Plate diffusion assay as a rapid method for dosimetry of mutagens.

In the feces of conventional rats, the amount of omega-muricholic and hyodeoxycholic acids vary according to the diet. To understand this phenomenon, we investigated the bacterial formation of these bile acids. The present paper reports the first isolation, from conventional rat feces, of a strain of Clostridium group III which transforms beta-muricholic acid, the main bile acid in germfree rats, into omega-muricholic acid.     

Analysis of bile acids in conventional and germfree rats.

The well-known bile acid analysis technique used by us and others (Grundy, Ahrens, and Miettinen. 1965. J. Lipid Res. 6:397-410) does not allow for the detection of hyodeoxycholic acid, a product of quantitative importance in rodent feces. Using updated methodology, it was established that hyodeoxycholic acid and omega-muricholic acid, both apparent conversion products of beta-muricholic acid, occur in apppreciable amounts in intestinal contents and feces of conventional Wistar type Lobund rats. In conventional rats, these bile acids comprise about 50% of fecal bile acids; they are not found in intestinal contents or feces of germfree rats. Others have demonstrated that hyodeoxycholic acid if formed by combined action of gut flora and liver. A new method for the separation of conjugated and free bile acids in biological samples was developed. Results with this method confirmed the total conjugation of bile acids in the germfree rat, and the almost total deconjugation that takes place in the cecum of the conventional rat.     

Formation of hyodeoxycholic acid from muricholic acid and hyocholic acid by an unidentified gram-positive rod termed HDCA-1 isolated from rat intestinal microflora.

From the rat intestinal microflora we isolated a gram-positive rod, termed HDCA-1, that is a member of a not previously described genomic species and that is able to transform the 3alpha,6beta, 7beta-trihydroxy bile acid beta-muricholic acid into hyodeoxycholic acid (3alpha,6alpha-dihydroxy acid) by dehydroxylation of the 7beta-hydroxy group and epimerization of the 6beta-hydroxy group into a 6alpha-hydroxy group. Other bile acids that were also transformed into hyodeoxycholic acid were hyocholic acid (3alpha, 6alpha,7alpha-trihydroxy acid), alpha-muricholic acid (3alpha,6beta, 7alpha-trihydroxy acid), and omega-muricholic acid (3alpha,6alpha, 7beta-trihydroxy acid). The strain HDCA-1 could not be grown unless a nonconjugated 7-hydroxylated bile acid and an unidentified growth factor produced by a Ruminococcus productus strain that was also isolated from the intestinal microflora were added to the culture medium. Germfree rats selectively associated with the strain HDCA-1 plus a bile acid-deconjugating strain and the growth factor-producing R. productus strain converted beta-muricholic acid almost completely into hyodeoxycholic acid.     

Metabolism of beta-muricholic acid in man

Labeled beta-muricholic acid was obtained from germfree rats given [24-14C]-chenodeoxycholic acid. It was crystallized with the same unlabeled bile acid extracted from germfree rat pooled biles. Five patients fitted with a T-tube after cholecystectomy were given orally 100 mg of the bile acid. Metabolites of beta-muricholic acid in bile, urine and feces were studied. Glyco- and tauro-beta-muricholic acid were the only metabolites detected in bile. The urinary bile acid pattern was complex and included free, glyco- and sulfoconjugated beta-muricholic acid, but no glucuronide was observed. Analysis of fecal bile acid showed very few metabolites: the 3 beta-epimer was identified; the 6 beta- and 7 beta-hydroxyls were apparently not transformed by human intestinal microflora.     

