Abstract
Background: To determine glutathione (reduced form: GSH, oxidized form: GSSG), which is known as an important biological antioxidant, the colorimetrical method utilizing DTNB is widely used. However, the method does not give us sufficient sensitivity, especially for the determination of GSSG, and the simultaneous detection of GSH and GSSG. Although LC-MS can analyze GSH and GSSG simultaneously with high-sensitivity, the access to LC-MS is limited due to an economical reason. We recently found that glutathione can be detected fluorometrically by using the König reaction, which is usually utilized for the detection of cyanide. In this study, we evaluated whether the technique can be applicable to the simultaneous determination of intracellular GSH and GSSG.
Methods: GSH and GSSG were separated by our HPLC system, and reacted with 0.03% chloramine T and pyridine-barbituric acid reagents to form fluorescent compounds on-line. The fluorescent compounds were detected at 583 nm (excitation) and 607 nm (emission). As an application of the technique to a biological sample, the change of GSH/GSSG ratios in rat pheochromocytoma cells, PC12, exposed to 250 or 500 μM paraquat for 24 h were evaluated.
Results & Discussion: Limits of quantification of the technique were 18.3 nM for GSH and 33.9 nM for GSSG. This indicated that our developing method was more sensitive than some LC-MS methods. Good linearity (R2>0.999) was obtained in ranges of 0.05-50 and 0.05-10 μM for GSH and GSSG, respectively. Precision and accuracy were 2.39-10.9% and 84.8-96.5%, respectively. The decrease in the GSH/GSSG ratio in PC12 cells was observed with the increase of paraquat concentration.
