Electron paramagnetic resonance (EPR) recording at “standard” 20 mW underestimates reactive oxygen species (ROS) in fresh rat blood using CPH and CMH as spin traps

Abstract

Use of spin traps during EPR measurements has enabled the quantitative estimate of [ROS], in both in vitro and ex vivo samples. As spin trap oxidation rate (OR) is proportional to [ROS], repeated measures over time are necessary to establish OR. Reliable [ROS] estimates require that at least two conditions are met during the measurement period: [ROS] should remain constant; and sample conditions, particularly temperature, should not change. To meet the first condition the measurement period must be as short as possible, so [ROS] can be considered near-constant. The second condition requires recording parameters that do not alter the sample itself during measurements. In this study, we optimized our EPR techniques to meet these requirements. Results To facilitate the measurement of [ROS] in a rat model of hemorrhage, serial heparinized blood samples were taken and immediately mixed with spin traps (CPH or CMH). The optimized timing was a 10 min incubation with OR measurements made from minutes 4 to 6. Using longer or shorter time windows resulted in underestimation of [ROS]. Most EPR investigations in vitro apply 20 mW power, which likely causes sample heating during EPR irradiation. Microwave power was very critical: using >2 mW power overestimated [ROS] while the typical 20 mW power destroyed the blood samples and recorded [ROS] decreased. Thus measured [ROS] critically depend on specific EPR parameters such as time window and especially the microwave power.