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.     

Potential bile acid metabolites. XVIII. Synthesis of stereoisomeric 3,6,12 alpha-trihydroxy-5 beta-cholanoic acids.

Two new 6-hydroxylated bile acids, 3 beta, 6 alpha, 12 alpha- and 3 beta, 6 beta, 12 alpha-trihydroxy-5 beta-cholanoic acids, were synthesized from deoxycholic acid. In addition, their C-3 epimers, 3 alpha, 6 alpha, 12 alpha- and 3 alpha, 6 beta, 12 alpha-trihydroxy acids, were prepared by a new route. The principal reactions used were 1) 6 beta-hydroxylation of 3-methoxy-3,5-dienes with m-chloroperbenzoic acid in aqueous dioxane; 2) catalytic hydrogenation of the resulting 6 beta-hydroxy-3-oxo-4-enes to the 6 beta-hydroxy-3-oxo-5 beta compounds with palladium on calcium carbonate catalyst in ethanol; and 3) stereoselective reduction of appropriate 3-oxo derivatives with potassium tri-sec-butylborohydride and tert-butylamine-borane complex. The thin-layer chromatographic, gas-liquid chromatographic, and high performance liquid chromatographic mobilities, and 1H- and 13C-nuclear magnetic resonance spectroscopic data of the four stereoisomers are presented. With this work all the 6-hydroxylated derivatives of lithocholic, deoxycholic, chenodeoxycholic, ursodeoxycholic, and cholic acids in the 5 beta series are now known and have been synthesized.     

Potential bile acid metabolites. 16. Synthesis of stereoisomeric 3 alpha,6,7,12 alpha-tetrahydroxy-5 beta-cholanoic acids

New synthetic routes to the four possible stereoisomeric 3 alpha,6,7,12 alpha-tetrahydroxy-5 beta-cholanoic acids (and their methyl esters), one of which (3 alpha,6 alpha 7 beta,12 alpha) is new, and some related compounds are described. In addition, the 5 alpha-epimer of the new acid was obtained. The final products were obtained in high purity for use as reference compounds in the analysis of bile acids in human biologic samples. The results of analysis of the prepared stereoisomers by proton and carbon 13 nuclear magnetic resonance spectroscopies are briefly discussed along with the thin-layer and gas-liquid chromatographic properties.     

Potential bile acid metabolites. 14. Hyocholic and muricholic acid stereoisomers

The complete set of the eight theoretically possible stereoisomeric 3,6,7-trihydroxy-5 beta-cholanic acids, four of which are new, related to hyocholic and muricholic acids were prepared from chenodeoxycholic acid. The principal reactions used were 1) cis-dihydroxylation of delta 6-compounds with osmium tetroxide/N-methylmorpholine N-oxide; 2) trans-dihydroxylation of 6 alpha, 7 alpha-epoxy compounds with boron trifluoride etherate in N,N-dimethyl-formamide; 3) inversion of equatorial 3 alpha-hydroxylated compounds to the corresponding 3 beta-epimers with diethyl azodicarboxylate/triphenylphosphine/formic acid; and 4) stereoselective reduction of 7-keto derivatives with zinc borohydride (or sodium borohydride) and by metallic potassium/tert-amyl alcohol.     

Potential bile acid metabolites. 13. Improved routes to 3 beta, 6 beta- and 3 beta, 6 alpha-dihydroxy-5 beta-cholanoic acids

New synthetic routes to three possible stereoisomers of hyodeoxycholic (3 alpha, 6 alpha-dihydroxy-5 beta-cholanic) acid are described. The principal reactions involved were inversion at C-3 of 3 alpha-hydroxy-6-oxo derivatives with diethyl azodicarboxylate-triphenylphosphine-formic acid and with N,N-dimethylformamide, without allomerization to the more stable 5 alpha form. On the basis of physical and chromatographic data, previously reported 3 beta, 6 alpha-dihydroxy-5 beta-cholanic acid and its methyl ester are shown to be C-3 epimeric mixtures. The 13C nuclear magnetic resonance spectra were of key importance in characterizing the stereoisomers and estimating their purity.     

