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 impose 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 sensitive, that is time consuming and laborious. A common technique to measure GSH in cultured cells is to add monochlorobimane to the culture medium where it readily enters cells to form a fluorescent GSH-monochlorobimane adduct that can be measured fluorometrically. This reaction is catalyzed by glutathione S-transferase. We reasoned that adding glutathione S-transferase and monochlorobimane to tissue homogenates would allow a rapid reliable method 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 HPLC approach while the other half was assayed using the monochlorobimane approach. The two methods were found to give identical results. We conclude that the monochlorobimane fluorescent method is sufficiently specific to reliably measure tissue GSH.
Kamencic
H
H
Department of Anatomy & Cell Biology, College of Pharmacy & Nutrition, Saskatoon, Saskatchewan, S7N 5E5, Canada.
Lyon
A
A
Paterson
P G
PG
Juurlink
B H
BH
D000818:Animals; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D005260:Female; D005978:Glutathione / Q000032:analysis*; D005982:Glutathione Transferase / Q000737:chemistry / Q000494:pharmacology; D008099:Liver / Q000378:metabolism; D011720:Pyrazoles / Q000737:chemistry* / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods*
D011720:Pyrazoles; C059597:monochlorobimane; D005982:Glutathione Transferase; D005978:Glutathione