Benchmark Test and Guidelines for DEER/PELDOR Experiments on Nitroxide-Labeled Biomolecules.

Distance distribution information obtained by pulsed dipolar EPR spectroscopy provides an important contribution to many studies in structural biology. Increasingly, such information is used in integrative structural modeling, where it delivers unique restraints on the width of conformational ensembles. In order to ensure reliability of the structural models and of biological conclusions, we herein define quality standards for sample preparation and characterization, for measurements of distributed dipole-dipole couplings between paramagnetic labels, for conversion of the primary time-domain data into distance distributions, for interpreting these distributions, and for reporting results. These guidelines are substantiated by a multi-laboratory benchmark study and by analysis of data sets with known distance distribution ground truth. The study and the guidelines focus on proteins labeled with nitroxides and on double electron-electron resonance (DEER aka PELDOR) measurements and provide suggestions on how to proceed analogously in other cases.

Effect of the tooth surface water on the accuracy of dose reconstructions in the X-band in vivo EPR dosimetry.

The X-band in vivo EPR tooth dosimetry is promising as a tool for the initial triage after a large-scale radiation accident. The dielectric losses caused by water on the tooth surface (WTS) are one of the major sources of inaccuracies in this method. The effect was studied by theoretical simulation calculations and experiments with water films of various thicknesses on teeth. The results demonstrate the possibility of sufficiently accurate measurements of the radiation-induced signal of the tooth enamel provided that the thickness of the water film on the tooth is below 60 µm. The sensitivity of the cavity decreases with increasing thickness of the water layer. The interference of WTS can be diminished by normalization of the radiation-induced signal to the signal of a reference sample permanently present in the cavity.

Electron Paramagnetic Resonance pO2 Image Tumor Oxygen-Guided Radiation Therapy Optimization.

Modern standards for radiation treatment do not take into account tumor oxygenation for radiation treatment planning. Strong correlation between tumor oxygenation and radiation treatment success suggests that oxygen-guided radiation therapy (OGRT) may be a promising enhancement of cancer radiation treatment. We have developed an OGRT protocol for rodents. Electron paramagnetic resonance (EPR) imaging is used for recording oxygen maps with high spatial resolution and excellent accuracy better than 1 torr. Radiation is delivered with an animal intensity modulated radiation therapy (IMRT) XRAD225Cx micro-CT/ therapy system. The radiation plan is delivered in two steps. First, a uniform 15% tumor control dose (TCD15) is delivered to the whole tumor. In the second step, an additional booster dose amounting to the difference between TCD98 and TCD15 is delivered to radio-resistant, hypoxic tumor regions. Delivery of the booster dose is performed using a multiport conformal beam protocol. For radiation beam shaping we used individual radiation blocks 3D-printed from tungsten infused ABS polymer. Calculation of beam geometry and the production of blocks is performed next to the EPR imager, immediately after oxygen imaging. Preliminary results demonstrate the sub-millimeter precision of the radiation delivery and high dose accuracy. The efficacy of the radiation treatment is currently being tested on syngeneic FSa fibrosarcoma tumors grown in the legs of C3H mice.

Overview of physical dosimetry methods for triage application integrated in the new European network RENEB.

PURPOSE: In the EC-funded project RENEB (Realizing the European Network in Biodosimetry), physical methods applied to fortuitous dosimetric materials are used to complement biological dosimetry, to increase dose assessment capacity for large-scale radiation/nuclear accidents. This paper describes the work performed to implement Optically Stimulated Luminescence (OSL) and Electron Paramagnetic Resonance (EPR) dosimetry techniques. MATERIALS AND METHODS: OSL is applied to electronic components and EPR to touch-screen glass from mobile phones. To implement these new approaches, several blind tests and inter-laboratory comparisons (ILC) were organized for each assay. RESULTS: OSL systems have shown good performances. EPR systems also show good performance in controlled conditions, but ILC have also demonstrated that post-irradiation exposure to sunlight increases the complexity of the EPR signal analysis. CONCLUSIONS: Physically-based dosimetry techniques present high capacity, new possibilities for accident dosimetry, especially in the case of large-scale events. Some of the techniques applied can be considered as operational (e.g. OSL on Surface Mounting Devices [SMD]) and provide a large increase of measurement capacity for existing networks. Other techniques and devices currently undergoing validation or development in Europe could lead to considerable increases in the capacity of the RENEB accident dosimetry network.

