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An optimized method for determination of intracellular glutathione in mouse macrophage cultures by fluorimetric high‐performance liquid chromatography
Author(s) -
Komatsu Hiroyoshi,
Obata Fumiya
Publication year - 2003
Publication title -
biomedical chromatography
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.4
H-Index - 65
eISSN - 1099-0801
pISSN - 0269-3879
DOI - 10.1002/bmc.250
Subject(s) - glutathione , chemistry , derivatization , intracellular , chromatography , autoxidation , dansyl chloride , extraction (chemistry) , high performance liquid chromatography , chloride , enzyme , biochemistry , organic chemistry
We have optimized a method for the determination of intracellular glutathione by high‐performance liquid chromatography, using fluorimetric detection. To minimize artifacts and provide an accurate determination of intracellular glutathione, cell extracts were prepared using extraction conditions specifically designed to inhibit autoxidation and enzymatic degradation of glutathione. The sensitivity of the method was enhanced by adjusting the dansyl chloride derivatization reaction with regard to parameters such as pH, reaction time and dansyl chloride concentration. Both oxidized and reduced forms of glutathione were quantified using the refined method in extracts of oxidatively stressed J774A.1 mouse macrophage cells and reflected an expected shift in cellular redox status. Copyright © 2003 John Wiley & Sons, Ltd.