Changes in in vivo metabolism of bile acids in rat after treatment with phenobarbital

The influence of phenobarbital on pool size and turnover of bile acids in rats have been investigated by administration of [24-¹⁴C] cholic acid and tritium labeled chenodeoxycholic acid. Phenobarbital treated rats had a smaller cholic acid pool compared to control rats (6.08±2.09 mg and 23.60±7.66 mg, respectively). The pool size of chenodeoxycholic acid, plus its metabolites (α- and β-muricholic acids), was of the same magnitude in the two groups of animals. Also the daily production of cholic acid was decreased in phenobarbital treated rats compared to control rats (2.12±0.46 mg and 7.24±1.66 mg, respectively). No significant difference was observed between the synthesis of chenodeoxycholic acid in the two groups of animals.     

Effects of phenobarbital upon bile acid synthesis in two strains of rats

Rats of the Wistar and Sprague-Dawley strains were injected with sodium phenobarbital (100 mg/kg body wt/day) for 8 days. Fecal bile acid excretion was measured on days 6 and 8 of the experiment, and biliary bile acid composition, hepatic microsomal cholesterol, 7α-hydroxylase, and 7α-hydroxy-4-cholesten-3-one 12α-hydroxylase were determined at the end of the study. In the Wistar rat, injection of phenobarbital produced a doubling of fecal bile acid output (controls, 5.3 mg/rat/day; treated rats, 10.6 mg/rat/day) and a two-three fold increase in cholesterol 7α-hydroxylase. The fecal bile acid output of Sprague-Dawley rats increased 20% in response to phenobarbital (controls, 9.5 mg/rat/day; treated rats, 11.6 mg/rat/day). The activity of cholesterol 7α-hydroxylase remained unchanged. In both strains, phenobarbital treatment produced a decrease in the proportion of cholic acid in total biliary bile acids (controls, 85%; treated groups, 65%). This was associated with a decrease of 7α-hydroxy-4-cholesten-3-one 12α-hydroxylase activity by ca. 50%. Biliary cholesterol concentrations were reduced in phenobarbital treated rats of both strains, but liver cholesterol concentrations remained unchanged. The drug produced a 25% increase in liver wt, on the average.     

Untersuchungen zur Organverteilung von Gallensäuren bei gesunden Ratten

Investigations on the Kinetics of Bile Acids in Normal Rats The kinetic of bile acids has been determined in normal rats after administration of ¹⁴C marked Cholic acid and ¹⁴C marked chenodesoxycholic acid. Cholic acid was administered intravenously to 30 rats to evaluate the distribution into organs after 2, 5, 10, 15 and 20 minutes. In 16 animals the biliary elimination has been assessed. In a further 6 animals a total body autoradiography has been performed after intravenous administration of ¹⁴C chenodesoxycholic acid. Already 10 minutes after i.v. administration of ¹⁴C cholic acid more than 50 % of the dose has passed into the intestine. At that time only 12% of the total activity remained in the liver with a conspicuous activity enrichment in the kidneys. The examinations with biliary drainage confirmed the fast flow off of the cholic acid with only little change in the temporal course with increasing cholic acid concentration. After administration of chenodesoxycholic acid the same kinetic has been found as for cholic acid, as confirmed by autoradiography. However, an additional enrichment of the nuclide in the CNS could be detected. These examinations show that the bile acid pool in normal rats is located in the small intestine, the liver only serves for rapid transfer for the bile acids and does not have a depot function.     

Effect of cholestyramine on bile acid metabolism in conventional rats

Effects of cholestyramine on biliary secretion of cholesterol, phospholipids and bile acids and fecal excretion of sterols and bile acids were examined in Wistar male rats. Six rats were fed a basal diet, and the other six were fed a basal diet supplemented with 5% cholestyramine for eight days. Bile flow and biliary secretion of bile acids and phospholipids (per hour per rat) decreased with cholestyramine treatment, while biliary cholesterol secretion (per hour per rat) remained unchanged. In the biliary bile acid composition, a marked increase of chenodeoxycholic acid with a concomitant decrease of β-muricholic acid was observed in cholestyramine-treated rats. Fecal excretion of total sterols and bile acids increased about three-and four-fold, respectively, after cholestyramine treatment. The increase of fecal bile acids derived from cholic acid was more predominant than that derived from chenodeoxylcholic acid, resulting in an increase of the cholic acid group/chenodeoxycholic acid group ratio.     

