triacetoneamine-n-oxyl and nitroxyl

To develop an estimation method of gadolinium magnetic resonance imaging (MRI) contrast agents, the effect of concentration of Gd compounds on the ESR spectrum of nitroxyl radical was examined. A solution of either 4-oxo-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPONE) or 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL) was mixed with a solution of Gd compound and the ESR spectrum was recorded. Increased concentration of gadolinium-diethylenetriamine pentaacetic acid chelate (Gd-DTPA), an MRI contrast agent, increased the peak-to-peak line widths of ESR spectra of the nitroxyl radicals, in accordance with a decrease of their signal heights. A linear relationship was observed between concentration of Gd-DTPA and line width of ESR signal, up to approximately 50 mmol/L Gd-DTPA, with a high correlation coefficient. Response of TEMPONE was 1.4-times higher than that of TEMPOL as evaluated from the slopes of the lines. The response was slightly different among Gd compounds; the slopes of calibration curves for acua[N,N-bis[2-[(carboxymethyl)[(methylcarbamoyl)methyl]amino]ethyl]glycinato(3-)]gadolinium hydrate (Gd-DTPA-BMA) (6.22 μT·L/mmol) and gadolinium-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid chelate (Gd-DOTA) (6.62 μT·L/mmol) were steeper than the slope for Gd-DTPA (5.45 μT·L/mmol), whereas the slope for gadolinium chloride (4.94 μT·L/mmol) was less steep than that for Gd-DTPA. This method is simple to apply. The results indicate that this method is useful for rough estimation of the concentration of Gd contrast agents if calibration is carried out with each standard compound. It was also found that the plot of the reciprocal square root of signal height against concentrations of contrast agents could be useful for the estimation if a constant volume of sample solution is taken and measured at the same position in the ESR cavity every time.

Topics: Animals; Contrast Media; Coordination Complexes; Cyclic N-Oxides; Electron Spin Resonance Spectroscopy; Gadolinium; Gadolinium DTPA; Magnetic Resonance Imaging; Male; Nitrogen Oxides; Rats; Rats, Wistar; Spin Labels; Triacetoneamine-N-Oxyl

The work in part 6 of this series (J. Phys. Chem. A 2009, 113, 4930), addressing the task of separating the effects of Heisenberg spin exchange (HSE) and dipole-dipole interactions (DD) on electron paramagnetic resonance (EPR) spectra of nitroxide spin probes in solution, is extended experimentally and theoretically. Comprehensive measurements of perdeuterated 2,2,6,6-tetramethyl-4-oxopiperidine-1-oxyl (pDT) in squalane, a viscous alkane, paying special attention to lower temperatures and lower concentrations, were carried out in an attempt to focus on DD, the lesser understood of the two interactions. Theoretically, the analysis has been extended to include the recent comprehensive treatment by Salikhov (Appl. Magn. Reson. 2010, 38, 237). In dilute solutions, both interactions (1) introduce a dispersion component, (2) broaden the lines, and (3) shift the lines. DD introduces a dispersion component proportional to the concentration and of opposite sign to that of HSE. Equations relating the EPR spectral parameters to the rate constants due to HSE and DD have been derived. By employing nonlinear least-squares fitting of theoretical spectra to a simple analytical function and the proposed equations, the contributions of the two interactions to items 1-3 may be quantified and compared with the same parameters obtained by fitting experimental spectra. This comparison supports the theory in its broad predictions; however, at low temperatures, the DD contribution to the experimental dispersion amplitude does not increase linearly with concentration. We are unable to deduce whether this discrepancy is due to inadequate analysis of the experimental data or an incomplete theory. A new key aspect of the more comprehensive theory is that there is enough information in the experimental spectra to find items 1-3 due to both interactions; however, in principle, appeal must be made to a model of molecular diffusion to separate the two. The permanent diffusion model is used to illustrate the separation in this work. In practice, because the effects of DD are dominated by HSE, negligible error is incurred by using the model-independent extreme DD limit of the spectral density functions, which means that DD and HSE may be separated without appealing to a particular model.

