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.     

Effect of age on plasma bile acids and lipid components in the rat

With increasing age, total plasma bile acid contents increased in rats over a period of 11 months, and also total plasma cholesterol and carcass fat contents increased in the same manner. Plasma showing high bile acid levels at 11 months was found by means of high performance liquid chromatography to contain cholic acid as one of the major components, chenodeoxycholic acid and trace deoxycholic acid. These results suggest that there are close relationships between the plasma bile acids and age-dependent changes of lipid components in the rat.     

Dietary squalene increases tissue sterols and fecal bile acids in the rat

Feeding 1% squalene increased markedly the concentrations of squalene and methyl sterols in each serum lipoprotein class, intestinal mucosa, liver and also in adipose tissue. It also increased cholesterol concentration of the liver and serum VLDL, and esterified cholesterol in serum LDL as well as fecal bile acids. The results suggest that absorbed dietary squalene contributes to some extent to the squalene content of adipose tissue, effectively increases the overall cholesterol synthesis and enhances cholesterol elimination preferentially as fecal bile acids.     

Cholanoic (bile) acids in hepatic and nonhepatic tissues of miniature swine

Preliminary information is given indicating that all tissues of the miniature swine examined thus far contain cholanoic (bile) acids. Thin layer and gas chromatographic analyses suggest that the cholanoic acids are conjugated with glycine and taurine. The amounts appear to be substantial and should be assessed in regard to studies of cholesterol turnover.     

Deconjugation of bile acids by human intestinal bacteria implanted in germ-free rats

Fecal bile acids in germ-free rats were analyzed after inoculation withBacteroides vulgatus, Bifidobacterium longum, Escherichia coli orClostridium ramosum. B. vulgatus preferentially deconjugated tauro-β-muricholic acid andB. longum taurocholic acid.C. ramosum deconjugated both bile acids, butE. coli deconjugated neither. 7α-Dehydroxylation of bile acids was negligible even after 18 days of inoculation, but a small amount of 7-oxo-bile acid, less than 5%, was formed. Fecal excretion of bile acids increased after inoculation withB. vulgatus, B. longum andC. ramosum, but not withE. coli.     

Composition of cecal bile acids in ex-germfree mice inoculated with human intestinal bacteria

Germfree (GF) mice were orally inoculated with human fecal suspension or various components of human fecal microbiota. Three weeks after the inoculation, cecal bile acid composition of these mice was examined. More than 80% of total bile acids was deconjugated in the cecal contents of ex-GF mice associated with human fecal dilutions of 10⁻² or 10⁻⁶, or anaerobic growth from a dilution of 10⁻⁶. In these ex-GF mice, deoxycholic acid accounted for about 20% of total bile acids. In the cecal contents of ex-GF mice associated only with clostridia, unconjugated bile acids made up less than 40% of total bile acids, about half of those in other ex-GF groups. However, the percentage of deoxycholic acid in these mice was the same as that in the other groups. These results indicate that dominant anaerobic bacterial combination is efficient for deconjugation of primary bile acids, and that clostridia in the human feces may play an important role in 7α-dehydroxylation of unconjugated primary bile acids in the intestine.     

Effects of bile salts on rat hepatic acyl CoA:Cholesterol acyltransferase

Acyl CoA:cholesterol acyltransferase (EC2.3.1.26, ACAT), responsible for intracellular esterification of cholesterol, may play an important role in cholesterol trafficking within the cell, and thus, in maintenance of cellular cholesterol homeostasis. Bile acids are potential regulators of cholesterol trafficking in the liver. Therefore, the effect of bile salts on hepatic ACAT activity was studied in the perfused rat liver. ACAT activity was increased after liver perfusion with either taurocholate or taurochenodeoxycholate. However, addition of these bile salts at physiological concentrationsin vitro had little effect on microsomal ACAT activity. The increase in hepatic ACAT activity due to perfusion with bile salts was accompanied by reduced accumulation of very low density lipoprotein cholesterol in the perfusate, but there was no effect on 3-hydroxy-3-methylglutaryl-CoA reductase activity. Hepatic ACAT activity was decreased after bile diversion for four hours in the intact animal. This treatment had no statistically significant effect on 3-hydroxy-3-methylglutaryl-CoA reductase activity. These data suggest that bile salts induce changes in hepatic compartmentation and traffic of cholesterol within the hepatocyte accompanied by response of ACAT activity to maintain cellular cholesterol homeostasis.     

