The anionic conjugates of bilirubin and bile acids stimulate ATP hydrolysis by S-(dinitrophenyl)glutathione ATPase of human erythrocyte.

These studies demonstrate that bilirubin-ditaurate (an analog of bilirubin-diglucuronide), lithocholic acid 3-O-sulfate, and lithocholic acid 3-O-glucuronide, which are believed to be transported from liver into bile through an active transport process stimulate ATP hydrolysis by purified dinitrophenylglutathione ATPase of human erythrocytes. The Km and Vmax values of the enzyme for these substrates are similar to those for dinitrophenylglutathione indicating the transport mechanisms for bilirubin conjugates, and anionic bile acid-conjugates from hepatocytes to bile and transport of GSH-conjugates from erythrocytes may be mediated by similar mechanisms.     

Comparison of the binding sites of GSH S-transferases of the Ya- and Yb-subunit classes: effect of glutathione on the binding of bile acids

We have previously observed that the Ya subunit-containing glutathione (GSH) S-transferases from rat liver exhibit a common high affinity binding site for lithocholic acid, bilirubin, and sulfobromophthalein (BSP) (1984. J. Lipid Res. 25: 1177-1183). Subsequently we found that cholic acid and its amidates bound to a site on the Ya subunit separate for the lithocholic acid/bilirubin site (1986. J. Lipid Res. 27: 955-966). We now have extended this work by showing that amidates of lithocholic acid as well as chenodeoxycholic acid and its amidates competitively displace [14C]lithocholic acid from the Ya subunit. GSH did not inhibit binding of any of the ligands to the high affinity Ya site, but did inhibit binding to the cholic acid site on the Ya subunit. We have also defined the binding sites and effects of GSH on the Yb class of subunits. Lithocholic, chenodeoxycholic, and cholic acids (and amidates) shared a common site on the Yb or Y'b subunit, whereas BSP and bilirubin were bound at a different site. Both the bile acid and organic anion sites on the Yb subunit were inhibited by GSH. The inhibition by GSH in all cases (Ya cholic acid site or Yb bile acid or bilirubin sites) was saturable, of the competitive type, and incomplete at maximal GSH concentrations, suggesting that when GSH binds to its distinct substrate site, it induces a conformational change in the proteins affecting the other binding sites.     

Synthesis of the 3-sulfates of S-acyl glutathione conjugated bile acids and their biotransformation by a rat liver cytosolic fraction

The 3-sulfates of the S-acyl glutathione (GSH) conjugates of five natural bile acids (cholic, chenodeoxycholic, deoxycholic, ursodeoxycholic, and lithocholic) were synthesized as reference standards in order to investigate their possible formation by a rat liver cytosolic fraction. Their structures were confirmed by proton nuclear magnetic resonance, as well as by means of electrospray ionization-linear ion-trap mass spectrometry with negative-ion detection. Upon collision-induced dissociation, structurally informative product ions were observed. Using a triple-stage quadrupole instrument, selected reaction monitoring analyses by monitoring characteristic transition ions allowed the achievement of a highly sensitive and specific assay. This method was used to determine whether the 3-sulfates of the bile acid-GSH conjugates (BA-GSH) were formed when BA-GSH were incubated with a rat liver cytosolic fraction to which 3'-phosphoadenosine 5'-phosphosulfate had been added. The S-acyl linkage was rapidly hydrolyzed to form the unconjugated bile acid. A little sulfation of the GSH conjugates occurred, but greater sulfation at C-3 of the liberated bile acid occurred. Sulfation was proportional to the hydrophobicity of the unconjugated bile acid. Thus GSH conjugates of bile acids as well as their C-3 sulfates if formed in vivo are rapidly hydrolyzed by cytosolic enzymes.     

