title: Increasing the throughput and productivity of Caco-2 cell permeability assays using liquid chromatography-mass spectrometry: application to resveratrol absorption and metabolism.
abstract: The Caco-2 cell monolayer permeability assay has become a standard model of human intestinal absorption and transport. This paper reviews recent progress in increasing the throughput of Caco-2 cell monolayer assays and in expanding the scope of this assay to include modeling intestinal drug metabolism. The state-of-the-art in Caco-2 cell monolayer permeability assays combines multi-well plates fitted with semi-permeable inserts on which Caco-2 cells have been cultured with liquid chromatography-mass spectrometry (LC-MS) or LC-tandem mass spectrometry (LC-MS-MS) for the quantitative analysis of test compounds and the identification of their intestinal metabolites. After reviewing the progress in increasing the throughput of Caco-2 cell monolayer assays for both modeling human intestinal permeability or transport and the metabolism of xenobiotic compounds, we demonstrate the application of LC-MS and LC-MS-MS to the measurement of resveratrol permeability and metabolism in the Caco-2 model. trans-Resveratrol (trans-3,5,4'-trihydroxystilbene) is a polyphenolic compound occurring in grapes, peanuts and other food sources, that is under investigation as a cancer chemoprevention agent. The apparent permeability coefficient for apical (AP) to basolateral (BL) movement of resveratrol was 2.0 x 10(-5)cm/sec. Resveratrol was not a substrate for P-glycoprotein or the multi-drug resistance associated proteins (MRP). No phase I metabolites were observed, but the phase II conjugates resveratrol-3-glucuronide and resveratrol-3-sulfate was identified based on LC-MS and LC-MS-MS analysis and comparison with synthetic standards. Although these data indicate that resveratrol diffuses rapidly across the intestinal epithelium, extensive phase II metabolism during absorption might reduce resveratrol bioavailability.
authors.affiliation: Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, 833 S. Wood St., Chicago, IL 60612, USA.
authors.forename: Yongmei
authors.initials: Y
authors.lastname: Li
authors.forename: Young Geun
authors.initials: YG
authors.lastname: Shin
authors.forename: Chongwoo
authors.initials: C
authors.lastname: Yu
authors.forename: Jerome W
authors.initials: JW
authors.lastname: Kosmeder
authors.forename: Wendy H
authors.initials: WH
authors.lastname: Hirschelman
authors.forename: John M
authors.initials: JM
authors.lastname: Pezzuto
authors.forename: Richard B
authors.initials: RB
authors.lastname: van Breemen
mesh_terms: D018938:Caco-2 Cells / Q000378:metabolism*; D002463:Cell Membrane Permeability; D002853:Chromatography, Liquid / Q000379:methods; D006801:Humans; D007408:Intestinal Absorption; D013058:Mass Spectrometry / Q000379:methods; D000077185:Resveratrol; D013267:Stilbenes / Q000378:metabolism / Q000493:pharmacokinetics*
chemical_list: D013267:Stilbenes; D000077185:Resveratrol