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Electron-spin relaxation is one of the determining factors in the efficacy of MRI contrast agents. Of all the parameters involved in determining relaxivity it remains the least well understood, particularly as it relates to the structure of the complex. One of the reasons for the poor understanding of electron-spin relaxation is that it is closely related to the ligand-field parameters of the Gd(3+) ion that forms the basis of MRI contrast agents and these complexes generally exhibit a structural isomerism that inherently complicates the study of electron spin relaxation. We have recently shown that two DOTA-type ligands could be synthesised that, when coordinated to Gd(3+), would adopt well defined coordination geometries and are not subject to the problems of intramolecular motion of other complexes. The EPR properties of these two chelates were studied and the results examined with theory to probe their electron-spin relaxation properties.

Carbonizates and active carbons (AC) are useful as sensors of oxygen. Adsorption of water vapor on such materials, prepared from the wood of a tropical tree, Peltogyne from Surinam, was investigated. Reproducible and reversible water vapor isotherms were obtained of preliminarily unheated samples, as well as of heated samples after several adsorption–desorption cycles. Water vapor adsorption revealed very fine (ultramicropores) structure in the investigated materials. Such ultramicropores can not be detected by traditional adsorption of nitrogen at 77 K because of a molecular sieve blocking effect. Several carbonizates at high water vapor pressure exhibited large weight reduction. This weight loss is the result of expulsion of organic impurities from the porous structure under the influence of adsorbed water. These data were confirmed by mass-spectrometric thermodesorption analysis. X-ray data and water vapor adsorption isotherms indicated that the strongly bound water stabilizes the carbonizate structure. When heated at 623 K, the AC exhibited desorption of strongly bound water and modified AC structure.

The original technique with the use of longitudinal electron paramagnetic resonance detection was applied to measure longitudinal and transversal electron relaxation times (T 1 andT 2) in specially prepared carbon chars containing paramagnetic centers (PCs) on the surface. The results not only confirmed the conclusions of our previous studies but enabled us to determine the exchange integral and clarify the mechanism of interaction between the PCs and adsorbed molecular oxygen O2. A spin relaxation mechanism was also suggested which takes into account both the exchange and dipolar interactions in the systems combining PC and O2. The value of the exchange integral between PCs as well as its functional dependence on the oxygen content were estimated.

With the use of a semi-empirical quantum chemical method AM1, it is shown that the availability of 4–6% oxygen heteroatoms of furan- and pyrone-type, and/or also 2–3% N of pyrrol-type in a carbon matrix should provide greatest electron-donor ability to carbons. The synthetic carbons HPSC and SCN, containing various states and amounts of O- and N-heteroatoms, were prepared and analyzed to confirm this prediction experimentally. Utilizing an electrochemical cell with divided electrode spaces, the reduction processes of a series of strong oxidizers and O 2 (on a spongy platinum electrode) in synthetic carbons with various surface chemical compositions were investigated directly by measuring a current in an external circuit. A good correlation between calculated data and electrochemical measurements was obtained. Two mechanisms of chemisorption of oxygen on evacuated carbons are suggested: homolytic (free radical) at small degrees of filling of a surface by oxygen, and heterolytic (at large degrees of filling) causing the fixation of oxygen on a surface in the form of a superoxide-ion O 2 − * .

The electron paramagnetic resonance (EPR) spectra of some paramagnetic materials exhibit a pO2 (partial pressure of oxygen)-dependent linewidth. By recording the EPR linewidth in vivo using low-frequency EPR spectrometers, it is possible to measure the partial pressure of oxygen in tissues. It has been found, however, that some of the paramagnetic materials with optimal spectroscopic properties in vitro may lose or change their responsiveness to oxygen in tissues. The aim of this study was to microencapsulate paramagnetic particles by biopolymers in order to stabilize their responsiveness to oxygen. Carbohydrate char particles (Bubinga) were encapsulated with different biopolymers: cellulose acetate or cellulose triacetate, silicone and polyurethane. The performance of the materials was evaluated in vitro and in vivo. X-band EPR spectroscopy was used to test the variation of the calibration curve (EPR linewidth as a function of the pO2) after incubation in saline and after prolonged residence in tissues. The stability of the responsiveness to PO2 in vivo was carried out by L-band EPR spectroscopy using mice that received injection of the oxygen sensors in the muscles. After residence in saline and prolonged residence in tissues, only the calibration curve of the silicone-coated (coating weight of 0.5% (w/w)) paramagnetic materials remained unchanged, while those of oxygen sensors coated with cellulose acetate, cellulose triacetate and polyurethane changed.

A modulation technique for direct measurement of extremely fast electron spin−lattice relaxation is described. The method is based on longitudinal magnetization detection. It is applied here to study the spin−lattice relaxation times T1e in aqueous solutions of perdeuterated nitroxide radical (T1e ≈ 200 ns) with various concentrations as well as to two Gd DOTA complexes (T1e ≈ 1.8−3.2 ns), which can be employed as paramagnetic contrast agents in magnetic resonance imaging.