Effect of diabetes on the metabolism of chenodeoxycholic acid in isolated perfused rat liver

The formation of alpha-muricholic acid and beta-muricholic acid from chenodeoxycholic acid was comparatively investigated in livers isolated from normal, streptozotocin-diabetic, and insulin-treated diabetic rats. [24-14C]Chenodeoxycholic acid or [24-14C]alpha-muricholic acid was infused into the perfused livers. There was no difference in biliary excretion of 14C among the different groups of rats after the infusion of each 14C-labelled bile acid. Biliary [14C]bile acids were chromatographed on a thin-layer plate and the distribution of radioactivity on the plate was measured by radioscanning. In the diabetic group, the formation ratio of alpha-muricholic acid and beta-muricholic acid from [24-14C]chenodeoxycholic acid and also that of beta-muricholic acid from [24-14C]alpha-muricholic acid were much smaller than in the normal group. Treatment of the diabetic group with insulin cancelled the difference in the infusion of each [24-14C]bile acid. The results indicate that not only 6 beta-hydroxylation of chenodeoxycholic acid to alpha-muricholic acid but also 7-epimerization of the latter acid to beta-muricholic acid is suppressed in an insulin-deficient state in rats.     

Effect of diabetes and of 7 alpha-hydroxycholesterol infusion on the profile of bile acids secreted by the isolated rat livers

The isolated livers from normal, streptozotocin-diabetic, and insulin-treated diabetic rats were perfused without and with infused 7 alpha-hydroxycholesterol. Biliary bile acids were extracted and analysed by gas chromatography. In each liver group, total bile acid concentration was more than four times greater with infused 7 alpha-hydroxycholesterol than without the sterol. Without infused 7 alpha-hydroxycholesterol, bile acids in the control group were composed mainly of beta-muricholic acid and to a lesser extent of cholic acid. In the diabetic group, the ratio between these two bile acids reversed. The ratio tended to be normalized by treatment with insulin. With infused 7 alpha-hydroxycholesterol, the control group secreted chenodeoxycholic acid at a considerable higher percentage besides major beta-muricholic acid and minor cholic acid. In the diabetic group, the ratio between the latter two bile acids reversed as was the case with the endogenous secretion, while the percentage of chenodeoxycholic acid remained then unchanged. The diminished percentage of beta-muricholic acid in the diabetic group was increased two times by treatment with insulin.     

Biotransformation of linoleic acid and bile acids by Eubacterium lentum.

Eubacterium lentum is a gram-positive, nonsporeforming, nonmotile, asaccharolytic anaerobe. In the present investigations, 3 E. lentum strains (group E) isolated from rat feces were compared with 30 E. lentum strains (groups A, B, C, and D) previously studied by Macdonald et al. (I. A. Macdonald, J. F. Jellet, D. E. Mahony, and L. V. Holdeman, Appl. Environ. Microbiol. 37:992-1000, 1979). All strains alkalized (pH 8 to 8.5) arginine-containing (2 to 15 mg/ml) culture media, and growth of the majority of the strains was stimulated by arginine. All strains converted linoleic acid into transvaccenic acid by shifting the 12,13-cis double bond of linoleic acid into an 11,12-trans(?) double bond followed by biohydrogenation of the 9,10-cis double bond. Hence, biohydrogenation of linoleic acid is a new general characteristic of E. lentum. The 33 strains were also studied for bile acid deconjugase and hydroxysteroid dehydrogenase (HSDH) activities. The 6 strains in group D were steroid inactive; the 27 strains in groups A, B, C, and E were steroid active. The steroid-active group contained bile acid deconjugase-producing strains (groups C and E, plus strain 116 in group A) and nondeconjugating strains. All nondeconjugating strains of groups A and B developed 7 alpha- and 12 alpha-HSDH activities and contained 3 alpha-HSDH-positive strains and 3 alpha-HSDH-negative strains. Deconjugating strains varied in HSDH activities.     

