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Simultaneous measurement of reactive oxygen species and reduced glutathione using capillary electrophoresis and laser‐induced fluorescence detection in cultured cell lines
Author(s) -
Parmentier Christine,
Wellman Maria,
Nicolas Alain,
Siest Gérard,
Leroy Pierre
Publication year - 1999
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/(sici)1522-2683(19991001)20:14<2938::aid-elps2938>3.0.co;2-m
Subject(s) - glutathione , reactive oxygen species , oxidative stress , chemistry , capillary electrophoresis , intracellular , fluorescence , rhodamine 123 , biophysics , chromatography , biochemistry , biology , physics , quantum mechanics , multiple drug resistance , enzyme , antibiotics
A capillary zone electrophoretic (CZE) method coupled with laser‐induced fluorescence (LIF) was developed for the simultaneous determination of two important intracellular parameters related to oxidative stress ( i.e. reactive oxygen species, ROS, and reduced glutathione, GSH). This rapid and sensitive method was applied to the study of oxidative stress in cultured V79 fibroblast cells. The fluorogenic reagents selected were: (i) dihydrorhodamine‐123 (DHR‐123) which is converted intracellularly by ROS to the fluorescent rhodamine‐123 dye (Rh‐123), and (ii) naphthalene‐2,3‐dicarboxaldehyde (NDA), which reacts quickly with GSH in cell extracts to produce a fluorescent adduct. Separation of Rh‐123, GSH‐NDA and γ‐glutamylcysteine‐NDA adducts was performed using an uncoated fused‐silica capillary and a 100 m M borate buffer, pH 9.2, at 20°C and at an applied voltage of 25 kV; LIF detection was operated using an argon laser. The cell line was also tested for its ability to alleviate oxidative stress induced by tert ‐butylhydroperoxide ( t ‐BuOOH). Exposure to t ‐BuOOH (up to 3 m M for 2 h) did not affect the intracellular ROS and GSH concentrations. At higher (4—10 m M ) t ‐BuOOH concentrations, an inverse relationship between the concentrations of ROS and GSH was obtained, showing that the present method can readily evaluate the gradual consumption of the primary cellular scavenger of ROS which occurs simultaneously with the increase of oxidative insult.