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. 9. 3,12-Dihydroxy- and 12 beta-hydroxy-5 alpha-cholanoic acids

Syntheses of the heretofore unreported 3 alpha, 12 beta-, 3 beta, 12 beta-dihydroxy-, and 12 beta-hydroxy-5 alpha-cholanic acids of the 5 alpha-series, their methyl esters, and some related derivatives are described. In addition, allodeoxycholic (3 alpha, 12 alpha-dihydroxy) acid was prepared by a new route. The principal reactions involved were the stereoselective reduction of C-12 ketones with an amino-borane reagent and of a C-3 ketone with K-Selectride, and inversion of a 3 beta-tosylate derivative with N,N-dimethylformamide.     

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. 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.     

Functional molecular mass of the 14C-azidobenzamidotaurocholic acid binding proteins in hepatocellular bile acid transport systems.

The apparent target size of 14C-azidobenzamidotaurocholate binding proteins in basolateral rat liver plasma membranes (blPm) was determined by analysis of the radiation induced decrease of the binding of this photoreactive taurocholate analog to blPm. Radiation causes a dose-dependent mono-exponential reduction of binding of ABATC to the protein subunits with molecular masses of 48-50 and 52-54 kDa in SDS-PAGE. The minimal functional molecular mass of the 48-50 and 52-54 kDa ABATC binding proteins was determined to be 99 +/- 8.2 and 93.2 +/- 7 kDa, respectively.     

Regulation of bile acid synthesis. IV. Interrelationship between cholesterol and bile acid biosynthesis pathways

Under most experimental conditions, the activities of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA reductase) and cholesterol 7 alpha-hydroxylase, change together in parallel directions. It has been suggested that newly synthesized cholesterol may be the preferred substrate for cholesterol 7 alpha-hydroxylase, which may account for the observed synchronous behavior of the two enzymes. To test this hypothesis, mevinolinic acid, a potent competitive inhibitor of HMG-CoA reductase, was administered as a single intravenous bolus (10 mg/kg) to rats with a chronic bile fistula. Bile acid synthesis was determined following inhibition of HMG-CoA reductase by mevinolinic acid over a 27-h time course and specific activities of HMG-CoA reductase and cholesterol 7 alpha-hydroxylase were determined in liver microsomes. At 3, 6, and 27 h after a bolus dose of mevinolinic acid, bile acid synthesis was reduced by 54 +/- 5%, 42 +/- 8%, and 23 +/- 13%, respectively, from preinfusion baseline. Within 30 min after administration of mevinolinic acid, HMG-CoA reductase activity was inhibited by at least 87%. At 0.5, 1.5, 3, 6, and 27 h after mevinolinic acid injection, cholesterol 7 alpha-hydroxylase activity was decreased by 6%, 25%, 54%, 41%, and 17%, respectively. By 27 h, the activities of both enzymes had returned to baseline levels. The reduction of bile acid synthesis correlated closely with the observed changes in the activities of cholesterol 7 alpha-hydroxylase. In vitro addition of mevinolinic acid (up to 20 microM) to rat liver microsomes failed to inhibit cholesterol 7 alpha-hydroxylase activity, suggesting no direct effect of mevinolinic acid on enzyme activity. When a bolus dose of mevinolinic acid was coupled with a continuous infusion of mevalonate, the product of the reaction catalyzed by HMG-CoA reductase, the mevinolinic acid-induced decrease in cholesterol 7 alpha-hydroxylase activity and bile acid synthesis was prevented. The results of this study provide evidence that, under the experimental conditions described, there is a linkage between the rates of cholesterol synthesis and the activities of cholesterol 7 alpha-hydroxylase. The data also emphasize the importance of the newly synthesized cholesterol in the regulation of cholesterol 7 alpha-hydroxylase activity.     

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.     

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.     

Testosterone metabolites in dog bile

Testosterone-1,2-3H was injected intravenously into a male dog with a bile fistula and bile and urine collected. The radioactivity was excreted preponderantly in bile (52% of the injected dose) in 6 hours; only 12% appeared in the urine. Methods to study the biliary metabolites of testosterone in this and other animals were developed. Satisfactory conjugate patterns were obtained by fractionation on DEAE-Sephadex A-25 columns using two different elution systems. In addition to an unchanged fraction, six different monoglucuronide fractions were separated. No other conjugates were isolated. Lipidex 5000 column chromatography, TLC and paper chromatography were used for the isolation and purification of aglycone metabolites, which were further identified by co-crystallization methods. The biliary metabolites of testosterone were epiandrosterone (3beta-hydroxy-5alpha-androstan-17-one), etiocholanlone (3alpha-hydroxy-5beta-androstan-17-one), 5alpha-androstan-3beta, 17beta-diol, 5beta-androstan-3alpha, 17beta-diol and 5beta-androstan-3beta,17beta-diol.     

Formation and enterohepatic circulation of metabolites of retinol and retinoic acid in bile duct-cannulated rats

Four hours after intraportal injection of retinoic acid-(14)C into bile duct-cannulated rats, less than 10% of the radioactivity was recovered in the liver, intestine, and kidneys. Within 6 hr, 40% of the radioactivity had appeared in bile. When suspensions of retinol-(14)C or retinal were similarly injected, 25-35% of the dose was excreted in bile within 24 hr and equivalent amounts were deposited in the liver as retinol ester. The isolated perfused liver also produced these bile metabolites and is probably the major site of their formation in vivo. The intestine may metabolize retinoic acid, however, since some metabolites were found in the intestinal wall and lumen, even in bile duct-cannulated rats. The bile metabolites of retinol-(14)C and retinoic acid-(14)C undergo extensive enterohepatic circulation. The bile radioactivity was not volatilized on boiling at acid pH, was not present in digitonin-precipitated sterols, and did not migrate with bile salts on reversed-phase paper chromatography. Anion-exchange chromatography resolved the metabolites of bile into three fractions containing nonionic compounds, acidic substances like retinoic acid, and more polar acidic derivatives.     