Reliability of arterial spin labelling measurements of perfusion within the quadriceps during steady-state exercise.

Arterial spin labelling (ASL) provides a potential method to non-invasively determine muscle blood flow and examine the impact of interventions such as supplementation and training. However, it's a method with intrinsically low signal, leading to limitations in accuracy and temporal resolution. To examine these limitations, the current study measured perfusion via ASL on three occasions in the rectus femoris of 10 healthy adults, during light and moderate exercise, over three different exercise durations. For data sampled over 9 min, light intensity exercise gave an average perfusion of 35.0 ± 5.1 ml/min.100g(-1) with a coefficient of variation (COV) of 16% and single intraclass correlation coefficient (ICC) of 0.67. For the moderate bout, perfusion was 51.3 ± 5.6 ml/min.100g(-1) (COV 10%, ICC 0.82). When the same data were analyzed over 5 min 24 s, perfusion was 37.8 ± 11.13 (COV 30%, ICC 0.13) during light and 49.5 ± 8.8 ml/min.100g(-1) (COV 18%, ICC 0.52) during moderate exercise. When sampling was reduced to 1 min 48 s, perfusion was 41.2 ± 13.7 (COV 33%, ICC 0.26) during light and 49.5 ± 13.6 ml/min.100g(-1) (COV 28%, ICC 0.04) during moderate exercise. For 9 min a significant perfusion difference was found between the exercise intensities; however, this was not the case for sampling over 5 min 24 s or 1 min 48 s. Such findings illustrate the potential of ASL to non-invasively monitor muscle perfusion under steady-state conditions, but highlight that extended exercise protocols are necessary in order to generate date of sufficient reliability to be able to discriminate intervention dependent perfusion differences.

Verification of the pure alanine in PMMA tube dosimeter applicability for dosimetry of radiotherapy photon beams: a feasibility study.

Alanine dosimeters in the form of pure alanine powder in PMMA plastic tubes were investigated for dosimetry in a clinical application. Electron paramagnetic resonance (EPR) spectroscopy was used to measure absorbed radiation doses by detection of signals from radicals generated in irradiated alanine. The measurements were performed for low-dose ranges typical for single-fraction doses often used in external photon beam radiotherapy. First, the dosimeters were irradiated in a solid water phantom to establish calibration curves in the dose range from 0.3 to 3 Gy for 6 and 18 MV X-ray beams from a clinical linear accelerator. Next, the dosimeters were placed at various locations in an anthropomorphic pelvic phantom to measure the dose delivery of a conventional four-field box technique treatment plan to the pelvis. Finally, the doses measured with alanine dosimeters were compared against the doses calculated with a commercial treatment planning system (TPS). The results showed that the alanine dosimeters have a highly sensitive dose response with good linearity and no energy dependence in the dose range and photon beams used in this work. Also, a fairly good agreement was found between the in-phantom dose measurements with alanine dosimeters and the TPS dose calculations. The mean value of the ratios of measured to calculated dose values was found to be near unity. The measured points in the in-field region passed dose-difference acceptance criterion of 3% and those in the penumbral region passed distance-to-agreement acceptance criterion of 3 mm. These findings suggest that the pure alanine powder in PMMA tube dosimeter is a suitable option for dosimetry of radiotherapy photon beams.

A proposed method for retrospective eye dose assessments for the purposes of resolving cataract compensation claims.

The 2011 International Commission on Radiological Protection (ICRP) statement on tissue reactions suggested a significant reduction in the threshold dose for radiation induced cataracts. This, combined with the potential for a long delay between exposure and cataract diagnosis, may result in an increased requirement to evaluate eye dose from past exposures in order to settle current compensation claims. This article highlights how compensation claims relating to radiation exposure are assessed within the UK legal system and suggests that in vivo Electro Paramagnetic Resonance (EPR) dosimetry of teeth has utility for the retrospective quantification of radiation doses to the eye. It was identified that in vivo EPR in its current form may be sufficiently sensitive to support cataract compensation claims, although further work is required to enable appropriate dose conversion coefficients to be quantified.

