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Magnetic nanoparticles are extensively studied for their use in diagnostics and medical therapy. The behavior of nanoparticles after adding them to cell culture is an essential factor (i.e., whether they attach to a cell membrane or penetrate the membrane and enter into the cell). The present studies aimed to demonstrate the application of electron spin resonance (ESR) as a suitable technique for monitoring of nanoparticles entering into cells during the endocytosis process. The model nanoparticles were composed of magnetite iron (II, III) oxide core functionalized with organic unit containing nitroxide radical 4-hydroxy-TEMPO (TEMPOL). The research studies included breast cancer cells, as well as model yeast and human microvascular endothelial cells. The results confirmed that the ESR method is suitable for studying the endocytosis process of nanoparticles in the selected cells. It also allows for direct monitoring of radical cellular processes.

Glutathione (GSH) is the most abundant non-protein thiol that buffers reactive oxygen species in the brain. GSH does not reduce nitroxides directly, but in the presence of ascorbates, addition of GSH increases ascorbate-induced reduction of nitroxides. In this study, we used electron paramagnetic resonance (EPR) imaging and the nitroxide imaging probe, 3-methoxycarbonyl-2,2,5,5-tetramethyl-piperidine-1-oxyl (MCP), to non-invasively obtain spatially resolved redox data from mouse brains depleted of GSH with diethyl maleate compared to control. Based on the pharmacokinetics of the reduction reaction of MCP in the mouse heads, the pixel-based rate constant of its reduction reaction was calculated as an index of the redox status in vivo and mapped as a "redox map". The obtained redox maps from control and GSH-depleted mouse brains showed a clear change in the brain redox status, which was due to the decreased levels of GSH in brains as measured by a biochemical assay. We observed a linear relationship between the reduction rate constant of MCP and the level of GSH for both control and GSH-depleted mouse brains. Using this relationship, the GSH level in the brain can be estimated from the redox map obtained with EPR imaging., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Purpose: Electron paramagnetic resonance spectroscopy promises quantitative images of important physiologic markers of animal tumors and normal tissues, such as pO(2), pH, and thiol redox status. These parameters of tissue function are conveniently reported by tailored nitroxides. For defining tumor physiology, it is vital that nitroxides are selectively localized in tumors relative to normal tissue. Furthermore, these paramagnetic species should be specifically taken up by cells of the tumor, thereby reporting on both the site of tumor formation and the physiological status of the tissue. This study investigates the tumor localization of the novel nitroxide, cis-3,4-di(acetoxymethoxycarbonyl)-2,2,5,5-tetramethyl-1-pyrrolidin-yloxyl 3 relative to the corresponding di-acid 4., Methods: We obtained images of nitroxide 3 infused intravenously into C3H mice bearing 0.5-cm(3) FSa fibrosarcoma on the leg, and compared these with images of similar tumors infused with nitroxide 4., Results: The ratio of spectral intensity from within the tumor-bearing region to that of normal tissue was higher in the mice injected with 3 relative to 4., Conclusion: This establishes the possibility of tumor imaging with a nitroxide with intracellular distribution and provides the basis for EPR images of animal models to investigate the relationship between crucial aspects of tumor microenvironment and malignancy and its response to therapy., (Copyright © 2013 Wiley Periodicals, Inc.)

Antioxidative effects of sesamin (a mixture of sesamin and episesamin) were evaluated in the liver, kidney and inferior vena cava of living rats using a radiofrequency ESR method. TEMPOL, 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl, was used as an in vivo redox probe, the half-life of which is believed to be correlated with the antioxidant status. The oral administration of sesamin (250 mg/kg rat weight) 3 h before ESR measurements shortened the half-life of TEMPOL in the liver by 10 - 15% as compared with the controls, but did not affect the other organs. This effect was maintained for at least 3 h after the administration, and then disappeared at 24 h, corresponding to the results of our preliminary pharmacokinetic studies. Changes in the reducing ability were observed only in the hepatic sites of the sesamin-treated rats. These findings suggest that sesamin exhibits effective antioxidant activity in the liver via modulation of the intracellular redox status related to TEMPOL reduction.

