High-rate intestinal conjugation of 4-methylumbelliferone during intravenous infusion in the rat in vivo

In recent years it has become quite well established that the gut mucosa can actively conjugate orally administered drugs during absorption from the gut lumen (1,2). Indeed, isolated rat intestinal mucosa cells have been shown to conjugate various phenolic substrates with glucuronic acid or sulfate at appreciable rates (3,4). In addition, conjugation could also be demonstrated in perfused intestinal loop preparations from the rat, in which the substrate was supplied in the lumen (5–7); the conjugates can subsequently be excreted to the luminal (gut) or the contra-luminal (blood) side. However, to our knowledge the contribution of the gastro-intestinal region to the overall conjugation $\text{in vivo}$ of intravenously administered substrates has not been studied. Experiments in which a substrate was supplied from the intravenous side in isolated perfused rat intestinal segments (6) and conjugation could be demonstrated, as well as the accumulation of L-DOPA conjugates in gut mucosa in the rat after i.v. administration of L-DOPA (8) suggested that indeed intestinal conjugation of substrates present in blood might be expected.In the course of an investigation on the extraction of the phenolic compound, 4-methylumbelliferone, by rat liver $\text{in situ}$, we happened to find a very substantial contribution of the gastro-intestinal region to the clearance by conjugation of 4-methylumbelliferone. In the rat, 4-methylumbelliferone is mainly conjugated with glucaronic acid (9,10). We show in this communication that during gastro-intestinal passage 37% of the unconjugated 4-methylumbelliferone is extracted from the incoming arterial blood and converted into the conjugates.     

Glucuronidation and excretion of nonylphenol in perfused rat liver.

Nonylphenol, an environmental estrogenic chemical, is reported to have adverse effects on the reproductive organs of animals. In this study, the metabolism of nonylphenol and that of other alkylphenols in the rat liver was investigated using liver perfusion. Alkylphenols (nonylphenol, hexylphenol, butylphenol, and ethylphenol) were glucuronidated by rat liver microsomes. Nonylphenol was found to be conjugated with glucuronic acid by an isoform of UDP-glucuronosyltransferase, UGT2B1, expressed in yeast AH22 cells. However, when nonylphenol was perfused into rat liver in situ, it was difficult for free nonylphenol and conjugated metabolite to be excreted into the bile or vein, and most of the perfused nonylphenol remained free and as a glucuronide conjugate in the liver tissue, even after 1 h of perfusion. After 1 h of perfusion of the other alkylphenols, most of them were excreted into the bile as glucuronides. Ethylphenol, which has the shortest alkyl chain, was excreted rapidly into both the bile and vein; however, the excretion rates of alkylphenols having longer alkyl chains tended to be slow. MRP-2-deficient Eisai hyperbilirubinemic rats could not secrete alkylphenol-glucuronides into the bile, indicating that alkylphenol-glucuronides are transported by MRP-2 to the bile in normal Sprague-Dawley rats. The results indicate that the kinetics of excretion of alkylphenol-glucuronides into the bile or vein depends on the length of alkyl chain and suggest that nonylphenol-glucuronide formed in the liver cannot be transported by MRP-2.     

Glucuronidation and sulfation of the tea flavonoid (-)-epicatechin by the human and rat enzymes.

(-)-Epicatechin (EC) is one of the flavonoids present in green tea, suggested to have chemopreventive properties in cancer. However, its bioavailability is not clearly understood. In the present study, we determined the metabolism of EC, focusing on its glucuronic acid and sulfate conjugation using human liver and intestinal microsomes and cytosol as well as recombinant UDP-glucuronosyltransferase (UGT) and sulfotransferase (SULT) isoforms in comparison with that occurring in the rat. Surprisingly, EC was not glucuronidated by the human liver and small intestinal microsomes. There was also no evidence of glucuronidation by human colon microsomes or by recombinant UGT1A7, which is not present in the liver or intestine. Interestingly, in the rat liver microsomes EC was efficiently glucuronidated with the formation of two glucuronides. In contrast, the human liver cytosol efficiently sulfated EC mainly through the SULT1A1 isoform. For the intestine, both SULT1A1 and SULT1A3 contributed. Other SULT isoforms contributed little. High-performance liquid chromatography of the sulfate conjugates showed one major sulfatase-sensitive peak with all tissues. An additional minor sulfatase-resistant peak was formed by the liver and intestinal cytosol as well as with SULT1A1 but not by the Caco-2 cytosol and SULT1A3. In the rat, EC sulfation was considerably less efficient than in the human liver. These results indicate that sulfation is the major pathway in EC metabolism in the human liver and intestine with no glucuronidation occurring. There was also a large species difference both in glucuronidation and sulfation of EC between rats and humans.     

