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Electron spin resonance of skin
Published in Roger L. McMullen, Antioxidants and the Skin, 2018
A spin trapping agent reacts with a radical, by way of an addition reaction, to form a spin adduct, which is a much more stable (persistent) radical. In regard to the formed spin adduct, the spin trapping agent is referred to as the spin trap and the radical portion of the newly formed compound as the radical addend. The resulting spin adduct is a much more long-lived radical than the highly reactive species we wish to detect, thereby allowing detection of this radical (spin adduct) using conventional ESR techniques. Generally, nitrones and nitroso compounds are used as spin traps, which become nitroxides upon electron donation (oxidation) to the radical species.1Figure 7.7 provides several examples for the addition of a radical to a nitrone spin trap. Nitroxides are fairly stable, because the unpaired electron is resonance stabilized. N-tert-butyl alpha-nitrone (PBN) was one of the original spin traps that were used to detect carbon- and oxygen-centered radicals. Later, alpha-(4-pyridyl-1-oxide)-N-tert-butyl-nitrone (POBN) was developed as an analog to PBN, which had better trapping capabilities. The spin trap, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), which is the most universally used for biological samples, can trap carbon-, oxygen-, and sulfur-centered free radicals.1 An example spectrum, demonstrating the use of the DMPO spin trap in skin is provided later in Figure 7.12.
Spin Trapping of Hydroxyl Radical
Published in Robert A. Greenwald, CRC Handbook of Methods for Oxygen Radical Research, 2018
Spin trapping has provided a wealth of information on the production of free radicals in biochemical and biological systems. Although other methods can provide similar information, spin trapping often reveals more, as an experiment may indicate the presence of more than one free radical or the formation of secondary radicals.
Electron Spin Resonance Spectroscopy
Published in Adorjan Aszalos, Modern Analysis of Antibiotics, 2020
George C. Yang, Adorjan Aszalos
Electron spin resonance (ESR) spectroscopy has been widely used as a sensitive tool for the detection and characterization of paramagnetic species generated during many chemical and biochemical reactions. With the exception of transition metals, such species are rarely observed to occur naturally. Chemical, electrochemical, and enzymatic reduction and/or oxidation of compounds of interest have been extensively utilized in the production of paramagnetic species from their nonparamagnetic precursors in biological systems. Additionally, two other techniques have been developed for the study of many biological phenomenon and reactions by ESR spectroscopy. These techniques involve the attachment of a stable free radical, usually called spin labels or spin probes (for example, the nitroxide radical), to a normally nonparamagnetic species; or the trapping of an unstable (short-lived) free radical using a spin trapping reagent. The spin labeling technique facilitates the study of molecular motion and local fluidity and local dielectric and electromagnetic environments. In spin trapping, a nonparamagnetic molecule (spin trap) reacts rapidly with a labile, free radical intermediate to form a stable paramagnetic adduct. The ESR spectrum of the resulting paramagnetic species (adduct) is then characterized by measurement of the hyperfine splitting constants and g factor. Excellent reviews on the theory and application of ESR spectroscopy in the biochemical field are Spin Labeling by L. J. Berliner, Biological Applications of Electron Spin Resonance Spectroscopy by H. Swartz, R. J. Bolton, and D. Borg, and Free Radicals by W. Pryor.
NiONPs-induced alteration in calcium signaling and mitochondrial function in pulmonary artery endothelial cells involves oxidative stress and TRPV4 channels disruption
Published in Nanotoxicology, 2022
Ophélie Germande, Magalie Baudrimont, Fabien Beaufils, Véronique Freund-Michel, Thomas Ducret, Jean-François Quignard, Marie-Hélène Errera, Sabrina Lacomme, Etienne Gontier, Stéphane Mornet, Megi Bejko, Bernard Muller, Roger Marthan, Christelle Guibert, Juliette Deweirdt, Isabelle Baudrimont
Superoxide anion production was measured using the spin probe 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH, Noxygen®) according to the manufacturer’s recommendations and as previously described (Deweirdt et al. 2017). EPR spin trapping is one of the specific techniques used to study free radical production such as the superoxide anion. However, the very short life span and the relatively low concentration of superoxide anion (O2˙ˉ) made the measurement difficult to develop. The CMH probe has the property of being soluble and makes it possible to measure intracellular O2˙ˉ (Dikalov et al. 2011; Konczol et al. 2012). Moreover, the CMH probe can be oxidized by O2˙ˉ to generate a stable nitroxide radical (CM•) easily detectable by EPR spectroscopy. After a 4 h-exposure to NiONPs, cells were incubated for 20 min with the spin-probe mix containing CMH (500 µM), diethyldithiocarbamate (5 µM), and deferoxamine (25 µM) in KHB solution. Then, HPAEC were scraped, homogenized, and frozen in a syringe in liquid nitrogen before EPR analysis. All the EPR spectra were recorded using the Spectrometer X Miniscope MS200 (Magnettech®, Germany). The EPR parameters have been previously described (Deweirdt et al. 2017). Following EPR spectra readings, protein quantities were measured by a Lowry test (Lowry reagent, Sigma Aldrich®), according to the manufacturer’s recommendations. The results were normalized to protein quantities and expressed as EPR signal amplitudes in arbitrary units (AU)/mg of protein.
Liposomal integration method for assessing antioxidative activity of water insoluble compounds towards biologically relevant free radicals: example of avarol
Published in Journal of Liposome Research, 2020
Đura Nakarada, Boris Pejin, Giuseppina Tommonaro, Miloš Mojović
To study the amount of NO• radicals in the system, the solution consisting of avarol-containing or 100% DPPC liposomes, sodium nitroprusside as a NO• radical generating system, and Fe(DTCS)2 complex as spin-trapping agent was used (Stefánsson et al.2005). In brief, 25 µl of sample which contained 19 µl of liposome suspension and 6 µl of Fe(DTCS)2 complex (final concentration 0.1 M) was transferred into the gas-permeable Teflon tube and finally 5 µl of sodium nitroprusside (final concentration 9 mM) was employed just before the EPR spectra was acquired. The NO-Fe(DTCS)2 adduct EPR signal was measured using following experimental settings: centre field 3500 G, microwave power 10 mW, microwave frequency 9.85 GHz, modulation frequency 100 kHz, modulation amplitude 1 G.