Measurement of Oxidized Glutathione Level in Rat Plasma Exposed to Paraquat Using High Performance Liquid Chromatography Method

Paraquat which is used as herbicide, decreases reduced glutathione (GSH) and increases oxidized glutathione (GSSG) via oxidative stress pathway and formation of ROS (Reactive Oxygen Species). High performance liquid chromatography (HPLC) with fluorescence detector is one of the best methods for assessment of glutathione in samples like plasma which contains low level of this substance for evaluation of many of diseases. The purpose of this study was to evaluate the changes of glutathione quantity due to effect of paraquat in rat plasma with the use of HPLC.

In this study the fluorescence substance 9-fluorenylmethylchloroformate(FMOC) was used for derivatization of amine group of glutathione. To do this, the rat obtained plasma, which had been exposed with four doses of paraquat (20, 30, 40, and 60 mg/kg), was mixed with acetonitryl and centrifuged at 5000 rpm for protein precipitation. The supernatant was completely dehydrated in a 40oC water bath under air flow with an air pump. 506l tap water, 50 6lborate buffer 0.5M, pH=9 and 100 6l FMOC 500 6g/ml were added and then was left at room temperature for 15 min for completion of derivatization. After that, 206l of this solution was injected into aHPLC with C18 column, mobile phase methanol: phosphate buffer (60:40 v/v) and ex=260nm, em=315 nm.

According to the obtained results, the amount of oxidized glutathione was increased significantly only in the dose of 60 mg/kg in comparison with the control group (p)