Development of isoindoline nitroxides for EPR oximetry in viable systems

Nitroxides are widely used as biophysical probes to study molecular motion, intracellular oxygen, pH, transmembrane potential, and cellular redox metabolism, etc. They may be rapidly metabolized to hydroxylamines by cells, which limits their use in viable systems. In this study, we have characterized relevant properties in cells of several isoindoline nitroxides that have been prepared to have different physicochemical properties: 1,1,3,3-tetramethylisoindolin-2-yloxyl (TMIO) and its analogs 5-carboxy-1,1,3,3-tetramethylisoindolin-2-yloxyl (CTMIO), 5-(N,N,N-trimethylammonio)-1,1,3,3-tetramethyl isoindolin-2-yloxyl iodide (QATMIO) and 2-hydroxy-1,1,3,3-tetramethylisoindoline hydrochloride (TMIOH.HCI). The oxygen sensitivity and metabolic kinetics of these were compared in CHO cells under different oxygen tensions with 1-oxyl-2,2,6,6-tetramethyl-4-piperidione (Tempone) and 3-carboxyl-2,2,5,5-tetramethyl-pyrrolidine-1-oxyl (PCA). Cytotoxicity was evaluated by the measurement of oxygen consumption rates, trypan blue exclusion, and clone formation. TMIO and its analogues have a higher relative oxygen sensitivity than Tempone and PCA with the oxygen sensitivity in electron paramagnetic resonance (EPR) spectrometry in the order of: TMIO=TMIOH=CTMIO>QATMIO=Tempone<PCA. The rates of metabolism of these nitroxides are moderate and depend on oxygen concentration, ring type, ring substituent, and membrane permeation. These nitroxides have low cytotoxicity. The results indicate that TMIO and its analogues are potentially useful for EPR studies of viable systems, especially for oximetry.     

Practical conditions and limitations for high-spatial-resolution multisite EPR oximetry

We have previously reported a high-spatial-resolution multisite electron paramagnetic resonance (EPR) oximetry method that is based on consecutive applications of magnetic field gradients with the same direction but different magnitudes. This method that could be called also two-gradient convolution EPR oximetry has no restrictions for the shape of solid paramagnetic materials implanted in tissue and is applicable for any particulate EPR oxygen-sensitive matieral with a Lorentzian line shape. To enhance the utilization of this method, a previously described algorithm was used to develop user-friendly Windows-based software. Practical conditions of application of the method were established using several different model systems. It has been shown that the spectral overlap from the adjacent sites can be neglected if the splitting between the corresponding lines exceeds the largest line width by at least a factor of 1.3. An additional requirement of the method is that the second field gradient should exceed by at least 30% the value of the first gradient. It was confirmed that the error in line width determination at L-band is proportional to the noise-to-signal ratio, and does not exceed 1% a noise-to-signal ratio of 0.1 in a typical in vivo experiment. We demonstrate that the line widths of up to 10 different sites can be determined.     

Nano-emulsions of fluorinated trityl radicals as sensors for EPR oximetry

This article reports the development and evaluation of two nano-emulsions (F45T-03/HFB and F15T-03/PFOB) containing fluorinated trityl radicals dissolved in perfluorocarbons. Preparation with a high-pressure homogenizer conferred sub-micronic size to both nano-emulsions. In vitro and in vivo EPR spectroscopy showed that the nano-emulsions had much greater oxygen sensitivity than the hydrophilic trityl, CT-03. In vivo experiments in rodents confirmed the ability of the nano-emulsions to follow the changes in oxygen concentration after induced ischemia. Histological evaluation of the tissue injected with the nano-emulsions revealed some acute toxicity for the F45T-03/HFB nano-emulsion but none for the F15T-03/PFOB nano-emulsion. These new formulations should be considered for further EPR oximetry experiments in pathophysiological situations where subtle changes in tissue oxygenation are expected.     