Overhauser-enhanced MRI (OMRI) offers the potentiality of detecting low-concentrated species generated by specific biological processes. However molecular imaging applications of OMRI need significant improvement in spatial localization. Here it is shown that 3D-OMRI of a free radical injected in tumor-bearing mice can be performed at high anatomical resolution at a constant field. A 30 mm cavity operating at 5.43 GHz was inserted in a C-shaped magnet for proton MRI at 0.194 T. Nude mice with or without brain-implanted C6 rat glioma were positioned in the cavity and injected with TOPCA (1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrole-3-carboxylic acid). OMRI was performed in 3D within several minutes in the brain region without high overheating of the animals. Voxel size was 0.5 × 0.5 × 1 mm³ , providing good delineation of brain regions. Signal amplifications ranged from 2 in tumors to 10 in vessels several minutes after TOPCA injection. Time-course of signal enhancement could be measured by 2D OMRI at 15 s time intervals in a localized thin slice. The method opens the way for molecular imaging of biological activities able to generate OMRI-visible free radicals., (Copyright © 2012 John Wiley & Sons, Ltd.)

For the imaging of low pH circumstances in vivo, a pH-sensitive radical-containing-nanoparticle (RNP), which has an intense electron paramagnetic resonance (EPR) signal, was designed and developed using a self-assembling amphiphilic block copolymer (PEG-b-PCTEMPO) composed of a hydrophilic poly(ethylene glycol) (PEG) segment and a hydrophobic poly(chloromethylstyrene) (PCMS) segment in which the chloromethyl groups were converted to 2,2,6,6-tetramethylpiperidinyloxys (TEMPOs) via the amination of PEG-b-PCMS block copolymer with 4-amino-TEMPO. This RNP formed core-shell-type micelles in the physiological environment, and the cumulant average diameter of the RNP was about 50 nm. The cytotoxicity and acute toxicity studies for the RNP revealed that the median inhibitory concentration (IC(50)) of TEMPO radicals in RNP core and median lethal dose (LD(50)) of RNP were >8 mmol N(TEMPO)/L and >600 mg/kg (>960 mumol N(TEMPO)/kg), respectively, indicating fairly low toxicity. The blood circulation of the RNP was evaluated using ICR mice. Contrary to the rapid clearance of low-molecular-weight TEMPO derivatives such as 4-hydroxy-TEMPO (TEMPOL) from the bloodstream, the EPR signal of the RNP remained for a fairly long period of time. Actually, the signal was observed in the blood for more than 2 h, as monitored by EPR spectroscopy. The compartmentalization of the TEMPO radicals in the RNP core improved the stability in the bloodstream. Since an amino group was introduced in each repeating unit of the PCTEMPO segment, the disintegration of the RNP was caused by the protonation of the amino groups in response to the acidic pH environment (pH < 6.0), as confirmed by the dynamic light scattering (DLS) measurements. In addition, a drastic change in the EPR spectra from broad to sharp triplet was observed, accompanying the disintegration. This change was based upon the mobility of the TEMPO moieties covalently conjugated in the hydrophobic segment, which was confirmed by the rotational correlation time of the TEMPO moieties on the PCTEMPO segment. Note that the peak intensity of the EPR signal increased at around the phase transition point (ca. pH = 6.0). When pH-sensitive RNP solutions at pH values 5.6 and 7.4 were visualized using an L-band EPR imaging system, the phantom images showed a remarkable on-off regulation in response to the acidic pH environment. These results demonstrate that pH-sensitive RNPs are expected to serve as nanoprobes for the in vivo EPR imaging of low pH circumstances.

A set of L-band electron spin resonance imaging (ESRI) equipment suitable for biological species was developed and an ESRI experiment model for viable skin samples was established. The mechanic process of nitroxide free radical TEMPO (2,2, 6, 6-tetramethyl-1-piperidinyloxy) penetrating through skin sample and the spin density distribution of TEMPO after it interacted with skin sample were detected by the developed ESRI method. Skin samples were extracted from mice back. The experimental samples were prepared by cutting the skin pieces into square shape of 2 x 2 cm2 and then the samples were divided into three groups by treating them with three different methods: Method A, simple treatment by simply cutting the hair; method B, 8% Na2S depilation treatment for 10 min; method C, 8% Na2S depilation and then 5% pancreatic digestion treatment for 2 hours. The liposoluble solvent DMSO (dimethyl sulfoxide) and distilled water were used as two kinds of solvent for the TEMPO liquor. The results indicated that the skin-penetration properties of TEMPO were significantly different among samples treated with different methods and the surface cornifin of skin offered remarkable resistance to TEMPO. The TEMPO liquor of water could hardly penetrate through skins, whereas about 20%-30% of the original TEMPO compounds that solved in liposoluble solvent DMSO could penetrate through the skin sample treated with method C after 16 hours of interaction. Furthermore, the penetration rate of TEMPO through the skin tissue was a strong time dependent process. The preliminary application results suggested that ESRI technique could provide an effective and applicable method for dynamically researching skin-penetration properties of some special kinds of materials such as paramagnetic compounds.

