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We have proposed a new method which can be applied in assessing the intracellular production of hydrogen peroxide. Using this assay we have examined the hydrogen peroxide generation during the L-glutamate induced oxidative stress in the HT22 hippocampal cells. The detection of hydrogen peroxide is based on two crucial reagents cis-[Cr(C 2 O 4 )(pm)(OH 2 ) 2 ] + (pm denotes pyridoxamine) and 2-ketobutyrate. The results obtained indicate that the presented method can be a promising tool to detect hydrogen peroxide in biological samples, particularly in cellular experimental models.

1,3-Diphenylisobenzofuran (DPBF) has been developed as a selective probe for the detection and quantitative determination of hydrogen peroxide in samples containing different reactive nitrogen and oxygen species (RNOS). DPBF is a fluorescent probe which, for almost 20 years, was believed to react in a highly specific manner toward some reactive oxygen species (ROS) such as singlet oxygen and hydroxy, alkyloxy or alkylperoxy radicals. Under the action of these individuals DPBF has been rapidly transformed to 1,2-dibenzoylbenzene (DBB). In order to check if DPBF can act as a unique indicator of the total amount of different RNOS, as well as oxidative stress caused by an overproduction of these individuals, a series of experiments was carried out, in which DPBF reacted with peroxynitrite anion, superoxide anion, hydrogen peroxide, hypochlorite anion, and anions commonly present under biological conditions, namely nitrite and nitrate. In all cases, except for hydrogen peroxide, the product of the reaction is DBB. Only under the action of H 2 O 2 9-hydroxyanthracen-10(9H)-one (oxanthrone) is formed. This product has been identified with the use of fluorescence spectroscopy, NMR spectroscopy, high performance liquid chromatography coupled with mass spectrometry, infrared spectroscopy, elemental analysis, and cyclic voltammetry (CV). A linear relationship was found between a decrease in the fluorescence intensity of DPBF and the concentration of hydrogen peroxide in the range of concentrations of 0.196-3.941 mM. DPBF responds to hydrogen peroxide in a very specific way with the limits of detection and quantitation of 88 and 122.8 μM, respectively. The kinetics of the reaction between DBBF and H 2 O 2 was also studied.

Hydrogen peroxide is a well-established precursor of reactive oxygen and nitrogen species that are known to contribute to oxidative stress-the crucial factor responsible for the course of a wide range of phy-sicochemical processes as well as the genesis of various diseases, such as cancer and neurodegenerative disorders. Thus, the development of sensitive and selective methods for the detection and quantitative determination of hydrogen peroxide is of great importance in monitoring the in vivo production of that species and elucidating its biological functions. This review highlights the progress that has been made in the development of fluorescent and luminescent probes (excluding nanoparticles) employed to monitor hydrogen peroxide under biological conditions. Attention was focused on probes developed in the past 10 years.

The purpose of this study was to examine the application of the coordinated cis-[Cr(C2O4)(pm)(OH)2]+ cation where pm denotes pyridoxamine, as a specific sensing ion for the detection of hydrogen peroxide (H2O2). The proposed method for H2O2 detection includes two key steps. The first step is based on the nonenzymatic decarboxylation of pyruvate upon reaction with H2O2, while the second step is based on the interaction of cis-[Cr(C2O4)(pm)(OH2)2]+ with the CO2 released in the previous step. Using this method H2O2 generated during glutamate-induced oxidative stress was detected in HT22 hippocampal cells. The coordination ion cis-[Cr(C2O4)(pm)(OH2)2]+ and the spectrophotometric stopped-flow technique were applied to determine the CO2 concentration in cell lysates, supernatants and cell-free culture medium. Prior to CO2 assessment pyruvate was added to all samples studied. Pyruvate reacts with H2O2 with 1:1 stoichiometry, and consequently the amount of CO2 released in this reaction is equivalent to the amount of H2O2.