Introduction

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UDP-glucuronosyltransferases (UGT, EC 2.4.1.17)² are a multigenic family of proteins actively involved in the final metabolism of a large variety of structurally unrelated substances. Drugs, pollutants, and toxic chemicals present in our environment are substrates of the enzymes. At least 70 cDNAs encoding animal and human UGTs have been isolated to date (Mackenzie et al., 1997). These enzymes catalyze the transfer of glucuronic acid from the high-energy donor, UDP-glucuronic acid, to the hydroxyl, carboxyl, amine, or thiol group of compounds, leading to the formation of water-soluble glucuronides that are excreted into bile or urine. UGTs are also actively involved in the metabolism of key endogenous substances (steroids, thyroid hormones, retinoids; Nebert, 1991). They regulate the concentration of these ligands, which participate to cell differentiation and growth.

From all substrates investigated so far, the molecules containing a carbohydrate moiety, such as the nucleoside analog antiviral drugs, constitute a peculiar class of compounds. The binding of glucuronic acid on the free hydroxyl groups of the hydrocarbon chain by glycosidic bonds between two carbohydrate entities leads to the formation of heteroside-like compounds. This is the case of the anti-HIV drug 3'-azido-3'-deoxythymidine (AZT), which is glucuronidated on the 5'-O of the ribose in humans (Haumont et al., 1990; Rajaonarison et al., 1991). The glucuronidation reaction leads to a pharmacologically inactive metabolite, which impairs its therapeutic efficiency. The same situation has also been reported for the nucleoside analog carbovir (Patanella et al., 1990). On the other hand, UGTs have been reported to catalyze the addition of a glucuronic acid group on endogenous compounds such as glycosides, glycolipids, or glycosaminoglycans (Chou et al., 1991; Lacarelle et al., 1993; Castle, 1993) and heparins (Lidholt and Lindahl, 1992). The function of these UGTs is beginning to be explored.

Until now, among the UGTs characterized, some of them in our laboratory, such as UGT2B4 or UGT2B1 (Fournel-Gigleux et al., 1991; Pritchard et al., 1994) no one has been reported to glucuronidate to an appreciable extent class of glycosidic drugs.

This study is therefore aimed to better understand glucuronidation on the hydroxyl group of carbohydrate-containing drugs and to determine the UGT isoforms involved in that process. The antithrombotic drug, LF 4.0212 (4-cyanophenyl 1,5 dithio--D-xylopyranoside), has been used as a model substrate (Fig. 1). This compound has been found to be a good acceptor of galactose transfer and, therefore, initiates glycosaminoglycan synthesis at the origin of the antithrombotic activity (Martin et al., 1996). Identification and characterization of the UGT isoforms able to glucuronidate the drug on the thioxyloside ring in humans and rat were achieved with several approaches: 1) use of metabolically competent, genetically engineered V79 cells expressing individual rat or human UGTs, 2) kinetics of the reaction and inhibitory effect of 7,7,7-triphenylheptanoic acid, a selective inhibitor of the glucuronidation reaction, and 3) study of the action of inducers on the glucuronidation of the drug in rat. The work will also define whether a single or distinct UGT isoform catalyzes the introduction of the glucuronic acid moiety on the different hydroxyl groups of LF 4.0212. 

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Chemical structure of LF 4.0212 and chromatograms of the HPLC resolution of the reaction products after incubation of rat liver microsomes with LF 4.0212.  A, LF 4.0212 (2 mM) was incubated with liver microsomes of rat and 2 mM UDP-glucuronic acid. B, identical incubation, but without UDP-glucuronic acid. The peaks were detected from their absorbance at 280 nm. Peak 1, UDP-glucuronic acid and UDP; peak 2, 2-O-glucuronide; peak 3, 3-O-glucuronide; peak 4, 4-O-glucuronide; peak 5, LF 4.0212. For clarity, a chromatogram obtained with rats treated with 3-methylcholanthrene is shown. This inducer was found to enhance the formation of the three glucuronide isomers (see text).

