Low-frequency Electron Paramagnetic Resonance Research Articles

Free Radicals in Licorice-Flavored Sweets Can Be Detected Noninvasively Using Low Frequency Electron Paramagnetic Resonance after Oral Administration to Mice

The observation of the fate of free radicals coming from food after oral administration could be important in evaluating their reactivity in vivo. The aim of this study was to demonstrate that it is feasible to detect directly in vivo free radicals coming from food with the use of low frequency electron paramagnetic resonance (EPR) spectroscopy. Because polyphenols are easily oxidized into stable radicals, we assumed that these radicals could be detected in food. We chose licorice, which contains several types of polyphenols. The presence of free radicals was demonstrated in licorice-flavored sweets. Using low frequency EPR spectroscopy, we detected these free radicals directly and noninvasively after oral administration to mice. These radicals were rather stable in the guts of the mice. This study is the first report demonstrating noninvasively the presence of free radicals in vivo coming from food.

In vivo EPR dosimetry of accidental exposures to radiation: experimental results indicating the feasibility of practical use in human subjects

Low frequency electron paramagnetic resonance (EPR) provides the potential advantage of making accurate and sensitive measurements of absorbed radiation dose in teeth in situ, i.e. without removing the teeth from the potential victim. The potential limiting factors for making such measurements are: (1) whether low frequency EPR is sufficiently sensitive to detect radiation-induced signal in human teeth; (2) whether sufficient sensitivity can be maintained under in vivo conditions. In this manuscript, we summarize results indicating that this approach is feasible. Using 1.2 GHz EPR spectroscopy, we found that the lower limit for these measurements in isolated human teeth is 0.2 Gy or lower. Measurements of radiation-induced EPR signals in the teeth of living rats were achieved with sufficient sensitivity to indicate that, when taking into consideration the larger mass of human teeth, similar measurements in human teeth in situ would provide sensitivity in the dose range for potential accidental exposures. We estimate that the current lower limit for detecting radiation doses in human teeth in situ (in vivo) is 0.5–1.0 Gy; this would be sufficient for determining if a person has been exposed to potentially life threatening doses of ionizing radiation. The limiting factor for sensitivity appears to be background signals rather than signal/noise, and there are feasible means to overcome this problem and further increase sensitivity. The additional instrumental developments required to make an effective in vivo EPR dosimetric spectrometer for the measurements in teeth in human subjects in situ, seem quite achievable.

Electrochemical Preparation and EPR Studies of Lithium Phthalocyanine: Evaluation of the Nucleation and Growth Mechanism and Evidence for Potential-Dependent Phase Formation

A very reliable and reproducible electrochemical preparative procedure to obtain oxygen-sensitive lithium phthalocyanine (LiPc) microcrystalline powder, a critical material for in vivo application of electron paramagnetic resonance (EPR) oximetry to measure the partial pressure of molecular oxygen pO2, is described. Important issues including the effect of preparative conditions on the resulting material and the influence of the deposition mechanism on crystal structure are investigated using cyclic voltammetry, chronoamperometry, X-ray diffraction (XRD), and high- and low-frequency EPR measurements. The electrochemical measurements reveal that electrodeposition of LiPc follows a nucleation pathway. Detailed electrocrystallization studies show that the nucleation mechanism is instantaneous and the three-dimensional growth is controlled by the diffusion of the reactant from the bulk solution. Critical evidence, for deposition potential-dependent electrochemical phase formation, is presented. The XRD studie...