Stimulation of bile acid synthesis by dibutyryl cyclic AMP in isolated rat hepatocytes

Freshly isolated rat hepatocytes were used to examine the effects of dibutyryl cyclic AMP on the incorporation of¹⁴C-acetate and¹⁴C-cholesterol into bile acids. After an initial lag period, both precursors were incorporated into cholic and chenodeoxycholic acids at a linear rate for the subsequent 60 min. An apparent stimulation of bile acid formation from¹⁴C-acetate by dibutyryl cyclic AMP was complicated by the concomitant inhibition of cholesterol synthesis. In experiments with¹⁴C-cholesterol, dibutyryl cyclic AMP (1 mM) increased the labeled cholic and chenodeoxycholic acids in the medium by 83 and 224%, respectively, but cellular levels of labeled bile acids were unchanged. As a result, the nucleotide stimulated the overall incorporation of¹⁴C-cholesterol into cholic acid by 39% and into chenodeoxycholic acid by 123%. The mean ratio of labeled cholic to chenodeoxycholic acid declined from 55∶45 in control cells to 41∶59 in cells incubated with dibutyryl cyclic AMP. The results demonstrate that label incorporation can be used to study the regulation of bile acid synthesis in isolated hepatocytes. We propose that dibutyryl cyclic AMP enhances bile acid production by phosphorylating, and thus stimulating the activity of, cholesterol 7α-hydroxylase, the rate-limiting enzyme in bile acid synthesis.     

Effects of clofibrate and tiadenol on the elimination of lipids and bile acids in rat bile

The aim of the work presented here was to compare the biliary elimination of cholesterol and the different bile acids of rats that had been made hypolipidemic by short-term treatments with clofibrate or tiadenol. Both treatments induced a significant decrease in cholesterol output in the bile. The analysis of the different bile acids showed a decrease in dihydroxylated acids elimination (especially CDC acid) without any difference between the 2 sexes. This decrease was associated with an increase in cholic acid excretion. These results are directly correlated with the dose of the administered hypolipidemic drug. The drugs caused a significant increase in the ratio of trihydroxylated acids to dihydroxylated acids. The maximal effect on the concentration of the biliary acids of the bile and on the output was obtained, for both drugs, with a treatment of 200 mg/kg/day. Clofibrate had a greater effect than tiadenol at this dose. Both drugs show a greater effect on lowering serum lipid levels in female animals when compared to males, whereas elimination of bile cholesterol and modifications of bile acids were greater in male animals than female animals.     

Bile acid metabolism in young-old parabiotic rats

Serum cholesterol, triglyceride and phospholipid levels, liver cholesterol concentration, bile flow, biliary cholesterol, phospholipid and bile acid secretion rates, fecal sterol and bile acid levels and their bile acid compositions were examined in young-old parabiotic rats and compared with those in young and old control rats and young-young parabiotic rats. Bile acid composition was expressed in terms of the cholic acid group/chenodeoxycholic acid group (CA/CDCA) ratio. Body weight (BW) gain decreased after parabiosis especially in old rats, but the liver weight (g/100 g BW), diet-intake, feces dry weight, liver cholesterol concentration and fecal sterol level were almost the same in all the groups. The biliary bile acid secretion rate was higher and the fecal bile acid level was lower in old rats than those in young rats but both the levels became comparable with those in young rats after parabiosis of old rats with young rats. Young rats, however, showed no changes in these levels after parabiosis. The serum cholesterol level and the biliary and fecal CA/CDCA ratios in old rats were higher than those in young rats but decreased after parabiosis with young rats, although they were still higher than those in young rats. The serum cholesterol level in young rats increased after parabiosis with old rats, but not after parabiosis with young rats, and the fecal bile acid level and the CA/CDCA ratio were not changed in either case. It is concluded from these findings that the serum cholesterol level and the CA/CDCA ratio increased with age and that these increases were prevented after parabiosis with young rats, while young rats, although their serum cholesterol level was increased, showed no increase in the CA/CDCA ratio after parabiosis with old rats.     