	Materials and Methods

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Chemicals. Uridine 5'-diphosphate-glucuronic acid (sodium salt) was purchased from Boehringer Mannheim (Mannheim, Germany). LF 4.0212 was a gift from Laboratoires Fournier (Daix, France). D-saccharic acid 1,4-lactone (saccharonolactone), -glucuronidase (bovine liver), digitonin, 3-methylcholanthrene, clofibrate, formic acid, and triethylamine were supplied by Sigma Chimie (St. Quentin Fallavier, France), and phenobarbital was supplied by Fluka (Buchs, Switzerland). -Glucuronidase (Escherichia coli) was purchased from Sanofi Diagnostics Pasteur (Paris, France). Trifluoroacetic acid, trichloroacetic acid, glycine, and dimethyl sulfoxide (DMSO) were provided by Merck (Darmstadt, Germany) and acetonitrile was provided by BDH (Poole, UK). The inhibitor of the glucuronidation reaction, 7,7,7-triphenylheptanoic acid, was synthesized as described previously (Fournel-Gigleux et al., 1991). The 2-, 3-, and 4-O-glucuronide isomers of LF 4.0212 were synthesized and purified by Laboratoires Fournier. They were used as HPLC authentic standards. All other chemicals were of the best purity commercially available (or HPLC grade).

Liver Samples. Five human hepatic samples from transplantable livers were a gift from Professor E. Singlas and J. Valayer (Hôpital Kremlin-Bicêtre, Paris, France). Upon removal, the liver samples were kept in liquid nitrogen. The microsomes were prepared according to the method of Dragacci et al. (1987) and were suspended in 100 mM Tris-HCl buffer (pH 8.0) containing 1 mM EDTA, 1 mM dithiothreitol, and 20% (v/v) glycerol.

Recombinant UGTs. Several recombinant V79 cell lines stably and individually expressing human (UGT1A6, UGT1A9, UGT2B4, and UGT2B11) and rat (UGT1A6 and 2B1) liver isoforms have been used to determine their potency to glucuronidate LF 4.0212. The UGT1A6 and 2B1 are 3-methylcholanthrene- and phenobarbital-inducible, respectively. Some of them (UGT1A6, UGT2B1, UGT2B4, and UGT2B11) have been established successfully in our laboratory (Fournel-Gigleux et al., 1991; Pritchard et al., 1994). The expression of each recombinant isoform was verified by immunoblots and in terms of specific activity with probe substrates, before testing LF 4.0212: planar and short phenols (1-naphthol, 4-methylumbelliferone) for the rat and human UGT1A6, chloramphenicol for UGT1A9, hyodeoxycholic acid for UGT2B4, 1-naphthol and 4-nitrophenol for UGT2B11, and morphine and ketoprofen for UGT2B1 (Pillot et al., 1993; Jin et al., 1993; Pritchard et al., 1994; Guengerich et al., 1997).

Enzyme Assays. The conditions for glucuronidation of LF 4.0212 with the various sources of UGTs were optimized in terms of time, protein concentration, detergent concentration, divalent ions, and incubation pH. In rat liver microsomes only, the enzyme was activated by preincubating microsomes on ice for 20 min with Triton X-100 at a detergent/protein weight ratio of 0.8. This step was omitted for cellular membranes of V79 expressing the UGT isoforms because a mechanical activation was achieved by sonication used to break the cells. Standard incubations were performed in Eppendorf tubes containing 100 mM Tris-HCl buffer (pH 7.4) with 5.0 mM MgCl₂ and 5.0 mM MnCl₂, microsomal proteins (10-100 µg), LF 4.0212 (2 mM) dissolved in 5 µl DMSO, 5 mM D-saccharic acid 1,4-lactone, and 1 to 2 mM UDP-glucuronic acid in a total volume of 100 µl. The mixture was incubated at 37°C for 20 to 90 min and the reaction was terminated by placing the tube in ice and adding 10 µl 6 N HCl. The final concentration in HCl (0.54 N) did not affect the stability of the glucuronide formed. The tubes were centrifuged for 10 min at 2500g and an aliquot of the supernatant was analyzed by HPLC. All assays were done in triplicate. Control experiments were always run by omitting UDP-glucuronic acid.

Identification of the Glucuronic Acid Conjugate.

-Glucuronidases Glucuronide formation was verified by the susceptibility of the products to hydrolysis by two -glucuronidases. For this purpose, -glucuronidase from bovine liver (200 µl, 1,200 U) dissolved in 200 mM sodium acetate buffer (pH 5.5) was added to 100 µl of the medium after 30-min incubation without D-saccharic acid 1,4-lactone. The reaction was conducted for an additional 4 h at 37°C; it was stopped by addition of 6 N HCl and the products were analyzed by HPLC. When the -glucuronidase from E. coli (54 U) was used, the enzyme was dissolved in 200 mM sodium phosphate buffer (pH 6.5) and the mixture was incubated for 30 min at 50°C. Control assays were simultaneously run under the same conditions, but without -glucuronidase to estimate the stability of the glucuronide under such conditions.

