Kinetic and inhibitor studies of 4-methylumbelliferone and 1-naphthol glucuronidation in human liver microsomes

Miners, John O.; Lillywhite, K. J.; Matthews, A. P.; Jones, Michael E.; Birkett, Donald

doi:10.1016/0006-2952(88)90140-2

Cited by 55 publications

(30 citation statements)

References 23 publications

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“…UGT1A6 is likely to be functionally orthologous among several species including humans, rats, mice, and rabbits (Iyanagi et al, 1986;Harding et al, 1988;Lamb et al, 1994). The 4-MU glucuronidation in human liver microsomes followed the Michaelis-Menten kinetics (Miners et al, 1988) and was catalyzed by several human UGTs (Uchaipichat et al, 2004). The different kinetic models between humans and rodents might be due to the different UGT enzymes.…”

Section: Discussionmentioning

confidence: 99%

Species Differences in UDP-Glucuronosyltransferase Activities in Mice and Rats

Shiratani

Katoh²,

Nakajima³

et al. 2008

Drug Metab Dispos

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ABSTRACT:UDP-glucuronosyltransferases (UGTs), expressed in various tissues including liver and intestine, catalyze phase II metabolic biotransformation. There is little information on species differences between mice and rats in UGT activities, especially in intestine. The purpose of the present study was to clarify the species differences between mice and rats in UGT activities using duodenal and liver microsomes. For estradiol 3-O-glucuronidation in duodenal microsomes, the kinetic data in mice were fit to the Hill equation. However, the Hill coefficient was low in rats (n ‫؍‬ 1.1), suggesting that rat estradiol 3-O-glucuronidation followed the Michaelis-Menten equation rather than the Hill equation. For 4-nitrophenol (4-NP) O-glucuronidation, the K m values were different between mice and rats. The intrinsic clearance (CL int ) values for mycophenolic acid (MPA) O-and morphine 3-O-glucuronidation in male mouse duodenum were 3-and 17-fold lower than those in rat, respectively. In male liver, the CL int values for 4-NP O-, propofol O-, MPA O-, and morphine 3-O-glucuronidation and the CL max value for 4-methylumbelliferone O-glucuronidation in mice were higher than those in rats. The CL max value for estradiol 3-O-glucuronidation in mice was lower than that in rats. Also, there were strain differences among C57BL/6J, BALB/c, C3H/HeJ, DBA/2, ddY, and ICR mice in UGT activities in duodenum. We clarified that the species differences in UGT activity evaluated by the CL int or CL max values in liver and duodenum varied according to the substrate.

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Section: Discussionmentioning

confidence: 99%

Species Differences in UDP-Glucuronosyltransferase Activities in Mice and Rats

Shiratani

Katoh²,

Nakajima³

et al. 2008

Drug Metab Dispos

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“…4MU and 1NP glucuronidation were measured using a previously published procedure (Miners et al, 1988). Briefly, incubations (total volume 0.6 ml) contained UDPGA (5 mM), MgCl 2 (5 mM), cell lysate (protein concentrations given in Table 1), 4MU or 1NP, and phosphate buffer (0.1 M, pH 7.4).…”

Section: Methodsmentioning

confidence: 99%

“…4MU and 1NP have been promoted in this regard since both form fluorescent glucuronides that are readily measurable (Bock et al, 1983;Miners et al, 1988). 4MU and 1NP are known to be metabolized by multiple human UGT isoforms, and hence these compounds represent convenient probes for comparing the glucuronidation activity and kinetics of tissues and recombinant enzymes.…”

mentioning

confidence: 99%

Human Udp-Glucuronosyltransferases: Isoform Selectivity and Kinetics of 4-Methylumbelliferone and 1-Naphthol Glucuronidation, Effects of Organic Solvents, and Inhibition by Diclofenac and Probenecid

et al. 2004

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ABSTRACT:The glucuronidation kinetics of the prototypic substrates 4-methylumbelliferone (4MU) and 1-naphthol (1NP) by human UDP-glucuronosyltransferases (UGT) 1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B7, 2B15, and 2B17 were investigated. Where activity was demonstrated, inhibitory effects of diclofenac, probenecid, and the solvents acetone, acetonitrile, dimethyl sulfoxide, ethanol, and methanol were characterized. All isoforms except UGT1A4 glucuronidated 4MU, whereas all but UGT 1A4, 2B15, and 2B17 metabolized 1NP. However, kinetic models varied with substrate (for the same isoform) and from isoform to isoform (with the same substrate). Hyperbolic (Michaelis-Menten), substrate inhibition, and sigmoidal kinetics were variably observed for both 4MU and 1NP glucuronidation by the various UGTs. K m or S 50 (sigmoidal kinetics) and V max values varied 525-(8-4204 M) and 5535-fold, respectively, for 4MU glucuronidation, and 1360-(1.3-1768 M) and 560-fold, respectively, for 1NP glucuronidation. The use of a two-site model proved useful for those reactions exhibiting non-MichaelisMenten glucuronidation kinetics. The organic solvents generally had a relatively minor effect on UGT isoform activity. UGT 1A6, 2B15, and 2B17 were most susceptible to the presence of solvent, although solvent-selective inhibition was occasionally observed with other isoforms. Diclofenac and probenecid inhibited all isoforms, precluding the use of these compounds for the reaction phenotyping of xenobiotic glucuronidation pathways in human tissues. Diclofenac and probenecid K i values, determined for selected isoforms, ranged from 11 to 60 M and 96 to 2790 M, respectively. Overall, the results emphasize the need for the careful design and interpretation of kinetic and inhibition studies with human UGTs.