In vivo EPR of topical delivery of a hydrophilic substance encapsulated in multilamellar liposomes applied to the skin of hairless and normal mice

In vivo low frequency EPR was used to measure the enhancement of topical delivery of hydrophilic substances by use of multilamellar liposomes. The contribution of transepidermal or/and transfollicular routes of transport was investigated using hairless and normal mice. Two liposome dispersions that previously had been shown to have different enhancement properties on ex vivo skin were used. The kinetics of the reduction of hydrophilic spin probe GluSL ( N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)-2,3,4,5,6-pentahydroxy-hexaneamide) applied to the skin encapsulated into the liposomes was measured. To distinguish the reduction of GluSL on the skin surface from its reduction inside the skin, the oxidizing agent potassium ferricyanide (KFeCN) was used. This does not penetrate into the skin and therefore it oxidizes hydroxylamines back to nitroxide only on the surface of the skin. We observed significant differences in the properties of the two types of liposomes with respect to their stability when in contact with skin and their transport characteristics. The results measured in vivo are consistent with those obtained ex vivo, indicating that in vivo L-band EPR is a powerful technique for following pharmacokinetics in the skin of live animals. The results also show that clearance by blood flow and possible alterations of skin after sacrifice of animal do not influence the results of penetration of liposome entrapped substances into the skin during the time of our experiment (typically around 60 min). The reduction of GluSL in the skin of hairless vs. normal mice was similar, indicating that the transfollicular penetration was not of major importance in vivo in this experimental model.

Kinetics of chromium(V) formation and reduction in fronds of the duckweed Spirodela polyrhiza — a low frequency EPR study

The uptake of chromate by the duckweed Spirodela polyrhiza was investigated with atomic absorption spectroscopy and the reduction of Cr(VI) to Cr(V) was measured using low frequency EPR spectroscopy. The biphasic kinetics of the uptake was fitted to parameters of a proposed kinetic model. Another model was developed to simulate chromate reduction. The first step of chromate reduction was found to be much faster than the uptake of Cr(VI) from the free space. Most probably, this step occurs already in the cell wall or on the cell membrane surface. Further reduction of Cr(V) to Cr(III) was estimated to be slower. The disappearance of the Cr(V) signal, following transfer of the plants into a Cr-free solution, lasted several tens of hours; the kinetics was mono- or biexponential depending on the length of Cr loading. The rate constants for Cr reduction in living plants were determined for the first time.

Spin trapping of nitric oxide by ferro-chelates: kinetic and in vivo pharmacokinetic studies

Biologically generated nitric oxide appears to play a pivotal role in the control of a diverse series of physiologic functions. Iron-chelates and low-frequency EPR spectroscopy have been used to verify in vivo production of nitric oxide. The interpretation of in vivo identification of nitric oxide localized at the site of evolution in real time is complicated by the varied kinetics of secretion. The quantitative efficiency of the spectroscopic measurement, so important in understanding the physiology of nitric oxide, remains elusive. The development of a more stable iron-chelate will help better define nitric oxide physiology. In this report, we present data comparing the commonly used ferro-di( N-methyl- d-glucamine-dithiocarbamate) (Fe 2+(MGD) 2) and the novel chelate ferro-di( N-(dithiocarboxy)sarcosine) (Fe 2+(DTCS) 2) quantifying the in vitro and in vivo stability of the corresponding spin trapped adducts, NO-Fe(MGD) 2 and NO-Fe(DTCS) 2. Finally, very low frequency EPR spectroscopy has been used to evaluate the pharmacokinetics of NO–Fe(MGD) 2 and NO–Fe(DTCS) 2 in mice in real time.

Evaluation of DEPMPO as a spin trapping agent in biological systems

Cellular toxicity, pharmacokinetics, and the in vitro and in vivo stability of the SO 3 •− spin adduct of the spin trap, 5-diethoxyphosphoryl-5-methyl-1-pyrroline-n-oxide (DEPMPO), was investigated, and the results were compared with those of the widely used spin trap 5,5-dimethyl-1-pyrroline- N-oxide (DMPO). Similar to DMPO, DEPMPO was quickly taken up (< 15 min) after intraperitoneal injection, and distributed evenly in the liver, heart, and blood of the mice. In the presence of ascorbate the in vitro stability of the adduct DEPMPO/SO 3 •− was 7 times better than DMPO/SO 3 •−. Under in vivo conditions, the spin adduct DEPMPO/SO 3 •− was 2–4 times more stable than DMPO/SO 3 •−, depending on the route of administration of the adducts. Using a low frequency EPR spectrometer, we were able to observe the spin trapped SO 3 •− radical both with DMPO and DEPMPO directly in the intact mouse. DEPMPO had a detectable spin adduct signal at a concentration as low as 1 mM, as compared to 5 mM for DMPO. We conclude that DEPMPO is potentially a good candidate for trapping radicals in functioning biological systems, and represents an improvement over the commonly used trap DMPO.