The mechanism of oxygen response in several newly synthesized oxygen-sensitive chars was studied with the use of EPR spectroscopy. The results suggest that the compounds contain two basic types of paramagnetic centers (PC). The change in oxygen concentration leads to a mutual and reversible transformation of PCs in chars, which is reflected in EPR parameters. The adsorbed molecular oxygen progressively disturbs the wave functions of the PCs and so breaks the Heisenberg exchange between them. At high oxygen concentration, the 2D dipole–dipole interaction between PCs at the surface comes into play and determines the EPR lineshape. A suggested model quantitatively describes the evolution of the basic EPR parameters of each PC as a function of oxygen concentration.

EPR oximetry using paramagnetic particles relies on the measurement of the EPR linewidth, which is directly related to the pO2. It was previously found that some of the paramagnetic materials with optimal EPR spectroscopic properties in vitro may lose their responsiveness to oxygen in tissues (change of the calibration curve of the EPR linewidth as a function of the pO2). We hypothesized that coating paramagnetic particle materials could improve the stability of response, as well as the biocompatibility. In this study, very thin films holding paramagnetic materials were prepared with different biopolymers (cellulose acetate, cellulose triacetate, cellulose nitrate, silicone, and polyurethane) that already are accepted for clinical applications. Their performance was evaluated in EPR oximetry by measuring the stability of the calibration curves (EPR linewidth as a function of pO2) after a prolonged period in an aqueous environment (1 week in saline) or in vivo (implantation for 3 weeks under the skin of mice). We found that one type of silicone film was able to stabilize the responsiveness of an intrinsically unstable carbon material (a wood char).

A strong particle size influence on NMR transverse proton relaxation in aqueous suspensions of several newly synthesized carbon based chars is reported. The results are quantitatively interpreted in terms of a two-stage molecular exchange model. A porous cage effect leads to slow exchange between molecules inside and outside pores, in contrast to fast molecular exchange at the solid–liquid interface, where a familiar two-site formalism can be applied. The dependence of surface properties on the relaxation data was detected. The transverse NMR relaxation time of water protons on the paramagnetic surface of chars is estimated to be T2s≈ 0.5×10−5 s, which is comparable with the EPR relaxation time of the free radical centers. Dynamic nuclear polarization (DNP) is used to estimate the paramagnetic shift Δv of water protons at the surface. The value Δv so obtained is much less than the transverse NMR relaxation rate of water protons on the paramagnetic surface Δ v ⪡ T 2 s −1 . This inequality can account for the independence of the NMR transverse relaxation times on the interval τ observed in CPMG measurements.

The Mark II W-band (94 GHz) EPR spectrometer with a low-noise millimeter-wave amplifier is described. The microwave bridge is of a high-sensitivity homodyne design. Signal-to-noise ratios were measured for a number of detectors with and without the low-noise amplifier. The signal-to-noise ratio was determined not only by the type of detector but also how well it was matched. Without a microwave preamplifier, a hot-electron bolometer provides the best signal-to-noise ratio. Addition of a low-noise microwave preamplifier to the CW homodyne bridge gives a 10 dB improvement in the noise figure of the receiver at a modulation frequency of 100 kHz. A greater improvement in the signal-to-noise ratio is seen at low modulation frequencies (1–10 kHz), making the low-noise amplifier useful for systems with large linewidths. This allows larger modulation amplitudes to be used without causing significant cavity heating or microphonics. The W-band spectrometer is capable of rapid sweeps from 0 to 7 T, as well as narrower (0.1 T) high-resolution sweeps. It is suitable for a wide variety of samples including liquids and samples cooled to sub-liquid-helium temperatures.

Multi-frequency EPR spectroscopy at 9.5, 35, 94, and 249 GHz has been employed to investigate the zero field splitting (ZFS) of high spin ions in liquids. In particular, experiments are reported on aqueous solutions of DTPA and DOTA chelates of Gd(III), and on the uncomplexed ion, which are relevant to the effectiveness of paramagnetic contrast agents for magnetic resonance imaging (MRI). The field dependence of the centroid of the resonance line, characterized by an effective g factor, geff, has been analysed in order to determine δ1, the trace of the square of the ZFS matrix. Analysis of the variation in transverse electron spin relaxation (T 2e) with experimental frequency provides yet another route to measure δ2 from EPR data. This analysis also gives δv, a correlation time describing the time-dependent ZFS effect. The ZFS parameters so obtained agree well with results obtained by the analysis of proton nuclear magnetic relaxation dispersion. At 94 GHz, partially resolved spectra from chelated and unc...

The gaseous signal molecule, nitric oxide (NO•), is generated enzymatically by NO synthase (NOS) from L-arginine. Overproduction of NO contributes to cell and tissue damage assequelaeof infection and stroke. Strategies to suppress NO synthesis rely heavily on guanidino-substituted L-arginine analogs (L-NAME, L-NA, L-NMMA, L-NIO) as competitive inhibitors of NOS, which are often used in high doses to compete with millimolar concentrations of intracellular arginine. We show that these analogs are also a source for non-enzymatically produced NO. Enzyme-independent NO release occurs in the presence of NADPH, glutathione, L-cysteine, dithiothreitol and ascorbate. This non-enzymatic synthesis of NO can produce potentially toxic, micromolar concentrations of NO and can oppose the effects of NOS inhibition. NO production driven by NOS inhibitors was demonstratedex vivoin the central nervous and peripheral tissues of gastropod molluscsAplysiaandPleurobranchaeausing electron paramagnetic resonance and spin-trapping techniques. These results have important implications for therapeutic regulation of NO homeostasis.

