Potent Method for the Simultaneous Determination of Glutathione and Hydrogen Peroxide in Mitochondrial Compartments of Apoptotic Cells with Microchip Electrophoresis-Laser Induced Fluorescence

摘要

The first application of microchip electrophoresis with laser-induced fluorescence (MCE-LIF) detection to simultaneously determine glutathione (GSH) and hydrogen peroxide (H_(2)O_(2)) in mitochondria was described. Organoselenium probe Rh-Se-2 and bis(p-methylbenzenesulfonate)dichlorofluorescein (FS) synthesized in our laboratory were utilized as fluorescent probes for GSH and H_(2)O_(2), respectively. Rh-Se-2, which is nonfluorescent, reacts with GSH to produce rhodamine 110 (Rh110) with high quantum yield. Similarly, nonfluorescent FS reacts with H_(2)O_(2) and produces dichlorofluorescein (DCF) accompanied by drastic fluorescence enhancement Both probes exhibit good sensitivity toward their respective target molecule determination. Fast, simple, and sensitive determination of GSH and H_(2)O_(2) was realized within 37 s using a running buffer of 50 mM mannitol, 40 mM HEPES (pH 7.4), and an electric field of 360 V/cm for separation. The linear ranges of the method were 3.3 X 10~(-9)-1.0 X 10~(-7) M/2.9 X 10~(-7)-1.0 X 10~(-4) M and 2.7 X 10~(-9)-4.0 X 10~(-7) M with detection limits (signal-to-noise ratio velence 3) of 1.3 nM (0.16 amol) and 1.0 nM (0.12 amol) for GSH and H_(2)O_(2), respectively. The relative standard deviations (RSDs) of migration time and peak area were less than 1.0percent and 4.0percent, respectively. The MCE-LIF assay was utilized to investigate the levels of GSH and H_(2)O_(2) in mitochondria isolated from HepG2 cells and were found to be 2.01 +- 0.21 mM and 5.36 +-0.45 (mu)M, respectively. The method was further extended to observe situations of the two species in mitochondria of HepG2 cells experiencing cell apoptosis that were induced by doxorubicin and photodynamic therapy (PDT).