Glucuronidation of propranolol and 4'-hydroxypropranolol. Substrate specificity and stereoselectivity of rat liver microsomal glucuronyltransferases

Liquid-chromatographic procedures were developed to assay the formation of diastereomeric glucuronides of propranolol (PG) and 4'-hydroxypropranolol (HPG) by rat liver microsomes, with identifications performed by GC/MS techniques. Propranolol was conjugated at a rate 10% of that determined for 4'-hydroxypropranolol. Glucuronyltransferase activity increased slightly (10-17%) in the presence of MgCl2. Inclusion of 0.04% Triton X-100 produced a 55% inhibition of PG formation, but increased HPG formation greater than 2-fold. Pretreatment of animals with phenobarbital resulted in a 4-fold increase in PG formation, but did not affect HPG formation unless MgCl2 was also present. Under these conditions, a 50-60% increase in HPG formation occurred. Pretreatment with 3-methylcholanthrene did not affect the formation of either glucuronide. (R)-(+)-Propranolol was glucuronidated 2-fold faster than the (S)-(-) enantiomer at substrate concentrations below 0.1 mM, and 1.3-fold faster at substrate concentrations above 2.0 mM. The estimated Vmax, 0.67 nmol/mg/min, was identical for both enantiomers. The dissociation constants were significantly different, however, being 0.57 mM for (R)-(+)-propranolol and 0.87 mM for (S)-(-)-propranolol. No stereoselectivity was observed in the formation of HPG when 4'-hydroxypropranolol was used as substrate, or when propranolol was incubated in the presence of an NADPH-generating system. Propranolol and 4'-hydroxypropranolol were used as substrate, or when propranolol was incubated in the presence of an NADPH-generating system. Propranolol and 4-hydroxypropranolol are apparently glucuronidated by different forms of rat liver glucuronyltransferase. Furthermore, propranolol glucuronidation occurs stereoselectively in vitro because of the different enzyme affinities for the enantiomers of the drug.     

Glucuronidation of dihydroartemisinin in vivo and by human liver microsomes and expressed UDP-glucuronosyltransferases.

The aim of this study was to elucidate the metabolic pathways for dihydroartemisinin (DHA), the active metabolite of the artemisinin derivative artesunate (ARTS). Urine was collected from 17 Vietnamese adults with falciparum malaria who had received 120 mg of ARTS i.v., and metabolites were analyzed by high-performance liquid chromatography-mass spectrometry (HPLC-MS). Human liver microsomes were incubated with [12-(3)H]DHA and cofactors for either glucuronidation or cytochrome P450-catalyzed oxidation. Human liver cytosol was incubated with cofactor for sulfation. Metabolites were detected by HPLC-MS and/or HPLC with radiochemical detection. Metabolism of DHA by recombinant human UDP-glucuronosyltransferases (UGTs) was studied. HPLC-MS analysis of urine identified alpha-DHA-beta-glucuronide (alpha-DHA-G) and a product characterized as the tetrahydrofuran isomer of alpha-DHA-G. DHA was present only in very small amounts. The ratio of the tetrahydrofuran isomer, alpha-DHA-G, was highly variable (median 0.75; range 0.09-64). Nevertheless, alpha-DHA-G was generally the major urinary product of DHA glucuronidation in patients. The tetrahydrofuran isomer appeared to be at least partly a product of nonenzymic reactions occurring in urine and was readily formed from alpha-DHA-G by iron-mediated isomerization. In human liver microsomal incubations, DHA-G (diastereomer unspecified) was the only metabolite found (V(max) 177 +/- 47 pmol min(-1) mg(-1), K(m) 90 +/- 16 microM). Alpha-DHA-G was formed in incubations of DHA with expressed UGT1A9 (K(m) 32 microM, V(max) 8.9 pmol min(-1) mg(-1)) or UGT2B7 (K(m) 438 microM, V(max) 10.9 pmol mg(-1) min(-1)) but not with UGT1A1 or UGT1A6. There was no significant metabolism of DHA by cytochrome-P450 oxidation or by cytosolic sulfotransferases. We conclude that alpha-DHA-G is an important metabolite of DHA in humans and that its formation is catalyzed by UGT1A9 and UGT2B7.     

