PT - JOURNAL ARTICLE AU - Schaefer, Olaf AU - Ohtsuki, Sumio AU - Kawakami, Hirotaka AU - Inoue, Tae AU - Liehner, Stephanie AU - Saito, Asami AU - Sakamoto, Atsushi AU - Ishiguro, Naoki AU - Matsumaru, Takehisa AU - Terasaki, Tetsuya AU - Ebner, Thomas TI - Absolute Quantification and Differential Expression of Drug Transporters, Cytochrome P450 Enzymes, and UDP-Glucuronosyltransferases in Cultured Primary Human Hepatocytes AID - 10.1124/dmd.111.042275 DP - 2012 Jan 01 TA - Drug Metabolism and Disposition PG - 93--103 VI - 40 IP - 1 4099 - http://dmd.aspetjournals.org/content/40/1/93.short 4100 - http://dmd.aspetjournals.org/content/40/1/93.full SO - Drug Metab Dispos2012 Jan 01; 40 AB - The levels of metabolizing enzymes and transporters expressed in hepatocytes are decisive factors for hepatobiliary disposition of most drugs. Induction via nuclear receptor activation can significantly alter those levels, with the coregulation of multiple enzymes and transporters occurring to different extents. Here, we report the use of a targeted liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) method for concurrent quantification of multiple cytochrome P450 (P450), UDP-glucuronosyltransferase (UGT), and transporter proteins in cultured primary human hepatocytes. The effects of culture format (i.e., sandwich culture versus conventional culture) and of dexamethasone (DEX) media concentrations on mRNA, protein, and activity levels were determined for three donors, and protein expression was compared with that in liver. In general, P450 and UGT expression was lower in hepatocyte cultures than that in liver, and CYP2C9 was found to be the most abundant P450 isoform expressed in cultured hepatocytes. The sandwich culture format and 0.1 μM DEX in media retained the protein expression in the hepatocytes closest to the levels found in liver. However, higher in vitro expression was observed for drug transporters, especially for multidrug resistance protein 1 and breast cancer resistance protein. Direct protein quantification was applied successfully to study in vitro induction in sandwich cultured primary hepatocytes in a 24-well format using the prototypical inducers rifampicin, omeprazole, and phenobarbital. We conclude that targeted absolute LC-MS/MS quantification of drug-metabolizing enzymes and transporters can broaden the scope and significantly increase the impact of in vitro drug metabolism studies, such as induction, as an important supplement or future alternative to mRNA and activity data.