Measurement of bile acid synthesis by 14CO2: the metabolism of propionyl CoA.

The relationship between 14CO2 evolution from the catabolism of [26 or 2714C] cholesterol to bile acids was studied in rats with biliary fistulae. When equal quantities of [26 or 2714C] cholesterol and [414C] cholesterol were administered, there was a significant linear relationship between 14CO2 expiration in the breath and [414C] bile acid excreted in the bile. Bile acid synthesis calculated as the ratio of 14CO2: molar specific activity of biliary cholesterol correlated highly with biliary bile acid excretion in the bile acid depleted rat. Phenobarbital, a known inducer of gamma-amino levulenic acid formation from succinyl CoA did not alter the relationship between the 14CO2 estimation of bile acid synthesis and biliary bile acid excretion, indicating that the relationship between [26 or 2714C] cholesterol side chain cleavage and 14CO2 formation was not altered. Phenobarbital, however, did cause a reduction in bile acid synthesis measured by 14CO2 evolution and by biliary bile acid excretion. The 14CO2 method underestimated bile acid excretion. 8.7% in untreated and phenobarbital treated rats respectively. Since 11% of the radioactivity which was expired as 14CO2 was isolated as bile acids, radioactivity cleaved as [1 or 314C] propionyl CoA may enter cholesterol-bile acid biosynthesis resulting in the underestimation of bile acid synthesis. To test whether radioactivity from propionyl CoA enters steroid biosynthesis [114C] propionate and [214C] propionate were given to untreated biliary fistula rats and the biliary lipids excreted in 60 hours were analyzed. Incorporation of radioactivity into cholesterol and bile acids was greater after the administration of [214C] propionate than after [114C] propionate than after [114C] propionate, suggesting that radioactivity from propionyl CoA may enter steroid biosynthesis by metabolic events in which the methylene and carboxyl carbon atoms are differentiated. Although the use of 14CO2 expiration from [26 or 2714C] cholesterol catabolism underestimates the rate of bile acid synthesis, it should have many applications because of the constant relationship between 14CO2 formation and cholesterol side chain cleavage.     

Electrochemical analysis of the carboxy-14C-labelled aliphatic carboxylic acid metabolites resulting from tracer studies.

14C02 output from carboxy-14C-labelled aliphatic carboxylic acids is measured in the micro-scale Kolbe reaction. Irrespective of whether rats were dosed with 1,1-dichloro[1-14C]ethylene or with chloro[1-14C]acetic acid, 1 mol.equiv. of the resulting thio[14C]diglycollic acid yields by electrolysis approx. 0.7 equiv. of 14CO2, which is interpreted in terms of the labelling of one of the carboxylic acid groups of thiodiglycollic acid. This observation provides important evidence concerning thiodiglycollic acid biosynthesis from 1.1-dichloroethylene.     

Hyocholic acid species improve glucose homeostasis through a distinct TGR5 and FXR signaling mechanism

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Separation and detection of uv-absorbing derivatives of fecal bile acid metabolites by high-performance liquid chromatography

Major fecal bile acid metabolites related to lithocholic acid were resolved by high-performance liquid chromatography (hplc). The uv-absorbing p-nitrobenzyl ester derivatives of lithocholic, isolithocholic, 3-keto-5β-cholanic, and 5β-cholanic acids were prepared using the reagent o,p-nitrobenzyl-N,N′-diisopropylisourea. Separation was achieved in less than 20 min on a microparticulate silica column using isocratic elution with 2% isopropanol in isooctane as the mobile phase. The p-chlorobenzoyl esters of methylated lithocholic and isolithocholic acids were also prepared but required purification by thin-layer chromatography before separation by hplc. These derivatives were eluted from a Porasil T column using 5% diisopropyl ether in isooctane as the mobile phase. Lithocholic and isolithocholic acids produced by microbial metabolism of [¹⁴COOH]taurolithocholic acid were separated and identified by preparing p-nitrobenzyl derivatives and monitoring the column effluent for both uv and radioactivity. This technique is a rapid and sensitive method for isolating bile acid metabolites.     

Potential of selected lactic acid bacteria to produce food compatible antifungal metabolites

The aim of this study was to assess the potential of lactic acid bacteria to inhibit the outgrowth of some common food-spoiling fungi. Culture supernatants of 17 lactic acid bacterial strains as well as of three commercial probiotic cultures were evaluated for antifungal activity using an agar-diffusion method. The method parameters were chosen in order to reveal compounds for potential use in food (bio)preservation. Thirteen strains showed antifungal activity of which five strains were very promising: Lactobacillus acidophilus LMG 9433, L. amylovorus DSM 20532, L. brevis LMG 6906, L. coryniformis subsp. coryniformis LMG 9196 and L. plantarum LMG 6907. Four of these five strains were further examined; it was found that the produced antifungal metabolites were pH-dependent. The exact chemical nature of these substances has not been revealed yet.