Relationships between cholesterogenesis,microsomal sterols and HMG-CoA reductase in the perfused rat liver

The relationships between cholesterogenesis and the activity of HMG-CoA reductase of microsomes prepared with or without sodium fluoride, and between changes of cholesterogenesis and microsomal sterols were studied in the isolated rat liver perfused with or without oleic acid in the presence of AY-9944. AY-9944 inhibits the conversion of 7-dehydrocholesterol, measured colorimetrically as “fast-acting” sterols, to cholesterol, measured colorimetrically as “slow-acting” sterols. The level of “fast-acting” sterols is used to estimate cholesterogenesis and changes in microsomal sterols. It was observed that the activity of HMG-CoA reductase of microsomes prepared with or without fluoride reflects the relative changes in cholesterogenesis of the perfused livers. In addition, the amount of “fast-acting” and “slow-acting” sterols in microsomes correlates with increases in the activity of HMG-CoA reductase and cholesterogenesis.     

Effect of dietary bile acids,cholesterol, and β-sitosterol upon formation of coprostanol and 7-dehydroxylation of bile acids by rat

During studies of sterol metabolism in the rat, the fecal neutral sterol fraction was analyzed by a combination of thin layer chromatography and gas liquid chromatography. On a stock diet of rat chow supplemented with 5% corn oil, the rats excreted 14.5 mg/day of total neutral sterols. Coprostanol comprised 35% (5 mg/day) of this fraction. When the diet was supplemented with 0.5% sodium taurochenodeoxycholate, the amount of coprostanol in the feces remained the same as in the controls (3.2 mg/day, 32%). The addition of 0.5% sodium taurocholate to the diet resulted in a fivefold reduction of coprostanol formation (0.6 mg/day, 8%). When 1.2% cholesterol was added to the stock diet, the amount of coprostanol present in the feces decreased to an average of 11% compared to controls, but the absolute amount formed was greater (35 mg/day). On a diet enriched with 0.8% β-sitosterol, the rats, on the average, converted 23% of the cholesterol to coprostanol. Feeding diets enriched with sodium taurochenodeoxycholate and sodium taurocholate reduced the 7-dehydroxylation of primary bile acids in the feces by 28% and 42%, respectively. The conversion of primary bile acids to secondary bile acids in the feces of control, cholesterol, and β-sitosterol fed rats was the same (64%).     

Bile acid metabolism in mammals: VI. Effect of ethionine upon bile acids of rat bile

Sex differences in the effect of ethionine upon rat liver metabolism prompted our investigation into possible sex differences in the effect of ethionine upon bile acid metabolism. The bile ducts of 24 rats, 12 male and 12 female, were cannulated. After 1 hr of bile collection, 6 rats of each sex were given ethionine, 1 mg/g body wt, by feeding tube. The bile acid composition of the bile collected during the subsequent 4 hr was analyzed by combined thin layer and gas chromatography. Ethionine induced a reduction in bile flow (3rd and 4th hr) and in bile acid concentration (4th hr) in female rats. The amino acid had no effect upon bile flow but did increase biliary secretion of bile acids (1st and 2nd hr) in male rats. Cholic acid accounted for the bulk of the reduction in total bile acid secretion in the female studies. The increase in total bile acid secretion in the male studies involved all bile acids. The effects of ethionine upon bile acid secretion were delayed in the female studies, immediate in the male. The changes in bile acid secretion involved only the taurine conjugates in the female studies, both taurine and glycine conjugates in the male. There are substantial sex differences in the effect of ethionine upon bile acid metabolism in the rat.     

Identification and quantitation of cholanoic acids in hepatic and extra-hepatic tissues of rat

Tissues of rats were examined for the presence of cholanoic acids. Quantitation of extraction, deconjugation, and isolation were verified by use of radioactive standards. Identification was made by thin layer and gas liquid chromatographic comparison to standards and mass spectrometry. All tissues examined were found to contain several conjugated cholanoic acids. Liver contained primarily cholic acid and peripheral tissues primarily dihydroxy compounds, mainly hyodeoxycholic acid.     

