Developments in Biodosimetry Methods for Triage With a Focus on X-band Electron Paramagnetic Resonance In Vivo Fingernail Dosimetry.

Instrumentation and application methodologies for rapidly and accurately estimating individual ionizing radiation dose are needed for on-site triage in a radiological/nuclear event. One such methodology is an in vivo X-band, electron paramagnetic resonance, physically based dosimetry method to directly measure the radiation-induced signal in fingernails. The primary components under development are key instrument features, such as resonators with unique geometries that allow for large sampling volumes but limit radiation-induced signal measurements to the nail plate, and methodological approaches for addressing interfering signals in the nail and for calibrating dose from radiation-induced signal measurements. One resonator development highlighted here is a surface resonator array designed to reduce signal detection losses due to the soft tissues underlying the nail plate. Several surface resonator array geometries, along with ergonomic features to stabilize fingernail placement, have been tested in tissue-equivalent nail models and in vivo nail measurements of healthy volunteers using simulated radiation-induced signals in their fingernails. These studies demonstrated radiation-induced signal detection sensitivities and quantitation limits approaching the clinically relevant range of ≤ 10 Gy. Studies of the capabilities of the current instrument suggest that a reduction in the variability in radiation-induced signal measurements can be obtained with refinements to the surface resonator array and ergonomic features of the human interface to the instrument. Additional studies are required before the quantitative limits of the assay can be determined for triage decisions in a field application of dosimetry. These include expanded in vivo nail studies and associated ex vivo nail studies to provide informed approaches to accommodate for a potential interfering native signal in the nails when calculating the radiation-induced signal from the nail plate spectral measurements and to provide a method for calibrating dose estimates from the radiation-induced signal measurements based on quantifying experiments in patients undergoing total-body irradiation or total-skin electron therapy.

POSSIBLE NATURE OF THE RADIATION-INDUCED SIGNAL IN NAILS: HIGH-FIELD EPR, CONFIRMING CHEMICAL SYNTHESIS, AND QUANTUM CHEMICAL CALCULATIONS.

Exposure of finger- and toe-nails to ionizing radiation generates an Electron Paramagnetic Resonance (EPR) signal whose intensity is dose dependent and stable at room temperature for several days. The dependency of the radiation-induced signal (RIS) on the received dose may be used as the basis for retrospective dosimetry of an individual's fortuitous exposure to ionizing radiation. Two radiation-induced signals, a quasi-stable (RIS2) and stable signal (RIS5), have been identified in nails irradiated up to a dose of 50 Gy. Using X-band EPR, both RIS signals exhibit a singlet line shape with a line width around 1.0 mT and an apparent g-value of 2.0044. In this work, we seek information on the exact chemical nature of the radiation-induced free radicals underlying the signal. This knowledge may provide insights into the reason for the discrepancy in the stabilities of the two RIS signals and help develop strategies for stabilizing the radicals in nails or devising methods for restoring the radicals after decay. In this work an analysis of high field (94 GHz and 240 GHz) EPR spectra of the RIS using quantum chemical calculations, the oxidation-reduction properties and the pH dependence of the signal intensities are used to show that spectroscopic and chemical properties of the RIS are consistent with a semiquinone-type radical underlying the RIS. It has been suggested that semiquinone radicals formed on trace amounts of melanin in nails are the basis for the RIS signals. However, based on the quantum chemical calculations and chemical properties of the RIS, it is likely that the radicals underlying this signal are generated from the radiolysis of L-3,4-dihydroxyphenylalanine (DOPA) amino acids in the keratin proteins. These DOPA amino acids are likely formed from the exogenous oxidation of tyrosine in keratin by the oxygen from the air prior to irradiation. We show that these DOPA amino acids can work as radical traps, capturing the highly reactive and unstable sulfur-based radicals and/or alkyl radicals generated during the radiation event and are converted to the more stable o-semiquinone anion-radicals. From this understanding of the oxidation-reduction properties of the RIS, it may be possible to regenerate the unstable RIS2 following its decay through treatment of nail clippings. However, the treatment used to recover the RIS2 also has the ability to recover an interfering, mechanically-induced signal (MIS) formed when the nail is clipped. Therefore, to use the recovered (regenerated) RIS2 to increase the detection limits and precision of the RIS measurements and, therefore, the dose estimates calculated from the RIS signal amplitudes, will require the application of methods to differentiate the RIS2 from the recovered MIS signal.

Dielectric-Backed Aperture Resonators for X-Band in vivo EPR Nail Dosimetry.

A new resonator for X-band in vivo EPR nail dosimetry, the dielectric-backed aperture resonator (DAR), is developed based on rectangular TE102 geometry. This novel geometry for surface spectroscopy improves at least a factor of 20 compared to a traditional non-backed aperture resonator. Such an increase in EPR sensitivity is achieved by using a non-resonant dielectric slab, placed on the aperture inside the cavity. The dielectric slab provides an increased magnetic field at the aperture and sample, while minimizing sensitive aperture resonance conditions. This work also introduces a DAR semi-spherical (SS)-TE011 geometry. The SS-TE011 geometry is attractive due to having twice the incident magnetic field at the aperture for a fixed input power. It has been shown that DAR provides sufficient sensitivity to make biologically relevant measurements both in vitro and in vivo Although in vivo tests have shown some effects of physiological motions that suggest the necessity of a more robust finger holder, equivalent dosimetry sensitivity of approximately 1.4 Gy has been demonstrated.

Multifrequency electron spin resonance study of the dynamics of spin labeled T4 lysozyme.

An extensive set of electron spin resonance spectra was obtained over a wide range of frequencies (9, 95, 170, and 240 GHz) and temperatures (2 to 32 degrees C) to explore the dynamic modes of nitroxide-labeled T4 lysozyme in solution. A commonly used nitroxide side chain (R1), or a methylated analogue with hindered internal motion (R2), was substituted for the native side chain at solvent-exposed helical sites, 72 or 131. The spectra at all four frequencies were simultaneously fit with the slowly relaxing local structure (SRLS) model. Good fits were achieved at all the temperatures. Two principle dynamic modes are included in the SRLS model, the global tumbling of the protein and the internal motion consisting of backbone fluctuations and side chain isomerizations. Three distinct spectral components were required for R1 and two for R2 to account for the spectra at all temperatures. One is a highly ordered and slow motional component, which is observed in the spectra of both R1 and R2; it may correspond to conformers stabilized by interaction with the protein surface. The fraction of this component decreases with increasing temperature and is more populated in the R2 spectra, possibly arising from stronger interaction of the nitroxide ring with the protein surface due to the additional methyl group. The other two components of R1 and the second component of R2 are characterized by fast anisotropic diffusion and relatively low ordering, most likely corresponding to conformers having little or no interactions with nearby residues. Ficoll of different concentrations was added to increase the solution viscosity, thereby slowing down the global tumbling of the protein. A significant effect of Ficoll on the internal motion of an immobilized component was apparent in R2 but not in R1. The ability of such multifrequency studies to separate the effects of faster internal modes of motion from slower overall motions is clearly demonstrated, and its utility in future studies is considered.