Formation and biliary excretion of glutathione conjugates of bile acids in the rat as shown by liquid chromatography/electrospray ionization-linear ion trap mass spectrometry

Acyl-adenylates and acyl-CoA thioesters of bile acids (BAs) are reactive acyl-linked metabolites that have been shown to undergo transacylation-type reactions with the thiol group of glutathione (GSH), leading to the formation of thioester-linked GSH conjugates. In the current study, we examined the transformation of cholyl-adenylate (CA-AMP) and cholyl-coenzyme A thioester (CA-CoA) into a cholyl-S-acyl GSH (CA-GSH) conjugate by rat hepatic glutathione S-transferase (GST). The reaction product was analyzed by liquid chromatography (LC)/electrospray ionization (ESI)-linear ion trap mass spectrometry (MS). The GST-catalyzed formation of CA-GSH occurred with both CA-AMP and CA-CoA. Ursodeoxycholic acid, lithocholic acid, and 2,2,4,4-²H₄-labeled lithocholic acid were administered orally to biliary fistula rats, and their corresponding GSH conjugates were identified in bile by LC/ESI-MS². These in vitro and in vivo studies confirm a new mode of BA conjugation in which BAs are transformed into their GSH conjugates via their acyl-linked intermediary metabolites by the catalytic action of GST in the liver, and the GSH conjugates are then excreted into the bile.     

Evidence for a common high affinity binding site on glutathione S-transferase B for lithocholic acid and bilirubin

Binding of lithocholic acid, bilirubin, and gossypol to glutathione S-transferase B (ligandin or transferase YaYc) was compared using four methods. Tryptophan quenching revealed a single high affinity site for bilirubin and gossypol but could not be used for lithocholic acid. Both displacement of the fluorescent probe, 1-anilino-8-naphthalenesulfonate, and spectral changes induced by bilirubin binding demonstrated a common high affinity site for which all three ligands compete. Similar results were obtained by equilibrium dialysis. The dissociation constants for the binding of both bilirubin and lithocholic acid were comparable with the various methods (range 0.2-0.7 microM). Thus, lithocholic acid and bilirubin share a high affinity binding site on gluthathione S-transferase B that appears to be separate from the binding site for substrates.     

Identification of S-acyl glutathione conjugates of bile acids in human bile by means of LC/ESI-MS

Previous work from this laboratory has reported the biotransformation of bile acids (BA) into the thioester-linked glutathione (GSH) conjugates via the intermediary metabolites formed by BA:CoA ligase and shown that such GSH conjugates are excreted into the bile in healthy rats as well as rats dosed with lithocholic acid or ursodeoxycholic acid. To examine whether such novel BA-GSH conjugates are present in human bile, we determined the concentration of the GSH conjugates of the five BA that predominate in human bile. Bile was obtained from three infants (age 4, 10, and 13 months) and the BA-GSH conjugates quantified by means of liquid chromatography (LC)/electrospray ionization (ESI)-linear ion trap mass spectrometry (MS) in negative-ion scan mode, monitoring characteristic transitions of the analytes. By LC/ESI-MS, only primary BA were present in biliary BA, indicating that the dehydroxylating flora had not yet developed. GSH conjugates of chenodeoxycholic and lithocholic acid were present in concentrations ranging from 27 to 1120 pmol/ml, several orders of magnitude less than those of natural BA N-acylamidates. GSH conjugates were not present, however, in the ductal bile obtained from 10 adults (nine choledocholithiasis, one bile duct cancer). Our results indicate that BA-GSH conjugates are formed and excreted in human bile, at least in infants, although this novel mode of conjugation is a very minor pathway.     

Comparison of biliary excretion and metabolism of lithocholic acid and its sulfate and glucuronide conjugates in rats

Biliary excretion and biotransformation of tracer doses of [14C]lithocholic acid and its sulfate and glucuronide intravenously injected into bile-drainaged rats were compared. Biliary excretion efficiency was in the order of unconjugate sulfate glucuronide and all conjugates were completely excreted into bile within 60 min after injection. Only tracer doses of radioactivity were found in the liver and urine. About 90% of radiolabeled bile acids in bile were conjugated with taurine immediately after injection of lithocholic acid, whereas lithocholic acid-glucuronide was only partly conjugated with taurine all the time (less than 6%) and excreted into bile mainly as native compound. In the first 10 min, 66% of lithocholic acid-sulfate was conjugated with taurine and it gradually proceeded up to 87%. Hydroxylation at C-6 and C-7 positions of lithocholic acid proceeded time-dependently up to 45%. No hydroxylation was observed with lithocholic acid-sulfate or glucuronide. Differences of biliary excretion rate of these conjugates may be one of the reasons for the delayed decrease of sulfated and glucuronidated bile acids in serum after bile drainage to patients with obstructive jaundice of during the recovery of acute hepatitis than non-esterified bile acids.     

