Decay Of Electron Paramagnetic Resonance Signal Research Articles

Cellular accumulation and antioxidant activity of acetoxymethoxycarbonyl pyrrolidine nitroxides

Nitroxides are widely used in biology as antioxidants, spin labels, functional spin probes for pH, oxygen and thiol levels, and tissue redox status imaging using electron paramagnetic resonance (EPR); however, biological applications of nitroxides is hindered by fast bioreduction to EPR-silent hydroxylamines and rapid clearance. In this work, we have studied pyrrolidine nitroxides with acetoxymethoxycarbonyl groups which can undergo hydrolysis by cellular esterases to hydrophilic carboxylate derivatives resistant to bioreduction. Nitroxides containing acetoxymethoxycarbonyl groups were rapidly absorbed by cells from the media, 3,4-bis-(acetoxymethoxycarbonyl)-proxyl (DCP-AM2) and 3-(2-(bis(2-(acetoxymethoxy)-2-oxoethyl)amino)acetamido)-proxyl (DCAP-AM2) showing the strongest EPR signal of the cellular fraction. Remarkably, the EPR parameters of 3,4-dicarboxy-proxyl (DCP) and its mono- and di-acetoxymethyl esters are different, and consequent intracellular hydrolysis of acetoxymethoxycarbonyl groups in DCP-AM2 can be followed by EPR. To elucidate intracellular location of the resultant DCP, the mitochondrial fraction has been isolated. EPR measurements showed that mitochondria were the main place where DCP was finally accumulated. TEMPO derivatives showed expectedly much faster decay of EPR signal in the cellular fraction, compared to pyrrolidine nitroxides. It was found that supplementation of endothelial cells with 50 nM of DCP-AM2 completely normalised the mitochondrial superoxide level. Moreover, administration of DCP-AM2 to mice (1.4 mg/kg/day) resulted in substantial nitroxide accumulation in the tissues and significantly reduced hypertension. We found that hydroxylamine derivatives of dicarboxyproxyl nitroxide DCP-AM-H can be used for the detection of superoxide in vivo in angiotensin II model of hypertension. Infusion of DCP-AM-H in mice leads to accumulation of persistent EPR signal of nitroxide in the blood and vascular tissue in angiotensin II-infused wild-type but not in SOD2 overexpressing mice. Our data demonstrate that acetoxymethoxycarbonyl group containing nitroxides accumulate in mitochondria and demonstrate site-specific antioxidant activity.

An EPR spin probe study of liposomes from sunflower and soybean phospholipids

Comparative properties of lecithin-based liposomes prepared from the mixed phospholipids of sunflower seeds, soybean and egg yolk were investigated by electron paramagnetic resonance (EPR) spectroscopy. For these investigations, stable nitroxide radicals, 1-oxyl-2,2,6,6-tetramethylpiperidin-4-yl 5,7-dimethyladamantane-1-carboxylate (DMAC-TEMPO), 5-doxylstearic acid (5-DSA) and 16-doxylstearic acid (16-DSA) were used as spin probes. Binding of the spin probes to the liposome membranes resulted in a substantial increase of the apparent rotational diffusion correlation times. The EPR spectra of the incorporated nitroxides underwent temperature-dependent changes. For every spin probe, values of apparent enthalpy and entropy of activation were calculated from the temperature dependence of rotational diffusion correlation times via Arrhenius equation. In case of DMAC-TEMPO, the data point to differences between the phospholipid bilayer of liposomes derived from sunflower and soy lecithin, and some similarity between the sunflower and egg yolk liposomes. Anisotropic hyperfine interaction constants of DMAC-TEMPO and 16-DSA included in the liposomes have been analyzed and attributed to different micropolarity of the surroundings of the spin probes. The kinetics of EPR signal decay of DMAC-TEMPO in the presence of 2,2′-azobis(2-amidinopropane) suggest the better stability of the sunflower liposomes to lipid peroxidation as compared to the liposomes prepared from soy lecithin.

Tetrathiatriarylmethyl radical with a single aromatic hydrogen as a highly sensitive and specific superoxide probe

