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Nora M Obrien - One of the best experts on this subject based on the ideXlab platform.

  • metabolism of quercetin 7 and quercetin 3 Glucuronides by an in vitro hepatic model the role of human β glucuronidase sulfotransferase catechol o methyltransferase and multi resistant protein 2 mrp2 in flavonoid metabolism
    Biochemical Pharmacology, 2003
    Co-Authors: Karen A Oleary, Fred A Mellon, Paul W. Needs, Andrea J Day, Nora M Obrien
    Abstract:

    Quercetin-3- and quercetin-7-Glucuronides are major products of small intestine epithelial cell metabolism (J. Nutr. 130 (2000) 2765) but it is not known if quercetin Glucuronides can be further processed in the liver or if they are excreted directly. Using the HepG2 hepatic cell model, we show that highly purified quercetin-7- and quercetin-3-Glucuronides can follow two pathways of metabolism: (i) methylation of the catechol functional group of both quercetin Glucuronides (44% of quercetin-7-glucuronide at a rate of 2.6 nmol/hr/10(6) cells, and 32% of quercetin-3-glucuronide at a rate of 1.9 nmol/hr/10(6) cells, over 48 hr) or (ii) hydrolysis of the glucuronide by endogenous beta-glucuronidase followed by sulfation to quercetin-3'-sulfate (7% of quercetin-7-glucuronide at a rate of 0.42 nmol/hr/10(6) cells and 10% of quercetin-3-glucuronide at a rate of 0.61 nmol/hr/10(6) cells, over 48 hr). In contrast, quercetin-4'-glucuronide was not metabolised, and interestingly this is not a major product of the small intestine absorption process. The conversion of the quercetin-7- and quercetin-3-glucuronide to the mono-sulfate conjugate shows intracellular deglucuronidation by beta-glucuronidase activity, allowing transient contact of the free aglycone with the cellular environment. Inhibition of methylation using a catechol-O-methyltransferase inhibitor shifted metabolism towards sulfation, as indicated by an increase in quercetin-3'-sulfate formation (increase in rate to 1.13 and 1.43 nmol/hr/10(6) cells for quercetin-7-glucuronide and quercetin-3-glucuronide, respectively). Efflux of quercetin metabolites from HepG2 cells (methylated glucuronide and sulfate conjugates) was not altered by verapamil, a p-glycoprotein inhibitor, but efflux was competitively inhibited by MK-571, a multidrug resistant protein inhibitor, indicating a role for multidrug resistant protein in the efflux of quercetin conjugates from HepG2 cells. These results show that HepG2 cells can absorb and turnover quercetin Glucuronides and that human endogenous beta-glucuronidase activity could modulate the intracellular biological activities of dietary antioxidant flavonoids.

  • metabolism of quercetin 7 and quercetin 3 Glucuronides by an in vitro hepatic model the role of human β glucuronidase sulfotransferase catechol o methyltransferase and multi resistant protein 2 mrp2 in flavonoid metabolism
    Biochemical Pharmacology, 2003
    Co-Authors: Karen Oleary, Paul W. Needs, Fred A Mellon, Nora M Obrien
    Abstract:

