Estimation of the in vivo decay rate of EPR signals for a nitroxide radical in rat brains by a region-selected intensity determination method

The region-selected intensity determination (RSID) method was proposed to obtain the temporal changes in electron paramagnetic resonance (EPR) signal intensity from a selected region by a stationary magnetic field gradient. To select the region, the subtraction field that was derived from the distance between the center and the projection of the selected region to the direction of the field gradient was applied to the main field. The directions of the stationary magnetic field gradient at a constant strength were systematically changed in a three-dimensional space after each acquisition of the spectrum. All spectra under the field gradient were accumulated and the resultant spectrum was deconvoluted by a spectrum without the field gradient. The center height of the deconvoluted spectrum indicates the signal intensity of the selected region. To verify this method, a phantom or in vivo study was conducted on a 700 MHz radio-frequency EPR spectrometer equipped with a bridged loop-gap resonator. In the temporal EPR measurements of phantoms including a nitroxide radical aqueous solution with and without ascorbic acid, the selected regions were alternatively changed at the position of the two phantoms. The signal intensity derived from the one phantom showed an exponential decay, and for the other phantom, no temporal changes. The spatial resolution of this method was estimated to be 2.7 mm by using a pinpoint phantom that included diphenylpicrylhydrazyl powder. In the in vivo temporal EPR measurements, the selected regions were alternatively changed at the cerebral cortex and the striatum of rats that had received a blood-brain barrier-permeative nitroxide radical. The decay rate of the signal intensity at each region obtained by this method was consistent with those previously reported.     

In Vivo Temporal EPR Measurements in the Lung of Mice by a Selected-Region Intensity Determination Method

Selected-region intensity determination (SRID) is a method for obtaining the temporal changes in electron paramagnetic resonance (EPR) signal intensity from a selected region without complicated procedures used in the previous imaging method. We used an in vivo 700 MHz radiofrequency EPR spectrometer equipped with a bridged loop-gap resonator and the SRID method to perform temporal EPR measurements of the lung area of mice which had received a nitroxide radical (3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl) administration via the intravenous route. The half-life and initial level of nitroxide radical in the lung or the mediastinum were calculated from temporal changes in the signal intensity. A mathematical model was devised to determine the nitroxide radical concentration in the lung, which is connected to other organs via the circulatory system. Using this model and the results of the EPR measurements, the degrees of influence of the nitroxide reduction in the lung and other organs were simulated. It was found that the reaction rate (=log2/half-life) obtained from the lung mainly reflected the reduction of nitroxide radical there. Authors' address: Hidekatsu Yokoyama, National Institute of Advanced Industrial Science and Technology, 2266-98 Anagahora, Shimoshidami, Moriyama-ku, 463-8560 Nagoya, Japan     

Reaction kinetics analysis to estimate in vivo decay rate of EPR signals of a nitroxide radical in the brain and the inferior vena cava of rats

To clarify the degree of the influence of the peripheral organs on the temporal changes in the electron paramagnetic resonance (EPR) signal intensity of a nitroxide radical permeating the blood-brain barrier, 3-hydroxymethyl-2,2,5,5-tetramethylprrolidine-1-oxyl (hydroxymethyl-PROXYL), in the brain, temporal changes in the EPR signal in the brain or inferior vena cava of rats were measured by an in vivo 700 MHz radio-frequency EPR spectrometer equipped with a bridged loop-gap resonator or a surface-coil-type resonator. In all measurements, good linearity was observed on semilogarithmic plots of the signal intensity against time after the hydroxymethyl-PROXYL injection. From these plots, the reaction rate and the initial level of hydroxymethyl-PROXYL in the brain and the vena cava were calculated. A mathematical model expressing the nitroxide radical concentration in the brain, which is connected to other organs via the circulatory system, was made. With this model and the results of the EPR measurements, the degrees of influence of the nitroxide reduction in the brain and the other organs were simulated. It was found that the reaction rate (equal to log2/half-life) of hydroxymethyl-PROXYL observed in the brain reflected the reduction of hydroxymethyl-PROXYL there and was not influenced by the reduction in other organs.     