Glucuronidation of carcinogenic arylamine metabolites by rat liver microsomes

Since 2-acetylaminofluorene (2-AAF), 4-acetylaminobiphenyl (4-AABP) and 2-aminonaphthalene (2-AN) display varying degrees of carcinogenicity in the rat, which is capable of N-acetylating arylamines, an attempt was made to correlate the difference in carcinogenicity of these compounds with the ease of O-glucuronidation of their hydroxamic acids by rat hepatic microsomes, a reaction believed to be a detoxification mechanism. UDP-glucuronosyltransferase activity of rat hepatic microsomes was activated by Triton X-100. Glucuronidation by Triton X-100 activated microsomes of the N-hydroxy derivative of 2-AN was approximately 1.5 and 1.8 times faster than the corresponding derivatives of 2-aminofluorene (2-AF) and 4-aminobiphenyl (4-ABP) respectively. However, glucuronidation of the N-hydroxy-N-acetyl derivative of 2-AN was 40 and 17 times faster than the corresponding derivatives of 2-AF and 4-ABP respectively. Aroclor 1254 and 3-methylcholanthrene, but not phenobarbital, acetanilide and butylated hydroxytoluene, induced the enzyme for the glucuronidation of 2-AN derivatives. The present study (1) demonstrates an inverse relationship between the carcinogenicity of 2-AN, 4-AABP and 2-AAF and the ease of glucuronidation of their hydroxamic acid derivatives, and (2) suggests that, in addition to N- and C-hydroxylation, glucuronidation may play an important role in determining the carcinogenicity of arylamines and arylacetamides in the rat.     

The role of the liver in the clearance ofl-dopa from plasma

The role of the liver in the plasma clearance of l-dopa in the rat was examined. Some published studies which ascribe an important role to the liver in l-dopa clearance are discussed and critically evaluated. Contrary evidence suggesting that the liver is not a significant site of l-dopa clearance in vivo ispresented. The plasma concentration of l-dopa during intravenous infusion of the drug was not significantly reduced after a single passage through the liver. All of the data discussed are consistent with the conclusion that the liver plays a minor role in l-dopa clearance in vivo.It is suggested that the small intestine is the major site of metabolism of orally administered l-dopa.     

Glucuronidation of the enantiomers of E-10-hydroxynortriptyline in human and rat liver microsomes

Conjugation of racemic E-10-hydroxynortriptyline (E-10-OH-NT) with glucuronic acid was studied in the liver microsomal fraction of rats and humans. The diastereomeric glucuronides of E-10-OH-NT were resolved and quantitated by HPLC. Only the (+)-enantiomer was glucuronidated in liver microsomes from humans. Rat liver microsomes catalyzed the formation of both glucuronides. Phenobarbital pretreatment of rats increased the glucuronidation of both enantiomers about five-fold. The formation rate of (+)-E-10-OH-NT glucuronide varied from 5.5 to 33.2 pmol/mg x min, in microsomes from 13 humans. High activity was found in individuals previously treated with pentobarbital. Inhibition experiments with human liver microsomes showed that amitriptyline is a potent competitive inhibitor of (+)-E-10-OH-NT glucuronidation. p-Nitrophenol, paracetamol and 2-hydroxydesipramine also inhibited this reaction.     