The heterogeneous 1H dynamic nuclear polarization (DNP) effect is studied at low magnetic fields for a system consisting of several newly synthesized carbon chars suspended in water. By using Fourier Transform pulsed-field-gradient spin-echo NMR spectroscopy, several different self-diffusion coefficients have been observed in aqueous char suspensions, corresponding to regions of differing water mobility in the porous structure. Proton spin-lattice relaxation data generally confirm the results of molecular diffusion measurements. Through utilization of the Torrey model, the influence of "cage effects" on DNP enhancement in porous media is discussed. Results suggest that short-range nuclear-electronic interactions in pores have a dominant effect on DNP enhancement in char suspensions.

The measurement of pO2 in vivo using EPR has some features that provide potential advantages for many applications. There is a strong possibility that EPR oximetry also will have significant applications in clinical medicine. The characteristics of EPR oximetry that appear to be especially useful often are complementary to existing techniques for measuring oxygen in tissues. These characteristics include capabilities of making repeated measurements from the same site, high sensitivity for low levels of oxygen, and non-invasive options. The existing EPR techniques are especially useful for studies in small animals, where the depth of measurements is not an over-riding issue. In larger animals and potentially in human subjects, the non-invasive techniques seem to be immediately applicable to study phenomena very near the surface (within 10 mm), while invasive techniques have some very promising uses for other sites. There also is a possibility of extending the non-invasive approach to greater depths by using lower frequency EPR. The clinical uses of EPR oximetry that seem especially promising are: long term monitoring of the status and response to treatment of peripheral vascular disease and optimizing cancer therapy by enabling it to be modified on the basis of the pO2 measured in the tumor. EPR oximetry is based on the effect of oxygen on EPR spectra, which provides a sensitive and accurate means to measure pO2 quantitatively in vivo. The in vivo applications of this technique have occurred because of the development of oxygen sensitive paramagnetic materials that are very sensitive to changes in the pO2 and, in some cases quite stable in tissues for long periods of time. These advances have made it feasible to make EPR measurements in intact animals. The physical basis and biological applications of in vivo EPR oximetry are reviewed, with particular emphasis on the use of EPR spectroscopy at 1200 MHz using particulate paramagnetic materials for the repetitive and non-invasive measurement of pO2 in tissues.

The interactions of two lipophilic magnetic resonance imaging paramagnetic contrast agents, gadolinium complexes of 1,4,7,10-tetraazacyclododecane-N-(n-pentyl)-N‘,N‘‘,N‘‘‘-triacetic acid (Gd-DOTAP) and 3,6,9-triaza-3,6,9-tris(carboxymethyl)-4-(4-ethoxybenzyl)undecandicarboxylic acid (Gd-EOB-DTPA), with model multilamellar liposomes prepared from 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) were assessed in three sets of EPR experiments. The first two experiments were carried out with phospholipids selectively labeled with a series of spin-labeled doxyl stearic acids. By means of counting the collisions of molecular oxygen with a spin-labeled site, local oxygen permeability across the bilayer was measured with the contrast agents present and compared with control experiments in which contrast agents were absent. The maximum increase of 6.5 ± 0.3% for oxygen permeability at 20 mM Gd-DOTAP concentration was observed at 30.8 °C for the label located at the twelfth carbon position of the acyl chain. This...

Magnetic resonance imaging often utilizes paramagnetic contrast agents (PCAs) to increase contrast between adjacent tissues. PCAs enhance the contrast by increasing the spin−lattice proton relaxation rate through processes known as inner-sphere, second-sphere, and outer-sphere mechanisms. Past studies on PCAs often described relaxation rates that are not caused by inner-sphere processes as outer-sphere, since comparatively little is known about second-sphere water. Utilizing vanadyl complexes (ethylenediaminetetraacetate (EDTA) and diethylenetriaminepentaacetate (DTPA)) that do not have an inner-sphere proton relaxation contribution and those with similar functional groups of different sizes, we find that the outer-sphere model does not adequately describe the relaxivity profiles. The observed relaxivity profiles are, however, consistent with a model that includes both second-sphere and outer-sphere contributions. Vanadyl ethoxybenzyl-diethylenetriaminepentaacetate (VOEOB-DTPA) exhibited relaxivity simila...

The temperature dependence of DNP enhancements in aqueous suspensions of hardwood chars by nonstationary DNP at low magnetic field has been investigated. With increasing temperature, the sign of the water proton DNP enhancement changed from negative to positive, indicating a change in the relative contributions from exchange (scalar) and dipole-dipole nuclear-electron coupling. A cross-over temperature was found at which the contributions of the dipole-dipole and exchange interactions canceled each other and the DNP effect disappeared. A model of molecular migration at the solid-liquid interface is constructed to account for these observations. Correlation of experimental and theoretical results has been achieved through consideration of the proton-electron spin dipole-dipole interaction and chemical exchange of solvent molecules. The increase with temperature in the contribution of the contact interaction to proton relaxation is a result mainly of a decrease in the lifetime τ h of water in the short-lived hydrated complex at the hardwood char interface. The enthalpy of activation E h = 13.5 kcal/mol for surface exchange diffusion as well as the lifetime of water molecules on the surface τ h (300K) = 1.9 × 10 −4 s and hyperfine scalar constant a = 5.3 × 10 −3 MHz are estimated. The results are characteristic of weak short-lived chemisorption bonds at the solid-liquid interface.

