In Vivo Extracellular pH Mapping of Tumors Using Electron Paramagnetic Resonance.

An electron paramagnetic resonance (EPR)-based method for noninvasive three-dimensional extracellular pH mapping was developed using a pH-sensitive nitroxyl radical as an exogenous paramagnetic probe. Fast projection scanning with a constant magnetic field sweep enabled the acquisition of four-dimensional (3D spatial +1D spectral) EPR images within 7.5 min. Three-dimensional maps of pH were reconstructed by processing the pH-dependent spectral information on the images. To demonstrate the proposed method of pH mapping, the progress of extracellular acidosis in tumor-bearing mouse legs was studied. Furthermore, extracellular pH mapping was used to visualize the spatial distribution of acidification in different tumor xenograft mouse models of human-derived pancreatic ductal adenocarcinoma cells. The proposed EPR-based pH mapping method enabled quantitative visualization of regional changes in extracellular pH associated with altered tumor metabolism.

Maintenance of redox balance by antioxidants in hemolymph of the greater wax moth Galleria mellonella larvae during encapsulation response.

The lipid peroxidation process in hemocytes, activities of phenoloxidase and key enzymatic antioxidants (superoxide dismutase, glutathione-S-transferase, catalase) and nonenzymatic antioxidants (thiols, ascorbate) in hemolymph of the greater wax moth Galleria mellonella L. (Lepidoptera: Pyralidae) were studied during the encapsulation process of nylon implants. It has been established that as soon as 15 min after piercing a cuticle with the implant, a capsule is formed on its surface. Active melanization of the capsule has been shown to last for 4 h. During the first hours after incorporating the implant, an increase in phenoloxidase activity and lipid peroxidation in the insect hemocytes has been revealed. Adhesion and degranulation on the surface of foreign object lead to the depletion of total hemocytes count (THC). Our results indicated that thiols and ascorbate molecules take part in the immediate antioxidant response, during later stages of encapsulation process hemolymph glutathione-S-transferase detoxifies and protects insect organism thereby restoring the internal redox balance. We suggest that nonenzymatic and enzymatic antioxidants of hemolymph plasma play a key role in the maintenance of redox balance during encapsulation of foreign targets.

Feasibility of in vivo three-dimensional T 2* mapping using dicarboxy-PROXYL and CW-EPR-based single-point imaging.

OBJECTIVES: The aim of this study was to demonstrate the feasibility of in vivo three-dimensional (3D) relaxation time T 2* mapping of a dicarboxy-PROXYL radical using continuous-wave electron paramagnetic resonance (CW-EPR) imaging. MATERIALS AND METHODS: Isotopically substituted dicarboxy-PROXYL radicals, 3,4-dicarboxy-2,2,5,5-tetra(2H3)methylpyrrolidin-(3,4-2H2)-(1-15N)-1-oxyl (2H,15N-DCP) and 3,4-dicarboxy-2,2,5,5-tetra(2H3)methylpyrrolidin-(3,4-2H2)-1-oxyl (2H-DCP), were used in the study. A clonogenic cell survival assay was performed with the 2H-DCP radical using squamous cell carcinoma (SCC VII) cells. The time course of EPR signal intensities of intravenously injected 2H,15N-DCP and 2H-DCP radicals were determined in tumor-bearing hind legs of mice (C3H/HeJ, male, n = 5). CW-EPR-based single-point imaging (SPI) was performed for 3D T 2* mapping. RESULTS: 2H-DCP radical did not exhibit cytotoxicity at concentrations below 10 mM. The in vivo half-life of 2H,15N-DCP in tumor tissues was 24.7 ± 2.9 min (mean ± standard deviation [SD], n = 5). The in vivo time course of the EPR signal intensity of the 2H,15N-DCP radical showed a plateau of 10.2 ± 1.2 min (mean ± SD) where the EPR signal intensity remained at more than 90% of the maximum intensity. During the plateau, in vivo 3D T 2* maps with 2H,15N-DCP were obtained from tumor-bearing hind legs, with a total acquisition time of 7.5 min. CONCLUSION: EPR signals of 2H,15N-DCP persisted long enough after bolus intravenous injection to conduct in vivo 3D T 2* mapping with CW-EPR-based SPI.

