title: Indirect detection of superoxide in RAW 264.7 macrophage cells using microchip electrophoresis coupled to laser-induced fluorescence.
abstract: 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.
authors.affiliation: Ralph N. Adams Institute for Bioanalytical Chemistry, University of Kansas, 2030 Becker Dr., Lawrence, KS, 66047-1620, USA.
authors.forename: Richard P S
authors.initials: RP
authors.lastname: de Campos
authors.forename: Joseph M
authors.initials: JM
authors.lastname: Siegel
authors.forename: Claudia G
authors.initials: CG
authors.lastname: Fresta
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Caruso
authors.forename: José A F
authors.initials: JA
authors.lastname: da Silva
authors.forename: Susan M
authors.initials: SM
authors.lastname: Lunte
mesh_terms: D000818:Animals; D015374:Biosensing Techniques / Q000295:instrumentation*; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D046209:Electrophoresis, Microchip / Q000295:instrumentation*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D005456:Fluorescent Dyes / Q000138:chemical synthesis; D007834:Lasers; D008657:Metabolic Clearance Rate / Q000187:drug effects / Q000502:physiology; D051379:Mice; D008856:Microscopy, Fluorescence / Q000295:instrumentation*; D000067996:RAW 264.7 Cells; D017382:Reactive Oxygen Species / Q000378:metabolism; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000295:instrumentation*; D013481:Superoxides / Q000378:metabolism*; D018511:Systems Integration; D013755:Tetradecanoylphorbol Acetate / Q000031:analogs & derivatives / Q000494:pharmacology
chemical_list: D005456:Fluorescent Dyes; D017382:Reactive Oxygen Species; D013481:Superoxides; C020922:4-O-methyl-12-O-tetradecanoylphorbol 13-acetate; D013755:Tetradecanoylphorbol Acetate