Use of nitroxides to measure redox metabolism in cells and tissues

Abstract

The metabolic and physical interactions of the paramagnetic nitroxides with cells and tissues are becoming effective tools for measuring biologically significant processes, including some not measured adequately by other means. We have exploited the physical (magnetic) interactions of oxygen with nitroxides to obtain the first reliable and facile measurements of intra-cellular oxygen, using the effects of oxygen on the relaxation times of nitroxides via Heisenberg exchange. With this technique we have demonstrated a significant gradient between extracellular and intracellular oxygen concentrations in our experimental cell suspension system. Previously, the existence of such a gradient has been debated hotly but not tested directly. We also have determined some of the principles of interactions between nitroxides and cells and have demonstrated the feasibility of using metabolic interactions of nitroxides with cells to measure hypoxia in vivo. The principal metabolism of nitroxides by cells is reversible reduction to the hydroxylamine. The rate of reduction depends on the physical characteristics of the nitroxides. Reduction occurs primarily in the intracellular compartment and therefore only nitroxides that can cross the cell membrane readily (e.g. small molecules that are lipid-soluble) can be reduced readily by cells. Once nitroxides get into cells, the rate of reduction depends on the structure of the nitroxide (e.g. those with the five-membered pyrrolidine ring reduce more slowly than those with the six-membered piperidine ring). For some nitroxides the rate of reduction is up to thirty times faster in severely hypoxic cells. This latter phenomenon makes feasible the use of nitroxides to detect hypoxic areas in vivo by using the nitroxides as ‘contrast agents’ for in vivo n.m.r. studies. In principle, regions of the body with hypoxic areas will have lower concentrations of nitroxides because in these regions the nitroxides become reduced to the non-paramagnetic hydroxylamines which do not affect n.m.r. images (paramagnetic molecules affect n.m.r. images by shortening the relaxation times of water protons – the relaxation times of water are the principal imaging parameters for most current in vivo n.m.r. techniques). Thus it appears feasible to use nitroxides to detect and follow processes in vivo that are associated with hypoxia; these include cancer, ischemia (i.e. drastically reduced blood flow) and inflammation.