Topics: Diffusion; Electron Spin Resonance Spectroscopy; Kinetics; Least-Squares Analysis; Models, Chemical; Nitrogen Oxides; Quantum Theory; Solutions; Squalene; Temperature; Thermodynamics; Triacetoneamine-N-Oxyl; Viscosity

Electron spin relaxation times of perdeuterated tempone (PDT) 1 and of a nitronyl nitroxide (2-(4-carboxy-phenyl)-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl) 2 in aqueous solution at room temperature were measured by 2-pulse electron spin echo (T(2)) or 3-pulse inversion recovery (T(1)) in the frequency range of 250 MHz to 34 GHz. At 9 GHz values of T(1) measured by long-pulse saturation recovery were in good agreement with values determined by inversion recovery. Below 9 GHz for 1 and below 1.5 GHz for 2,T(1)~T(2), as expected in the fast tumbling regime. At higher frequencies T(2) was shorter than T(1) due to incomplete motional averaging of g and A anisotropy. The frequency dependence of 1/T(1) is modeled as the sum of spin rotation, modulation of g and A-anisotropy, and a thermally-activated process that has maximum contribution at about 1.5 GHz. The spin lattice relaxation times for the nitronyl nitroxide were longer than for PDT by a factor of about 2 at 34 GHz, decreasing to about a factor of 1.5 at 250 MHz. The rotational correlation times, τ(R) are calculated to be 9 ps for 1 and about 25 ps for 2. The longer spin lattice relaxation times for 2 than for 1 at 9 and 34 GHz are due predominantly to smaller contributions from spin rotation that arise from slower tumbling. The smaller nitrogen hyperfine couplings for the nitronyl 2 than for 1 decrease the contribution to relaxation due to modulation of A anisotropy. However, at lower frequencies the slower tumbling of 2 results in a larger value of ωτ(R) (ω is the resonance frequency) and larger values of the spectral density function, which enhances the contribution from modulation of anisotropic interactions for 2 to a greater extent than for 1.

Topics: Anisotropy; Deuterium; Electron Spin Resonance Spectroscopy; Indicators and Reagents; Nitrogen Oxides; Oxygen; Solutions; Spin Labels; Triacetoneamine-N-Oxyl; Water

We propose the use of the pulse electron double resonance (ELDOR) method to determine the effective saturation factor of nitroxide radicals for dynamic nuclear polarization (DNP) experiments in liquids. The obtained values for the nitroxide radical TEMPONE-D,(15)N at different concentrations are rationalized in terms of spin relaxation and are shown to fulfil the Overhauser theory.

Topics: Electron Spin Resonance Spectroscopy; Magnetic Resonance Spectroscopy; Nitrogen Isotopes; Nitrogen Oxides; Triacetoneamine-N-Oxyl

EPR line shifts due to spin exchange of perdeuterated 2,2,6,6-tetramethyl-4-oxopiperidine-1-oxyl (14N-PDT) in aqueous solutions and the same probe isotopically substituted with 15N (15N-PDT) were measured from 293 to 338 and 287 to 353 K, respectively. Nonlinear least-squares fits of the EPR spectra yielded the resonance fields of the nitrogen hyperfine lines to high precision from which the shifts were deduced. The shifts are described by two terms: one linear and the other quadratic in the electron spin-exchange frequency, omegae. The quadratic term is due to spin exchange that occurs when two spin probes diffuse together and collide. A linear term is predicted for spin exchanges that occur upon re-encounter of the same two probes while they occupy the same "cage" before diffusing apart. The quadratic term has no adjustable parameters, while the linear term has one: the mean time between re-encounters, tauRE. The theory is cast in terms of the spin-exchange-induced line broadening that can be measured from each spectrum independently of the line shifts, thereby removing the explicit dependence of omegae on the temperature and the spin-probe concentration. In this form, theoretically, the value of the linear term is about a factor of 2 larger for 15N-PDT than for 14N-PDT for all temperatures; however, tauRE must be the same. Experimentally, we find that both of these expectations are fulfilled, providing strong support that the linear term is indeed due to re-encounter collisions. Values of tauRE derived from 14N-PDT and 15N-PDT are of the same order of magnitude and show the same trend with temperature as a hydrodynamic estimate based on the Stokes-Einstein equation.

Topics: Electron Spin Resonance Spectroscopy; Free Radicals; Least-Squares Analysis; Nitrogen Isotopes; Nitrogen Oxides; Piperidines; Triacetoneamine-N-Oxyl

High-level ab initio g and A tensor components have been calculated for PD-tempone and tempo-palmitate (TP) radical spin probes dissolved in n-pentyl and n-hexyl cyanobiphenyl liquid crystals. Solvent effects have been included in the proposed approach by means of the polarizable continuum model, allowing for solvent anisotropy. An in-depth analysis of the electronic structure of probes was performed to choose a suitable model for TP and make the calculations more accessible. Computed magnetic tensor components have been compared with corresponding values measured in the rigid limit. The quality of the results suggests the use of quantum-mechanical data to determine the order parameter of the nematic from experimental electron-spin resonance measurements.

