Analysis of reactive oxygen species generated by neutrophils using a chemiluminescence probe L-012

Reactive oxygen species (ROS) play important roles in the defense mechanism against infection and in the pathogenesis of various diseases. Although chemical properties of ROS generated by leukocytes have been studied extensively, methods available for their analysis are not sufficiently sensitive. We found that 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4-(2H,3H)dione (L-012) reacted with various types of ROS generated by activated neutrophils in human blood and oral cavity, and from peritoneal cavity of the rat, and developed strong chemiluminescence (CHL). Under physiological conditions, opsonized zymosan-dependent CHL intensity of L-012 in human blood and rat peritoneal neutrophils was about 100 and 10 times higher than that of luminol and luciferin analog MCLA, respectively. Phorbol ester-activated CHL of oral neutrophils was also higher with L-012 than that with luminol and MCLA. The presence of either superoxide dismutase, catalase, uric acid, deferoxamine, or azide decreased CHL intensity of L-012 by 52, 57, 57, 63, and 91%, respectively. Kinetic analysis revealed that L-012 developed CHL predominantly by reacting with hydroxyl radical and hypochlorite. Thus, highly sensitive L-012 permits studies on ROS generation by complex biological systems, such as leukocytes, and on the role of ROS in the pathogenesis of various diseases.