Combining a Magnetic Field Modulation Coil with a Surface-Coil-Type EPR Resonator

It is thought that the design of magnetic field modulation coils is one of the factors limiting enlargement of the sample size in electron paramagnetic resonance (EPR) measurements. In this study, we miniaturized the magnetic field modulation coil and combined it with a surface-coil-type resonator (SCR). The inductor of the SCR was a circular single-turn one-loop coil (diameter, 1 mm), and the magnetic field modulation coil was a twin-loop coil consisting of two solenoid coils each made of 15 turns of copper wire on a cylindrical bobbin with an axial length of 3 mm and an elliptical cross section (major axis, 7 mm; minor axis, 3 mm). The former was located on the latter via a spacer (thickness, 3 mm) in such a way that the directions of their axes coincided. Their combined size was about 10 mm wide, 10 mm deep, and 6 mm high. The transmission lines of the SCR were set on resonance at about 700 MHz. EPR measurements of a phantom (comprising agar that included a nitroxide radical and physiological saline solution), made with a miniaturized modulation coil combined with the SCR, exhibited a sensitivity similar to that for the conventional method. Authors' address: Hidekatsu Yokoyama, Department of Pharmaceutical Sciences, International University of Health and Welfare, 2600-1 Kitakanemaru, Ohtawara 324-8501, Japan     

EPR imaging to estimate the in vivo intracerebral reducing ability of mature rats after neonatal hypoxic-ischemic brain injury

A rat model of neonatal hypoxic-ischemic encephalopathy (Rice's model) was obtained by unilateral ligation of the common carotid artery of 7-day-old rats with hypoxia (exposure to 8% oxygen). To estimate the in vivo intracerebral reducing ability of the mature rats (8 weeks old) of Rice's model, temporal electron paramagnetic resonance (EPR) imaging of the brain of a rat receiving a blood-brain barrier-permeable nitroxide radical, 3-hydroxymethyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl, was performed. In this imaging technique, the decay rate of the EPR signal intensity in a selected region of the brain is indicative of region-specific reducing ability. The effect of neonatal treatment of an antioxidant agent, 3-methyl-1-phenyl-2-pyrazolin-5-one (MCI-186), after a hypoxic-ischemic insult was also tested. It was found that the reducing ability had been depleted in the contralateral hemisphere of Rice's model rats; this depletion was suppressed by administering MCI-186.     

Development of surface-coil-type resonators consisting of an irradiation and receiver coil system for EPR measurements at 700 MHz

Surface-coil-type resonators (SCRs) consisting of irradiation and receiver coils that are highly isolated from each other were developed for electron paramagnetic resonance (EPR) measurements. Their sample space was open to free space. For these coils, a circular single-turn one-loop receiver coil and a square single-turn twin-loop irradiation coil were fabricated. The transmission lines were set to resonate at about 700 MHz. A phantom (agar, including a nitroxide radical and physiological saline solution) is located on the receiver coil, and the irradiation coil is under the receiver coil. In this condition, the isolation between the receiver and irradiation SCRs was about 40 dB at the resonant frequency. When radiowaves that were divided from the line to the irradiation SCR were applied to the line from the receiver SCR at the appropriate phase and power to cancel the direct coupling between both SCRs, the isolation increased to more than 70 dB. In the conventional SCR, the noise level increased at high incident power. Because such an increase in the noise was not observed in the irradiation-receiver SCR system, high sensitivity at high incident power was obtained.     

Permethyl-β-Cyclodextrin Spin-Labeled with Nitronyl Nitroxide: Synthesis and EPR Study

A method for synthesis of new covalently linked spin-labeled cyclodextrin (CD) via the attachment of nitronyl nitroxide 2-(4-hydroxyphenyl)-4,4,5,5-tetramethyl-4,5-dihydro-1H-imidazole-3-oxide-1-oxyl to permethylated β-cyclodextrin is described. Electron spin echo envelope modulation (ESEEM) studies demonstrate that the new spin-labeled CD exhibits dynamic equilibrium between conformations with radical fragment capping the cavity of CD and radical fragment located outside the cavity. In solution, nitronyl nitroxide attached to CD retains its sensitivity to nitric oxide (NO), as reaction with NO leads to formation of iminonitroxide fragment evidenced by continuous-wave (CW) electron paramagnetic resonance (EPR). At the same time, CW EPR study of the reaction with ascorbic acid shows that the described binding of nitronyl nitroxide to CD does not provide higher stability of radical towards the reduction, and the corresponding rate constants are close to those obtained for free nitronyl nitroxide. Plausible explanations of these observations are discussed.     