Electron paramagnetic resonance imaging (EPRI) enables noninvasive spatial mapping of free radical metabolism and has recently been shown to provide in vivo physiologic information regarding alterations in the redox state of tumors and neoplastic tissues. With the use of nitroxide spin probes, it has been shown that certain tumors possess a highly reduced state. To determine whether EPRI can be used for early detection and visualization of gastric carcinoma based on its altered redox metabolism, studies were performed in a rat gastric cancer model induced by 1-methyl-3-nitro-1-nitrosoguanidine. Using a specialized 750 MHz resonator and EPRI instrument, a technique was developed for imaging nitroxide radicals in the whole stomach. In vivo three-dimensional EPRI of the stomach of rats with continuous intravenous administration of nitroxide 3-carboxamido-2,2,5,5-tetramethylpyrrolidine-N-oxyl (3-carbamoyl-proxyl) [3-CP] was performed. Whereas electron paramagnetic resonance images from untreated controls provide a uniform visualization of the stomach mucosa and wall, in the treated rats with gastric cancer, holes were present in the image at the locations of tumors. With localized spectroscopy, it was confirmed that the tumor regions were devoid of signal, and this was largely due to the presence of a more reduced state with rapid reduction of nitroxide. Pharmacokinetic studies indicated that 3-CP in tumors was rapidly reduced to an undetectable level, whereas the 3-CP levels in normal stomach tissue persisted. Near-infrared reflectance measurements of indocyanine green dye uptake indicated that there were no significant differences in tumor versus normal mucosal perfusion. From these results, we concluded that gastric cancer tumors could be distinguished from normal tissue based primarily on the marked difference in their rate of radical metabolism. Because alterations in cellular redox state and radical metabolism are of critical importance in tumor biology and treatment, this methodology should provide an important new tool for the study and visualization of gastric carcinoma and may also be of use in other cancer models.

We present a method for measuring the transmembrane pH difference (deltapH=pHin-pHout) in chloroplasts with a spin label TEMPAMINE (4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl) accumulating inside the thylakoids in response to generation of deltapH. Experiments with chloroplasts suspended in the media of different osmolarity demonstrated that most of TEMPAMINE (TA) molecules taken up by chloroplasts were localized in the bulk of the thylakoid lumen. The DeltapH value was determined from the relationship deltapH=lg([H+]in/[H+]out) approximately equal to lg(Cin/Cout), where Cin and Cout are the concentrations of TA inside and outside the thylakoids, respectively. To quantify the internal concentration Cin, we used the threshold nature of the concentration-dependent broadening of the EPR signal from TA. It was demonstrated that spin-exchange interactions between TA molecules caused an observable broadening of the signal only when the concentration of TA exceeded the threshold level, [TA]theta approximately 2.0-2.2mM. The concentration dependencies of the signal parameters (the peak-to-peak amplitude, App, and the linewidth, deltaHpp) were described within a model of the non-homogeneous broadening of an unresolved hyperfine multiplet from the protons of TA molecule. If the concentration of TA inside the thylakoids went beyond the threshold level, the spin-exchange broadening of the EPR signal was accompanied by a reversible decrease in the signal height (parameter deltaA). By measuring the signal behavior at different levels of microwave power, we were able to discriminate between the line broadening effects caused by concentrating TA molecules inside the thylakoids or the light-induced changes in the concentration of oxygen. We developed a general algorithm for determination of the deltapH value and the internal volume of thylakoids, Vin, from the non-linear dependence of parameter deltaA on the concentration C0 of TA in a chloroplast suspension. Advantages of this method are: (i) it avoids the use of a broadening agent; (ii) it allows the internal volume of thylakoids to be evaluated; and (iii) the concentrations of TA used to measure the deltapH are below the range of concentrations that could cause the uncoupling electron transport to ATP synthesis in chloroplasts. Results of our measurements are consistent with the literature data on deltapH determinations by other methods.

