Proton-electron Double-resonance Imaging Research Articles

Direct and indirect assessment of cancer metabolism explored by MRI.

Magnetic resonance-based approaches to obtain metabolic information on cancer have been explored for decades. Electron paramagnetic resonance (EPR) has been developed to pursue metabolic profiling and successfully used to monitor several physiologic parameters such as pO2 , pH, and redox status. All these parameters are associated with pathophysiology of various diseases. Especially in oncology, cancer hypoxia has been intensively studied because of its relationship with metabolic alterations, acquiring treatment resistance, or a malignant phenotype. Thus, pO2 imaging leads to an indirect metabolic assessment in this regard. Proton electron double-resonance imaging (PEDRI) is an imaging technique to visualize EPR by using the Overhauser effect. Most biological parameters assessed in EPR can be visualized using PEDRI. However, EPR and PEDRI have not been evaluated sufficiently for clinical application due to limitations such as toxicity of the probes or high specific absorption rate. Hyperpolarized (HP) 13 C MRI is a novel imaging technique that can directly visualize the metabolic profile. Production of metabolites of the HP 13 C probe delivered to target tissue are evaluated in this modality. Unlike EPR or PEDRI, which require the injection of radical probes, 13 C MRI requires a probe that can be physiologically metabolized and efficiently hyperpolarized. Among several methods for hyperpolarizing probes, dissolution dynamic nuclear hyperpolarization is a widely used technique for in vivo imaging. Pyruvate is the most suitable probe for HP 13 C MRI because it is part of the glycolytic pathway and the high efficiency of pyruvate-to-lactate conversion is a distinguishing feature of cancer. Its clinical applicability also makes it a promising metabolic imaging modality. Here, we summarize the applications of these indirect and direct MR-based metabolic assessments focusing on pO2 and pyruvate-to-lactate conversion. The two parameters are strongly associated with each other, hence the acquired information is potentially interchangeable when evaluating treatment response to oxygen-dependent cancer therapies.

In Vivo Application of Proton-Electron Double-Resonance Imaging.

Proton-electron double-resonance imaging (PEDRI) employs electron paramagnetic resonance irradiation with low-field magnetic resonance imaging so that the electron spin polarization is transferred to nearby protons, resulting in higher signals. PEDRI provides information about free radical distribution and, indirectly, about the local microenvironment such as partial pressure of oxygen (pO2), tissue permeability, redox status, and acid-base balance. Recent Advances: Local acid-base balance can be imaged by exploiting the different resonance frequency of radical probes between R and RH+ forms. Redox status can also be imaged by using the loss of radical-related signal after reduction. These methods require optimized radical probes and pulse sequences. High-power radio frequency irradiation is needed for optimum signal enhancement, which may be harmful to living tissue by unwanted heat deposition. Free radical probes differ depending on the purpose of PEDRI. Some probes are less effective for enhancing signal than others, which can reduce image quality. It is so far not possible to image endogenous radicals by PEDRI because low concentrations and broad line widths of the radicals lead to negligible signal enhancement. PEDRI has similarities with electron paramagnetic resonance imaging (EPRI) because both techniques observe the EPR signal, directly in the case of EPRI and indirectly with PEDRI. PEDRI provides information that is vital to research on homeostasis, development of diseases, or treatment responses in vivo. It is expected that the development of new EPR techniques will give insights into novel PEDRI applications and vice versa. Antioxid. Redox Signal. 28, 1345-1364.

Proton-Electron Double-Resonance Imaging of pH using phosphonated trityl probe.

Variable Radio Frequency Proton-Electron Double-Resonance Imaging (VRF PEDRI) enables extracting a functional map from a limited number of images acquired at pre-selected EPR frequencies using specifically designed paramagnetic probes with high quality spatial resolution and short acquisition times. In this work we explored potential of VRF PEDRI for pH mapping of aqueous samples using recently synthesized pH-sensitive phosphonated trityl radical, pTR. The ratio of Overhauser enhancements measured at each pixel at two different excitation frequencies corresponding to the resonances of protonated and deprotonated forms of pTR probe allows for a pH map extraction. Long relaxation times of pTR allow for pH mapping at EPR irradiation power as low as 1.25 W during 130 s acquisition time with spatial resolution of about 1 mm. This is particularly important for in vivo applications enabling one to avoid sample overheating by reducing RF power deposition.

