Abstract
This study characterized the mechanism by which bovine serum albumin (BSA) reduces the Km for phenytoin (PHY) hydroxylation and the implications of the “albumin effect” for in vitro-in vivo extrapolation of kinetic data for CYP2C9 substrates. BSA and essentially fatty acid-free human serum albumin (HSA-FAF) reduced the Km values for PHY hydroxylation (based on unbound substrate concentration) by human liver microsomes (HLMs) and recombinant CYP2C9 by approximately 75%, with only a minor effect on Vmax. In contrast, crude human serum albumin increased the Km with both enzyme sources. Mass spectrometric analysis of incubations containing HLMs was consistent with the hypothesis that BSA sequesters long-chain unsaturated acids (arachidonic, linoleic, oleic) released from membranes. A mixture of arachidonic, linoleic and oleic acids, at a concentration corresponding to 1/20 of the content of HLMs, doubled the Km for PHY hydroxylation by CYP2C9, without affecting Vmax. This effect was reversed by addition of BSA to incubations. Ki values for arachidonic acid inhibition of human liver microsomal- and CYP2C9-catalyzed PHY hydroxylation were 3.8 and 1.6 μM, respectively. Similar effects were observed with heptadecanoic acid, the most abundant long-chain unsaturated acid present in Escherichia coli membranes. Extrapolation of intrinsic clearance (CLint) values for each enzyme source determined in the presence of BSA and HSA-FAF accurately predicted the known CLint for PHY hydroxylation in vivo. The results indicate that previously determined in vitro Km values for CYP2C9 substrates are almost certainly overestimates, and accurate in vitro-in vivo extrapolation of kinetic data for CYP2C9 substrates is achievable.
Footnotes
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This study was supported by a grant from the National Health and Medical Research Council of Australia. A.R. is the recipient of a Flinders University Research Scholarship.
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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.107.019885.
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ABBREVIATIONS: CLint, intrinsic clearance; CLH, hepatic clearance; HLM, human liver microsome; IV-IVE, in vitro-in vivo extrapolation; BSA, bovine serum albumin; PHY, phenytoin; HSA-FAF, essentially fatty acid-free human serum albumin; HPPH, hydroxy-phenytoin; TBA, 5-ethyl-5-p-tolylbarbituric acid; HSA, human serum albumin; MS, mass spectrometry; HPLC, high-performance liquid chromatography; OxR, NADPH cytochrome P450 oxidoreductase; r, rat; P450, cytochrome P450; AUC, area under the plasma concentration-time curve.
- Received November 25, 2007.
- Accepted February 5, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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