A comparison of the glutathione S-transferases of trout and rat liver.

1. Cytosol from trout liver, gills and intestinal caeca has substantial glutathione S-transferase activity. 2. Gel-exclusion and ion-exchange chromatography suggest that trout liver has several glutathione S-transferases with different molecular weights and ionic charges. 3. A component capable of binding lithocholic acid eluted together with glutathione S-transferase activity. Some of the transferase activity did not elute together with binding activity. 4. The enzymic activity from trout liver was less stable at 37 degrees C than that from rat liver. 5. The glutathione S-transferases of fish liver have a similar specific activity to those of rat liver but different molecular properties.     

Identification of an anion-transport ATPase that catalyzes glutathione conjugate-dependent ATP hydrolysis in canalicular plasma membranes from normal rats and rats with conjugated hyperbilirubinemia (GY mutant).

Rat liver canalicular plasma membranes were found to contain a 37-kDa protein that is immunologically cross-reactive with the dinitrophenyl glutathione-stimulated ATPase previously identified in human tissues. The protein, which was partially purified by affinity chromatography, exhibited ATPase activity dependent on dinitrophenyl glutathione, bilirubin ditaurate, and other dianionic compounds. The localization of this protein in the canalicular membrane and its measured enzymatic activity indicate that it is involved in the transport of glutathione derivatives and other dianionic organic compounds. A rat mutant in which the above transport activities are impaired contained the protein in amounts similar to those in a normal control.     

Interactions of bilirubin and other ligands with ligandin.

Circular dichroism methods were used to study the structure of rat ligandin and the binding of organic anions to the protein. Ligandin has a highly ordered secondary structure with about 40%alpha helix, 15% beta structure, and 45% random coil. Bilirubin binding occurred primarily at a single high affinity site on the protein. The binding constant for bilirubin (5 X 10-7 Mminus 1) was the highest among the ligands studied. The bilirubin-ligandin complex exhibited a well-defined circular dichroic spectrum with two major overlapping ellipticity bands of opposite sign in the bilirubin absorption region. This spectrum was virtually a mirror image of that of human or rat serum albumin-bilirubin complexes. Studies on the direct transfer of bilirubin from ligandin to rat serum albumin showed that sasociation constants of bilirubin-ligandin complexes were approximately tenfold less than those of the bilirubin-albumin system. Ligandin exhibited a broad specificity with respect to the typeof ligand bond. A series of organic anions inclucing dyes used clinically for liver function tests, fatty acids, hormones, heme derivatives, bile acids, and other ligands that were considered likely to interact with ligandin, were examined. Most induced ellipticity changes consistent with competitive displacement of bilirubin from ligandin and relative affinities of these compounds for ligandin were determined based on their effectiveness in desplacing the bilirubin. Some substances such as glutathione, conjugated sulfobromophthaleins and lithocholic acid bound to ligandin but induced anomalous spectral shifts, when added to ligandin-bilirubin complexes. Other compounds, including some that act as substrates for the glutathione transferase activity exhibited by ligandin, revealed no apparent competitive effects with respect to the bilitubin binding site.     

6alpha-hydroxylation of taurochenodeoxycholic acid and lithocholic acid by CYP3A4 in human liver microsomes

