Measurement of Reduced Glutathione Using High-Pressure Liquid Chromatography

As outlined in Chapter 8 , glutathione in the intact cell is maintained predominantly in its reduced form by the cytosolic enzyme, glutathione reductase. Cell lysis can lead to rapid oxidation to the oxidized form, GSSG, and degradation by γ-glutamyl transpeptidase. In order to obtain a true measurement of the amount of reduced glutathione (GSH) in living cells we have utilized the method of Cotgreave and Moldeus ( 1 ) in which GSH is derivatized using monobromobimane (MBBr), which can freely cross the cell membrane (Fig. 1 ). The GSH-MBBr adduct is then extracted and the amount formed measured by high-performance liquid chromatography (HPLC) with fluorescence detection.   Fig. 1.  Reaction of monobromobimane with glutathione.