Photoaffinity labeling studies of the rat renal sodium bile salt cotransport system

The uptake of a photolabile taurocholate derivative, (7,7-azo-3 alpha, 12 alpha-dihydroxy-5 beta-cholan-24-oyl)-2-aminoethanesulfonate, 7,7-azo-TC, into rat renal brush-border membrane vesicles was stimulated by Na+ and inhibited by taurocholate indicating an interaction with the Na+/bile salt cotransport system. Irradiation of membrane vesicles in the presence of 7,7-azo-TC inhibited Na+-dependent taurocholate uptake irreversibly. Photoaffinity labeling with [3H]7,7-azo-TC resulted in a predominant incorporation of radioactivity into a polypeptide with apparent molecular weight of 99,000. These results suggest that the proteins involved in Na+/bile salt cotransport are similar in renal and ileal brush-border membranes, but differ from those in hepatocytes.     

Photolabile derivatives of bile salts. Synthesis and suitability for photoaffinity labeling

In an approach to the identification of bile salt-binding carriers, the photoactivable bile acid derivatives A) 3 beta-azido, 7 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oic acid, B) 7,7-azo-3 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oic acid, and C) 11 xi-azido-12-oxo-3 alpha,7 alpha-dihydroxy-5 beta-cholan-24-oic acid were synthesized in unconjugated and taurine-conjugated form. Photolysis of the 3 beta-azido derivatives was studied using a light source with a maximum emission at 300 nm and established a half-life time of 18.5 min. The photochemistry of the 7,7-azo derivatives was investigated using light with a maximum at 350 nm and had a half-life time of 2.2 min. The 11 xi-azido-12-oxo derivatives were photolyzed with light having a maximum at 300 nm resulting in a half-life time of 8.5 min. The suitability of the 7,7-azo derivatives for photoaffinity labeling was demonstrated by photolyses in 14C-labeled methanol and acetonitrile. The generated carbene reacted with the solvents under covalent bond formation of 6 to 12%. The efficiency of all synthesized photolabile derivatives for photoaffinity labeling of bile salt binding proteins was demonstrated.     

3-Diazirine-derivatives of bile salts for photoaffinity labeling

New carbene-generating photolabile bile salt derivatives, 3,3-azo-7 alpha,12 alpha-dihydroxy-5 beta [7 beta-3H]cholan-24-oic acid and (3,3-azo-7 alpha,12 alpha-dihydroxy-5 beta [7 beta-3H]cholan-24-oyl)-2- aminoethanesulfonic acid were synthesized with high specific radioactivity. These 3-diazirine-derivatives could be activated to the corresponding carbenes by irradiation with ultraviolet light at 350 nm with a half-life time of 2 min. The 3-diazirine derivatives behaved in enterohepatic circulation like the natural bile salts. The uptake of [3H]taurocholate into isolated hepatocytes was competitively inhibited by (3,3-azo-7 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oyl)-2- aminoethanesulfonic acid indicating that the 3,3-azo-derivative of taurocholate shares the hepatic transport systems for natural bile salts. It was demonstrated that the radioactively labeled 3-diazirine bile salt derivatives are useful probes for photoaffinity labeling of bile salt binding proteins especially in intact cells and tissues.     

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.     

The effect of cholesterol feeding on gallbladder bile acids of the rabbit. Evidence that lithocholic acid is a primary bile acid in the rabbit.

The bile acid in gallbladder bile of rabbits fed a normal diet or one containing 2% (w/w) cholesterol have been determined by gas chromatography-mass spectrometry. The predominant bile acids in normally fed rabbits were 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholan-24-oic acid (cholic acid), 3 alpha, 12 alpha-dihydroxy-5 alpha-cholan-24-oic acid (allodeoxycholic acid) and 3 alpha, 12 alpha-dihydroxy-5 beta-cholan-24-oic acid (deoxycholic acid) with very much smaller amounts of 3 alpha-hydroxy-5 beta-cholan-24-oic acid (lithocholic acid) and 3 alpha, 12 beta-dihydroxy-5 beta-cholan-24-oic acid. In the cholesterol-fed animals the lithocholate became a predominant bile acid. Sulphated bile acids accounted for less than 1% of the total bile acids. It is proposed that lithocholic acid may be a primary bile acid in the cholesterol-fed rabbit, formed by an alternative pathway of biosynthesis involving hepatic mitochondria.     

