Transformation of bile acids by Eubacterium lentum.

A group of fecal isolates identified as Eubacterium lentum elaborated 3 alpha-, 7 alpha-, and 12 alpha-dehydrogenases and also an epimerizing enzyme(s) for the 3 alpha-hydroxy group. The activities of the enzymes, however, were variably manifested according to the kind of bile acid substrate and the oxygen tension under which the reaction occurred.     

Transformation of bile acids by Clostridium perfringens.

Thirty-five strains of Clostridium perfringens were examined for their ability to transform bile acids, both in growing cultures and by washed whole cells. All of the strains oxidized the 3 alpha-hydroxy group to an oxo group, and all except three converted the same alpha-hydroxy group into a beta-configuration. The oxidative 3 alpha-dehydrogenation was barely detectable under anaerobic cultural conditions but was clearly demonstrated in an aerated system using washed whole cells, with a pH optimum between 7.0 and 9.0. The epimerizing reaction amounting to 10 to 20% conversion was observed in anaerobic cultures and also with resting cells, irrespective of oxygen supply. Both reactions were carried out with seven conventional 3 alpha-hydroxy bile acids, thus producing a series of 3-oxo and 3 beta-hydroxy derivatives that could be examined for gas-liquid chromatographic and mass spectrometric behavior. No evidence for the occurrence of 7 alpha- and 12 alpha-hydroxysteroid dehydrogenase activities among the test strains was found. A highly potent deconjugating hydrolase was elaborated by all of the strains.     

Inhibition of glutathione S-transferase by bile acids.

The effects of bile acids on the detoxification of compounds by glutathione conjugation have been investigated. Bile acids were found to inhibit the total soluble-fraction glutathione S-transferase activity from rat liver, as assayed with four different acceptor substrates. Dihydroxy bile acids were more inhibitory than trihydroxy bile acids, and conjugated bile acids were generally less inhibitory than the parent bile acid. At physiological concentrations of bile acid, the glutathione S-transferase activity in the soluble fraction was inhibited by nearly 50%. This indicates that the size of the hepatic pool of bile acids can influence the ability of the liver to detoxify electrophilic compounds. The A, B and C isoenzymes of glutathione S-transferase were isolated separately. Each was found to be inhibited by bile acids. Kinetic analysis of the inhibition revealed that the bile acids were not competitive inhibitors of either glutathione or acceptor substrate binding. The microsomal glutathione S-transferase from guinea-pig liver was also shown to be inhibited by bile acids. This inhibition, however, showed characteristics of a non-specific detergent-type inhibition.     

Deconjugation of bile acids by intestinal lactobacilli.

Lactobacillus species normally found in the intestinal tract of humans varied in the ability to deconjugate bile acids, whereas laboratory strains of Lactobacillus acidophilus deconjugated both glycocholate and taurocholate. All isolates of L. acidophilus from human feces deconjugated taurocholate, whereas only one of six deconjugated glycocholate. None of 13 isolates identified as L. casei deconjugated taurocholate, whereas 9 deconjugated glycocholate. The deconjugating system of L. acidophilus appeared to be constitutive, required low oxidation-reduction potential, and was most active at pH 6. No degradation beyond deconjugation was detected.     

Deconjugation of bile acids by intestinal lactobacilli.

Lactobacillus species normally found in the intestinal tract of humans varied in the ability to deconjugate bile acids, whereas laboratory strains of Lactobacillus acidophilus deconjugated both glycocholate and taurocholate. All isolates of L. acidophilus from human feces deconjugated taurocholate, whereas only one of six deconjugated glycocholate. None of 13 isolates identified as L. casei deconjugated taurocholate, whereas 9 deconjugated glycocholate. The deconjugating system of L. acidophilus appeared to be constitutive, required low oxidation-reduction potential, and was most active at pH 6. No degradation beyond deconjugation was detected.     

Chemical properties of bile acids. IV. Acidity constants of glycine-conjugated bile acids

The dissociation constants for the carboxyl group of a series of glycine (N-acyl)-conjugated and unconjugated bile acids were determined by potentiometric titration using dimethylsulfoxide-water and methanol-water mixtures of varying proportions. The pKa values in water were calculated by extrapolating the experimental values determined in different mole fractions of the organic solvent mixtures. The following values were obtained: 3.9 +/- 0.1 for glycine-conjugated bile acids and 5.0 +/- 0.1 for unconjugated bile acids, as general pKa values for the two classes of bile acids, respectively. The amidation of bile acids with glycine lowers the pKa value because of the proximity of the amide bond to the terminal carboxyl group. Bile acid dissociation constants are independent of the substituents in the steroid nucleus, since inductive effects of the hydroxyl groups on the steroid nucleus are too distant from the acidic group at the end of the side chain to influence its ionization.     