Superoxide (O2•−) plays crucial roles in normal physiology and disease; however, its measurement remains challenging because of the limited sensitivity and/or specificity of prior detection methods. We demonstrate that a tetrathiatriarylmethyl (TAM) radical with a single aromatic hydrogen (CT02-H) can serve as a highly sensitive and specific O2•− probe. CT02-H is an analogue of the fully substituted TAM radical CT-03 (Finland trityl) with an electron paramagnetic resonance (EPR) doublet signal due to its aromatic hydrogen. Owing to the neutral nature and negligible steric hindrance of the hydrogen, O2•− preferentially reacts with CT02-H at this site with production of the diamagnetic quinone methide via oxidative dehydrogenation. Upon reaction with O2•−, CT02-H loses its EPR signal and this EPR signal decay can be used to quantitatively measure O2•−. This is accompanied by a change in color from green to purple, with the quinone methide product exhibiting a unique UV–Vis absorbance (ε=15,900M−1cm−1) at 540nm, providing an additional O2•− detection method. More than five-fold higher reactivity of CT02-H for O2•− relative to CT-03 was demonstrated, with a second-order rate constant of 1.7×104M−1s−1 compared to 3.1×103M−1 s−1 for CT-03. CT02-H exhibited high specificity for O2•− as evidenced by its inertness to other oxidoreductants. The O2•− generation rates detected by CT02-H from xanthine/xanthine oxidase were consistent with those measured by cytochrome c reduction but detection sensitivity was 10- to 100-fold higher. EPR detection of CT02-H enabled measurement of very low O2•− flux with a detection limit of 0.34nM/min over 120min. HPLC in tandem with electrochemical detection was used to quantitatively detect the stable quinone methide product and is a highly sensitive and specific method for measurement of O2•−, with a sensitivity limit of ∼2×10−13mol (10nM with 20-μl injection volume). Based on the O2-dependent linewidth broadening of its EPR spectrum, CT02-H also enables simultaneous measurement of O2 concentration and O2•− generation and was shown to provide sensitive detection of extracellular O2•− generation in endothelial cells stimulated either by menadione or with anoxia/reoxygenation. Thus, CT02-H is a unique probe that provides very high sensitivity and specificity for measurement of O2•− by either EPR or HPLC methods.

Temperature-dependent free radical reaction in water

Temperature-dependent free radical reactions were investigated using nitroxyl radicals as redox probes. Reactions of two types of nitroxyl radicals, TEMPOL (4-hydroxyl-2,2,6,6-tetramethylpiperidine-N-oxyl) and carbamoyl-PROXYL (3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-N-oxyl), were tested in this paper. Heating a solution containing a nitroxyl radical and a reduced form of glutathione (GSH) caused temperature-dependent decay of electron paramagnetic resonance (EPR) signal of the nitroxyl radical. Heating a solution of the corresponding hydroxylamine form of the nitroxyl radical showed EPR signal recovery. The GSH-dependent reduction of nitroxyl radicals at 70°C was suppressed by antioxidants, spin trapping agents, and/or bubbling N2 gas, although heating carbamoyl-PROXYL with GSH showed temporarily enhanced signal decay by bubbling N2 gas. Since SOD could restrict the GSH-dependent EPR signal decay of TEMPOL, O2•− is related with this reaction. O2•− was probably generated from dissolved oxygen in the reaction mixture. Oxidation of the hydroxylamines at 70°C was also suppressed by bubbling N2 gas. Heating a solution of spin trapping agent, DMPO (5,5-dimethyl-1-pyrroline-N-oxide) showed a temperature-dependent increase of the EPR signal of the hydroxyl radical adduct of DMPO. Synthesis of hydroxyl radical adduct of DMPO at 70°C was suppressed by antioxidants and/or bubbling N2 gas. The results suggested that heating an aqueous solution containing oxygen can generate O2•−.

Intracellular Hypoxia of Tumor Tissue Estimated by Noninvasive Electron Paramagnetic Resonance Oximetry Technique Using Paramagnetic Probes

Electron paramagnetic resonance (EPR) oximetry at 700 MHz operating frequency employing a surface coil resonator is used to assess tissue partial pressure of oxygen (pO(2)) using paramagnetic media whose linewidth and decay constant are related to oxygen concentration. Differences in extracellular and intracellular pO(2) in squamous cell carcinoma (SCC) tumor tissue were tested using several types of water-soluble paramagnetic media, which localize extracellularly or permeate through the cell membrane. The nitroxide carboxy-PROXYL (CxP) can only be distributed in blood plasma and extracellular fluids whereas the nitroxides carbamoyl-PROXYL (CmP) and TEMPOL (TPL) can permeate cell membranes and localize intracellularly. EPR signal decay constant and the linewidth of the intravenously administered nitroxides in SCC tumor tissues implanted in mouse thigh and the contralateral normal muscle of healthy mice breathing gases with different pO(2) were compared. The pO(2) in the blood can depend on the oxygen content in the breathing gas while tissue pO(2) was not directly influenced by pO(2) in the breathing gas. The decay constants of CmP and TPL in tumor tissue were significantly larger than in the normal muscles, and lower linewidths of CmP and TPL in tumor tissue was observed. The SCC tumor showed intracellular hypoxia even though the extracellular pO(2) is similar to normal tissue in the peripheral region.

