Recent developments in detection of superoxide radical anion and hydrogen peroxide: Opportunities, challenges, and implications in redox signaling

Abstract

In this review, some of the recent developments in probes and assay techniques specific for superoxide (O2•−) and hydrogen peroxide (H2O2) are discussed. Over the last decade, significant progress has been made in O2•− and H2O2 detection due to syntheses of new redox probes, better understanding of their chemistry, and development of specific and sensitive assays. For superoxide detection, hydroethidine (HE) is the most suitable probe, as the product, 2-hydroxyethidium, is specific for O2•−. In addition, HE-derived dimeric products are specific for one-electron oxidants. As red-fluorescent ethidium is always formed from HE intracellularly, chromatographic techniques are required for detecting 2-hydroxyethidium. HE analogs, Mito-SOX and hydropropidine, exhibit the same reaction chemistry with O2•− and one-electron oxidants. Thus, mitochondrial superoxide can be unequivocally detected using HPLC-based methods and not by fluorescence microscopy. Aromatic boronate-based probes react quantitatively with H2O2, forming a phenolic product. However, peroxynitrite and hypochlorite react more rapidly with boronates, forming the same product. Using ROS-specific probes and HPLC assays, it is possible to screen chemical libraries to discover specific inhibitors of NADPH oxidases. We hope that rigorous detection of O2•− and H2O2 in different cellular compartments will improve our understanding of their role in redox signaling.

Mitochondria-Targeted Triphenylphosphonium-Based Compounds: Syntheses, Mechanisms of Action, and Therapeutic and Diagnostic Applications

“…241–247 In general, rigorous characterization of specific products generated from either biomolecules (proteins, DNA, or lipids) or exogenous probes (exomarkers) is required to completely understand the redox processes. 248–250 This methodology can provide specific detection but does not provide real-time monitoring.…”

“…The chromatographic techniques (HPLC, LC-MS/MS) are currently the only way for simultaneous determination of probe uptake, profiling, and specific quantification of the oxidation products formed from MitoSOX and other probes. 247,259,262 …”

Recent Developments in the Probes and Assays for Measurement of the Activity of NADPH Oxidases

“…Despite many artifacts, lucigenin and luminol were the most frequently used probes for measuring Nox activity. However, the situation recently changed, due to a better understanding of the chemistry of the probes, including the reaction mechanism, stoichiometry and kinetics, and the development of new probes and assays (26–29). These rigorous approaches have also enabled the establishment of the protocol for high-throughput screening (HTS) assays for faster development of new inhibitors of NADPH oxidases.…”

Taking up the cudgels for the traditional reactive oxygen and nitrogen species detection assays and their use in the cardiovascular system

“…There are also a number of useful review articles published including guidelines how to use the traditional (and the new) assays for RONS detection, highlighting the specific pitfalls and drawbacks of them [28][29][30][31][32].…”

“…much more sensitive electrochemical detection of the DHE products) or new DHE analogs allowing sitespecific detection of superoxide (e.g. mito-SOX for mitochondrial superoxide and hydropropidine for extracellular superoxide) or the most advanced new spin traps providing higher stability in biological samples and thereby allow the EPR-based detection of superoxide and/ or peroxynitrite-derived free radicals in cells and tissues (some candidates might be found in [32,[135][136][137]). High performance liquid chromatography (HPLC)-based detection of superoxide formation in the extracellular space, cytosol and mitochondria can be established by hydropropidine, a positively-charged water-soluble analogue of dihydroethidium (DHE) for extracellular superoxide formation [138], DHE for cytosolic superoxide formation and mitoSOX, a mitochondria-targeted DHE analogue for mitochondrial superoxide formation (Fig.…”