Hydrophilic bile acids: Prevention and dissolution experiments in two animal models of cholesterol cholelithiasis

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.     

Dietary fat alters the distribution of cholesterol between vesicles and micelles in hamster bile

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.     

Effect of a synthetic androgen on biliary lipid secretion in the female hamster

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 increments 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 methyl-testosterone 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.     

Dietary induction of cholesterol gallstones in hamsters from three different sources

Cholesterol gallstones were produced in young male, golden Syrian hamsters, obtained from three different suppliers, by administering a nutritionally adequate, semipurified diet for periods of either 5 or 10 weeks. The major components of the lithogenic diet were casein, cornstarch, butterfat, corn oil and 0.3% cholesterol. The hamsters were obtained from Sesco, Harlan Sprague-Dawley (Engle hamster) and Charles River (Lakeview hamster). There were profound differences among the three groups with respect to gallstone formation and cholesterol metabolism: The highest incidence of gallstones occurred in Sesco hamsters, 44.4% and 63.6% after 5 and 10 weeks on the lithogenic diet, respectively. In the Engle hamster, after a 5-week feeding, cholesterol crystals and gallstones were absent. When the feeding period was extended to 10 weeks, cholesterol gallstones were present in 45.5% of the animals. In the Lakeview hamsters, neither gallstones nor cholesterol crystals were found in the gallbladder after a 5-week period. After 10 weeks, cholesterol gallstones were found in only a single hamster. In all groups, the lithogenic diet produced large increases of liver, serum and biliary cholesterol concentrations and increased liver weights. When the animals were fed for 5 weeks, only the bile of Sesco hamsters became supersaturated. Supersaturated bile was induced in all groups after a 10-week feeding of the lithogenic diet with cholesterol saturation ranging from 1.47 to 1.97. These data indicate that it is possible to induce cholesterol gallstones in hamsters by means of a nutritionally adequate, semipurified diet of moderate cholesterol content. The source of the animals appears to be an important variable, because there were significant differences among the hamsters of differing origins, in cholesterol metabolism and rates of gallstone formation.     

Distribution of cholesterol among its carriers in the bile of male and female hamsters

The distribution of cholesterol among its carriers was studied in the bile of male and female hamsters. Sasco hamsters (Sasco Inc., Omaha, NE) were fed a semipurified diet with 0.0% cholesterol and 4% butterfat (group 1, males; group 4, females); a semipurified diet with 0.3% cholesterol and 1.2% plamitic acid (group 2, males; group 5, females); and a semipirified diet with 0.3% cholesterol and 4% safflower oil (group 3, males; group 6, females). At the end of six weeks, gallstones were found only in male hamsters receiving both cholesterol and dietary fat (fatty acid) (incidence of cholesterol stones: 90% in group 2; 22% in group 3). The biliary cholesterol carriers were separated and isolated from the bile of the hamsters by gel filtration chromatography, using the method of Pattinson [Pattinson, N.R., Willis, K.E., and Frampton, C.M. (1991)J. Lipid Res. 32, 205–214]. In those male hamsters that formed cholesterol gallstones, significant amounts of cholesterol were present in the void volume which contained large cholesterol phospholipid vesicles (void volume vesicles) (23% in group 2 and 15% in group 3). Smaller cholesterol/phospholipid vesicles were eluted next (fractions 30–45) and contained 15% of biliary cholesterol in group 2 and 21% in group 3. The remainder of the cholesterol was associated with mixed cholesterol/phospholipid/bile salt micelles. The cholesterol/phospholipid ratio was larger in both the void volume vesicles and small vesicles (2.40 and 1.48 in group 2; 2.56 and 1.33 in group 3, respectively) compared to the micelles (about 0.3 in groups 2 and 3). In contrast, the bile of the female hasmters contained few vesicles (3% small vesciles in group 5) and the cholesterol/phospholipid ratio of these vesicles was lower (0.94). Hamsters fed cholesterol-free diets (groups 1 and 4) had no biliary cholesterol/phospholipid vesilces; and cholesterol was present in micelles. The results suggest that both the gender and the diet of the hamsters affected the distribution of biliary cholesterol between vesicles and micelles. The development of cholelithiasis in this animal model appears to depend on the rapid nucleation of cholesterol-rich phospholipid vesicles in bile.     