The aim of the present study was to identify the enzymes in human liver catalyzing hydroxylations of bile acids. Fourteen recombinant expressed cytochrome P450 (CYP) enzymes, human liver microsomes from different donors, and selective cytochrome P450 inhibitors were used to study the hydroxylation of taurochenodeoxycholic acid and lithocholic acid. Recombinant expressed CYP3A4 was the only enzyme that was active towards these bile acids and the enzyme catalyzed an efficient 6alpha-hydroxylation of both taurochenodeoxycholic acid and lithocholic acid. The Vmax for 6alpha-hydroxylation of taurochenodeoxycholic acid by CYP3A4 was 18.2 nmol/nmol P450/min and the apparent Km was 90 microM. Cytochrome b5 was required for maximal activity. Human liver microsomes from 10 different donors, in which different P450 marker activities had been determined, were separately incubated with taurochenodeoxycholic acid and lithocholic acid. A strong correlation was found between 6alpha-hydroxylation of taurochenodeoxycholic acid, CYP3A levels (r2=0.97) and testosterone 6beta-hydroxylation (r2=0.9). There was also a strong correlation between 6alpha-hydroxylation of lithocholic acid, CYP3A levels and testosterone 6beta-hydroxylation (r2=0.7). Troleandomycin, a selective inhibitor of CYP3A enzymes, inhibited 6alpha-hydroxylation of taurochenodeoxycholic acid almost completely at a 10 microM concentration. Other inhibitors, such as alpha-naphthoflavone, sulfaphenazole and tranylcypromine had very little or no effect on the activity. The apparent Km for 6alpha-hydroxylation of taurochenodeoxycholic by human liver microsomes was high (716 microM). This might give an explanation for the limited formation of 6alpha-hydroxylated bile acids in healthy humans. From the present results, it can be concluded that CYP3A4 is active in the 6alpha-hydroxylation of both taurochenodeoxycholic acid and lithocholic acid in human liver.     

Bilirubin conjugates in bile of man, rat and dog. Semi-quantitative analysis of bile composition by thin-layer chromatography.

1. Conjugated bile pigments, separated in two fractions by semi-quantitative t.l.c. performed on silicic acid with phenol/water as the developing solvent, were treated with diazotized ethyl anthranilate. Resulting dipyrrylazo derivatives were analysed by quantitative t.l.c. 2. The tentative structure elucidation of tetrapyrrolic bilirubin conjugates and semi-quantitative evaluation of rat bile, post-obstructive human bile and dog bile composition is presented. 3. Homogeneous and mixed hexuronic acid diesters of bilirubin containing glucuronic acid constitute 51% of the total conjugates in normal rat bile, 45% of those in human post-obstructive bile and 38% of those in obstructed rat biles. 4. Monoconjugated bilirubin amounts to 33% of total conjugated bile pigments in normal rat bile, and 17 and 14% in post-obstructive hepatic human bile and gall-bladder bile of dog respectively. After loading with unconjugated bilirubin a greater amount of monoconjugates (56%) occur in the rat bile, whereas bilirubin diglucuronide excretion is decreased (34%). 5. In gall-bladder bile of normal dog, 40% of glucose-containing diconjugates, 32% of homogeneous and/or mixed hexuronic acid (mainly glucuronic acid) diesters of bilirubin and 14% of xylose-containing diconjugates are estimated. 6. Increased amounts of bilirubin conjugates, including some with unidentified uronic acid groups, were observed in cholestatic rat biles and quantities of conjugates with glucuronic acid were decreased.     

Identification of bile acid S-acyl glutathione conjugates in rat bile by liquid chromatography/electrospray ionization–linear ion trap mass spectrometry

Acyl-adenylates and acyl-CoA thioesters of bile acids (BAs) are reactive acyl-linked metabolites that have been shown to acylate the thiol group of glutathione (GSH); the reaction is catalyzed by glutathione S-transferase (GST) and the product is a thioester-linked BA-GSH conjugate. Such GSH conjugates are present in bile in lithocholic acid and ursodeoxycholic acid dosed-rats. To determine whether such novel BA-GSH conjugates are present in the bile of normal rats, we first synthesized the GSH conjugates of the major and minor biliary BAs of the rat and defined their MS and proton NMR properties. We then analyzed the BA-GSH composition in the bile of anesthetized biliary fistula rats by means of liquid chromatographic separation and electrospray ionization–linear ion trap mass spectrometric detection in negative- and positive-ion scan modes, monitoring characteristic transitions of the analytes. GSH conjugates of cholic, ω-muricholic, hyodeoxycholic, deoxycholic, 12-oxolithocholic, and lithocholic acids were present with concentrations in the range of 1.4–2.8 nmol/ml, some four orders of magnitude less than those of natural BA N-acyl amidates. Our results indicate that BA-GSH conjugates are formed and excreted in bile in the healthy rat, although this novel mode of BA conjugation is a very minor pathway.     

