Nitric oxide localized to spinal cords of mice with experimental allergic encephalomyelitis: an electron paramagnetic resonance study

Experimental allergic encephalomyelitis (EAE) is a demyelinating autoimmune disorder that can be induced in susceptible mice by T lymphocytes sensitized to central nervous system (CNS) myelin components and is a prime animal model for the human CNS demyelinating disorder, multiple sclerosis (MS). Although CNS inflammation in which T lymphocytes and activated macrophages are the predominant cell types is observed in mice with EAE and in humans with MS, the exact mechanisms underlying the CNS damage and demyelination are not understood. Nitric oxide (NO), a gaseous free radical, has recently been shown to be a cytolytic product of activated macrophages. Using electron paramagnetic resonance spectroscopy, the nitric oxide free radical complexed with iron-sulfur proteins has been identified in affected spinal cords of mice with EAE, concurrent with the diminution of iron-sulfur proteins. These results indicate NO may play a role in the disease process of EAE, and perhaps MS.     

Reactions of reducing xenobiotics with oxymyoglobin. Formation of metmyoglobin, ferryl myoglobin and free radicals: an electron spin resonance and chemiluminescence study

The oxygen-haem centre of oxymyoglobin reacts with reducing xenobiotics such as hydroxylamines and phenols with the concomitant formation of metmyoglobin and oxidation of the respective xenobiotic. Metmyoglobin formation rates were measured by visible spectroscopy with xenobiotic concentrations ranging from 100 microM to 30 mM. Analogous to previous results obtained with oxyhaemoglobin, the first step in the reaction of hydroxylamines with oxymyoglobin leads to the formation of the one-electron oxidation product of hydroxylamine, a nitroxyl radical detectable by electron spin resonance. A variety of paramagnetic secondary products were also found. The terminal oxidation product of hydroxylamine and hydroxyurea was the myoglobin-nitric oxide complex, one showing similar spectral characteristics to the analogous haemoglobin-nitric oxide adduct found in our previous experiments. On the other hand, the amount of low-spin ferric complexes obtained from metmyoglobin and an excess of the respective hydroxylamine was considerably lower than the corresponding results with methaemoglobin. A second important reaction intermediate was the compound I-type ferryl haem-species detected by a recently-published chemiluminescence assay. Partial spectral resolution of the emitted light using a set of cut-off filters indicated that maximum light emission occurred above 600 nm, most probably involving excited porphyrin states. The intensity of oxymyoglobin-related light emission was considerably higher than that reported earlier with oxyhaemoglobin. This indicates a difference in the excitation mechanism which leads to the formation of the compound I-type ferry haem species.     

Ascorbyl free radical as a real-time marker of free radical generation in briefly ischemic and reperfused hearts. An electron paramagnetic resonance study

The role of free radicals in myocardial reperfusion injury remains controversial. We have developed a new method using ascorbyl free radical (AFR) as a real-time, quantitative marker of free radical generation during myocardial reperfusion. A total of 35 dogs were studied. Twelve open-chest dogs underwent either 5 minutes (n = 5) or 20 minutes (n = 7) of coronary artery occlusion and 30 minutes of reperfusion. Seven additional animals undergoing 20 minutes of coronary occlusion also received the antioxidant enzymes superoxide dismutase and catalase, beginning 10 minutes before occlusion through the end of reperfusion. Exogenous ascorbate was infused intravenously, and the concentration of AFR in the great cardiac vein was continuously measured by electron paramagnetic resonance spectroscopy. Preocclusion AFR concentration was similar in the three groups. Upon reperfusion, AFR rose significantly in each animal group (P < .05). However, the AFR rise in the 20-minute-occlusion group, 38 +/- 17%, was significantly greater than in the 5-minute-occlusion group, 27 +/- 14% (P < .002). In addition, in the animals that received superoxide dismutase and catalase, the rise in the AFR was markedly attenuated, 13 +/- 6% (P < .002). Two dogs that received ascorbate but did not undergo coronary artery occlusion/reperfusion sequences showed no change in coronary venous AFR signal, indicating the stability of the signal over time. Five dogs received ascorbate while undergoing interventions to alter coronary venous flow: intravenous saline, dobutamine, dipyridamole, and nitroglycerin. Coronary venous AFR changes were minimal despite large coronary flow alterations, indicating that the AFR signal is independent of changes in coronary venous flow.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of oxygen at physiological levels on the detection of free radical intermediates by electron paramagnetic resonance