In vivo temporal EPR imaging of the brain of rats by using two types of blood-brain barrier-permeable nitroxide radicals

In vivo temporal EPR imaging was conducted on the brain of rats that received one of two kinds of blood-brain barrier-permeable nitroxide radicals via the tail vein-one is a water-soluble 3-hydroxymethyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (hydroxymethyl-PROXYL); and the other is a non-water-soluble 3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (PCAM). From temporal EPR imaging data, temporal changes in the distribution of the nitroxide radical in the cerebral cortex, striatum, and hippocampus in the brain were investigated. It was found that the half-lives of the three parts in the brain of hydroxymethyl-PROXYL are longer and their EPR signal intensities are greater than those of PCAM.     

Determination of absolute concentration of nitroxide radical by radio-frequency EPR imaging

The absolute concentrations of a nitroxide radical in samples in a loop-gap resonator (LGR) were determined by using a radio-frequency (about 720 MHz) electron paramagnetic resonance (EPR) imaging system. EPR imaging of phantoms containing a nitroxide radical, 3-carbamoyl-2,2,5,5-tetramethylpyrrolidin-1-yloxy (carbamoyl-PROXYL), dissolved in various concentrations of an aqueous sodium chloride solution was made to investigate the influence of dielectric losses and sample position within the LGR. As it was found that these influences on the signal intensity were sufficiently small (less than 6%), it is possible to use identical radical solutions in which the radical is dissolved in a known concentration as an internal marker. Two phantoms containing aqueous solutions of 3 mM (as a marker) and 1, 2, 3, 4, or 5 mM (as a sample) carbamoyl-PROXYL were placed together in the LGR. From EPR images of these phantoms, the absolute concentration of the sample could be calculated by using the gray-scale value (i.e., the signal intensity) of the marker and sample within a small margin of error (about 4%).     

Rate constant measurements for initial addition reactions of radicals at the propagation step of photo‐polymerization as studied by pulsed EPR spectroscopy

Pulsed EPR spectroscopy was employed to determine reaction rate constants at an early stage of addition reactions in radical polymerizations triggered by four initiator radicals, which were generated by photodissociation of four parent molecules. Two monomers (tert‐butylacrylate and tert‐butylmethacrylate) were examined as reactant. Stern–Volmer analysis on the measured decay time of electron spin echo intensity of reacting radicals provides rate constants for addition reactions. We focused on rate constants for the second step reaction between monomer and adduct radical that is produced by the first step addition reaction between initiator radical and monomer. The rate constant measured by pulsed EPR was evaluated by theoretical calculations in the light of (1) enthalpy difference between product radical and reactants and (2) charge transfer interaction between reacting radical and monomer. Copyright © 2016 John Wiley & Sons, Ltd.     

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 (9.4 GHz) electron paramagnetic resonance (EPR) bile flow monitoring (BFM) and 300 MHz 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.     

In vivo temporal EPR study using a region-selected intensity determination method to estimate cerebral reducing ability in rats treated with olanzapine

Region-selected intensity determination (RSID) is a method for obtaining the temporal changes in electron paramagnetic resonance (EPR) signal intensity from a target region, without the use of complicated procedures employed in the conventional imaging methods. An in vivo 700-MHz radio frequency EPR spectrometer equipped with a bridged loop-gap resonator was used with the RSID method to estimate intracerebral reducing ability in the rat following acute administration of olanzapine (OZP) or haloperidol (HPD). To this end, temporal changes in EPR signal intensity of target regions (the striatum and the prefrontal cortex) of rats which had received a blood-brain-barrier-permeable nitroxide radical (3-hydroxymethyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl) via an intravenous route were observed. The half-lives of EPR signal intensity in both regions of OZP- or HPD-treated rats were significantly longer than in control animals. This indicated that reducing abilities of the striatum and cerebral cortex decreased in the rats to which either OZP or HPD had been acutely administered.     