Bile acids of snakes of the subfamily Viperinae and the biosynthesis of C-23-hydroxylated bile acids in liver homogenate fractions from the adder, Vipera berus (Linn.).

1. Analysis of bile salts of four snakes of the subfamily Viperinae showed that their bile acids consisted mainly of C-23-hydroxylated bile acids. 2. Incubations of 14C-labelled sodium cholate (3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholan-24-oate) and deoxycholate (3 alpha, 12 alpha-dihydroxy-5 beta-cholan-24-oate) with whole and fractionated adder liver homogenates were carried out in the presence of molecular oxygen and NADPH or an NADPH-generating system. The formation of C-23-hydroxylated bile acids, namely bitocholic acid (3 alpha, 12 alpha, 23xi-trihydroxy-5 beta-cholan-24-oic acid) and 3 alpha, 7 alpha, 12 alpha, 23 xi-tetrahydroxy-cholanic acid (3 alpha, 7 alpha, 12 alpha, 23 xi-tetrahydroxy-5 beta-cholan-24-oic acid), was observed mainly in the microsomal fraction and partly in the mitochondrial fraction. 3. Biosynthetic pathways of C-23-hydroxylated bile acids are discussed.     

Fluorescent derivatives of bile salts. II. Suitability of NBD-amino derivatives of bile salts for the study of biological transport.

Interaction of unconjugated and taurine-conjugated NBD-amino-dihydroxy-5 beta-cholan-24-oic acids bearing the fluorophor in the 3 alpha, 3 beta, 7 alpha, 7 beta, 12 alpha, or 12 beta position with albumin results in a small hypsochromic shift of the emission maximum and an increase in quantum yield, suggesting binding by hydrophobic interactions. The different unconjugated fluorescent bile salt derivatives are metabolized by intact rat liver in different ways. The unconjugated 3 beta-NBD-amino derivative is completely transformed to its taurine conjugate and secreted as such, whereas the 3 alpha-NBD-amino derivative is completely transformed to a polar fluorescent compound not identical with its taurine conjugate. The unconjugated 7 alpha- and 7 beta-NBD-amino derivatives are only partially conjugated with taurine and mainly secreted in unmetabolized form. The unconjugated 12 alpha- and 12 beta-NBD-amino derivatives are not at all transformed to their taurine conjugates, but are partially metabolized to unidentified compounds. They are predominantly secreted as the unmetabolized compounds. In contrast to the unconjugated derivatives, all taurine-conjugated fluorescent bile salt derivatives are secreted into bile unmetabolized. With the exception of the 3 alpha-compound, all synthesized taurine-conjugated fluorescent derivatives interfere with the secretion of cholyltaurine. Differential photoaffinity labeling studies using (7,7-azo-3 alpha,12 alpha- dihydroxy-5 beta-cholan-24-oyl)-2'-[2'-3H(N)]aminoethanesulfonate as a photolabile derivative revealed that in liver cells all fluorescent bile salt derivatives interact with the same polypeptides as the physiological bile salts. The hepatobiliary transport of taurine-conjugated NBD-amino bile salt derivatives is, due to hydrophobic interactions, accompanied by an increase in fluorescence intensity which is favorable for the study of biological bile salt transport by fluorescence microscopy.     

Synthesis of 3 alpha,6 beta,7 alpha,12 beta- and 3 alpha,6 beta,7 beta,12 beta-tetrahydroxy-5 beta-cholanoic acids.

