Monochlorobimane fluorometric method to measure tissue glutathione

Glutathione (GSH) is the principal intracellular low-molecular-weight thiol and plays a critical role in the cellular defense against agents that impo se oxidative stress. A common technique to measure GSH uses reversed-phase high-performance liquid chromatography (HPLC) following derivatization with 5,5'-dithiobis(2-nitrobenzoic acid), a technique, although reliable and se nsitive, that is time consuming and laborious. A common technique to measur e GSH in cultured cells is to add monochlorobimane to the culture medium wh ere it readily enters cells to form a fluorescent GSH-monochlorobinane addu ct that can be measured fluorometrically. This reaction is catalyzed by glu tathione S-transferase. We reasoned that adding glutathione S-transferase a nd monochlorobimane to tissue homogenates would allow a rapid reliable meth od to measure GSH. The accuracy of the new test was assessed in homogenates of rat livers. One-half of each homogenate was assayed for GSH using a HPL C approach while the other half was assayed using the monochlorobimane appr oach. The two methods were found to give identical results. We conclude tha t the monochlorobimane fluorescent method is sufficiently specific to relia bly measure tissue GSH (C) 2000 Academic Press.