Optimization of magnetic field sweep and field modulation amplitude for continuous-wave EPR oximetry

For continuous-wave electron paramagnetic resonance spectroscopy, what settings of magnetic field sweep width and field modulation amplitude yield the best accuracy in estimated linewidth? Statistical bounds on estimation error presented in this work provide practical guidance: set the sweep width and modulation amplitude to 8 and 4 times the half-width half-maximum linewidth, Γ, respectively. For unknown linewidths in the range [Γ(min),Γ(max)] the worst-case estimation error is minimized by using settings designed for Γ(max). The analysis assumes a Lorentzian lineshape and a constant modulation amplitude across the extent of the irradiated paramagnetic probe. The analytical guidelines are validated using L-band spectroscopy with a particulate LiNc-BuO probe.     

Fast and accurate characterization of biological membranes by EPR spectral simulations of nitroxides

A method by which it is possible to characterize the membranes of biological samples on the basis of the EPR spectral lineshape simulation of membrane-dissolved nitroxide spin probes is described. The presented simulation procedure allows the determination of the heterogeneous structure of biological membranes and fluidity characteristics of individual membrane domains. The method can deal with isotropic and anisotropic orientations of nitroxides introduced into the biological samples described by restricted fast motion with a correlation time between 0.01 and 10 ns. The linewidths of the Lorentzian lineshapes are calculated in a restricted fast-motion approximation. In the special case of samples with high concentrations of nitroxides or in the presence of paramagnetic ions, the lineshapes are calculated directly from the exchange-coupled Bloch equations. The parameters describing ordering, relaxation, polarity, and the portions of the individual spectral components are extracted by optimizing the simulated spectra to the experimental spectrum with either a Simplex or a Monte Carlo algorithm. To improve the algorithm's efficiency, a new way of characterizing the goodness of fits is introduced. The new criterion is based on the standard least-squares function, but with special weighting of the partial sums. Its benefits are confirmed with membrane spectral simulation. Two classes of examples-simulation and optimizations of synthetic spectra to evaluate the accuracy of the optimization algorithms and simulation and optimization of EPR spectra of nitroxides in liposome suspensions in the presence of a broadening agent and in human leukocytes are shown.     

High spatial resolution multi-site EPR oximetry. The use of convolution-based fitting method

We describe a new method to enhance the spatial resolution of multi-site electron paramagnetic resonance (EPR) oximetry. The method is suitable for any shape (density distribution function) of a solid paramagnetic material implanted in tissue. It corrects distortions of lineshapes caused by the gradient and thus overcomes limitations of previous multi-site EPR oximetry methods that restricted the ratio of the particle size to the distance between sites. The new method is based on consecutive applications of magnetic field gradients with the same direction but with a different magnitude and uses a convolution-based fitting algorithm to derive Lorentzian EPR linewidths of each individual peak of the EPR spectrum. The method is applicable for any particulate EPR oxygen sensitive materials whose EPR spectra can be approximated by a Lorentzian function or a superposition of Lorentzian functions. By incorporating this model of the lineshape in the data processing, we are able to decrease significantly the number of parameters needed for the calculations and to recover the oxygen concentration, even from quite noisy spectra. We (i) describe our method and the data-processing algorithm, (ii) demonstrate our approach in model and in vivo experiments, and (iii) discuss the limitations.     

Evaluation of oxygen-response times of phthalocyanine-based crystalline paramagnetic spin probes for EPR oximetry

The goal of the present study was to evaluate the temporal response of particulate-based EPR oximetry probes to changes in partial pressure of oxygen (pO(2)). In order to accurately evaluate the oxygen-response time, we developed a method for rapid modulation of pO(2) in a chamber containing the probe using an oscillator-driven speaker-diaphragm setup. The apparatus was capable of producing sinusoidal changes in pO(2) at frequencies up to 300 Hz or more. The pressure-modulation setup was used to evaluate the temporal response of some of the most commonly used phthalocyanine-based particulate probes. For validation, the time-response of the probes was compared to that of a high sensitivity pressure sensor. The results revealed that some particulate probes could respond to changes in pO(2) with a temporal response of 3.3 ms (300 Hz). The observations were interpreted in the light of their crystalline packing in favor of oxygen diffusion. The results of the present study should enable the selection of probes for oximetry applications requiring high temporal resolution.     