Effects of bile acid oxazolines on gallstone formation in prairie dogs

The effects of 2 bile acid analogs, chenodeoxy-oxazoline [2-(3α,7α-dihydroxy-24-nor-5β-cholanyl)-4,4-dimethyl-2-oxazoline] and ursodeoxy-oxazoline [2-(3α, 7β-dihydroxy-24-nor-5β-cholanyl)-4,4-dimethyl-2-oxazoline] were examined in the prairie dog model of cholesterol cholelithiasis. Gallstones and biliary cholesterol crystals were induced in 5 out of 6 male prairie dogs fed a semisynthetic diet containing 0.4% cholesterol for 8 weeks. Six animals maintained on a low cholesterol control diet (0.08% cholesterol) exhibited neither gallstones nor biliary cholesterol crystals. The addition of 0.06% chenodeoxy-oxazoline to the lithogenic diet did not prevent induced cholelithiasis or the appearance of cholesterol crystals in bile. In contrast, 0.06% dietary ursodeoxy-oxazoline prevented gallstones in 5 out of 6 prairie dogs (but cholesterol crystals were present in the bile of 4 of these animals). Histologically, most of the livers from the prairie dogs fed the cholesterol-supplemented semisynthetic diet showed bile duct proliferation, inflammatory infiltration and fibrosis along the portal tracts. These pathologic changes were generally not ameliorated by adding chenodeoxy-oxazoline or chenodeoxy-oxazoline plus chenodeoxycholic acid to the diet. Portal tract pathology was markedly reduced in most animals by adding ursodeoxy-oxazoline to the cholesterol-supplemented diet. The pathologic changes overall could best be correlated with the presence of gallstones, but not with the incidence of biliary cholesterol crystals.     

Palmitic acid enhances cholesterol gallstone incidence in sasco hamsters fed cholesterol enriched diets

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. The cholesterol saturation index increased from 0.56 to 1.32 in the butterfat groups and from 0.56 to 1.30 in the palmitic acid groups upon raising the dietary cholesterol from 0.0 to 0.3%. Biliary total bile acid concentration did not vary significantly within all groups; however, the addition of cholesterol produced an increase in the ratio of chenodeoxycholic acid to cholic acid. It is concluded that in Sasco hamsters the saturated fatty acid, palmitic acid, when substituted for butterfat in a nutritionally adequate lithogenic diet, is capable of increasing gallstone incidence to almost 100% during a 6-wk feeding period.     

Effect of previous nutritional status on the formation of cholesterol gallstones in the prairie dog

In the prairie dog model of cholesterol cholelithiasis, a high incidence of gallstones is achieved by feeding a semipurified lithogenic diet containing 0.4% cholesterol for 2 mo. On occasion, we noted a decrease in the percentage of animals with gallstones from 90–100% to 50–55%. To explain this phenomenon, we studied the effect of dietary history on gallstone formation. After weaning, animals were fed either rodent chow or alfalfa plus corn (mo 0–3) followed by a cross-over experiment at mo 4–6. Gallstone formation then was studied by feeding the lithogenic diet from mo 7 to 8. At sacrifice, the incidences of gallstones, biliary lipids and tissue cholesterol levels were correlated with dietary history. The incidence of gallstones was 100% only in animals fed the alfalfa-corn diet from weaning to 3 mo. In addition, the feeding of the alfalfa-corn diet at mo 4–6 increased gallstone incidence from 65% to 86%. The lithogenic index of all groups was highest when the animals received only alfalfa-corn prior to the lithogenic stimulus. The activity of hepatic HMG-CoA reductase was elevated in animals fed alfalfa-corn from weaning to 8 mo, suggesting that this diet stimulates hepatic cholesterol synthesis, leading to increased biliary cholesterol secretion. It is concluded that previous nutritional conditioning affects the incidence of gallstones. The prairie dog is a useful model of cholesterol cholelithiasis, but the dietary history of the animals plays an important role in lithogenesis.     

