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.     

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.     

In vivo incorporation of labeled fatty acids in rat liver lipids after oral administration

Striking differences were found in the compartmentalization of fatty acids into liver lipid fractions. The saturated fatty acids—lauric, myristic, palmitic and stearic—were incorporated into phosphoglycerides at faster rates with increasing chain lengths, while triglyceride incorporation was almost uniform. The degree of incorporation of the unsaturated fatty acids into phosphoglycerides (structural) compared to triglyceride (storage and energy) was the converse of their oxidation rates. The incorporation of oleic, linoleic and α-linolenic acids was mainly into triglyceride, whereas dihomo-γ-linolenic acid and arachidonic acid were preferentially incorporated into phosphoglycerides. The data suggest that distribution of each fatty acid is different depending on its destination for structural or energy function.     

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.     

The metabolism of linoleic and arachidonic acids in rat testis

Linoleic and arachidonic acids, labeled with¹⁴C and injected intratesticularly, were used to study with time the interconversion of polyunsaturated fatty acids in rat testis and their incorporation into the major lipid classes. With both substrates¹⁴C activity was readily incorporated into longer chain, more highly unsaturated fatty acids. After the injection of 1-¹⁴C-linoleic acid the major portion of the¹⁴C was found in palmitic, linoleic, 8,11,14-eicosatrienoic, 5,8,11,14-eicosatetraenoic, 7,10,13,16-docosatetraenoic and 4,7,10,13,16-docosapentaenoic acids. Hydrogenation of the total fatty acids isolated from rat testes after intratesticular injection of 1-¹⁴C-linoleate revealed that the polyenoic acids hydrogenating to lignoceric acid (previously characterized as 9,12,15,18-tetracosatetraenoate and 6,9,12,15,18-tetracosapentaenoate) had a relatively high specific activity. After the injection of 1-¹⁴C-arachidonate significant¹⁴C activity was found in palmitate, 7,10,13,16-docosatetraenoate, 4,7,10,13,16-docosapentaenoate, 9,12,15,18-tetracosatetraenoate and 6,9,12,15,18-tetracosapentaenoate. The biosynthesis of the ω⁶ polyunsaturated fatty acids in rat testis is discussed in relation to these data. Investigation of the distribution of label in the complex lipid fractions demonstrated the majority of the¹⁴C activity to be present in phosphatides and triglycerides after injection of either of these¹⁴C substrates with only small quantities being present as nonesterified acids. At the time periods studied the polyenoic acids of triglycerides had a higher specific activity than the corresponding acids of phosphatides with the exception of linoleate. Presented in part at the Meeting of the American Institute of Nutrition, Atlantic City, April 1968 and at the AOCS Meeting in New York, April 1969. These data were taken from a thesis submitted by R. B. Bridges in partial fulfillment of the requirements for the Ph.D. degree, Vanderbilt University.     

Effect of candicidin on cholesterol and bile acid metabolism in the rat

Sterol metabolism studies were carried out in rats maintained on a diet containing a polyene antibiotic, candicidin, (30 mg/kg/day) for 2-1/2 months. Compared to the controls, the candicidintreated animals had a smaller food intake and weight gain during this period. There was no difference between the 2 groups in serum cholesterol levels, biliary cholesterol or bile acid concentrations. However, in the experimental group, liver cholesterol content decreased by 27% and hepatic HMG-CoA reductase increased by 36%. Candicidin administration produced an 84% increase in neutral sterol output without change in bile acid output. Cholesterol absorption was reduced 80% by candicidin feeding. The weight of ventral prostate was reduced 33% by candicidin administration. Prostatic HMG-CoA reductase levels were 3 times higher than those of the liver, but enzyme activity was unchanged by candicidin treatment.     

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.     

Action of three bile acids on hepatic and intestinal cholesterogenesis in the rat

Incorporation of [1¹⁴−C] acetate into cholesterol by subcellular particles from the liver and the small intestine of rats with a biliary diversion and a duodenal perfusion of sodium taurocholate, taurochenodeoxycholate or taurodehydrocholate, was studied in vitro. In the liver, taurochenodeoxycholate prevented the increase of cholesterol synthesis induced by biliary drainage. Taurocholate had no action on cholesterol synthesis at any time, day or night. Intestinal synthesis of cholesterol was reduced by taurocholate and taurochenodeoxycholate but was not modified by taurodehydrocholate infusion.     