Feeding natural hydrophilic bile acids inhibits intestinal cholesterol absorption: studies in the gallstone-susceptible mouse

We explored the influence of the hydrophilic-hydrophobic balance of a series of natural bile acids on cholesterol absorption in the mouse. Male C57L/J mice were fed standard chow or chow supplemented with 0.5% cholic; chenodeoxycholic; deoxycholic; dehydrocholic; hyocholic; hyodeoxycholic; alpha-, beta-, or omega-muricholic; ursocholic; or ursodeoxycholic acids for 7 days. Biliary bile salts were measured by reverse-phase HPLC, and hydrophobicity indices were estimated by Heuman's method. Cholesterol absorption efficiency was determined by a plasma dual-isotope ratio method. In mice fed chow, natural proportions of tauro-beta-muricholate (42 +/- 6%) and taurocholate (50 +/- 7%) with a hydrophobicity index of -0.35 +/- 0.04 produced cholesterol absorption of 37 +/- 5%. Because bacterial and especially hepatic biotransformations of specific bile acids occurred, hydrophobicity indices of the resultant bile salt pools differed from fed bile acids. We observed a significant positive correlation between hydrophobicity indices of the bile salt pool and percent cholesterol absorption. The principal mechanism whereby hydrophilic bile acids inhibit cholesterol absorption appears to be diminution of intraluminal micellar cholesterol solubilization. Gene expression of intestinal sterol efflux transporters Abcg5 and Abcg8 was upregulated by feeding cholic acid but not by hydrophilic beta-muricholic acid nor by hydrophobic deoxycholic acid. We conclude that the hydrophobicity of the bile salt pool predicts the effects of individual fed bile acids on intestinal cholesterol absorption. Natural alpha- and beta-muricholic acids are the most powerful inhibitors of cholesterol absorption in mice and might act as potent cholesterol-lowering agents for prevention of cholesterol deposition diseases in humans.     

Effect of bile duct ligation on bile acid and cholesterol metabolism in rats

The effects of bile duct ligation on bile acid and cholesterol metabolism were examined in male Wistar strain rats. Quantitative and qualitative changes of bile acids and cholesterol in serum and urine occurred; beta-muricholic acid predominantly increased in serum and urine and the ratio of urinary cholic acid and beta-muricholic acid changed from about 5:3 on day 1 to about 1:8 on day 5 under biliary obstruction. The form of the increased urinary bile acids was mainly taurine-conjugated and partly sulfated. Under conditions of bile duct ligation on day 5, 14C-labeled 3 beta-hydroxy-5-cholenoic, lithocholic, and chenodeoxycholic acids were intragastrically administered to the rats after pretreatment with antibiotics and the metabolites of these three acids were investigated. 3 beta-Hydroxy-5-cholenoic acid was most efficiently converted to beta-muricholic acid. The present study strongly suggested the presence of an alternative metabolic pathway induced by bile duct ligation, which caused the change in composition of urinary bile acids, and especially the marked increase in beta-muricholic acid formation. A possible alternative pathway for bile acid biosynthesis under biliary obstruction in rats is postulated.     

Effects of bile salt supplementation on biliary secretion in estrogen-treated rats

The aim of the present study was to determine whether bile acid feeding to rats can reverse ethinyl estradiol-induced cholestasis. Animals received ethinyl estradiol (2 mg/kg/day) for 6 days or were coinfused with estrogen plus various bile acids (60 mg/kg/day). Cholestasis could be significantly prevented by tauroursodeoxycholic acid, was partly corrected by ursodeoxycholic acid, and was unchanged by chenodeoxycholic acid. Total bile salt secretion was increased in every group. The secretion of the major primary bile acids (cholic acid and beta-muricholic acid) was restored to a large extent in rats supplemented with tauroursodeoxycholate but not in chenodeoxycholate-fed rats. In the former group, the canalicular transport of taurocholate and the bile salt pool size were identical with those of control rats. The hydrophilic-hydrophobic balance of the administered bile salt species appears to be an essential factor in the restoration of bile secretion, the more hydrophilic bile salt having the more hepatoprotective effect.     