Functional electron paramagnetic resonance imaging of ischemic rat heart: Monitoring of tissue oxygenation and pH.

PURPOSE: Electron paramagnetic resonance (EPR) imaging in the spectral-spatial domain with application of soluble paramagnetic probes provides an opportunity for spatially resolved functional measurements of living objects. The purpose of this study was to develop EPR methods for visualization of oxygenation and acidosis of ischemic myocardium. METHODS: EPR oxygen measurements were performed using isotopically substituted (2) H,(15) N-dicarboxyproxyl. The radical has an EPR line width of 320 mG and oxygen-induced line broadening of 0.53 mG/mm Hg, providing oxygen sensitivity down to 5 µM. pH measurements were performed using previously developed pH-sensitive imidazoline nitroxide. The radical has an EPR spectrum with pH-dependable hyperfine splitting, pK = 6.6, providing pH sensitivity of approximately 0.05 U in the physiological range. RESULTS: EPR imaging of isolated and perfused rat hearts was performed in the two-dimensional + spectral domain. The spatial resolution of the measurements was about 1.4 mm. Marked tissue hypoxia was observed in the ischemic area of the heart after occlusion of the left anterior descending coronary artery. Tissue oxygenation was partly restored upon reperfusion. EPR mapping of myocardial pH indicated acidosis of the ischemic area down to pH 6.7-6.8. CONCLUSION: This study demonstrates the capability of low-field EPR and the nitroxide spin probes for mapping of myocardial oxygenation and pH. The developed approaches might be used for noninvasive investigation of microenvironment on living objects. Magn Reson Med 76:350-358, 2016. © 2015 Wiley Periodicals, Inc.

Effect of Sterical Shielding on the Redox Properties of Imidazoline and Imidazolidine Nitroxides.

The oxidant properties of the series of 2,2,5,5-tetraalkyl imidazoline and imidazolidine nitroxides were investigated. An increase in the number of bulky alkyl substituents leads to a decrease in the rate of reduction with ascorbate, which makes the electrochemical reduction potential more negative and shifts the equilibrium in the mixture of nitroxide and reference hydroxylamine (3-carboxy-1-hydroxy-2,2,5,5-tetramethylpyrrolidine-1-oxyl-1-(15)N) toward the starting compounds. The effect of structural factors on these reactions was analyzed by means of multiple regression using the Fujita steric constant Es and the inductive Hammett constant σI. Satisfactory statistical outputs were obtained in all of the biparameter correlations, denoting that the oxidant properties of the nitroxides are determined by steric and electronic effects of the substituents. The data imply that bulky substituents can stabilize nitroxide and/or destabilize hydroxylamine.

Phosphonated trityl probes for concurrent in vivo tissue oxygen and pH monitoring using electron paramagnetic resonance-based techniques.

Previously we proposed the concept of dual function pH and oxygen paramagnetic probes based on the incorporation of ionizable groups into the structure of persistent triarylmethyl radicals, TAMs (J. Am. Chem. Soc.2007, 129, 7240-7241). In this paper, we synthesized an asymmetric monophosphonated TAM probe with the simplest doublet hfs pattern ideally suited for dual function electron paramagnetic resonance (EPR)-based applications. An extraordinary low line width of the synthesized deuterated derivative, p1TAM-D (ΔHpp ≤ 50 mG, Lorentz line width, ≤20 mG) results in high sensitivity to pO2 due to oxygen-induced line broadening (ΔLW/ΔpO2 ≈ 0.5 mG/mmHg or ≈400 mG/mM); accuracy of pO2 measurement, ≈1 mmHg). The presence of a phosphono group in the p1TAM-D structure provides pH sensitivity to its EPR spectra in the physiological range of pH from 5.9 to 8.2 with the ratio of signal intensities of protonated and deprotonated states being a reliable pH marker (accuracy of pH measurements, ± 0.05). The independent character of pH and [O2] effects on the EPR spectra of p1TAM-D provides dual functionality to this probe. The L-band EPR studies performed in breast tumor-bearing mice show a significant difference in extracellular pH and pO2 between tumor and normal mammary gland tissues, as well as the effect of animal breathing with 100% O2 on tissue oxygenation. The developed dual function phosphonated p1TAM-D probe provides a unique tool for in vivo concurrent tissue oxygen and pH monitoring.