Chemical synthesis of 3 alpha,6 beta,7 alpha,12 beta- and 3 alpha,6 beta,7 beta,12 beta-tetrahydroxy-5 beta-cholan-24-oic acids is described. 3 alpha,12 beta-Dihydroxy-5 beta-chol-6-en-24-oic acid used as the starting material in the synthesis was prepared via oxidation of 3 alpha,12 alpha-dihydroxy-5 beta-chol-6-en-24-oic acid 3-hemisuccinate at C-12 followed by reduction with potassium/tertiary amyl alcohol. alpha-Epoxidation of the ester diacetate of 3 alpha,12 beta-dihydroxy-5 beta-chol-6-en-24-oic acid with m-chloroperbenzoic acid followed by cleavage of the epoxide with acetic acid and alkaline hydrolysis yielded 3 alpha,6 beta,7 alpha,12 beta-tetrahydroxy-5 beta-cholan-24-oic acid (overall yield 25%). N-Methylmorpholine-N-oxide-catalyzed osmium tetroxide oxidation of the ester diacetate of 3 alpha,12 beta-dihydroxy-5 beta-chol-6-en-24-oic acid followed by alkaline hydrolysis yielded 3 alpha,6 beta,7 beta,12 beta-tetrahydroxy-5 beta-cholan-24-oic acid (overall yield 33%). The structures of the synthesized bile acids were confirmed from their proto nuclear magnetic resonance and mass spectral fragmentation patterns.     

Synthesis of potential cholelitholytic agents: 3 alpha,7 alpha,12 alpha-trihydroxy-7 beta-methyl-5 beta-cholanoic acid, 3 alpha,7 beta,12 alpha-trihydroxy-7 alpha-methyl-5 beta-cholanoic acid, and 3 alpha,12 alpha-dihydroxy-7 xi-methyl-5 beta-cholanoic acid

This report describes the chemical synthesis of six new bile acid analogs, namely, 3 alpha,7 alpha,12 alpha-trihydroxy-7 beta-methyl-5 beta-cholanoic acid (7 beta-methyl-cholic acid), 3 alpha,7 beta,12 alpha-trihydroxy-7 alpha-methyl-5 beta-cholanoic acid (7 alpha-methyl-ursocholic acid), 3 alpha,12 alpha-dihydroxy-7 xi-methyl-5 beta-cholanoic acid (7 xi-methyl-deoxycholic acid), 3 alpha,12 alpha-dihydroxy-7-methyl-5 beta-chol-7-en-24-oic acid, 3 alpha,12 alpha-dihydroxy-7-methyl-5 beta-chol-6-en-24-oic acid, and 3 alpha,12 alpha-dihydroxy-7-methylene-5 beta-cholan-24-oic acid. The carboxyl group of the starting material 3 alpha,12 alpha-dihydroxy-7-oxo-5 beta-cholanoic acid was protected by conversion to its oxazoline derivative. A Grignard reaction of the bile acid oxazoline with CH3MgI followed by acid hydrolysis gave two epimeric trihydroxy-7-methyl-cholanoic acids and three dehydration products. The latter were purified by silica gel column chromatography and silica gel-AgNO3 column chromatography of their methyl ester derivatives. Catalytic hydrogenation of 3 alpha,12 alpha-dihydroxy-7-methyl-5 beta-chol-6-en-24-oic acid and 3 alpha,12 alpha-dihydroxy-7-methylene-5 beta-cholan-24-oic acid gave 3 alpha,12 alpha-dihydroxy-7 xi-methyl-5 beta-cholanoic acid. The configuration of the 7-methyl groups and the position of the double bonds were assigned by proton nuclear magnetic resonance spectroscopy and the chromatographic and mass spectrometric properties of the new compounds. These compounds were synthesized for the purpose of exploring new and potentially more effective cholelitholytic agents. The hydrophilic bile acids 7 beta-methyl-cholic acid and 7 alpha-methyl-ursocholic acid are of particular interest because they should be resistant to bacterial 7-dehydroxylation.     

Identification of bile acids in the serum and urine in cholestasis. Evidence for 6alpha-hydroxylation of bile acids in man.