The temporal changes in the electron paramagnetic resonance (EPR) signal intensities of a nitroxide radical, 4-hydroxy 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL), in the kidney in rat puromycin aminonucleoside (PAN) nephrosis were investigated in vivo and in vitro. The rats of the PAN nephrosis group received intraperitoneal injections of PAN at 75 mg/kg body weight while those of control group received saline. The in vivo renal half-lives of TEMPOL were calculated from the decay curve of EPR signal intensities after the intravenous injection of the TEMPOL solution. The mitochondrial half-lives were obtained from the decay curve of the EPR signals after mixing the mitochondrial fraction of the kidney and TEMPOL solution. The in vivo half-lives of TEMPOL of the kidney from 7 to 14 d after PAN administration were significantly longer than those of the controls. The mitochondrial half-lives of TEMPOL on the 9th day after the PAN administration prolonged remarkably compared to the controls (378 +/- 69 vs. 676 +/- 183 s, p <.01). These findings indicate that the in vivo and mitochondrial reducing activity in PAN treated rats decreased markedly, because the half-life of TEMPOL in the kidney reflects the renal reducing activity.

In vivo ESR spectroscopy using a low frequency microwave of approximately 1 GHz has been developed to measure non-invasive ESR spectra in animals given paramagnetic compounds, in which a loop-gap-type resonator was used and ESR spectra were measured at the animal's head or abdomen. Therefore, the concentrations of paramagnetic species in both the blood and organs were compositely contributed to the spectra. When we understand the kinetics of paramagnetic species in detail, it is essentially important to know how these kinetics are expressed in each organ. For this purpose, a surface-coil-type resonator, which enabled local ESR measurement in specific organs, has been developed. By using this method, we studied the real-time pharmacokinetics of spin clearance curves detected in the organs of mice given 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (4-hydroxy-TEMPO) intravenously (i.v.), by monitoring the inferior vena cava, liver and kidney. Quantified dearance curves in the organs were analyzed on the basis of a two-compartment model, and pharmacokinetic parameters were estimated based on the curve-fitting. The obtained pharmacokinetic parameters were found to depend on the measurement site, and the distribution and elimination processes of the spin probe were successfully separated between the blood and organs of mice.

In vivo electron spin resonance (ESR) spectroscopy and whole body imaging were used to investigate the toxicity of biological reactions and organ specific oxidative changes associated with the development of acute asbestosis. Pathogen-free mice were exposed to 100 microg of crocidolite asbestos suspended in 50 microL of a 0.9% NaCl solution by aspiration. The bio-assay group had broncho-alveolar lavage (BAL) and serum draws performed on control and treated mice at 1, 3, and 7 days post-instillation. The ESR spectroscopic measurements and whole body imaging were performed with a separate group of mice at the same time points. Bio-assays included measurements of albumin, lactate dehydrogenase (LDH), N-acetyl-beta-D-glucoaminidase (NAG), and catalase in acellular lavage fluids, and total antioxidants status in blood serum. ESR spectroscopic and imaging measurements were performed after intraperitoneal injection of 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-15N-1-oxyl (TEMPOL) or 3-carbamoylproxyl (3-CP) nitroxides at a final concentration of 344 mg/kg body weight. Albumin showed a significant increase in BAL fluid at the 3 day exposure time point. The presence of this protein in lavage fluid indicates that the gas/blood barrier has been damaged in the lung. LDH in BAL fluid also exhibited a significant increase at 3 days post-exposure, an indication of enhanced cell membrane damage in the lung. Similar results were observed for NAG, a lysosomal enzyme, implying activation of phagocytic cells. Contemporaneously with the development of acute asbestosis at day 3 post-exposure, there were significant increases in the levels of total antioxidants in the serum and catalase in the BAL fluid. Significant impairment in the ability of asbestos exposed animals to clear TEMPOL radical during acute disease progression was evident at days 1 and 3 post exposure. ESR image measurements provided information on the location and distribution of the 3-CP label within the lungs and heart of the mouse and its clearance over time. Bioassays in concert with ESR spectroscopy and imaging presented in this study provide congruent data on the early acute phase of pulmonary injury and oxidant generation in response to asbestos exposure and their decline after 7 days. The increased levels of total antioxidants in the serum and catalase in BAL fluid correlated with the reduction in the clearance rate for TEMPOL, suggesting that a change in the redox status of the lung is associated with lung injury induced by asbestos.

