Simultaneous multi-site EPR spectroscopy in vivo

A gradient technique to measure electron paramagnetic resonance spectra simultaneously at several different locations is described. The technique is based on the use of point probes containing paramagnetic centers. The value of the magnetic field gradient is chosen to be sufficient to separate the EPR signals from the different paramagnetic probes yet at the same time small enough to change only minimally the shape of individual signals. The conditions to apply this technique are considered in detail. When experimental data have a high signal-to-noise ratio, the lineshape distortion induced by the gradient can be corrected with the aid of a known distribution function of paramagnetic centers within the probe. The maximum entropy deconvolution algorithm is successfully applied for the correction of significantly distorted lines. The technique is experimentally tested and applied to measure the concentration of oxygen in hypertrophied rat myocardium and normal rat kidney in vivo by low frequency EPR (L-band, 1.2 GHz). No types of EPR oxygen-sensitive probes-lithium phthalocyanine crystals and synthetic carbohydrate chars-were used.     

Use of 3-acetoxymethoxycarbonyl-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl as an EPR oximetry probe: potential for in vivo measurement of tissue oxygenation in mouse brain.

Measurement of oxygen concentration and distribution in the brain is essential for understanding the pathophysiology of stroke. Low-frequency electron paramagnetic resonance (EPR) spectroscopy with a paramagnetic probe is an attractive imaging modality that potentially can be used to map O(2) concentration in the brain. We examined two nitroxides, 3-methoxycarbonyl-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl [2] and 3-acetoxymethoxycarbonyl-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl [3], as pro-imaging agents to deliver 3-carboxy-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl [1] across the blood-brain barrier (BBB). In primary cultured neurons, nitroxide [3] but not [2] was hydrolyzed by intracellular esterases to [1], which, being anionic at physiologic pH, was well retained intracellularly. In contrast, [2] was not well retained by neurons. In vivo pharmacokinetic and pharmacodynamic studies in mice suggested that esterase-labile nitroxide [3] crossed the BBB, and was converted to [1] and retained. Retention occurred in brain tissue and not in the extensive vasculature, as evidenced by the fact that removal of blood by whole-body saline perfusion did not eliminate the nitroxide EPR signal from the brain. The EPR linewidths of [1] and [3] were more O(2)-sensitive than that of the commonly-used oximetry probe 4-oxo-2,2,6,6-tetramethylpiperidine-d(16)-1-(15)N-oxyl [4]. Moreover, we used [3] in vivo to estimate O(2) concentration in mouse brains. These results indicate that nitroxide [3] could be useful for mapping O(2) distribution in the brain following stroke.     

Location of anthralin radical generation in mouse skin by UV-A irradiation: an estimation using microscopic EPR spectral-spatial imaging.

In vivo location of the anthralin radical generated in mouse skin by ultraviolet A (UV-A) irradiation was estimated by microscopic electron paramagnetic resonance (EPR) spectral-spatial imaging. An X-band EPR spectrometer equipped with specially designed high-power imaging coils and a TE-mode cavity was employed. The maximum field gradient used in this study was 6.77 mT/mm. Anthralin was applied to the dorsal skin of live mice, which were then exposed to UV-A irradiation. A broad singlet EPR spectrum (peak-to-peak line width = 0.6 mT and g = 2.004) was obtained. Microscopic EPR spectral-spatial imaging of the skin tissue showed that the anthralin radical was located mainly in the epidermis (27 microm from the skin surface). This result was consistent with the finding that the proportions of the radical in the dermis and epidermis were about 15% and 85%, respectively.     