Suitability of primary monolayer cultures of adult rat hepatocytes for studies of cholesterol and bile acid metabolism

Monolayer cultures of hepatocytes isolated from cholestyramine-fed rats and incubated in serum-free medium converted exogenous [4-14C]cholesterol into bile acids at a 3-fold greater rate than did cultures of hepatocytes prepared from untreated rats. Cholic acid and beta-muricholic acid identified and quantitated by gas-liquid chromatography and thin-layer chromatography were synthesized by cultured cells for at least 96 h following plating. The calculated synthesis rate of total bile acids by hepatocytes prepared from cholestyramine-fed animals was approximately 0.058 micrograms/mg protein/h. beta-Muricholic acid was synthesized at approximately a 3-fold greater rate than cholic acid in these cultures. Cultured hepatocytes rapidly converted the following intermediates of the bile acid pathway; 7 alpha-hydroxy[7 beta-3H]cholesterol, 7 alpha-hydroxy-4-[6 beta-3H] cholesten-3-one, and 5 beta-[7 beta-3H]cholestane-3 alpha, 7 alpha, 12 alpha-triol into bile acids. [24-14C]Chenodeoxycholic acid and [3H]ursodeoxycholic acid were rapidly biotransformed to beta-muricholic acid. 3-Hydroxy-3-methylglutaryl-coenzyme A reductase activity measured in microsomes of cultured hepatocytes decreased during the initial 48 h following plating, but remained relatively constant for the next 72 h. In contrast, cholesterol 7 alpha-hydroxylase activity appeared to decrease during the first 48 h, followed by an increase over the next 48 h. Despite the apparent changes in enzyme activity in vitro, the rate of bile acid synthesis by whole cells during this time period remained constant. It is concluded that primary monolayer cultures of rat hepatocytes can serve as a useful model for studying the interrelationship between cholesterol and bile acid metabolism.     

Difference between cholic acid and chenodeoxycholic acid in dependence upon cholesterol of hepatic and plasmatic sources as the precursor in rats

Some difference in functional pool of cholesterol acting as the precursor of bile acids is pointed out between cholic acid and chenodeoxycholic acid. In order to elucidate this problem further, some experiments were performed with rats equilibrated with [7(n)-3H, 4-(14)C] cholesterol by subcutaneous implantation. The bile duct was cannulated in one series of experiments and ligated in another. After the operation 14C-specific radioactivity of serum cholesterol fell, but reached practically a new equilibrium within three days. 14C-Specific radioactivity of serum cholesterol as well as of biliary bile acids in bile-fistula rats and urinary bile acids in bile duct-ligated rats was determined during a three days-period in the new equilibrated state. The results were as follows: (1) 14C-Specific radioactivity of cholic acid and chenodeoxycholic acid in bile was lower than that of serum cholesterol, and 14C-specific radioactivity of cholic acid was clearly lower than that of chenodeoxycholic acid. (2) 14C-Specific radioactivity of cholic acid and beta-muricholic acid in urine was lower than that of serum cholesterol, and 14C-specific radioactivity of cholic acid was lower than that of beta-muricholic acid. (3) Biliary as well as urinary beta-muricholic acid lost tritium label at 7-position entirely during the course of formation from [7(n)-3H, 4-(14)C]cholesterol.     

New markers for Eubacterium lentum.

Of 37 strains of Eubacterium lentum and phenotypically similar organisms, 26 (70%) synthesized a corticoid 21-dehydroxylase and/or a 3 alpha-hydroxysteroid dehydrogenase. It appeared that the corticoid 3 alpha-hydroxysteroid dehydrogenase was identical to the bile acid 3 alpha-hydroxysteroid dehydrogenase. Steroid-metabolizing enzymes were found both in E. lentum and in phenotypically similar organisms. E. lentum is characterized by nitrate reduction and enhanced growth in the presence of arginine. Many phenotypically similar organisms possess either one or the other of the two markers. In contrast, using the steroid-metabolizing enzymes as markers, a "steroid-active" and a "steroid-inactive" group were established with minimal overlapping of metabolic characteristics. Synthesis of the steroid enzymes was positively correlated with production of gas from H2O2 and formation of H2S. A simple method for the detection of corticoid 21-dehydroxylase and 3 alpha-hydroxysteroid dehydrogenase, one or both of which were present in 92% of the steroid-active group, is described.     