Dynamic SIMS ion microscopy imaging of individual bacterial cells for studies of isotopically labeled molecules

A CAMECA IMS-3f SIMS ion microscope instrument capable of 500 nm spatial resolution was used for imaging single bacterial cells in the soil treated with ¹³C-labeled phenol in desired treatments. After formaldehyde fixation, the soil samples containing bacterial cells were smeared on the silicon substrate. The O₂⁺ primary ion beam was used for the detection of negative secondary ions. Images of individual bacterial cells were recorded on a CCD camera in masses 24, 25, 26, and 27 signals. The respective mass images represented contributions from the molecular ions of interest, for example ²⁴(¹²C₂)⁻, ²⁵(¹³C¹²C)⁻, ²⁶(¹²C¹⁴N)⁻, ²⁷(¹³C¹⁴N)⁻, as well as interfering mass species associated with a particular mass. However, in cells treated with ¹³C-labeled phenol the enhancement of signals in masses 25 and 27 images due to increased signals of ²⁵(¹³C¹²C)⁻ and ²⁷(¹³C¹⁴N)⁻, respectively, unequivocally indicated the presence of ¹³C-labeled phenol in bacterial cells in direct comparison to the samples treated only with unlabeled phenol. The ratio images of masses 25/24 and 27/26 from individual cells revealed a cell-to-cell heterogeneity in the labeling with ¹³C-phenol. This study indicates that the ion microscope can be applied effectively for studies of labeled molecules in microbiology and biogeochemistry.     

The effect of donor self-association on EPR studies of transient complex formation between nitroxide radicals and solvent molecules

Nitroxide radical-hydrogen donor systems in which the donor can self-associate are examined by EPR techniques. Data for CH₃OH and CH₃COOH and the nitroxide radical TMPO are first fitted by using methods developed for transient complex formation between hydrogen donors and nitroxides. TheK _assoc and hyperfine coupling constants obtained are inconsistent with previous correlations with thepK _a of the hydrogen donor. An alternative method of analysis that incorporates donor self-association is developed and applied to the systems studied.     

Comparing continuous wave progressive saturation EPR and time domain saturation recovery EPR over the entire motional range of nitroxide spin labels

The measurement of spin-lattice relaxation rates from spin labels, such as nitroxides, in the presence and absence of spin relaxants provides information that is useful for determining biomolecular properties such as nucleic acid dynamics and the interaction of proteins with membranes. We compare X-band continuous wave (CW) and pulsed or time domain (TD) EPR methods for obtaining spin-lattice relaxation rates of spin labels across the entire range of rotational motion to which relaxation rates are sensitive. Model nitroxides and spin-labeled biological species are used to illustrate the potential complications that arise in extracting relaxation data under conditions typical to biological experiments. The effect of super hyperfine (SHF) structure is investigated for both CW and TD spectra. First and second harmonic absorption and dispersion CW spectra of the nitroxide spin label, TEMPOL, are all fit simultaneously to a model of SHF structure over a range of microwave amplitudes. The CW spectra are novel because all harmonics and microwave phases were acquired simultaneously using our homebuilt CW/TD spectrometer. The effect of the SHF structure on the pulsed free induction decay (FID) and pulsed saturation recovery spectrum is shown for both protonated and deuterated TEMPOL. We present novel pulsed saturation recovery measurements on biological molecules, including spin-lattice relaxation rates of spin-labeled proteins and spin-labeled double-stranded DNA. The impact of structure and dynamics on relaxation rates are discussed in the context of each of these examples. Collisional relaxation rates with oxygen and transition metal paramagnetic relaxants are extracted using both continuous wave and time domain methods. The extent of the errors inherent in the CW method and the advantages of pulsed methods for unambiguously measuring collisional relaxation rates are discussed. Spin-lattice relaxation rates, determined by both CW and pulsed methods, are used to determine the electrostatic potential on the surface of a protein.     