A novel electron paramagnetic resonance spin-probe technique demonstrates the relation between the production of hydroxyl radicals and ischemia–reperfusion injury☆

Many previous studies have suggested an increase in hydroxyl radical (OH) production after myocardial ischemia-reperfusion; however, traditional techniques have not been able to conclusively prove this phenomenon. We investigated whether the production of OH was increased during myocardial reperfusion using a novel electron paramagnetic resonance (EPR) technique using an OH-specific spin probe. An OH scavenger, 3-methyl-1-phenyl-2-pyrazolin-5-one (MCI-186), was used to examine the relationship between OH production and post-ischemic functional recovery or the degree of myocardial injury. We used an isolated rabbit-heart preparation perfused with support-rabbit blood, and the heart was reperfused after normothermic global ischemia. Heart samples were reacted with the OH-specific spin probe, 4-hydroxy-2,2,6,6-tetramethyl-piperidine-N-oxyl (hydroxyl-TEMPO). The rate of decay of the EPR signal showed OH production. We investigated the rate of EPR signal decay and cardiac function. The rate of signal decay was significantly increased just after reperfusion compared with that of pre-ischemia (2.00×10(-2)±0.77×10(-2)min(-1) vs 0.11×10(-2)±0.02×10(-2)min(-1), p<0.01). Administration of MCI-186 reduced the rate of decay to 0.86×10(-2)±0.14×10(-2)min(-1) just after reperfusion (p<0.01). Cardiac function was significantly improved 60 min after reperfusion using MCI-186 compared without MCI-186 (left ventricular developed pressure was 95±9 mm Hg vs 60±6 mm Hg and the first derivative of the left ventricular pressure (dP/dt) was 1843±200 mm Hg s(-1) vs 1182±127 mm Hg s(-1)). A novel EPR spin-probe technique demonstrated the relation between the production of OH and ischemia-reperfusion injury. We confirmed that OH production influenced cardiac function and myocardial ischemia-reperfusion injury.

Competition of nitroxyl contrast agents as an in vivo tissue redox probe: Comparison of pharmacokinetics by the bile flow monitoring (BFM) and blood circulating monitoring (BCM) methods using X‐band EPR and simulation of decay profiles

Nitroxyl radicals used as tissue redox-sensitive contrast agents in electron paramagnetic resonance (EPR) and/or NMR imaging should satisfy the following two conditions: 1) the molecules disperse into tissues rapidly, and 2) paramagnetic loss occurs by simple reduction of the radical. The pharmacokinetic trends of several nitroxyl contrast agents were compared with the results obtained by bile flow monitoring (BFM) and blood circulation monitoring (BCM) methods using X-band EPR. The nitroxyl radicals (TEMPO, TEMPONE (oxo-TEMPO), and amino-TEMPO) showed additional EPR signals in the bile that were attributed to metabolites formed during transport from blood to bile through the liver. However, the highly hydrophilic CAT-1 (trimethylammonium-TEMPO), which has low membrane permeability, showed minimal concentration in the bile. Probes that have carboxyl moiety, such as carboxy-TEMPO and carboxy-PROXYL, can be transported via anion transporter into hepatic cells. The EPR signal decay profiles of the nitroxyl radicals were simulated based on the experimental data. The simulation, which we previously applied to mouse blood, was modified to simultaneously fit the experimental results of BFM and BCM obtained with rats. The simulation data showed the simplicity/complexity of the pharmacokinetic mechanisms and that carbamoyl-PROXYL and TEMPOL (hydroxy-TEMPO) are suitable contrast agents for assessing tissue redox status.

Importance of volume limitation for tissue redox status measurements using nitroxyl contrast agents: A comparison of X-band EPR bile flow monitoring (BFM) method and 300 MHz in vivo EPR measurement

Methods proposed for in vivo redox status estimation, X-band (9.4GHz) electron paramagnetic resonance (EPR) bile flow monitoring (BFM) and 300MHz in vivo EPR measurement, were compared. The spin probe 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (carbamoyl-PROXYL) was utilized for both methods, due to its suitable lipophilicity. EPR signal decay of a nitroxyl spin probe in the bile flow and in the liver region (upper abdomen) of several rat groups with different selenium status were measured by both the BFM and the in vivo EPR method, respectively. The nitroxyl radical clearance measured with in vivo EPR method may be affected not only by the redox status in the liver but also by information from other tissues in the measured region of the rat. On the other hand, the time course of nitroxyl radical level in the bile flow of rats was found to be a reliable index of redox status. Measurement site and/or volume limitation, which was achieved by the BFM method in this paper, is quite important in estimating reasonable EPR signal decay information as an index of tissue/organ redox status.

Triplet—Triplet Absorption in Sensitized Solvent Decomposition: An EPR Study

The decay of the steady-state electron paramagnetic resonance (EPR) signal from naphthalene triplet-state molecules in ether and other glasses under continuous near-ultraviolet irradiation at 77°K has been examined in some detail. It has been shown that free radicals are produced by sensitized solvent decomposition at a rate proportional to the rate of the disappearance of the triplet-state EPR signal. Also, it has been demonstrated that the latter decay process has a first-order dependence upon naphthalene concentration and that it exhibits a dependence upon excitation intensity, which is consistent with a stepwise two-photon absorption mechanism. The experimental results are discussed in terms of a model that involves the resonant transfer of energy from a highly excited triplet state of naphthalene to the solvent. Excitation of the excited triplet state takes place via the absorption of a photon by a naphthalene molecule in its lowest triplet state. The free radicals are produced by the decomposition of the sensitized solvent molecule. It is believed that the naphthalene molecule is not destroyed in this process but that the EPR signal decay arises from a radical—molecule interaction which effectively shortens the lifetime of the triplet state.