Janusz Koscielniak

Absolute oxygen tension (pO2) in murine fatty and muscle tissue as determined by EPR

The absolute partial pressure of oxygen (pO2) in the mammary gland pad and femoral muscle of female mice was measured using EPR oximetry at 700 MHz. A small quantity of lithium phthalocyanine (LiPc) crystals was implanted in both mammary and femoral muscle tissue of female C3H mice. Subsequent EPR measurements were carried out 1–30 days after implantation with or without control of core body temperature. The pO2 values in the tissue became stable 2 weeks after implantation of LiPc crystals. The pO2 level was found to be higher in the femoral muscle than in the mammary tissue. However, the pO2 values showed a strong dependence on the core body temperature of the mice. The pO2 values were responsive to carbogen (95% O2, 5% CO2) breathing even 44–58 days after the implantation of LiPc. The LiPc linewidth was also sensitive to changes in the blood supply even 60 days after implantation of the crystals. This study further validates the use of LiPc crystals and EPR oximetry for long‐term non‐invasive assessment of pO2 levels in tissues, underscores the importance of maintaining normal body core temperature during the measurements, and demonstrates that mammary tissue functions at a lower pO2 level than muscle in female C3H mice. Magn Reson Med, 2005. Published 2005 Wiley‐Liss, Inc.

300 MHz continuous wave electron paramagnetic resonance spectrometer for small animal in vivo imaging

Design and construction of an electron paramagnetic resonance (EPR) spectrometer, operating in the continuous wave mode in the radio frequency (rf) region, and capable of performing spectroscopy and in vivo imaging of paramagnetic spin probes is described. A resonant frequency of 300 MHz was chosen to provide the required sensitivity at nontoxic levels of commonly used spin probes and penetration of the rf in small animals. Three major components, the magnet, the radio frequency signal detection bridge, and the data acquisition module are described in this article. Integration of a rapid scan capability to reduce imaging time is also described. Two- and three-dimensional EPR images of the spin probe distribution in phantom objects as well as from in vivo experiments are reported. From the EPR images, morphology of some internal organs could be recognized. EPR images of the spin probe distribution in mice suggest differences in perfusion of the spin probe between normal and tumor regions. Addition of a spe...

Biological Reduction of Aromatic Nitroso Compounds: Evidence for the Involvement of Superoxide Anions

The in vitro formation of phenylhydronitroxide and 2-methylphenylhydronitroxide free radicals from nitrosobenzene (NB) and 2-nitrosotoluene (NT), respectively, in either red blood cells (RBC) or RBC hemolysates, was confirmed by electron spin resonance spectroscopy (ESR). Free radicals were generated nonenzymatically from reaction of the respective nitroso compounds with a number of biological reducing agents as corroborated by model studies of NB or NT with NAD(P)H. Under aerobic conditions, phenylhydronitroxide and 2-methylphenylhydronitroxide underwent a subsequent one-electron transfer to oxygen, which then resulted in the formation of superoxide anion (O2-). The latter product was confirmed by the superoxide dismutase (SOD)-inhibitable reduction of cytochrome c (cyt c). Apparently, oxygen is needed for continuous formation of the hydronitroxide radical derivatives. On the other hand, under anaerobic conditions, no phenylhydronitroxide radical was generated from NB in the presence of NADH, but the formation of phenylhydroxylamine from NB was detected by the absorption spectrometry. These results suggest that oxygen is a preferential electron acceptor for hydronitroxide radical derivatives.

DUAL DIODE DETECTOR FOR HOMODYNE EPR MICROWAVE BRIDGES

After low noise FET amplifiers found widespread use in both X‐band and low‐frequency systems, it became possible to use multidiode mixers for signal detection. Here, a novel dual diode mixer is described for use in homodyne microwave bridges for both cw and pulsed electron paramagnetic resonance spectrometers. The structure is composed of a 90° hybrid coupler and separate detectors in two arms. The device provides common mode rejection of noise‐like regular balanced mixers but, by making diode dc currents accessible, offers big advantages for tuning of the bridge when low Q sample probes are used. Although the use of this detector does not affect sensitivity directly, tuning precision and increased long‐term stability leads to unexpected improvements.

In vivo ESR observation of bioradical metabolites in living animals

L-band ESR spectroscopy with a loop-gap resonator demonstrated the first in vivo detection of a “bioradical”, generated from the metabolism of nitrosobenzene in live mice, A broad three-line ESR spectrum (aN = 11.6 G) was detected in the buttocks or stomach region of a mouse after intramuscular or intraperitoneal injection of 0.2 mmol/kg nitrosobenzene. The signal intensity reached a maximum at 20 to 30 min and remained constant well beyond twelve hours. When muscle tissue was doped with nitrosobenzene and excised within 5 min, a similar three-line spectrum was also detected at X-band, which was preceded by the rapid growth and subsequent decay of a spectrum identical to that of the phenylhydronitroxide radical. A model system containing nitrosobenzene and unsaturated fatty acids yielded an identical three-line spectrum which came from the radical adduct of nitrosobenzene across double bonds. These results suggest that one of the first possible targets of nitroso compounds in vivo may be regions of polyunsaturated fatty acid clusters in fat or membranes. From the success in detecting “bioradicals” generated by pollutants in vivo, two- and three-dimensional visualization of localized bioradicals seems quite feasible.