Electron spray liquid chromatography-mass spectrometry. The glucuronides biosynthesized by human and rat liver microsomes were further characterized by electron spray mass spectrometry. The glucuronides were separated on an HPLC system (see below). Mass spectrometry was carried out using a Fisons Instrument VG Platform (Micromass Ltd., Manchester, UK). Cone voltage was set at 20 V and the temperature source was 120°C. Data were collected in both positive and negative modes and reprocessed by means of Mass Lynx software (Micromass).

Chromatographic Conditions. The HPLC system consisted of a solvent delivery pump (Merck 655 A-11), an injection valve fitted with a 100 µl loop (Rheodyne, Cotati, CA), and a computing integrator D-2000 (Merck). Spectrophotometric detection was operated with a UV variable wavelength detector (LKB, Broma, Sweden) set at 280 nm. The analytical MN Nucleosil 120-5 C₁₈ column (250 × 4.6 mm, 5 µm) was provided by Macherey-Nagel (Hoerdt, France). The mobile phase was composed of 20% (v/v) acetonitrile, 0.015% (v/v) formic acid, and 0.030% (v/v) triethylamine in water (apparent pH 3.5). The mobile phase was filtered through a 0.45-µm microfilter (Sartorius, Palaiseau, France) and used at a flow rate of 1.0 ml/min. The chromatograms were run at room temperature. Quantitation of LF 4.0212 glucuronide was performed by comparison with calibration curves established upon injection of increasing amounts of pure LF 4.0212. The glucuronide and the parent drug were found to present the same molar extinction coefficient. The response (peak area) was linear from 0.12 to 15 nmol LF 4.0212 injected. The detection limit was 0.02 nmol glucuronide under our experimental conditions.

	Results

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Structural Identification of LF 4.0212 Glucuronides and Regioselectivity of the Glucuronidation. In no case did we observe evidence of degradation of LF 4.0212 during the incubation. This was tested specifically by incubating this compound with microsomes from either human or rat liver, or from V79 cells for 90 min at 37°C under our standard incubation conditions, except that UDP-glucuronic acid was omitted.

Incubation of LF 4.0212 by rat and human liver microsomes. A typical chromatogram profile resulting from the incubation of the drug with liver microsomes of rats is shown in Fig. 1. Three glucuronides were formed at retention times 7.8, 8.1, and 12.9 min. (Fig. 1A). These peaks disappeared when incubations were run without UDP-glucuronic acid (Fig. 1B). They corresponded to the 2-O-, 4-O-, and 3-O-glucuronide isomers, respectively, by comparison of retention times of authentic glucuronides (peaks 2, 4, and 3). The chromatogram also indicated that the 3-O-glucuronide isomer was the main metabolite biosynthesized by the rat liver microsomes. All three glucuronides formed from rat liver microsomes were degraded by the E. coli and the bovine -glucuronidase (not shown). The other major peaks corresponded to UDP-glucuronic acid and free LF 4.0212 (peaks 1 and 5, retention times 2.6 and 20.7 min, respectively).

Electrospray liquid chromatography-mass spectrometry. A pseudomolecular ion [M-H]^-corresponding to the most intense peak was observed for the 2-O glucuronide in the negative mode at a m/z 458 (Fig. 2A). Similar spectra were obtained with the 3- and 4-O glucuronide isomers (not shown). The peak at m/z 193 was tentatively assigned to glucuronic acid. By comparison, whatever the isomer considered, the positive mode led to a more complicated spectrum of the LF 4.0212 glucuronide, with two peaks of interest at m/z values 561 and 266, respectively, corresponding to an adduct between the glucuronide and triethylamine and to the aglycone with a loss of a water molecule (Fig. 2B).

View larger version (11K): [in this window] [in a new window]   Fig. 2.   Electrospray liquid chromatography-mass spectrometry of 2-O-glucuronide of LF 4.0212 in negative (A) or positive (B) mode. Similar spectra were obtained with the 3-O and 4-O-glucuronide isomers.

Kinetics of LF 4.0212 Glucuronidation and UGT Isoforms Catalyzing the Reaction.