Platelet-neutrophil interactions. 12S,20- and 5S,12S-dihydroxyeicosapentaenoic acids: two novel neutrophil metabolites from platelet-derived 12S-hydroxyeicosapentaenoic acid

Dietary marine n-3 polyunsaturated fatty acids have demonstrated an antiinflammatory potential in epidemiologic and intervention studies in humans. Proposed mechanisms, involving only leukocytes, fall short of explaining this potential completely. Enriched by dietary means with eicosapentaenoic acid (EPA), stimulated human platelets release substantial amounts of eicosapentaenoic acid and 12S-hydroxyeicosapentaenoic acid (12S-HEPE) in addition to 12S-hydroxyeicosatetraenoic acid (12S-HETE) derived from arachidonic acid. Human neutrophils metabolize 12S-HETE to 5S,12S-DiHETE when stimulated, whereas unstimulated neutrophils produce 12S,20-DiHETE. This study was undertaken to characterize metabolism of 12S-HEPE in human neutrophils. We demonstrate herein for the first time that 12S-HEPE is metabolized by human neutrophils. In unstimulated neutrophils 20-hydroxylation to 12S,20-DiHEPE occurs, whereas in stimulated neurtrophils 5-lipoxygenation to 5S,12S-DiHEPE takes place. The structures of these metabolites were characterized by their relative retention times on reversed-phase high pressure liquid chromatography, by their UV absorbance spectra, and by gas-liquid chromatography-mass spectrometry. With increasing amounts of 12S-HEPE, stimulated neutrophils produced increasing amounts of 5S,12S-DiHEPE, which is virtually inactive biologically. Concomitantly, production of the potent chemokinetic and chemoattractant arachidonic acid derivative leukotriene B4 decreased. Thus, 12S-HEPE can compete with endogenous arachidonic acid for 5-lipoxygenation in stimulated human neutrophils. 12,20-DiHEPE, LTB5, and 5S,12S-DiHEPE were detectable after coincubating EPA-enriched platelets with unenriched neutrophils, and arachidonic acid-derived 5-lipoxygenase products were decreased. We conclude that 12S-HEPE can participate in platelet-neutrophil interactions in a manner similar to 12S-HETE. By providing competing substrates for neutrophil 5-lipoxygenase, platelets might contribute to the antiinflammatory potential of dietary n-3 fatty acids through platelet-neutrophil interaction.     

Hepatic metabolism of short-chain bile acids. Inversion of the 3-hydroxyl group of isoetianic acid (3 beta-hydroxy-5 beta-androstane-17 beta-carboxylic acid) by the adult rat

The stereospecificity of mechanisms for hepatic transport of short-chain bile acids has been examined by following the hepatic metabolism and biliary secretion of 3 beta-hydroxy-5 beta-androstane-17 beta-carboxylic acid (isoetianic acid) administered in two different labeled forms to rats prepared with an external biliary fistula. While 93% of the administered [2,2,4,4-3H]isoetianic acid was recovered in bile after 20 h, only 18% of a similar dose of [3 alpha-3H]isoetianic acid was secreted in bile over the same time period. The recovered radioactivity of the latter compound was mainly associated with bile water. With the [2,2,4,4-3H]isoetianic acid, the bulk of the biliary isotope was determined to be in the form of two glucuronide conjugates. Spectral analysis identified these metabolites as the hydroxyl-linked (major) and carboxyl-linked (minor) beta-glucuronides, not of the 3 beta-hydroxy compound administered, but of 3 alpha-hydroxy-5 beta-androstane-17 beta-carboxylic acid (etianic acid), i.e., the products of hydroxyl group inversion. It is concluded that isoetianic acid is efficiently cleared from plasma and conjugated with glucuronic acid after its epimerization to etianic acid. The prevalent, but not complete, loss of the 3-tritium atom and the retention of the 2- and 4-tritium atoms probably indicates a 3-oxo-5 beta-androstane-17 beta-carboxylic acid intermediate with partial return of the label via a limited labeled pool of reduced nicotinamide cofactor.     