CIDEP Created by the Quenching of Photo-Excited Tryptophan at Protein Surface: A Challenge to CIDEP Probing of Protein Structural Changes

Quenching of the triplet excited state of molecular tryptophan by nitroxide radical in 1,4-dioxane and water solutions was investigated by means of time-resolved electron paramagnetic resonance (EPR) and Fourier-transform (FT)-EPR. The chemically induced dynamic electron polarization (CIDEP) signals with net emissive phase were recorded at these quenching events and were analyzed through radical-triplet pair mechanism. The CIDEP time profiles were well reproduced by Bloch and kinetic equations, assuming radical-triplet pair mechanism with the appropriate quenching rate constants. From a comparison of the simulation and the experiment, CIDEP enhancement factor in 1,4-dioxane was determined to be −30 × P _eq, where P _eq is the spin polarization of nitroxide at thermal equilibrium. Net emissive CIDEP was also observed by FT-EPR measurements on the nitroxide quenching of the triplet excited state of tryptophan residue in α-lactalbumin. Magnitude of CIDEP created in α-lactalbumin/nitroxide system depends on the pH condition of α-lactalbumin solution, which is related to protein folding dynamics. We argue the CIDEP mechanism at the α-lactalbumin surface and propose a possibility of a novel CIDEP method to probe a protein surface and structural changes.     

Modified surface-coil-type resonators for EPR measurements of a thin membranelike sample

Surface-coil-type resonators (SCRs) equipped with a circular single-tum coil (conventional SCR), a circular spiral coil (spiral SCR), and a plate-type single-turn coil (plate-type SCR) were fabricated. By using these SCRs, the electron paramagnetic resonance (EPR) sensitivities of thin membranelike samples were investigated. For a non-dielectric-loss phantom, filter paper containing 1,1-diphenyl-2-picrylhydrazyl was used. For a high-dielectric-loss phantom, gauze containing an aqueous solution of 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (carbamoyl-PROXYL) was used. For a biological sample, a pea leaf impregnated with the carbamoyl-PROXYL solution was used. The sensitivity (signal-to-noise ratio) of the spiral and plate-type SCRs for the non-dielectric-loss phantom was significantly greater than that of the conventional SCR. Under these conditions, the sensitivity of the spiral SCR was relatively higher than that of the plate-type SCR. For the high-dielectric-loss phantom, the sensitivity of the plate-type SCRs was significantly greater than that of the conventional SCR, but there were no differences in sensitivity between the spiral and conventional SCRs. The sensitivity of the plate-type SCR in the EPR measurement of a pea leaf was significantly greater than that of the conventional SCR. These findings show that the spiral and plate-type SCRs are suitable for measuring EPR of thin membranelike samples, especially when the former is used for the non-dielectric-loss sample and the latter for high-dielectric-loss sample, including the leaf.     

Time-resolved EPR studies on magnetic interactions between excited triplet (tetraphenylporphinato) zinc and doublet nitroxide radical

By time-resolved electron paramagnetic resonance (TREPR), four (tetraphenylporphinato) zinc (ZnTPP) complexes coordinated by an axial ligand containing a nitroxide radical (NRX; X=4, 5, 8, and 10, denotes the bond number from zinc to nitroxide nitrogen) have been studied in terms of magnetic interactions between the photoexcited triplet state of ZnTPP and NRX. The TREPR spectrum of ZnTPP coordinated by NR10 is almost the same as the one of ZnTPP coordinated by pyridine, indicating that the electron exchange interaction,J, between ZnTPP and the doublet nitroxide is negligibly small. On the other hand, TREPR spectra of the NR4 and NR5 complexes are assigned to the Q₁ state constituted by the ZnTPP and the nitroxide radical. In the case of the ZnTPP-NR8 complex, both T₁ and Q₁ TREPR signals are seen, which may originate from two conformations or degenerate T₁ states of ZnTPP. This EPR study is useful for understanding the photophysical and photochemical properties of chromophores.     

Noninvasive mapping of the redox status in septic mouse by in vivo electron paramagnetic resonance imaging

Increased reactive oxygen species (ROS) contribute to numerous brain disorders, and ROS generation has been examined in diverse experimental models of lipopolysaccharide (LPS)-induced inflammation. The in vivo electron paramagnetic resonance (EPR)/nitroxide spin probe method has been used to analyze the redox status in animal models modulated by ROS generation. In this study, a blood–brain barrier (BBB)-permeable nitroxide spin probe, 3-hydroxymethyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (HMP), was used as a redox-sensitive nitroxide probe. Magnetic resonance images of mouse head after the injection of HMP showed that HMP was distributed throughout all regions of the mouse head including the brain, suggesting that HMP can reveal redox information in all regions of the mouse head. After the injection of HMP through the mouse tail vein 6 h after the injection of LPS, three-dimensional (3D) EPR images were obtained each minute under a field scanning of 0.3 s and with 81 projections. The reduction reaction of HMP in septic mouse heads was remarkably accelerated compared to that in control mice, and this accelerated reaction was inhibited by aminoguanidine and allopurinol, which inhibit enzymatic activities of induced nitric oxide synthase and xanthine oxidase, respectively. Based on the pharmacokinetics of HMP in mouse heads, the half-life mapping of HMP was performed in LPS-treated mouse head. Half-life maps clearly show a difference in the redox status induced by ROS generation in the presence or absence of inhibitors of ROS-generating enzymes. The present results suggest that a 3D in vivo EPR imaging system combined with BBB-permeable HMP is a useful noninvasive tool for assessing changes in the redox status in rodent models of brain disease under oxidative stress.     

