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 5.0 ng/mL for GSH and 1.0 ng/mL for GSSG with lower limits of detection at 0.5 ng/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.
Original language | English |
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Pages (from-to) | 85-94 |
Number of pages | 10 |
Journal | Free Radical Biology and Medicine |
Volume | 97 |
DOIs | |
State | Published - Aug 1 2016 |
Bibliographical note
Publisher Copyright:© 2016 Elsevier Inc. All rights reserved.
Funding
This work is supported by NIH training grant T32 ES007266 , the Edward P. Evans Foundation , and the the NCI Cancer Center Support Grant P30 CA177558 .
Funders | Funder number |
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National Institutes of Health (NIH) | |
National Childhood Cancer Registry – National Cancer Institute | P30 CA177558 |
National Institutes of Health/National Institute of Environmental Health Sciences | T32ES007266 |
Edward P Evans Foundation |
Keywords
- Glutathione
- HSC's
- LC-MS/MS
- Method validation
ASJC Scopus subject areas
- Biochemistry
- Physiology (medical)