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    Microchip Electrophoresis with Amperometric Detection Method for Profiling Cellular Nitrosative Stress Markers

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    Issue Date
    2015-07-07
    Author
    Gunasekara, Dulan B.
    Siegel, Joseph Michael
    Caruso, Giuseppe
    Hulvey, Matthew K.
    Lunte, Susan M.
    Publisher
    Royal Society of Chemistry
    Type
    Article
    Article Version
    Scholarly/refereed, author accepted manuscript
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    Abstract
    The overproduction of nitric oxide (NO) in cells results in nitrosative stress due to the generation of highly reactive species such as peroxynitrite and N2O3. These species disrupt the cellular redox processes through the oxidation, nitration, and nitrosylation of important biomolecules. Microchip electrophoresis (ME) is a fast separation method that can be used to profile cellular nitrosative stress through the separation of NO and nitrite from other redox-active intracellular components such as cellular antioxidants. This paper describes a ME method with electrochemical detection (ME-EC) for the separation of intracellular nitrosative stress markers in macrophage cells. The separation of nitrite, azide (interference), iodide (internal standard), tyrosine, glutathione, and hydrogen peroxide (neutral marker) was achieved in under 40 s using a run buffer consisting of 7.5 to 10 mM NaCl, 10 mM boric acid, and 2 mM TTAC at pH 10.3 to 10.7. Initially, NO production was monitored by the detection of nitrite (NO2−) in cell lysates. There was a 2.5- to 4-fold increase in NO2− production in lipopolysaccharide (LPS)-stimulated cells. The concentration of NO2− inside a single unstimulated macrophage cell was estimatedto be 1.41 mM using the method of standard additions. ME-EC was then used for the direct detection of NO and glutathione in stimulated and native macrophage cell lysates. NO was identified in these studies based on its migration time and rapid degradation kinetics. The intracellular levels of glutathione in native and stimulated macrophages were also compared, and no significant difference was observed between the two conditions.
    URI
    http://hdl.handle.net/1808/22625
    DOI
    https://doi.org/10.1039/c4an00185k
    Collections
    • Chemistry Scholarly Works [553]
    Citation
    Gunasekara, Dulan B., Joseph M. Siegel, Giuseppe Caruso, Matthew K. Hulvey, and Susan M. Lunte. "Microchip Electrophoresis with Amperometric Detection Method for Profiling Cellular Nitrosative Stress Markers." The Analyst 139.13 (2014): 3265-273.

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    Contact KU ScholarWorks
    785-864-8983
    KU Libraries
    1425 Jayhawk Blvd
    Lawrence, KS 66045
    785-864-8983

    KU Libraries
    1425 Jayhawk Blvd
    Lawrence, KS 66045
    Image Credits
     

     

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