METABOLISM OF NITROXIDES BY LYMPHOCYTES AND MACROPHAGES

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Nitroxides are stable free radicals which are reduced and reoxidized (metabolized) by cells and organisms. Nitroxides have the potential to act as contrast agents for magnetic resonance imaging but before this potential can be realized, it is necessary to understand the factors which affect their metabolism. The concentration of oxygen is one such factor; there may be others. The overall aim of this proposal is to study the metabolism of nitroxides in lymphocytes and macrophages. These cells are found in the circulatory system where nitroxides would most likely be introduced for imaging studies. In addition, lymphocytes play an important role in the immune response and undergo dramatic physiological changes upon stimulation. These changes may be observed as changes in the metabolism of nitroxides. In such a case, nitroxides would be 'immunologically responsive' contrast agents. The work proposed here will use electron paramagnetic resonance (EPR) to study nitroxide metabolism. Cells will be maintained in a thermostated bath in which oxygen concentration and pH can be regulated. Cells from this bath will be pumped into the cavity of the EPR spectrometer where the nitroxide signal will be measured. Nitroxides and metabolic inhibitors can be added directly to the cells in the bath; sample of cells can be withdrawn for analysis and viability studies. The specific aims of this proposal are to determine rates of nitroxide metabolism in whole blood, isolated T and B lymphocytes and macrophages, and activated lymphocytes. The molecular mechanisms involved in nitroxide metabolism will be studied as well as the toxicity of the nitroxides used and the concentrations at which they inhibit lymphocyte activation. A variety of nitroxides which are uncharged, partly, or completely ionized, water- or lipid-soluble, and which have different ring structures (piperidine, pyrrolidine, and oxazolidine) will be used. Also, hydrocarbon nitroxides which may be less toxic than other lipid-soluble nitroxides will be synthesized.