Role of the carboxyl terminal stop transfer sequence of UGT1A6 membrane protein in ER targeting and translocation of upstream lumenal domain.

We investigated the role of the stop transfer sequence of human UGT1A6 in ER assembly and enzyme activity. We found that this sequence was able to address and translocate the upstream lumenal domain into microsomal membranes in vitro co- and posttranslationally. The signal activity of this sequence was further demonstrated in HeLa cells by its ability to target and maintain the CD4 protein deleted from both the N-terminal signal peptide and C-terminal transmembrane domain into the ER. We showed that total or partial deletion of the stop transfer sequence of UGT1A6 severely impaired enzyme activity highlighting its importance in both membrane assembly and function.

The heparan sulfate sulfotransferase 3-OST3A (HS3ST3A) is a novel tumor regulator and a prognostic marker in breast cancer.

Heparan sulfate (HS) proteoglycan chains are key components of the breast tumor microenvironment that critically influence the behavior of cancer cells. It is established that abnormal synthesis and processing of HS play a prominent role in tumorigenesis, albeit mechanisms remain mostly obscure. HS function is mainly controlled by sulfotransferases, and here we report a novel cellular and pathophysiological significance for the 3-O-sulfotransferase 3-OST3A (HS3ST3A), catalyzing the final maturation step of HS, in breast cancer. We show that 3-OST3A is epigenetically repressed in all breast cancer cell lines of a panel representative of distinct molecular subgroups, except in human epidermal growth factor receptor 2-positive (HER2+) sloan-kettering breast cancer (SKBR3) cells. Epigenetic mechanisms involved both DNA methylation and histone modifications, producing different repressive chromatin environments depending on the cell molecular signature. Gain and loss of function experiments by cDNA and siRNA transfection revealed profound effects of 3-OST3A expression on cell behavior including apoptosis, proliferation, response to trastuzumab in vitro and tumor growth in xenografted mice. 3-OST3A exerted dual activities acting as tumor-suppressor in lumA-michigan cancer foundation (MCF)-7 and triple negative-MD Anderson (MDA) metastatic breast (MB)-231 cells, or as an oncogenic factor in HER2+-SKBR3 cells. Mechanistically, fluorescence-resonance energy transfer-fluorescence-lifetime imaging microscopy experiments indicated that the effects of 3-OST3A in MCF-7 cells were mediated by altered interactions between HS and fibroblast growth factor-7 (FGF-7). Further, this interplay between HS and FGF-7 modulated downstream ERK, AKT and p38 cascades, suggesting that altering 3-O-sulfation affects FGFR2IIIb-mediated signaling. Corroborating our cellular data, a clinical study conducted in a cohort of breast cancer patients uncovered that, in HER2+ patients, high level expression of 3-OST3A in tumors was associated with reduced relapse-free survival. Our findings define 3-OST3A as a novel regulator of breast cancer pathogenicity, displaying tumor-suppressive or oncogenic activities in a cell- and tumor-dependent context, and demonstrate the clinical value of the HS-O-sulfotransferase 3-OST3A as a prognostic marker in HER2+ patients.

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.

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.

The UDP glycosyltransferase gene superfamily: recommended nomenclature update based on evolutionary divergence.

