Glutathione Peroxidase Activity Assay with Colorimetric Method and Microplate Reading Format and Comparison with Chemiluminescence Method

Glutathione peroxidase (GPX) has a key role in the protection of organisms from oxidative damage. Many diseases and disorders are associated with changes in GPX activity. Therefore, its activity assay can be crucial in prevention, diagnosis and treatment of them. Several companies produce GPX activity assay kit but it is being imported to Iran which is expensive and time-consuming. This research has been done to investigate a simple, rapid and inexpensive method for GPX activity assay. In this study, GPX reduced cumene hydroperoxide while oxidized glutathione (GSH) to GSSG. The generated GSSG was reduced to GSH with consumption of NADPH by glutathione reductase. The decrease of NADPH absorption which was proportional to GPX activity measured at 340 nm with microplate reading format. Sensitivity, precision and accuracy have been examined. The results obtained by the colorimetric method compared with chemiluminescence method and correlation coefficient has been determined. Sensitivity of this method was 15 mU/ml. The coefficient of variation percent for intra and inter assay was less than 9.7 %. According to parallelism and recovery tests, ratio % and recovery %  ranged from 91% to 112% and correlation coefficient between the two methods was 0.9898 (n=60). Data from this study showed that the method has an acceptable sensitivity, precision and accuracy and can be suitable for both clinical and research studies. Indeed, this study is the first step of domestic commercial kit production purpose

Total Antioxidant Capacity Determination with Colorimetric Method and Microplate Reading Format and Comparison with Chemilumin Essence Assay

Total antioxidant capacity (TAC) is related to a variety of molecules that protect biological systems against reactive oxygen species (ROS). The importance of total antioxidants results in the requirement of TAC assay methods. This study is done to design a simple, rapid and inexpensive method for TAC measurement. In this method, ABTS (2, 2′-azino-bis [3-ethylbenzthiazoline-6-sulfonic acid]) was incubated with met myoglobin and H2O2 to produce green radical cation ABTS+. Antioxidants were suppressed the color production, thereby reducing the intensity of the color that was proportional to the concentration of antioxidants, measured at 600nm with a microplate reading format. Trolox was served as a standard or control antioxidant. Sensitivity, precision and accuracy were examined to check the validity of the method. Sensitivity of this method was 0.10 U/ml. The coefficient of variation percent for intra and inter assay was less than 9.6%. According to parallelism and recovery tests, ratio% and recovery% ranged from 90.9-108% and correlation coefficient in comparison with the sensitive chemiluminescence method was 0.9897 (n=60). Data from this study showed that this method has a good sensitivity, precision and accuracy and can be suitable for clinical and research uses

Glutathione Peroxidase Activity Assay with Colorimetric Method and Microplate Reading Format and Comparison with Chemiluminescence Method

Glutathione peroxidase (GPX) has a key role in the protection of organisms from oxidative damage. Many diseases and disorders are associated with changes in GPX activity. Therefore, its activity assay can be crucial in prevention, diagnosis and treatment of them. Several companies produce GPX activity assay kit but it is being imported to Iran which is expensive and time-consuming. This research has been done to investigate a simple, rapid and inexpensive method for GPX activity assay. In this study, GPX reduced cumene hydroperoxide while oxidized glutathione (GSH) to GSSG. The generated GSSG was reduced to GSH with consumption of NADPH by glutathione reductase. The decrease of NADPH absorption which was proportional to GPX activity measured at 340 nm with microplate reading format. Sensitivity, precision and accuracy have been examined. The results obtained by the colorimetric method compared with chemiluminescence method and correlation coefficient has been determined. Sensitivity of this method was 15 mU/ml. The coefficient of variation percent for intra and inter assay was less than 9.7 %. According to parallelism and recovery tests, ratio % and recovery %  ranged from 91% to 112% and correlation coefficient between the two methods was 0.9898 (n=60). Data from this study showed that the method has an acceptable sensitivity, precision and accuracy and can be suitable for both clinical and research studies. Indeed, this study is the first step of domestic commercial kit production purpose