Topics: Anisotropy; Cyclic N-Oxides; Electron Spin Resonance Spectroscopy; Electrons; Models, Molecular; Models, Statistical; Models, Theoretical; Molecular Conformation; Nitrogen Oxides; Palmitic Acid; Quantum Theory; Reproducibility of Results; Software; Triacetoneamine-N-Oxyl

Earlier we described a novel cytochrome P450 (CYP) catalyzed metabolism of the 2,2,6,6-tetramethylpiperidine (2,2,6,6-TMPi) moiety in human liver microsomes to a ring-contracted 2,2-dimethylpyrrolidine (2,2-DMPy) [Yin, W., et al. (2003) Drug Metab. Dispos. 31, 215-223]. In the current report, evidence is provided for the involvement of 2,2,6,6-TMPi hydroxylamines and their one-electron oxidation products, the nitroxide radicals, as intermediates in this pathway. Nitroxide radicals could be converted to their corresponding 2,2-DMPy metabolites by "inactivated CYP3A4", as well as by a number of other heme proteins and hemin, suggesting that this is a heme-catalyzed process. The conversion of nitroxide radicals to the 2,2-DMPy products by CYP3A4 or hemin was accompanied by the generation of acetone in incubations, providing evidence that the three-carbon unit from 2,2,6,6-TMPi was lost as acetone. With one model 2,2,6,6-TMPi nitroxide radical, evidence for an alternate pathway, which resulted in the formation of an intermediate that incorporated two oxygen atoms from water of the incubation medium before collapsing to the 2,2-DMPy product, was also obtained. To account for both pathways, a mechanism involving interaction of the nitroxide radicals with heme iron (Fe(III)), followed by a homolytic scission of the N-O bond and transfer of the nitroxide oxygen to heme iron to form a perferryl-oxygen complex, is proposed. The nitrogen-centered 2,2,6,6-TMPi radical thus formed then precipitates the contraction of the piperidine ring via C2-C3 bond cleavage, and the resulting product further oxidizes to an exocyclic iminium ion (by the perferryl-oxygen complex); the latter may undergo capture by water from the incubation medium and eliminate the three-carbon unit via N-dealkylation. It remains to be determined whether this novel interaction of nitroxide radicals with heme iron has any relevance in regard to the known biological properties of these stable radical species.

Topics: Aerobiosis; Anaerobiosis; Carbon Monoxide; Cyclic N-Oxides; Cytochrome P-450 CYP3A; Cytochrome P-450 Enzyme System; Deferoxamine; Free Radicals; Heme; Hemin; Humans; Iron; Iron Chelating Agents; Microsomes, Liver; NADP; Nitrogen Oxides; Oxygen Isotopes; Piperidones; Recombinant Proteins; Spectrometry, Mass, Electrospray Ionization; Triacetoneamine-N-Oxyl

Modulation of radiation- and metal ion-catalyzed oxidative-induced damage using plasmid DNA, genomic DNA, and cell survival, by three nitroxides and their corresponding hydroxylamines, were examined. The antioxidant property of each compound was independently determined by reacting supercoiled DNA with copper II/1,10-phenanthroline complex fueled by the products of hypoxanthine/xanthine oxidase (HX/XO) and noting the protective effect as assessed by agarose gel electrophoresis. The nitroxides and their corresponding hydroxylamines protected approximately to the same degree (33-47% relaxed form) when compared to 76.7% relaxed form in the absence of protectors. Likewise, protection by both the nitroxide and corresponding hydroxylamine were observed for Chinese hamster V79 cells exposed to hydrogen peroxide. In contrast, when plasmid DNA damage was induced by ionizing radiation (100 Gy), only nitroxides (10 mM) provide protection (32.4-38.5% relaxed form) when compared to radiation alone or in the presence of hydroxylamines (10 mM) (79.8% relaxed form). Nitroxide protection was concentration dependent. Radiation cell survival studies and DNA double-strand break (DBS) assessment (pulse field electrophoresis) showed that only the nitroxide protected or prevented damage, respectively. Collectively, the results show that nitroxides and hydroxylamines protect equally against the damage mediated by oxidants generated by the metal ion-catalyzed Haber-Weiss reaction, but only nitroxides protect against radiation damage, suggesting that nitroxides may more readily react with intermediate radical species produced by radiation than hydroxylamines.

