We propose to develop methods to measure intracelluar oxygen concentrations and then to apply these methods to measure the effect of intracellular oxygen concentrations on physiological and pathological processes. Our initial efforts will be to complete development of the methodology to measure intracellular oxygen. Although detailed knowledge of the concentration of intracellular oxygen is of crucial importance in understanding many physiological and pathological processes, the actual intracellular concentration of oxygen has rarely been measured because of a lack of suitable methods. We have carried our preliminary studies that indicate that our approach is feasible and sensitive. These preliminary studies indicate that extracellular oxygen concentrations may not be parallel to concentrations of intracellular oxygen. Our approach is based on using electron spin resonance to measure the effects of oxygen on the relaxation of free radicals. We shall use two different methods: the effect of oxygen on the superhyperfine spectra of nitroxide spin labels and the effect of oxygen on the microwave power saturation of melanin. These methods are complimentary, and together provide a wide range of potential applications and a means to validate further the results obtained by each approach. The physiological processes and effects of environmental conditions to be studied in relation to intracellular oxygen and in comparison to oxygen concentrations in the extracellular compartment include: external oxygen concentrations; temperature; metabolism; cell anchorage; the cell cycle; and the carrying out of specialized functions of cells in tissues such as muscle and nerve. The pathological processes to be studied in relation to intracellular oxygen include: ionizing radiation; irradiation by visible light; UV irradiation; and lipid peroxidation.
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