Optimization of axial RF field distribution in low-frequency EPR loop-gap resonators

A novel coupling method that optimizes the axial RF distribution of low-frequency EPR loop-gap resonators is presented. It consists of a resonant coupling loop positioned at the centre of a two-section loop-gap resonator. This arrangement ensures a symmetrical distribution of the radio frequency field along the axis of the resonator. The design of a central coupling system suitable for EPR resonators operating at about 220 MHz is described. Experimental results show that with the central coupling system the RF field is symmetrical and has a very good axial homogeneity (100% of the resonator length).

Carbon-centered radicals as oxygen sensors for in vivo electron paramagnetic resonance: screening for an optimal probe among commercially available charcoals.

It is known that some charcoals possess paramagnetic centers with an electron paramagnetic resonance (EPR) linewidth which can be broadened by oxygen. In order to identify potential candidates as sensors for in vivo EPR oximetry, we carried out a systematic study among commercially available charcoals. A total of 34 charcoals were tested. The steps used for the screening were: (1) to check the presence of paramagnetic centers in the material; (2) to measure the EPR linewidth in nitrogen and in air on the dry material and on a aqueous suspension of particles; (3) to calibrate the oxygen sensitive materials (EPR linewidth vs. pO2); (4) to test the sensitivity and stability of the response to changes of pO2 in a simple model of hypoxia induced in mice. Seventeen charcoals contained paramagnetic centers detectable by low-frequency EPR (1.1 GHz). The EPR spectrum consist of one single line which is typical of carbon-centered radicals (g-factor approximately 2). Eight charcoals presented sufficient interesting EPR properties (linewidth in nitrogen < 0.1 mT, linewidth in air for an aqueous suspension of particles > 0.15 mT) to be further characterized in vivo. Only three charcoals presented a stable, reproducible, and sensitive response to pO2 for more than 2 months. These three coals should be considered as good candidates to be used as oxygen sensor using in vivo EPR spectroscopy.

PH-sensitive imaging by low-frequency EPR: a model study for biological applications

The use of pH-sensitive nitroxides, in conjunction with low-frequency EPR, offers a unique opportunity for non-invasive assessment of pH values (in the range 0 to 14) in living animals. In the present study, we have investigated the potential use of pH-sensitive nitroxide free radicals in conjunction with EPR imaging techniques at low and very low frequencies (280 MHz - 2.1 GHz). In particular, we have measured the hyperfine splitting (hfs) of a pH-sensitive probe at three different EPR frequencies: 280 MHz, 1.1 GHz and 2.1 GHz. We have also developed EPR imaging experiments with phantoms simulating in vivo conditions, using pH-sensitive probes at 280 MHz (spatial-spatial) and 1.1 GHz (spectral-spatial). Finally, we discuss the actual sensitivity/resolution limits of the EPR imaging techniques at low frequencies. Practical applications of this method in the biomedical field are suggested for the continuous and non-invasive localization of pH in vivo.

In vivo detection of free radicals in real time by low-frequency electron paramagnetic resonance spectroscopy.

During his studies on the properties of oxygen, Priestley () noted that this gas, an essential ingredient for life processes, appears to “burn out the candle of life too quickly.” More than two centuries would elapse, however, before this observation would be associated with Grubbé’s () accounts of redness and irritation on the hands of his workers testing X-ray tubes. By 1954, Gerschman et al. () suggested that free radicals were the common element linking the observed toxicity of oxygen to the harmful effects of ionizing radiation. The implication of this hypothesis seemed remote at that time. However, within a decade, the search for biologically generated free radicals would lead to the discovery of superoxide and an enzyme that attenuated cellular levels of this free radical (,). In the intervening years, free radicals have been recognized as common intermediates in cellular metabolism (,), found to play an essential role in host immune response () and demonstrated to regulate many essential physiologic functions ().