    Abstract Quercetin-3- and quercetin-7-Glucuronides are major products of small intestine epithelial cell metabolism (J. Nutr. 130 (2000) 2765) but it is not known if quercetin Glucuronides can be further processed in the liver or if they are excreted directly. Using the HepG2 hepatic cell model, we show that highly purified quercetin-7- and quercetin-3-Glucuronides can follow two pathways of metabolism: (i) methylation of the catechol functional group of both quercetin Glucuronides (44% of quercetin-7-glucuronide at a rate of 2.6 nmol/hr/10 6 cells, and 32% of quercetin-3-glucuronide at a rate of 1.9 nmol/hr/10 6 cells, over 48 hr) or (ii) hydrolysis of the glucuronide by endogenous β-glucuronidase followed by sulfation to quercetin-3′-sulfate (7% of quercetin-7-glucuronide at a rate of 0.42 nmol/hr/10 6 cells and 10% of quercetin-3-glucuronide at a rate of 0.61 nmol/hr/10 6 cells, over 48 hr). In contrast, quercetin-4′-glucuronide was not metabolised, and interestingly this is not a major product of the small intestine absorption process. The conversion of the quercetin-7- and quercetin-3-glucuronide to the mono-sulfate conjugate shows intracellular deglucuronidation by β-glucuronidase activity, allowing transient contact of the free aglycone with the cellular environment. Inhibition of methylation using a catechol- O -methyltransferase inhibitor shifted metabolism towards sulfation, as indicated by an increase in quercetin-3′-sulfate formation (increase in rate to 1.13 and 1.43 nmol/hr/10 6 cells for quercetin-7-glucuronide and quercetin-3-glucuronide, respectively). Efflux of quercetin metabolites from HepG2 cells (methylated glucuronide and sulfate conjugates) was not altered by verapamil, a p -glycoprotein inhibitor, but efflux was competitively inhibited by MK-571, a multidrug resistant protein inhibitor, indicating a role for multidrug resistant protein in the efflux of quercetin conjugates from HepG2 cells. These results show that HepG2 cells can absorb and turnover quercetin Glucuronides and that human endogenous β-glucuronidase activity could modulate the intracellular biological activities of dietary antioxidant flavonoids.

  • flavonoid Glucuronides are substrates for human liver β glucuronidase
    FEBS Letters, 2001
    Co-Authors: Karen Oleary, Paul W. Needs, Nora M Obrien
    Abstract:

    Abstract Quercetin Glucuronides are the main circulating metabolites of quercetin in humans. We hypothesise that the potential availability of the aglycone within tissues depends on the substrate specificity of the deconjugating enzyme β-glucuronidase towards circulating flavonoid Glucuronides. Human tissues (small intestine, liver and neutrophils) exhibited β-glucuronidase against quercetin Glucuronides. The various quercetin Glucuronides were deconjugated at similar rates, but liver cell-free extracts were the most efficient and the activity was completely inhibited by saccharo-1,4-lactone (a β-glucuronidase inhibitor). Furthermore, pure recombinant human β-glucuronidase hydrolysed various flavonoid Glucuronides, with a 20-fold variation in catalytic efficiency (kcat/Km=1.3×103 M−1 s−1 for equol-7-O-glucuronide and 26×103 M−1 s−1 for kaempferol-3-O-glucuronide). Similar catalytic efficiencies were obtained for quercetin O-Glucuronides substituted at different positions. These results show that flavonoid Glucuronides can be deconjugated by microsomal β-glucuronidase from various human cells.

Karen Oleary - One of the best experts on this subject based on the ideXlab platform.

  • metabolism of quercetin 7 and quercetin 3 Glucuronides by an in vitro hepatic model the role of human β glucuronidase sulfotransferase catechol o methyltransferase and multi resistant protein 2 mrp2 in flavonoid metabolism
    Biochemical Pharmacology, 2003
    Co-Authors: Karen Oleary, Paul W. Needs, Fred A Mellon, Nora M Obrien
    Abstract:

    Abstract Quercetin-3- and quercetin-7-Glucuronides are major products of small intestine epithelial cell metabolism (J. Nutr. 130 (2000) 2765) but it is not known if quercetin Glucuronides can be further processed in the liver or if they are excreted directly. Using the HepG2 hepatic cell model, we show that highly purified quercetin-7- and quercetin-3-Glucuronides can follow two pathways of metabolism: (i) methylation of the catechol functional group of both quercetin Glucuronides (44% of quercetin-7-glucuronide at a rate of 2.6 nmol/hr/10 6 cells, and 32% of quercetin-3-glucuronide at a rate of 1.9 nmol/hr/10 6 cells, over 48 hr) or (ii) hydrolysis of the glucuronide by endogenous β-glucuronidase followed by sulfation to quercetin-3′-sulfate (7% of quercetin-7-glucuronide at a rate of 0.42 nmol/hr/10 6 cells and 10% of quercetin-3-glucuronide at a rate of 0.61 nmol/hr/10 6 cells, over 48 hr). In contrast, quercetin-4′-glucuronide was not metabolised, and interestingly this is not a major product of the small intestine absorption process. The conversion of the quercetin-7- and quercetin-3-glucuronide to the mono-sulfate conjugate shows intracellular deglucuronidation by β-glucuronidase activity, allowing transient contact of the free aglycone with the cellular environment. Inhibition of methylation using a catechol- O -methyltransferase inhibitor shifted metabolism towards sulfation, as indicated by an increase in quercetin-3′-sulfate formation (increase in rate to 1.13 and 1.43 nmol/hr/10 6 cells for quercetin-7-glucuronide and quercetin-3-glucuronide, respectively). Efflux of quercetin metabolites from HepG2 cells (methylated glucuronide and sulfate conjugates) was not altered by verapamil, a p -glycoprotein inhibitor, but efflux was competitively inhibited by MK-571, a multidrug resistant protein inhibitor, indicating a role for multidrug resistant protein in the efflux of quercetin conjugates from HepG2 cells. These results show that HepG2 cells can absorb and turnover quercetin Glucuronides and that human endogenous β-glucuronidase activity could modulate the intracellular biological activities of dietary antioxidant flavonoids.

  • flavonoid Glucuronides are substrates for human liver β glucuronidase
    FEBS Letters, 2001
    Co-Authors: Karen Oleary, Paul W. Needs, Nora M Obrien
    Abstract:

    Abstract Quercetin Glucuronides are the main circulating metabolites of quercetin in humans. We hypothesise that the potential availability of the aglycone within tissues depends on the substrate specificity of the deconjugating enzyme β-glucuronidase towards circulating flavonoid Glucuronides. Human tissues (small intestine, liver and neutrophils) exhibited β-glucuronidase against quercetin Glucuronides. The various quercetin Glucuronides were deconjugated at similar rates, but liver cell-free extracts were the most efficient and the activity was completely inhibited by saccharo-1,4-lactone (a β-glucuronidase inhibitor). Furthermore, pure recombinant human β-glucuronidase hydrolysed various flavonoid Glucuronides, with a 20-fold variation in catalytic efficiency (kcat/Km=1.3×103 M−1 s−1 for equol-7-O-glucuronide and 26×103 M−1 s−1 for kaempferol-3-O-glucuronide). Similar catalytic efficiencies were obtained for quercetin O-Glucuronides substituted at different positions. These results show that flavonoid Glucuronides can be deconjugated by microsomal β-glucuronidase from various human cells.

Moshe Finel - One of the best experts on this subject based on the ideXlab platform.

  • human efflux transport of testosterone epitestosterone and other androgen Glucuronides
    The Journal of Steroid Biochemistry and Molecular Biology, 2020
    Co-Authors: Erkka Jarvinen, Heidi Kidron, Moshe Finel
    Abstract:

    Abstract Several drug-metabolizing enzymes are known to control androgen homeostasis in humans. UDP-glucuronosyltransferases convert androgens to glucuronide conjugates in the liver and intestine, which enables subsequent elimination of these conjugated androgens via urine. The most important androgen is testosterone, while others are the testosterone metabolites androsterone and etiocholanolone, and the testosterone precursor dehydroepiandrosterone. Epitestosterone is another endogenous androgen, which is included as a crucial marker in urine doping tests. Since glucuronide conjugates are hydrophilic, efflux transporters mediate their excretion from tissues. In this study, we employed the membrane vesicle assay to identify the efflux transporters for Glucuronides of androsterone, dehydroepiandrosterone, epitestosterone, etiocholanolone and testosterone. The human hepatic and intestinal transporters MRP2 (ABCC2), MRP3 (ABCC3), MRP4 (ABCC4), BCRP (ABCG2) and MDR1 (ABCB1) were studied in vitro. Of these transporters, only MRP2 and MRP3 transported the androgen Glucuronides investigated. In kinetic analyses, MRP3 transported Glucuronides of androsterone, epitestosterone and etiocholanolone at low Km values, between 0.4 and 4 μM, while the Km values for Glucuronides of testosterone and dehydroepiandrosterone were 14 and 51 μM, respectively. MRP2 transported the Glucuronides at lower affinity, as indicated by Km values over 100 μM. Interestingly, the MRP2-mediated transport of androsterone and epitestosterone Glucuronides was best described by sigmoidal kinetics. The inability of BCRP to transport any of the androgen Glucuronides investigated is drastically different from its highly active transport of several estrogen conjugates. Our results explain the transporter-mediated disposition of androgen Glucuronides in humans, and shed light on differences between the human efflux transporters MRP2, MRP3, MRP4, BCRP and MDR1.

  • efflux transport of nicotine cotinine and trans 3 hydroxycotinine Glucuronides by human hepatic transporters
    Basic & Clinical Pharmacology & Toxicology, 2019
    Co-Authors: Erkka Jarvinen, Noora Sjostedt, Jan B. Koenderink, Heidi Kidron, Moshe Finel
    Abstract:

    Nicotine is the addiction causing alkaloid in tobacco, and it is used in smoking cessation therapies. Although the metabolic pathways of nicotine are well known and mainly occur in the liver, the transport of nicotine and its metabolites is poorly characterized. The highly hydrophilic nature and urinary excretion of nicotine glucuronide metabolites indicate that hepatic basolateral efflux transporters mediate their excretion. We aimed here to find the transporters responsible for the hepatic excretion of nicotine, cotinine and trans-3'-hydroxycotinine (OH-cotinine) Glucuronides. To this end, we tested their transport by multidrug resistance-associated proteins 1 (MRP1, ABCC1) and MRP3-6 (ABCC3-6), which are located on the basolateral membranes of hepatocytes, as well as MRP2 (ABCC2), breast cancer resistance protein (BCRP, ABCG2) and multidrug resistance protein 1 (MDR1, P-gp, ABCB1) that are expressed in the apical membranes of these cells. ATP-dependent transport of these Glucuronides was evaluated in inside-out membrane vesicles expressing the transporter of interest. In addition, potential interactions of both the Glucuronides and parent compounds with selected transporters were tested by inhibition assays. Considerable ATP-dependent transport was observed only for OH-cotinine glucuronide by MRP3. The kinetics of this transport activity was characterized, resulting in an estimated Km value of 895 micromol/L. No significant transport was found for nicotine or cotinine Glucuronides by any of the tested transporters at either 5 or 50 micromol/L substrate concentration. Furthermore, neither nicotine, cotinine nor OH-cotinine inhibited MRP2-4, BCRP or MDR1. In this study, we directly examined, for the first time, efflux transport of the three hydrophilic nicotine glucuronide metabolites by the major human hepatic efflux transporters. Despite multiple transporters studied here, our results indicate that an unknown transporter may be responsible for the hepatic excretion of nicotine and cotinine Glucuronides.