This review represents an update of the nomenclature system for the UDP glucuronosyltransferase gene superfamily, which is based on divergent evolution. Since the previous review in 1991, sequences of many related UDP glycosyltransferases from lower organisms have appeared in the database, which expand our database considerably. At latest count, in animals, yeast, plants and bacteria there are 110 distinct cDNAs/genes whose protein products all contain a characteristic 'signature sequence' and, thus, are regarded as members of the same superfamily. Comparison of a relatedness tree of proteins leads to the definition of 33 families. It should be emphasized that at least six cloned UDP-GlcNAc N-acetylglucosaminyltransferases are not sufficiently homologous to be included as members of this superfamily and may represent an example of convergent evolution. For naming each gene, it is recommended that the root symbol UGT for human (Ugt for mouse and Drosophila), denoting 'UDP glycosyltransferase,' be followed by an Arabic number representing the family, a letter designating the subfamily, and an Arabic numeral denoting the individual gene within the family or subfamily, e.g. 'human UGT2B4' and 'mouse Ugt2b5'. We recommend the name 'UDP glycosyltransferase' because many of the proteins do not preferentially use UDP glucuronic acid, or their nucleotide sugar preference is unknown. Whereas the gene is italicized, the corresponding cDNA, transcript, protein and enzyme activity should be written with upper-case letters and without italics, e.g. 'human or mouse UGT1A1.' The UGT1 gene (spanning > 500 kb) contains at least 12 promoters/first exons, which can be spliced and joined with common exons 2 through 5, leading to different N-terminal halves but identical C-terminal halves of the gene products; in this scheme each first exon is regarded as a distinct gene (e.g. UGT1A1, UGT1A2, ... UGT1A12). When an orthologous gene between species cannot be identified with certainty, as occurs in the UGT2B subfamily, sequential naming of the genes is being carried out chronologically as they become characterized. We suggest that the Human Gene Nomenclature Guidelines (http://www.gene.acl.ac.uk/nomenclature/guidelines.html++ +) be used for all species other than the mouse and Drosophila. Thirty published human UGT1A1 mutant alleles responsible for clinical hyperbilirubinemias are listed herein, and given numbers following an asterisk (e.g. UGT1A1*30) consistent with the Human Gene Nomenclature Guidelines. It is anticipated that this UGT gene nomenclature system will require updating on a regular basis.

Expression of a human liver cDNA encoding a UDP-glucuronosyltransferase catalysing the glucuronidation of hyodeoxycholic acid in cell culture.

A cDNA encoding a human liver UDPGT (HLUG 25) transcribed and translated in vitro showed that the encoded protein was synthesized as a precursor and was cleaved and glycosylated when dog pancreatic microsomes were present during translation. The UDPGT cDNA was transiently expressed in mammalian cell culture (COS-7 cells) resulting in the biosynthesis of a polypeptide of 52 kDa. This expressed UDPGT glycoprotein catalysed the glucuronidation of hyodeoxycholic acid forming an ether glucuronide. These results suggest that this UDPGT isoenzyme may be responsible for the glucuronidation of 6 alpha-hydroxy bile acids in human liver.

Expression of a functionally active human hepatic UDP-glucuronosyltransferase (UGT1A6) lacking the N-terminal signal sequence in the endoplasmic reticulum.

UDP-glucuronosyltransferase 1A6 (UGT1A6) is a membrane glycoprotein of the endoplasmic reticulum playing a key role in drug metabolism. It is synthesized as a precursor with an N-terminal cleavable signal peptide. We demonstrate that deletion of the signal peptide sequence does not prevent membrane targeting and integration of this human isoform when expressed in an in vitro transcription-translation system, as shown by N-glycosylation, resistance to alkaline treatment and protease protection. Furthermore, UGT1A6 lacking the signal peptide (UGT1A6delta sp) was targeted to the endoplasmic reticulum in mammalian cells as shown by immunofluorescence microscopy and was catalytically active with kinetic constants for 4-methylumbelliferone glucuronidation similar to that of the wild-type. These results provide evidence that the signal peptide is not essential for the membrane assembly and activity of UGT1A6 suggesting that additional topogenic element(s) mediate(s) this process.

The chemical modification of human liver UDP-glucuronosyltransferase UGT1*6 reveals the involvement of a carboxyl group in catalysis.