A high precision method for quantitative measurements of reactive oxygen species in frozen biopsies.

OBJECTIVE: An electron paramagnetic resonance (EPR) technique using the spin probe cyclic hydroxylamine 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH) was introduced as a versatile method for high precision quantification of reactive oxygen species, including the superoxide radical in frozen biological samples such as cell suspensions, blood or biopsies. MATERIALS AND METHODS: Loss of measurement precision and accuracy due to variations in sample size and shape were minimized by assembling the sample in a well-defined volume. Measurement was carried out at low temperature (150 K) using a nitrogen flow Dewar. The signal intensity was measured from the EPR 1st derivative amplitude, and related to a sample, 3-carboxy-proxyl (CP•) with known spin concentration. RESULTS: The absolute spin concentration could be quantified with a precision and accuracy better than ±10 µM (k = 1). The spin concentration of samples stored at -80°C could be reproduced after 6 months of storage well within the same error estimate. CONCLUSION: The absolute spin concentration in wet biological samples such as biopsies, water solutions and cell cultures could be quantified with higher precision and accuracy than normally achievable using common techniques such as flat cells, tissue cells and various capillary tubes. In addition; biological samples could be collected and stored for future incubation with spin probe, and also further stored up to at least six months before EPR analysis, without loss of signal intensity. This opens for the possibility to store and transport incubated biological samples with known accuracy of the spin concentration over time.

EPR spectroscopic investigation of psoriatic finger nails.

BACKGROUND: Nail lesions are common features of psoriasis and found in almost half of the patients. However, there is no feasible spectroscopic method evaluating changes and severity of nail psoriasis. EPR (electron paramagnetic resonance) might be feasible for evaluating nail conditions in the patients of psoriasis. METHODS: Finger nails of five cases with nail psoriasis, (three females and two males) were examined. Nail samples were subjected to the EPR assay. The small piece of the finger nail (1.5 × 5 mm(2)) was incubated in ~50 µM 5-DSA (5-doxylstearic acid) aqueous solutions for about 60 min at 37°C. After rinsing and wiping off the excess 5-DSA solution, the nail samples were measured by EPR. RESULTS: EPR spectra were analyzed using the intensity ratio (Fast/Slow) of the two motions at the peaks of the lower magnetic field. We observed two distinguishable sites on the basis of the EPR results. In addition, the modern EPR calculation was performed to analyze the spectra obtained. The nail psoriasis-related region is 2~3 times higher than that of the control. CONCLUSION: The present EPR results show that there are two distinguishable sites in the nail. In the case of nail psoriasis, the fragile components are 2~3 times more than those of the control. Thus, the EPR method is thought to be a novel and reliable method of evaluating the nail psoriasis.

Dose response of xylitol and sorbitol for EPR retrospective dosimetry with applications to chewing gum.

The purpose of this investigation was to study the radiation-induced electron paramagnetic resonance signal in sweeteners xylitol and sorbitol for use in retrospective dosimetry. For both sweeteners and chewing gum, the signal changed at an interval of 1-84 d after irradiation with minimal changes after 4-8 d. A dependence on storage conditions was noticed and the exposure of the samples to light and humidity was therefore minimised. Both the xylitol and sorbitol signals showed linearity with dose in the measured dose interval, 0-20 Gy. The dose-response measurements for the chewing gum resulted in a decision threshold of 0.38 Gy and a detection limit of 0.78 Gy. A blind test illustrated the possibility of using chewing gums as a retrospective dosemeter with an uncertainty in the dose determination of 0.17 Gy (1 SD).

Assessment of a standardized ROS production profile in humans by electron paramagnetic resonance.