New markers for Eubacterium lentum.

Of 37 strains of Eubacterium lentum and phenotypically similar organisms, 26 (70%) synthesized a corticoid 21-dehydroxylase and/or a 3 alpha-hydroxysteroid dehydrogenase. It appeared that the corticoid 3 alpha-hydroxysteroid dehydrogenase was identical to the bile acid 3 alpha-hydroxysteroid dehydrogenase. Steroid-metabolizing enzymes were found both in E. lentum and in phenotypically similar organisms. E. lentum is characterized by nitrate reduction and enhanced growth in the presence of arginine. Many phenotypically similar organisms possess either one or the other of the two markers. In contrast, using the steroid-metabolizing enzymes as markers, a "steroid-active" and a "steroid-inactive" group were established with minimal overlapping of metabolic characteristics. Synthesis of the steroid enzymes was positively correlated with production of gas from H2O2 and formation of H2S. A simple method for the detection of corticoid 21-dehydroxylase and 3 alpha-hydroxysteroid dehydrogenase, one or both of which were present in 92% of the steroid-active group, is described.     

Metabolism of 3 alpha, 7 alpha-dihydroxy-5 beta-cholestanoic acid by rat liver in vivo and in vitro.

The metabolism of 3 alpha, 7 alpha-dihydroxy-5 beta-cholestanoic acid was studied in the bile fistula rats and in preparations from rat liver homogenates. In the bile fistula rats, the main products were chenodeoxycholic acid, alpha-muricholic acid, and beta-muricholic acid. Only small amounts of cholic acid were formed. Incubations of 3 alpha, 7 alpha-dihydroxy-5 beta-cholestanoic acid with microsomes and NADPH yielded as the main product 3 alpha, 6 beta, 7 alpha-trihydroxy-5 beta-cholestanoic acid. The formation of small amounts of 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestanoic acid was shown. The major product in incubations of 3 alpha, 7 alpha-dihydroxy-5 beta-cholestanoic acid with microsomes and the 100,000 g supernatant fluid fortified with ATP was identified as 3 alpha, 7 alpha, 24 xi-trihydroxy-5 beta-cholestanoic acid. This compound was converted into chenodeoxycholic acid and its metabolites in the bile fistula rat.     

Effect of bile acid deconjugation on the fecal excretion of steroids

The effect of microbiological deconjugation of bile acids on total bile acid and neutral sterol fecal excretion by adult male rats has been studied. A screening method utilizing mice allowed selection of a Clostridium perfringens type A strain, which accelerated cholesterol catabolism in mice. When this species of bacteria was associated with germfree rats, the fecal bile acids were excreted as free bile acids (deconjugated), however the quantities of bile acids excreted were not increased compared with those of germfree rats. Conventional rats excrete twice as much bile acids (all deconjugated) as do the germfree and C. perfringens-associated rats. It is, therefore, unlikely that the microbiological deconjugation of bile acids is responsible for the increased fecal excretion of bile acids seen in conventional rats. The C. perfringens-associated rats excreted identical kinds and quantities of fecal neutral sterols as did the germfree rats.     

