title: An ultra performance liquid chromatographic method for determining phytosterol uptake by Caco-2 cells.
abstract: A simple method for the determination of cellular uptake of phytosterols by Caco-2 cells has been developed by ultra performance liquid chromatography with ultraviolet detection (UPLC-UV). UPLC-UV was established using an ODS column, acetonitrile/H(2)O (9:1, v/v) as a mobile phase, and a detection wavelength at 210 nm. As analytes, β-sitosterol, campesterol, stigmasterol, and brassicasterol were selected based on the abundance in foods and the similarity of their structures. A linear relation was observed between the peak area and the amount of sterol injected from 50 to 2000 pmol (r>0.999) with a relative standard deviation (RSD) of less than 2.5% (n=6). This method was applied to the determination of cellular uptake of phytosterols by Caco-2 cells. Recovery tests showed that phytosterols were extracted from the cell lysates by chloroform and determined by UPLC-UV with a recovery rate of more than 80.2% and an RSD of less than 11.3% (n=3). When Caco-2 cells were incubated with phytosterols at 37°C, their uptake was increased with time in a concentration-dependent manner. This method will be useful for the simultaneous determination of cellular phytosterols in an in vitro intestine model.
authors: Department of Analytical Chemistry, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo 192-0392, Japan.
authors: Nanako
authors: N
authors: Ito
authors: Takaaki
authors: T
authors: Ohtsubo
authors: Fumiyo
authors: F
authors: Kusu
authors: Hideki
authors: H
authors: Hakamata
mesh_terms: D001693:Biological Transport, Active; D018938:Caco-2 Cells; D002775:Cholestadienols / Q000032:analysis / Q000378:metabolism; D002784:Cholesterol / Q000031:analogs & derivatives / Q000032:analysis / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D007700:Kinetics; D010840:Phytosterols / Q000032:analysis* / Q000378:metabolism*; D012855:Sitosterols / Q000032:analysis / Q000378:metabolism; D013265:Stigmasterol / Q000032:analysis / Q000378:metabolism
chemical_list: D002775:Cholestadienols; D010840:Phytosterols; D012855:Sitosterols; C003532:brassicasterol; C021273:campesterol; C025473:gamma-sitosterol; D002784:Cholesterol; D013265:Stigmasterol