In Vivo Proton–Electron Double-Resonance Imaging of Extracellular Tumor pH Using an Advanced Nitroxide Probe

A variable radio frequency proton-electron double-resonance imaging (VRF PEDRI) approach for pH mapping of aqueous samples has been recently developed (Efimova et al. J. Magn. Reson. 2011, 209, 227-232). A pH map is extracted from two PEDRI acquisitions performed at electron paramagnetic resonance (EPR) frequencies of protonated and unprotonated forms of a pH-sensitive probe. To translate VRF PEDRI to an in vivo setting, an advanced pH probe was synthesized. Probe deuteration resulted in a narrow spectral line of 1.2 G compared to a nondeuterated analogue line width of 2.1 G allowing for an increase of Overhauser enhancements and reduction in rf power deposition. Binding of the probe to the cell-impermeable tripeptide, glutathione (GSH), allows for targeting to extracellular tissue space for monitoring extracellular tumor acidosis, a prognostic factor in tumor pathophysiology. The probe demonstrated pH sensitivity in the 5.8-7.8 range, optimum for measurement of acidic extracellular tumor pH (pH(e)). In vivo VRF PEDRI was performed on Met-1 tumor-bearing mice. Compared to normal mammary glands with a neutral mean pH(e) (7.1 ± 0.1), we observed broader pH distribution with acidic mean pH(e) (6.8 ± 0.1) in tumor tissue. In summary, VRF PEDRI in combination with a newly developed pH probe provides an analytical approach for spatially resolved noninvasive pHe monitoring, in vivo.

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.

In vivo proton electron double resonance imaging of mice With fast spin echo pulse sequence

To develop and evaluate a two-dimensional (2D) fast spin echo (FSE) pulse sequence for enhancing temporal resolution and reducing tissue heating for in vivo proton electron double resonance imaging (PEDRI) of mice. A four-compartment phantom containing 2 mM TEMPONE was imaged at 20.1 mT using 2D FSE-PEDRI and regular gradient echo (GRE)-PEDRI pulse sequences. Control mice were infused with TEMPONE over ~1 min followed by time-course imaging using the 2D FSE-PEDRI sequence at intervals of 10-30 s between image acquisitions. The average signal intensity from the time-course images was analyzed using a first-order kinetics model. Phantom experiments demonstrated that EPR power deposition can be greatly reduced using the FSE-PEDRI pulse sequence compared with the conventional gradient echo pulse sequence. High temporal resolution was achieved at ~4 s per image acquisition using the FSE-PEDRI sequence with a good image SNR in the range of 233-266 in the phantom study. The TEMPONE half-life measured in vivo was ~72 s. Thus, the FSE-PEDRI pulse sequence enables fast in vivo functional imaging of free radical probes in small animals greatly reducing EPR irradiation time with decreased power deposition and provides increased temporal resolution.

In vivo monitoring of pH, redox status, and glutathione using L‐band EPR for assessment of therapeutic effectiveness in solid tumors

Approach for in vivo real-time assessment of tumor tissue extracellular pH (pH(e)), redox, and intracellular glutathione based on L-band EPR spectroscopy using dual function pH and redox nitroxide probe and disulfide nitroxide biradical, is described. These parameters were monitored in PyMT mice bearing breast cancer tumors during treatment with granulocyte macrophage colony-stimulating factor. It was observed that tumor pH(e) is about 0.4 pH units lower than that in normal mammary gland tissue. Treatment with granulocyte macrophage colony-stimulating factor decreased the value of pH(e) by 0.3 units compared with PBS control treatment. Tumor tissue reducing capacity and intracellular glutathione were elevated compared with normal mammary gland tissue. Granulocyte macrophage colony-stimulating factor treatment resulted in a decrease of the tumor tissue reducing capacity and intracellular glutathione content. In addition to spectroscopic studies, pH(e) mapping was performed using recently proposed variable frequency proton-electron double-resonance imaging. The pH mapping superimposed with MRI image supports probe localization in mammary gland/tumor tissue, shows high heterogeneity of tumor tissue pH(e) and a difference of about 0.4 pH units between average pH(e) values in tumor and normal mammary gland. In summary, the developed multifunctional approach allows for in vivo, noninvasive pH(e), extracellular redox, and intracellular glutathione content monitoring during investigation of various therapeutic strategies for solid tumors.

ChemInform Abstract: Solution‐State Dynamic Nuclear Polarization

AbstractReview: 194 refs.

Standard-based method for proton–electron double resonance imaging of oxygen

Proton–electron double resonance imaging (PEDRI) has been utilized for indirect determination of oxygen concentrations in aqueous samples and living systems. Due to the complexity of the problem, there are seven oxygen related parameters that need to be measured to determine the distribution of oxygen. We present an improved approach in which image intensities from only two PEDRI acquisitions with different EPR irradiation powers are required to determine the distribution of a paramagnetic probe and oxygen in an analyzed sample. This is achieved using three reference samples with known concentrations of a paramagnetic probe and oxygen placed inside the resonator together with the measurement sample. An EPR-off image, which has low signal intensity at low magnetic field (0.02 T) is not required for the calculations, significantly reducing the total time of the experiments and the noise while enhancing the accuracy of these oxygen measurements. The Finland trityl radical was used as the paramagnetic probe and oxygen concentrations could be accurately measured and imaged over the physiological range from 0 to 240 μM.

