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.     

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.     

Dissolution of cholesterol gallstones by bile acids in the prairie dog

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.     

Dietary fats rich in saturated fatty acids (12∶0, 14∶0, and 16∶0) enhance gallstone formation relative to monounsaturated fat (18∶1) in cholesterol-fed hamsters

To test the possibility that dietary palmitic acid (16∶0) may be lithogenic, different fats were blended to exchange 18∶1 in olive oil with either 16∶0 in palm stearin, 12∶0+14∶0 in coconut oil, or 14∶0+16∶0 in butterfat. Dietary 18∶2 was held constant at 1.2% energy (en) (with extra safflower oil as needed) in these four purified diets containing low fat (11% of total energy) and 0.4% cholesterol. A fifth, high-fat diet provided 40% of the total energy as the 16∶0-rich blend. All hamsters fed the low-fat, 16∶0-rich blend for six weeks developed cholesterol gallstones (8/8). Although the gallstone incidence was lower for the 12∶0+14∶0-rich diet (5/8), the severity of stone formation in affected hamsters was equal to that in the low-fat, 16∶0-rich group. Mucin accumulation in gallbladder bile was often associated with cholesterol gallstones in diets containing 16∶0, but was minimal in 18∶1-rich and 12∶0+14∶0-rich groups. Neither the lithogenic index (all>1.0), plasma lipids, nor liver cholesterol was a selective predictor of stone formation. The high-fat, 16∶0-rich diet actually decreased cholesterol stone incidence (3/8) and severity, but yielded a high incidence of pigment stones (5/8). Thus, saturated fat and 16∶0per se were not responsible for the exaggerated lithogenesis. Because the antilithogenic 18∶1-rich diet also normalized the 18∶2 intake (1.2% en) relative to previous butter diets (0.3% en), the potential importance of essential fatty acids (EFA) deficiency in the model was tested in a second study by feeding graded amounts of 18∶2 (0.3, 0.6, 0.9, and 1.2% en) as safflower oil in four low-fat, butter-rich diets (11% en as fat) without alleviating gallstone incidence or severity. These studies indicate that substitution of 18∶1 for saturated fatty acids in low-fat diets reduces gallstone formation without affecting the lithogenic index. Furthermore, intake of 18∶2 at or below the EFA requirement does not appear to be a major factor in this model.     

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.     

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.     

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.     

Interrelationship of stearic acid content and triacylglycerol composition of lard,beef tallow and cocoa butter in rats

We investigated modes whereby stearic acid (18∶0) exerts a neutral or cholesterol-lowering effect using dietary fats which provided graded levels of 18∶0 and distinct triacylglycerol (TAG) profiles. Male Sprague-Dawley rats (150–175 g) were fed diets containing 0.2% cholesterol and 16% fat from corn oil, or from 1% corn oil plus 15% lard (13.2% 18∶0), beef tallow (19.2% 18∶0) or cocoa butter (34.7% 18∶0) for 3 wk, and then killed in a fasted or fed state. Chylomicron (CM) fatty acid profiles suggested reduced absorption of 18∶0 with greater 18∶0 intake. CM TAG profiles indicated a reduction or loss of two TAG species compared to the TAG profiles of the stearate-rich diets: 1-palmitoyl-2-oleoyl-3-stearoyl glycerol (POS) and 1,3-distearoyl-2-oleoyl glycerol (SOS). Hepatic total cholesterol concentrations were 54–77% lower (P<0.01) in the cocoa butter-fed than the lard- and beef tallow-fed groups. The cocoa butter group showed a significantly lower ratio of high-density lipoprotein esterified/free cholesterol than all other groups. Hepatic stearoyl-CoA and oleoyl-CoA concentrations, the substrate and product for hepatic δ9 desaturase, were not significantly different for corn oil-fed and cocoa butter-fed groups in spite of a large difference in 18∶0 intake. These data suggest that the neutral or cholesterol-lowering effect of 18∶0 is not due to hepatic conversion of stearic to oleic acid, and that POS and SOS are poorly absorbed from stearate-rich dietary fats.     

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.     

