Roger Lester

Biosynthesis and chemical synthesis of carboxyl-linked glucuronide of lithocholic acid

The glucuronidation of lithocholic acid (LA) by phenobarbital-induced male Fischer 344 rat liver microsomes supplemented with UDP-glucuronic acid was studied. A single radioactive metabolite was formed and its structure was determined by high pressure liquid chromatography/particle beam/mass spectrometry (HPLC/PB/MS), both with and without prior methylation and acetylation of the sample. The reaction product was rigorously identified as the 1-O-acyl-beta-D-glucuronide of LA by comparison with a chemically synthesized standard. The chemical synthesis of the acyl glucuronide of LA was accomplished via a condensation reaction using benzyl 2,3,4-tri-O-benzyl-D-glucopyranuronate. The latter compound was prepared in two steps from benzyl 2,3,4-tri-O-benzyl-1-O-methyl-alpha-D-glucopyranuronate via the 1-O-acetyl derivative. The stereoselective beta coupling of LA with 2,3,4-tri-O-benzyl-D-glucopyranuronate was achieved by the Mitsunobu reaction, in the presence of the free hydroxyl function of LA, using triphenylphosphine and diisopropyl azodicarboxylate in THF followed by preparative TLC. The benzylic ester and ether groups were cleaved by hydrogenation with Pd on charcoal as the catalyst. Positive identification of the glucuronide was established by HPLC/PB/MS and 1H-NMR spectra. No side products formed by acyl migration were detected, but the free acyl glucuronide underwent rapid transesterification in methanol.

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 K m and V ma? 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.

Bile acid glucuronidation by rat liver microsomes and cDNA-expressed UDP-glucuronosyltransferases

Four rat UDP-glucuronosyltransferases (UGTs), UGT2B1, UGT2B2, UGT2B3 and UGT2B6, synthesized in COS-7 cells from appropriate cDNA clones were screened for activity towards a range of bile acids, neutral steroids and retinoic acid. For comparison, as well as optimization of enzymatic assays and product identification, rat liver microsomal preparations from Sprague-Dawley, Fischer 344 and phenobarbital-induced Fischer 344 male rats were also used as enzyme sources. Only two of the expressed proteins, UGT2B1 and UGT2B2, were active in bile acid glucuronidation. UGT2B1 exhibited a high substrate specificity for the carboxyl function of bile acids, whereas UGT2B2 demonstrated less specificity, accepting both hydroxyl and carboxyl functions of bile acids. The preferred substrates for both cloned enzymes were mono-hydroxylated bile acids, followed by di-hydroxylated 6-OH compounds. The levels of UGT activity were sufficient to allow for the identification of the biosynthesized products. The data presented here demonstrate that bile acid glucuronidation is carried out, at least in part, by members of the UGT2B subfamily. Similar results have been obtained previously for neutral steroid glucuronidation. UGT2B3 and UGT2B6 was not involved in BA glucuronidation; none of the cloned enzymes was active toward retinoic acid.

Characterization of UDP-glucuronic acid transport in rat liver microsomal vesicles with photoaffinity analogs