Vulnerability of keto bile acids to alkaline hydrolysis.

Rigorous alkaline hydrolysis of the two primary (cholic and chenodeoxycholic) and of the two preponderant secondary (deoxycholic and lithocholic) bile acids found in bile led to excellent recoveries. Such was not the case with 11 different keto bile acid standards. Recoveries for a number of standards were unacceptably low and a variety of artefactural products were tentatively identified by gas-liquid chromatography. Keto bile acids bearing a keto gropu on C-3 were particularly vulnerable. In view of thee findings, quantitative and qualitative data reported on biological specimens submitted to saponification in ethanol, methanol, or even in water are of questionable significance.     

A convenient synthesis of 3-keto bile acids by selective oxidation of bile acids with silver carbonate-Celite.

A number of 3-keto bile acids were synthesized by the selective oxidation of bile acid methyl esters with silver carbonate-Celite in refluxing toluene. The pure 3-keto bile acids were isolated simply by filtering the reaction mixture and concentrating the filtrate. The relation of the bile acid structure to the oxidation rate is also discussed.     

Enhancement of the autocatalytic activation of trypsinogen to trypsin by bile and bile acids

The activation of trypsinogen to trypsin in the small intestine can occur by the action of enterokinase or, alternatively, as an autocatalytic process catalysed by trypsin itself. We have found that bile salts and human bile cause a significant enhancement of the autocatalytic activation of trypsinogen. This effect is dependent on the calcium ion concentration and is most marked around pH 5.4 and 7.8. An optimum concentration exists for each bile salt at which the greatest enhancement occurs. At this concentration, certain bile salts have been shown to produce activation effects of up to 55-fold. It is suggested that this activation of the autocatalytic process by bile plays an important role in protein digestion in the small intestine, since it has been shown previously that duodenal trypsin levels are abnormally low in patients with an impairment of bile secretion.     

Bile acids. 38. Conversion of 5 -cholestane-3 ,7 -diol to allo bile acids by the rat

5alpha-[4-(14)C, 3alpha-(3)H]Cholestane-3beta,7alpha-diol was prepared from individual samples of 5alpha-[3alpha-(3)H]cholestane-3beta,7alpha-diol and 5alpha-[4-(14)C]cholestane-3beta,7alpha-diol, each derived from 3beta-acetoxycholest-5-en-7-one. Bile was collected for 11 days from adult male rats, with cannulated bile ducts, that had received intraperitoneally 0.90-0.92 mg of the doubly labeled diol. Bile from the first 10 hr, containing 63% of the administered (14)C and 6% of the (3)H, was hydrolyzed, and the bile acids were separated by acetic acid partition chromatography. Allochenodeoxycholic and allocholic acids contained at least 20.6% and 48.6%, respectively, of the (14)C retained in the biliary acids. Small amounts of (14)C (2.5% and 1.9%, respectively) were present in the 3beta isomers of these acids, but the tritium content totaled more than half of that found in the bile acid fraction. No evidence was obtained for presence of the extensive quantities of the allomuricholates.     

Aerobic catabolism of bile acids.

Seventy-eight stable cultures obtained by enrichment on media containing ox bile or a single bile acid were able to utilize one or more bile acids, as well as components of ox bile, as primary carbon sources for growth. All isolates were obligate aerobes, and most (70) were typical (48) or atypical (22) Pseudomonas strains, the remainder (8) being gram-positive actinomycetes. Of six Pseudomonas isolates selected for further study, five produced predominantly acidic catabolites after growth on glycocholic acid, but the sixth, Pseudomonas sp. ATCC 31752, accumulated as the principal product a neutral steroid catabolite. Optimum growth of Pseudomonas sp. ATCC 31752 on ox bile occurred at pH 7 to 8 and from 25 to 30 degrees C. No additional nutrients were required to sustain good growth, but growth was stimulated by the addition of ammonium sulfate and yeast extract. Good growth was obtained with a bile solids content of 40 g/liter in shaken flasks. A near-theoretical yield of neutral steroid catabolites, comprising a major (greater than 50%) and three minor products, was obtained from fermentor growth of ATCC 31752 in 6.7 g of ox bile solids per liter. The possible commercial exploitation of these findings to produce steroid drug intermediates for the pharmaceutical industry is discussed.     

Separation of bile acids of rat bile by thin-layer chromatography

The common bile acids of rat bile (chenodeoxycholic, hyodeoxycholic, cholic, alpha-muricholic, and beta-muricholic acids) are completely separated by a new thin-layer chromatographic system.