Turnover of bile acids in the hypercholesterolemic rat as influenced by saturation of dietary fat

Injections of [24-¹⁴C] chenodeoxycholate and³H-cholate were made by heart puncture into 300 g male rats that bore T-cannulas in their bile ducts. The animals had been raised on diet A, containing glucose, cholesterol and cholate, or diet B, containing sucrose and cholesterol; each of the diets contained 5% safflower oil or 5% beef tallow as variables. From analysis of bile samples collected from the T at intervals over a 5 day period, it was observed that the safflower oil group fed diet B had a 17% shorter cholate half-life, a 29% larger cholate pool size and 52% higher rate of cholate synthesis than those fed beef tallow in the same diet. The safflower group fed diet A also had a larger cholate pool size, but synthesis and half-life were obscured by cholate feeding. Chenodeoxycholate turnover data were not obtainable because the decay curves were bimodal for all treatments and hence did not conform to a simple pool model. It is concluded that dietary safflower oil causes more rapid formation of cholate than does dietary beef tallow in the cholesterol-fed rat. Journal Paper No. 4952 AES, Purdue University.     

Effect of 7-methylated bile acids and bile alcohols on cholesterol metabolism in hamsters

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

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.     

Composition of bile acids in ruminants

The bile acids found in sheep bile, beef bile, beef feces, sheep fetus bile, and beef fetus bile have been analyzed by using conventional techniques. Animals maintained on natural and purified diets were used. The bile acids are a complex mixture of isomeric hydroxy- and keto-5β-cholanoic acids which were substituted at one or several of the carbon atoms 3, 7, and 12. Cholic acid is the predominant bile acid found in these species. Deoxycholic acid was the major product formed from cholic acid when the animals were on a natural diet but the concentration of 3α, 12α-dihydroxy-7-keto-5β-cholanoic acid was elevated in the animals that were maintained on a high concentrated purified diet (without roughage). The fetus bile was found to contain nearly all of the bile acids found in the bile of the mature animal but in different concentrations.     

Distribution of bile acids in rats

Distribution and biliary and fecal excretion of bile acids were examined in Wistar strain male rats of about 300 g body weight. The pool size of the rats on ordinary diet was 40 mg/rat, biliary secretion was 14 mg/hr, and fecal excretion was 10 mg/day. Bile acids were mainly located in the small and large intestinal contents, 87% and 10%, respectively; but a portion was found in the intestinal wall and the liver. Rats fed 2% cholesterol-supplemented diet for a week showed similar values for pool size and biliary secretion with the rats on ordinary diet, but higher values for fecal excretion and distribution ratio in the large intestinal contents. Cholic acid was a major component in the bile, small intestinal wall, small intestinal content and liver, while the bile acid composition ratios were roughly similar to each other, although a relatively large amount of α-muricholic acid was found in the intentinal wall and liver. Both the wall and content compositions of the large intestine were similar to that of the feces, in which lithocholic, deoxycholic, α- and β-muricholic acids were the main components, although the ratios of α- and β-muricholic acids in the large intestinal wall were larger than those in the intestinal contents or feces. The high concentrations of these bile acids may indicate a difference of transport velocity across the cell membrane, but the mechanism is not known.     

Regulation of 3-hydroxy-3-methylglutaryl CoA reductase by analogs of cholesterol and bile acids in cultured intestinal mucosa

Sodium fusidate and its glycine conjugate, which have the same detergent properties as bile acids, significantly (p<0.05) stimulate HMG-CoA reductase of cultured intestine below the critical micellar concentration (CMC) without affecting brush border enzymes. Above CMC, both amphiphiles are cytotoxic. At concentrations between 1 and 5 mM, sodium fusidate decreased cholesterol contents of cultured mucosa (p<0.05), the increase in synthesis only partially compensating for the sterol loss. Oxygenated sterols, 7-keto- and 25-hydroxycholesterol, also depleted mucosal cholesterol at 0.5 mM, exerting their effect differently by inhibiting HMG-CoA reductase (p<0.01). In contrast to their marked effect on total mucosal cholesterol contents, brush border cholesterol was unaffected by both cholesterol and bile acid analogs.     