The treatment of UDP-glucuronosyltransferase UGT1*6 stably expressed in V79 cells with three carboxyl-specific reagents, dicyclohexylcarbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and N-ethyl-5-phenylisoxazolium-3'-sulfonate (Woodward's reagent K), resulted in a fast, dose-dependent decrease of the 4-methylumbelliferone glucuronidation. The inactivation reactions followed pseudo-first order kinetics. The pKa of the modified residue was close to 5.0. A partial protection against inactivation by Woodward's reagent was observed at pH 7.4 in the presence of UDP-glucuronic acid, UDP, and, to a lesser extent, in the presence of 4-methylumbelliferone. Dicyclohexylcarbodiimide significantly decreased the Vmax, without affecting the apparent Km towards UDP-glucuronic acid and 4-methylumbelliferone. The results support the involvement of a carboxyl group in the catalytic process.

Expression of the human UDP-glucuronosyltransferase UGT1*6 in Escherichia coli. Influence of bacterial signal peptides on the production and localization of the recombinant protein.

The membrane-bound human liver UDP-glucuronosyltransferase UGT1*6 was expressed in Escherichia coli. Exchange of the natural signal peptide by the bacterial signal peptides of pclB or OmpT proteins considerably increased the level of expression and, as the natural signal peptide, targeted the protein to the membranes. The extent of maturation of SpelB-UGT1*6 precursor was about 30%. No processing of sOmpT-UGT1*6 occurred but the processing rate of this precursor could be significantly increased by mutagenesis of the first two amino acid residues of the mature sequence. These expression vectors allowed us to produce high levels of recombinant mature UGT1*6 required for further structural studies.

The metabolic chiral inversion and dispositional enantioselectivity of the 2-arylpropionic acids and their biological consequences.

The 2-arylpropionic acids are currently an important group of non-steroidal anti-inflammatory agents. They contain a chiral centre, and in vitro studies on inhibition of prostaglandin synthesis show that their activity resides almost exclusively in the S(+)-isomers. However, this stereoselectivity of action is not manifest in vivo, due to the thus-far-unique unidirectional metabolic inversion of the chiral centre from the inactive R(-)-isomers to the S(+)-antipodes. Available evidence strongly suggests that this reaction proceeds via the formation of the acyl CoA thioesters of the 2-arylpropionates, but the participation of enzyme(s) in the inversion process remains uncertain. Although the chiral inversion is seemingly a general feature of the fate of 2-arylpropionates, there do occur important combinations of acid and species where the reaction is not extant. The stereochemistry of the chiral centre of these acids also influences other aspects of their disposition, including the oxidative metabolism of the aryl/arylakyl moiety, glucuronidation of the -COOH group and plasma protein binding, and the importance of certain of these becomes more evident when renal function is impaired. The biological consequences of the metabolic chiral inversion and enantioselective disposition of the 2-arylpropionates have been summarized in terms of their implications for the development and use of safer and more effective drugs of this class.

Coordinate induction by antioxidants of UDP-glucuronosyltransferase UGT1A6 and the apical conjugate export pump MRP2 (multidrug resistance protein 2) in Caco-2 cells.

Treatment of Caco-2 cells with the antioxidants quercetin or t-butylhydroquinone led to induced protein levels of UDP-glucuronosyltransferase UGT1A6 (ca. 3-fold over controls) and of the apical conjugate export pump multidrug resistance protein 2 (MRP2; 1.9-fold over controls). In contrast to UGT1A6, MRP2 (symbol ABCC2) was not inducible by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Immunocytochemistry demonstrated that MRP2 was only expressed at the brush border domain of Caco-2 cell monolayers. The results indicate that UGT1A6 and MRP2 are coordinately induced by antioxidants, facilitating chemoprotection against phenolic toxins and excretion of conjugates into the intestinal lumen.

Comparative study of clofibric acid and bilirubin glucuronidation in human liver microsomes.

