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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

Department of Clinical Pharmacology, Flinders University and Flinders Medical Centre, Bedford Park, Adelaide, Australia

This study characterized the mechanism by which bovine serum albumin (BSA) reduces the K_m 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 K_m 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 V_max. In contrast, crude human serum albumin increased the K_m 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 K_m for PHY hydroxylation by CYP2C9, without affecting V_max. This effect was reversed by addition of BSA to incubations. K_i 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 (CL_int) values for each enzyme source determined in the presence of BSA and HSA-FAF accurately predicted the known CL_int for PHY hydroxylation in vivo. The results indicate that previously determined in vitro K_m values for CYP2C9 substrates are almost certainly overestimates, and accurate in vitro-in vivo extrapolation of kinetic data for CYP2C9 substrates is achievable.

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				K. K. Wolf, K. R. Brouwer, G. M. Pollack, and K. L. R. Brouwer Effect of Albumin on the Biliary Clearance of Compounds in Sandwich-Cultured Rat Hepatocytes Drug Metab. Dispos., October 1, 2008; 36(10): 2086 - 2092. [Abstract] [Full Text] [PDF]