Phenolic 9,10-secosteroids as products of the catabolism of bile acids by a Pseudomonas sp.

The obligate aerobe, Pseudomonas putida ATCC 31752, efficiently utilises bile acids as a source of carbon and energy for growth and maintenance. When aeration is considerably restricted, a consequence to the catabolism of the bile acids in a fermentor is an accumulation of certain steroidal catabolites. Evidence is presented to show that among these are hydroxy-9,10-seco-1,3,5(10)-androstratriene-9,17-diones and those from four of the common bile acids, cholic, chenodeoxycholic, hyodeoxycholic and deoxycholic acids have been isolated and their structures determined. The product of catabolism of hyodeoxycholic acid appears to exist in a hemi-acetal form which readily forms an acetal during isolation procedures. All but one of these are described for the first time.     

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.     

Potential bile acid metabolites. 17. Synthesis of 2 beta-hydroxylated bile acids

The 2 beta-hydroxylated derivatives of lithocholic, chenodeoxycholic, deoxycholic, and cholic acids were synthesized from the respective parent bile acids by established procedures. The principal reactions involved were (1) bromination of 3-oxo formylated bile acids in N,N-dimethylformamide, (2) rearrangement and substitution of the resulting 4 beta-bromo-3-oxo derivatives to the 2 beta-acetoxy-3-oxo compounds with potassium acetate, and (3) reduction to the 2 beta-acetoxy-3 alpha-hydroxy compounds with tert-butylamine-borane complex. As for the prepared 2 beta-hydroxylated bile acids with a diequatorial trans-glycol structure, proton and carbon-13 nuclear magnetic resonance spectroscopic and gas-liquid chromatographic/mass spectrometric properties are discussed.     

A proposed nomenclature for bile acids.

A proposal is made for a system of nomenclature of the more common unconjugated and conjugated bile acids. Acceptable trivial names for bile acids are tabulated, and guidelines are proposed for using these existing trivial names as roots to create acceptable semi-systematic names for other bile acids, as well as for new natural bile acids that will be discovered in the future. The term alpha-hyocholic is recommended to replace hyocholic, and beta-hyocholic to replace omega-muricholic. The term murideoxycholic acid is recommended for 3 alpha,6 beta-dihydroxy-5 beta-cholan-24-oic acid. Proposals are also made for bile acids with epimeric hydroxy groups, for unsaturated bile acids, and for bile acids with oxo- and/or hydroxy-oxo- substituents on the nucleus and/or on the side chain. For conjugated bile acids, the term "aminoacyl amidates" is recommended to replace "amidates" for bile acids conjugated in N-acyl linkage with amino acids. Nomenclature for other types of conjugates (sulfates, glucuronides, glucosides) is included as well as abbreviations. It is recommended that the historic tradition of naming a newly discovered bile acid after the species from which it was isolated be abandoned, and that in the future such a bile acid should be named using the principles contained in this paper.     

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.     

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.     

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.     

Influence of dietary bile acids on formation of bile acids in rat

Various taurine-conjugated bile acids were fed to rats at the 1%-level in the diet for 3 or 7 days and the effect on several hydroxylations involved in the biosynthesis and metabolism of bile acids was studied. The hydroxylations studied were all catalyzed by the microsomal fraction of liver homogenate fortified with NADPH. The 7α-hydroxylation of cholesterol was inhibited by feeding taurocholic acid, taurocheno-deoxycholic acid and taurodeoxycholic acid for 3 as well as 7 days. No marked inhibition was obtained with taurohyodeoxycholic acid or taurolithocholic acid. The 12α-hydroxylation of 7α-hydroxy-4-cholesten-3-one was inhibited after 3 as well as 7 days by all bile acids except taurohyodeoxycholic acid. With this acid a marked stimulation of 12α-hydroxylation was observed. The effects of the different bile acids on the 7α-hydroxylation of taurodeoxycholic acid were not very marked. The 6β-hydroxylation of lithocholie acid and taurochenodeoxycholic acid was stimulated by taurocholic acid and taurodeoxycholic acid. The reaction was inhibited by taurochenodeoxycholic acid, at least after 7 days. Taurohyodeoxycholic acid inhibited the 6β-hydroxylation slightly and taurolithocholic acid had no effect. The results were discussed in the light of present knowledge concerning mechanisms of regulation of formation and metabolism of bile acids and it was suggested that the mechanisms may be more complex than previously thought.     

NAH plasmid-mediated catabolism of anthracene and phenanthrene to naphthoic acids.

Pseudomonas fluorescens 5R contains an NAH7-like plasmid (pKA1), and P. fluorescens 5R mutant 5RL contains a bioluminescent reporter plasmid (pUTK21) which was constructed by transposon mutagenesis. Polymerase chain reaction mapping confirmed the localization of lux transposon Tn4431 300 bp downstream from the start of the nahG gene. Two degradation products, 2-hydroxy-3-naphthoic acid and 1-hydroxy-2-naphthoic acid, were recovered and identified from P. fluorescens 5RL as biochemical metabolites from the biotransformation of anthracene and phenanthrene, respectively. This is the first report which provides direct biochemical evidence that the naphthalene plasmid degradative enzyme system is involved in the degradation of higher-molecular-weight polycyclic aromatic hydrocarbons other than naphthalene.     

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.     

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.     

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.