Abstract

Introduction

Materials & Methods

Results

Discussion & Conclusion

References

Discussion
Board

GSH is the principal intracellular low-molecular weight thiol and plays a critical role in the cellular defence of agents that impose oxidative stress. It acts as an electron donor for glutathione peroxidase and can also scavenge reactive oxigen species directly. A common technique in measuring GSH is derivatization with 5,5`-dithiobis-(2-nitrobenzoic acid) [DTNB], using reversed-phase high performance liquid chromatography (HPLC) to separate the derivates followed by ultraviolet detection. This technique although very reliable and sensitive is laborious. A technique to measure GSH in cultured cells is to add monochlorobimane (mCB) to the culture medium where it readily enters cells to form a fluorescent GSH-mCB adduct that can be measured with a fluorometer. This reaction is catalyzed by glutathione transferase. We reasoned that adding glutathione transferase and mCB to cell homogenates ought to, in principle, allow a rapid reliable method to measure GSH. To test this we prepared homogenates from rat livers. One-half of each homogenate was assayed for GSH using HPLC approach while the other half was assayed using the mCB approach. The two methods were found to give identical results. We conclude that the mCB method to measure GSH is as sensitive and reliable as the HPLC method.

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Poster Number PAkamencic0562
Keywords: oxidative stress, glutathione, hplc, monochlorobimane, assay