Abstract
In humans, orally administered phenytoin, 5,5-diphenylhydantoin, is mainly excreted as 5-(4′-hydroxyphenyl)-5-phenylhydantoin (4′-HPPH)O-glucuronide. Phenytoin is oxidized to 4′-HPPH by CYP2C9 and to a minor extent by CYP2C19, and then 4′-HPPH is metabolized to 4′-HPPH O-glucuronide by UDP-glucuronosyltransferase (UGT). In the present study, 4′-HPPHO-glucuronidation in human liver microsomes was investigated. The metabolite formed by incubation with human liver microsomes, 4′-HPPH, and UDP-glucuronic acid was identified as 4′-HPPHO-glucuronide by liquid chromatography-tandem mass spectrometry analysis. The 4′-HPPHO-glucuronosyltransferase activity in human liver microsomes was not saturated at concentrations up to 500 μM of 4′-HPPH. Any commercially available recombinant human UGTs (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, UGT2B7, and UGT2B15) expressed in baculovirus-infected insect cells did not show detectable 4′-HPPHO-glucuronide. The 4′-HPPHO-glucuronidation in pooled human liver microsomes was inhibited by β-estradiol as a typical substrate for UGT1A1 (IC50 = 21.1 μM) and imipramine as a typical substrate for UGT1A4 (IC50 = 57.7 μM). The inhibitory effects of propofol as a specific substrate for UGT1A9 (IC50 = 167.1 μM) and emodin as a substrate for UGT1A8 and UGT1A10 (IC50 = 287.6 μM) were not prominent. The interindividual difference in the 4′-HPPHO-glucuronidation in 14 human liver microsomes was 28.5-fold (0.023–0.656 nmol/min/mg of protein). The 4′-HPPHO-glucuronosyltransferase activity in 11 human liver microsomes was significantly (r = 0.609,P < 0.05) correlated with the 4-nitrophenol glucuronosyltransferase activity, which is catalyzed by UGT1A6 and UGT1A9. These results suggest that multiple UGT1As such as UGT1A1, UGT1A4, UGT1A6, and UGT1A9 are involved in 4′-HPPHO-glucuronidation in human liver microsomes, although the percentage contribution of each UGT1A could not be estimated. Large interindividual differences in the glucuronidation of 4′-HPPH might be responsible for the nonlinearity of the phenytoin plasma concentration or adverse reactions in humans.
Footnotes
- Abbreviations used are::
- 4′-HPPH
- 5-(4′-hydroxyphenyl)-5-phenylhydantoin
- UGT
- UDP-glucuronosyltransferase
- HPLC
- high performance liquid chromatography
- LC-MS/MS
- liquid chromatography-tandem mass spectrometry
- ESI
- electrospray ionization
- Received June 13, 2002.
- Accepted August 12, 2002.
- The American Society for Pharmacology and Experimental Therapeutics
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