title: Simultaneous quantitation of oxidized and reduced glutathione via LC-MS/MS: An insight into the redox state of hematopoietic stem cells.
abstract: Cellular redox balance plays a significant role in the regulation of hematopoietic stem-progenitor cell (HSC/MPP) self-renewal and differentiation. Unregulated changes in cellular redox homeostasis are associated with the onset of most hematological disorders. However, accurate measurement of the redox state in stem cells is difficult because of the scarcity of HSC/MPPs. Glutathione (GSH) constitutes the most abundant pool of cellular antioxidants. Thus, GSH metabolism may play a critical role in hematological disease onset and progression. A major limitation to studying GSH metabolism in HSC/MPPs has been the inability to measure quantitatively GSH concentrations in small numbers of HSC/MPPs. Current methods used to measure GSH levels not only rely on large numbers of cells, but also rely on the chemical/structural modification or enzymatic recycling of GSH and therefore are likely to measure only total glutathione content accurately. Here, we describe the validation of a sensitive method used for the direct and simultaneous quantitation of both oxidized and reduced GSH via liquid chromatography followed by tandem mass spectrometry (LC-MS/MS) in HSC/MPPs isolated from bone marrow. The lower limit of quantitation (LLOQ) was determined to be 5.0ng/mL for GSH and 1.0ng/mL for GSSG with lower limits of detection at 0.5ng/mL for both glutathione species. Standard addition analysis utilizing mouse bone marrow shows that this method is both sensitive and accurate with reproducible analyte recovery. This method combines a simple extraction with a platform for the high-throughput analysis, allows for efficient determination of GSH/GSSG concentrations within the HSC/MPP populations in mouse, chemotherapeutic treatment conditions within cell culture, and human normal/leukemia patient samples. The data implicate the importance of the modulation of GSH/GSSG redox couple in stem cells related diseases.
authors.affiliation: Department of Toxicology and Cancer Biology, University of Kentucky, College of Medicine, Lexington, KY, United States.
authors.forename: Dustin
authors.initials: D
authors.lastname: Carroll
authors.affiliation: Hematology and Oncology Comprehensive Cancer Center, Wake Forest Baptist Medical Center, Wake Forest University, Winston-Salem, NC, United States.
authors.forename: Diana
authors.initials: D
authors.lastname: Howard
authors.affiliation: Department of Molecular and Cellular Biochemistry, University of Kentucky, College of Medicine, Lexington, KY, United States.
authors.forename: Haining
authors.initials: H
authors.lastname: Zhu
authors.affiliation: Department of Chemistry, Eastern Kentucky University, Richmond, KY, United States.
authors.forename: Christian M
authors.initials: CM
authors.lastname: Paumi
authors.affiliation: Department of Toxicology and Cancer Biology, University of Kentucky, College of Medicine, Lexington, KY, United States.
authors.forename: Mary
authors.initials: M
authors.lastname: Vore
authors.affiliation: Department of Microbiology and Molecular Genetics, University of Kentucky, College of Medicine, Lexington, KY, United States.
authors.forename: Subbarao
authors.initials: S
authors.lastname: Bondada
authors.affiliation: Department of Toxicology and Cancer Biology, University of Kentucky, College of Medicine, Lexington, KY, United States.
authors.forename: Ying
authors.initials: Y
authors.lastname: Liang
authors.affiliation: Division of Cancer Biostatistics, Markey Cancer Center, University of Kentucky, College of Medicine, Lexington, KY, United States.
authors.forename: Chi
authors.initials: C
authors.lastname: Wang
authors.affiliation: Department of Toxicology and Cancer Biology, University of Kentucky, College of Medicine, Lexington, KY, United States.
authors.forename: Daret K
authors.initials: DK
authors.lastname: St Clair
mesh_terms: D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods*; D005978:Glutathione / Q000302:isolation & purification* / Q000378:metabolism; D019803:Glutathione Disulfide / Q000302:isolation & purification* / Q000378:metabolism; D006412:Hematopoietic Stem Cells / Q000378:metabolism; D006801:Humans; D061986:MCF-7 Cells; D051379:Mice; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D005978:Glutathione; D019803:Glutathione Disulfide
keywords: Glutathione; HSC’s; LC-MS/MS; Method validation