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.     

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.     

Fluorescent derivatives of bile salts. I. Synthesis and properties of NBD-amino derivatives of bile salts.

In order to visualize bile salt transport, fluorescent bile salt derivatives were synthesized by introduction of the relatively small fluorescent 4-nitrobenzo-2-oxa-1,3-diazol (NBD)-amino group in either the 3-, 7-, or 12-position of the steroid structure, thus providing a complete set of diastereomeric derivatives, 3 alpha-NBD-amino-7 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oic acid, 3 beta-NBD-amino-7 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oic acid, 7 alpha-NBD-amino-3 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oic acid, 7 beta-NBD-amino-3 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oic acid, 12 alpha-NBD-amino-3 alpha,7 alpha-dihydroxy-5 beta-cholan-24-oic acid, 12 beta-NBD-amino-3 alpha,7 alpha-dihydroxy-5 beta-cholan-24-oic acid, as well as their taurine conjugates. Their optical properties with absorption maxima at about 490 nm and emission maxima at 550 nm make them suitable for fluorescent microscopic studies. Fluorescence of the NBD-derivatives is strongly dependent on polarity of the solvent, on the concentration of the bile salt derivatives, and only slightly on temperature.     

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.     

Enzyme profiles of mammalian bile.

The activities of subcellular marker enzymes in bile and liver homogenate from several mammalian species have provided information on the specificity of protein release during bile formation. The presence of significant amounts of the plasma membrane enzymes alkaline phosphodiesterase I and leucyl-beta-naphthylamidase in bile, in addition to alkaline phosphatase and 5'-nucleotidase, and the relative absence of intracellular enzymes lends support to the view that bile salt liberation from the hepatocyte is accompanied by a partial solubilization of the plasma (canalicular) membrane without extensive damage to the whole hepatocyte.     

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.     

Bilirubin conjugates of human bile. Isolation of phenylazo derivatives of bile bilirubin

A method is presented that allows the isolation of eight different phenylazo derivatives of bile bilirubin. In step I of the isolation procedure, three bilirubin fractions (bilirubin fractions 1, 2 and 3) from human hepatic bile are separated by reverse-phase partition chromatography on silicone-treated Celite with the use of a solvent system prepared from butan-1-ol and 5mm-phosphate buffer, pH6.0. Azo coupling is then performed with diazotized aniline. The three azo pigment mixtures are subjected to step II, in which the above chromatography system is used again. With each azo pigment mixture this step brings about the separation of a non-polar and a polar azo pigment fraction (azo 1A and azo 1B, azo 2A and azo 2B, and azo 3A and azo 3B from bilirubin fractions 1, 2 and 3 respectively). Approximately equal amounts of non-polar and polar pigments are obtained from bilirubin fractions 1 and 2, whereas bilirubin fraction 3 yields azo 3B almost exclusively. In step IIIA the non-polar azo pigment fractions are fractionated further by adsorption chromatography on anhydrous sodium sulphate with the use of chloroform followed by a gradient of ethyl acetate in chloroform. Three azo pigments are thus obtained from both azo 2A (azo 2A(1), azo 2A(2) and azo 2A(3)) and azo 3A (azo 3A(1), azo 3A(2) and azo 3A(3)). The 2A pigments occur in approximately the following proportions: azo 2A(1), 90%; azo 2A(2), 10%; azo 2A(3), traces. The pigments are purified by crystallization, except for the A(3) pigments, which are probably degradation products arising from the corresponding A(2) pigments. In step IIIB the polar azo pigment fractions are subjected to reverse-phase partition chromatography on silicone-treated Celite with the use of a solvent system prepared from octan-1-ol-di-isopropyl ether-ethyl acetate-methanol-0.2m-acetic acid (1:2:2:3:4, by vol.). Azo pigment fractions 2B and 3B each yield six azo pigments (azo 2B(1) to azo 2B(6) and azo 3B(1) to azo 3B(6) respectively) together with small amounts of products of hydrolysis (azo 2A(B) and azo 3A(B)). Only one azo B pigment is obtained from bilirubin fraction 1, and this azo pigment is probably of the B(2) type. The yields of the azo 3B pigments suggest that these pigments are present in approximately the following proportions: azo 3B(1), 0-0.4%; azo 3B(2), traces; azo 3B(3), traces; azo 3B(4), 10%; azo 3B(5), 50%; azo 3B(6), 40%. Azo pigments 2B(1) to 2B(6) are estimated to occur in similar proportions. Since pairs of correspondingly numbered azo pigments from bilirubin fractions 1, 2 and 3 do not separate on rechromatography together (e.g. azo 2A(1) co-chromatographs with azo 3A(1), and azo 2B(6) co-chromatographs with azo 3B(6)), it is concluded that such pigments are chemically identical. The structures of the isolated phenylazo derivatives are discussed in an accompanying paper (Kuenzle 1970c).     

Ursodeoxycholate: a common bile acid in gallbladder bile of Japanese subjects.

Bile acid composition was investigated in normal gallbladder-bile collected from the Japanese patients suffering from the diseases other than hepatobiliary tracts.In addition to cholate, chenodeoxycholate, deoxycholate and lithocholate, ursodeoxycholate was detected as a predominant bile acid in all cases tested and its quantity was higher than that of lithocholate in most cases.A simplified method has been developed for the quantitative determination of bile acids. They were derived to their methyl ester-trimethylsilyl ethers and determined by gas-liquid chromatography on a column of 3% poly-phenyldiethanol amine succinate-80-100 mesh Chromosorb WHP. Average recoveries of added amounts of standard bile acids were found to range from 97 to 100%.