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.

The kinetics of a nitroxide radical in the renal parenchyma and pelvis in rats were investigated by employing an in vivo EPR imaging system equipped with a surface-coil-type resonator (SCR). The exposed kidney of a living rat was inserted into the single-turn coil of the SCR, with the renal major axis aligned with the direction of alternative magnetic field (B(1)). After the injection of nitroxide radical via the tail vein, EPR measurements were repeated. From the temporal EPR images of the kidney on the 2-D projection to the plane which is perpendicular to the direction of B(1,) the decay rate of nitroxide radical in the renal parenchyma and pelvis was estimated. The parenchymal decay rate was found to be significantly shorter than that for the pelvis.

Free radical species in animals have been measured by X-band ESR spectrometric method on a block of organs or a portion of homogenized samples. However, a nondestructive in vivo ESR measurement has been realized by using a recently developed L-band ESR spectrometry. With this L-band ESR method, we measured ESR spectra in animals, who received stable nitroxide radicals. L-band ESR spectra were observed at the upper abdomen of mice as well as at the heads of mice and rats at various ages immediately after the intravenous injections of nitroxide radicals such as 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (4-hydroxy-TEMPO) and 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (3-carbamoyl-PROXYL), in which ESR measurements of the radicals were performed noninvasively at the real time. On the basis of the observed time-dependent free radical clearance curves, the following important results were obtained: (1) Free radical clearances were able to analyze by the pharmacokinetic method. (2) The radicals at the head of mice, given 4-hydroxy-TEMPO, were determined quantitatively by a new analytical method using L-band ESR for the first time. (3) The elimination of the radical was found to be saturated in mice. (4) The clearance rate constant of 4-hydroxy-TEMPO detected at the head of mice was decreased in dose- and age-dependent manners. While, no age-dependent clearance rate constant of 4-hydroxy-TEMPO was observed at the upper abdomen of mice. (5) Ratios of the amount of the detected radicals to that of the administered radicals were decreased age-dependently, but they were independent of the dose of the radicals, suggesting the age-dependent decrease of distribution capacity ratio of the radical at the head of animals. (6) Clearance rate constants of 4-hydroxy-TEMPO and 3-carbamoyl-PROXYL, that were estimated by X- and L-band ESR for the collected blood of mice and rats, were found to be remarkably smaller than those in whole living animals observed by in vivo L-band ESR method. The results suggest that the clearance of the nitroxide radical is relevant to the alteration of the radical in animals following the change of organ distribution and metabolism. (7) Both the radical and its corresponding hydroxylamine, which is the reduced form of the radical, were detectable by X-band ESR method in the collected urine of mice and rats without and with an oxidizing agent, respectively. On the basis of the results on L-band ESR spectrometry, the first quantitative pharmacokinetic analysis of stable spin probes in animals is proposed.

Piperidine nitroxides have considerable clinical potential, both as antioxidant therapeutic compounds and contrast agents in magnetic resonance imaging. However, their development has thus far been limited by their rapid bioreduction in vivo. Recently, it was reported that polynitroxyl albumin (PNA) can reverse the bioreduction of the reduced 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (Tempol) in the rat heart, enabling the performance of high resolution EPR imaging for prolonged time (Kuppusamy et al., Biochemistry 35, 7051-7057 (1996)). In this report, the efficacy of PNA in maintaining Tempol concentrations in vivo in mice was demonstrated, using L-band (1.25 GHz) EPR spectroscopy and imaging. The EPR signal of intravenous Tempol had a half-life of 1.0+/-0.2 min and became undetectable within 6 min. Subcutaneous Tempol, however, decayed at a slower rate (half-life, 5.0+/-0.5 min) suggesting that Tempol had been bioreduced to the corresponding hydroxylamine form, Tempol-H. Subcutaneously injected PNA restored 20% of the Tempol signal in the vicinity of the PNA deposit. In vivo topical EPR imaging demonstrated that the Tempol signal was restored at the site of PNA injection, but not at locations remote from the PNA injection site. The ability of PNA to maintain Tempol in its paramagnetic state in vivo should enable a wide range of therapeutic and diagnostic applications of piperidinyl nitroxides.