Evidence for distinct precursor pools for biliary cholesterol and primary bile acids in cebus and cynomolgus monkeys

The abnormal metabolism and distribution of plasma lipoproteins have been associated with atherosclerosis and gallstones. To better understand the process of cholesterol excretion, a study was designed to determine whether the contribution of lipoprotein free¹⁴C-cholesterol (as LDL or HDL) to biliary cholesterol or primary bile acids differs in two species of nonhuman primates, cebus and cynomolgus monkeys, having opposite plasma LDL/HDL ratios. Since amino acid conjugation might influence bile acid synthesis or secretion, the taurine and glycine conjugates of newly synthesized primary bile acids, cholic acid (CA) and chenodeoxycholic acid (CDCA), were measured in the species capable of conjugating with taurine or glycine (cynomolgus). After total bile acid pool washout, monkeys were infused with human LDL or HDL labeled with free¹⁴C-cholesterol, and the specific activities (SA) of biliary cholesterol and primary bile acid conjugates were determined. In both species, regardless of the lipoprotein infused, the SA of biliary cholesterol and CA were greater than those for total bile acids and CDCA, respectively. In cynomolgus, the SA of glycine conjugates was higher for CA than CDCA, while the SA of taurine conjugates was greater for CDCA than CA. Under these conditions, (i) infused lipoprotein free cholesterol (as either LDL or HDL) contributed more to biliary cholesterol than to bile acids and more to CA than to CDCA; (ii) glycine conjugated preferentially with CA rather than CDCA, while taurine was the preferred conjugate for CDCA. Further, whereas the two primary bile acids had similar rates of synthesis and turnover in cynomolgus, basal bile acid synthesis was much greater in cebus and the CDCA turnover appeared disproportionately large.     

Hypolipidemic effects of β-cyclodextrin in the hamster and in the genetically hypercholesterolemic rico rat

The effect of increasing amounts of a cyclic oligosaccharide, β-cyclodextrin (BCD), included in the diet on plasma cholesterol and triglycerides, was investigated in two animal models, namely in male genetically hypercholesterolemic Rico rats and in male Syrian hamsters. The distribution of bile acids in the gastrointestinal tract and in the feces of hamsters was also determined. In the Rico rats and hamsters, plasma cholesterol and triglycerides decreased linearly with increasing doses of BCD. In these two species, 20% BCD as compared to control diet lowered cholesterolemia (−35%) and triglyceridemia (−70%). In the hamster, the BCD diet caused a marked decrease in cholesterol and triglycerides in chylomicrons and very low density lipoprotein, and in high density lipoproteins cholesterol. Composition and amounts of bile acids were modified in the gastrointestinal tract of hamsters receiving 10% BCD as compared to the control group. The total bile acid content of the gallbladder of treated hamsters was fourfold higher than in the control group, and the bile contained a large amount of hydrophilic bile acids. This trend was also observed in the small intestine, in which percentages and total quantities of cholic plus deoxycholic acids (cholic pathway) were higher than those of chenodeoxycholic plus ursodeoxycholic plus lithocholic acids (chenodeoxycholic pathway). The bile acid contents of the cecum and colon of treated hamsters were 2.7-fold higher than those of control animals, but the bile acid composition was similar in the two groups of hamsters. Fecal excretion of bile acids was 3.3-fold higher in the treated group than in the control group, and the percentage of lithocholic acid was markedly increased and close to that observed in the colon. The turnover of the chenodeoxycholic pool was twice as fast in treated hamsters as in control hamsters, whereas that of cholic acid was not significantly modified. These results suggest that BCD does not alter the microbial degradation of bile acids, but rather stimulates their synthesis and increases their pool size. BCD prevents the intestinal absorption of lithocholic acid and washes this cytotoxic bile acid from the colon. The hypocholesterolemic effect of BCD appears to be due to stimulation of bile acid synthesis.     