Sex differences in sulfation and glucuronidation of phenol, 4-nitrophenol and N-hydroxy-2-acetylaminofluorene in the rat in vivo

Sulfation and glucuronidation of phenol, 4-nitrophenol (4NP) and N-hydroxy-2-acetylaminofluorene (N-OH-AAF) were studied in adult (60 days) male and female rats. Within 3 hours almost 50% of a dose of phenol was excreted in urine as phenyl sulfate; male rats excreted slightly more phenyl sulfate than females. This probably was due to a slower excretion of phenyl sulfate by the females. No sex difference in glucuronidation of phenol was found. Over a period of 24 hours male and female rats excreted almost 35% of a dose of 4NP as 4NP-sulfate in urine and almost 40% as 4NP-glucuronide. No differences in the excretion of 4NP-conjugates were found between sexes. However, almost twice as much of a dose of N-OH-AAF was excreted after 4 hours as the N-O-glucuronide in bile and urine in female than in males. On the other hand, females excreted less of the AAF-glutathione conjugates that are derived from the reaction of AAF-N-sulfate with glutathione in vivo [Meerman et al., Chem.-Biol. Interactions, 39, 149, 1982] in bile, than males. This indicates that sulfation of N-OH-AAF is less active in females than in males. Most likely, sulfation of the phenols is catalyzed by a different sulfotransferase than that of N-OH-AAF.     

Dose-dependent shifts in the sulfation and glucuronidation of phenolic compounds in the rat in vivo and in isolated hepatocytes: The role of saturation of phenolsulfotransferase

The role of enzyme-kinetic parameters of sulfotransferase and UDP-glucuronyltransferase in the balance between sulfation and glucuronidation of various phenolic substrates was studied in the rat in vivo after i.v. administration and in isolated hepatocytes. A pronounced shift from sulfation to glucuronidation was observed in vivo upon increasing the dose of two phenols, harmol and phenol. Similar shifts were found when these compounds were incubated with isolated hepatocytes. However, the shift from sulfation to glucuronidation was small when 4-chlorophenol, or absent when 4-t-butylphenol were given in vivo. Such shifts were also absent when 4-chlorophenol and 4-t-butylphenol were incubated at increasing concentrations with isolated hepatocytes. The in vivo results with the various phenols were very similar to the conjugation patterns found in isolated hepatocytes. This suggests that these conjugations in hepatocytes are regulated by similar factors as in the intact animal.In isolated hepatocytes at most 16 per cent of the available pool of inorganic sulfate was consumed during the incubation. Since Cheng and Levy have shown [J. biol. Chem.255, 2637 (1980)] that uptake of inorganic sulfate by hepatocytes is very rapid, the present results suggest that the limitation of sulfation of harmol and phenol at increasing dose was caused by saturation of the overall sulfation process by the acceptor substrate, rather than by depletion of inorganic sulfate.     

Assessment of the drug inhibitor specificity of the human liver 4-methylumbelliferone UDP-glucuronosyltransferase activity

The data suggest that the 4MU-UDPGT activity of human liver microsomes probably contributes to the glucuronidation of a limited number of clinically used drugs. However, confirmation of this ultimately requires studies to be performed with purified isozymes, cDNAs expressed in cell culture, or specific inhibitory antibodies.     

Glucuronidation of propofol and its analogs by human and rat liver microsomes

Propofol (2,6-diisopropylphenol), widely used an intravenous anesthetic, is rapidly metabolized to its glucuronide in the in vivo studies. Kinetic parameters for the glucuronidation of propofol and its analogs, such as 2,5-diisopropylphenol, 2-tert-butyl-6-methylphenol, 2-tert-butyl-5-methylphenol, 2,6-dimethylphenol and 2,5-dimethylphenol, were determined in vitro using human and rat liver microsomes. 2,5-Dimethylphenol and 2-tert-butyl-6-methylphenol exhibited the highest and lowest glucuronidation rates, respectively. Substitutes at the 2,6-positions gave lower glucuronidation rates than those at the 2,5-positions in both the human and rat microsomes. 2,5-Diisopropylphenol was glucuronidated at a lower rate in human than propofol. The affinity of uridine 5'-diphosphate (UDP)-glucuronosyltransferase for disubstituted phenols, such as propofol, 2,5-diisopropylphenol, 2,5-dimethylphenol, and 2-tert-butyl-6-methylphenol, gave higher Km values in human liver microsomes than in rat ones, and lower Vmax values showed similar relationship, expect for Vmax in propofol. The alkyl group at the 6 position showed a higher Km for glucuronidation by a steric hindrance in the human and rat microsomes. Our results propose that the glucuronidation of propofol and its analogs may not be explained by only a steric hindrance.     