需行全身照射患者辐射损伤血浆代谢谱的研究

目的利用代谢组学的方法发现与人辐射损伤密切相关的特征血浆代谢物,初步探讨辐射损伤涉及的代谢通路。方法收集2012年1月至2014年5月期间40名患者全身照射（TBI）辐照前后的血浆样本,通过气相色谱-质谱（GC-MS）联用的代谢组学方法研究TBI对人血浆代谢物的影响,并筛选出与TBI损伤密切相关的特征血浆代谢物。结果 TBI后,血浆中葡萄糖、肉豆蔻酸、草酸、3-羟基丁酸、尿素、天冬氨酸、缬氨酸、亮氨酸、赖氨酸和苏氨酸含量显著增加（P<0.05）,胆固醇、丙酮酸、丙酸、乳酸、丙氨酸、甘氨酸、肌醇、山梨糖酐、乙二醇和次黄嘌呤含量显著降低（P<0.05）。结论 TBI可引起人体血浆中代谢物水平显著变化,主要涉及氨基酸代谢、糖代谢、脂代谢等代谢通路。     

Changes in mitochondrial functioning with electromagnetic radiation of ultra high frequency as revealed by electron paramagnetic resonance methods

Abstract

Purpose: To study the effects of electromagnetic radiation (EMR) of ultra high frequency (UHF) in the doses equivalent to the maximal permitted energy load for the staffs of the radar stations on the biochemical processes that occur in the cell organelles.

Materials and methods: Liver, cardiac and aorta tissues from the male rats exposed to non-thermal UHF EMR in pulsed and continuous modes were studied during 28 days after the irradiation by the electron paramagnetic resonance (EPR) methods including a spin trapping of superoxide radicals.

Results: The qualitative and quantitative disturbances in electron transport chain (ETC) of mitochondria are registered. A formation of the iron-nitrosyl complexes of nitric oxide (NO) radicals with the iron-sulphide (FeS) proteins, the decreased activity of FeS-protein N2 of NADH-ubiquinone oxidoreductase complex and flavo-ubisemiquinone growth combined with the increased rates of superoxide production are obtained.

Conclusions: (i) Abnormalities in the mitochondrial ETC of liver and aorta cells are more pronounced for animals radiated in a pulsed mode; (ii) the alterations in the functioning of the mitochondrial ETC cause increase of superoxide radicals generation rate in all samples, formation of cellular hypoxia, and intensification of the oxide-initiated metabolic changes; and (iii) electron paramagnetic resonance methods could be used to track the qualitative and quantitative changes in the mitochondrial ETC caused by the UHF EMR.     

牙釉质EPR剂量测定方法的主要影响因素分析

EPR(电子顺磁共振)剂量测定方法是回顾性测定个人辐射剂量的主要方法,它能够准确估算出很久以前发生的辐射照射事件的吸收剂量值,其理论基础是在牙釉质中,辐射所致自由基水平随着辐射剂量的增加而增加。目前,将EPR方法用于低剂量测定还存在一些困难,为了降低测量阈值,减小误差,优化方法,需要进一步研究该方法的影响因素。本文重点分析了影响牙釉质EPR剂量方法测量阈值和测量结果不确定度的主要因素,并对解决这些问题的修正方法进行了讨论。     

Competition of nitroxyl contrast agents as an in vivo tissue redox probe: comparison of pharmacokinetics by the bile flow monitoring (BFM) and blood circulating monitoring (BCM) methods using X-band EPR and simulation of decay profiles.

Nitroxyl radicals used as tissue redox-sensitive contrast agents in electron paramagnetic resonance (EPR) and/or NMR imaging should satisfy the following two conditions: 1) the molecules disperse into tissues rapidly, and 2) paramagnetic loss occurs by simple reduction of the radical. The pharmacokinetic trends of several nitroxyl contrast agents were compared with the results obtained by bile flow monitoring (BFM) and blood circulation monitoring (BCM) methods using X-band EPR. The nitroxyl radicals (TEMPO, TEMPONE (oxo-TEMPO), and amino-TEMPO) showed additional EPR signals in the bile that were attributed to metabolites formed during transport from blood to bile through the liver. However, the highly hydrophilic CAT-1 (trimethylammonium-TEMPO), which has low membrane permeability, showed minimal concentration in the bile. Probes that have carboxyl moiety, such as carboxy-TEMPO and carboxy-PROXYL, can be transported via anion transporter into hepatic cells. The EPR signal decay profiles of the nitroxyl radicals were simulated based on the experimental data. The simulation, which we previously applied to mouse blood, was modified to simultaneously fit the experimental results of BFM and BCM obtained with rats. The simulation data showed the simplicity/complexity of the pharmacokinetic mechanisms and that carbamoyl-PROXYL and TEMPOL (hydroxy-TEMPO) are suitable contrast agents for assessing tissue redox status.     