Hepatic microsomal glucuronoconjugation of the hypolipidemic drug clofibric acid was characterized in human liver and compared to the acylglucuronide formation of an endogenous substrate, bilirubin. The affinity of UDP-glucuronosyltransferase for bilirubin was 15-fold higher than for clofibric acid; the Vmax for the transformation of the two substrates were similar. The analysis of the specific activity in 32 liver biopsies showed that glucuronidation of clofibric acid or bilirubin were comparable in man and in rat. However, UDP-glucuronosyltransferase activity towards clofibric acid exhibited a large interindividual variation in man. Sex or age did not influence the glucuronidation of bilirubin and clofibric acid. Among the drugs given to the patients only clofibrate was able to increase the bilirubin conjugation. No effect of alcohol or smoking on the conjugation of the two substrates was observed. The absence of correlation between UDP-glucuronosyltransferase activities towards clofibric acid and bilirubin together with the specific induction of bilirubin glucuronidation by clofibrate suggested that these arylcarboxylic substrates were conjugated by separate forms of UDP-glucuronosyltransferase in human.

Characterization of distinct forms of cytochromes P-450, epoxide metabolizing enzymes and UDP-glucuronosyltransferases in rat skin.

Study of drug metabolizing enzyme activity was undertaken in skin microsomal and cytosolic fractions of male and female rats. The presence of several isoforms was revealed from their activities towards selected substrates and from their cross immunoreactivity using antibodies raised against purified hepatic or renal cytochromes P-450, epoxide hydrolase and UDP-glucuronosyltransferases. Cytochrome P-450 content was precisely quantified by second derivative spectrophotometry, 23.1 and 16.5 pmol/mg protein in males and females, respectively. The monooxygenase activity associated to cytochromes P-450IIB1 and P-450IA1 was determined through O-dealkylation of ethoxy-; pentoxy- and benzoxyresorufin. The activity ranged between 4 and 2 nmol/min/mg protein for male and female rats, respectively. These results and Western blot analysis indicated that rat skin microsomes contain both monooxygenase systems associated with cytochromes P-450IIB1 and P-450IA1. By contrast lauric acid hydroxylation, supported by cytochrome P-450IVA1, was not detectable. Activities of epoxide metabolizing enzymes (microsomal and cytosolic epoxide hydrolases; glutathione S-transferase) were also characterized in skin. Microsomes catalysed the hydratation of benzo(a)pyrene-4,5-oxide and cis-stilbene oxide at the same extent, whatever the sex, although the specific activity was 10 times lower than in liver. The hydratation of trans-stilbene oxide by soluble epoxide hydrolase was four times lower than in the liver. Conjugation of cis-stilbene oxide with glutathione in skin and liver proceeded at essentially similar rates, as the specific activity of glutathione S-transferase in skin was only two times less than that measured in hepatic cytosol. Glucuronidation of 1-naphthol, bilirubin but not of testosterone could be followed in the microsomal fraction. Revelation by Western blot indicated that both the isoforms involved in conjugation of phenols and bilirubin were present in skin microsomes. By contrast, the isoform catalysing the conjugation of testosterone was apparently missing. When immunoblotting was carried out using specific antibodies raised against the renal isoforms, the same result was obtained. In addition, an intense staining corresponding to a 57 kD-protein was observed.

Effect of 1-benzylimidazole on cytochromes P-450 induction and on the activities of epoxide hydrolases and UDP-glucuronosyltransferases in rat liver.