In this study, we examined the effect of sepsis on the electrical properties of isolated ventricular myocytes. Sepsis was induced by cecal ligation and puncture (CLP). The control rats were sham-operated. Membrane potentials and ionic currents in isolated cardiac myocytes were measured by the tight-seal, whole-cell patch-clamp technique. The results show that the resting membrane potentials of heart cells were significantly lower in the septic group (18 hr post-CLP) than those in the control group. However, there was no significant difference in action potential duration of 50% and 90% repolarization between the two groups of cells. In voltage-clamp experiments, isoproterenol (10 nM), a beta-adrenergic agonist, caused an increase in L-type calcium current (ICa,L) in a similar magnitude in myocytes isolated from the control and septic rats. Furthermore, isoproterenol failed to modify the time constants for ICa,L inactivation and the overall shape of current-voltage relationship for both groups of cells. These results indicate that formation of a G omega seal and subsequent tight-seal whole-cell recording with patch-clamp technique can be performed in heart cells derived from CLP-induced septic rats, and that septic rat heart is capable of responding effectively to beta-adrenergic stimulation.     

Phase transitions of rat stratum corneum lipids by an electron paramagnetic resonance study and relationship of phase states to drug penetration

In order to relate barrier function to stratum corneum structure and the thermal transitions of corneum lipids, samples from hairless rat skin were investigated by using ESR and drug penetration techniques. The phase transition of stratum corneum lipids was estimated using a deeper probe (16-doxyl-stearic acid) inserted in the lipid bilayers and measuring the rotational correlation time, tau(c). Results of ESR study showed that stratum corneum lipids underwent thermal transitions at 39.3 +/- 1.6 degrees C and 63.6 +/- 2.6 degrees C roughly similar to the data obtained by differential scanning calorimetry measurements. Cholesterol oxidase treatment decreased the fluidity of the lipids at lower temperatures. The treatment of stratum corneum with laurocapram (1%) and isopropyl myristate (IPM, 2%) little changed both phase transition temperatures, although the treatment highly increased the molecular motion of the lipids. The flux (J(s)) of lipophilic drugs (beta-estradiol, indomethacin and betahistine) through the skin was enhanced with increasing temperatures, with an increase in the diffusion constant within skin and a decrease in the lag time. There was a good relationship between log J(s) or log permeability coefficient (K(p)) and 1/tau(c) in the temperature range of 45 to 64 degrees C. The calculated activation energy (delta E) for diffusion of these drugs across skin was 17-40 kcal/mol. Judging from our data, stratum corneum lipids of rat probably exist as the gel, crystalline state below 39 degrees C, the mesomorphic state between 39 and 64 degrees C and the fluid, liquid-crystalline state at temperatures of 64 degrees C or above. These results are in line with the permeability of these lipophilic drugs through the intercellular lipids disordered is highly increased.     

Radiation-induced radical formation in beta-glycerophosphate single crystals studied by electron paramagnetic resonance and electron nuclear double resonance spectroscopy: formation of an allylic-type radical

The purpose of this study was to better understand students' perceptions of and preferences for inclusion or pull-out service delivery models. Thirty-two students with and without learning disabilities who had participated in both models during the past 2 or 3 years were interviewed individually. Key questions assessed their perceptions of which model was most conducive to academic learning and which was most likely to yield social benefits, and the reasons for their beliefs. Results indicated that students' views varied. Overall, more children identified pull-out as the model of choice, but many children were confident that inclusion was meeting their academic and social needs. We interpret the results of this study as providing support for maintaining a continuum of service delivery options and for considering the placement of each child individually, based on his or her unique needs.     

Applications of electron paramagnetic resonance spectroscopy to study interactions of iron proteins in cells with nitric oxide