Direct EPR irradiation of a sample using a quartz oscillator operating at 250 MHz for EPR measurements

Direct irradiation of a sample using a quartz oscillator operating at 250 MHz was performed for EPR measurements. Because a quartz oscillator is a frequency fixed oscillator, the operating frequency of an EPR resonator (loop-gap type) was tuned to that of the quartz oscillator by using a single-turn coil with a varactor diode attached (frequency shift coil). Because the frequency shift coil was mobile, the distance between the EPR resonator and the coil could be changed. Coarse control of the resonant frequency was achieved by changing this distance mechanically, while fine frequency control was implemented by changing the capacitance of the varactor electrically. In this condition, EPR measurements of a phantom (comprised of agar with a nitroxide radical and physiological saline solution) were made. To compare the presented method with a conventional method, the EPR measurements were also done by using a synthesizer at the same EPR frequency. In the conventional method, the noise level increased at high irradiation power. Because such an increase in the noise was not observed in the presented method, high sensitivity was obtained at high irradiation power.     

Resolving Conformational and Rotameric Exchange in Spin-Labeled Proteins Using Saturation Recovery EPR

The function of many proteins involves equilibria between conformational substates, and to elucidate mechanisms of function it is essential to have experimental tools to detect the presence of conformational substates and to determine the time scale of exchange between them. Site-directed spin labeling (SDSL) has the potential to serve this purpose. In proteins containing a nitroxide side chain (R1), multicomponent electron paramagnetic resonance (EPR) spectra can arise either from equilibria involving different conformational substates or rotamers of R1. To employ SDSL to uniquely identify conformational equilibria, it is thus essential to distinguish between these origins of multicomponent spectra. Here we show that this is possible based on the time scale for exchange of the nitroxide between distinct environments that give rise to multicomponent EPR spectra; rotamer exchange for R1 lies in the ≈0.1–1 μs range, while conformational exchange is at least an order of magnitude slower. The time scales of exchange events are determined by saturation recovery EPR, and in favorable cases, the exchange rate constants between substates with lifetimes of approximately 1–70 μs can be estimated by the approach.     

Contributions of Exchange and Dipole–Dipole Interactions to the Shape of EPR Spectra of Free Radicals in Diluted Solutions

This report presents a comprehensive analysis of the contributions of the Heisenberg exchange and dipole–dipole interactions in diluted solutions of nitroxide radicals to the shape of their electron paramagnetic resonance (EPR) spectra taking into account all coherence transfer processes. It is shown that these contributions interfere. The approaches to obtain the molecular-kinetic and exchange integral parameters by analyzing the EPR spectrum dependence on the radical concentration and the solvent viscosity are discussed.     

In vivo EPR imaging by using an acyl-protected hydroxylamine to analyze intracerebral oxidative stress in rats after epileptic seizures

EPR imaging by using an acyl-protected hydroxylamine, 1-acetoxy-3-carbamoyl-2,2,5,5-tetramethylpyrrolidine (ACP), in the head of a living rat after kainic acid (KA)-induced epileptic seizures was performed. ACP is a stable non-radical compound, but is easily deprotected with intracellular esterase to yield a hydroxylamine, which is oxidized by intracellular oxidative stress to yield an EPR-detectable nitroxide radical. From in vivo image data, the average values of EPR signal intensity from the hippocampus, striatum, and cerebral cortex were computed. There was no significant difference in cortical signal intensity between the control and KA-treated rats. The signal intensities from the hippocampus and striatum for the KA-treated rats were significantly higher than those for the control. The in vitro study showed that almost the same quantity of ACP moved into all regions of the brain of the control and KA-treated rats. These findings indicate that following a KA-induced seizure, the oxidative stress in the hippocampus and striatum is enhanced, but not so in the cerebral cortex.