Topics: Animals; Antioxidants; Cell Line; Cell Survival; Copper; Cyclic N-Oxides; DNA; DNA Damage; Dose-Response Relationship, Drug; Dose-Response Relationship, Radiation; Electrophoresis; Hydrogen Peroxide; Hydroxylamines; Nitrogen Oxides; Oxidation-Reduction; Phenanthrolines; Plasmids; Radiation-Protective Agents; Spin Labels; Triacetoneamine-N-Oxyl

The primary objective of this study is the investigation of bioreduction kinetics of hydrophilic spin probes, 2,2,6,6, -tetramethyl-4-oxo-piperidinyl-1-oxyl (Tempone), and spin-labeled antibiotic gentamicin by gram-negative bacteria maintained at various oxygen tensions, with emphasis on the effect of probe penetration rate. This information was used to evaluate the effect of ultrasound on the penetration of hydrophilic compounds, including antibiotics, into Pseudomonas aeruginosa and Escherichia coli cells. Penetration of spin-labeled compounds into the cells was assessed by the reduction rate of the nitroxyl moiety measured by EPR. In cell suspensions, both Tempone and spin-labeled gentamicin were localized predominantly in the aqueous phase surrounding the cells. However, a gradual reduction of the probes in contact with the cells indicated that the probes penetrated through the outer membrane and periplasmic space into the cytoplasmic membrane, where the electron transport chains and other metabolic activities of gram-negative bacteria are localized. The kinetics of probe reduction depended on oxygen tension and presence of electron transport chain blockers. It was found that probe penetration rate through the outer cell membrane affected the rate of probe reduction; damaging the permeability barrier by cell incubation with EDTA or by powerful insonation above the cavitation threshold increased the rate of probe reduction. In contrast, insonation below the cavitation threshold did not affect the rate of probe reduction. These findings imply that the recently observed synergistic effect between hydrophilic antibiotics and low frequency ultrasound in killing gram-negative bacteria did not result from the enhanced antibiotic penetration through bacterial cell walls.

Topics: Cell Membrane; Cell Membrane Permeability; Cell Wall; Edetic Acid; Electron Spin Resonance Spectroscopy; Electron Transport; Escherichia coli; Gentamicins; Kinetics; Nitrogen; Nitrogen Oxides; Oxidation-Reduction; Oxygen; Periplasm; Pseudomonas aeruginosa; Sodium Azide; Sonication; Spin Labels; Triacetoneamine-N-Oxyl; Vitamin K

We describe real-time measurement of myocardial oxygen consumption during ischemia in the intact heart. Measurement of extracellular oxygen concentration during myocardial ischemia by spin label oximetry has been limited by ischemia-induced reduction of the neutral, water-soluble nitroxide TEMPONE. We have overcome this problem by encapsulating the nitroxides. Isolated immature (7-10 d old) rabbit hearts (n = 8) were perfused aerobically within the cavity of a loop gap resonator with bicarbonate buffer containing an oxygen-sensitive, lipid-soluble nitroxide (14N-TEMPO laurate in FC-43 perfluorocarbon micelles) and a much less oxygen-sensitive and positively charged nitroxide (15N-TEMPO choline in multilamellar vesicles) as an internal standard. The ratio of the ESR signal amplitudes of these nitroxides was used as a sensitive index of oxygen concentration. Sequestration of the nitroxides decreased their reduction rate by ascorbate in comparison with nonsequestered nitroxides. Hearts were subjected to 60 min of global no-flow ischemia at 20 degrees C. Extracellular oxygen content (mean +/- SD) during aerobic perfusion was 1195 +/- 55 mumol/liter. The electron spin resonance signal from TEMPO laurate increased with the onset and progression of ischemia, consistent with a decrease in extracellular oxygen, while the signal for TEMPO choline was relatively unchanged. Extracellular oxygen content after 40 and 60 min of ischemia was reduced to 393 +/- 27 mumol/liter (p < .05) and 61 +/- 5 mumol/liter (p < .05), respectively. We conclude that spin-label oximetry can directly and precisely measure myocardial oxygen consumption at constant temperature during ischemia in the intact heart.