The formation of lithocholic acid, chenodeoxycholic acid and α- and β-muricholic acids from cholesterol incubated with rat-liver mitochondria

1. When rat-liver mitochondria were incubated with [4-(14)C]cholesterol in the presence of a soluble supernatant fraction, various steroids more polar than cholesterol were formed. These included 3beta-hydroxycholest-5-en-26-oic acid, 3beta-hydroxychol-5-enoic acid, lithocholic acid, chenodeoxycholic acid and alpha- and beta-muricholic acids. 2. All the radioactive C(24) bile acids recovered were in conjugated form, probably as taurine conjugates. 3. The formation of 3beta-hydroxychol-5-enoic acid from cholesterol shows that liver mitochondria are capable of carrying out the oxidative removal of the isopropyl unit of the side chain before any modification has occurred in the ring system.     

Partial purification of two lithocholic acid-binding proteins from rat liver 100 000g supernatants.

1. The partial purification of two lithocholic acid-binding proteins from liver 100 000g supernatants is described. 2. Gel-filtration, (NH4)2SO4 fractionation, Ca3(PO4)2 fractionation and ion-exchange chromatography were used. 3. Both proteins exhibited glutathione S-transferase activity; one may be the non-specific anion-binding protein ligandin. 4. Glutathione S-transferase activity of one of the binding proteins was inhibited by lithocholic acid.     

Identification of two lithocholic acid-binding proteins. Separation of ligandin from glutathione S-transferase B.

1. Two lithocholic acid-binding proteins in rat liver cytosol, previously shown to have glutathione S-transferase activity, were resolved by CM-Sephadex chromatography. 2. Phenobarbitone administration resulted in induction of both binding proteins. 3. The two proteins had distinct subunit compositions indicating that they are dimers with mol.wts. 44 000 and 47 000. 4. The two lithocholic acid-binding proteins were identified by comparing their elution volumes from CM-Sephadex with those of purified ligandin and glutathione S-transferase B prepared by published procedures. Ligandin and glutathione S-transferase B were eluted separately, as single peaks of enzyme activity, at volumes equivalent to the two lithocholic acid-binding proteins. 5. Peptide 'mapping' revealed structural differences between the two proteins.     

Dinitrophenyl S-glutathione ATPase purified from human muscle catalyzes ATP hydrolysis in the presence of leukotrienes.

Dinitrophenyl S-glutathione (Dnp-SG) ATPase has been purified from human muscle to apparent homogeneity using Dnp-SG affinity chromatography and immunoaffinity chromatography using antibodies raised against human erythrocyte Dnp-SG ATPase. The enzyme purified from human muscle showed a subunit M(r) value of about 38 kDa in denaturing gels. The M(r) value of the native enzyme as determined by Sephadex G-200 gel filtration was found to be about 80 kDa, which indicates that it is a dimer. The N-terminus of the enzyme was blocked. Its immunological and kinetic properties were similar to Dnp-SG ATPase of human erythrocytes. Besides catalyzing the ATP hydrolysis in the presence of Dnp-SG, the muscle enzyme also catalyzed ATP hydrolysis in the presence of various leukotrienes, namely LTC4.LTD4, LTE4, and N-acetyl LTE4. The specific activity of the enzyme toward LTC4 was relatively higher than other GSH-xenobiotic conjugates. The muscle enzyme exhibits a low Km value for all leukotrienes as compared to Dnp-SG, indicating high affinity of the enzyme for leukotrienes as activators. The enzyme also catalyzed ATP hydrolysis in the presence of GSH conjugates of endogenously generated fatty acid epoxides. Our results might suggest that Dnp-SG ATPase is involved in the transport of GSH conjugates, leukotrienes, and other organic anions in muscle, erythrocytes, liver, and probably other tissues.     