  • enzyme assisted synthesis and structure characterization of glucuronic acid conjugates of losartan candesartan and zolarsartan
    Bioorganic Chemistry, 2008
    Co-Authors: Anna Alonen, Moshe Finel, Johanna Jansson, Sirkku Kallonen, Alexandros Kiriazis, Olli Aitio, Risto Kostiainen
    Abstract:

    Abstract Three angiotensin II receptor antagonists—losartan, candesartan, and zolarsartan—were investigated. All the compounds, which are structural analogues, are metabolized via conjugation to glucuronic acid. Interestingly, both O - and N -glucuronidation take place, so that regioisomers are formed. One ether O -glucuronide, two acyl O -Glucuronides, and five tetrazole- N -Glucuronides were biosynthesized, in milligram scale, from the three sartan aglycones. Liver microsomes from bovine, moose, rat, and pig and recombinant human UDP-glucuronosyltransferases were used as catalysts. The synthesized compounds were identified as sartan Glucuronides by mass spectrometry, while the sites of glucuronidation were determined by nuclear magnetic resonance spectroscopy. Drug metabolites are needed as standards for pharmaceutical research and, as the present study shows, they can easily be produced with enzymes as catalyst.

Paul W. Needs - One of the best experts on this subject based on the ideXlab platform.

  • deconjugation kinetics of glucuronidated phase ii flavonoid metabolites by β glucuronidase from neutrophils
    Drug Metabolism and Pharmacokinetics, 2010
    Co-Authors: Roger Bartholome, Paul W. Needs, Guido R M M Haenen, Peter C H Hollman, Aalt Bast, Pieter C Dagnelie, Dirk Roos, Jaap Keijer, Paul A Kroon, Ilja C W Arts
    Abstract:

    Summary : Flavonoids are inactivated by phase II metabolism and occur in the body as Glucuronides. Mammalian β -glucuronidase released from neutrophils at inflammatory sites may be able to deconjugate and thus activate flavonoid Glucuronides. We have studied deconjugation kinetics and pH optimum for four sources of β -glucuronidase (human neutrophil, human recombinant, myeloid PLB-985 cells, Helix pomatia ) with five flavonoid Glucuronides (quercetin-3-glucuronide, quercetin-3'-glucuronide, quercetin-4'-glucuronide, quercetin-7-glucuronide, 3'-methylquercetin-3-glucuronide), 4-methylumbelliferyl- β - D -glucuronide, and paranitrophenol-glucuronide. All substrate-enzyme combinations tested exhibited first order kinetics. The optimum pH for hydrolysis was between 3.5-5, with appreciable hydrolysis activities up to pH 5.5. At pH 4, the K m ranged 44-fold from 22 μ M for quercetin-4'-glucuronide with Helix pomatia β -glucuronidase, to 981 μ M for para-nitrophenol-glucuronide with recombinant β -glucuronidase. V max (range: 0.735-24.012mmol· min − 1· unit − 1 [1 unit is defined as the release of 1 μ M 4-methylumbelliferyl- β -D-glucuronide per min]) and the reaction rate constants at low substrate concentrations (k) (range: 0.002-0.062 min − 1 ·(unit/L) − 1 were similar for all substrates-enzyme combinations tested. In conclusion, we show that β -glucuronidase from four different sources, including human neutrophils, is able to deconjugate flavonoid Glucuronides and non-flavonoid substrates at fairly similar kinetic rates. At inflammatory sites in vivo the pH, neutrophil and flavonoid glucuronide concentrations seem favorable for deconjugation. However, it remains to be confirmed whether this is actually the case.

  • metabolism of quercetin 7 and quercetin 3 Glucuronides by an in vitro hepatic model the role of human β glucuronidase sulfotransferase catechol o methyltransferase and multi resistant protein 2 mrp2 in flavonoid metabolism
    Biochemical Pharmacology, 2003
    Co-Authors: Karen A Oleary, Fred A Mellon, Paul W. Needs, Andrea J Day, Nora M Obrien
    Abstract:

