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

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 (O-2 (aEuro cent a')) 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 O-2 (aEuro cent a') 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/O-2 (aEuro cent a') ratios in macrophage cell lysates under physiological and proinflammatory conditions were determined. The NO/O-2 (aEuro cent a') 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/O-2 (aEuro cent a') ratio was also investigated.