Gas-liquid chromatography of bile acids

Methods are reviewed for the preparation, gasliquid chromatographic (GLC) separation, identification, and quantitative estimation of the trifluoroacetyl derivatives of bile acid methyl esters. Of the stationary phases tried (SE-30, QF-1 and XE-60) methylfluoroalkyl silicone (QF-1) was best suited for analysis of the trifluoroacetates. This phase (1–2% QF-1 on 100–120 mesh Gas Chrom P) allowed an orderly resolution of conformational isomers and consistently gave GLC columns (stainless steel tubes, 1/8 in. O.D. × 3 ft) from which the bile acid derivatives could be recovered in high yield. Applications to biological materials are illustrated with bile acid samples from animal biles and from human duodenal drainage and feces. Identifications of the major bile acids made by the GLC of the trifluoroacetates were confirmed by results obtained with bile acid methyl esters and bile acid methyl ester acetates on QF-1 and the other liquid phases investigated. For most mixtures of bile acids, however, it appears that GLC of methyl esters and methyl ester trifluoroacetates on QF-1 is sufficient for a reliable recognition of common bile acids. Overall accuracy of the estimates was of the order of ± 5%, but it varied with the nature and concn of the component.     

Inhibition and induction of bile acid synthesis by ketoconazole effects on bile formation in the rat

The effects of ketoconazole, an antimycotic agent, and metyrapone, an inhibitor of mixed function oxidases, on bile acid synthesis were compared in the rat bothin vitro andin vivo. In rat liver microsomes, ketoconazole was much more potent than metyrapone in inhibiting the activity of cholesterol 7α-hydroxylase, the rate-limiting enzyme in the synthesis of bile acids. The I₅₀ values were 0.42 μM and 0.91 mM for ketoconazole and metyrapone, respectively. Intraduodenal administration of ketoconazole caused a rapid, dose-dependent reduction of bile acid synthesis in eight-day bile diverted rats. A single dose of 50 mg/kg reduced bile acid synthesis to 5% of control value; the same dose of metyrapone caused a reduction to only 85%. Inhibition of bile acid synthesis by ketoconazole was followed by a marked overshoot. At 28 hr after injection of 50 mg/kg of the drug, formation of bile acids was stimulated maximally by 45% compared to control value and remained elevated for more than 20 hr thereafter. Synthesis of all primary bile acids was affected to the same extent. Cholesterol 7α-hydroxylase activity in livers of ketoconazole treated (30 mg/kg) rats with an intact enterohepatic circulation was increased by 70% at 16 hr after i.p. injection of the drug. During the very large decrease of biliary bile acid output with ketoconazole, bile flow rate was relatively increased, due to stimulation of the bile acid-independent fraction of bile flow. The latter effect can probably be explained as caused by biliary secretion of osmotically active metabolites of ketoconazole.     

Effect of lecithin on the release of 5′-nucleotidase from liver plasma membrane of rat by bile acids

Bile acids solubilize proteins from liver plasma membrane both in vivo and in vitro. The ability to solubilize the proteins is dependent on the species of bile acid. In this paper, the effect of phospholipid on the solubilization of a membrane-bound enzyme by bile acids was investigated in vitro. Taurocholate (TC) and tauroursodeoxycholate (TUDC) solubilized the enzyme, 5′-nucleotidase, from the liver plasma membrane of the rat in a concentration-dependent manner, although there was a great difference in their effect; at 40 mM, TC solubilized 55.4% of the original 5′-nucleotidase activity of the membrane, but TUDC only 5.7%. While lecithin alone had no solubilizing effect, its addition to the bile acids provoked a 10-fold increase in the solubilizing effect of TUDC, but virtually no change for TC, essentially equalizing the solubilizing effect of the two. Both TC-rich and TUDC-rich bile were obtained from rats infused with the respective bile acids via the jugular vein after their endogenous bile acid pool had been depleted. The solubilization effect of these biles was quite similar to the bile acid-lecithin mixtures. These findings demonstrate that lecithin enhances the ability of the bile acids to solubilize the membrane protein and eliminates the difference in the two bile acid species in their solubilizing ability.