    Quercetin-3- and quercetin-7-Glucuronides are major products of small intestine epithelial cell metabolism (J. Nutr. 130 (2000) 2765) but it is not known if quercetin Glucuronides can be further processed in the liver or if they are excreted directly. Using the HepG2 hepatic cell model, we show that highly purified quercetin-7- and quercetin-3-Glucuronides can follow two pathways of metabolism: (i) methylation of the catechol functional group of both quercetin Glucuronides (44% of quercetin-7-glucuronide at a rate of 2.6 nmol/hr/10(6) cells, and 32% of quercetin-3-glucuronide at a rate of 1.9 nmol/hr/10(6) cells, over 48 hr) or (ii) hydrolysis of the glucuronide by endogenous beta-glucuronidase followed by sulfation to quercetin-3'-sulfate (7% of quercetin-7-glucuronide at a rate of 0.42 nmol/hr/10(6) cells and 10% of quercetin-3-glucuronide at a rate of 0.61 nmol/hr/10(6) cells, over 48 hr). In contrast, quercetin-4'-glucuronide was not metabolised, and interestingly this is not a major product of the small intestine absorption process. The conversion of the quercetin-7- and quercetin-3-glucuronide to the mono-sulfate conjugate shows intracellular deglucuronidation by beta-glucuronidase activity, allowing transient contact of the free aglycone with the cellular environment. Inhibition of methylation using a catechol-O-methyltransferase inhibitor shifted metabolism towards sulfation, as indicated by an increase in quercetin-3'-sulfate formation (increase in rate to 1.13 and 1.43 nmol/hr/10(6) cells for quercetin-7-glucuronide and quercetin-3-glucuronide, respectively). Efflux of quercetin metabolites from HepG2 cells (methylated glucuronide and sulfate conjugates) was not altered by verapamil, a p-glycoprotein inhibitor, but efflux was competitively inhibited by MK-571, a multidrug resistant protein inhibitor, indicating a role for multidrug resistant protein in the efflux of quercetin conjugates from HepG2 cells. These results show that HepG2 cells can absorb and turnover quercetin Glucuronides and that human endogenous beta-glucuronidase activity could modulate the intracellular biological activities of dietary antioxidant flavonoids.

  • metabolism of quercetin 7 and quercetin 3 Glucuronides by an in vitro hepatic model the role of human β glucuronidase sulfotransferase catechol o methyltransferase and multi resistant protein 2 mrp2 in flavonoid metabolism
    Biochemical Pharmacology, 2003
    Co-Authors: Karen Oleary, Paul W. Needs, Fred A Mellon, Nora M Obrien
    Abstract:

    Abstract Quercetin-3- and quercetin-7-Glucuronides are major products of small intestine epithelial cell metabolism (J. Nutr. 130 (2000) 2765) but it is not known if quercetin Glucuronides can be further processed in the liver or if they are excreted directly. Using the HepG2 hepatic cell model, we show that highly purified quercetin-7- and quercetin-3-Glucuronides can follow two pathways of metabolism: (i) methylation of the catechol functional group of both quercetin Glucuronides (44% of quercetin-7-glucuronide at a rate of 2.6 nmol/hr/10 6 cells, and 32% of quercetin-3-glucuronide at a rate of 1.9 nmol/hr/10 6 cells, over 48 hr) or (ii) hydrolysis of the glucuronide by endogenous β-glucuronidase followed by sulfation to quercetin-3′-sulfate (7% of quercetin-7-glucuronide at a rate of 0.42 nmol/hr/10 6 cells and 10% of quercetin-3-glucuronide at a rate of 0.61 nmol/hr/10 6 cells, over 48 hr). In contrast, quercetin-4′-glucuronide was not metabolised, and interestingly this is not a major product of the small intestine absorption process. The conversion of the quercetin-7- and quercetin-3-glucuronide to the mono-sulfate conjugate shows intracellular deglucuronidation by β-glucuronidase activity, allowing transient contact of the free aglycone with the cellular environment. Inhibition of methylation using a catechol- O -methyltransferase inhibitor shifted metabolism towards sulfation, as indicated by an increase in quercetin-3′-sulfate formation (increase in rate to 1.13 and 1.43 nmol/hr/10 6 cells for quercetin-7-glucuronide and quercetin-3-glucuronide, respectively). Efflux of quercetin metabolites from HepG2 cells (methylated glucuronide and sulfate conjugates) was not altered by verapamil, a p -glycoprotein inhibitor, but efflux was competitively inhibited by MK-571, a multidrug resistant protein inhibitor, indicating a role for multidrug resistant protein in the efflux of quercetin conjugates from HepG2 cells. These results show that HepG2 cells can absorb and turnover quercetin Glucuronides and that human endogenous β-glucuronidase activity could modulate the intracellular biological activities of dietary antioxidant flavonoids.

