Detection and Analysis of Reactive Oxygen Species (ROS): Buffer Components Are Not Bystanders

Reactive oxygen species (ROS) play critical roles in pathophysiological processes. Therefore, there are widespread interests in determining ROS concentrations under various conditions. However, “the application and interpretation of these measurements are fraught with challenges and limitations,” as indicated by a recent consensus paper in Nature Metabolism. These challenges and limitations are partially due to the large number of factors that could impact experimental outcome, making most side-by-side comparisons difficult. In an effort to address some seemingly idiosyncratic fluctuations in our own experiments, we examined buffer effects on ROS concentration determination through kinetic and HPLC experiments. Specifically, we examined the effects of the following buffer compounds: HEPES, Tris, MES, citrate, ammonium acetate, and PBS and found that most organic buffer components can rapidly consume NaOCl (the second most abundant ROS) and/or directly interact with certain ROS probes such as a boronate for H2O2 determination, leading to substantial variations in experimental outcome. We use these examples to draw attention to the profound effects of buffer components on ROS concentration determination. We hope that the work described will stimulate additional studies in examining similar factors that impact ROS research. Because of their highly reactive nature, very few chemical entities are truly bystanders in ROS studies. Together with our recent finding of DMSO’s effect on ROS concentration determination, we hope the results described will lead to improved rigor in designing ROS experiments by considering factors that were previously considered as nothing but bystanders or benign.