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Simultaneous analysis of oxidized and reduced glutathione in cell extracts by capillary zone electrophoresis
Author(s) -
Yang Qing,
Krautmacher Carsten,
Schilling David,
Pittelkow Mark R.,
Naylor Stephen
Publication year - 2002
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.129
Subject(s) - chemistry , glutathione , capillary electrophoresis , chromatography , glutathione disulfide , quantitative analysis (chemistry) , detection limit , acetic acid , phosphoric acid , thiol , biochemistry , enzyme , organic chemistry
Glutathione (GSH) and glutathione disulfide (GSSG) levels in cells constitute a thiol redox system. They can be used as an indicator of oxidative stress of the cell. In this study, a capillary zone electrophoresis (CZE) method is described that enables quantitation of GSH and GSSG from cellular extracts. The CZE buffer used was 20 m M ammonium acetate containing 5% (v/v) acetic acid at pH 3.1 in conjunction with a polybrene coated capillary operated in reverse polarity mode. Effects of different acids used to prepare cell samples were investigated on CZE performance. The acids include meta phosphoric acid (MPA), trichloroacetic acid (TCA), phosphoric acid (PA) and sulfosalicylic acid (SSA) and are used to stabilize GSH and GSSG before performing CZE analysis. The method features a limit of detection of 4 µ M and a limit of quantitation of 12 µ M for both GSSG and GSH and recoveries of 94% for GSH and 100% for GSSG. Quantitative analysis of GSSG and GSH in HaCaT cell extracts (5% SSA, w/v) was performed with this method and changes in the ratio of GSH to GSSG in N‐ethylmaleimide treated cell sample was observed by comparing with control cell samples. Copyright © 2002 John Wiley & Sons, Ltd.