In vivo EPR imaging of the distribution and metabolism of nitroxide radicals in human skin

While altered cellular free radical and redox metabolism are critical factors in many human diseases, it has not been previously possible to both measure and image these processes in humans. The development and application of electron paramagnetic resonance instrumentation capable of in vivo spectroscopy and imaging of free radicals in human skin are reported. The instrumentation uses a specially designed topical resonator and a 2.2-GHz microwave bridge. Noninvasive measurements of the distribution and metabolism of the topically applied nitroxide, (15)N-perdeuterated tempone (100 mM), in forearm skin were performed. A single broad peak due to the concentrated label at the skin surface was initially observed, followed by a sharp doublet from the diluted label that permeated the skin. The penetration of the label into the skin and its metabolic clearance were modeled using kinetic equations. It was observed that the penetration process from the skin surface into the dermis and subcutaneous regions, as well as its clearance from these regions, could be described by single exponential functions. Phantom imaging experiments using the nitroxide showed that a spatial resolution of up to 50 microm could be achieved. The skin imaging measurements showed two bands in the distribution of the label along the skin depth. The first band appeared in the outer 400 microm of the skin, the epidermis region, whereas the second band was centered at a depth of 1000 microm in the subcutaneous region with a thickness about 400 microm. These two bands decayed and merged into a single band with time. The results are important in the understanding of the permeability and metabolism of free radicals in human skin.     

A new laser concept for isotopically selective analysis of noble gases

A new laser approach for the isotopically selective analysis of noble gases is presented. This approach uses noble gas atoms prepared in the 1s ⁵ metastable state. Hyperfine levels in the 1s ⁵ and 2p ⁹ states form two-level systems in Ne, Ar, Kr, and Xe which can be excited by a commercially available single-frequency laser system. Absorption of photons from such a laser, and the resulting momentum transfer, can be used to selectively deflect the desired isotope from a supersonic atomic beam into a detection area. Light from the same laser can then be used to selectively count atoms of the desired isotope using the photon-burst technique. Thus, enrichment and selective detection are accomplished with a single laser in a single pass through the apparatus.The problem of analyzing for⁸⁵Kr in a sample of noble gases extracted from the air is examined in detail. This is a stringent test of the selectivity of this approach because⁸⁵Kr has the same nuclear spin, and thus similar hyperfine splittings, as naturally occurring⁸³Kr. Calculations indicate that isotopic selectivity of the new approach is easily adequate to resolve⁸⁵Kr in a 10¹⁰ excess of⁸³Kr.     

CW EPR, echo-detected EPR, and field-step ELDOR study of molecular motions of nitroxides ino-terphenyl glass: Dynamical transition, dynamical heterogeneity and β-relaxation

Continuous-wave electron paramagnetic resonance (CW EPR), echo-detected (ED) EPR, and field-step electron-electron double resonance (FS ELDOR) were simultaneously applied to study molecular motions of nitroxide spin probes of two different types in glassyo-terphenyl. A strong linear temperature dependence of the overall splitting of the CW EPR lineshape was found for nitroxide Tempone and only a weak one for a phenyl-ring-containing imidasoline nitroxide. The linear temperature dependence of the splitting is explained within the model of harmonic librations. The assessed libration frequency for Tempone is of the order of 3·10¹² rad/s. The observed remarkable difference between the two nitroxides is explained by the different strength of interactions between guest and host molecules and by dynamical heterogeneity of the glass. The nonlinear temperature dependence above 250 K is attributed to the onset of anharmonic motion that is postulated in a number of neutron scattering and Mössbauer spectroscopy studies for molecular glasses and proteins (the so-called dynamical transition). Above 245 K also ED EPR spectra change drastically, which may be explained on the same ground. Magnetization transfer was observed in FS ELDOR for nitroxide Tempone, with a time constant around 10^?5 s. It was found to be almost temperature-independent between 160 K and 265 K and was attributed to the Johari-Goldstein β-relaxation process. For the phenyl-ring-containing imidasoline nitroxide this transfer was not observed, which may be explained again by the dynamical heterogeneity of the glass and by small effectivity of the β-relaxation process in this case.     

