Amplex® Red (10-acetyl-3,7-dihydroxyphenoxazine) is a fluorogenic probe widely used to detect and quantify hydrogen peroxide in biological systems. Detection of hydrogen peroxide is based on peroxidase-catalyzed oxidation of Amplex® Red to resorufin. In this study we investigated the mechanism of one-electron oxidation of Amplex® Red and we present the spectroscopic characterization of transient species formed upon the oxidation. Oxidation process has been studied by a pulse radiolysis technique with one-electron oxidants (N3•, CO3•−, •NO2 and GS•). The rate constants for the Amplex® Red oxidation by N3• (2k = 2.1·109 M−1s−1, at pH = 7.2) and CO3•− (2k = 7.6·108 M−1s−1, at pH = 10.3) were determined. Two intermediates formed during the conversion of Amplex® Red into resorufin have been characterized. Based on the results obtained, the mechanism of transformation of Amplex® Red into resorufin, involving disproportionation of the Amplex® Red-derived radical species, has been proposed. The results indicate that peroxynitrite-derived radicals, but not peroxynitrite itself, are capable to oxidize Amplex® Red to resorufin. We also demonstrate that horseradish peroxidase can catalyze oxidation of Amplex® Red not only by hydrogen peroxide, but also by peroxynitrite, which needs to be considered when employing the probe for hydrogen peroxide detection.

Background: Nitroxyl (HNO) is a reactive nitrogen species implicated in cardioprotection. Results: Nitroxyl reacts with oxygen to form an oxidizing and nitrating species, peroxynitrite. Conclusion: In the presence of oxygen, HNO donors may be a source of peroxynitrite. Significance: Peroxynitrite formation should be taken into account in the extracellular milieu when exposing cells to HNO donor under aerobic conditions.

Aromatic boronic acids react rapidly with peroxynitrite (ONOO−) to yield phenols as major products. This reaction was used to monitor ONOO− formation in cellular systems. Previously, we proposed that the reaction between ONOO− and arylboronates (PhB(OH)2) yields a phenolic product (major pathway) and a radical pair PhB(OH)2O•−…•NO2 (minor pathway). [Sikora A. et al., Chem Res Toxicol 24, 687-97, 2011]. In this study, we investigated the influence of a bulky triphenylphosphonium (TPP) group on the reaction between ONOO− and mitochondria-targeted arylboronate isomers (o-, m-, and p-MitoPhB(OH)2). Results from the electron paramagnetic resonance (EPR) spin-trapping experiments unequivocally showed the presence of a phenyl radical intermediate from meta and para isomers, and not from the ortho isomer. The yield of o-MitoPhNO2 formed from the reaction between o-MitoPhB(OH)2 and ONOO− was not diminished by phenyl radical scavengers, suggesting a rapid fragmentation of the o-MitoPhB(OH)2O•− radical anion with subsequent reaction of the resulting phenyl radical with •NO2 in the solvent cage. The DFT quantum mechanical calculations showed that the energy barrier for the dissociation of o-MitoPhB(OH)2O•− radical anion is significantly lower than that of m-MitoPhB(OH)2O•− and p-MitoPhB(OH)2O•− radical anions. The nitrated product, o-MitoPhNO2, is not formed by nitrogen dioxide radical generated by myeloperoxidase in the presence of nitrite anion and hydrogen peroxide, indicating that this specific nitrated product may be used as a diagnostic marker product for ONOO−. Incubation of o-MitoPhB(OH)2 with RAW 264.7 macrophages activated to produce ONOO− yielded the corresponding phenol o-MitoPhOH as well as the diagnostic nitrated product, o-MitoPhNO2. We conclude that the ortho isomer probe reported here is most suitable for specific detection of ONOO− in biological systems.

Over the last forty years, there has been tremendous progress in understanding the biological reactions of reactive oxygen species (ROS) and reactive nitrogen species (RNS). It is widely accepted that the generation of ROS and RNS is involved in physiological and pathophysiological processes. To understand the role of ROS and RNS in a variety of pathologies, the specific detection of ROS and RNS is fundamental. Unfortunately, the intracellular detection and quantitation of ROS and RNS remains a challenge. In this short review, we have focused on the mechanistic and quantitative aspects of their detection with the use of selected fluorogenic probes. The challenges, limitations and perspectives of these methods are discussed.

This article presents the results of the interlaboratory comparison (ILC) study of the following four characteristics of ceramic tile adhesives (CTAs): initial tensile adhesion strength, tensile adhesion strength after heat ageing, tensile adhesion strength after immersion in water, and tensile adhesion strength after freeze–thaw cycles. The results showed that the objective of the ILC was achieved—the z-score analysis carried out following ISO 13528 allowed for classifying all results obtained by 23 laboratories out of 27 as satisfactory. The results of the remaining four laboratories were rated worse. Despite the achieved goal, the ILC notes high heterogeneity of the results in terms of failure patterns, as well as significant differences between the lowest and the highest values of tensile adhesion strength for various measurement conditions. The results of the ILC were discussed in terms of the possibility of including them in the risk analysis conducted by the manufacturer. The results of the ILC are also valuable information for market surveillance authorities, who, in the authors’ opinion, should be more cautious about results on samples taken from the market. The ILC results for CTAs are also a valuable recommendation for a possible revision of EN 12004.