Prevention of cholesterol cholelithiasis by dietary unsaturated fats in hormone-treated female hamsters

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 acidfed 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.     

Dietary protein effects on cholelithiasis in hamsters: Interaction with amino acids and bile acids

The objective of this experiment was to study the effects of dietary cottonseed protein and casein on plasma and biliary lipids, plasma amino acids and gallstones in hamsters. Thirty-four male hamsters (60 ± 5 g) were fed either the lithogenic “Dam Diet” (containing 20% casein, 74.3% sucrose and 5.7% vitamin-mineral mix) or a similar diet that contained 20% cottonseed protein for 30 days. Both diets contained protein as a protein isolate. The concentration of alpha-aminobutyric acid was significantly elevated in the casein-fed group. Significant differences in the total plasma cholesterol or lipoprotein cholesterol concentrations were not observed between the two dietary groups. A significant elevation in the absolute concentration of biliary cholesterol was observed in the casein-fed hamsters. Cottonseed protein-fed animals exhibited a significantly elevated concentration of bile acids. The ratio of glycochenodeoxycholic:glycocholic acid was significantly higher in the cotton-seed protein-fed group. This study reports that an elevated concentration of biliary cholesterol with a concomitant decrease in bile acid concentration yields a condition favorable to gallstone formation. It is proposed that cottonseed protein may have a specific effect on the bile acid pool by increasing the ratio of glycochenodeoxycholic acid:glycocholic acid which, in turn, prevents formation of cholesterol gallstones.     

Age,sex and source of hamster affect experimental cholesterol 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.     

Dietary protein effects on gallstone formation

Semipurified lithogenic diets for hamsters contain casein as the protein source. Since substitution of soy protein isolate for casein reduces serum cholesterol concentrations in several species, we studied replacement of casein by soy protein for effects on gallstone formation. Feeding soy protein consistently resulted in a significantly reduced incidence of gallstones. Switching to a soy-based diet after induction of gallstones resulted in dissolution of a significant percentage of the stones. Partial substitution of soy for casein gave results intermediate between 100% casein and 100% soy. The lysine/arginine ratio of the proteins may be responsible for the observed differences in cholelithiasis. The reduction in lithogenicity associated with feeding soy protein appears to be mediated primarily through decreased secretion of cholesterol into bile.     

Effects of dietary fat and fatty acids on sterol balance in hamsters

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.     

Arachidyl amido cholanoic acid (aramchol) is a cholesterol solubilizer and prevents the formation of cholesterol gallstones in inbred mice

We have recently synthesized fatty acid bile acid conjugates (FABAC) that were able to reduce and retard cholesterol crystallization in model and human biles. When given orally, they prevented the formation of cholesterol crystals in the bile of hamsters. The aim of the present study was to determine whether the FABAC are cholesterol solubilizers, whether they can dissolve pre-existing crystals, whether they can prevent the formation of cholesterol gallstones, and to investigate the optimal type of bond between the fatty acid and bile acid. The presence of cholesterol crystals was determined by light microscopy, and the total crystal mass of precipitated crystals was measured by chemical means. Inbred (C57J/L) mice on a lithogenic diet were used to evaluate cholesterol crystal formation, dissolution, and gallstone formation in vivo. Arachidyl amido cholanoic acid (Aramchol) was the FABAC used in the present experiments. At equimolar amounts, the cholesterol-solubilizing capacity of Aramchol was higher than that of taurocholate and similar to that of phosphatidylcholine. The addition of Aramchol dissolved approximately 50% of pre-existing crystals in model bile solutions. The same phenomenon was demonstrated in human bile ex vivo, with a dose-response effect. All inbred mice developed cholesterol crystals in bile after 10-14 d on the lithogenic diet. Thereafter, supplementation of the diet with Aramchol progressively reduced the proportion of mice with crystals to 25% after 28 d. On the lithogenic diet, 100% of inbred mice developed cholesterol gallstones in the gallbladder by day 21. None of the mice whose diet was supplemented with 0.5 mg or 1.0 mg of Aramchol/d developed stones or crystals. FABAC are a new class of molecules that are cholesterol solubilizers and which are able to dissolve cholesterol crystals in bile. Upon oral administration, they dissolve pre-existing cholesterol crystals and prevent the formation of gallstones in gallstone-susceptible mice.     

