Effect of Anticoagulants and Storage of Coupling Reagent on the Activity Assay of Extracellular Glutathione Peroxidase in Human Plasma

Abstract

Extracellular glutathione peroxidase (eGPx) is an antioxidant enzyme present in extracellular space by reducing hydrogen peroxide and lipid hydroperoxide. Changes of its level were associated with some disease conditions. In this study, we described the details of GPx activity assay for human plasma and compared activities of eGpx in EDTA plasma, heparin plasma and serum from eight healthy volunteers. We also investigated effect of freezing the coupling reagent for one to five weeks on activities of eGpx. The imprecision of assay was below 5% and the enzymatic reaction rate was linear in the range of 0.01-0.03 of absorbance change. Results also showed that eGPx activities were significantly higher in heparin plasma but not different between serum and EDTA plasma. Moreover, the coupling reagent could be stored at -80℃, but not at -20℃, for at least five weeks without affecting results of eGPx activities. Taken together, we have established detailed assay procedures for analysis of eGPx activity in plasma with low imprecision and easy preparation of reagent without the need of using commercial kits.