Rats were given vitamin E (Vit-E), idebenone (ID), or vitamin C (Vit-C) in their food for 2 or 4 weeks. After feeding, the ability of rats to reduce 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (Tempol) in terms of the half-life of Tempol was examined as a specific marker. Tempol was repeatedly injected intravenously, and its half-life was serially evaluated by an in vivo electron spin resonance (ESR) technique. The radical-reducing ability in rats was enhanced differently by Vit-E, ID, and Vit-C, i.e., slow onset of the ability after Vit-E and ID (lipid-soluble antioxidants) and fast onset after Vit-C (a water-soluble antioxidant).

The use of RF (100 to 300 MHz) PEDRI and CW-EPR techniques allows the in vivo study of large animals such as whole rats and rabbits. Recently a PEDRI instrument was modified to also allow CW-EPR spectroscopy with samples of similar size and under the same experimental conditions. In the present study, this CW-EPR and PEDRI apparatus was used to assess the feasibility of the detection of a pyrrolidine nitroxide free radical (2,2,5,5,-tetramethylpyrrolidine-1-oxyl-3-carboxylic acid, PCA) in the abdomen of rats. In particular, we have shown that after the PCA administration (4 mmol kg(-1) b.w.): (i) the PCA EPR linewidth does not show line broadening due to concentration effects; (ii) a similar PCA up-take phase is observed by EPR and PEDRI; and (iii) the PCA half-lives in the whole abdomen of rats measured with the CW-EPR (T1/2=26+/-4 min, mean+/-sd, n=10) and PEDRI (T1/2=29+/-4 min, mean+/-sd, n=4) techniques were not significantly different (p > 0.05). These results show, for the first time, that information about PCA pharmacokinetics obtained by CW-EPR is the same as that from PEDRI under the same experimental conditions.

Electron paramagnetic resonance imaging (EPRI) is currently being developed at frequencies between 200 MHz and 2 GHz. EPRI can map the in vivo distribution of paramagnetic species, such as water soluble free radicals; nitroxide free radicals are commonly used. EPR images reflect the complexity of metabolic actions on the exogenous delivered nitroxides. Their reduction rate in vivo is affected by parameters such as oxygen concentration, pH and biodistribution. This paper illustrates the main features of low frequency EPRI and reconstruction techniques. Examples of EPR imaging, such as two-dimensional (2D) spatial mapping of the distribution of a nitroxide free radical in phantoms and in whole rat, are given.

The flexible surface-coil-type resonator (FSCR) operating in a 700 MHz microwave electron spin resonance (ESR) system was applied to measure the nitroxide radicals at a specific area in rats. The FSCR was composed of a single-turn coil with a diameter of 5 mm and two flexible coaxial lines of 450 mm in length. For an endoscope-like application, the FSCR was inserted into the rectum of the rat and sequential changes in the ESR signals of the intravenously injected nitroxide radical (4-hydroxy-2,2,6,6-tetramethyl-piperidine-1-oxyl; TEMPOL or 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-yloxy; carbamoyl-PROXYL) were measured. The ESR signal intensity of the nitroxide decreased according to first-order kinetics. For a stethoscope-like application, the FSCR was placed at several sites on the abdominal skin of the rats receiving a subcutaneous injection of carbamoyl-PROXYL, and diffusion and/or metabolism of the radical in the skin was observed.

The nitroxide group 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) was covalently bound to a dextran via an N-acetamido linkage to produce a novel spin probe, TEMPO-dextran (TEMPO-DX), which circulates for long time periods in an animal without metabolic degradation. TEMPO-DX was stable in mice, while small TEMPO analogs quickly disappeared after administration. Since dextran was reduced somewhat in size during synthesis, the resultant spin-labeled dextran could be excreted through the kidney. A strong L-band electron spin resonance signal was obtained shortly after intravenous administration of TEMPO-DX into the tail vein of a mouse, from which three-dimensional images of specific organs were calculated. The signal was found to persist for well over 1 h.