Despite the growing interest in the role of reactive oxygen species (ROS) in health and disease, reliable quantitative noninvasive methods for the assessment of oxidative stress in humans are still lacking. EPR technique, coupled to a specific spin probe (CMH: 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine) is here presented as the method of choice to gain a direct measurement of ROS in biological fluids and tissues. The study aimed at demonstrating that, differently from currently available "a posteriori" assays of ROS-induced damage by means of biomolecules (e.g., proteins and lipids) spin-trapping EPR provides direct evidence of the "instantaneous" presence of radical species in the sample and, as signal areas are proportional to the number of excited electron spins, lead to absolute concentration levels. Using a recently developed bench top continuous wave system (e-scan EPR scanner, Bruker) dealing with very low ROS concentration levels in small (50 µL) samples, we successfully monitored rapid ROS production changes in peripheral blood of athletes after controlled exercise and sedentary subjects after antioxidant supplementation. The correlation between EPR results and data obtained by various enzymatic assays (e.g., protein carbonyls and thiobarbituric acid reactive substances) was determined too. Synthetically, our method allows reliable, quick, noninvasive quantitative determination of ROS in human peripheral blood.

A novel analytical method to evaluate directly catalase activity of microorganisms and mammalian cells by ESR oximetry.

Electron spin resonance (ESR) oximetry technique was applied for analysis of catalase activity in the present study. Catalase activity was evaluated by measuring oxygen from the reaction between hydrogen peroxide (H(2)O(2)) and catalase-positive cells. It was demonstrated that the ESR spectra of spin-label probes, 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPOL), 4-oxo-2,2,6,6-tetramethyl-1-piperidinyloxy (4-oxo-TEMPO) and 4-maleimido-2,2,6,6-tetramethyl-1-piperidinyloxy (4-maleimido-TEMPO) in the presence of H(2)O(2) were broadened with the concentrations of catalase. It was possible to make a calibration curve for catalase activity by peak widths of the spectra of each spin-label probe, which are broadened dependently on catalase concentrations. The broadened ESR spectra were also observed when the catalase-positive micro-organisms or the mammalian cells originally from circulating monocytes/macrophages were mixed with TEMPOL and H(2)O(2). Meanwhile, catalase-negative micro-organisms caused no broadening change of ESR spectra. The present study indicates that it is possible to evaluate directly the catalase activity of various micro-organisms and mammalian cells by using an ESR oximetry technique.

A signal-to-noise standard for pulsed EPR.

A 2 mm diameter by 10mm long cylinder of fused SiO2 (quartz) gamma-irradiated to 1 kGy with 60Co contains about 2x10(16) spins/cm3. It is proposed as a standard for monitoring signal-to-noise (S/N) performance of X-band pulsed EPR spectrometers. This sample yields S/N of about 25 on modern spin echo spectrometers, which permits measurement of both signal and noise under the same conditions with an 8-bit digitizer.

Calibration and validation of an optical sensor for intracellular oxygen measurements.

Calibration of fluorescent optical sensors for accurate, quantitative intracellular measurements in vivo suffers from lack of a representative medium that appropriately simulates the molecular complexity of the cytosol. We present a novel protocol for accurate intracellular oxygen sensing via fluorescence lifetime imaging microscopy (FLIM) using cell lysate-FLIM measurements to correct the in vitro calibration of a fluorescent oxygen sensor, and we describe electron paramagnetic resonance (EPR) validation studies. Lysate-FLIM studies provided biochemical information, while EPR provided a "gold standard" for intracellular oxygen estimation. Oxygen levels were evaluated in living human normal squamous and adenocarcinoma esophageal epithelial cells, and good agreement was observed between oxygen levels derived from the optical protocol and EPR. The proposed protocol introduces the concept of a living cell line as a reference for estimating unknown oxygen levels in other cell lines and accounts for high degrees of variability between different cell lines.

Significantly improved sensitivity of Q-band PELDOR/DEER experiments relative to X-band is observed in measuring the intercoil distance of a leucine zipper motif peptide (GCN4-LZ).