Niemann-Pick C2 Protein Expression Regulates Lithogenic Diet-Induced Gallstone Formation and Dietary Cholesterol Metabolism in Mice

Niemann-Pick C2 protein (NPC2) is a lysosomal soluble protein that is highly expressed in the liver; it binds to cholesterol and is involved in intracellular cholesterol trafficking, allowing the exit of lysosomal cholesterol obtained via the lipoprotein endocytic pathway. Thus, this protein may play an important role in controlling hepatic cholesterol transport and metabolism. The aim of this work was to study the relevance of NPC2 protein expression in hepatic cholesterol metabolism, biliary lipid secretion and gallstone formation by comparing NPC2 hypomorph [NPC2 (h/h)] and wild-type mice fed control, 2% cholesterol, and lithogenic diets. NPC2 (h/h) mice exhibited resistance to a diet-induced increase in plasma cholesterol levels. When consuming the chow diet, we observed increased biliary cholesterol and phospholipid secretions in NPC2 (h/h) mice. When fed the 2% cholesterol diet, NPC2 (h/h) mice exhibited low and high gallbladder bile cholesterol and phospholipid concentrations, respectively. NPC2 (h/h) mice fed with the lithogenic diet showed reduced biliary cholesterol secretion, gallbladder bile cholesterol saturation, and cholesterol crystal and gallstone formation. This work indicates that hepatic NPC2 expression is an important factor in the regulation of diet-derived cholesterol metabolism and disposal as well as in diet-induced cholesterol gallstone formation in mice.     

Dietary fat and fatty acids modulate cholesterol cholelithiasis in the hamster

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.     

Cholelithiasis in hamsters: Effects of cholic acid and calcium on gallstone formation

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.     

A hydrophilic bile acid effects partial dissolution of cholesterol gallstones in the prairie dog

Gallstone formation and dissolution were studied in a prairie dog model of cholesterol (CH) cholelithiasis. Gallstones were induced in 49 prairie dogs by feeding 1.2% CH in a nutritionally adequate semisynthetic diet for 6 wk (period 1). At 6 wk, gallstones had developed in all animals examined. The diets were modified by reducing the amounts of CH to 0.4, 0.2, 0.1 and 0.0% (diets 1–4); hyodeoxycholic acid (HDA; 30 mg/kg/day) was added to these diets (diets 5–8). All animals were fed the modified experimental diets for an additional 8 wk (period 2). At week 14, spontaneous gallstone dissolution had not occurred, even in the groups given no added dietary CH during period 2 (group 4). Addition of HDA to the diet tended to reduce the incidence of biliary CH crystals and the size and number of CH gallstones. Biliary CH remained elevated and the lithogenic indices in all groups were found to be greater than 1.0 at the end of the experiment. Liver and plasma CH levels tended to be lower in the groups fed HDA. In these groups, HDA and 6βHDA became the major biliary bile acids. This study demonstrates that HDA achieved partial dissolution of gallstones in bile supersaturated with CH.     