In vivo measurement of regional oxygenation and imaging of redox status in RIF-1 murine tumor: effect of carbogen-breathing.

The purpose of this study was to noninvasively monitor tumor oxygenation and redox status during hyperoxygenation treatment, such as carbogen-breathing, in a murine tumor model using in vivo electron paramagnetic resonance (EPR) spectroscopy and imaging techniques. The study was performed using implanted lithium phthalocyanine (LiPc) microcrystals as the oximetry probe and 3-carbamoylproxyl (3-CP) as the redox probe in RIF-1 tumors implanted in the upper hind leg of C3H mice. Repetitive measurements of pO(2) from the same tumors as a function of tumor growth (8-24 mm in size) showed that the tumors were hypoxic and that the tumor pO(2) values were decreasing with tumor growth. Carbogen-breathing mostly showed an increase in the tumor oxygenation, although there were considerable variations in the magnitude of change among the tumors. The pharmacokinetic studies with 3-CP showed a significant decrease in the overall tumor reduction status in the carbogen-breathing mice. Spatially resolved (imaging) pharmacokinetic data over the tumor volume were obtained to visualize the distribution of the redox status within the tumor. The redox images of the tumor in the air-breathing mice showed significant heterogeneity in the magnitude and spatial distribution of reducing equivalents. On carbogen-breathing the tissue reduction status decreased considerably, with a concomitant decrease in the heterogeneity of distribution of the redox status. The results suggest that 1) carbogen-breathing considerably enhances tissue oxygenation and significantly decreases the redox status in RIF-1 tumor, and 2) changes in the magnitude and distribution of the redox status within the tumor volume during carbogen-breathing are correlated with the increased tissue oxygenation.     

Modelling hematological parameters after total body irradiation

Abstract

Purpose: The time- and dose-dependent reconstitution of hematopoiesis after radiation exposure is strongly related to the stem cell population and can be used to predict hematological parameters. These parameters allow further insight into the hematopoietic system and might lead to the development of novel stem cell transplantation models.

Materials and methods: CD4-/- C57Bl/6 mice, transgenic for human CD4 and HLA-DR3, were irradiated in a single (3, 6, 8 and 12 Gy) and fractionated (6 × 1 Gy, 6 × 1.5 Gy, 6 × 2 Gy; twice daily) dose regimen. Blood was analyzed weekly for red blood cells (RBC), hemoglobin concentration (Hb), hematocrit (HCT) and white blood cells (WBC). Organ and tissue damage after irradiation were examined by histopathology.

Results: The recovery curves for RBC, Hb, HCT and WBC showed the same velocity (< 1 week) for all radiation doses (3–12 Gy) starting at different, dose-dependent times. The only dose-dependent parameter was defined by the beginning of the recovery process (dose-dependent shift) and higher doses were related to a later recovery of the hematopoietic system. The RBC, Hb and HCT recovery was followed by a saturation curve reaching a final concentration independent of the radiation dose. Histological analysis of the bone marrow in the single dose cohort showed a dose-dependent reduction of the cellularity in the bone marrow cavities. The fractioned radiation dose cohort resulted in a regeneration of all bone marrow cavities.

Conclusion: Specific functions were developed to describe the reconstitution of hematological parameters after total body irradiation.     

Feasibility and assessment of non-invasive in vivo redox status using electron paramagnetic resonance imaging

