Abstract
Caffeine (1,3,7-trimethylxanthine) has previously been shown to undergo metabolic switching in vivo when the N-1 or the N-7 methyl groups were trideuteromethylated [Horning et al. (1976) Proceedings of the Second International Conference on Stable Isotopes, pp 41–54]. We have examined the effect of replacing the N-3 methyl group with a trideuteromethyl group. The corresponding isotope effects can then be used to distinguish the kinetic mechanism by which four primary metabolites can be formed from one substrate by one cytochrome P450 (P450). We have synthesized 3-CD3-caffeine and 3-CD3-7-CD3-caffeine as well as trideuteromethylated analogs of each of the in vitro metabolites formed by cytochrome P4501A2. The observed competitive isotope effects for the metabolites, which do not result from deuterium abstraction (theobromine, theophylline), demonstrate that the nondissociative mechanism applies to caffeine metabolism by cytochrome P4501A2. Thus, there must be equilibration of the kinetically distinguishable activated P450-substrate complexes at rates competitive with hydrogen abstraction. The true isotope effects for the N-3 demethylation of caffeine were derived from the ratios of the amount of paraxanthine relative to the amount of theobromine or theophylline. The resultant ratios indicate that these isotope effects are essentially intrinsic. Observation of the isotope effects on N-3 demethylation was facilitated by branching to the minor in vitro metabolites as well as water formation. Product release is not rate-limiting for this system.
Footnotes
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This research was supported by National Institute of Health Grants R01 GM25418 and P01 GM32165 (S.D.N. and K.L.K.), the University of Washington-National Institute of Environmental Health Sciences (NIEHS)-sponsored Center for Ecogenetics and Environmental Health: NIEHS P30ES07033, National Research Award in Pharmacological Sciences GM07750, and a Hope Barnes Fellowship (K.A.R.).
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.105.006031.
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ABBREVIATIONS: PX, paraxanthine or 1,7-dimethylxanthine; TB, theobromine or 3,7-dimethylxanthine; TP, theophylline or 1,3-dimethylxanthine; TMU, 1,3,7-trimethyluric acid; P450, cytochrome P450; d0, commercially available d0 compounds (2H0, nondeuterated); d3, trideuteromethyl functionality (CD3; 2H3); d6, two trideuteromethyl functionalities (2H6); EOS, the active oxygen intermediate of cytochrome P450 complexed to substrate; ES, P450-substrate complex at the beginning of the catalytic cycle; GC-MS, gas chromatography-mass spectrometry; HPLC, high-performance liquid chromatography; DMF, dimethylformamide; HL103, human liver microsomes high in CYP1A2 content; Dk, kH/kD or the intrinsic isotope effect; P, primary isotope effect; PS2, product of one primary and two secondary isotope effects; S, secondary isotope effect; DV, (Vmax)H/(Vmax)D; DV/K, (Vmax/Km)H/(Vmax/Km)D.
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↵1 Current affiliation: Amgen, Thousand Oaks, California.
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↵2 Current affiliation: AstraZeneca, Department of Drug Metabolism and Pharmacokinetics, Macclesfield, United Kingdom.
- Received June 14, 2005.
- Accepted August 31, 2005.
- The American Society for Pharmacology and Experimental Therapeutics
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