title: Application of exogenous mixture of glutathione and stable isotope labeled glutathione for trapping reactive metabolites in cryopreserved human hepatocytes. Detection of the glutathione conjugates using high resolution accurate mass spectrometry.
abstract: Metabolites (including reactive metabolites) of troglitazone were generated by incubation with cryopreserved human hepatocytes and trapped in the presence of an exogenous mixture of unlabeled and stable isotope labeled (SIL: [1,2-(13)C, (15)N]-glycine) glutathione (GSH/SIL-GSH). The incubation samples were analyzed using liquid chromatography-high resolution accurate mass spectrometry (LC-HRAMS) implemented on a LTQ Orbitrap mass spectrometer. The GSH conjugates of the reactive metabolites were detected via a characteristic mono-isotopic pattern (peaks separated by 3.0037u). Analysis of the incubation samples led to detection of a number of previously described GSH conjugates, as well as two novel methylated GSH conjugates, which were partially characterized based on accurate mass measurements and MS/MS data. The addition of exogenous GSH led to an increase in the apparent level of detected GSH conjugates. Kinetic isotopic measurements showed that the rates of incorporation of exogenous GSH are conjugate-specific. In conclusion, this approach, based on the use of a mixture of GSH/SIL-GSH, allows facile capture and detection of reactive metabolites in human hepatocytes. Moreover, the data suggest that routine addition of glutathione to the assay medium may be advisable for experiments with cryopreserved hepatocytes.
authors.affiliation: Absorption Systems, Exton, PA 19341, USA. imezine@absorption.com
authors.forename: Igor
authors.initials: I
authors.lastname: Mezine
authors.forename: Chris
authors.initials: C
authors.lastname: Bode
authors.forename: Bethany
authors.initials: B
authors.lastname: Raughley
authors.forename: Sid
authors.initials: S
authors.lastname: Bhoopathy
authors.forename: Kenneth J
authors.initials: KJ
authors.lastname: Roberts
authors.forename: Albert J
authors.initials: AJ
authors.lastname: Owen
authors.forename: Ismael J
authors.initials: IJ
authors.lastname: Hidalgo
mesh_terms: D002853:Chromatography, Liquid; D015925:Cryopreservation; D005978:Glutathione / Q000737:chemistry* / Q000378:metabolism; D022781:Hepatocytes / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007553:Isotope Labeling; D015394:Molecular Structure; D053719:Tandem Mass Spectrometry
chemical_list: D005978:Glutathione
keywords: Glutathione conjugation; Human hepatocytes; LC–HRAMS; Reactive metabolites; Stable isotope label; Troglitazone