  • flavonoid Glucuronides are substrates for human liver β glucuronidase
    FEBS Letters, 2001
    Co-Authors: Karen Oleary, Paul W. Needs, Nora M Obrien
    Abstract:

    Abstract Quercetin Glucuronides are the main circulating metabolites of quercetin in humans. We hypothesise that the potential availability of the aglycone within tissues depends on the substrate specificity of the deconjugating enzyme β-glucuronidase towards circulating flavonoid Glucuronides. Human tissues (small intestine, liver and neutrophils) exhibited β-glucuronidase against quercetin Glucuronides. The various quercetin Glucuronides were deconjugated at similar rates, but liver cell-free extracts were the most efficient and the activity was completely inhibited by saccharo-1,4-lactone (a β-glucuronidase inhibitor). Furthermore, pure recombinant human β-glucuronidase hydrolysed various flavonoid Glucuronides, with a 20-fold variation in catalytic efficiency (kcat/Km=1.3×103 M−1 s−1 for equol-7-O-glucuronide and 26×103 M−1 s−1 for kaempferol-3-O-glucuronide). Similar catalytic efficiencies were obtained for quercetin O-Glucuronides substituted at different positions. These results show that flavonoid Glucuronides can be deconjugated by microsomal β-glucuronidase from various human cells.

Jeremy K Nicholson - One of the best experts on this subject based on the ideXlab platform.

  • kinetic studies on the intramolecular acyl migration of β 1 o acyl Glucuronides application to the Glucuronides of r and s ketoprofen r and s hydroxy ketoprofen metabolites and tolmetin by 1h nmr spectroscopy
    Xenobiotica, 2005
    Co-Authors: E Skordi, Ian D Wilson, John C Lindon, Jeremy K Nicholson
    Abstract:

    Conjugation of carboxylate drugs with D-glucuronic acid is of considerable interest because of the inherent reactivity of the resulting beta-1-O-acyl Glucuronides. These conjugates can degrade by spontaneous hydrolysis and internal acyl migration. beta-1-O-acyl Glucuronides and their acyl migration products can also react covalently with macromolecules with potential toxicological consequences. The spontaneous degradation of the diastereoisomeric beta-1-O-acyl glucuronide metabolites of the racemic drug ketoprofen, two of its ring-hydroxylated metabolites and of tolmetin beta-1-O-acyl glucuronide was investigated by (1)H-NMR spectroscopy in buffer solutions, at pH 7.4 and 37 degrees C. A plot of the logarithm of the peak integrals against time revealed first-order kinetics. Degradation rates and half-lives were calculated for each glucuronide using first-order reaction equations. Tolmetin glucuronide had the fastest degradation rate, whilst all of the ketoprofen-related Glucuronides had similar degradation rates. The degradation of the diastereoisomeric Glucuronides was stereoselective, with the rate for the (S)-isomer always slower compared with the (R)-isomer by approximately a factor of 2.

