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

Abstract

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 = 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 1.0% and 4.0%, 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 microM, 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).