Response of docosapentaenoic acids of rat heart phospholipids to dietary fat

Groups of male Holtzman strain rats were fed from weanling one of the following diets: 20% hydrogenated soybean fat (20% HF), and 20% HF plus 2%, 3% and 4% corn oil, respectively, for 20 weeks. The animals were killed, and the heart phospholipid fractions isolated by chromatographic procedures. The levels and distribution of the docosapolyenoic acids, especially 22∶5ω3, were compared among the animals fed the corn oil supplemented and nonsupplemented diets. Although dietary linolenate (18∶3ω3) level was very low in the nonsupplemented diet, 22∶5ω3 accounted for 8.4% of the total fatty acids of heart total phospholipids when this diet was fed-half the level of total eicosatetraenoic acids. The amounts of 22∶5ω3 were decreased by corn oil supplementation of the diet and got down to the “normal” range of 2.0–2.5% at corn oil supplementation levels greater than 2%. The docosapolyenoic acids were confined largely to the phosphatidylcholine and phosphatidylethanolamine classes of phospholipids. These findings are discussed from the standpoint of the structural role of the phospholipids in the heart subcellular fractions.     

A diet containing myristoleic plus palmitoleic acids elevates plasma cholesterol in young growing swine

The objective of this study was to test the effect of a novel fatty acid mixture, enriched with myristoleic and palmitoleic acids, on plasma lipoprotein cholesterol concentrations. Weanling pigs were assigned to one of six groups and each group received a diet differing in fatty acid composition. Diets were fed for 35 days and contained 10 g added cornstarch/100 g (to provide baseline data) or 10 g added fatty acids/100 g. For those diets containing added fatty acids, extracted lipids contained 36% myristoleic plus palmitoleic acid combined (14∶1/16∶1 diet), 52% palmitic acid (16∶0 diet), 51% stearic acid (18∶0 diet), 47% oleic acid (18∶1 diet), or 38% linoleic acid (18∶2 diet). Witht the exception of the cornstarch diet, all diets contained approximately 30% myristic acid. There were no significant differences in weight gain across treatment groups (P=0.22). All diets caused a significant increase in triglycerides and in total, low density lipoprotein, high density lipoprotein, and very low density lipoprotein cholesterol. The increase in total plasma cholesterol from pretreatment values was greatest in pigs fed the 14∶1/16∶1 and 18∶1 diets. However, the increase in low density lipoprotein cholesterol from the pretreatment concentration was greatest in the 14∶1/16∶1-fed pigs. Increases in very low density lipoprotein cholesterol above pretreatment concentrations were lowest in 16∶0-fed pigs and greatest in 18∶1-fed pigs. Dietary fatty acids elicited changes in plasma fatty acids which generally were reflective of the diets, although the 18∶0 diet did not alter plasma fatty acid concentrations and the 16∶0 diet increased plasma 16∶0 only at the end of the study. These results demonstrated that the combination of myristoleic plus palmitoleic acids increased plasma cholesterol in young pigs, suggesting that fatty acid chain length, rather than degree of unsaturation, is primarily responsible for the effects of fatty acids on circulating lipoprotein cholesterol concentrations.     

Δ15, 18-tetracosadienoic acid content of sphingolipids from platel and erythrocytes of animals fed diets high in saturated or polyunsaturated fats

The effect of diets high (15%) in saturated (beef tallow) or polyunsaturated (corn or cottonseed oil) fatty acids on the fatty acid composition of sphingomyelin from canine erythrocytes and platelets and sphingomyelin and neutral glycosphingolipids of swine erythrocytes was determined. Sphingolipids of platelets and erythrocytes from animals fed high levels of corn or cottonseed oil exhibited a dramatic alteration in their fatty acid composition, most notable of which was a 50% reduction in nervonic acid (24∶1ω9) as compared to levels observed in control or tallow fed animals. This decrease was compensated for by a quantitatively similar increase in a C₂₄ dienoic acid. The long chain dienoic acid was isolated by silver nitrate thin layer chromatography and determined by analysis of its oxidation products to be Δ15, 18-tetracosadienoic acid (24∶2ω6). When the animals were fed the diets high in polyunsaturates, the 24∶2ω6 represented 13, 20, and 9% of the sphingomyelin fatty acids from canine erythrocytes, platelets, and swine erythrocytes, respectively, and 5% of the neutral glycosphingolipid fatty acids of swine erythrocytes. In contrast, the 24∶2ω6 represented less than 4% of the total cellular sphingolipid fatty acids in animals fed the control or high beef tallow diets. The 24∶1ω9 in the sphingolipids of the animals fed the polyunsaturated diet was roughly equal to that of 24∶2ω6, whereas in the sphingolipids of animals fed the control or saturated fat (beef tallow) diet, the 24∶1ω9 was twice these values. Since sphingomyelin is a membrane component, the increase in unsaturation (24∶2ω6) in its fatty acid moiety induced by dietary polyunsaturates may affect membrane fluidity and may alter membrane properties. Dr. Nelson’s current affiliation is with the Lipid Metabolism Branch, Division of Heart and Vascular Diseases, National Heart, Lung, and Blood Institute.     