Effects of triton WR 1339 and orotic acid on lipid metabolism in rats

In order to investigate the effect of hepatic cholesterol flux on biliary bile acids, Triton WR 1339 and orotic acid were administered to rats, and the biliary cholesterol, phospholipids and bile acids were analyzed together with serum lipoproteins and hepatic lipids. Triton, which raised serum very low density lipoprotein and lipid levels and decreased serum high density lipoprotein liver lipid levels, increase the biliary cholic acid group/chenodeoxycholic acid group ratio (CA/CDCA) in the bile without affecting the total amount of bile acids and the other biliary lipids. Orotic acid, which decreased serum lipid and lipoprotein concentrations and increased liver lipid levels, increased the biliary excretion of cholesterol and phospholipids, but produced no significant change in the total amount of bile acids and in the CA/CDCA ratio in bile.     

Cholesterol synthesis and degradation in normal rats fed a cholesterol-free diet with excess cystine

Feeding a diet with excess cystine to rats resulted in hypercholesterolemia. To understand the mechanism of the hypercholesterolemia’ cholesterol synthesis and degradation’ bile acid content of bile’ and fecal steroids were determined. The in vivo incorporation of tritiated water into hepatic cholesterol’ and activity of hepatic 3-hydroxy-3-methylglutaryl-CoA reductase in rats fed a high-cystine diet were significantly higher than those in rats fed a control diet. The activity of hepatic cholesterol 7α-hydroxylase was similar between two groups. Little effect of cystine supplementation was found on fecal sterol excretion although there were some changes in biliary excretion of cholic acid derivatives. These results indicate that hypercholesterolemia caused by feeding of a high-cystine diet may be due to the stimulation of hepatic cholesterol synthesis.     

Effect of glucose administration on cholesterol and bile acid metabolism in rats

Glucose administered to fasted rats caused a marked stimulation in hepatic cholesterogenesis and cholesterol 7 alpha-hydroxylation, and an increase in biliary excretion of cholesterol and total bile acids. The excretion of cholic acid was not incluenced during the first few hr after glucose administration, but was significantly increased after 5 hr. Chenodeoxycholic acid showed a similar change, but the increase was only ca. one tenth of that of cholic acid. The excretion of deoxycholic acid was markedly increased by 1 hr, but gradually decreased thereafter. Pretreatment with neomycin abolished the increase in deoxycholic acid by fasting and glucose administration. Other bile acid components showed no significant change. It thus was presumed that cholesterol endogenously synthesized in the liver was metabolized mainly to cholic acid. In contrast, exogenous cholesterol was metabolized mainly to chenodeoxycholic acid. During the period of the acute enhancement of cholic acid formation from the endogenous cholesterol, biliary excretion of deoxycholic acid was increased. This probably occurred through the depression of 7 alpha-rehydroxylation of deoxycholic acid, or through the enhancement of microbial formation of deoxycholic acid in the lumen, and through the increase of intestinal absorption.     

Effect of biliary obstruction on canine plasma and biliary lipids

The common bile duct was obstructed in 17 dogs. Reciprocal changes were noted for the plasma and biliary lipid concentrations of each after obstruction. As the plasma lecithin and free cholesterol concentrations increased, the biliary lipid concentrations declined. After biliary obstruction the reflux of biliary lecithin into the plasma of these animals was demonstrated with both labeled and unlabeled lecithin. The plasma lipid abnormalities seen after acute biliary obstruction were closely simulated by the reflux of lecithin alone from the biliary tree. The isolated reflux of biliary tract taurocholate produced a distinct lowering of plasma phospholipid and cholesterol concentrations, quite different from the plasma lipid alteration noted with acute biliary obstruction. Similar to observations in human obstruction, some of the plasma lipid was in mesophase form after these animals were obstructed.     