Evaluation of spin labels for in-cell EPR by analysis of nitroxide reduction in cell extract of Xenopus laevis oocytes

Spin-label electron paramagnetic resonance (SL-EPR) spectroscopy has become a powerful and useful tool for studying structure and dynamics of biomacromolecules. However, utilizing these methods at physiological temperatures for in-cell studies is hampered by reduction of the nitroxide spin labels and thus short half-lives in the cellular environment. Consequently, reduction kinetics of two structurally different nitroxides was investigated in cell extracts of Xenopus laevis oocytes using rapid-scan cw-experiments at X-band. The five member heterocyclic ring nitroxide PCA (3-carboxy-2,2,5,5-tetramethylpyrrolidinyl-1-oxy) under investigation features much higher stability against intracellular reduction than the six member ring analog TOAC (2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-carboxilic acid) and is therefore a suitable spin label type for in-cell EPR. The kinetic data can be described according to the Michaelis–Menten model and thus suggest an enzymatic or enzyme-mediated reduction process.     

In vivo temporal EPR imaging for estimating the kinetics of a nitroxide radical in the renal parenchyma and pelvis in rats

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

Real-time monitoring of drug-induced changes in the stomach acidity of living rats using improved pH-sensitive nitroxides and low-field EPR techniques

New improved pH-sensitive nitroxides were applied for in vivo studies. An increased stability of the probes towards reduction was achieved by the introduction of the bulky ethyl groups in the vicinity of the paramagnetic NO fragment. In addition, the range of pH sensitivity of the approach was extended by the synthesis of probes with two ionizable groups, and, therefore, with two pKa values. Stability towards reduction and spectral characteristics of the three new probes were determined in vitro using 290 MHz radiofrequency (RF)- and X-band electron paramagnetic resonance (EPR), longitudinally detected EPR (LODEPR), and field-cycled dynamic nuclear polarization (FC-DNP) techniques. The newly synthesized probe, 4-[bis(2-hydroxyethyl)amino]-2-pyridine-4-yl-2,5,5-triethyl-2,5-dihydro-1H-imidazol-oxyl, was found to be the most appropriate for the application in the stomach due to both higher stability and convenient pH sensitivity range from pH 1.8 to 6. LODEPR, FC-DNP and proton-electron double resonance imaging (PEDRI) techniques were used to detect the nitroxide localization and acidity in the rat stomach. Improved probe characteristics allowed us to follow in vivo the drug-induced perturbation in the stomach acidity and its normalization afterwards during 1 h or longer period of time. The results show the applicability of the techniques for monitoring drug pharmacology and disease in the living animals.     

Interstitial Mn as a new donor in GaP and GaAs: an EPR study

We report the observation of a new electron paramagnetic resonance centre in neutron-irradiated GaP and a similar new EPR centre in Mn-doped GaAs. Both centres have been identified as interstitial Mn and act as a donor. To our knowledge this is the first observation by EPR of an interstitial transition-metal impurity in a III-V compound. The implication of this new finding on the issue of Mn diffusion is discussed.     

A new method for the determination of oxygen solubilities by EPR

Free radicals dissolved in oxygen-containing solutions give rise to EPR spectra characterized by very broad lines due to Heisenberg spin exchange. In the method herein proposed oxygen is consumed at a constant rate, within the cavity of an EPR spectrometer, by alkyl radicals generatedin situ by thermal decomposition of an aliphatic azo compound in the presence of a nitroxide probe. The effect of decreasing the oxygen concentration is to reduce the width, and therefore to increase the height of the spectral lines of the nitroxide, which reach a maximum when oxygen has been completely consumed. From the knowledge of the rate of generation of the alkyl radicals, the oxygen solubility in a given solvent can be easily determined.