Determination of Reactive Oxygen Species in Isolated Erythrocytes

Determination of reactive oxygen species (ROS) in erythrocytes have been extensively studied due to its important pathophysiological role in many diseases. Also, studies on the potential of many antioxidants compounds are important to improve human healthy. This paper describes a simple and fast method to quantify ROS in erythrocytes with induction of oxidation and antioxidant protection. Tert-butyl hydroperoxide (tBOOH)-induced ROS was measured in erythrocytes isolated from 60 healthy adults between 21 and 39 years old, using microplates methods in which 2,7-dichlorofluorescein (DCF) was produced. Fluorescence was monitored at 530 nm after excitation at 495 nm. Data points were taken after 15 and 30 min of incubation at room temperature and at 37°C. Ascorbic acid was used to evaluate the protection of an antioxidant compound. Results were analyzed using the Statistica 10.0 (StatSoft). No outlier was identified and all parameters were normally distributed. Data were expressed as mean ± standard deviation and one-way analysis of variance (ANOVA) and Tukey test were performed. Results were considered statistically significant when p<0.05. Different concentrations of t-BOOH solutions induced ROS generation with significant differences in all concentrations. Time and temperature interfere in the method that has to be well standardized. Dose-dependent protection of ascorbic acid on ROS production was observed. ROS-induced oxidative stress in erythrocytes and antioxidant protection can be determinate using an easy and fast method, with little blood volume under microplate methods.