Measurement of Vascular Reactive Oxygen Species Production by Chemiluminescence

Reactive oxygen species (ROS) play important roles in the pathogenesis of vascular disease states. In particular, superoxide anion participates in endothelial dysfunction mainly owing to its rapid interaction with NO, but also as it causes direct biological effects and serves as a progenitor for many other ROS. Detection of ROS in intact tissues and cells is much more difficult than in chemical systems. We describe advantages and potential pitfalls of chemiluminescent methods of vascular ROS detection. Lucigenin and luminol-enhanced chemiluminescent methods are described in the detection of vascular superoxide and peroxynitrite production and NAD(P)H oxidase activity. We also describe the use of new chemiluminescent probes, including cypridina luciferin analogs (coelenterazine; CLA and MCLA) and pholasin. The validity of some of these chemiluminescent methods (in particular lucigenin-enhanced chemiluminescence) recently has been questioned. It has been suggested that lucigenin itself, especially at high concentrations (>50 �mol/L), may produce superoxide via redox cycling. Using intact human vascular rings and vascular homogenates, we show that lucigenin, particularly at lower concentrations (5 �mol/L), provides an accurate assessment of the rate of superoxide production as assessed by close correlations with the SOD inhibitable ferricytochrome c reduction assay. Chemiluminescent techniques provide a useful approach for vascular ROS measurements, but should be always interpreted in the context of measurements obtained using other complementary techniques.