Bile acid synthesis. Metabolism of 3 beta-hydroxy-5-cholenoic acid to chenodeoxycholic acid

Metabolism of 3 beta-hydroxy-5-cholenoic acid to chenodeoxycholic acid has been found to occur in rabbits and humans, species that cannot 7 alpha-hydroxylate lithocholic acid. This novel pathway for chenodeoxycholic acid synthesis from 3 beta-hydroxy-5-cholenoic acid led to a reinvestigation of the pathway for chenodeoxycholic acid from 3 beta-hydroxy-5-cholenoic acid in the hamster. Simultaneous infusion of equimolar [1,2-3H]lithocholic acid and 3 beta-hydroxy-5-[14C]cholenoic acid indicated that the 14C enrichment of chenodeoxycholic acid was much greater than that of lithocholic acid. Thus, in all these species, a novel 7 alpha-hydroxylation pathway exists that prevents the deleterious biologic effects of 3 beta-hydroxy-5-cholenoic acid.     

Microbiological degradation of bile acids. The preparation of some hypothetical metabolites involved in cholic acid degradation.

1. To identify the intermediates involved in the degradation of cholic acid, the further degradation of (4R)-4-[4alpha-(2-carboxyethyl)-3aalpha-hexahydro-7abeta-methyl-5-oxoindan-1beta-yl]valeric acid (IVa) by Arthrobacter simplex was attempted. The organism could not utilize this acid but some hypothetical intermediate metabolities of compound (IVa) were prepared for later use as reference compounds. 2. The nor homologue (IIIa) and the dinor homologue (IIIb) of compound (IVa) were prepared by exposure of 3-oxo-24-nor-5beta-cholan-23-oic acid (I) and (20S)-3beta-hydroxy-5-pregnene-20-carboxylic acid (II) to A. simplex respectively. These compounds correspond to the respective metabolites produced by the shortening of the valeric acid side chain of compound (IVa) in a manner analogous to the conventional fatty acid alpha- and beta-oxidation mechanisms. Their structures were confirmed by partial synthesis. 3. The following authentic samples of reduction products of the oxodicarboxylic acids (IIIa), (IIIb) and (IVa) were also synthesized as hypothetical metabolities: (4R)-4-[3aalpha-hexahydro-5alpha-hydroxy-4alpha-(3-hydroxypropyl)-7abeta-methylindan-1beta-yl]valeric acid (Vb) and its nor homologue (VIIa) and dinor homologue (IXa);(4R)-4-[3Aaalpha-hexahydro-5alpha-hydroxy-4alpha-(3-hydroxypropyl)-7abeta-methylindan-1beta-yl]-pentan-1-ol (Vc); and their respective 5beta epimers (Ve), (VIIc), (IXc) and (Vf). 4. In connexion with the non-utilization of compound (IVa) by A. simplex, the possibility that not all the metabolites formed from cholic acid by a certain micro-organism can be utilized by the same organism is considered.     

Bile acid sulfates in serum bile acids determination.

Some bile acid sulfates were synthesized and characterized. The configuration of sulfate groups at C-3, C-7 and C-12 positions was confirmed by Nuclear Magnetic Resonance analysis. These sulfates were utilized in a study of their chemical behaviour in different analytical procedures currently used for serum bile acids determination. Procedures for bile acids extraction from serum with ethanol or Amberlite XAD-2 result in an important loss of the most polar sulfated bile acids. Complete separation of unsulfated from sulfated bile acids on Sephadex LH-20 is not achieved when deconjugation of the most polar bile acid sulfate is slow but does not produce artifacts. Enzymatic determination of bile acids gives positive response with some bile acid sulfates. The current procedures of serum bile acids determination are discussed in consideration of these results.     

Concurrent occurrence of 3 beta,12 alpha-dihydroxy-5-cholenoic acid associated with 3 beta-hydroxy-5-cholenoic acid and their preferential urinary excretion in liver diseases

