PT  - JOURNAL ARTICLE
AU  - Andrew Rowland
AU  - David J. Elliot
AU  - Kathleen M. Knights
AU  - Peter I. Mackenzie
AU  - John O. Miners
TI  - The “Albumin Effect” and in Vitro-in Vivo Extrapolation: Sequestration of Long-Chain Unsaturated Fatty Acids Enhances Phenytoin Hydroxylation by Human Liver Microsomal and Recombinant Cytochrome P450 2C9
AID  - 10.1124/dmd.107.019885
DP  - 2008 May 01
TA  - Drug Metabolism and Disposition
PG  - 870--877
VI  - 36
IP  - 5
4099  - http://dmd.aspetjournals.org/content/36/5/870.short
4100  - http://dmd.aspetjournals.org/content/36/5/870.full
SO  - Drug Metab Dispos2008 May 01; 36
AB  - 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. The American Society for Pharmacology and Experimental Therapeutics