S. Colacicchi

Whole mouse nitroxide free radical pharmacokinetics by low frequency electron paramagnetic resonance

The in vivo uptake distribution and reduction of the oxygen-sensitive nitroxide spin label PCA in the mouse monitored by low frequency electron paramagnetic resonance (EPR) spectroscopy are reported. Spectra were obtained from the head and liver regions of pentobarbital anesthetized mice during different circulatory and ventilatory conditions. Identical clearances were found in these regions during normoxia. Moderate hypoxia (10% O2-90% N2) did not significantly affect the spin label reduction rate.

New experimental procedures for in vivo L-band and radio frequency EPR spectroscopy/imaging

Electron paramagnetic resonance spectroscopy/imaging experiments are currently being performed on small animals and isolated organs. The exogenous paramagnetic probes commonly used are nitroxide (aminoxyl) free radicals. Nitroxide reduction and clearance were evaluated to understand in vivo distribution and metabolism. Pharmacokinetics data were collected with the same nitroxide at three different frequencies in an external rat blood circuit, in a mouse head and in a whole rat body. A 280 MHz spectrometer made it possible to obtain images of a whole rat body after nitroxide intravenous injection. At L-band (1500 MHz), a synchronization technique was used to obtain images of a dynamic phantom utilized to mimic the model of the isolated perfused rat heart. Different experimental procedures are discussed and the problems which affect sensitivity and resolution are evaluated.

An EPR study of lipid vesicles as paramagnetic agent vectors

Paramagnetic nitroxides have been proposed as probes in electron paramagnetic resonance (EPR) imaging and in clinical diagnosis. However, nitroxides are rapidly reducedin vivo to hydroxylamines, diamagnetic EPR-inactive species. Reduction occurs in blood via soluble agents such as ascorbic acid, as well as in the cells via enzymatic and non-enzymatic endocellular systems. To prevent the reduction, a water soluble nitroxide, i.e., potassium peroxylamine disulfonate, is entrapped in reverse phase evaporation vesicles. The loaded liposomes have a high entrapment capacity, and vesicles with the encapsulated agent are stable for days, even at room temperature. The vesiclesin vitro can almost completely prevent the reduction of the entrapped nitroxide by ascorbic acid. In blood of a rat, enriched with a homogenate of rat liver proteins, the vesicles are able to greatly prolong the life time of the nitroxide. In particular, the encapsulated nitroxide has a half-life of more than one hour, compared to two minutes for free nitroxide under the same conditions. Due to these protective effects, the lipid vesicles might be useful as a delivery system for paramagnetic agents.

EPR study of Fremy’s salt nitroxide roduction by ascorbic acid; influence of the bulk pH values

EPR and UV spectroscopy were used to investigate the efficiency of ascorbic acid in reducing Fremy’s salt. Our data indicates that the first proton electron transfer from ascorbate occurs within the mixing time. Even after the disappearance of the UV signal of the ascorbate, EPR measurements showed that the reaction goes forward, indicating a biphasic redox process. The slower time-course of this second phase was related to the initial concentrations of the reductant. Experiments performed at four different pH values demonstrated that the reduction was a function of the bulk solution pH. At the lower pH, after a fast initial reduction, the Fremy’s salt EPR signal remained constant, while at physiological or higher pH a further reduction was found.The reaction rates demonstrate that the reducing power of ascorbic acid towards Fremy’s salt strongly depends on its dissociation state.

Static magnetic field effect on the Fremy's salt-ascorbic acid chemical reaction studied by continuous-wave electron paramagnetic resonance.

Static magnetic field effect in the framework of the radial pair mechanism (RPM) theory was studied on the biologically significant chemical reaction between ascorbic acid and Fremys salt. The data indicate that the reaction rate depends on the applied magnetic field strength. The time scale of the studied reaction and the improved continuous-wave electron paramagnetic resonance system allowed for the first time the direct comparison of the amplitude differences between exposed and control samples in the strictly same boundary conditions. Until now the RPM was studied in a different time scale, focusing only on faster reactions by time-resolved techniques or by spectrophotometer measurement. The magnetic field effects presently measured can not be extended tout court to living systems; however the understanding of magnetic field sensitivity in basic chemical reaction in vitro could help clarifying the underlying basic step of interaction between magnetic fields and biological systems.

EPR study of the effect, induced by zidovudine (AZT), on the membrane lipid dynamics in leukemic cell

Zidovudine (AZT) was the first drug approved by the FDA for treatment of HIV infections. To investigate the AZT effects on the physical properties of K562 cell membranes, experiments were performed by measuring an order parameter value in these cells, previously labelled with 16-DSA (16-doxyl-stearic acid) or 5-DSA (5-doxyl-stearic acid). The EPR spectra of the labelled fatty acid were used to detect the alteration in their freedom of motion and to provide indications of membrane fluidity. Two different parameters were calculated from EPR spectra for the 16-DSA and 5-DSA. Preliminary data indicate that, for both the probes used, these parameters were not significantly different in the control cells with respect to the AZT-treated ones. Control measurements, performed to test the sensitivity of the technique using the DMSO agent, showed a significant increase in the K562 membrane fluidity.

First observation of very long-lasting emissive polarization in EPR spectra of Fremy's salt when reduced by ascorbic acid. An unusual aspect of the radical pair mechanism

Emissive EPR spectral lines were observed in experiments dedicated to investigating the efficiency of ascorbic acid in reducing Fremys salt. The emission was detected by CW spectroscopy and was exceptionally long lasting, being observed as long as 20 minutes. Fremys salt emission lines were observed at the end of the redox reaction. The Fremys salt absorption signal was totally quenched, and was replaced by an emission signal. This feature was observed under different experimental conditions. The inversion proceeded simultaneously for the three lines, which keep their relative intensities and hyperfine separation. The emission signal was also detected together with the normal absorption signal of ascorbyl free radical. No emission spectra were observed by using other nitroxides, or by using as reducing agent 1,4-dithio-DL-threitol instead of ascorbic acid. The observed spin polarization is accounted for on the basis of a Radical Pair Mechanism (RPM). The amount of polarization is related to the diffusion constant D, which decreases with increasing viscosity. By adding glycerol to the solution, enhanced polarisation was indeed observed, as expected, which confirms the proposed RPM polarisation mechanism. We report here for the first time, to our knowledge, the phenomenon of long-lasting emissive EPR lines.

EPR spectroscopy of tooth enamel: the tooth radicals and the microcrystal alignment

The paramagnetic radicals induced by radiation in dental enamel are very important because they can be related to the crystalline structure of hydroxyapatite. The R-value, that is, the ratio of the amplitude of the lines of the EPR signal due to radiation, is a measure of the degree of microcrystal alignment in human tooth enamel. The aim of this study is to underline the importance of a correct evaluation of the R-value, by using the current method to increase reproducibility in EPR spectroscopy of tooth enamel. Data with and without correction show a significant difference and, consequently, they give rise to a different valuation of microcrystal alignment.

A minimal cavity and its capacitive coupling for in vivo EPR measurements on mice.

A very simple ring resonator for in vivo L-band EPR spectroscopy was built and characterised. It employs a special capacitive coupling that allows measurents to be made on large biological samples which are not possible with other resonators. In spite of its intrinsic low Q it has a sensitivity almost equivalent to that obtained from high Q resonators. These features were tested down to a nitroxide concentration of 10 μM in high conductivity phantoms.