Dual-function pH and oxygen phosphonated trityl probe.

Triarylmethyl radicals (TAMs) are used as persistent paramagnetic probes for electron paramagnetic resonance (EPR) spectroscopic and imaging applications and as hyperpolarizing and contrast agents for magnetic resonance imaging (MRI) and proton-electron double-resonance imaging (PEDRI). Recently we proposed the concept of dual-function pH and oxygen TAM probes based on the incorporation of ionizable groups into the TAM structure ( J. Am. Chem. Soc. 2007 , 129 , 7240 - 7241 ). In this paper we report the synthesis of a deuterated derivative of phosphonated trityl radical, pTAM. The presence of phosphono substitutes in the structure of TAM provides pH sensitivity of its EPR spectrum in the physiological range from 6 to 8, the phosphorus hyperfine splitting acting as a convenient and highly sensitive pH marker (spectral sensitivity, 3Δa(P)/ΔpH ≈ 0.5 G/pH unit; accuracy of pH measurements, ±0.05). In addition, substitution of 36 methyl protons with deuterons significantly decreased the individual line width of pTAM down to 40 mG and, as consequence, provided high sensitivity of the line-width broadening to pO(2) (ΔH/ΔpO(2) ≈ 0.4 mG/mmHg; accuracy of pO(2) measurements, ≈1 mmHg). The independent character of pH and [O(2)] effects on the EPR spectra of pTAM provides dual functionality to this probe, allowing extraction of both parameters from a single EPR spectrum.

Electron paramagnetic resonance monitoring of ischemia-induced myocardial oxygen depletion and acidosis in isolated rat hearts using soluble paramagnetic probes.

A new low-field electron paramagnetic resonance approach for noninvasive measurements of myocardial oxygen tension and tissue acidity was developed. The approach was applied to monitor myocardial pO(2) and pH in a model of global no-flow ischemia (30 min) and reperfusion in isolated perfused rat hearts. The myocardial oxygen measurements were performed using deuterated Finland trityl radical probe. A rapid decrease in myocardial pO(2) from 160 mmHg to about 2 ± 1 mmHg was observed within the first minute of ischemia followed by incomplete restoration of pO(2) to 50 mmHg during 30 min of reperfusion. The lower oxygen concentration after ischemia was attributed to the 50% reduction in coronary flow after ischemia as a consequence of myocardial ischemia and reperfusion damage. Myocardial pH measurements using a specially designed imidazoline pH-sensitive nitroxide showed severe myocardial acidification to pH 6.25 during 30 min of ischemia. Preconditioning of the hearts with two 5-min periods of ischemia significantly reduced the acidification of myocardial tissue during sustained ischemia. Noninvasive electron paramagnetic resonance monitoring of myocardial oxygenation and pH may provide important insights into the mechanisms of ischemia and reperfusion injury and a background for development of new therapeutic approaches.

Synthesis of a chiral C2-symmetric sterically hindered pyrrolidine nitroxide radical via combined iterative nucleophilic additions and intramolecular 1,3-dipolar cycloadditions to cyclic nitrones.

A sterically hindered bis-spirocyclic C(2)-symmetric chiral pyrrolidine-type nitroxide has been successfully synthesized starting from an l-tartaric derived nitrone. Starting from a pyrrolidine flanked by two methylene groups, complete quaternization of the two α-carbon atoms has been accomplished through iteration of completely regio- and stereoselective intramolecular cycloaddition reactions and organometallic additions to key nitrone intermediates, formed in turn by oxidation procedures. This method appears to be very useful for building up bulky spirocyclic moieties adjacent to a nitroxide group and provides an important supplementation to traditional methods of nitroxide synthesis. The synthesized chiral nitroxide showed a very high stability to reduction with ascorbate (k ≈ 8 × 10(-3) M(-1) s(-1)).

High fidelity triangular sweep of the magnetic field for millisecond scan EPR imaging.