The endoplasmic reticulum (ER) of rat liver contains several well characterized UDP-glucuronosyltransferases (UGTs), membrane-bound proteins of 50-54 kDa, and also less well identified UDP-glucosyltransferases, with nucleotide binding sites located on the lumenal surface. There is evidence that the substrates for these enzymes, UDP-glucuronic acid (UDP-GlcUA) and UDP-glucose (UDP-Glc), biosynthesized in the cytosol, are transported into the lumen of the ER via unknown mechanisms, the characteristics of which are poorly defined. A new approach for the study of the transport process has been devised using two active-site directed photoaffinity analogs, [beta-32P]5-azido-UDP-GlcUA and [beta-32P]5-azido-UDP-Glc. Photoincorporation of these probes into the lumenally oriented UGTs of intact rat liver microsomal vesicles was used as an indicator of transport. In intact vesicles, [32P]5N3UDP-GlcUA was efficiently incorporated into UGTs in a time, temperature and concentration dependent manner. In contrast, [32P]5N3UDP-Glc apparently was not transported effectively; maximal photolabeling of the 50-54 kDa proteins by this probe was dependent on detergent disruption of the vesicles. Vesicular uptake of and subsequent photolabeling of the 50-54 kDa proteins by [32P]5N3UDP-GlcUA were inhibited by UDP-GlcUA and 5N3UDP-GlcUA while UDP-Glc, 5N3UDP-Glc, UDP-xylose and UDP-N-acetylglucosamine were less inhibitory, suggesting a high degree of specificity for the uptake/photolabeling process. The anionic transport inhibitors DIDS and SITS inhibited [32P]5N3UDP-GlcUA photoincorporation into UGTs in intact vesicles, but also inhibited photolabeling of these and other enzymes in detergent disrupted vesicles. These data suggest the presence in rat liver microsomal vesicles of a specific, carrier-mediated transport process for UDP-GlcUA which is distinct from the mechanism of UDP-Glc transport.

Photoaffinity labeling for evaluation of uridinyl analogs as specific inhibitors of rat liver microsomal UDP-glucuronosyltransferases

The UDP-glucuronosyltransferases (UGT) involved in glucuronidation of endogenous and exogenous toxic compounds transfer the glucuronic acid residue from UDP-glucuronic acid (UDP-GlcUA), to various acceptor groups. A series of compounds that contain N-acyl phenylaminoalcohol derivatives linked to uridine or isopropylideneuridine were tested as UGT inhibitors. The potency of these inhibitors was determined by studying their effect on the photoaffinity labeling of rat liver microsomal UGTs by two photoaffinity probes, [beta-32P]5-azido-UDP-glucuronic acid (5N3UDP-GlcUA) and [beta-32P]5-azido-UDP-glucose (5N3UDP-Glc) and on the enzymatic formation of the two glucuronide conjugates (3-O- and carboxyl-specific) of lithocholic acid. All but one of the compounds tested proved to have an inhibitory effect on UGTs, both in the photoaffinity labeling system and in the enzymatic glucuronidation assay. In the photoaffinity labeling system, the inhibitors containing the isopropylidene moiety were less effective than their unprotected derivatives; however, the protected forms were, with one exception, more potent inhibitors of enzymatic activity. The photoaffinity labeling of UGTs with [beta-32P]5N3UDP-Glc was more susceptible to inhibition by all derivatives than that with [beta-32P]5N3UDP-GlcUA. The effect of one inhibitor, PP50B, on the two enzymatic activities involved in LA glucuronidation was extensively tested. A double-reciprocal plot suggested a competitive inhibition for UDP-GlcUA with an apparent Ki of 35 microM for LA 3-O-glucuronide formation and 94 microM for the carboxyl-linked glucuronide of the same substrate.

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.

Characterization of a new class of inhibitors of the recombinant human liver UDP-glucuronosyltransferase, UGT1*6.

The inhibitory effect of a series of novel structurally related compounds on the human UDP-glucuronosyltransferase UGT1*6 stably expressed in a V79 cell line was investigated. The inhibitors contain a lipophilic N-acyl phenylaminoalcohol residue and a uridine moiety connected by a spacer varying for each compound. The effects of these compounds on the glucuronidation reaction measured with 4-methylumbelliferone as substrate were determined. The best inhibitor of the series, D-DPMSU, had an IC50 of 39 microM in the assay conditions. Low Ki values were found toward both UDP-glucuronic acid and 4-methylumbelliferone (17 and 21 microM, respectively). The inhibition was competitive toward both substrates. A similar strong and competitive inhibitory effect was observed with two other inhibitors, DHPASU and DHPASiU. Another compound, D-DPASiU, showed a pure competitive inhibition towards UDP-glucuronic acid, but a non-competitive inhibition towards the acceptor substrate. These data and the optimization of the structures of the inhibitors by molecular modeling suggest that D-DPMSU and DHPASiU compounds may be transition state analog inhibitors of the recombinant UGT1*6 enzyme.