In this qualitative study of the pattern of bile acid excretion in cholestasis, methods are described for the isolation of bile acids from large volumes of urine and plasma. The bile acids were subjected to a group separation and identified by combined gas chromatography-mass spectrometry. The techniques were developed to allow identification of the minor components of the bile acid mixture. Four bile acids that have not previously been described in human urine and plasma were detected, namely 3beta, 7alpha-dihydroxy-5beta-cholan-24-oic acid, 3alpha, 6alpha-dihydroxy-5beta-cholan-24-oic acid (hyodeoxycholic acid), 3alpha, 6alpha, 7alpha-trihydroxy-5beta-cholan-24-oic acid (hyocholic acid) and 3alpha, 7beta, 12alpha-trihydroxy-5beta-cholan-24-oic acid. In addition three C27 steroids were found; 26-hydroxycholesterol and a trihydroxy cholestane, probably 5 beta-cholestane-3alpha, 7alpha, 26-triol were found in the sulphate fraction of plasma and urine. In the plasma sample, a sulphate conjugate of 24-hydroxycholesterol was found. The presence of these compounds probably reflects the existence of further pathways for bile acid metabolism. It is not yet known whether this is a consequence of the cholestasis or whether they are also present in normal man, at much lower concentrations.     

Synthesis of 24-nor-5 beta-cholan-23-oic acid derivatives: a convenient and efficient one-carbon degradation of the side chain of natural bile acids

An efficient procedure for obtaining nor-bile acids from natural (C24) bile acids is described. Treatment of formylated bile acids with sodium nitrite in a mixture of trifluoroacetic anhydride with trifluoroacetic acid gives, through a "second order" Beckmann rearrangement, 24-nor-23-nitriles. These compounds, on alkaline hydrolysis, afford the corresponding nor-bile acids in high yields. The sequence was successfully applied to the synthesis of 3 alpha-hydroxy-24-nor-5 beta-cholan-23-oic (norlithocholic) acid, 3 alpha,6 alpha- (norhyodeoxycholic), 3 alpha,7 alpha- (norchenodeoxycholic), 3 alpha,7 beta- (norursodeoxycholic), and 3 alpha,12 alpha-dihydroxy-24-nor-5 beta-cholan-23-oic (nordeoxycholic) acids, as well as 3 alpha,7 alpha,12 alpha-trihydroxy-24-nor-5 beta-cholan-23-oic (norcholic) acid. 13C-NMR spectra of their methyl esters are reported. The procedure provides a more rapid alternative to the Barbier-Wieland degradation for shortening by one methylene group the side chain of natural (C24) bile acids.     

Anodic electrochemical oxidation of cholic acid

Regioselectivity in the anodic electrochemical oxidation of cholic acid with different anodes is described. The oxidation with PbO(2) anode affords the dehydrocholic acid in quantitative yield after 22 h. 3alpha,12alpha-Dihydroxy-7-oxo-5beta-cholan-24-oic acid (59%) and 3alpha-hydroxy-7,12-dioxo-5beta-cholan-24-oic acid (51%) are obtained stopping the reaction at lower time. The rate of the OH-oxidation is C7 > C12 > C3. The electro-oxidation with platinum foil anode gives selectively the 7-ketocholic acid in 40% yield. On the other hand, the graphite plate anode, varying the reaction conditions, produces selectively the dehydrocholic acid in quantitative yield or the 3alpha,12alpha-dihydroxy-7-oxo-5beta-cholan-24-oic acid (96%) while the 3alpha,7alpha-dihydroxy-12-oxo-5beta-cholan-24-oic acid (34%) is obtained together with the other oxo acids.     