Linearity of the magnetic field sweep is important for high resolution continuous wave EPR imaging. Driving the field with triangular wave function is the most efficient way to scan EPR projections. However, the magnetic field sweep profile can be significantly distorted during fast millisecond projection scan. In this work, we introduce a method to generate highly linear and properly symmetrical triangular sweeps of the magnetic field using calibrated harmonics of the triangular wave function. First, the frequency response function of the EPR magnet and its power circuitry was obtained. For this, the field sweeping coil was driven with sinusoidal signals of different frequencies and the actual magnetic field inside the magnet was recorded. To cover wide range of frequencies, the measurements were carried out independently using gaussmeter, Hall-effect linear sensor integrated circuit, and an inductance coil. For each frequency, the system gain and the phase delay were determined. These data were used to adjust the amplitudes and the phases of individual harmonics of the triangular wave function. After the calibration, the maximum deviation of the magnetic field from the linear function was 0.05% of sweep width for 4 ms scan. The maximum discrepancy between the forward and the reverse scan was less than 0.04%. Sweep overhead time for changing the scan direction was 5%. The proposed approach allows generation of high fidelity triangular magnetic field sweeps with accuracy better than 0.1% for the range of the magnetic field sweep widths up to 48 G and scan duration from 10 s down to 1 ms.

4-Dialkylamino-2,5-dihydroimidazol-1-oxyls with Functional Groups at the Position 2 and at the Exocyclic Nitrogen: The pH-Sensitive Spin Labels.

Local acidity and electrostatic interactions are associated both with catalytic properties and the adsorption activity of various materials, and with the vital functions of biomolecules. The observation of acid-base equilibria in stable free radicals using EPR spectroscopy represents a convenient method for monitoring pH changes and the investigation of surface electrostatics, the advantages of which are especially evident in opaque and turbid samples and in porous materials such as xerogels. Imidazoline nitroxides are the most commonly used pH-sensitive spin probes and labels due to the high sensitivity of the parameters of the EPR spectra to pH changes, their small size, and their well-developed chemistry. In this work, several new derivatives of 4-(N,N-dialkylamino)-2,5-dihydrioimidazol-1-oxyl, with functional groups suitable for specific binding, were synthesized. The dependence of the parameters of their EPR spectra on pH was studied. Several showed a pKa close to 7.4, following the pH changes in a normal physiological range, and some demonstrated a monotonous change of the hyperfine coupling constant by 0.14 mT upon pH variation by four units.

Algebraic reconstruction of 3D spatial EPR images from high numbers of noisy projections: An improved image reconstruction technique for high resolution fast scan EPR imaging.

A novel method for reconstructing 3D spatial EPR images from large numbers of noisy projections was developed that minimizes mean square error between the experimental projections and those from the reconstructed image. The method utilizes raw projection data and zero gradient spectrum to account for EPR line shape and hyperfine structure of the paramagnetic probe without the need for deconvolution techniques that are poorly suited for processing of high noise projections. A numerical phantom was reconstructed for method validation. Reconstruction time for the matrix of 1283 voxels and 16,384 noiseless projections was 4.6 min for a single iteration. The algorithm converged quickly, reaching R2 ~ 0.99975 after the very first iteration. An experimental phantom sample with nitroxyl radical was measured. With 16,384 projections and a field gradient of 8 G/cm, resolutions of 0.4 mm were achieved for a cubical area of 25 × 25 × 25 mm3. Reconstruction was sufficiently fast and memory efficient making it suitable for applications with large 3D matrices and fully determined system of equations. The developed algorithm can be used with any gradient distribution and does not require adjustable filter parameters that makes for simple application. A thorough analysis of the strengths and limitations of this method for 3D spatial EPR imaging is provided.

A 750-MHz Electronically Tunable Resonator Using Microstrip Line Couplers for Electron Paramagnetic Resonance Imaging of a Mouse Tumor-Bearing Leg.