EPR spin-trapping experiments are usually carried out at X-band (9.5 GHz) because of the good concentration sensitivity and ready availability of this method. The radical precursors are then characterized from an analysis of isotropic hyperfine coupling and comparison of these coupling factors with those for the reference spin adducts. These experiments encounter two major challenges: (i) spin adducts from many carbon-centered free radicals have g factors that are nearly the same (resulting in strongly overlapping spectra at 9.5 GHz), and (ii) measurable hyperfine couplings correspond to interactions of the electron spin with just the nearest nuclei. Therefore, very little or no information is obtained on the overall structure of the spin adduct molecule. Some of these difficulties can be overcome by carrying out spin-trapping experiments at 10-fold higher frequency, 95 GHz (W-band). Examples of two spin adducts with nearly the same isotropic g factors (Δgiso = 1.2 × 10-4) are the benzene solutions of ph...

A new method for the experimental determination of the cross-relaxation (CR) transition rates in liquid solutions of paramagnetic compounds has been developed utilizing a pulsed dynamic nuclear polarization (DNP) technique. In contrast to NMR relaxation, which is proportional to the sum of relaxation rates, the DNP effect is determined by the ratio of transition rates in the nucleus−electron coupled spin system. By use of pulsed DNP, the NMR relaxation rates and DNP data can be obtained in the same experiment. As a result, a set of independent equations for CR and dipole−dipole (DD) transition rates can be derived. The solution of these equations defines individual cross-relaxation and DD transition rates, as well as molecular−kinetic information, and avoids the necessity of performing complicated variable frequency and temperature measurements. A pulsed DNP relaxometer operating at a proton frequency of 0.5 MHz was constructed. The 4-oxo-2,2,6,6-tetramethyl-1-piperidinyloxyl (TEMPONE) stable free radical...

We studied the molecular dynamics of vanadyl−chelate complexes covalently attached to the surface of cascade polymers, dendrimers. The rotational correlation times of the ion−chelate complex were determined from computer simulations of their EPR spectra. The chelate 2-(4-isothiocyanatobenzyl)-6-methyldiethylenetriaminepentaacetic acid was covalently attached to ammonia core poly(amidoamine) (PAMAM) cascade polymers via a thiourea (TU) linkage, resulting in PAMAM−TU−DTPA cascade polymers. X-band EPR spectra of their vanadyl complexes were taken, and the A and g matrices were determined from the rigid limit spectra using the SIMPOW program. Spectra were fitted with modification of the slow-motional line-shape theory. Our results indicate that the rotational correlation times of the surface chelate increase with molecular weight and resemble those of “internal” segmental motions found in PAMAMs. For this macromolecular system, the rotational correlation times alone cannot account for differences in the relax...

Electron paramagnetic resonance spectra of model magnetic resonance imaging contrast agents vanadyl ethylenediaminetetraacetate and vanadyl diethylenetriaminepentaacetate in 46% w/v sucrose solutions have been acquired from near physiological temperature to near freezing to study their rotational dynamics in a viscous environment. The sucrose solution simulates environments that may arise in vivo and also when the contrast agents are bound either covalently or noncovalently (e.g., via hydrogen bonds) to another molecule either by chance or by design to improve the relaxivity and the specificity. We report that an isotropic Brownian model, which described the rotational dynamics of these agents in water, does not fully portray the rotational dynamics in this environment, especially in the intermediate or slow tumbling regime. However, an axial anisotropic rotational model simulated the experimental spectra well and can account for the trends exhibited by the data. The dynamics results suggest that the moti...

We have developed an electron paramagnetic resonance (EPR) method for the nondestructive detection and quantification of intracellular NO in real time. Based upon this technique, we have obtained evidence for the metabolism of this bioregulatory molecule by mitochondria. Line-broadening of the EPR signal of a coal derivative, fusinite, was calibrated as a function of NO concentration in aqueous solution. The methodology was validated using two compounds which release NO in a controlled and predictable manner with first-order rate constants k 1 = 5.0 · 10 −3 s −1 and k′ 1 = 3.4 · 10 −4 s −1 (35° C). Fusinite was internalized in Chinese hamster ovary cells (CHO) by phagocytosis, after which the cells were allowed to consume the available O 2 producing an hypoxic environment. The NO released from one of the NO donors, added to the culture fluid at an initial concentration of 50 μM, was directly measured in the intracellular environment as line-broadening of the fusinite EPR signal. The linewidth diminished with time, indicating that NO was being converted to a non-paramagnetic species by the cells with an apparent zero-order rate constant of 5 · 10 8 NO molecules cell −1 min t-1 (20° C). Addition of cyanide to the culture medium (5 mM final concentration) inhibited this disappearance of NO. NO also was converted in the presence of isolated mitochondria in the absence of oxygen. These observations suggest that under hypoxic conditions, there exists in CHO cells a metabolic pathway for the conversion of NO to diamagnetic species, which involves interactions with mitochondria.

