A Conceptual Guide to the Measurement of Different Reactive Oxygen and Reactive Nitrogen Species in Cardiovascular Tissue

This long overdue comprehensive guide to the “Measurement of Reactive Oxygen Species (ROS), Reactive Nitrogen Species (RNS), and Redox-dependent Signaling in the Cardiovascular System” provides specific suggestions for choosing appropriate and reliable assay methods to measure different ROS and RNS. Although primarily aimed at helping researchers in the field of cardiovascular research, this conceptual guideline will help investigators and clinicians involved in a wide range of research areas ensure specificity and reliability in the measurement of ROS and RNS molecules. The clear instructions in this AHA Scientific Statement are informative, timely, and practical in that it provides specific recommendations for the assay(s) to be used for each ROS and RNS molecule such as superoxide (O2•-), hydrogen peroxide (H2O2), hydroxyl radical (•OH), nitric oxide (NO), and peroxyntirite (OONO-). Additional useful suggestions on the use of specific methods for ROS/RNS measurements in different conditions such as in cells, tissues, and biological fluids are commendable.

Given the complexity of the short-lived, highly reactive, and dynamic nature of the molecules, one may come to erroneous conclusions of ROS/RNS levels using an assay method that lacks specificity or sensitivity. This article along with its Introduction and Background sections will help avoid that and may even serve as a conceptual guideline to a newcomer in the field of oxidant research. A comprehensive list of major ROS and RNS molecules in Table I is very helpful. Inclusion of several Figures will guide the investigators to pick from a “Choice of assays to measure superoxide and hydrogen peroxide” (Figure 1) and will provide with a “Decision tree for measuring RNS” (Figure 2). The addition of a list of assays that can be used to quantify (1) ROS and RNS directly, and (2) ROS/RNS indirectly by (a) measuring secondary products of ROS/RNS, and (b) measuring ROS/RNS-modified protein and/or DNA (Figure 9) has made the review complete and useful. Indication of the most selective and sensitive assays available to detect each ROS/RNS molecule and the recommendation to use more than one assay method will help the investigators avoid experimental error.

Introductory discussions on the roles of unpaired electron(s) in different molecules and how molecules without unpaired electron may become reactive, on the association between ROS/RNS and cardiovascular disease, and on the lack of conclusive evidence suggesting involvement of ROS/RNS in the pathogenesis, are timely and do reiterate our current understanding. However, the notion regarding the usage of weak antioxidants as the probable cause behind the failure of clinical trials is controversial and may have been avoided. An emphasis on the importance of sub-cellular localization of ROS/RNS and discussion on our current lack of targeting technologies for these sub-cellular compartments will help guide future directions and developments of effective technologies. Discussion on the biomarkers that are currently used to measure “oxidative stress” in humans is very informative; authors’ comments on the indirect nature of the current biomarkers and their lack of sensitivity reiterate the need for rigorous research in this field. Further emphasis on the temporospatial nature of ROS/RNS molecules and how ratiometric analytical tools can help quantify those changes may be considered for future updates.

One specific criterion among many that makes this article outstanding is that it provides the researchers with sufficient information to decide upon the use of a specific probe for each species of ROS or RNS depending on the cell, tissue, or biological fluids involved. The section devoted to spin-trapping methods aimed at measuring superoxide, NO, or hydroxyl radicals covers several different conditions in cardiovascular research. For example, measurement of superoxide generation during myocardial ischemia and reperfusion injury in a Langendorff perfused heart using spin-trap has been very well-described. It also covered principles behind the methodologies used to measure superoxide and hydroxyl anion generation by cardiovascular enzymes including mitochondria in vitro and ex vivo.

A large array of assay methods, techniques, and commercially available ROS/RNS assay kits has been developed during the last decade. Many of these methodologies lack precision, sensitivity, and selectivity, and some are even named or termed inappropriately, resulting in confusion and potential inaccuracies in the research field. Although this article is not aimed at providing the experimental details for each method, it provides appropriate background information for the assays currently available. This Scientific Statement provides general methodological guidelines, points out each method’s strength and limitations, and guides to reproducible techniques for quantifying each ROS/RNS content in the cells, tissues, and biological body fluids. Finally, it lists all the available assays categorically under a “direct ROS measure” and indirect/by-product of ROS measure, which will lessen confusion in the field. This comprehensive guideline will certainly help the readers make an informed choice of their assay method for their respective queries and experimental conditions. It will also help young investigators have a better realization about the differences between the content/level of ROS/RNS and the injuries resulted from ROS/RNS in a given system