  • hplc 1h nmr spectroscopic studies of the reactive alpha 1 o acyl isomer formed during acyl migration of s naproxen beta 1 o acyl glucuronide
    Chemical Research in Toxicology, 2001
    Co-Authors: Olivia Corcoran, Steen Honore Hansen, J Troke, Rasmus W Mortensen, Jeremy K Nicholson
    Abstract:

    A widely held view in drug metabolism and pharmacokinetic studies is that the initial 1-isomer to 2-isomer step in the intramolecular acyl migration of drug ester Glucuronides is irreversible, and that alpha-1-O-acyl isomers do not occur under physiological conditions. We investigated this hypothesis using high-performance liquid chromatography directly coupled to proton nuclear magnetic resonance spectroscopy (HPLC/1H NMR) and mass spectrometry (LC/MS) to probe the migration reactions of S-naproxen beta-1-O-acyl glucuronide, in phosphate buffer at pH 7.4, 37 degrees C. We report the first direct observation of the alpha-1-O-acyl isomer of a drug ester glucuronide (S-naproxen) formed in a biosystem via the facile acyl migration of the corresponding pure beta-1-O-acyl glucuronide. The unequivocal identification of the reactive product was achieved using stopped-flow one-dimensional HPLC/1H NMR and two-dimensional 1H-1H total correlation spectroscopy (1H-1H TOCSY). Parallel LC/ion-trap mass spectrometry yielded the confirmatory glucuronide masses. Moreover, "dynamic" stopped-flow HPLC/1H NMR experiments revealed transacylation of the isolated alpha-1-O-acyl isomer to a mixture of alpha/beta-2-O-acyl isomers; the reverse reaction from the isolated alpha/beta-2-O-acyl isomers to the alpha-1-O-acyl isomer was also clearly demonstrated. This application of "dynamic" stopped-flow HPLC/1H NMR allows key kinetic data to be obtained on a reactive metabolite that would otherwise be difficult to follow by conventional HPLC and NMR methods where sample preparation and off-line separations are necessary. These data challenge the widely held view that the alpha-1-O-acyl isomers of drug ester Glucuronides do not occur under physiological conditions. Furthermore, the similar formation of alpha-1-O-acyl isomers from zomepirac and diflunisal beta-1-O-acyl Glucuronides has recently been confirmed (Corcoran et al., unpublished results). Such reactions are also likely to be widespread for other drugs that form ester Glucuronides in biological systems. Ultimately, the presence of significant quantities of the kinetically labile alpha-1-O-acyl glucuronide isomer may also have toxicological implications in terms of reactivity toward cellular proteins.

  • 750 mhz hplc nmr spectroscopic studies on the separation and characterization of the positional isomers of the Glucuronides of 6 11 dihydro 11 oxodibenz b e oxepin 2 acetic acid
    Analytical Chemistry, 1996
    Co-Authors: Ulla G Sidelmann, Ian D Wilson, John C Lindon, Eva M Lenz, Manfred Spraul, Martin Hofmann, J Troke, Paul N Sanderson, Jeremy K Nicholson
    Abstract:

    Ester Glucuronides (beta-1-O-acyl-D-glucopyranuronates) of many drugs can undergo a series of acyl migration reactions, resulting in positional isomers and anomers which can react with serum proteins with possible toxicological consequences. We have investigated the acyl migration of the ester Glucuronides of the model drug 6,-11-dihydro-11-oxodibenz[b,e]oxepin-2-acetic acid in pH 7.4 buffer using directly coupled 750 MHz stopped-flow HPLC-NMR spectroscopy. Using a reversed phase isocratic HPLC method with 21% acetonitrile and 79% D2O in the mobile phase, it was possible to separate and hence identify the individual positional isomers of the model drug glucuronide by 750 MHz HPLC-NMR. The order of elution of the isomers from the C18 column was 4alpha-, 4beta-, aglycon, 1beta-, 3beta-, 3alpha-, 2alpha-, 2beta- (alpha- and beta- referring to the anomerization state at C1 on the glucuronide ring and the numbers referring to the carbon number on the glucuronide ring to which the drug moiety has migrated). It is shown that directly coupled ultra-high-field HPLC-NMR spectroscopy offers a unique analytical advantage for obtaining structural information of interconverting compounds in equilibrium mixtures, and this method will be of value in the study of reactive drug Glucuronides of toxicological importance.