. In rabbit experiments, these compounds produced arterial injury resulting in arteriosclerosis. Purified cholesterol only minimally inhibited cholesterol biosynthesis and had no effect on cultured aortic smooth muscle cells. This raises the possibility that plasma lipoproteins containing β-apoprotein (i.e. LDL and VLDL), which are considered to be atherogenic, may carry more oxidation products than HDL which is not atherogenic [ 3 H] 25-hydroxycholesterol and [ 14 C]cholesterol were given orally to 10 squirrel monkeys ( Saimiri sciureus ) and blood samples were collected via femoral puncture 24 h after administration. Lipoproteins were separated by ultracentrifugation and the radioactivity in each fraction was counted. Results show that the distribution of labeled cholesterol in VLDL, LDL, and HDL was almost identical to that of unlabeled cholesterol. Most of the radio-activity of 25-hydroxycholesterol was located in LDL & VLDL (55.1% and 34.7%, respectively), only 10.2% was present in HDL. If the radioactivity of 25-hydroxycholesterol were calculated on the basis of the apoprotein content of the lipoprotein micelle, the relative capacity of VLDL and LDL to carry 25-hydroxycholesterol was even greater and more significant than that of HDL (90 × and 42 ×, respectively).

Dietary cholic acid (0.1%) and/or calcium (2.6% as calcium carbonate) were added to a semipurified diet containing cholesterol and ethynyl estradiol to determine whether the incidence of pigment and/or cholesterol gallstones would be changed. Male golden Syrian hamsters were fed the experimental diets for 96 days (Group 1, control; Group 3, cholic acid plus calcium) or only an average of 60 days (Group 2, 0.1% cholic acid). Animals in Group 2 became ill (weight loss, low food intake, diarrhea) possibly due to cholic acid (or deoxycholic acid) toxicity. Cholesterol gallstones and crystals were absent in all experimental groups. The incidence of pigment gallstones was: control, Group 1, 12/16; 0.1% cholic acid, Group 2, 3/13; and 0.1% cholic acid plus calcium, Group 3, 11/22. Cholic acid with or without calcium produced an elevation of both liver and plasma cholesterol: Group 2, 80.1 mg/g and 501 mg/dl; Group 3, 103.7 mg/g and 475 mg/dl vs Group 1, 65 mg/g and 209 mg/dl, respectively. The lithogenic indices of the bile were lower in Groups 2 and 3 compared to Group 1, controls, 0.45 and 0.58 vs 1.16, respectively. The extent of the portal tract pathology could not be correlated with the presence or absence of pigment gallstones or with the levels of lithocholic acid in the hamster bile. In summary, when semipurified diets were supplemented with ethynyl estradiol and cholic acid, with and without calcium supplementation, no cholesterol gallstones formed and the incidence of pigment gallstones was not altered.

C26 Bile of the 24-nor-5beta-cholestane series were prepared starting from methyl cholate. A Grignard reaction of methyl magnesium iodide with methyl cholate yielded 24-nor-5beta-cholestane-3alpha, 7alpha, 12alpha, 25-tetrol which was dehydrated TO FORM a mixture of 24-nor-5beta-cholest-23-ene-3alpha, 7alpha, 12 alpha-triol and the corresponding delta25 compound. Oxidation of the former with OsO4 yielded 24-nor-5beta-cholestane-3alpha, 7alpha, 12alpha, 23epsilon, 25pentol, while catalytic hydrogenation of a mixture of the delta23 and delta25 triols resulted in the formation of 24-nor-5beta-cholestane-3-alpha-, 7alpha-12alpha-triol. The structures of these new compounds were confirmed by infrared and nuclear megnetic resonance spectrometry and by gas-liquid chromatography-mass spectrometry.