Human hepatic microsomes The optimal conditions for the biosynthesis of the 2-O-glucuronide isomer of LF 4.0212 with human liver microsomes were established before the determination of the apparent kinetic constants. The biosynthesis of the 2-O-glucuronide isomer was linear up to 60 min for a protein concentration range up to 20 µg in the incubation medium. Glucuronidation had a broad pH optimum between 7 and 8. Maximal formation required the presence of both 5 mM Mn²⁺ and 5 mM Mg²⁺, compared with 70% maximal biosynthesis with 10 mM Mg²⁺ alone or 25% only with no added divalent metal ion. Therefore, a concentration of 5 mM Mn²⁺ and 5 mM Mg²⁺ and a pH 7.4 were used for the subsequent experiments.

Rat liver microsomes. For synthesis of all three products, the glucuronidation reaction was linear with respect to time of incubation (up to 60 min) and protein concentration (up to 0.100 mg). 10 mM Mg²⁺ or 10 mM Mn²⁺ were equally effective in stimulating the glucuronidation rate.

Use of genetically engineered V79 expressing UGT cDNAs. Using microsomes from nontransfected V79 fibroblasts, the only product was the 4-O-glucuronide isomer of LF 4.0212 (Table 3). Its biosynthesis was linear for 60 min. It had a broad pH optimum, 7.5 to 8.8, and was highest with 10 mM MnCl₂. Interestingly, membrane fractions from all available recombinant V79 cell constructs had similar specific activities for synthesis of the 4-O-glucuronide isomer, ranging from 0.11 to 0.28 nmol/min·mg, as shown in Table 3.

Effect of triphenylheptanoic acid on glucuronidation of LF 4.0212. We previously reported that 7,7,7-triphenylheptanoic acid was an effective inhibitor of UGTs (Fournel-Gigleux et al., 1989; Said et al., 1992). This compound was therefore tested to determine if the glucuronidation of LF 4.0212 was sensitive to its inhibitory effect. The results summarized in Fig. 3 show that 7,7,7-triphenylheptanoic acid (0.10 mM) strongly inhibited by 82% the glucuronidation on the 2-hydroxyl position catalyzed by human liver microsomes. 7,7,7-Triphenylheptanoic acid also inhibited the same activity on the recombinant UGT 1A9 and 2B4 expressed in V79 cell lines. By contrast, it was ineffective, at the same concentration, in inhibiting the glucuronidation at the 4-hydroxy position of the drug catalyzed by the nontransfected cells as well as by the transfected cells (Fig. 3). Indeed, the compound activated the 4-O-glucuronidation by 1.3- to 1.7-fold (Fig. 3). The potential inhibitory effect of 7,7,7-triphenylheptanoic acid on the glucuronidation of LF 4.0212 by rat liver microsomes on the three O-positions was also investigated (Fig. 3). The compound (0.10 mM) could fully abolish the 2-O and also the 3-O-glucuronidation. However, interestingly, the formation of the 4-O-glucuronide was only decreased by 48% with the inhibitor in this case. In that respect, this result contrasted with that found with V79 cells.

View larger version (33K): [in this window] [in a new window]   Fig. 3.   Differential effect of 7,7,7-triphenylheptanoic acid on the formation of 2-O, 3-O, and 4-O-glucuronides. The activity (means ± S.D., three determinations) was measured with human and rat liver microsomes, with membranes of V79 expressing the human liver recombinant UGT 1A9 and 2B4, and with membranes of nontransfected V79 fibroblasts, in the absence () or in the presence (+) of 7,7,7-triphenylheptanoic acid. The inhibitor (0.10 mM in DMSO) was added 1 min before LF 4.0212 (2 mM) and UDP-glucuronic acid (2 mM). ND, no glucuronide detectable; limit of detection, 0.020 nmol glucuronide.

	Discussion

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The in vitro glucuronidation of LF 4.0212 was characterized in rat and humans. These two species presented radically different patterns of glucuronidation of the drug in terms of regioselectivity and metabolic rate. The 2-O-glucuronide isomer was the only metabolite found in humans, but rat could glucuronidate on all three positions, the main metabolite being the 3-O-glucuronide isomer. Additionally, human liver microsomes were much more active in glucuronidating LF 4.0212. The results indicate that the rat is not a suitable animal model to evaluate the glucuronidation of the drug in humans. This is also true for the glucuronidation of other carbohydrate-containing drugs, because we previously reported (Haumont et al., 1990) that rat glucuronidated AZT at a low rate compared to humans. These differences in the regioselectivity and in the efficiency of the glucuronidation process suggest that distinct UGT isoforms are involved in the glucuronidation of LF 4.0212 in rat and humans.