ATP-dependent transport of taurocholate across the hepatocyte canalicular membrane mediated by a 110-kDa glycoprotein binding ATP and bile salt

Direct photoaffinity labeling of liver plasma membrane subfractions enriched in sinusoidal and canalicular membranes using [35S]adenosine 5'-O-(thiotriphosphate) ([35S]ATP gamma S) allows the identification of ATP-binding proteins in these domains. Comparative photoaffinity labeling with [35S]ATP gamma S and with the photolabile bile salt derivative (7,7-azo-3 alpha, 12 alpha-dihydroxy-5 beta-[3 beta-3H]-cholan-24-oyl-2'- aminoethanesulfonate followed by immunoprecipitation with a monoclonal antibody (Be 9.2) revealed the identity of the ATP-binding and the bile salt-binding canalicular membrane glycoprotein with the apparent Mr of 110,000 (gp110). The isoelectric point of this glycoprotein was 3.7. Transport of bile salt was studied in vesicles enriched in canalicular and sinusoidal liver membranes. Incubation of canalicular membrane vesicles with [3H] taurocholate in the presence of ATP resulted in an uptake of the bile salt into the vesicles which was sensitive to vanadate. ATP-dependent taurocholate transport was also observed in membrane vesicles from mutant rats deficient in the ATP-dependent transport of cysteinyl leukotrienes and related amphiphilic anions. Substrates of the P-glycoprotein (gp170), such as verapamil and doxorubicin, did not interfere with the ATP-dependent transport of taurocholate. Reconstitution of purified gp110 into liposomes resulted in an ATP-dependent uptake of [3H]taurocholate. These results demonstrate that gp110 functions as carrier in the ATP-dependent transport of bile salts from the hepatocyte into bile. This export carrier is distinct from hitherto characterized ATP-dependent transport systems.     

Bile salts of the green turtle Chelonia mydas (L.)

1. Bile salts of the green turtle Chelonia mydas (L.) were analysed as completely as possible. 2. They consist of taurine conjugates of 3 alpha, 7 alpha, 12 alpha, 22 xi-tetrahydroxy-5 beta-cholestan-26-oic acid (tetrahydroxysterocholanic acid) and 3 alpha 12 alpha, 22 xi-trihydroxy-5 beta-cholestan-26-oic acid, with minor amounts of 3 alpha, 7 alpha, 12 alpha-trihydroxy-5beta-cholan-24-oic acid (cholic acid), 3alpha, 12 alpha-dihydroxy-5beta-cholan-24-oic acid (deoxycholic acid) and possibly other bile acids. 3. Cholic acid and deoxycholic acid represent the first known examples of bile acids common to chelonians and other animal forms: they may indicate independent evolution in chelonians to C24 bile acids. 4. The discovery of a 7-deoxy C27 bile acid is the first evidence that C27 bile acids or their conjugates have an enterohepatic circulation.     

Effect of bile acid analogs on 7 alpha-dehydroxylase activity in Eubacterium sp. V.P.I. 12708

7 beta-Methyl-chenodeoxycholic acid (7-MeCDC, 3 alpha, 7 alpha-dihydroxy-7 beta-methyl-5 beta-cholan-24-oic acid), 7 alpha-methyl-ursodeoxycholic acid (7-MeUDC, 3 alpha, 7 beta-dihydroxy-7 alpha-methyl-5 beta-cholan-24-oic acid), 7 xi-methyl-lithocholic acid (7-MeLC, 3 alpha-hydroxy-7 xi-methyl-5 beta-cholan-24-oic acid) and ursodeoxycholylsarcosine (UDCS) were tested as inhibitors of bacterial bile acid 7 alpha-dehydroxylase activity. At a concentration of 50 microM, 7-MeCDC and 7-MeUDC inhibited enzyme activity by 66% and 12%, respectively. 7 alpha-Dehydroxylase activity was not inhibited in the presence of 7-MeLC and UDCS. None of the four bile acid analogs tested inhibited the growth of Eubacterium sp. V.P.I. 12708 at concentrations up to 100 microM.     