Nitric oxide and species derived from it have a wide range of biological functions. Some applications of electron paramagnetic resonance (EPR) spectroscopy are reviewed, for observing nitrosyl species in biological systems. Nitrite has long been used as a food preservative owing to its bacteriostatic effect on spoilage bacteria. Nitrosyl complexes such as sodium nitroprusside, which are added experimentally as NO-generators, themselves produce paramagnetic nitrosyl species, which may be seen by EPR. We have used this to observe the effects of nitroprusside on clostridial cells. After growth in the presence of sublethal concentrations of nitroprusside, the cells show they have been converted into other, presumably less toxic, nitrosyl complexes such as (RS)2Fe(NO)2. Nitric oxide is cytotoxic, partly due to its effects on mitochondria. This is exploited in the destruction of cancer cells by the immune system. The targets include iron-sulfur proteins. It appears that species derived from nitric oxide such as peroxynitrite may be responsible. Addition of peroxynitrite to mitochondria led to depletion of the EPR-detectable iron-sulfur clusters. Paramagnetic complexes are formed in vivo from hemoglobin, in conditions such as experimental endotoxic shock. This has been used to follow the course of production of NO by macrophages. We have examined the effects of suppression of NO synthase using biopterin antagonists. Another method is to use an injected NO-trapping agent, Fe-diethyldithiocarbamate (Fe-DETC) to detect accumulated NO by EPR. In this way we have observed the effects of depletion of serum arginine by arginase. In brains from victims of Parkinson's disease, a nitrosyl species, identified as nitrosyl hemoglobin, has been observed in substantia nigra. This is an indication for the involvement of nitric oxide or a derived species in the damage to this organ.     

New method to measure the carbamoylating activity of nitrosoureas by electron paramagnetic resonance spectroscopy

Neuronal nuclear fraction N1 was isolated from cerebral cortices of 15-day-old rabbits, and nuclear subfractions prepared, in order to study the location of nuclear lyso platelet-activating factor (lyso-PAF) acetyltransferase and alkylglycerophosphate (AGP) acetyltransferase, and factors that affect the loss of these two nuclear activities. Subfractionation of prelabelled N1 indicated that the nuclear envelope had the highest percentage of the radioactive acetylated products alkylacetylglycerophosphate (AAGP) and PAF, and the distribution of these phospholipids reflected phospholipid distributions in the nuclear subfractions. The majority (95%) of radioactive AAGP and PAF was also recovered in Triton X-100 extracts of prelabelled nuclei, suggesting that these acetylated lipids are located in nuclear membranes rather than in the nuclear matrix/chromatin. Of the nuclear subfractions, the envelope had the highest AGP and lyso-PAF acetyltransferase specific activities which were close to corresponding values seen in the parent N1 fraction. Thus the nuclear AGP and lyso-PAF acetyltransferases were principally localized to the nuclear membranes. Differentials in activity loss were seen for the two acetyltransferase activities. In the nuclear envelope fractions, the lyso-PAF acetyltransferase was the more susceptible to oxidation reactions which could be reversed or blocked by the use of reducing agents. In preincubations, N1 showed greater losses in lyso-PAF acetyltransferase activity than in AGP acetyltransferase activity, losses which were not attributable to oxidation. Addition of cytosolic fraction S3 to preincubations promoted losses for each acetyltransferase in N1, and gave evidence for cytosolic and endogenous nuclear contributions to the activity loss. Addition of okadaic acid to the preincubations did not prevent the decline of either acetyltransferase in intact nuclei, but did diminish the loss of nuclear lyso-PAF acetyltransferase activity promoted by S3 addition, and also blocked the loss of this acetyltransferase seen in preincubations of isolated nuclear envelopes. This suggests that nuclear lyso-PAF acetyltransferase is susceptible to okadaic acid-sensitive nuclear and cytosolic protein phosphatase activities, while AGP acetyltransferase may lose activity by the action of other phosphatases or by other mechanisms within the nucleus.     

Current status of electron spin resonance (ESR) for in vivo detection of free radicals

Much outstanding progress concerning the application of ESR spectroscopy/imaging in the biomedical field has been made in recent years. The literature in this field has already been specifically covered by several reviews. The aim of this article is to provide an overview of the most important findings, obtained in the last four years, in the detection and localization of different exogenous free radicals, as well as of endogenous free radicals in diverse experimental animal models.     

Why spin = 1, 2 species have no electron paramagnetic resonance signal under normal conditions: possible detection by electron paramagnetic resonance at frequency close to D value?

A universal EPR simulation program has been created by the author, which is based on the following spin Hamiltonian equation: [equation: see text] where D and E are the axial and rhombic zero-field splitting parameters, respectively. The program can be used for simulation of EPR spectra with half-integer electronic spin (S = n/2, n = 3, 5, 7, 9) systems. In this article, the integer spin (S = n/2, n = 2, 4) systems are also considered. The EPR simulation results show that when D > frequency, no EPR signal can be seen from EPR simulation; when D approximately frequency, whichever X/Q/W-band is used, the EPR signal can be seen on the basis of the simulated EPR results presented.     