Intrahepatic conversion of a glutathione conjugate to its mercapturic acid. Metabolism of 1-chloro-2,4-dinitrobenzene in isolated perfused rat and guinea pig livers

Because of the low hepatic activity of gamma-glutamyl-transferase in the rat, the liver is generally considered to play only a minor role in the degradation of glutathione conjugates, a limiting step in mercapturic acid formation. Recent findings indicate, however, that the liver has a prominent role in glutathione catabolism, particularly in species other than rat. To examine the contributions of liver to mercapturic acid biosynthesis, mercapturate formation was compared in isolated perfused livers from rats and guinea pigs dosed with either 0.3 or 3.0 mumol of 1-chloro-2,4-dinitrobenzene (CDNB). Chemically synthesized glutathione conjugate, mercapturic acid, and intermediary metabolites of CDNB were used as standards in the high performance liquid chromatography analysis of bile and perfusate samples. Biliary excretion accounted for almost all of the recovered metabolites. A marked species difference was observed in the pattern of CDNB metabolism. Rat livers dosed with 0.3 mumol of CDNB excreted 55% of total biliary metabolites as the glutathione conjugate and 8.2% as the mercapturic acid, whereas guinea pig livers excreted only 4.8% as the glutathione conjugate and 47% as the mercapturate. Mercapturic formation was also dose-dependent, with a larger fraction formed at the 0.3- versus the 3.0-mumol dose (8.2 versus 3.7% in the rat; 47 versus 19% in the guinea pig). Hepatic conversion of the glutathione conjugate to the mercapturic acid was markedly inhibited in both species after retrograde intrabiliary infusion of acivicin, an inhibitor of gamma-glutamyltransferase activity. These findings provide direct evidence for intrahepatic biosynthesis of mercapturic acids. Thus, glutathione conjugates synthesized within hepatocytes are secreted into bile and broken down to cysteine conjugates; the latter are then presumably reabsorbed by the liver, N-acetylated to form the mercapturic acid and re-excreted into bile.     

A novel binding site for bile acids on human serum albumin

Binding sites of bile acids on human serum albumin were studied using various probes: dansylsarcosine (site I probe), 7-anilinocoumarin-4-acetic acid (ACAA, site II probe), 5-dimethylaminonaphthelene-1-sulfonamide (DNSA, site III probe), cis-parinaric acid (probe for fatty acid binding site) and bilirubin. Bile acids competitively inhibited the binding of dansylsarcosine to human serum album whereas bile acids enhanced the binding of ACAA, DNSA, cis-parinaric acid and bilirubin. Considering the concentrations of bile acids required to inhibit the binding of dansylsarcosine to human serum albumin, the secondary binding site of bile acids may correspond to site I. Dissociation constants (K_d) of the primary binding sites of lithocholic and chenodeoxycholic acid to human serum albumin were approximately 0.2 and 4 μM, respectively, which was measured by equilibrium dialysis at 37° C. All the bile acids and their sulfates and glucuronides inhibited the binding of chenodeoxycholic acid to human serum albumin. Lithocholic and chenodeoxycholic acid and their sulfates and glucuronides exhibited more inhibition than cholic acid and its conjugates. In conclusion, bile acids may bind to a novel binding site on human serum albumin.     

Human liver steroid sulphotransferase sulphates bile acids.

The sulphation of bile acids is an important pathway for the detoxification and elimination of bile acids during cholestatic liver disease. A dehydroepiandrosterone (DHEA) sulphotransferase has been purified from male and female human liver cytosol using DEAE-Sepharose CL-6B and adenosine 3',5'-diphosphate-agarose affinity chromatography [Falany, Vazquez & Kalb (1989) Biochem. J. 260, 641-646]. Results in the present paper show that the DHEA sulphotransferase, purified to homogeneity, is also reactive towards bile acids, including lithocholic acid and 6-hydroxylated bile acids, as well as 3-hydroxylated short-chain bile acids. The highest activity towards bile acids was observed with lithocholic acid (54.3 +/- 3.6 nmol/min per mg of protein); of the substrates tested, the lowest activity was detected with hyodeoxycholic acid (4.2 +/- 0.01 nmol/min per mg of protein). The apparent Km values for the enzyme are 1.5 +/- 0.31 microM for lithocholic acid and 4.2 +/- 0.73 microM for taurolithocholic acid. Lithocholic acid also competitively inhibits DHEA sulphation by the purified sulphotransferase (Ki 1.4 microM). No evidence was found for the formation of bile acid sulphates by sulphotransferases different from the DHEA sulphotransferase during purification work. The above results suggest that a single steroid sulphotransferase with broad specificity encompassing neutral steroids and bile acids exists in human liver.