Identification of different transport systems for bile salts in sinusoidal and canalicular membranes of hepatocytes

The preservation of the functional polarity of hepatocytes in liver snips (1 x 2 x 4 mm) was demonstrated by fluorescent microscopic studies using the sodium salt of (N-[7-(4-nitrobenzo-2-oxa-1,3-diazol)]-3 beta-amino-7 alpha,12 alpha- dihydroxy-5 beta-cholan-24-oyl)-2-aminoethanesulfonic acid. This fluorescent bile salt derivative is not only taken up by hepatocytes of several cell layers at the surface of the snips but also secreted into bile canaliculi. The intact hepatobiliary transport of bile salts by hepatocytes of liver snips demonstrates that they are a useful system for the investigation of those transcellular transport processes which require the integrity of hepatic structure. Photoaffinity labelling of liver snips with the sodium salt of (7,7-azo-3 alpha,12 alpha-dihydroxy-5 beta-[3 beta-3H]cholan- 24-oyl)-2-aminoethanesulfonic acid revealed that the bile-salt-binding membrane polypeptides with apparent Mr values of 54,000 and 48,000 are exclusively located in the sinusoidal membrane, whereas a single bile-salt-binding polypeptide with an apparent Mr of 100,000 is located in the bile-canalicular membrane. Photoaffinity labelling of liver snips at 4 degrees C, when transcellular bile-salt transport is insignificant, resulted in the labelling of the two sinusoidal membrane polypeptides and practically no labelling of the polypeptide with an apparent Mr of 100,000. This latter polypeptide was also not labelled when Ca2 deprivation abolished bile secretion completely. These results indicate that the directed hepatobiliary transport of bile salts in hepatocytes is accomplished by transport systems which are different for sinusoidal uptake and canalicular secretion.     

Isolation and characterization of the putative canalicular bile salt transport system of rat liver

Through labeling with the sodium salt of the photolabile bile salt derivative (7,7-azo-3 alpha,12 alpha-dihydroxy-5 beta-[3 beta-3H]cholan-24-oyl)- 2-aminoethanesulfonic acid, a bile salt-binding polypeptide with an apparent molecular weight of 100,000 was identified in isolated canalicular but not basolateral (sinusoidal) rat liver plasma membranes. This labeled polypeptide was isolated from octyl glucoside-solubilized canalicular membranes by DEAE-cellulose and subsequent wheat germ lectin Sepharose chromatography. The purified protein still contained covalently incorporated radioactive bile salt derivative and exhibited a single band with an apparent molecular weight of 100,000 on sodium dodecyl sulfate-gels. Antibodies were raised in rabbits and their monospecificity toward this canalicular polypeptide demonstrated by immunoblot analysis. No cross-reactivity was found with basolateral membrane proteins. The antibodies inhibited taurocholate uptake into isolated canalicular but not basolateral membrane vesicles. In addition, the antibodies also decreased efflux of taurocholate from canalicular vesicles. If the canalicular bile salt-binding polypeptide was immunoprecipitated from Triton X-100-solubilized canalicular membranes and subsequently deglycosylated with trifluoromethanesulfonic acid, the apparent molecular weight was decreased from 100,000 to 48,000 (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). These studies confirm previous results in intact liver tissue and strongly indicate that a canalicular specific glycoprotein with an apparent molecular weight of 100,000 is directly involved in canalicular excretion of bile salts.     

Mechanisms of hepatic transport of cyclosporin A: an explanation for its cholestatic action?