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.     

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.     

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%).     

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.     

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.     

Studies on the in vivo metabolism of retinoic acid in the rat

A time study of the metabolism of 6,7-¹⁴C-retinoic acid after intraperitoneal injection of physiological levels (17 μg, 0.39 μc) into vitamin A deficient rats, which had been repleted with retinoic acid for two weeks up to two days before injection, resulted in a rapid metabolism to more polar compounds in the small intestine and its contents and a slower metabolism to primarily different materials in the liver and kidney. The major route of metabolism resulted in the urinary excretion of 60% of the injected dose in 24 to 27 hr. Urinary metabolites of 15-¹⁴C-retinoic acid were eluted from silicic acid at a similar concentration of solvents as the ring labeled metabolites although only 32% of the injected dose was recovered in 24 hr. Compounds chromatographically similar to the urinary metabolites were observed at various times in the liver, kidney and small intestine plus contents in addition to retinoic acid and other metabolites. The relative amounts of the metabolites in the different tissues studied varied as a function of the tissue and the time of analysis after injection. Most of the radioactivity from all tissues was extractable into methanol. A liver subcellular distribution of the radioactivity derived from the intraperitoneal injection of 650 μg of 6,7-¹⁴C-retinoic acid (25.9 μc) after 3 hr indicated a minimal level of association of radioactivity (150–250 dpm/mg protein) with all fractions and a greater association of radioactivity with the lysosomal-microsomal fraction (300–350 dpm/mg protein) and the 60–100% ammonium sulfate precipitable (750–800 dpm/mg protein) and 100% ammonium sulfate soluble fractions (422 dpm/mg protein) of the soluble supernatant.     

The metabolism of polyunsaturated fatty acids in rat sertoli and germinal cells

The metabolism of [1-¹⁴C]linoleic, [1-¹⁴C]arachidonic and [3-¹⁴C]docosa-4,7,10,13,16-pentaenoic acids was investigated after intratesticular injection of the labeled compounds and isolation of rat Sertoli and germinal cells. Following injection of either¹⁴C-linoleate or¹⁴C-arachidonate, the specific activity (sp act) of docosa-4,7,10,13,16-pentaenoic acid of Sertoli cells was greater than that of the germinal cells. The data suggest that the Sertoli cells are more active in the biosynthesis of the 22-carbon pentaene than the germinal cells. Differences between these 2 cell types were also noted in the distribution of the incorporated¹⁴C among the various lipid classes. Following intratesticular injection of¹⁴C-docosapentaenoic acid, a greater proportion of the recovered¹⁴C in Sertoli cells than in germinal cells was present in 20-carbon fatty acids, suggesting a greater activity in Sertoli cells in the metabolism of the pentaene. The major portion of the recovered¹⁴C in both cell types was present in triacylglycerols during early time periods and in phospholipids after 24 hr. The possibility of transfer of biosynthesized docosapentaenoic acid from Sertoli to germinal cells is discussed.     

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.     

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.     

Lecithin inhibits fatty acid and bile salt absorption from rat small intestine in vivo

During digestion of a fatty meal, long chain free fatty acids (FFA) and lecithin are among the lipids solubilized in intestinal contents as mixed micelles with bile salts. We hypothesized that if lecithin were not hydrolyzed, the mixed micelles would be abnormal, and absorption of FFA and bile salts would be depressed. To test this hypothesis, isolated segments of rat small intestine were infused in vivo with micellar solutions of 2 mMolar linoleic acid and 10 mMolar taurocholate to which was added 3 mMolar 1-palmitoyl, 2-oleoyl lecithin (a common lecithin in bile and food), or 1-palmitoyl lysolecithin (the hydrolytic product of lecithin). Absorption of FFA and bile salt was measured under steady state conditions using a single-pass technique. Lecithin depressed the rate of FFA absorption by 40% (p<0.025) in jejunal and ileal segments whereas lysolecithin was associated with normal rates of FFA absorption. Lecithin also reduced taurocholate absorption from the ileum by 30% (p<0.05). These data support the idea that lecithin may depress FFA and bile salt absorption from the small intestine in pancreatic insufficiency. The following trivial names are used: lecithin (1,2-diacyl-sn-glycero-3-phosphorylcholine); lysolecithin (1-acyl-sn-glycero-3-phosphorylcholine).