Studies on human uterine cervix and rat uterus using S-, X- and Q-band electron-spin-resonance spectroscopy.

In previous studies we have reported on the detection of a strong e.s.r. signal in samples of normal human cervix; the signal is much reduced or absent in samples of invasive cancer of the cervix. In order to identify the species responsible for the strong signal, we have used X-, S- and Q-band e.s.r. spectroscopy. The major signal that is detectable in ground-up samples of cervix preserved at -196 degrees C has features consistent with the presence of a peroxy free radical. Good agreement with the experimental findings was obtained by computer simulation, using values for the g-tensor of gx = 2.002, gy = 2.005 and gz = 2.036. The peroxy radical is produced on grinding the normal cervix samples to a powder under liquid N2, and appears to be formed by modification of a pre-existing oxygen-containing complex. Control experiments eliminated the possibility that the strong signals seen in frozen powders prepared from normal cervix were artefacts only of the grinding procedure. Experiments with rats in vivo and with cervix samples in vitro are consistent with the conclusion that the peroxy radical is formed by disturbing the cyclo-oxygenase system that is involved in prostaglandin synthesis.     

Rapid assessment of liposomal stability in blood by an aqueous nitroxide spin label

An electron spin resonance method using an aqueous nitroxide spin label, 2,2,6,-tetramethyl-piperidine-N-oxyl-4-trimethyl-ammonium, for rapid assessment of liposome stability in blood is presented. The retention of the nitroxide in liposomes is measured by its electron spin resonance signal intensity, a procedure which does not require separation of the sample from the blood. Any nitroxide that is released from the liposomes is reduced by external ascorbic acid which is added to the sample. The method permits kinetic studies on the integrity of liposomes in the presence of destabilizing factors such as detergent, blood, or alteration in temperature.     

Oxygen gradients in CHO cells: measurement and characterization by electron spin resonance

The concentration of oxygen within cells is important in many physiological and pathological processes, but such oxygen-dependent processes are generally studied as a function of the concentration of extracellular oxygen, due to a lack of suitable methods. Using a newly developed technique based on ESR spectroscopy, we show that respiration stimulation of a cell suspension can result in a significant difference between average intracellular and extracellular concentrations of oxygen. These results indicate that studies of oxygen-dependent phenomena in cells may require measurement of intracellular oxygen concentrations and imply that there are mechanisms in cells that restrict the free diffusion of oxygen.     

EPR method for the measurement of cellular sulfhydryl groups.

An EPR method that can measure the concentration of sulfhydryl groups in intact cells has been developed using a specially designed stable nitroxyl biradical. The biradical, RS-SR, contains a disulfide bond and readily undergoes thiol-disulfide exchange reactions with thiols resulting in a characteristic EPR spectrum which can be analyzed to provide a quantitative measure of sulfhydryl groups. The data obtained from the EPR method are in good agreement with those obtained from the conventional optical method using Ellman's reagent. The advantages of the EPR method are that the measurement can be carried out on intact cells or any other highly colored, absorbing and/or scattering solutions and the sensitivity is such that only a few cells (approximately 100) are needed for each quantitative measurement.