Three British coals and their iodine treated counterparts were examined in order to investigate the increase in electron paramagnetic resonance (e.p.r.) signal intensity that occurs upon iodine uptake. The iodine is not removed by vacuum. Scanning electron microscopy (SEM) results show that the iodine exists in a highly dispersed form. Correlation of elemental analysis and e.p.r. spin density measurements indicates two to ten new spins added per 100 added I 2 molecules. A study of e.p.r. intensity as a function of temperature reveals nearly identical trends for untreated and iodine treated coals. High frequency e.p.r. resolves additional signal at lower fields in the iodine treated samples. Such observations contradict the notion of donor-acceptor complex formation between iodine and aromatic constituents of the coal. Although there is considerable evidence of iodine participation in the formation of this additional signal, it would appear to be of a non-bonding nature.

Half-field EPR transitions (g = 4.00) were observed in some synthetic carbohydrate chars prepared by heat treatment at temperatures less than 620°C. The half-field line has a lineshape similar to that of the main resonance and about the same intensity at transverse and longitudinal microwave excitation. Its relative intensity decreases with increasing char heat-treatment temperature. In contrast, no g = 4 transitions were detected for chars synthesized at temperatures greater than 620°C. The ratio of intensity of the half-field transition to that of the main resonance is about 10−6 and does not change from 4.5–55K. At 4.5K, the g = 4.00 line has about half of the linewidth of the main resonance. The observed half-field resonance can be explained by the existence of isolated aromatic radical clusters or by a low-dimensional spin-exchange network within the solid.

A new technique to study cellular respiration under steady-state conditions is described. For detection of oxygen concentration the technique utilizes lithium phthalocyanine crystals whose, electron paramagnetic resonance (EPR) spectra are highly sensitive to oxygen. A gas permeable poly(tetrafluoroethylene) (TFE) capillary (i.d. = 0.81 mm; o.d. = 0.86 mm) is used to control the oxygen flux to the sample of cultured neural cells (N1E-115) of 50 microliters volume. The measured oxygen permeability of the TFE capillary exhibits Arrhenius law behavior (Ep = 21.2 kJ/mole) in the temperature range 15-43 degrees C. The observed increase in the respiratory rate of the N1E-115 cells with temperature can be described with an apparent activation energy of Ea = 110 +/- 20 kJ/mol. Cellular depolarization with 35 mM KCl increases the respiratory rate of the cells but gives the same activation energy. The results confirm a hypothesis that respiratory depolarizers affect only the total enzyme concentration and do not change the apparent Ea of the substrate-enzyme decomposition reaction. The theoretical treatment of oxygen diffusion in the system provides guidelines for designing steady-state cellular respiration experiments.

The magnetization and static magnetic susceptibility of fusinite and synthetic carbohydrate chars are reported for magnetic fields up to 5.5 T and temperature range 2-320 K. The low-field magnetic susceptibility data exhibited Curie-Weiss behavior over the whole temperature range. The Weiss constant characterizes the overall exchange as antiferromagnetic, of the order of 1 K for all samples studied. The magnetic field dependence was fitted to a modified Brillouin function in order to determine the effective spin states of the paramagnetic species. Under the assumption of complete quenching of the orbital momentum, the spin quantum number was derived from the characteristic shape of the Brillouin function as well as from the ratio of the Curie constant to the saturation magnetization

The peak-to-peak line width (LW) of the first derivative electron spin resonance (EPR) spectrum of the coal maceral fusinite is reversibly broadened by O2. The extent of broadening per unit of partial pressure of oxygen (pO2) is unusually large, exceeding that of nitroxides by almost two orders of magnitude. This paramagnetic property of fusinite, combined with its very stable physicochemical properties and low toxicity, is shown to be of utility in the measurement of pO2 in vitro and in vivo. Fusinite particles are endocytosed by chinese hamster ovary (CHO) cells in vitro; this is useful for intracellular O2 measurements with commercially available EPR spectrometers operating at 9.1-9.3 GHz. For measurement of oxygen in vivo using low frequency EPR (1.1-1.3 GHz), fusinite provides a sensitive and persistent means to measure pO2 in tissues. Particles implanted into the gastrocnemius muscle of A/J mice remained interstitially in the same position for months with undiminished sensitivity to pO2 and no specific toxic effects.

There are many advantages to carrying out electron paramagnetic resonance experiments at very high frequencies, either as part of a multifrequency strategy for solving problems or for special characteristics of high frequencies. These special characteristics include the potential for high point sensitivity, enhanced resolution, separation of similar species, altered sensitivity to motion, suppression of motional effects, and many others. This paper describes a three-millimeter-wavelength (W-band, 94 GHz) EPR spectrometer built for a multi-user facility and illustrates with some examples, most of them being disordered systems. One significant example is the oxidized primary reaction center, P 865 + , isolated from the red photosynthetic bacteriaRhodobacter Sphaeroides R-26. The W-band technique applied to both centers isolated from bacteria grown from either deuterated or ordinary growth media allows extraction of the fullg anisotropy in these centers and sets the stage for multifrequency EPR spectroscopy to yield a full analysis of the various contributions to linewidths in these systems.

