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Research ArticleArticle

TRANS-3′-HYDROXYCOTININE O- AND N-GLUCURONIDATIONS IN HUMAN LIVER MICROSOMES

Hiroyuki Yamanaka, Miki Nakajima, Miki Katoh, Ayano Kanoh, Osamu Tamura, Hiroyuki Ishibashi and Tsuyoshi Yokoi
Drug Metabolism and Disposition January 2005, 33 (1) 23-30; DOI: https://doi.org/10.1124/dmd.104.001701
Hiroyuki Yamanaka
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Miki Nakajima
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Miki Katoh
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Ayano Kanoh
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Osamu Tamura
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Hiroyuki Ishibashi
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Tsuyoshi Yokoi
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Abstract

Trans-3′-hydroxycotinine is a major metabolite of nicotine in humans and is mainly excreted as O-glucuronide in smoker's urine. Incubation of human liver microsomes with UDP-glucuronic acid produces not only trans-3′-hydroxycotinine O-glucuronide but also N-glucuronide. The formation of N-glucuronide exceeds the formation of O-glucuronide in most human liver microsomes, although N-glucuronide has never been detected in human urine. Trans-3′-hydroxycotinine N-glucuronidation in human liver microsomes was significantly correlated with nicotine and cotinine N-glucuronidations, which are catalyzed mainly by UDP-glucuronosyltransferase (UGT)1A4 and was inhibited by imipramine and nicotine, which are substrates of UGT1A4. Recombinant UGT1A4 exhibited substantial trans-3′-hydroxycotinine N-glucuronosyltransferase activity. These results suggest that trans-3′-hydroxycotinine N-glucuronidation in human liver microsomes would be mainly catalyzed by UGT1A4. In the present study, trans-3′-hydroxycotinine O-glucuronidation in human liver microsomes was thoroughly characterized, since trans-3′-hydroxycotinine O-glucuronide is one of the major metabolites of nicotine. The kinetics were fitted to the Michaelis-Menten equation with a Km of 10.0 ± 0.8 mM and a Vmax of 85.8 ± 3.8 pmol/min/mg. Among 11 recombinant human UGT isoforms expressed in baculovirus-infected insect cells, UGT2B7 exhibited the highest trans-3′-hydroxycotinine O-glucuronosyltransferase activity (1.1 pmol/min/mg) followed by UGT1A9 (0.3 pmol/min/mg), UGT2B15 (0.2 pmol/min/mg), and UGT2B4 (0.2 pmol/min/mg) at a substrate concentration of 1 mM. Trans-3′-hydroxycotinine O-glucuronosyltransferase activity by recombinant UGT2B7 increased with an increase in the substrate concentration up to 16 mM (10.5 pmol/min/mg). The kinetics by recombinant UGT1A9 were fitted to the Michaelis-Menten equation with Km = 1.6 ± 0.1 mM and Vmax = 0.69 ± 0.02 pmol/min/mg of protein. Trans-3′-hydroxycotinine O-glucuronosyltransferase activities in 13 human liver microsomes ranged from 2.4 to 12.6 pmol/min/mg and were significantly correlated with valproic acid glucuronidation (r = 0.716, p < 0.01), which is catalyzed by UGT2B7, UGT1A6, and UGT1A9. Trans-3′-hydroxycotinine O-glucuronosyltransferase activity in human liver microsomes was inhibited by imipramine (a substrate of UGT1A4, IC50 = 55 μM), androstanediol (a substrate of UGT2B15, IC50 = 169 μM), and propofol (a substrate of UGT1A9, IC50 = 296 μM). Interestingly, imipramine (IC50 = 45 μM), androstanediol (IC50 = 21 μM), and propofol (IC50 = 41 μM) also inhibited trans-3′-hydroxycotinine O-glucuronosyltransferase activity by recombinant UGT2B7. These findings suggested that trans-3′-hydroxycotinine O-glucuronidation in human liver microsomes is catalyzed by mainly UGT2B7 and, to a minor extent, by UGT1A9.

Footnotes

  • This study was supported by an SRF grant for Biomedical Research in Japan and by Philip Morris Incorporated.

  • doi:10.1124/dmd.104.001701.

  • ABBREVIATIONS: UGT, UDP-glucuronosyltransferase; UDPGA, UDP-glucuronic acid; LC-MS/MS, liquid chromatography-mass/mass spectrometry; m/z, mass/charge.

    • Received July 31, 2004.
    • Accepted October 4, 2004.
  • The American Society for Pharmacology and Experimental Therapeutics
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Drug Metabolism and Disposition: 33 (1)
Drug Metabolism and Disposition
Vol. 33, Issue 1
1 Jan 2005
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Research ArticleArticle

TRANS-3′-HYDROXYCOTININE O- AND N-GLUCURONIDATIONS IN HUMAN LIVER MICROSOMES

Hiroyuki Yamanaka, Miki Nakajima, Miki Katoh, Ayano Kanoh, Osamu Tamura, Hiroyuki Ishibashi and Tsuyoshi Yokoi
Drug Metabolism and Disposition January 1, 2005, 33 (1) 23-30; DOI: https://doi.org/10.1124/dmd.104.001701

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Research ArticleArticle

TRANS-3′-HYDROXYCOTININE O- AND N-GLUCURONIDATIONS IN HUMAN LIVER MICROSOMES

Hiroyuki Yamanaka, Miki Nakajima, Miki Katoh, Ayano Kanoh, Osamu Tamura, Hiroyuki Ishibashi and Tsuyoshi Yokoi
Drug Metabolism and Disposition January 1, 2005, 33 (1) 23-30; DOI: https://doi.org/10.1124/dmd.104.001701
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