Simultaneous determination of intracellular reduced and oxidized glutathiones by the König reaction.

The König reaction is commonly used for the detection of cyanide and its derivatives, including thiocyanate and selenocyanate. We found that this reaction can be used to quantify glutathione fluorometrically, and applied it to the simultaneous determination of reduced and oxidized glutathiones (GSH and GSSG) using a conventional LC system with isocratic elution. The limits of detection were 6.04 nM and 9.84 nM for GSH and GSSG, respectively, and the limits of quantification were 18.3 nM and 29.8 nM, respectively. We also determined GSH and GSSG levels in PC12 cells exposed to paraquat, an oxidative stressor, and observed a decrease in GSH/GSSG ratio, as expected. Total GSH levels quantified by this method and by the conventional colorimetric method with 5,5'-dithiobis(2-nitrobenzoic acid) were comparable. Our new application of the König reaction offers a reliable and useful method to simultaneously quantify intracellular GSH and GSSG.