Nitric oxide: a biological effector. Detection using electron paramagnetic resonance

The synthesis and preliminary biological characterization of two isomeric technetium labeled complexes (2,5,5,9-tetramethyl-4,7-diaza-7-(3' (R)-quinuclidinylcarboxymethyl)-2,9-decanedithiolato oxo 99/99mtechnetium(V), [99/99mTc]-1 and [99/99mTc]-2) designed to exhibit affinity to muscarinic cholinergic receptors are described. In vitro binding assays were conducted in mouse brain homogenates (whole brain-cerebellum) at 37 degrees C by the centrifugation method, where non-specific binding was defined by atropine (1 microM). The measured affinity (KD) of [99Tc]-1 for mAChR was 1.9 +/- 0.5 microM (mean +/- SEM; n = 3) and [99Tc]-2 was 4.5 +/- 0.5 microM (mean +/- SEM; n = 3). Scatchard analysis indicated that Bmax values were 10.6 +/- 0.5 and 16.9 +/- 0.5 pmol/mg tissue, respectively. In competition assays, [99Tc]-1 exhibited an apparent affinity (KI) of 16.5 microM (n = 2) against [125I] iododexetimide, whereas [99Tc]-2 exhibited an affinity (KI) of 105 microM. In vivo, 0.3% of the injected dose of [99mTc]-1 and [99mTc]-2 accumulated in the brain at 5 min after injection. These values indicate technetium analogues of neuroreceptor binding ligands can be synthesized and retain some affinity for the receptor.     

An application of electron paramagnetic resonance to evaluate nitric oxide and its quenchers

Electron paramagnetic resonance (EPR) spectrometry has been used for detection of free radicals, including nitric oxide (NO). The method was applied to evaluate if lazaroids, nonspecific scavengers of oxygen-derived free radicals, can reduce another radical species, namely, NO. When solutions containing 0 to 10 microM lazaroids (U78517F, U83836E, and U74500A) were mixed with NO (approximately 8.0 microM), and with a spin-trap agent specific to NO (10 microM carboxy-2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3 oxide, c-PTIO), the spectrum of NO-c-PTIO determined by the EPR decreased with increasing concentrations of lazaroids. An identical study with 1 to 1000 microM methylprednisolone or dexamethasone, which are prototypes of lazaroids, failed to reduce NO. In a separate study, rat red blood cells preloaded with U78517F were exposed to NO. EPR spectrometry demonstrated that hemoglobin in these cells were less nitrosylated compared with the cells not preloaded with U78517F. An ability of lazaroids to quench NO in vivo was evaluated by an intravenous injection of sodium nitrite (4 mumol/kg body weight) into rats pretreated with 10 mg/kg lazaroids (U78517F and U74500A). The nitrosylation of RBC hemoglobin was markedly attenuated in rats pretreated with lazaroids compared with those with vehicle. Thus, these lipophilic compounds protected cells from NO-induced nitrosylation both in vitro and in vivo. Lazaroids may be applicable as anti-NO agents.     

Detection of free radicals in blood by electron spin resonance in a model of respiratory failure in the rat

Previous work has suggested that a free radical mechanism is involved in some types of muscle fatigue and that there can be free radicals released extracellularly. Because muscle fatigue may be an important factor in respiratory failure, the authors tested the hypothesis that increased concentrations of free radicals could be detected in the blood of animals undergoing severe resistive loading to respiratory failure. An ex vivo spin trapping technique with alpha-phenyl-N-tert-butylnitrone (PBN) was used to investigate the possible formation of free radicals in systemic blood samples by electron spin resonance (ESR) spectrometry. After 2.5-3 h of severe inspiratory resistive loading with 70% supplemental inspired oxygen, free radical levels in the form of PBN-adducts were found to rise significantly over the control group breathing room air and the control group breathing 70% oxygen (p < 0.05, N = 8). There were no significant differences between control groups breathing room air and control groups breathing 70% oxygen. This study presents direct evidence that free radicals are produced ex vivo and that they can be detected in the systemic circulation due to excessive resistive loading of the respiratory muscles.     

Binding of nitric oxide to intact human erythrocytes as monitored by electron paramagnetic resonance

Human blood was diluted in isotonic saline at pH 7.4 and deoxygenated. After gentle exposure to nitric oxide gas (NO), the red blood cells (erythrocytes) remained intact. Increasing the cell volume fraction allowed detection of strong electron paramagnetic (EPR) signals, even at ambient temperature (293 K). These spectra were compared to those recorded at 77 K. With maximal NO exposure, a relatively featureless and stable spectrum was recorded. Reduced exposure produced a spectrum, which gradually transformed into the final one, with more structure. The spectral features with unfrozen samples reflected the degree of resolution of the hyperfine triplet component observed at 77 K. This hyperfine coupling was independent of the temperature. The spectra reveal rearrangement of the NO ligand between the subunits of hemoglobin. At subsaturation levels, binding to the alpha subunit, with its iron pentacoordinated in the T-state, dominates.     