Low-Frequency EPR of the Copper in Particulate Methane Monooxygenase from Methylomicrobium albus BG8

Low-Frequency EPR of the Copper in Particulate Methane Monooxygenase from <i>Methylomicrobium </i><i>albus</i> BG8

Detection of Free Radical Metabolite Formation Usingin VivoEPR Spectroscopy: Evidence of Rat Hemoglobin Thiyl Radical Formation Following Administration of Phenylhydrazine

The spin-trapping technique in conjunction with a low-frequency electron paramagnetic (or spin) resonance (EPR or ESR) spectrometer was used to detect the hemoglobin thiyl free radical in living rats using a whole body resonator. The hemoglobin thiyl free radical was formed following the intragastric administration of phenylhydrazine at the LD50dose of 188 mg/kg. The hemoglobin thiyl free radical was then trapped by preinjected 5,5-dimethyl-1-pyrrolineN-oxide (DMPO), which formed the DMPO/hemoglobin thiyl-free radical adduct in the blood. The time course of thein vivoformation and disappearance of the spin adduct was followed. The DMPO/hemoglobin thiyl free radical was detected in blood samples using 9.5 GHz (X-band) and 1.1 GHz (L-band) EPR at room temperature and 77 K. Pretreatment of rats with ascorbate and diethylmaleate (DEM) decreased the signal intensity of the DMPO/hemoglobin thiyl free radical spin adduct. The incubation of ascorbate or DEM at 37°C with rat blood containing preformed DMPO/hemoglobin thiyl radical adduct showed that there was no effect of DEM on the free radical concentration, while ascorbate reduced the radical adduct. This study provided direct evidence of the formation of the DMPO/hemoglobin thiyl free radicalin vivoand enabled us to study this formation in living animals free of any artifacts that can occur when usingex vivomethods.

Low frequency epr surface probe based on dielectric resonator

A new surface probe for low frequency in vivo EPR studies was designed and constructed based on a dielectric resonator. Dielectric resonators previously have been used in X-band EPR for small samples to take advantage of its well separated microwave magnetic and electric field distributions and high Q. In this work we have built a dielectric resonator for use as a surface probe for in vivo EPR at low frequency (1.2 GHz). The core of the probe was a commercially available dielectric ceramic resonator supported by a PTFE ring. The resonator was coupled to a 50 Ω line by an inductive loop which was interfaced by a quarter wave length transformer. The quarter wave length transformer improved the matching and stabilized the resonant frequency. The resonator was surrounded by a silver coated brass shielding. The shielding blocked the penetration of the field modulation and therefore there was no contribution from any intrinsic EPR signal from the dielectric material. The Q factor before loading samples was 3,600 which was much higher than the Q factors of ∼ 1000 obtained by other types of surface probes. The use of animals or biological samples decreases the Q factor to about 330 but this is at least two times higher than occurs with other types of surface probes. The sensitivity of the dielectric surface probe in air decreased more slowly than with other types of surface probes. This would be especially useful for in vivo studies in which the animal has an irregular surface at the site(s) of measurement and/or there is a significant air gap between the probe and the animal. This dielectric resonator can be inexpensively constructed and is robust, and sensitive for in vivo EPR spectroscopy and imaging.

