Simultaneous and ultrarapid determination of reactive oxygen species and reduced glutathione in apoptotic leukemia cells by microchip electrophoresis.
A microchip electrophoresis method coupled with laser-induced fluorescence (LIF) detection was established for simultaneous determination of two kinds of intracellular signaling molecules (reactive oxygen species, ROS, and reduced glutathione, GSH) related to apoptosis and oxidative stress. As the probe dihydrorhodamine-123 (DHR-123) can be converted intracellularly by ROS to the fluorescent rhodamine-123 (Rh-123), and the probe naphthalene-2,3-dicarboxaldehyde (NDA) can react quickly with GSH to produce a fluorescent adduct, rapid determination of Rh-123 and GSH was achieved on a glass microchip within 27 s using a 20 mM borate buffer (pH 9.2). The established method was tested to measure the intracellular ROS and GSH levels in acute promyelocytic leukemia (APL)-derived NB4 cells. An elevation of intracellular ROS and depletion of GSH were observed in apoptotic NB4 cells induced by arsenic trioxide (As(2)O(3)) at low concentration (1-2 microM). Buthionine sulfoximine (BSO), in combination with As(2)O(3) enhanced the decrease of reduced GSH to a great extent. The combined treatment of As(2)O(3) and hydrogen peroxide (H(2)O(2)) led to an inverse relationship between the concentrations of ROS and GSH obtained, showing the proposed method can readily evaluate the generation of ROS, which occurs simultaneously with the consumption of the inherent antioxidant.
Qin
Jianhua
J
Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China.
Ye
Nannan
N
Yu
Linfen
L
Liu
Dayu
D
Fung
Yingsing
Y
Wang
Wei
W
Ma
Xiaojun
X
Lin
Bingcheng
B
D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D000077237:Arsenic Trioxide; D001152:Arsenicals / Q000627:therapeutic use; D019328:Buthionine Sulfoximine / Q000627:therapeutic use; D046209:Electrophoresis, Microchip / Q000379:methods*; D005456:Fluorescent Dyes; D005978:Glutathione / Q000032:analysis*; D006801:Humans; D007834:Lasers; D015473:Leukemia, Promyelocytic, Acute / Q000188:drug therapy / Q000378:metabolism*; D008904:Miniaturization; D010087:Oxides / Q000627:therapeutic use; D017382:Reactive Oxygen Species / Q000032:analysis*; D015203:Reproducibility of Results; D012235:Rhodamines; D015398:Signal Transduction / Q000502:physiology; D013050:Spectrometry, Fluorescence; D014407:Tumor Cells, Cultured
D001152:Arsenicals; D005456:Fluorescent Dyes; D010087:Oxides; D017382:Reactive Oxygen Species; D012235:Rhodamines; C058319:dihydrorhodamine 123; D019328:Buthionine Sulfoximine; D005978:Glutathione; D000077237:Arsenic Trioxide