In order to meet the need for a good new EPR intensity andg-value standard whose paramagnetic species are carbon-based radicals, several materials were investigated, including coal, fusinite (a coal maceral), and several carbohydrate chars. Of the prototypical standards prepared, a chemically-treated fusinite is recommended as most suitable because of its chemical stability, spin density, EPR signal line shape and line width, microwave power saturation characteristics, availability, and homogeneity. Effects of dilution with KBr, KCl, and polymer are negligible, although the line width is broadened in the presence of paramagnetic gases. Several model standard compounds have been prepared in a polymer matrix to minimize changes in packing density over time.

A gradient technique to measure electron paramagnetic resonance spectra simultaneously at several different locations is described. The technique is based on the use of point probes containing paramagnetic centers. The value of the magnetic field gradient is chosen to be sufficient to separate the EPR signals from the different paramagnetic probes yet at the same time small enough to change only minimally the shape of individual signals. The conditions to apply this technique are considered in detail. When experimental data have a high signal-to-noise ratio, the lineshape distortion induced by the gradient can be corrected with the aid of a known distribution function of paramagnetic centers within the probe. The maximum entropy deconvolution algorithm is successfully applied for the correction of significantly distorted lines. The technique is experimentally tested and applied to measure the concentration of oxygen in hypertrophied rat myocardium and normal rat kidney in vivo by low frequency EPR (L-band, 1.2 GHz). Two types of EPR oxygen-sensitive probes--lithium phthalocyanine crystals and synthetic carbohydrate chars--were used.

Introduction of a Gaussian weighting function into the phase encoding of the spin-echo imaging method via variable tip angle excitation, with concomitant optimization of the recycle delay, is shown to increase the sensitivity of T2-weighted imaging by up to 40%. In addition, the Gibbs artifact associated with sharp boundaries is reduced considerably without the need for postprocessing of the data. © 1991 Academic Press, Inc.

A new method able to measure the concentration of oxygen in complex biological systems, including in vivo, has been developed using low-frequency EPR and newly characterized free radicals that are very sensitive to the concentration of oxygen. The free radicals (fusinite and lithium phthalocyanine) are very stable in tissues (for at least 150 days), apparently nontoxic, and can reflect oxygen concentrations that are less than the Km of cytochrome oxidase (0.1 microM or lower). Their biological stability is indicated by the fact that repeated measurements with fusinite of the concentration of oxygen in skeletal muscle have been made in the same animal for more than 150 days without any change in sensitivity or signs of toxicity.

Nano-scaled ceramic particles, such as layered double hydroxides (LDHs) have been developed to preserve and deliver drugs or genes in biological cells. Unstable molecules were intercalated into LDHs displacing the Cl - ions, shielding the drug or gene's anionic charge and enabling penetration into the cell. In the slightly acidic environment of the cell, the ceramic nano-platelets of ∼ 100 nm diameter dissolved, releasing the intercalates in a controlled manner. Indeed, the chelate diethylenetriamine-N, N, N', N", N"-pentaacetate (DTPA), complexed with Gd 3 + or VO 2 + showed their characteristic EPR spectra with a slow tumbling environment in the LDH matrix. Preliminary safety studies with rats showed that there were no systemic effects as demonstrated by clinical chemistry and histopathology. In vitro studies indicated that pristine LDH nanohybrid material had no cytotoxic or growth inhibitory effects. LDH intercalated with disodium pamidronate (Aredia ) have successfully been synthesized. From pharmacokinetic in vivo studies, we can suggest the possibility of using LDH bioresorbable nanoparticles as carriers for slow-release, intravenous drug administration.

Objective—To determine effects of reactive oxygen metabolites (ROMs), with and without flunixin meglumine, on equine right ventral colon (RVC) in vitro. Animals—18 healthy horses and ponies. Procedures—In 3 groups of 6 animals each, short-circuit current and conductance were measured in RVC mucosa in Ussing chambers. The 3 groups received physiologic saline (0.9% NaCl) solution, IV, 10 minutes before euthanasia and tissue incubation in Krebs-Ringer-bicarbonate (KRB) solution; flunixin meglumine (1.1 mg/kg, IV) 10 minutes before euthanasia and tissue incubation in KRB solution; or physiologic saline solution, IV, 10 minutes before euthanasia and incubation in KRB solution with 2.7 × 10−5M flunixin meglumine. Incubation conditions included control (no addition) and ROM systems, including addition of 1mM xanthine and 80 mU of xanthine oxidase (to produce the superoxide radical), 1mM H2O2, and 1mM H2O2 and 0.5mM ferrous sulfate (to produce the hydroxyl radical). Results—All ROMs that were added or generated significantly increased the short-circuit current except in tissues coincubated with flunixin meglumine, and they induced mild epithelial vacuolation and apoptosis, but did not disrupt the epithelium nor change conductance, lactate dehydrogenase release, or [3H]mannitol flux. Conclusions and Clinical Relevance—Responses to ROMs could be attributed to increased chloride secretion and inhibited neutral NaCl absorption in equine RVC, possibly by stimulating prostaglandin production. The ROMs examined under conditions of this study could play a role in prostaglandin-mediated colonic secretion in horses with enterocolitis without causing direct mucosal injury.