Effect of bile acid feeding on hepatic steroid 12 α-hydroxylase activity in hamsters

The effects of feeding cholic acid, chenodeoxycholic acid and ursodeoxycholic acid on the activity of the hepatic steroid 12α-hydroxylase, gallbladder bile acid composition, fecal neutral sterol output, cholesterol synthesis and bile acid synthesis were determined in female hamsters. The 12α-hydroxylase activity was inhibited to 56% by cholic acid, to 62% by chenodeoxycholic acid, and to 78% by ursodeoxycholic acid compared with the control. Bile acid composition was altered by feeding of cholic acid and chenodeoxycholic acid to be rich in the given bile acids. Fecal neutral sterol output increased about twice by feeding chenodeoxycholic acid and ursodeoxycholic acid, whereas cholic acid had no significant effect. Body cholesterol synthesis increased to 217% by chenodeoxycholic acid and to 274% by ursodeoxycholic acid, whereas effect of cholic acid was not significant. Bile acid synthesis was suppressed to 48% of control only by chenodeoxycholic acid. A positive correlation between the 12α-hydroxylase activity and the bile acid synthesis was observed in the control, chenodeoxycholatefed and ursodeoxycholate-fed animals. In conclusion, ursodeoxycholic acid might have less inhibitory effect on the steroid 12α-hydroxylase and the bile acid synthesis than chenodeoxycholic acid.     

The effect of alfalfa-corn diets on cholesterol metabolism and gallstones in prairie dogs

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.     

Differential effects of ursodeoxycholic acid and ursocholic acid on the formation of biliary cholesterol crystals in mice

The preventive effect of 3α,7β,12α-trihydroxy-5β-cholanoic acid (ursocholic acid) and ursodeoxycholic acid on the formation of biliary cholesterol crystals was studied in mice. Cholesterol crystals developed with 80% incidence after feeding for five weeks a lithogenic diet containing 0.5% cholesterol and 0.25% sodium cholate. When 0.25% ursocholic acid or ursodeoxycholic acid was added to the lithogenic diet, the incidence as well as the grade (severity) of the gallstones were reduced. Plasma and liver cholesterol levels were decreased by ursodeoxycholic acid but not by ursocholic acid. Gallbladder cholesterol and phospholipid levels were decreased by both bile acids. The biliary bile acid level was decreased by ursocholic acid but not by ursodeoxycholic acid. After feeding ursocholic acid, its level in the bile was about 25% and the levels of cholic acid and β-muricholic acid decreased. Fecal sterol excretion was not changed by ursocholic acid, but was increased by ursodeoxycholic acid. After feeding ursocholic acid, fecal excretion of deoxycholic acid, cholic acid, and ursocholic acid increased. No differences were found between mice, with or without gallstones, in plasma and liver cholesterol levels, biliary phospholipid and bile acid levels, fecal sterol and bile acid levels, and biliary and fecal bile acid composition. The results suggest that the lower incidence of crystal formation after treatment with ursocholic acid is probably by a different mechanism than with ursodeoxycholic acid. In the mouse model, ursodeoxycholic acid exerts its effect at least partially, by decreasing cholesterol absorption. Ursocholic acid is well absorbed and excreted into bile and transformed into deoxycholic acid by the intestinal microflora in mice.     

Effects of feeding chenodeoxycholic acid on metabolism of cholesterol and bile acids in germ-free rats