OBJECTIVE: The purpose of this work was to develop an electronically tunable resonator operating at 750 MHz for continuous-wave electron paramagnetic resonance (CW-EPR) imaging of a mouse tumor-bearing leg. METHODS: The resonator had a multi-coil parallel-gap structure with a sample space of 16 mm in diameter and 20 mm in length. Microstrip line couplers were used in conjunction with varactor diodes to enable resonance frequency adjustment and to reduce the nonlinear effects of the varactor diodes. The resonator was modeled by the finite-element method and a microwave circuit simulation was performed to clarify its radiofrequency characteristics. RESULTS: A tunable resonator was evaluated in terms of its resonance frequency, tunable frequency band, and conversion efficiency of the RF magnetic field. The developed resonator provided a tunable frequency band of 4 MHz at a central frequency of 747 MHz and a conversion efficiency of 34 µT/W1/2. To demonstrate the application of this tunable resonator to EPR imaging, three-dimensional EPR images of a sample solution and a mouse tumor-bearing leg were obtained. CONCLUSION: The developed tunable resonator satisfied our initial requirements for in vivo EPR imaging and may be able to be further improved using the present finite-element and circuit models if any problems arise during future practical applications. SIGNIFICANCE: This work may help to promote EPR imaging of tumor-bearing mice in cancer-related studies.

Fast backprojection-based reconstruction of spectral-spatial EPR images from projections with the constant sweep of a magnetic field.

In this paper, we introduce a procedure for the reconstruction of spectral-spatial EPR images using projections acquired with the constant sweep of a magnetic field. The application of a constant field-sweep and a predetermined data sampling rate simplifies the requirements for EPR imaging instrumentation and facilitates the backprojection-based reconstruction of spectral-spatial images. The proposed approach was applied to the reconstruction of a four-dimensional numerical phantom and to actual spectral-spatial EPR measurements. Image reconstruction using projections with a constant field-sweep was three times faster than the conventional approach with the application of a pseudo-angle and a scan range that depends on the applied field gradient. Spectral-spatial EPR imaging with a constant field-sweep for data acquisition only slightly reduces the signal-to-noise ratio or functional resolution of the resultant images and can be applied together with any common backprojection-based reconstruction algorithm.

Superoxide and hydrogen peroxide formation during enzymatic oxidation of DOPA by phenoloxidase.

Generation of superoxide anion and hydrogen peroxide during enzymatic oxidation of 3-(3,4-dihydroxyphenyl)-DL-alanine (DOPA) has been studied. The ability of DOPA to react with O2*- has been revealed. EPR spectrum of DOPA-semiquinone formed upon oxidation of DOPA by O2*- was observed using spin stabilization technique of ortho-semiquinones by Zn2+ ions. Simultaneously, the oxidation of DOPA by O2*- was found to produce hydrogen peroxide (H2O2). The analysis of H2O2 formation upon oxidation of DOPA by O2*- using 1-hydroxy-3-carboxy-pyrrolidine (CP-H), and SOD as competitive reagents for superoxide provides consistent values of the rate constant for the reaction between DOPA and O2*- being equal to (3.4+/-0.6)x10(5) M(-1) s(-1). The formation of H2O2 during enzymatic oxidation of DOPA by phenoloxidase (PO) has been shown. The H2O2 production was found to be SOD-sensitive. The inhibition of H2O2 production by SOD was about 25% indicating that H2O2 is produced both from superoxide anion and via two-electron reduction of oxygen at the enzyme. The attempts to detect superoxide production during enzymatic oxidation of DOPA using a number of spin traps failed apparently due to high value of the rate constant for DOPA interaction with O2*-.

Influence of fungal infection on the DOPA-semiquinone and DOPA-quinone production in haemolymph of Galleriamellonella larvae.

The formation of reactive oxygen metabolites in haemolymph of Galleria mellonella larvae was studied by ESR spectroscopy. The inhibition of the production of the reactive oxygen metabolites of DOPA in haemolymph under the action of fungal infection was shown using spin trap 1-hydroxy-3-carboxy-pyrrolidine. This inhibition correlated with decrease of phenoloxidase activity in haemolymph of infected insects. Simultaneously, the decrease of production of DOPA-semiquinone was detected using method of spin stabilization by diamagnetic metal ions. Moreover, it was shown that the formation of DOPA-quinone was slowed down in haemolymph of infected insects. Our results suggest that the DOPA-derived quinones/semiquinones may be involved in immune response of insects as part of its defense mechanism.