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.     

Effects of diets high in saturated fat and cholesterol on the lipid composition of canine platelets

The phospholipid composition of platelets from dogs on various experimental diets was determined. Thyroidectomized foxhounds were fed a control diet or the control diet supplemented with (1) beef tallow, (2) beef tallow and cholesterol, or (3) beef tallow, cholesterol, and safflower oil for 23 weeks prior to isolation of platelets. Platelets from animals fed the control diet contained 36.7% phosphatidylcholine (PC), 22.8% phosphatidylethanolamine (PE), 18.4% sphingomyelin (Sph), 11.8% phosphatidylserine (PS), 6.3% phosphatidylinositol (PI), and 2.2% lysophosphatidylcholine. The PE was 77.6% in the plasmalogen form. No highly significant changes in the phospholipid class composition resulted from the experimental diets. Cholesterol supplementation of the diets, however, caused consistent alterations in the fatty acid compositions of the platelet phospholipids including increases in the percentages of 18∶1ω9 (oleic acid), 18∶2ω6 (linoleic acid), and 20∶3ω6 (homo-gamma linolenic acid) and a decrease in the percentage of 20∶4ω6 (arachidonic acid). Addition of safflower oil to the tallow-cholesterol diet partially reversed these effects. These cholesterol-induced alterations in fatty acid composition could be due to exchange with plasma lipids, de novo synthesis, or altered platelet metabolism. The mechanism remains to be determined. Der. Nelson’s current affiliation is the Lipid Metabolism Branch, Division of Heart and Vascular Diseases, National Heart, Lung, and Blood Institute.     

Effects of dietary fish oil on biliary phospholipids and prostaglandin synthesis in the cholesterol-fed pairie dog

Cholesterol gallstone formation in the prairie dog is accompanied by an increase in the percentage of biliaryphospholipids containing arachidonic acid, and an increase in gallbladder prostaglandin (PG) synthesis, but the pathogenetic significance of these changes is unclear. Dietary supplementation with eicosapentaenoic acid (EPA), an omega-3 fatty acid which is commonly found in fish oil, decreases prostaglandin synthesis in some tissues by replacing arachidonic acid, and by competitively inhibiting prostaglandin synthesis. We studied the effect of dietary fish oil on gallbladder PG synthesis, and the relative abundance of various molecular species of phosphatidylcholines and phosphatidylethanolamines in bile and gallbladder epithelium in the cholesterol-fed prairie dog. Prairie dogs were maintained for 4 weeks on one of four diets: i) control, ii) cholesterol-supplemented (0.34%), iii) menhaden oil (50 g/kg chow), or iv) cholesterol plus menhaden oil. Supplementation with menhaden oil resulted in a replacement of arachidonic and linoleic acids with EPA and docosahexaenoic acids in the phospholipids of bile and gallbladder mucosa. In cholesterol-fed animals, supplementation with menhaden oil prevented increased gallbladder PG synthesis. Menhaden oil also reduced the incidence of cholesterol monohydrate crystals among cholesterol-fed animals (9/20 with cholesterol plus menhaden oil vs 21/22 with cholesterol alone), but the improvement could not clearly be attributed to decreased PG synthesis since supplementation with menhaden oil also increased the total phospholipid concentration in bile, and decreased the degree of cholesterol saturation. These results demonstrate that dietary supplementation with omega-3 fatty acids significantly influences biliary phospholipids, and decreases the incidence of cholesterol monohydrate crystal formation in this animal model.     