Purpose:
To test the feasibility of electron paramagnetic resonance imaging (EPRI) to provide non-invasive images of tissue redox status using redox-sensitive paramagnetic contrast agents. Material and Methods:
Nitroxide free radicals were used as paramagnetic agents and a custom-built 300 MHz EPR spectrometer/imager was used for all studies. A phantom was constructed consisting of four tubes containing equal concentrations of a nitroxide. Varying concentrations of hypoxanthine/xanthine oxidase were added to each tube and reduction of the nitroxide was monitored by EPR as a function of time. Tumor-bearing mice were intravenously infused with a nitroxide and the corresponding reduction rate was monitored on a pixel-by-pixel basis using 2D EPR of the tumor-bearing leg and normal leg serving as control. For animal studies, nitroxides were injected intravenously (1.25 mmol/kg) and EPR projections were collected every 3 min after injection using a magnetic field gradient of 2.5 G/cm. The reduction rates of signal intensity on a pixel-by-pixel basis were calculated and plotted as a redox map. Redox maps were also collected from the mice treated with diethylmaleate (DEM), which depletes tissue thiols and alters the global redox status. Results:
Redox maps obtained from the phantoms were in agreement with the intensity change in each of the tubes where the signals were decreasing as a function of the enzymatic activity, validating the ability of EPRI to accurately access changes in nitroxide reduction. Redox imaging capability of EPR was next evaluated in vivo. EPR images of the nitroxide distribution and reduction rates in tumor-bearing leg of mice exhibited more heterogeneity than in the normal tissue. Reduction rates were found to be significantly decreased in tumors of mice treated with DEM, consistent with the depletion of thiols and the consequent alteration of the redox status. Conclusion:
Using redox-sensitive paramagnetic contrast agents, EPRI can non-invasively discriminate redox status differences between normal tissue and tumors.     

Strategies for improved temporal and spectral resolution in in vivo oximetric imaging using time-domain EPR.

A radiofrequency (RF) time-domain electron paramagnetic resonance (EPR) instrument operating at 300, 600, and 750 MHz was used to image tumor hypoxia with high spatial and temporal resolution. A high-speed signal-averaging Peripheral Component Interconnect (PCI) board with flexibility in the input signal level and the number of digitized samples per free induction decay (FID) was incorporated into the receive arm of the spectrometer. This enabled effective and fast averaging of FIDs. Modification of the phase-encoding protocol, and replacement of the General Purpose Interface Bus (GPIB)-based handshake with a PCI-based D/A board for direct control of the gradient amplifier decreased the gradient settling and communication overhead times by nearly two orders of magnitude. Cyclically-ordered phase sequence (CYCLOPS) phase cycling was implemented to correct for pulse imperfections and cancel out unwanted constant signals. These upgrades considerably enhanced the performance of the imager in terms of image collection time, sensitivity, and temporal resolution. We demonstrated this by collecting a large number of 2D images successively and rapidly. The results show that it is feasible to achieve accurate, 2D pO(2) maps of tumor hypoxia with 1-mm(2) resolution and minimal artifacts using a set of multigradient images within an acceptable measuring time of about 3 s, and 3D maps can be obtained in less than 1 min.     

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.     

Benefits of online in vivo dosimetry for single-fraction total body irradiation

Use of a patient test dose before single-fraction total body irradiation (TBI) allows review of in vivo dosimetry and modification of the main treatment setup. However, use of computed tomography (CT) planning and online in vivo dosimetry may reduce the need for this additional step. Patients were treated using a supine CT-planned extended source-to-surface distance (SSD) technique with lead compensators and bolus. In vivo dosimetry was performed using thermoluminescent dosimeters (TLDs) and diodes at 10 representative anatomical locations, for both a 0.1-Gy test dose and the treatment dose. In total, 28 patients were treated between April 2007 and July 2013, with changes made in 10 cases (36%) following test dose results. Overall, 98.1% of measured in vivo treatment doses were within 10% of the prescribed dose, compared with 97.0% of test dose readings. Changes made following the test dose could have been applied during the single-fraction treatment itself, assuming that the dose was delivered in subportions and online in vivo dosimetry was available for all clinically important anatomical sites. This alleviates the need for a test dose, saving considerable time and resources.     

Development of biocompatible oxygen-permeable films holding paramagnetic carbon particles: evaluation of their performance and stability in EPR oximetry.