The effects of lattice water on free radical yields in X-irradiated crystalline pyrimidines and purines: a low-temperature electron paramagnetic resonance investigation

The hydration layer of DNA increases the target size of DNA with respect to the formation of direct-type damage by ionizing radiation. The mechanisms that give rise to this increase are being investigated by EPR spectroscopy. To determine these mechanisms, it is necessary to distinguish between the change in sample mass and changes in packing/conformation brought about by the change in the level of hydration. Certain model compounds that crystallize as hydrates provide a system where the effects of mass and packing can be discerned. Three such hydrate crystals were used in this work: barbituric acid dihydrate (BA:2H2O), inosine dihydrate (IR:2H2O) and thymine monohydrate (T:H2O). The free radical yields (+/-25%) in the native crystals at 7-11 K are 0.08, 0.03 and 0.02, respectively. Removal of the lattice water leaves behind an ordered lattice and results in free radical yields of 0.08, 0.03 and < 0.004, respectively. Thus removal of the lattice water does not affect the free radical yield in BA:2H2O or IR:2H2O but decreases the free radical yield in T:H2O by an order of magnitude. Based on these observations and the known crystal packing, we conclude that the hydrogen bonding network is a major factor in determining the distribution and yield of trapped free radicals. We ascribe this to the importance of proton transfer processes which act to reduce the probability of radical combination. Consistent with this conclusion are the types of free radicals trapped in these crystalline materials before and after dehydration. From these results, we argue that a major determinant of free radical yields in solidstate samples of DNA constituents is molecular packing. In addition, the absence of HO. radicals trapped in single crystals of BA:2H2O provides an upper limit for the yield of trapped HO. of less than 10(-4) mumol/J. This supports the thesis that at < 77 K direct ionization of those waters associated directly with a pyrimidine or purine results in hole transfer to that molecule. Hydroxyl radical formation on a water adjacent to a DNA base is predicted to be negligible.     

Characterization of calcium-binding sites in the kidney stone inhibitor glycoprotein nephrocalcin with vanadyl ions: electron paramagnetic resonance and electron nuclear double resonance spectroscopy

Nephrocalcin (NC) is a calcium-binding glycoprotein of 14,000 molecular weight. It inhibits the growth of calcium oxalate monohydrate crystals in renal tubules. The NC used in this study was isolated from bovine kidney tissue and purified with the use of DEAE-cellulose chromatography into four isoforms, designated as fractions A-D. They differ primarily according to the content of phosphate and gamma-carboxy-glutamic acid. Fractions A and B are strong inhibitors of the growth of calcium oxalate monohydrate crystal, whereas fractions C and D inhibit crystal growth weakly. Fraction A, with the highest Ca(2+)-binding affinity, was characterized with respect to its metal-binding sites by using the vanadyl ion (VO2+) as a paramagnetic probe in electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) spectroscopic studies. By EPR spectrometric titration, it was shown that fraction A of NC bound VO2+ with a stoichiometry of metal:protein binding of 4:1. Also, the binding of VO2+ to NC was shown to be competitive with Ca2+. Only protein residues were detected by proton ENDOR as ligands, and these ligands bound with complete exclusion of solvent from the inner coordination sphere of the metal ion. This type of metal-binding environment, as derived from VO(2+)-reconstituted NC, differs significantly from the binding sites in other Ca(2+)-binding proteins.     

The application of PEDRI to the study of free radicals in vivo

Proton-electron double-resonance imaging (PEDRI) has considerable value for study of the distribution and elimination pathways of nitroxide free radicals (NFRs). This has been illustrated by its use in studies of kidney function in the living rat in which the NFR proxyl carboxylic acid (PCA) has been employed as a 'tracer'. The technique, at its present stage of development, can demonstrate location of PCA in enough detail to observe the passage through kidney cortex and medulla differentially, and to see the NFR within the major abdominal blood vessels. These studies are helping towards an understanding of the metabolic fate of PCA, as well as providing information about kidney performance after challenge with a nephrotoxin. In addition, nitric oxide complexes, formed in vivo by providing rats with a nitrite-rich diet, have been observed ex vivo using PEDRI and field-cycled DNP.