The current approaches used for the analysis of electron paramagnetic resonance spectra of Gd3+ complexes suffer from a number of drawbacks. Even the elaborate model of [Rast et al., J. Chem. Phys. 113, 8724 (2000)] where the electron spin relaxation is explained by the modulation of the zero-field splitting (ZFS), by molecular tumbling (the so called static contribution), and deformations (transient contribution), is only readily applicable within the validity range of the Redfield theory [Advances in Magnetic Resonance, edited by J.-S. Waugh (Academic, New York, 1965), Vol. 1, p. 1], that is, when the ZFS is small compared to the Zeeman energy and the rotational and vibrational modulations are fast compared to the relaxation time. Spin labels (nitroxides and transition metal complexes) have been studied for years in systems that violate these conditions. The theoretical framework commonly used in such studies is the stochastic Liouville equation (SLE). The authors shall show how the physical model of Rast et al. can be cast into the SLE formalism, paying special attention to the specific problems introduced by the [Uhlenbeck and Ornstein, Phys. Rev. 36, 823 (1930)] process used to model the transient ZFS. The resulting equations are very general and valid for arbitrary correlation times, magnetic field strength, electron spin S, or symmetry. The authors demonstrate the equivalence of the SLE approach with the Redfield approximation for two well-known Gd3+ complexes.

Particulate materials with a strong, exchange-narrowed electron paramagnetic resonance (EPR) absorption line, can be used as oxygen sensors in EPR oximetry. Advantages of this kind of sensor include a single, nearly Lorentzian line shape which depends on T2e, size flexibility (from less than a micron to several millimeters), very low toxicity, and good chemical stability in biological environments. Two basic types of particulate sensors have been developed: carbon-based sensors derived from coal or synthesized from wood, and lithium phthalocyanine (LiPc). Both types of material report on local oxygen concentration through a variation in EPR line width with [O2], an approach sometimes called T2 oximetry. Interaction between oxygen molecules and unpaired electrons in the sensor is thought to proceed via Heisenberg spin exchange. Microscopy reveals very different morphology for LiPc and chars, the former being a crystalline structure and the latter being a complex network of channels and pores. These structural differences manifest themselves in the different behaviors of the two classes of sensor materials. Models of coal and char behavior, involving electron spin exchange, can explain the line shape behavior of these materials under various oxygen concentrations. Magnetic resonance and dynamic nuclear polarization can be employed along with other physical methods to develop a detailed picture of char surface area, porosity, and the interaction of the surface with water.

The diffusion and permeation of liquids into coal has important ramifications for a wide variety of industrial processes including coal liquefaction, combustion, coal transport in water slurries, and sulfur extraction. Each of these processes depends upon the maximum internal surface area of the coal coming into contact with the permeating liquid. In contrast to other techniques using indirect methods to follow permeation, nuclear magnetic resonance imaging can be used in a completely non-invasive and non-destructive way directly to visualize and measure the changes in internal structure as coal is swollen with organic and inorganic solvents. Two-dimensional and three-dimensional imaging techniques have been used to follow the ingress of a variety of solvents into samples of coal. The images obtained show that considerable inhomogeneities exist within the coal sample with regard to the rate of solvent penetration.

Advanced electronic magnetic resonance (EMR) as well as nuclear magnetic resonance (NMR) methods have been used to examine properties of coals, chars, and molecular species related to constituents of coal. During the span of this grant, progress was made on construction and applications to coals and chars of two high frequency EMR systems particularly appropriate for such studies--48 GHz and 95 GHz electron magnetic resonance spectrometer, on new low-frequency dynamic nuclear polarization (DNP) experiments to examine the interaction between water and the surfaces of suspended char particulates in slurries, and on a variety of proton nuclear magnetic resonance (NMR) techniques to measure characteristics of the water directly in contact with the surfaces and pore spaces of carbonaceous particulates.

Rational development of new selective paramagnetic contrast agents (PCAs) requires a detailed understanding of their interactions with biological macromolecules. This report shows how some of these interactions can be studied with electron paramagnetic resonance (EPR) through examples of Gd3+ complexes interactions with model phospholipid membranes. It is shown that the spin label EPR method can be used to detect: (i) presence and possible location of lipophilic contrast agents in the model membranes, (ii) changes and distortions in membrane organization upon interaction with the PCAs, and (iii) changes in the local polarity of the bilayer and its phase behavior due to addition of Gd3+ complexes. This work demonstrates that interaction of Gd3+ complexes with phospholipid bilayers can be observed directly from changes in their continuous wave (CW) EPR spectra obtained at frequencies higher than X-band (9.5 GHz), where signals arising from aqueous and lipid-bound Gd3+ complexes become resolved. Analysis of frequency dependence of the effective g-factors of the EPR signal provides estimates of zero-field splitting (ZFS) parameter for these complexes at physiological conditions and information on how this parameter is affected by interaction with lipids. Multifrequency EPR experiments at high magnetic fields are also useful in providing data on the frequency dispersion of electronic relaxation caused mainly by a modulation of the electron-electron dipolar interaction (ZFS) of these high spin ions.