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“…For all enzymes, except UGT2B4 and UGT2B28, the conversion of 4-MU to 4-MUG was measured according to the fluorometric procedure of Miners et al (1988), as modified by Uchaipichat et al (2004). The concentration of 4-MU present in incubations corresponded to the known K m (or S 50 ) for each enzyme (Sorich et al, 2002;Uchaipichat et al, 2004).…”

Section: Methodsmentioning

confidence: 99%

The Glucuronidation of Δ⁴-3-Keto C19- and C21-Hydroxysteroids by Human Liver Microsomal and Recombinant UDP-glucuronosyltransferases (UGTs): 6α- and 21-Hydroxyprogesterone Are Selective Substrates for UGT2B7

Bowalgaha¹,

Elliot²,

Mackenzie³

et al. 2006

Drug Metab Dispos

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ABSTRACT:The stereo-and regioselective glucuronidation of 10 ⌬ 4 -3-keto monohydroxylated androgens and pregnanes was investigated to identify UDP-glucuronosyltransferase (UGT) enzyme-selective substrates. Kinetic studies were performed using human liver microsomes (HLMs) and a panel of 12 recombinant human UGTs as the enzyme sources. Five of the steroids, which were hydroxylated in the 6␤-, 7␣-, 11␤-or 17␣-positions, were not glucuronidated by HLMs. Of the remaining compounds, comparative kinetic and inhibition studies indicated that 6␣-and 21-hydroxyprogesterone (OHP) were glucuronidated selectively by human liver microsomal UGT2B7. 6␣-OHP glucuronidation by HLMs and UGT2B7 followed Michaelis-Menten kinetics, whereas 21-OHP glucuronidation by these enzyme sources exhibited positive cooperativity. UGT2B7 was also identified as the enzyme responsible for the high-affinity component of human liver microsomal 11␣-OHP glucuronidation. In contrast, UGT2B15 and UGT2B17 were the major forms involved in human liver microsomal testosterone 17␤-glucuronidation and the high-affinity component of 16␣-OHP glucuronidation. Activity of UGT1A subfamily enzymes toward the hepatically glucuronidated substrates was generally low, although UGT1A4 and UGT1A9 contribute to the low-affinity components of microsomal 16␣-and 11␣-OHP glucuronidation, respectively. Interestingly, UGT1A10, which is expressed only in the gastrointestinal tract, exhibited activity toward most of the glucuronidated substrates.The results indicate that 6␣-and 21-OHP may be used as selective "probes" for human liver microsomal UGT2B7 activity and, taken together, provide insights into the regio-and stereoselectivity of hydroxysteroid glucuronidation by human UGTs.

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Kinetic and inhibitor studies of 4-methylumbelliferone and 1-naphthol glucuronidation in human liver microsomes

Cited by 55 publications

References 23 publications

Species Differences in UDP-Glucuronosyltransferase Activities in Mice and Rats

Species Differences in UDP-Glucuronosyltransferase Activities in Mice and Rats

Human Udp-Glucuronosyltransferases: Isoform Selectivity and Kinetics of 4-Methylumbelliferone and 1-Naphthol Glucuronidation, Effects of Organic Solvents, and Inhibition by Diclofenac and Probenecid

The Glucuronidation of Δ⁴-3-Keto C19- and C21-Hydroxysteroids by Human Liver Microsomal and Recombinant UDP-glucuronosyltransferases (UGTs): 6α- and 21-Hydroxyprogesterone Are Selective Substrates for UGT2B7

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Kinetic and inhibitor studies of 4-methylumbelliferone and 1-naphthol glucuronidation in human liver microsomes

Cited by 55 publications

References 23 publications

Species Differences in UDP-Glucuronosyltransferase Activities in Mice and Rats

Species Differences in UDP-Glucuronosyltransferase Activities in Mice and Rats

Human Udp-Glucuronosyltransferases: Isoform Selectivity and Kinetics of 4-Methylumbelliferone and 1-Naphthol Glucuronidation, Effects of Organic Solvents, and Inhibition by Diclofenac and Probenecid

The Glucuronidation of Δ4-3-Keto C19- and C21-Hydroxysteroids by Human Liver Microsomal and Recombinant UDP-glucuronosyltransferases (UGTs): 6α- and 21-Hydroxyprogesterone Are Selective Substrates for UGT2B7

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The Glucuronidation of Δ⁴-3-Keto C19- and C21-Hydroxysteroids by Human Liver Microsomal and Recombinant UDP-glucuronosyltransferases (UGTs): 6α- and 21-Hydroxyprogesterone Are Selective Substrates for UGT2B7