Fluorogenic Analysis of H₂O₂ in Biological Materials

Abstract

The biological importance of hydrogen peroxide and other “reactive oxygen species” (ROS) has become greatly appreciated in recent years. It has become apparent that certain ROS, in particular hydrogen peroxide (H₂O₂) and nitric oxide (•NO), are ubiquitously used as intra- or intercellular messengers (1). Because these ROS are short-lived in vivo, their steady-state concentrations remain low, and their accurate quantitation poses a significant technical challenge. Moreover, it is often necessary to monitor changes in ROS in real time, for instance during the time course of hormonal stimulation of cultured cells (2). The most common methods of measuring H₂O₂ rely upon peroxide-dependent oxidation of reduced xanthene dyes such as 2′,7′-dichlorodihydrofluorescein (H₂DCF, also called dichlorofluorescin) or dihydrorhodamine 123 (H₂RD123) (Fig. 1). These dyes were originally used to measure peroxidase activities (3) but the assays were easily modified to allow peroxide determination (2,4–7). Although the reduced dyes are not highly fluorescent, their oxidation products are, and can be monitored continuously using a fluorescence spectrometer, microplate reader, or confocal microscope. Although flexible and convenient, the fluorogenic determination of H₂O₂ must be performed with due consideration of factors that may interfere with the chemistry of the assay.