The aim of this investigation was to study the influence of chenodeoxycholic acid administration on cholesterol and bile acid synthesis in germ-free rats. Seven rats were fed a basal diet and 2 groups of 4 rats received the same diet supplemented with 0.4 and 1% chenodeoxycholic acid, respectively. After 6 weeks, feces were collected in one 3- and one 4-day pool for analysis of cholesterol and bile acids. When the sampling period was finished, the rats were killed and the liver microsomal fractions isolated. The activities of HMG CoA reductase and cholesterol 7α-hydroxylase were determined, the 7α-hydroxylase by a mass fragmentographic method. The 2 dominating bile acids in the untreated rats were cholic acid and β-muricholic acid. During treatment with chenodeoxycholic acid, 60–70% of this bile acid was converted into α- and β-muricholic acid, indicating a high activity of the 6β-hydroxylase. The excretion of cholic acid was almost completely inhibited and the 7α-hydroxylase activity was decreased ca 75% in the rats fed 1% chenodeoxycholic acid. The activity of the hepatic HMG CoA reductase was unchanged. The fecal excretion of cholesterol increased 2–3 times. An accumulation of cholesterol was seen in the rats treated with 1% chenodeoxycholic acid, which was probably a result of the decreased catabolism of cholesterol to bile acids.     

Effects of β-lactam antibiotics on intestinal microflora and bile acid metabolism in rats

Wistar male rats were treated for six days with broad spectrum β-lactam antibiotics, latamoxef, and cefotaxime. On the seventh day, the number of fecal anaerobic microbes decreased, total fecal bile acids decreased, and bile acid pools increased. Secondary bile acids such as β-hyocholic, hyodeoxycholic, lithocholic, and deoxycholic acids decreased in the feces while the primary bile acids, cholic, β-muricholic, and chenodeoxycholic acids, became predominant. Coprostanol, a microbial metabolite of cholesterol, also disappeared from the feces during the treatment. The cecum enlarged to almost twice the size of that in control rats, whereas the liver weight was not significantly changed. After treatment was stopped, the number of fecal microbes returned to the initial counts within a week, but restoration of bile acid and cholesterol metabolism required at least three weeks.     

Effect of Castration and hormonal supplementation on cholesterol cholelithiasis in the male hamster

This study examined the effect of castration and dietary hormonal supplementation on cholesterol cholelithiasis in male hamsters. Animals fed a standard lithogenic diet developed cholesterol gallstones (17%) after 6 wk, while castrated hamsters did not form any stones. Addition of a synthetic androgen, methyltestosterone, to the lithogenic diet induced cholelithiasis in castrated animals (50%). The biles of normal and castrated-hormone supplemented hamsters had cholesterol saturation indices of 1.0 and 1.1, respectively, while the bile of the castrated animals remained unsaturated (0.6). The ratio of cholic acid/chenodeoxycholic acid in bile increased after castration, but returned to normal levels following hormonal supplementation. Biliary cholesterol carriers were separated by ultracentrifugation. Animals in the stone-forming groups (normal and castrated-hormone treated) had a significant proportion of their biliary cholesterol in vesicles (44 and 46%, respectively); castrated hamsters had less cholesterol in vesicle form (9%). The molar ratio of cholesterol/phospholipid in vesicles was reduced after castration (0.93 vs. 0.42) and increased by hormonal supplementation (1.89). In conclusion, when compared to normal male hamsters fed a standard lithogenic diet, castration reduced the cholesterol saturation of bile, lowered the vesicular/micellar ratio in bile, and inhibited cholesterol cholelithiasis. Dietary androgen supplementation increased the lithogenicity of bile, resulting in stone formation in castrated animals.     

Biliary cholesterol absorption in normal and L-thyroxin-fed rats

Infusion of bile containing labeled cholesterol into bile fistula rats has permitted an in vivo study of the movements and of the absorption of biliary cholesterol in the digestive tract. The specific activities of cholesterol were similar in the micelles and the sediment of the luminal content after a 6 hr infusion, indicating rapid exchange of cholesterol between these fractions. In animals fed a basal diet, the biliary cholesterol absorption was higher (83%) than that of dietary cholesterol (70%). Bile cholesterol is essentially absorbed in the jejunum while the absorption of cholesterol from the diet takes place all along the small intestine but preferentially in its second and third quarters. Both alimentary cholesterol and bile cholesterol enter the top cells of the villi in preference to those of the crypts. In L-thyroxin-fed rats, a parallel decrease in biliary and dietary cholesterol absorption was observed. The increase in the intestinal transit of cholesterol and epithelium cell renewal of the jejunum accounted for this observation.