The influence of 1-benzylimidazole on the activities of hepatic monooxygenases cytochromes P-450 dependent, epoxide hydrolases and UDP-glucuronosyltransferases was investigated in male Wistar rats. Several doses (25, 75 and 100 mg/kg/day) were administered gastrically during 5 days in order to evaluate the dose-related induction. The treatment caused a dose-dependent hepatomegaly. 1-Benzylimidazole decreased the plasma level in triglycerides by 60-70%; by contrast the cholesterol content was not changed during the time course of the experiment. Lauric acid hydroxylase, benzphentamine N-demethylase, 7-ethoxyresorufin O-deethylase, 7-ethoxycoumarin O-deethylase activities were increased 3.5-, 4-, 13- and 46-fold, respectively with the highest dose. By immunoblotting, an enhancement in the protein bands corresponding to cytochromes P-450c and P-450b could be simultaneously observed, whatever the dose administered, thus suggesting an induction process. However, 1-benzylimidazole failed to bind with high affinity to the cytosolic Ah receptor. On the other hand, measurement of the activity of the microsomal epoxide hydrolase with benzo(a)pyrene-4,5-oxide as substrate and quantitation of the enzyme protein by immunoassay revealed that the increase in the activity after treatment with the compound was the result of enzyme activation only. By contrast, cytosolic epoxide hydrolase was not affected by 1-benzylimidazole. This compound also stimulated three distinct forms of UDP-glucuronosyltransferase. The activities towards 4-methylumbelliferone, 1-naphthol, morphine or a monoterpenoid alcohol, nopol, supported by two different isozymes were significantly increased only with the highest dose; meanwhile bilirubin glucuronidation was 2-fold enhanced, whatever the dose used. These observations emphasize the variety of the effects of 1-benzylimidazole on drug-metabolizing enzymes.

Effect of hypolipidemic compounds on lauric acid hydroxylation and phase II enzymes.

Treatment of male Fischer 344 rats with various hypolipidemic drugs of different peroxisome proliferating potency (1-benzylimidazole, acetylsalicylic acid, clofibrate, tiadenol) led to an induction of liver lauric acid hydroxylase, whereas probucol, which is not a peroxisome proliferator, did not induce this enzyme. Activity of bilirubin UDP-glucuronosyltransferase was increased by all the compounds tested. The highest increase was observed after treatment with acetylsalicylic acid (2.3-fold). High correlation (r = 0.953) was observed between the activities of lauric acid hydroxylase and the corresponding activities of cytosolic epoxide hydrolase reported previously. The amount of microsomal epoxide hydrolase was not changed by any of the compounds. Whereas clofibrate and tiadenol decreased glutathione S-transferase activity with 1-chloro-2,4-dinitrobenzene as substrate, 1-benzylimidazole and probucol increased this activity. With 4-hydroxynonenal as a substrate qualitatively the same results were obtained with the exception that probucol did not affect the enzyme activity. When glutathione S-transferase activity was measured with cis-stilbene oxide as substrate only the more than five-fold increase after treatment with 1-benzylimidazole was significantly different from control values. Activity of dihydrodiol dehydrogenase was increased after treatment of rats with 1-benzylimidazole (1.5-fold), whereas application of tiadenol led to a decrease of enzyme activity. Feeding of male guinea pigs with clofibrate did not change the activity of peroxisomal beta-oxidation, cytosolic epoxide hydrolase or lauric acid hydroxylase. However, treatment with tiadenol caused an increase of these activities.

Paracetamol glucuronidation by recombinant rat and human phenol UDP-glucuronosyltransferases.

Stably expressed human and rat phenol UDP-glucuronosyltransferases (UGTs) of the UGT1 complex (HlugP1, HlugP4 and 3-methylcholanthrene-inducible rat UGT1A1, the latter considered to be an orthologous enzyme to HlugP1) have been used to investigate the role of UGTs in paracetamol glucuronidation. Kinetic analysis of recombinant UGTs was compared to that of total UGT activities in liver microsomes. Paracetamol was found to be an overlapping substrate of several UGTs. It shows higher affinity for HlugP1 and rat UGT1A1 (apparent Km values of 2 and 3 mM, respectively) than for HlugP4 (Km = 50 mM) and other UGTs present in liver microsomes (Km values of > 12 mM). Glucuronidation of paracetamol with HlugP1 contrasts with that of 6-hydroxychrysene and of 4-methylumbelliferone, which are conjugated with higher affinity by HlugP4 than by HlugP1. Due to the wide tissue distribution of rat UGT1A1, paracetamol glucuronidation was also investigated in extrahepatic rat and human tissues. Paracetamol UGT activity was present and inducible by 2,3,7,8-tetrachlorodibenzo-p-dioxin in rat kidney, lung and spleen. It was also detected in human kidney. A selective cDNA probe for exon 1 of HlugP1 cross-reacted with mRNA from both human liver and kidney. The results demonstrate that paracetamol is conjugated by HlugP1 and its rat orthologue UGT1A1 with higher affinity than by HlugP4 and other UGTs.