Measurement of Differences in pO 2 in Response to Perfluorocarbon/Carbogen in FSa and NFSa Murine Fibrosarcomas with Low-Frequency Electron Paramagnetic Resonance Oximetry

We have used very low-frequency electron paramagnetic resonance (EPR) oximetry to measure the change in oxygen concentration (delta pO2) due to change in breathing atmosphere in FSa and NFSa fibrosarcomas implanted in the legs of C3H mice infused with perfluoro-octylbromine (PFOB). Measurements in each tumor were made before and after the administration of the high-density (47% v/v) perfluorocarbon PFOB, perflubron (Alliance Pharmaceutical Corporation, San Diego, CA). Measurements in each tumor were also made, after the administration of the PFOB, both before (PFOB/air) and after the administration of carbogen (95% O2 + 5% CO2, PFOB/carbogen). Large changes (delta p02) relative to PFOB/air oxygenation were seen with the administration of PFOB/carbogen. No significant difference in oxygen concentration was seen between air-breathing mice with and without PFOB. The mean delta pO2 for FSa tumors was 13 +/- 6 torr, while the mean for NFSa fibrosarcomas was 28 +/- 7 torr. There were such large intertumor differences that the trend toward a smaller change in the more hypoxic FSa tumors was not significant (P = 0.13). This paper describes a novel method of measuring differences in oxygenation in tumor tissues. The results of such measurements indicate large differences in pO2 response to different breathing atmospheres in PFOB-infused tumors of similar histology. The intertumor delta pO2 differences may correlate with differences in radiation response.

Non-invasive in vivo characterization of release processes in biodegradable polymers by low-frequency electron paramagnetic resonance spectroscopy

Using stable free radicals (nitroxides) whose spectra reflect microviscosity and pH, low-frequency electron paramagnetic resonance (EPR) spectroscopy was used to characterize the release pattern of subcutaneous implants of poly( d,l-lactide-co-glycolide) (PLGA) continuously and non-invasively in living mice. No significant changes occurred during the first days after implantation. After about 1 week, the recorded EPR spectra gave direct evidence for the formation of compartments with high mobility and increasing acidity in the delivery system. The contribution of the mobile part of the spectrum increased with time, but no remarkable decay of the overall signal intensity was observed during the second week. The EPR signals decayed rapidly after 3 weeks. The experimental data are consistent with bulk hydrolysis as the dominating mechanism of release and are not consistent with a surface-controlled pattern of degradation. The formation of acidic compartments in the delivery system may have significant effects on drug stability, drug solubility, bioavailability, pharmacokinetics, and ultimately on therapeutic efficiency. In particular, the finding of areas of low pH within the polymer raise the possibility that hydrolysable drugs may undergo degradation in the implant prior to their release. Our results demonstrate that EPR is a valuable tool for characterizing such drug delivery systems in vivo.

High Frequency (139.5 GHz) Electron Paramagnetic Resonance Characterization of Mn(II)−H217O Interactions in GDP and GTP Forms of p21 ras

As a molecular switch, the ras protein p21 undergoes structural changes that couple recognition sites on the protein surface to the guanine nucleotide-divalent metal ion binding site. X-ray crystallographic studies of p21 suggest that coordination between threonine-35 and the divalent metal ion plays an important role in these conformational changes. Recent ESEEM studies of p21 in solution, however, place threonine-35 more distant from the metal and were interpreted as weak or indirect coordination of this residue. We report high frequency (139.5 GHz) EPR spectroscopy of p21.Mn(II) complexes of two guanine nucleotides that probes the link between threonine-35 and the divalent metal ion. By analysis of high-frequency EPR spectra, we determine the number of water molecules in the first coordination sphere of the manganous ion to be four in p21.Mn(II).GDP, consistent with prior low-frequency EPR and X-ray crystallographic studies. In the complex of p21 with a GTP analog, p21.Mn(II).GMPPNP, we determine the hydration number to be 2, also consistent with crystal structures. This result rules out indirect coordination of threonine-35 in the solution structure of p21.Mn(II).GMPPNP, and implicates direct, weak coordination of this residue as suggested by Halkides et al. [(1994) Biochemistry 33,4019]. The 17O hyperfine coupling constant of H2(17)O is determined as 0.25 mT in the GDP from and 0.28 mT in the GTP form. These values are similar to reported values for 17O-enriched aquo ligands and some phosphato ligands in Mn(II) complexes. The high magnetic field strength (4.9 T) employed in these 139.5 GHz EPR measurements leads to a narrowing of the Mn(II) EPR lines that facilitates the determination of 17O hyperfine interactions.