The hepatic transport of the immunosuppressive Cyclosporin A (CyA) was studied using liposomal phospholipid membranes, freshly isolated rat hepatocytes and bile canalicular plasma membrane vesicles from rat liver. The Na(+)-dependent, saturable uptake of the bile acid 3H-taurocholate into isolated rat liver cells was apparently competitively inhibited by CyA. However, the uptake of CyA into the cells was neither saturable, nor temperature-dependent nor Na(+)-dependent, nor could it be inhibited by bile salts or CyA-derivatives, indicating passive diffusion. In steady state depolarization fluorescence studies, CyA caused a concentration-dependent decrease of anisotropy, indicating a membrane fluidizing effect. Ion flux experiments demonstrated that CyA dramatically increases the permeability of Na+ and Ca2+ across phospholipid membranes in a dose- and time-dependent manner, suggesting a iontophoretic activity that might have a direct impact on cellular ion homeostasis and regulation of bile acid uptake. Photoaffinity labeling with a [3H]-labeled photolabile CyA-derivative resulted in the predominant incorporation of radioactivity into a membrane polypeptide with an apparent molecular weight of 160,000 and a minor labeling of polypeptides with molecular weights of 85,000-90,000. In contrast, use of a photolabile bile acid resulted in the labeling of a membrane polypeptide with an apparent molecular weight of 110,000, representing the bile canalicular bile acid carrier. The photoaffinity labeling as well as CyA transport by canalicular membrane vesicles were inhibited by CyA and the p-glycoprotein substrates daunomycin and PSC-833, but not by taurocholate, indicating that CyA is excreted by p-glycoprotein. CyA uptake by bile canalicular membrane vesicles was ATP-dependent and could not be inhibited by taurocholate. CyA caused a decrease in the maximum amount of bile salt accumulated by the vesicles with time. However, initial rates of [3H]-taurocholate uptake within the first 2.5 min remained unchanged at increasing CyA concentrations. In summary, the data indicate that CyA does not directly interact with the hepatic bile acid transport systems. Its cholestatic action may rather be the result of alterations in membrane fluidity, intracellular effects and an interaction with p-glycoprotein.     

Isolation and chemical synthesis of a major, novel biliary bile acid in the common wombat (Vombatus ursinus): 15alpha-hydroxylithocholic acid

The major bile acids present in the gallbladder bile of the common Australian wombat (Vombatus ursinus) were isolated by preparative HPLC and identified by NMR as the taurine N-acylamidates of chenodeoxycholic acid (CDCA) and 15alpha-hydroxylithocholic acid (3alpha,15alpha-dihydroxy-5beta-cholan-24-oic acid). Taurine-conjugated CDCA constituted 78% of biliary bile acids, and (taurine-conjugated) 15alpha-hydroxylithocholic acid constituted 11%. Proof of structure of the latter compound was obtained by its synthesis from CDCA via a Delta14 intermediate. The synthesis of its C-15 epimer, 15beta-hydroxylithocholic acid (3alpha,15beta-dihydroxy-5beta-cholan-24-oic acid), is also reported. The taurine conjugate of 15alpha-hydroxylithocholic acid was synthesized and shown to have chromatographic and spectroscopic properties identical to those of the compound isolated from bile. It is likely that 15alpha-hydroxylithocholic acid is synthesized in the wombat hepatocyte by 15alpha-hydroxylation of lithocholic acid that was formed by bacterial 7alpha-dehydroxylation of CDCA in the distal intestine. Thus, the wombat appears to use 15alpha-hydroxylation as a novel detoxification mechanism for lithocholic acid.     

Identification of a novel bile acid in swans, tree ducks, and geese: 3alpha,7alpha,15alpha-trihydroxy-5beta-cholan-24-oic acid

By HPLC, a taurine-conjugated bile acid with a retention time different from that of taurocholate was found to be present in the bile of the black-necked swan, Cygnus melanocoryphus. The bile acid was isolated and its structure, established by (1)H and (13)C NMR and mass spectrometry, was that of the taurine N-acyl amidate of 3alpha,7alpha,15alpha-trihydroxy-5beta-cholan-24-oic acid. The compound was shown to have chromatographic and spectroscopic properties that were identical to those of the taurine conjugate of authentic 3alpha,7alpha,15alpha-trihydroxy-5beta-cholan-24-oic acid, previously synthesized by us from ursodeoxycholic acid. By HPLC, the taurine conjugate of 3alpha,7alpha,15alpha-trihydroxy-5beta-cholan-24-oic acid was found to be present in 6 of 6 species in the subfamily Dendrocygninae (tree ducks) and in 10 of 13 species in the subfamily Anserinae (swans and geese) but not in other subfamilies in the Anatidae family. It was also not present in species from the other two families of the order Anseriformes. 3alpha,7alpha,15alpha-Trihydroxy-5beta-cholan-24-oic acid is a new primary bile acid that is present in the biliary bile acids of swans, tree ducks, and geese and may be termed 15alpha-hydroxy-chenodeoxycholic acid.