Drug metabolizing enzymes related to laboratory medicine: cytochromes P-450 and UDP-glucuronosyltransferases.

Many studies on drug metabolism have been carried out during the last decades using protein purification, molecular cloning techniques and analysis of polymorphisms at phenotype and genotype levels. These researchers led to a better understanding of the role of drug metabolizing enzymes in the biotransformation of drugs, pollutants or foreign compounds and of their use in laboratory medicine. The metabolic processes commonly involved in the biotransformation of xenobiotics have been classified into functionalization reaction (phase I reactions), which implicate lipophilic compounds. These molecules are modified via monooxygenation, dealkylation, reduction, aromatization, hydrolysis and can be substrates for the phase II reactions, often called conjugation reactions as they conjugate a functional group with a polar, endogenous compound. This review, devoted to cytochromes P-450 (CYP) and UDP-glucuronosyltransferases (UGT), describes essentially the genetic polymorphisms found in humans, their clinical consequences and the methods to assess the phenotypes or genotypes, with a view to studying the interindividual differences in drug monooxygenation and drug glucuronidation. Variations in drug glucuronidation reported here focused essentially on variations due to physiological factors, induction, drug interactions and genetic factors in disorders such as Gilbert's Syndrome and Crigler-Najjar type I and II diseases.

Establishment of a V79 transfected cell line highly producing recombinant human gamma-glutamyltransferase.

gamma-Glutamyltransferase (GGT) is a glutathione-metabolizing enzyme whose activity variations in serum and organs are valuable markers of preneoplastic processes, alcohol abuse and induction by drugs. To elucidate the implication of GGT in various metabolic pathways, we established a stable transfected V79 cell line highly producing the human GGT. A full length cDNA, encoding the human hepatoma Hep G2 GGT, was subcloned into an expression vector under the control of the SV40 early promoter and was used to transfect V79 cells. A cell line was selected, exhibiting a GGT activity of 2 units per mg of protein, one of the highest levels reported to date. The recombinant GGT purified from this cell line showed the expected heterodimeric structure, with two subunits existing as sialylated and differentially glycosylated isoforms, with mean molecular masses of 80 and 29 kDa. Catalytic features were found to be identical to those of human serum and Hep G2 GGTs. Thus, the newly engineered cell line should be useful for the production of human GGT and as a potential alternative model for pharmacological studies.

A method for the determination of UDP-glucuronosyltransferase activity toward arylcarboxylic acids.

A fast and reliable method for the assay of UDP-glucuronosyltransferase (UGT) activity toward aglycones containing a carboxylic acid function is described. The procedure involves incubation with UDP-[U-14C]-glucuronic acid, the common substrate for the reaction, solid-phase separation of the radiolabeled acylglucuronides and unreacted cofactor, and quantification by liquid scintillation counting. The technique was validated for each of the seven substrates tested by reversed-phase HPLC, and was then applied successfully to the determination of optimal conditions for the activation of the carboxylic acid-UGT, and the estimation of kinetic constants for the glucuronidation of clofibric acid, 2-naphthylacetic acid, naproxen, and 4,4,4-triphenylbutanoic acid in rat liver microsomes. From the results obtained, we believe that this is an assay which, with only minor modification, could be applied to a wide range of carboxylic acid substrates for which, until now, specific and sensitive assays have been largely unavailable.