Variable radio frequency proton–electron double-resonance imaging: Application to pH mapping of aqueous samples

Proton–electron double-resonance imaging (PEDRI) offers rapid image data collection and high resolution for spatial distribution of paramagnetic probes. Recently we developed the concept of variable field (VF) PEDRI which enables extracting a functional map from a limited number of images acquired at pre-selected EPR excitation fields using specific paramagnetic probes (Khramtsov et al., J. Magn. Reson. 202 (2010) 267–273). In this work, we propose and evaluate a new modality of PEDRI-based functional imaging with enhanced temporal resolution which we term variable radio frequency (VRF) PEDRI. The approach allows for functional mapping (e.g., pH mapping) using specifically designed paramagnetic probes with high quality spatial resolution and short acquisition times. This approach uses a stationary magnetic field but different EPR RFs. The ratio of Overhauser enhancements measured at each pixel at two different excitation frequencies corresponding to the resonances of protonated and deprotonated forms of a pH-sensitive nitroxide is converted to a pH map using a corresponding calibration curve. Elimination of field cycling decreased the acquisition time by exclusion periods of ramping and stabilization of the magnetic field. Improved magnetic field homogeneity and stability allowed for the fast MRI acquisition modalities such as fast spin echo. In total, about 30-fold decrease in EPR irradiation time was achieved for VRF PEDRI (2.4 s) compared with VF PEDRI (70 s). This is particularly important for in vivo applications enabling one to overcome the limiting stability of paramagnetic probes and sample overheating by reducing RF power deposition.

Variable Field Proton–Electron Double-Resonance Imaging: Application to pH mapping of aqueous samples

A new concept of Variable Field Proton–Electron Double-Resonance Imaging (VF PEDRI) is proposed. This allows for functional mapping using specifically designed paramagnetic probes (e.g. oxygen or pH mapping) with MRI high quality spatial resolution and short acquisition time. Studies performed at 200 G field MRI with phantoms show that a pH map of the sample can be extracted using only two PEDRI images acquired in 140 s at pre-selected EPR excitation fields providing pH resolution of 0.1 pH units and a spatial resolution of 1.25 mm. Note that while concept of functional VF PEDRI was demonstrated using the pH probe, it can be applied for studies of other biologically relevant parameters of the medium such as redox state, concentrations of oxygen or glutathione using specifically designed EPR probes.

Research Status of ultra low field magnetic resonance imaging

Ultra low field magnetic resonance imaging is one of the latest research directions in MRI. Compared with conventional MRI, the size of imaging magnet used could be in mT level or even uT level, which not only reduces the cost and size of the equipment, but also makes it possible to obtain special images that are impossible to get by ordinary MRI equipments. These features provide the ultra low field magnetic resonance imaging technique with very bread application prospects. Recent developments of the technique are reviewed with the focus being put on prepolarized MRI, proton-electron double resonance imaging, laser MRI and superconducting quantum interference device MRI. Key words: Ultra low field; Prepolarized MRI; Proton-electron double resonance imaging; Laser MRI; Superconducting quantum interference device; Magnetic resonance imaging

Field-Cycled Proton-Electron Double-Resonance Imaging (FC-PEDRI) of a 14N nitroxide free radical at low magnetic field

Field-Cycled Proton-Electron Double-Resonance Imaging (FC-PEDRI) of a 14N nitroxide free radical at low magnetic field

Enhanced Overhauser contrast in proton–electron double-resonance imaging of the formation of an alginate hydrogel

Proton–electron double-resonance imaging (PEDRI) was recently employed to monitor the process of formation of a calcium alginate hydrogel at a field of 16 mT. Here, under the same experimental conditions, images obtained through this technique are compared to images obtained by conventional T 2-weighted method. The results confirm that the image contrast obtained using PEDRI, thanks to the Overhauser effect, exhibits an improved sensitivity with respect to changes in water mobility as previously suggested in the literature. Furthermore, by increasing the echo time interval for the T 2-weighted images, important features of the gelling dynamics obtained via PEDRI could not be reproduced.

