A Highly Sensitive Chemiluminometric Assay for Real-Time Detection of Biological Hydrogen Peroxide Formation

Hydrogen peroxide (H₂O₂) is a major reactive oxygen species (ROS) produced by various cellular sources, especially mitochondria. At high levels, H₂O₂ causes oxidative stress, leading to cell injury, whereas at low concentrations, this ROS acts as an important second messenger to participate in cellular redox signaling. Detection and measurement of the levels or rates of production of cellular H₂O₂ are instrumental in studying the biological effects of this major ROS. While a number of assays have been developed over the past decades for detecting and/or quantifying biological H₂O₂formation, none has been shown to be perfect. Perhaps there is no perfect assay for sensitively and accurately quantifying H₂O₂ as well as other ROS in cells, wherein numerous potential reactants are present to interfere with the reliable measurement of the specific ROS. In this context, each assay has its own advantages and intrinsic limitations. This article describes a highly sensitive assay for real-time detection of H₂O₂ formation in cultured cells and isolated mitochondria. This assay is based on the luminol/horseradish peroxidase-dependent chemiluminescence that is inhibitable by catalase. The article discusses the usefulness and shortcomings of this chemiluminometric assay in detecting biological H₂O₂ formation induced by beta-lapachone redox cycling with both cells and isolated mitochondria.