Carbon chars have been synthesized in our laboratory from a variety of starting materials, by means of a highly controlled pyrolysis technique. These chars exhibit electron paramagnetic resonance (EPR) line shapes which change with the local oxygen concentration in a reproducible and stable fashion; they can be calibrated and used for oximetry. Biological stability and low toxicity make chars good sensors for in vivo measurements. Scalar and dipolar interactions of water protons at the surfaces of chars may be utilized to produce dynamic nuclear polarization (DNP) of the 1H nuclear spin population in conjunction with electron Zeeman pumping. Low-frequency EPR, DNP and DNP-enhanced MRI all show promise as oximetry methods when used with carbon chars.

Advanced electronic magnetic resonance (EMR) methods are used to examine properties of coals, chars, and molecular species related to constituents of coal. We have achieved substantial progress on upgrading the high field (HF) EMR (W-band, 95 GHz) spectrometers that are especially advantageous for such studies. Particularly, we have built a new second W-band instrument (Mark II) in addition to our Mark I. Briefly, Mark II features: (i) an Oxford custom-built 7 T superconducting magnet which is scannable from 0 to 7 T at up to 0.5 T/min; (ii) water-cooled coaxial solenoid with up to ±550 G scan under digital (15 bits resolution) computer control; (iii) custom-engineered precision feed-back circuit, which is used to drive this solenoid, is based on an Ultrastab 860R sensor that has linearity better than 5 ppm and resolution of 0.05 ppm; (iv) an Oxford CF 1200 cryostat for variable temperature studies from 1.8 to 340 K. During this grant period we have completed several key upgrades of both Mark I and II, particularly microwave bridge, W-band probehead, and computer interfaces. We utilize these improved instruments for HF EMR studies of spin-spin interaction and existence of different paramagnetic species in carbonaceous solids.

Magnetic resonance imaging was used to examine the equine metacarpophalangeal joint. Thirty-two saggital images generated by partial volume imaging were transferred to a computer for three dimensional reconstruction and analysis. All the tissues constituting the metacarpophalangeal joint were readily identified. The most significant increase finding regarded the soft tissues on the palmar aspect of the metacarpophalangeal joint and their interactions with the proximal sesamoid bones. the equine metacarpophalangeal joint has not previously been evaluated using 3-dimensional imaging software.

Advanced electronic magnetic resonance (EMR) methods are used to examine properties of coals, chars, and molecular species related to constituents of coal. During this grant period, progress was made on setting up a separate high frequency EMR system particularly appropriate for such studies and exploring the use of low-frequency dynamic nuclear polarization (DNP) to examine the interaction between fluids such as water and the surface of suspended char particles.

It is well known that continuous-wave EPR spectra of nitroxide probes (labels) introduced into phospholipid bilayers are sensitive to molecular oxygen. However, accurate determination of oxygen broadening from these experiments is complicated by the complex shapes of EPR spectra, which are strongly influenced by anisotropic restricted motion of the probe molecules. An accurate method is presented to extract the oxygen broadening from the spectra measured with and without oxygen and at the same temperature. The method is based on a fast convolution algorithm with Levenberg–Marquardt optimization. This method was previously applied to EPR oximetry with nitroxides exhibiting rotational motion in the fast limit. It is shown that for several membrane spin probes, the oxygen broadening can be described as homogeneous; thus, a one-linewidth-parameter fitting model is appropriate. The method is applied to measure permeability profiles of model membranes composed from 1,2-dimyristoyl-sn-glycero-3-phosphocholine above and below the main phase transition. For both membrane phases, the broadening of doxyl- and sterol-type labels is found to be homogeneous, a finding consistent with the model of Heisenberg exchange between molecular oxygen and spin probes. As an example, the method is applied to study the ethanol effect on local oxygen permeability of a phospholipid bilayer. It is shown that ethanol concentrations as low as 1% (v/v) increase oxygen permeability of the bilayer. The effect is larger at the surface of the membrane than at its center, indicating that ethanol molecules interact primarily within the polar head region of the bilayer.

EPR oximetry is used for in vivo and in vitro measurements of oxygen in biological systems, including experimental animals. The accuracy of oxygen measurements in T2 (line width) EPR oximetry is significantly improved if least-squares simulation is used to extract the line width parameters. The oxygen effect on the EPR spectra of nitroxide solutions and aqueous suspensions of fusinite can be described as an additional homogeneous broadening that modifies the EPR spectrum of the oxygen-free probe. This allows one to use a one-parameter line width model in most cases. The simulations were carried out with the use of a fast-convolution algorithm followed by Levenberg-Marquardt optimization. The validity of error estimates provided by this method was tested on sets of experimental spectra taken under common conditions. It is shown that the accuracy of oxygen measurements in line width (T2) oximetry is determined not only by the probe sensitivity (rate of line width change versus oxygen concentration), but also by the signal-to-noise ratio, inhomogeneous contribution to the line shape (e.g., unresolved proton superhyperfine structure), and the spectral window. The accuracy of oxygen measurements is compared for aqueous solutions of two nitroxide radicals with different superhyperfine structure and for aqueous suspensions of fusinite.