EPR oximetry using paramagnetic particles relies on the measurement of the EPR linewidth, which is directly related to the pO2. It was previously found that some of the paramagnetic materials with optimal EPR spectroscopic properties in vitro may lose their responsiveness to oxygen in tissues (change of the calibration curve of the EPR linewidth as a function of the pO2). We hypothesized that coating paramagnetic particle materials could improve the stability of response, as well as the biocompatibility. In this study, very thin films holding paramagnetic materials were prepared with different biopolymers (cellulose acetate, cellulose triacetate, cellulose nitrate, silicone, and polyurethane) that already are accepted for clinical applications. Their performance was evaluated in EPR oximetry by measuring the stability of the calibration curves (EPR linewidth as a function of pO2) after a prolonged period in an aqueous environment (1 week in saline) or in vivo (implantation for 3 weeks under the skin of mice). We found that one type of silicone film was able to stabilize the responsiveness of an intrinsically unstable carbon material (a wood char).     

Estimation of X-ray beam quality by electron paramagnetic resonance (EPR) spectroscopy

A novel dosimetry-based technique using EPR spectroscopy to determine X-ray beam quality is proposed. The radiation-sensitive material is made of a mixture of two polycrystalline substances with different X-ray absorption properties.

The composite samples, consisting of polycrystalline lithium formate monohydrate and calcium formate, were prepared as pellets, X-irradiated, and analyzed with EPR spectroscopy. The ratio of the EPR signal amplitudes of the two constituents can serve as a measure of the X-ray beam quality given by the equivalent photon energy. The calculation of the signal amplitude ratio involves a reconstruction of the composite EPR spectrum. The logarithm of the signal amplitude ratio appears to be linearly correlated with the logarithm of the equivalent photon energy. The linear relationship can be used as a calibration for estimating the equivalent photon energy from the composite EPR spectrum.

The composite material was used to investigate the changes in the equivalent photon energy in a Perspex phantom with increasing depth. When a 220 kV X-ray beam with an equivalent photon energy of about 100 keV was used, changes in the EPR signal amplitude ratio revealed a buildup of scattered photons with increasing depth in the phantom. This change could be related to the equivalent photon energy using the logarithmic calibration curve. It was found that the equivalent photon energy at the depth of 13 cm in the phantom was 25% lower than on its surface.

The proposed method can be used for estimating equivalent photon energy in both standardized and non-standardized situations, the latter corresponding to beam setups where use of filters and ionization chambers is difficult or impossible. Also, the system can provide a means for measuring photon energy in X-irradiated phantoms.     

CT analysis of lung density changes in patients undergoing total body irradiation prior to bone marrow transplantation

Sequential changes in lung density measured by CT are potentially sensitive and convenient monitors of lung abnormalities following total body irradiation (TBI). Methods have been developed to compare pre- and post-TBI CT of lung. The average local features of a cross-sectional lung slice are extracted from three peripheral regions of interest in the anterior, posterior, and lateral portions of the CT image. Also, density profiles across a specific region may be obtained. These may be compared first for verification of patient position and breathing status and then for changes between pre- and post-TBI. These may also be compared with radiation dose profiles through the lung. A preliminary study on 21 leukemia patients undergoing total body irradiation indicates the following: (a) Density gradients of patients' lungs in the antero-posterior direction show a marked heterogeneity before and after transplantation compared with normal lungs. The patients with departures from normal density gradients pre-TBI correlate with later pulmonary complications. (b) Measurements of average peripheral lung densities have demonstrated that the average lung density in the younger age group is substantially higher: pre-TBI, the average CT number (1,000 scale) is -638 +/- 39 Hounsfield unit (HU) for 0-10 years old and -739 +/- 53 HU for 21-40 years old. (c) Density profiles showed no post-TBI regional changes in lung density corresponding to the dose profile across the lung, so no differentiation of a radiation-specific effect has yet been possible. Computed tomographic density profiles in the antero-posterior direction are successfully used to verify positioning of the CT slice and the breathing level of the lung.     

Noninvasive measurement of the pH inside the gut by using pH-sensitive nitroxides. An in vivo EPR study

The use of pH-sensitive probes permits the measurement of the proton activity in biological systems by EPR spectroscopy. To illustrate the potential of this technique for in vivo purposes, the authors took advantage of the oral administration of nitroxides to monitor the pH value inside the stomach of mice after administration of different antacidics. The results indicate that EPR can be a valuable tool to characterize the pH in vivo in a continuous and noninvasive way.