Two kinetically-distinct components of UDP-glucuronic acid transport in rat liver endoplasmic reticulum

Previous studies have documented the presence of protein-mediated transport of UDP-glucuronic acid (UDP-GlcUA) in rat liver endoplasmic reticulum (ER). Measurement of uptake at varying concentrations of high specific activity [beta-32P]UDP-GlcUA has revealed the presence of a two component UDP-GlcUA transporting system. Transport at low substrate concentrations occurred predominantly via a high affinity component (K(m) = 1.6 microM), whereas a low affinity component (K(m) = 38 microM) predominated at high substrate concentrations. The K(m) for the high affinity system is in agreement with that previously published, while the low affinity component is a new finding. The uptake of UDP-GlcUA was temperature-sensitive, time dependent, and saturable for both components. The high affinity transport was affected by trans-stimulation and cis-inhibition by UDP-N-acetylglucosamine (UDP-GlcNAc); however, the same concentrations of UDP-GlcNAc had less effect on the low affinity system. In order to further study the two transport components, various inhibitors of anion transport carriers were tested. The high affinity component was strongly inhibited by 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid (SITS) and furosemide, while the low affinity system was less sensitive to these reagents. Dose-dependent inhibition by 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS) was found for both transport systems. Probenecid was found to be a weak inhibitor of both components of the UDP-GlcUA uptake. Finally, the major metabolite of 3-azido-3-deoxythymidine, 3-azido-3-deoxythymidine monophosphate (AZTMP), was able to inhibit the uptake of UDP-GlcUA by both components. The results indicate the presence of two carrier-mediated UDP-glucuronic acid transporting components in rat liver ER.

Hepatic metabolism of 3-oxoandrost-4-ene-17β-carboxylic acid in the adult rat: formation of carboxyl-linked glucuronides both in vivo and in vitro

The hepatic metabolism of 3-oxoandrost-4-ene-17 beta-carboxylic acid (etienic acid), a probable acidic catabolite of deoxycorticosterone, was investigated using rats prepared with an external biliary fistula. Metabolic products were identified by GC-MS after hydrolysis with beta-glucuronidase and by proton nuclear magnetic resonance after chromatographic purification of protected glucuronides. About 80% of the injected dose was secreted into bile in 20 hours. Three fully reduced etianic acids (3 alpha-hydroxy-5 alpha-, 3 beta-hydroxy-5 alpha-, 3 alpha-hydroxy-5 beta-androstan-17 beta-carboxylic acids) were identified as were several of their di- and trihydroxylated congeners. Glucuronides of these reduced and/or hydroxylated metabolites constituted over half of the recovered dose, with carboxyl-linked glucuronides predominating over 3-hydroxyl-linked glucuronides. The mode of glucuronidation correlated well with the ability of liver microsomes to form the corresponding compounds in vitro from the set of four 3,5-diastereomeric etianic acids.

Disorders of Biliary Secretion

More than 0.5 liter of bile is produced per day in humans. The chemical composition of bile is complex. In addition to inorganic electrolytes, bile contains high (millimolar) concentrations of organic compounds, including glutathione, dipeptides, and amino acids (Table 26.1). While the above compounds are freely water-soluble, equally high concentrations of bilirubin, cholesterol, and phospholipids are present in bile. The latter material is kept in a micellar and/or vesicular form by another major biliary component, the bile acids. The physical chemistry of the ternary bile acid-cholesterol-phospholipid system is beyond the scope of the present chapter; it should be mentioned, however, that an imbalance in the secretion of these three components sets the stage for biliary stone formation. Finally, bile contains proteins, e.g., IgA. Except for the inorganic electrolytes, all of the compounds mentioned above are present in bile at concentrations that significantly exceed their concentrations in plasma. This indicates that biliary secretion is an active process. The latter conclusion is also consistent with the generally accepted model of bile formation.