Electron spin resonance (ESR) measurements after intracerebroventricular injection of a nitroxide radical were carried out in rats (n = 6) that received oral idebenone for 2 weeks and in control rats (n = 5), using an in vivo rapid scan ESR spectrometer. The half-life of nitroxide, which was estimated from the change in the peak height (delta M = +1) of the ESR signals from the head, was used as a marker for the elimination of the nitroxide radical. The half-life in the rats treated with idebenone was significantly shorter than it was in the controls (p < 0.05). This finding indicates that the treatment with idebenone can enhance the intracerebral-eliminating ability of the nitroxide radical.

A novel, very-low-frequency electron paramagnetic resonance (EPR) technique is used to image the distribution of several nitroxides with distinct pharmacologic compartment affinities in the abdomens of living mice. Image acquisition is sufficiently rapid to allow a time sequence of the distribution for each compound. The spectra and concentrations of these nitroxides are imaged with the use of spectral-spatial imaging to distinguish a single spatial dimension. Liver and bladder of the mouse anatomy are distinguished by this technique. After an intraperitoneal injection of the spin-label probes, a shift in the distribution of the compounds from the upper abdomen (primarily liver) to the lower abdomen (primarily bladder) is observed. The time dependence of the shift in regional distribution depends on the structural properties of the side chain attached to the spin label. These results indicate that this application of in vivo electron paramagnetic resonance imaging will provide a new method of magnetic resonance imaging for determination of pharmacodynamics in the body of an intact animal.

Images of nitroxide radicals were obtained from the brains of living rats following intracarotid injection of imaging agent, [2-(14-carboxytetradecyl)-2-ethyl-4,4-dimethyl-3-oxazolidinylox y] (16 DS) using L-band electron spin resonance computed tomography (ESR-CT). The image patterns obtained from the 3-carbamoyl-2,2,5,5-tetramethyl-1-pyrrolinyloxy injected rats showed the agent in tissues of the cranium but not within the brain. The uptake of 16 DS was found in the cortex, hippocampus, striatum, midbrain, pons medulla oblongata and cerebellum, and nuclei, mitochondria, synaptosomes of the cerebrum after intracarotid injection of 16 DS using an x-band ESR spectrometer. These results suggest that 16 DS penetrates the blood brain barrier.

A radio frequency (RF) (280 MHz) electron paramagnetic resonance (EPR) spectroscopy and imaging apparatus has been used to localize a pyrrolidine nitroxide free radical in the rat abdomen and thorax. The nitroxide 2,2.5.5,-tetramethylpyrrolidine-1-oxyl-3- carboxylic acid (PCA) had a whole body monoexponential decay with half-life of 13.3 +/- 0.7 (n = 4), 19.4 +/- 0.2 (n = 3), and 23 +/- 2 (n = 6) min for 1, 2, and 3 mmol/kg PCA, respectively. Up to seven one-dimensional longitudinal projections were collected on six rats in the presence of a 8 mT/m field gradient. With an injection dose of 3 mmol/kg, PCA half-lives were 19 +/- 1, 17 +/- 2, and 22 +/- 2 min (n = 6) in the lower abdomen, in the liver, and in the thorax, respectively. Thorax half-life was significantly longer than liver half-life. Sequential two-dimensional images of PCA distribution in a plane longitudinal to the rat body were obtained from eight spectra in the presence of a gradient of 12 mT/m (acquisition time 5 min; spatial resolution 8 mm). After 7 min, the nitroxide was detectable in the left side of the thorax area, but it was mostly localized in the liver. PCA was more uniformly distributed in the image collected after 17 min.

Low-field in vivo electron spin resonance (ESR) has been used to follow the course of metabolism and distribution of a paramagnetic spin probe, 3-carboxamido-2,2,5,5-tetramethylpyrrolidine-1-oxyl. Sprague-Dawley rats (250-300 g) were catheterized in the jugular vein and given serial doses of the nitroxide spin probe above. The decrease in the tail blood nitroxide ESR spectrum with the time was followed. This reflects both normal distribution/excretion and a significant metabolic step--reversible reduction of the nitroxide group to its hydroxylamine. The studies serve as important and useful comparisons to the nitroxide reduction kinetics in vitro while offering the important advantage of avoiding those non-steady-state artifacts frequently expected in ex vivo procedures. This study represents the first report of systematic in vivo pharmacokinetics of a paramagnetic probe.