Use of EPR oximetry with India ink to measure the pO2 in the liver in vivo in mice.

The partial pressure of oxygen (pO2) of the liver in vivo in unanesthetized mice was determined using electron paramagnetic resonance (EPR) oximetry with India ink. The EPR spectra were obtained using a low-frequency (1.2 GHz) EPR spectrometer with a loop gap cavity resonator. The line width of the India ink used in this experiment was reversibly broadened by oxygen and was particularly sensitive to pO2 below 30 torr. After the administration of India ink into the tail vein, the India ink particles were taken up mainly by Kupffer cells in the liver and in part by phagocytes in the spleen. The pO2 measured in the normal liver was about 14 torr and was constant for the 2-week experimental period. The pO2 decreased when measured at 1, 2, and 6 days after treatment with hepatotoxin (carbon tetrachloride (CCl4)); within 2 weeks, it returned almost to the initial level. Measurements by EPR at sacrifice of controls and CCl4-treated mice indicated that more than 90% of the India ink went to the liver; the spleen contained 4.7% of total amount in control mice and 8.8% in CCl4-treated mice when measured 2 weeks after the treatment. These data indicate the usefulness of India ink for measuring the pO2 of the liver in vivo and that the pO2 in the Kupffer cells is decreased when the liver is damaged by CCl4.

Assessment of cerebral pO2 by EPR oximetry in rodents: effects of anesthesia, ischemia, and breathing gas.

This report describes experiments designed to assess and illustrate the effectiveness of a new method for the measurement of cerebral interstitial pO2 in conscious rodents. It is based on the use of low frequency electron paramagnetic resonance (EPR) spectroscopy with lithium phthalocyanine as the oxygen sensitive probe. Magnetic resonance imaging was used to document placement of the probe in the brain, and to assess potential cerebral changes associated with the placement. The technique provided accurate and reproducible measurements of localized pO2 in the brains of conscious rodents under a variety of physiological conditions and for time periods of at least 2 weeks. Using this approach we quantitated the depressing effects on cerebral pO2 of three representative anesthetics, isoflurane, ketamine/xylazine, and sodium pentobarbital. The effects of changing the content of oxygen in the breathing gas was investigated and found to change the cerebral pO2. In experiments with gerbils, crystals of lithium phthalocyanine were implanted in each side of the brain and using a one-dimensional magnetic field gradient, simultaneous measurement of pO2 values from normal and ischemic (ischemia induced by unilateral ligation of a carotid artery) hemispheres of the brain were obtained. These results demonstrate that EPR oximetry with lithium phthalocyanine is a versatile and useful method in the measurement of cerebral pO2 under various physiological and pathophysiological conditions.

Development of an Electronically Tunable L-Band Resonator for EPR Spectroscopy and Imaging of Biological Samples

While interest in the EPR spectroscopy and imaging of biological samples triggered the development of numerous resonator and microwave-bridge designs operating at high RF and L-band frequencies, a number of important technical problems remain, Two of these problems are the occurrence of sample-induced drifts in resonance frequency of the resonator and the intrinsic limitations in sensitivity of low-frequency EPR measurements, In an effort to address these problems, an approach was developed for obtaining fixed-frequency EPR measurements, utilizing a low-phase-noise fixed-frequency oscillator as a microwave source and an electronically tunable ceramic re-entrant L-band resonator, which was locked to the oscillator via automatic frequency control (AFC). The tunable microwave resonator described in this paper employs a voltage-controlled piezoelectric actuator for adjustment of the resonance frequency via variations of the equivalent capacitance of the resonator, Design considerations and data, as well as schematic drawings and test results, are presented, The reported approach provides true fixed-frequency operation under a wide range of sample and environment conditions and over prolonged periods of time, The use of this tunable resonator design in conjunction with an ultra-low-noise fixed-frequency oscillator should enable enhanced sensitivity in EPR measurements of large lossy biological samples.