Superoxide, a naturally produced reactive oxygen species (ROS) in the human body, is involved in many pathological and physiological signaling processes. However, if superoxide formation is left unregulated, overproduction can lead to oxidative damage to important biomolecules, such as DNA, lipids, and proteins. Superoxide can also lead to the formation of peroxynitrite, an extremely hazardous substance, through its reaction with endogenously produced nitric oxide. Despite its importance, quantitative information regarding superoxide production is difficult to obtain due to its high reactivity and low concentrations in vivo. MitoHE, a fluorescent probe that specifically reacts with superoxide, was used in conjunction with microchip electrophoresis (ME) and laser-induced fluorescence (LIF) detection to investigate changes in superoxide production by RAW 264.7 macrophage cells following stimulation with phorbol 12-myristate 13-acetate (PMA). Stimulation was performed in the presence and absence of the superoxide dismutase (SOD) inhibitors, diethyldithiocarbamate (DDC) and 2-metoxyestradiol (2-ME). The addition of these inhibitors resulted in an increase in the amount of superoxide specific product (2-OH-MitoE+) from 0.08 ± 0.01 fmol (0.17 ± 0.03 mM) in native cells to 1.26 ± 0.06 fmol (2.5 ± 0.1 mM) after PMA treatment. This corresponds to an approximately 15-fold increase in intracellular concentration per cell. Furthermore, the addition of 3-morpholino-sydnonimine (SIN-1) to the cells during incubation resulted in the production of 0.061 ± 0.006 fmol (0.12 ± 0.01 mM) of 2-OH-MitoE+ per cell on average. These results demonstrate that indirect superoxide detection coupled with the use of SOD inhibitors and a separation method is a viable method to discriminate the 2-OH-MitoE+ signal from possible interferences.
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This work was performed with financial support from National Science Foundation (CHE-1411993), National Institutes of Health (R01NS042929 and COBRE P20GM103638), Fundação de Amparo à Pesquisa do Estado de Sāo Paulo, Conselho Nacional de Desenvolvimento Científico e Tecnológico(CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, and National Institute of Science and Technology on Bioanalysis (INCTBio – Brazil). R.P.S.C. received support from the CNPq scholarship through the Science Without Borders program. J.M.S. was supported by a Madison and Lila Self Graduate Fellowship. We would also like to thank Ryan Grigsby for help with microchip fabrication and Nancy Harmony for editorial support.
Published in the topical collection Capillary Electrophoresis of Biomolecules with guest editor Lisa Holland.
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de Campos, R.P.S., Siegel, J.M., Fresta, C.G. et al. Indirect detection of superoxide in RAW 264.7 macrophage cells using microchip electrophoresis coupled to laser-induced fluorescence. Anal Bioanal Chem 407, 7003–7012 (2015). https://doi.org/10.1007/s00216-015-8865-1
- Bioanalytical methods
- Microchip electrophoresis