The effects of dietary phospholipids enriched with phosphatidylethanolamine on bile and red cell membrane lipids in humans

The role of phospholipids in biliary cholesterol solubilization and crystallization has only recently begun to be appreciated. Phospholipid vesicles are believed to be the metastable carrier from which cholesterol nucleates. Cholesterol crystallization is influenced by the phospholipid species in bile. Feeding rats and hamsters with diets enriched in phospholipids or their precursors, especially ethanolamine, resulted in reduced cholesterol saturation of bile. Although whole phospholipids are normal dietary constituents, the effects and safety of phospholipid components have not been tested in humans. In the present study, we have evaluated the effects of a dietary phospholipid mixture, enriched with phosphatidylethanolamine, on human bile and red blood cell membrane lipid composition. Five ambulatory volunteers having a chronic indwelling T-tube, with an intact enterohepatic circulation, were investigated. Thirty-six grams of phospholipids (54% phosphatidylethanolamine, 54% linoleyl acyl chains) were added to their daily diet for fourteen days. Biliary nucleation time, cholesterol carriers, as well as plasma, red blood cell membrane, and bile lipid compositions, were monitored. Following phospholipid supplementation, the proportion of linoleyl chains (18:2) in biliary phospholipids increased significantly from 31.1±1.2 to 37.7±5.3%, while that of oleyl chains (18:1) decreased from 11.4±1.6 to 9.6±1.1%. These changes were accompanied by an increase of linoleate and its metabolite, arachidonate, in red cell membranes. Phospholipid feeding did not cause any side effects, and no significant changes in biliary nucleation time, cholesterol, phospholipid, or bile salt concentrations, or in the distribution of cholesterol within micelles or vesicles. We conclude that phospholipid feeding is safe, and can be effective as a vehicle for lecithin fatty acyl chain modulation of bile and lipid membranes. These findings may provide a basis for a controlled modulation of biliary phospholipids to increase cholesterol solubility in bile.     

Dietary fat alters biliary lipid secretion in the hamster

Dietary fat has been found to alter the incidence of cholesterol gallstones in hamsters: butterfat intensifies while safflower oil reduces lithiasis. We now report how dietary fat affects bile flow and biliary lipid secretion in this model. Male hamsters were fed one of three experimental diets: a control diet (containing 0.3% cholesterol); control diet +4.0% butterfat; or control diet +4.0% safflower oil. After three weeks, bile samples were collected via an external biliary fistula. The endogenous bile acid pool was depleted for 120 min followed by increasing rates of taurocholate infusion for 160 min. Basal secretion of biliary lipids was measured during the bile acid depletion period. Basal bile flow and bile acid output were not significantly different in the three groups. Dietary butterfat increased basal cholesterol output compared to the control diet (0.037 vs. 0.025 μmol/min·kg, respectively); safflower oil did not change cholesterol output (0.027 μmol/min·kg). Hamsters fed butterfat or safflower oil secreted more phospholipid (0.171 and 0.178 μmol/min·kg, respectively) than controls (0.131 μmol/min·kg). The cholesterol/phospholipid output ratio of the butterfat group was higher than the safflower oil group (0.220 vs. 0.153, respectively). Effects of dietary fat on several relationships between bile flow and biliary lipid secretion were analyzed by linear regression using the data for the entire bile collection period (bile acid depletion and taurocholate infusion). Butterfat and safflower oil did not change either bile acid dependent or bile acid independent bile flow. Hamsters fed butterfat had a higher linkage coefficient (slope) of cholesterol vs. bile acid output than the safflower oil group (0.023 vs. 0.009, respectively). The linkage coefficient of phospholipid vs. bile acid output of the butterfat group was higher than the controls (0.278 vs. 0.185, respectively). In summary, butterfat induced a high cholesterol and phospholipid secretion with a high cholesterol/phospholipid output ratio; safflower oil induced a high phospholipid secretion with a low cholesterol/phospholipid output ratio. Butterfat and safflower oil have different effects on biliary lipid secretion. These differences in biliary lipid secretion may explain, in part, how butterfat and safflower oil differ in affecting gallstone formation in hamsters.