3 beta-Hydroxy-(delta 5-3 beta-ol), 3 beta,12 alpha-dihydroxy-(delta 5-3 beta,12 alpha-ol), 3 beta,7 alpha-dihydroxy-(delta 5-3 beta,7 alpha-ol) and 3 beta,7 beta-dihydroxy-(delta 5-3 beta,7 beta-ol) 5-cholenoic acids were identified in patients with liver diseases by gas-liquid chromatography-mass spectrometry (GLC-MS). Of these unusual 3 beta-hydroxy-5-en-metabolites, delta 5-3 beta-ol and delta 5-3 beta,12 alpha-ol were found as major components in the urine of patients with liver diseases (cholestasis, liver cirrhosis, chronic hepatitis, acute hepatitis). Other 3 beta-dihydroxy-5-en-metabolites, delta 5-3 beta,7 alpha-ol and delta 5-3 beta,7 beta-ol, were found as minor components in the urine. The levels of delta 5-3 beta-ol and delta 5-3 beta,12 alpha-ol in urine were correlated with their levels in serum, with total bile acids in the urine, and with liver function, implying that the degree of their increment correlated well with the severity of liver diseases. The most abundant amounts of delta 5-3 beta-ol and delta 5-3 beta,12 alpha-ol were found in the urine as sulfate conjugates in comparison with bile, portal and hepatic venous sera, and liver tissue of the patients. The biliary excretion and hepatic extraction of these 3 beta-hydroxy-5-en-unsaturated bile acids were more impaired and inefficient than those of cholic and chenodeoxycholic acids.     

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.     

Inhibition of 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acid oxidation and of bile acid secretion in rat liver by fatty acids

In isolated rat hepatocytes, fatty acids inhibited the side chain oxidation, but not the uptake, of exogenously added 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestan-26-oic acid (THCA). THCA did not inhibit fatty acid oxidation. In liver homogenates, fatty acids inhibited THCA activation to its CoA ester (THC-CoA) and THCA oxidation. THCA did not influence fatty acid activation or oxidation. Comparison of the THC-CoA concentrations present in the incubation mixtures during THCA oxidation, with substrate concentration curves determined for THC-CoA oxidation, indicated that the inhibition of THCA oxidation by fatty acids was at least partly exerted at the activation step. The inhibition of THCA activation by fatty acids was noncompetitive. Palmitoyl-CoA at concentrations found in the incubation mixtures during THCA oxidation in the presence of palmitate inhibited THC-CoA oxidation, but not sufficiently to fully explain the fatty acid-induced inhibition of THCA oxidation. The inhibition of THC-CoA oxidation by palmitoyl-CoA did not seem to be competitive. Acyl-CoA oxidase, the first enzyme of peroxisomal beta-oxidation (which catalyzes the side chain oxidation of THCA), was enhanced 15-fold in liver homogenates from clofibrate-treated rats when palmitoyl-CoA was the substrate, but the oxidase activity remained unaltered when THC-CoA was the substrate. In the perfused liver, oleate, infused after a wash-out period of 60 min, markedly inhibited bile acid secretion. The results 1) suggest that fatty acids inhibit THCA metabolism both at the activation step and at the peroxisomal beta-oxidation sequence and that separate enzymes may be involved in both the activation and peroxisomal beta-oxidation of fatty acids and THCA and 2) raise the question whether fatty acids might (indirectly?) affect overall bile acid synthesis via their inhibitory effect on THCA metabolism.     

Effects of feeding bile acids and a bile acid sequestrant on hepatic bile acid composition in mice

An improved ultra performance liquid chromatography-tandem mass spectrometry (UPLC/MS/MS) method was established for the simultaneous analysis of various bile acids (BA) and applied to investigate liver BA content in C57BL/6 mice fed 1% cholic acid (CA), 0.3% deoxycholic acid (DCA), 0.3% chenodeoxycholic acid (CDCA), 0.3% lithocholic acid (LCA), 3% ursodeoxycholic acid (UDCA), or 2% cholestyramine (resin). Results indicate that mice have a remarkable ability to maintain liver BA concentrations. The BA profiles in mouse livers were similar between CA and DCA feedings, as well as between CDCA and LCA feedings. The mRNA expression of Cytochrome P450 7a1 (Cyp7a1) was suppressed by all BA feedings, whereas Cyp7b1 was suppressed only by CA and UDCA feedings. Gender differences in liver BA composition were observed after feeding CA, DCA, CDCA, and LCA, but they were not prominent after feeding UDCA. Sulfation of CA and CDCA was found at the 7-OH position, and it was increased by feeding CA or CDCA more in male than female mice. In contrast, sulfation of LCA and taurolithocholic acid (TLCA) was female-predominant, and it was increased by feeding UDCA and LCA. In summary, the present systematic study on BA metabolism in mice will aid in interpreting BA-mediated gene regulation and hepatotoxicity.