Fecal neutral steroids and bile acids from germfree rats

The amount and composition of fecal neutral sterols and bile acids excreted by adult male germfree and conventional rats have been determined. The amounts of neutral sterols excreted were 12.8 (germfree) and 19.5 (conventional) mg/kg of body wt per day. The germfree rats excreted cholesterol and lathosterol (methostenol was not assayed); the conventional rats excreted coprostanol and coprostanone in addition. The amounts of bile acids excreted were 11.3 (germfree) and 21.4 (conventional) mg/kg of body wt per day. The bile acids excreted by the rats were tentatively identified as tauro--muricholate, tauro-alpha-muricholate, and tauro-cholate, besides an unidentified component. The conventional rats excreted the corresponding unconjugated acids as well as many other unconjugated bile acids. No significant correlation was found between the amount of coprosterols and the total amount of neutral sterols excreted by the conventional rats. This suggests that bacterial reduction of cholesterol is not an important mechanism of increasing neutral sterol excretion of conventional rats as compared to germfree rats. Evidence is presented that suggests that this difference in neutral sterol excretion is due to changes in intestinal secretion and sloughing between the two types of animal. The factors reponsible for the differences in bile acid excretion have not been identified.     

In vivo formation of omega-oxidized metabolites of leukotriene C4 in the rat

[3H8]Leukotriene C4 was administered to germfree rats and to conventional rats having a bile duct cannula. Several radioactive metabolites were isolated. Two polar biliary metabolites from conventional rats were identified as N-acetyl-omega-carboxy-leukotriene E4 and N-acetyl-omega-hydroxy-leukotriene E4. A polar fecal metabolite from germfree rats was found to be N-acetyl-omega-carboxy-leukotriene E4. Chemical identities were established using UV spectroscopy and cochromatographies with authentic compounds in several HPLC systems. The fecal metabolite was further characterized by reductive desulfurization followed by gas-liquid-radiochromatography. The yield of the two biliary metabolites was 5% of the administered tritium after three hours and the yield of fecal N-acetyl-omega-carboxy-leukotriene E4 was 3.5% after three days.     

Bile acid metabolism in isolated rat hepatocytes: studied by gas-liquid chromatography-mass spectrometry-selected ion monitoring

Bile acid contents in isolated rat hepatocytes were determined by gas-liquid chromatography-mass spectrometry-selected ion monitoring with the use of deuterium-labeled internal standards. This allowed us first to monitor the actual amounts of not only major but also minor bile acid components present with sufficient sensitivity and specificity and to follow the changes of individual bile acids in cultured rat hepatocytes simultaneously. In freshly isolated rat hepatocytes, cholic and beta-muricholic acids were the major components, comprising 35 and 46% of the total bile acids, respectively. These two bile acids were found to be most actively synthesized during the first 2 h of incubation and continued to increase thereafter for up to 6 h (the end of the period studied). In contrast, chenodeoxycholic and alpha-muricholic acids, which are the precursors of beta-muricholic acid, showed slight increases only in the first hour of incubation and decreased thereafter. These results suggested that the conversion to beta-muricholic acid from chenodeoxycholic acid via alpha-muricholic acid occurred rapidly in cultured rat hepatocytes. The secondary bile acids such as deoxycholic, hyodeoxycholic, and 3 alpha, 12 beta-dihydroxy-5 beta-cholanoic acids declined steadily from the start of incubation, which supported the findings that further hydroxylation of these dihydroxy bile acids occurs in rat liver.     

Influence of nicotinamide administration on the bile-acid pattern in rats given streptozotocin

This study was carried out in order to exclude the possibility that streptozotocin (STZ) as such may be directly responsible for the alteration in the metabolism of bile acids. The STZ-diabetic rats had a higher percentage of cholic acid and a lower percentage of chenodeoxycholic acid and beta-muricholic acid compared to the controls. Although the rats were given STZ, yet there was no alteration in the bile acid pattern when they were protected against diabetes by simultaneous administration of nicotinamide. Nicotinamide itself had no influence on the composition of bile acids. Treatment of the STZ-diabetic rats with insulin cancelled the altered composition of bile acids partially. From these results it became clear that the alteration of the bile-acid metabolism in the STZ-treated rats was caused not by a direct effect of STZ itself but by an absolutely or relatively insulin-deficient state induced by STZ.