Surprisingly in humans, the glucuronidation on the thioxyloside ring proceeded with a very high rate when compared with that observed on the ribose moiety of AZT. We found (Haumont et al., 1990) that AZT glucuronidation had a V_max (0.98 nmol/min·mg protein) 36-fold lower than that reported in this study for the formation of the 2-O-glucuronide isomer. LF 4.0212 was a better substrate than AZT (V_max/K_m 30 and 13 µl/min/mg, respectively). Compared with glucuronidation of AZT on the nonreactive 5' hydroxyl of the ribose, the presence of the two electronegative sulfur atoms of the thioxyloside ring and of the thioglycoside bond could, by an attractive effect, increase the acidity of the vicinal 2-hydroxyl group, thus facilitating its glucuronidation. In that respect, the specific activity found in humans can be favorably compared with that reported for phenolic molecules, such as 1-naphthol and 4-methylumbelliferone, that are considered as good substrates of UGTs and are glucuronidated at high rates when compared with glucuronidation of carboxylic acids or amines (Magdalou et al., 1992; Huskey et al., 1994; Little et al., 1997).

Attempts to determine the isoform(s) involved in the glucuronidation of LF 4.0212 were performed by the aid of standard inducers of UGTs in rats and by genetically engineered V79-expressing single proteins. Phenobarbital, 3-methylcholanthrene, and clofibrate are substances known to stimulate the expression of distinct UGT isoforms belonging to families 1 and 2. 3-Methylcholanthrene induces preferentially the proteins of family 1 that glucuronidate planar phenols (1-naphthol, reduced naftazone; Okulicz-Kozaryn et al., 1981; Herber et al., 1995), whereas phenobarbital has a broader stimulating effect on the glucuronidation of bulky phenols, carboxylic acids such as nonsteroidal anti-inflammatory drugs (Magdalou et al., 1990), and aliphatic hydroxylated molecules catalyzed by isoforms of families 1 and 2. Finally, the peroxisome proliferator, clofibrate, induces specifically the bilirubin UGTs encoded by the UGT1 gene locus (Fournel et al., 1987; Magdalou et al., 1994). All these substances were powerful inducers, but exerted a nonspecific effect on LF 4.0212 glucuronidation, as the formation of all three position isomers was enhanced upon treatment. Interesting was the action of clofibrate, which strongly enhanced (11-times) the formation of the 2-O-glucuronide isomer. This hypolipidemic drug stimulates bilirubin glucuronidation 3- to 4-fold in rat (Fournel et al., 1987). The large extent of the induction observed in this study could indicate that bilirubin UGTs would be involved in the process. However, we found that Gunn rats, which present a genetic defect in the UGT isoforms that conjugate bilirubin and planar phenols, were still able to glucuronidate LF 4.0212 on the 2 position, although at a lower rate (50% less) than the congenic normal rats (results not shown). Altogether, these data indicated that, in rat, several UGTs differentially sensitive to the inducers, including bilirubin UGT, were probably involved in the glucuronidation of LF 4.0212.

Metabolically competent V79s have been found to be invaluable tools to determine which UGT isoform catalyzes a glucuronidation reaction (Remmel and Burchell, 1993; Huskey et al., 1994) because each cell line expresses one individual protein. These genetically engineered cells lines shared with the nontransfected V79 cells the ability to glucuronidate LF 4.0212 on the 4 position. Of all the human isoforms, only UGT1A9 and UGT2B4 catalyzed the formation of the 2-O-glucuronide isomer. The results indicated that in humans, both UGTs belonging to families 1 and 2 could glucuronidate the drug on the 2 position. The UGT1A9 isoform, which glucuronidates bulky phenols, could accommodate the nonplanar thioxyloside ring within the active site of the protein. Comparison of the apparent kinetic constants found with this recombinant enzyme and human liver microsomes revealed that the K_m values were similar, suggesting that UGT1A9 is a potential candidate as the main isoform involved in this process in humans. Interestingly, UGT 2B4 was also active in glucuronidating LF 4.0212 on the 2-hydroxyl group. We previously found that this isoform exhibited a very strict substrate specificity (Fournel-Gigleux et al., 1991; Pillot et al., 1993). The only substrate previously known in our hands to be glucuronidated by UGT2B4 was the bile acid, hyodeoxycholic acid. The enzyme was also regioselective, because hyodeoxycholic acid, which has three potential glucuronidation sites, was glucuronidated only on the 6- position of the molecule. The same situation stands for LF 4.0212, which appears therefore as a new, alternate substrate of UGT2B4. These data indicate that UGT isoforms belonging to both families 1 (UGT1A9) and 2 (UGT2B4) could glucuronidate the drug on the 2-hydroxyl group of LF 4.0212. In contrast, the results unambiguously show that the UGT1A6, which presents a strict specificity toward short and planar phenols (Fournel-Gigleux et al., 1991; Ouzzine et al., 1994) could not form the 2-O-glucuronide isomer. The same conclusion was drawn for UGT2B11, despite the fact that its substrate specificity is much broader toward phenols, aliphatic hydroxylated compounds, or hydroxysteroids (Jin et al., 1993, 1997).

