Analytical and Bioanalytical Chemistry

, Volume 409, Issue 19, pp 4529–4538 | Cite as

Microchip electrophoresis with laser-induced fluorescence detection for the determination of the ratio of nitric oxide to superoxide production in macrophages during inflammation

  • Giuseppe Caruso
  • Claudia G. Fresta
  • Joseph M. Siegel
  • Manjula B. Wijesinghe
  • Susan M. Lunte
Paper in Forefront

Abstract

It is well known that excessive production of reactive oxygen and nitrogen species is linked to the development of oxidative stress-driven disorders. In particular, nitric oxide (NO) and superoxide (O2•−) play critical roles in many physiological and pathological processes. This article reports the use of 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate and MitoSOX Red in conjunction with microchip electrophoresis and laser-induced fluorescence detection for the simultaneous detection of NO and O2•− in RAW 264.7 macrophage cell lysates following different stimulation procedures. Cell stimulations were performed in the presence and absence of cytosolic (diethyldithiocarbamate) and mitochondrial (2-methoxyestradiol) superoxide dismutase (SOD) inhibitors. The NO/O2•− ratios in macrophage cell lysates under physiological and proinflammatory conditions were determined. The NO/O2•− ratios were 0.60 ± 0.07 for unstimulated cells pretreated with SOD inhibitors, 1.08 ± 0.06 for unstimulated cells in the absence of SOD inhibitors, and 3.14 ± 0.13 for stimulated cells. The effect of carnosine (antioxidant) or Ca2+ (intracellular messenger) on the NO/O2•− ratio was also investigated.

Graphical Abstract

Simultaneous detection of nitric oxide and superoxide in macrophage cell lysates

Keywords

Bioanalytical methods Inflammation Macrophages Microchip electrophoresis Nitric oxide Superoxide 

Supplementary material

216_2017_401_MOESM1_ESM.pdf (211 kb)
ESM 1(PDF 211 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  • Giuseppe Caruso
    • 1
    • 2
  • Claudia G. Fresta
    • 1
    • 2
  • Joseph M. Siegel
    • 1
    • 3
  • Manjula B. Wijesinghe
    • 1
    • 3
  • Susan M. Lunte
    • 1
    • 2
    • 3
  1. 1.Ralph N. Adams Institute for Bioanalytical ChemistryUniversity of KansasLawrenceUSA
  2. 2.Department of Pharmaceutical ChemistryUniversity of KansasLawrenceUSA
  3. 3.Department of ChemistryUniversity of KansasLawrenceUSA

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