Effects of dietaryTrans-fat on biliary and fecal steroid excretion and serum lipoproteins in rats

Rats were fed cholesterol-free or cholesterol-enriched diets containing olive oil or partially hydrogenated corn oil at the 10% level for ca. 30 days (c-18∶1, 77.0% in the former diet andc-18∶1, 24.7% andt-18∶1, 42.5% in the latter). The linoleic acid content of these fat diets was made equivalent (1.7 energy%). After feeding cholesterol-free diets,trans fat compared tocis fat showed(a) no untoward effects on growth parameters, (b) a reduction of serum cholesterol levels without influencing concentrations of serum apolipoproteins A-I, B and E, (c) no effects on the bile flow and the concentration of biliary cholesterol and bile acids, (d) an increasing trend of fecal excretion of neutral and acidic steroids, both in terms of mg/day and mg/g feces, and (e) rather equivocal change in the composition of fecal, but not biliary steroids. Similar response patterns were also observed when cholesterol-enriched diets were fed except for a decrease in serum apo B and an ineffectiveness to increase fecal acidic steroids. Together with the results obtained from experiments simultaneously performed with safflower oil and completely hydrogenated corn oil, it seems that the steroid metabolism can be specificallymodified by the geometry of dietary fats.     

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.     

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.     

Serum and aortic levels of phytosterols in rabbits fed sitosterol or sitostanol ester preparations

Campesterol is present in all the phytosterol-containing dietary hypocholesterolemic agents in current use. Campesterol is absorbed more efficiently than sitosterol, and the question of its possible atherogenicity has been raised. To test this possibility, rabbits were fed either a semipurified, cholesterol-free diet that has been shown to be atherogenic for this species or the same diet augmented with 0.5 g of phytosterol-rich diet preparations (spreads) containing either sitosterol or sitostanol. The diets contained 295 mg phytosterol per 100 g. After 60 d, serum cholesterol levels in the two phytosterol groups were 78±4 mg/dL (sitosterol) and 76±4 mg/dL (sitostanol), respectively. The serum cholesterol level of rabbits fed the control diet was 105±8 mg/dL. Serum campesterol (μg/mL) levels were higher than sitosterol or sitostanol levels in all groups. Aortic phytosterols were present in nanogram quantities compared to cholesterol, which was present in microgram quantities. The ratio of campesterol/sitosterol/sitostanol in the aortas was: control, 1.00∶0.43∶0.02; sitosterol, 1∶00∶0.32∶0.01; sitostanol, 1∶00∶0.34∶0.11. Aortic campesterol was present at 4% the concentration of aortic cholesterol, sitosterol at 1.4%, and sitostanol at 0.14%. Aortic lesions were not present in any of the animals.     

Rat testicular lipids and dietary isomeric fatty acids in essential fatty acid deficiency

Weanling rats were fed essential fatty acid-deficient diets, either completely fat-free, or with partially hydrogenated fish oil (PHFO, 28 wt %), or with fractions derived from PHFO containing primarily positional isomers oftrans-eicosenoate (20∶1, 3 wt %) ortrans-docosenoate (22∶1, 3 wt %). Control animals were fed a peanut oil-containing diet (28 wt %). After 5 or 15 weeks on the diet, the content of neutral and phosphorus-containing lipids in the testes was determined. The fatty acid distribution in major lipid classes was analyzed for animals fed the diets for 15 weeks. The testicular stage of maturation or degeneration was assessed by histology. The group fed PHFO exhibited signs of complete testicular degeneration, or lack of maturation, already after 5 weeks, whereas the animals on the diets with the very long chain monoenoic acids suffered severe degenerations only after 15 weeks. In the PHFO-fed rats, a sharp decline in the concentration of testicular triacylglycerols was observed. In all of the essential fatty acid-deficient groups, an increase in testicular sphingomyelin was observed. Cholesterol levels were fairly similar among all dietary groups. The total testicular fatty acids of the PHFO-fed animals contained somewhat more eicosadienoic acid than found in the other groups, and somewhat less (n−9)-acids. In all EFA-deficient groups, (n−6)-acids were lowered, in particular in triacylglycerols and phosphatidyl cholines. The PHFO group did not show a lower (n−6)-concentration than the other deficient groups, in spite of the more severe symptoms of deficiency. There was no evidence of a major accumulation of long chain isomeric fatty acids in the degenerated testes of the PHFO-, 20∶1, and 22∶1-fed groups.