Pulsed electron double resonance (PELDOR)/double electron-electron resonance (DEER) spectroscopy is a very powerful structural biology tool in which the dipolar coupling between two unpaired electron spins (site-directed nitroxide spin-labels) is measured. These measurements are typically conducted at X-band (9.4 GHz) microwave excitation using the four-pulse DEER sequence and can often require up to 12 h of signal averaging for biological samples (depending on the spin-label concentration). In this work, we present for the first time a substantial increase in DEER sensitivity obtained by collecting DEER spectra at Q-band (34 GHz), when compared to X-band. The huge boost in sensitivity (factor of 13) demonstrated at Q-band represents a 169-fold decrease in data collection time, reveals a greatly improved frequency spectrum and higher-quality distance data, and significantly increases sample throughput. Thus, the availability of Q-band DEER spectroscopy should have a major impact on structural biology studies using site-directed spin labeling EPR techniques.

EPR line shifts and line shape changes due to spin exchange of nitroxide free radicals in liquids: 6. Separating line broadening due to spin exchange and dipolar interactions.

EPR spectra of perdeuterated 2,2,6,6-tetramethyl-4-oxopiperidine-1-oxyl (PDT) are studied as functions of molar concentration, c, and temperature, T, in water and 70 wt % glycerol in water. The increase of the intrinsic line width averaged over the three hyperfine lines, B(tot), varies linearly with c with zero intercept in both solvents at all temperatures; therefore dB(tot)/dc is independent of c. The spin exchange induced dispersion, from which the spin exchange frequency, omega(e), may be computed, increases linearly with B(tot), passing through the origin in water and in 70% glycerol at high temperatures; however, at low temperatures, where dipolar interactions broaden the spectra, linearity does not prevail until B(tot) > 1 G due to a contribution of dipolar interactions to the dispersion. The broadening constant due to spin exchange, dB(e)/dc, is found from the slope of the linear region, permitting a computation of the dipolar constant, dB(dip)/dc = dB(tot)/dc - dB(e)/dc. Thus, the separation of concentration broadening into spin exchange and dipolar contributions is effected without having to appeal to some supposed temperature dependence of the two interactions. The fractional broadening by spin exchange, Omega(T), is near unity at high temperatures in both solvents, decreasing to zero in 70% glycerol at 273 K. Omega(T) is a continuous function of the inverse rotational correlation time of PDT but is discontinuous as a function of T/eta where eta is the shear viscosity. Omega(T) = 0.5, where spin exchange and dipolar interactions contribute equally to the line width occurs at T/eta = 20 +/- 1 K/cP in 70% glycerol. Hydrodynamic predictions of dB(e)/dc via the Stokes-Einstein (SE) equation are remarkably accurate in 70% glycerol comparable with the results in a series of alkanes. In water, dB(e)/dc is linear with T/eta with zero intercept as required by the SE; however, with slope a factor of 0.73 smaller. dB(dip)/dc is reasonably predicted by the SE only at very small values of eta/T very quickly following an approximately logarithmic dependence rather that the linear prediction. Values of dB(dip)/dc approach a plateau above eta/T = 0.20 cP/K that is about one-half the solid state limit. Line shifts due to spin exchange are not yet useful to deduce values of Omega(T) due to a lack of knowledge of the time between re-encounters; however, they may be used to verify the values determined from line broadening and spin exchange induced dispersion. Some effects at low temperatures in 70% glycerol suggest that the effects of dipolar interaction are inadequately described by the widely accepted theory.

Medical reference dosimetry using EPR measurements of alanine: development of an improved method for clinical dose levels.