The rat liver phenobarbital-inducible UGT2B1 could only form one of the three glucuronides (4-O-glucuronide isomer). However, part of this activity (0.283 nmol/min·mg protein) was due to the basal endogenous activity found with nontransfected V79 cells (see below). In comparison, V79 cells expressing the rat liver, 3-methylcholanthrene-inducible UGT1A6 was also active for glucuronidating the drug on the 4-hydroxyl position with activity similar to that found in nontransfected cells.

The enzyme expressed in V79 cells did not catalyze the glucuronidation of LF 4.0212 on the 2- or 3-hydroxyl positions as found in rat liver microsomes. UGT2B1 is actively involved in the formation of both ether and ester glucuronides from aliphatic or aromatic origin, the best substrates being morphine or the nonsteroidal anti-inflammatory drugs of the series of 2-phenylpropionic acid (profens). The enzyme could glucuronidate AZT on the 5' hydroxyl position, but at a very low rate (activity less than 0.01 nmol/min·mg protein; Pritchard et al., 1994).

Interestingly, the presence of an endogenous UGT able to form the 4-O-glucuronide isomer was detected in nontransfected V79 cell. The choice of V79 hamster lung fibroblasts as host cells for expression UGT cDNA was primarily dictated by the nondetectable or the very low glucuronidation activity at the level of the detection limit toward structurally unrelated xenobiotics. The nontransfected cells could actively glucuronidate LF 4.0212 on the 4-hydroxyl position with apparent kinetic constants similar to those found in the genetically engineered cell lines. The apparent K_m, which was very low, and the V_max values were similar in nontransfected V79 cells and in cells expressing UGT1A6 or UGT1A9. These data suggest that glucuronidation of the 4-hydroxyl of LF 4.0212 is catalyzed by UGT form(s) expressed in the host fibroblastic V79 cell line with a high affinity for the drug. UGT1A6 and UGT1A9 would not significantly contribute to the formation of 4-O-glucuronide isomer. This endogenous UGT exhibited distinct properties. No immunoreactive band was observed in nontransfected V79 cells using several types of antibodies raised against the full-length sequence of the rat liver UGT isoforms (Coughtrie et al., 1988) or against the N-terminal end of the human UGT1A6 and 2B4 (Fournel-Gigleux et al., 1991; Pritchard et al., 1994). Because it is not recognized by these probes directed against several domains of the protein, this UGT could be structurally different. The synthesis of LF 4.0212 4-O-glucuronide was not inhibited by 7,7,7-triphenylheptanoic acid, which has been found to be a very effective inhibitor of bilirubin and phenol glucuronidation in rat and human liver microsomes as well as of recombinant UGTs expressed in V79 cells (Said et al., 1992; Senay et al., 1997). Thus, the inability of this compound to inhibit the endogenous UGT also favors the existence of a different protein in nontransfected V79 cells. The same situation occurs in rat liver microsomes, for which 7,7,7-triphenylheptanoic acid could only partially inhibit the formation of 4-O-glucuronide. We believe that LF 4.0212, whose structure mimics the trisaccharidic moiety (xylose-galactose-galactose), could be used as a model substrate to study the class of UGTs that is involved in the biosynthesis of glycosaminoglycans. These enzymes are known to incorporate glucuronic acid on such trisaccharides, leading to the formation of glycosidic polymers (Kitagawa et al., 1998).