Glucuronidation of 3'-azido-3'-deoxythymidine by rat and human liver microsomes

The glucuronidation of 3'-azido-3'-deoxythymidine (AZT) by rat and human liver microsomes has been studied in vitro. The AZT-glucuronide was preliminarily identified through specific hydrolysis by beta-glucuronidase and rigorous product identification was performed by high-field proton nuclear magnetic resonance and fast-atom-bombardment mass spectrometry. A beta-linked 5'-O-glucuronide was the exclusive product formed in liver microsomes. Rat and human liver microsomal uridine 5'-diphosphoglucuronyltransferase activities toward AZT were investigated. These studies revealed that AZT had a lower Km and a 5-6-fold higher relative catalytic efficiency for uridine 5'-diphosphoglucuronyltransferase in human as compared to rat liver microsomes which may play a role in the quantitative differences observed in the degree of AZT glucuronidation between rat and human.     

Glucuronidation and sulfation in subcellular fractions and in the isolated perfused rabbit lung: influence of ethanol

Glucuronidation and sulfation of 1-naphthol, 7-hydroxycoumarin, 4-nitrocatechol and phenolphthalein were studied in rabbit lung and liver. Pulmonary UDP-glucuronyltransferase and sulfotransferase activities in subcellular fractions were approximately 20-50% of those determined in the liver. Ethanol did not markedly induce these enzymes in either tissue. Glucuronidation and sulfation of 1-naphthol and 7-hydroxycoumarin were also studied in the isolated perfused rabbit lung as an intact cell model. Neither glucuronidation nor sulfation of 1-naphthol was observed. The absence of conjugate formation was due neither to the presence of beta-glucuronidase and/or sulfatase, nor to alternative biotransformation pathways. About 35% of the initial 7-hydroxycoumarin was conjugated, the majority being sulfate conjugate (14.4 nmol/h) with only minor amounts (0.12%) of the glucuronide. These results indicate the importance of studying both whole organ and in vitro metabolism.     

Glucuronidation of amitriptyline in man in vivo

The urinary excretion of amitriptyline (AT) as N-glucuronide was studied in healthy volunteers after single oral doses of AT and in patients on continuous treatment with AT. In the volunteers, 8 +/- 3% of a 25 mg dose of AT was recovered in urine as glucuronide during 108 hr. No difference between slow and rapid debrisoquine hydroxylators with respect to the excretion of AT glucuronide was seen. 0.08 to 1.68% of the given AT dose was recovered in urine in unchanged form. The excretion of unchanged AT correlated with the debrisoquine metabolic ratio (rs = 0.61; p less than 0.01). In 5 patients on continuous treatment with AT (125-150 mg/day), 8 +/- 5% of the daily dose was recovered in 24-hr urine as AT glucuronide. The present study shows that direct glucuronidation is a minor metabolic pathway of AT in man in vivo both after single low doses and during continuous treatment with therapeutic doses.     

Age-dependent propranolol clearance in perfused rat liver

The effect of age on the hepatic clearance of propranolol was studied by perfusing the liver isolated from 3- to 104-week-old rats. Propranolol levels in the recirculating perfusate declined biexponentially with time in all age groups. When the liver isolated from 7-week-old rats was perfused with propranolol (1 microgram/ml, 100 ml), hepatic clearance of this drug by the perfused liver (CLperf) increased from 0.589 to 1.14 ml X min-1 X (g liver)-1 with the increase of the perfusion flow rate from 1.0 to 2.0 ml X min-1 X (g liver)-1, confirming evidence of "perfusion-limited" hepatic clearance for this drug. Furthermore, there was no initial concentration(dose)-dependence in CLperf up to 2.5 micrograms/ml (i.e. 250 micrograms/organ). The effect of age on CLperf was then investigated by perfusing the isolated liver with 1.0 micrograms/ml propranolol at 2.0 ml X min-1 X (g liver)-1. Elimination of this drug from the perfusion medium was relatively rapid in 5- to 7-week-old rats, yielding the highest CLperf in these relatively young rats [approximately 1.0 to 1.1 ml X min-1 X (g liver)-1]. In contrast, CLperf values in both immature and older rats were 0.5 ml X min-1 X (g liver)-1 or less. The in vitro intrinsic hepatic clearance estimated in 5- and 7-week-old rats was about ten times as high as that in 104-week-old rats.