BACKGROUND: Electron spin resonance (EPR) is used to determine the absorbed dose of alanine dosimeters exposed to clinical photon beams in a solid-water phantom. Alanine is potentially suitable for medical reference dosimetry, because of its near water equivalence over a wide energy spectrum, low signal fading, non-destructive measurement and small dosimeter size. MATERIAL AND METHODS: A Bruker EMX-micro EPR spectrometer with a rectangular cavity and a measurement time of two minutes per dosimeter was used for reading of irradiated alanine dosimeters. Under these conditions a new algorithm based on scaling of known spectra was developed to extract the alanine signal. RESULTS: The dose accuracy, including calibration uncertainty, is less than 2% (k=1) above 4 Gy (n=4). The measurement uncertainty is fairly constant in absolute terms (approximately 30 mGy) and the relative uncertainty therefore rises for dose measurements below 4 Gy. Typical reproducibility is <1% (k=1) above 10 Gy and <2% between 4 and 10 Gy. Below 4 Gy the uncertainty is higher. A depth dose curve measurement was performed in a solid-water phantom irradiated to a dose of 20 Gy at the maximum dose point (d(max)) in 6 and 18 MV photon beams. The typical difference between the dose measured with alanine in solid water and the dose measured with an ion chamber in a water tank was about 1%. A difference of 2% between 6 and 18 MV was found, possibly due to non-water equivalence of the applied phantom. DISCUSSION: Compared to previously published methods the proposed algorithm can be applied without normalisation of phase shifts caused by changes in the g-value of the cavity. The study shows that alanine dosimetry is a suitable candidate for medical reference dosimetry especially for quality control applications.

Spin-probes designed for measuring the intrathylakoid pH in chloroplasts.

Nitroxide radicals are widely used as molecular probes in different fields of chemistry and biology. In this work, we describe pH-sensitive imidazoline- and imidazolidine-based nitroxides with pK values in the range 4.7-7.6 (2,2,3,4,5,5-hexamethylperhydroimidazol-1-oxyl, 4-amino-2,2,5,5-tetramethyl-2,5-dihydro-1H-imidazol-1-oxyl, 4-dimethylamino-2,2-diethyl-5,5-dimethyl-2,5-dihydro-1H-imidazol-1-oxyl, and 2,2-diethyl-5,5-dimethyl-4-pyrrolidyline-1-yl-2,5-dihydro-1H-imidazol-1-oxyl), which allow the pH-monitoring inside chloroplasts. We have demonstrated that EPR spectra of these spin-probes localized in the thylakoid lumen markedly change with the light-induced acidification of the thylakoid lumen in chloroplasts. Comparing EPR spectrum parameters of intrathylakoid spin-probes with relevant calibrating curves, we could estimate steady-state values of lumen pHin established during illumination of chloroplasts with continuous light. For isolated bean (Vicia faba) chloroplasts suspended in a medium with pHout=7.8, we found that pHin approximately 5.4-5.7 in the state of photosynthetic control, and pHin approximately 5.7-6.0 under photophosphorylation conditions. Thus, ATP synthesis occurs at a moderate acidification of the thylakoid lumen, corresponding to transthylakoid pH difference DeltapH approximately 1.8-2.1. These values of DeltapH are consistent with a point of view that under steady-state conditions the proton gradient DeltapH is the main contributor to the proton motive force driving the operation of ATP synthesis, provided that stoichiometric ratio H+/ATP is n> or =4-4.7.

A discrepancy in superoxide scavenging activity between the ESR-spin trapping method and the luminol chemiluminescence method.

In a previous study of ours, the superoxide scavenging activity of aqueous extracts from dinophycean red tide flagellates was detected by an electron spin resonance (ESR)-spin trapping method, but not by an L-012 (luminol analog)-dependent chemiluminescence (CL) method. To investigate the discrepancy between the two methods, the effect of ferric-protein complexes on superoxide scavenging activity was examined. The reduced signal intensity of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO)-OOH due to superoxide dismutase (SOD) did not change with the addition of horseradish peroxidase (HRP), while the reduced CL response due to SOD was restored by the addition of different concentrations of HRP. Since HRP is a ferric-protein complex, the effects of other ferric-protein complexes, catalase and hemoglobin, on the reduced CL response due to SOD were examined, and similar results were obtained. As is the case with SOD, the reduced CL response activity due to an aqueous extract from a raphidophycean red tide flagellate, Chattonella ovata, was also enhanced by HRP, catalase, and hemoglobin. ESR spectra analyzed at 77 K indicated that aqueous extracts of Gymnodinium impudicum and Alexandrium affine, both of which are dinophycean red tide flagellates, contained a ferric-protein complex, and that an extract of C. ovata did not. These results suggest that the presence of such a ferric-protein complex is a causative factor in the discrepancy between the ESR and luminol CL methods when determining superoxide scavenging activity.