Modified Alderman‐Grant resonator with high‐power stability for proton electron double resonance imaging

Proton-electron double resonance imaging (PEDRI) requires a dual EPR/NMR resonator. In order to saturate electron spins, long high-power RF EPR pulses are needed. Dissipated power causes resonator and sample heating, resulting in instability of the resonator frequency and coupling, which in turn causes a reduction in sensitivity. To overcome this problem, we designed, built, and tested a modified Alderman-Grant (MAG) resonator for in vivo and in vitro applications. We improved the temperature stability by using fused quartz in critical structural components of the resonator and employing a design that minimized moving parts. All of the components of the resonator and matching circuit, including coupling capacitors, were rigidly positioned on the surface of the quartz tube. Electrical coupling circuitry between the feeding cable and resonator was employed to provide independent coupling and tuning adjustments, which improved the ease and speed of operation. The equivalent schematic of the matching circuit is presented along with calculations of critical parameters. The resonator exhibits no frequency shift with 20 W of continuous power irradiation, and can handle intermittent power of 60 W for up to 3 min without detuning, enabling stable and reliable data acquisition for PEDRI of biological samples.

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 N O 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 p K a 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-1 H-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.

In vivo proton electron double resonance imaging of the distribution and clearance of nitroxide radicals in mice

Proton electron double resonance imaging (PEDRI) is an emerging technique that utilizes the Overhauser effect to enable in vivo and in vitro imaging of free radicals in biological systems. Nitroxide spin probes enable measurement of tissue redox state based on their reduction to diamagnetic hydroxylamines. PEDRI instrumentation at 0.02 T was applied to assess the ability to image the in vivo distribution, clearance, and metabolism of nitroxide radicals in living mice. Using phantoms of 2,2,5,5-tetramethyl-3-carboxylpyrrolidine-N-oxyl (PCA) in normal saline the dependence of the enhancement on RF power and spin probe concentration was determined. Enhancements of up to -23 were obtained in phantoms with 2 mM levels. Maximum enhancement of -7 was observed in vivo. Coronal images of nitroxide-infused mice enabled visualization of the kinetics of spin probe uptake and clearance in different organs including the great vessels, heart, lungs, kidneys, and bladder with an in-plane spatial resolution of 0.6 mm. PEDRI of living mice was also performed using 3-carbamoyl-proxyl and 2,2,6,6-tetramethyl-4-oxopiperidine-N-oxyl to compare the different rate of clearance and metabolism among different nitroxide probes. PCA, due to its intravascular compartmentalization, provided the sharpest contrast for the vascular system and highest enhancement values in the PEDRI images among the three nitroxides.

Proton electron double resonance imaging of free radical distribution in environmental science applications—first results and perspectives

In this contribution, we explore the potential of proton electron double resonance imaging (PEDRI) in environmental science (hydrogeological) applications. After a discussion of the hydrogeological motivation for studies of free radical transport in environmental matrices, we present results from first experiments that show the principal applicability of the PEDRI technique to sediment samples. Field-cycled (FC) relaxation time contrast is identified as a possible source of artifacts in samples in which strong concentration gradients of the free radical phase are present. Furthermore, an outlook is given on how PEDRI can help in observations of the local interplay among contaminants, water and nonaqueous liquid phases.

Field-cycled PEDRI imaging of free radicals with detection at 450 mT

This paper describes the design, construction and use of a field-cycled proton-electron double-resonance imaging (FC-PEDRI) system for the detection and imaging of free radicals. The unique feature of this imager is its use of a 450-mT detection magnetic field in order to achieve good image quality and sensitivity. The detection magnetic field is provided by a superconducting magnet, giving high stability and homogeneity. Field cycling is implemented by switching on and off the current in an internal, coaxial, resistive secondary magnet that partially cancels the superconducting magnet's field at the sample; the secondary magnet is actively shielded to avoid eddy currents. EPR irradiation takes place at approximately 5 mT, following which the field is switched to 450 mT in 40 ms for NMR signal detection. Full details of the imager's subsystems are given, and experiments to image the distribution of stable free radical contrast agents in phantoms and in anesthetized rats are described.

Spin pH and SH probes: enhancing functionality of EPR‐based techniques

Along with significant progress in low‐frequency electron spin resonance (ESR, also called electron paramagnetic resonance, EPR), other techniques such as Longitudinally‐Detected ESR (LODESR), proton electron double‐resonance imaging (PEDRI) and field‐cycled dynamic nuclear polarization (FC‐DNP) have been developed for in vivo applications. However their potential is still far from maximally defined, in part, because of the need for new specific function‐directed spin probes. An application of stable nitroxides of imidazoline and imidazolidine types provides unique possibility to measure local values of pH and thiol content in various biological systems, including in vivo studies. These applications are based on the observation of specific chemical reactions of these nitroxides with protons or thiols, followed by significant changes in their EPR spectra. To increase sensitivity of pH probes for low‐frequency EPR spectroscopy we evaluated two alternative approaches: (i) application of isotopically substituted labels, and (ii) acquisition of EPR spectra at high modulation amplitude. Spatial and spectral‐spatial imaging (pH‐mapping) using PEDRI and L‐band EPR imagers were performed both on phantom samples and in vivo. The applications of the pH and SH probes in model systems, biological fluids, and in vivo in living animals are discussed.