Topics: Animals; Electron Spin Resonance Spectroscopy; Feasibility Studies; Free Radicals; In Vitro Techniques; Liposomes; Micelles; Myocardial Ischemia; Nitrogen Oxides; Oxidation-Reduction; Oximetry; Oxygen; Perfusion; Rabbits; Spin Labels; Triacetoneamine-N-Oxyl

Sonodynamic therapy is a promising new modality for cancer treatment based on the synergistic effect on tumor cell killing by combination of a drug (typically a photosensitizer) and ultrasound. The mechanism of sonodynamic action was suggested to involve photoexcitation of the sensitizer by sonoluminescent light, with subsequent formation of singlet oxygen. In this work we studied the aqueous sonochemical reactions of the gallium-porphyrin derivative ATX-70, one of the most active sonodynamic agents found, using 50 kHz ultrasound. The experiments were carried out in the presence of 2,2,6,6-tetramethyl-4-piperidone hydrochloride (TMP), which reacts with singlet oxygen or .OH radicals to give the EPR-detectable nitroxide 2,2,6,6-tetramethyl-4-piperidone-N-oxyl (TMP-NO). Recently it has been suggested that the enhancement of TMP-NO yields in the presence of aqueous solutions of ATX-70 exposed to ultrasound was evidence for the formation of singlet oxygen in the system. Our results show that the surfactant cetyltrimethylammonium bromide (CTAB) can mimic the ATX-70-induced increase in the TMP-NO signal, but it fails to reproduce the behavior of ATX-70 in D2O: while the yields of TMP-NO in the presence of ATX-70 increase in D2O, the opposite effect was found with the surfactant CTAB. However, our data show that the increased TMP-NO yields in D2O are paralleled by an increased concentration of ATX-70 dimer, a form that is inactive in the photochemical generation of singlet oxygen. Our finding that the ATX-70-dependent enhancement of the TMP-NO signal was highest at approximately 20% O2, in both N2/O2 and argon/O2 mixtures, and decreased with increasing oxygen concentration is not compatible with the singlet oxygen mechanism. Finally, our results on the temperature dependence of the ATX-70-induced formation of TMP-NO are not consistent with the photochemical excitation of ATX-70 by sonoluminescent light: the ATX-70-dependent enhancement of TMP-NO signal increased with temperature in the range 10-25 degrees C, while the intensity of sonoluminescence of aqueous solutions both in multiple-bubble fields and in single-bubble experiments is known to decrease with increasing temperature.

Topics: Antineoplastic Agents; Deuterium; Electron Spin Resonance Spectroscopy; Free Radicals; Nitrogen Oxides; Oxygen; Piperidones; Porphyrins; Triacetoneamine-N-Oxyl; Ultrasonics; Water

Low-temperature electron paramagnetic resonance (EPR) spectroscopy and spin traps were used to measure paramagnetic species generation in rat hearts and isolated mitochondria. The hearts were freeze-clamped at 77 K during control perfusion by the Langendorff procedure, after 20-30 min of normothermic ischemia or 10-30 s of reperfusion with oxygenated perfusate. All EPR spectra measured at 4.5-50 K exhibited signals of both mitochondrial free radical centers and FeS proteins. The analysis of spectral parameters measured at 243 K showed that free radicals in heart tissue were semiquinones of coenzyme Q10 and flavins. The appearance of a typical "doublet" signal at g = 1.99 in low-temperature spectra indicated that a part of ubisemiquinones formed a complex with a high potential FeS protein of succinate dehydrogenase. Ischemia decreased the free radical species in myocardium approximately 50%; the initiation of reflow of perfusate resulted in quick increase of the EPR signal. Mitochondria isolated from hearts during control perfusion and after 20-30 min of ischemia were able to produce superoxide radicals in both the NADH-coenzyme Q10 reductase and the bc1 segments of the respiratory chain. The rate of oxyradical generation was significantly higher in mitochondria isolated from ischemic heart.

Topics: Animals; Coronary Disease; Electron Spin Resonance Spectroscopy; Flavoproteins; Free Radicals; In Vitro Techniques; Male; Myocardial Reperfusion; Myocardium; Nitrogen Oxides; Rats; Rats, Inbred Strains; Superoxides; Triacetoneamine-N-Oxyl; Ubiquinone