Electron Spin Resonance Measurements and Studies of Intracellular Oxygen Concentrations and Nitroxide Metabolism
Pals, Mark Alan
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https://hdl.handle.net/2142/70719
Description
Title
Electron Spin Resonance Measurements and Studies of Intracellular Oxygen Concentrations and Nitroxide Metabolism
Author(s)
Pals, Mark Alan
Issue Date
1988
Doctoral Committee Chair(s)
Swartz, Harold M.
Department of Study
Physiology and Biophysics
Discipline
Biophysics
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biophysics, General
Abstract
The concentration of molecular oxygen within cells plays a crucial role in many physiological and pathological processes. There are, however, very few data on actual intracellular oxygen concentrations, due largely to the lack of a suitable measurement technique. We have employed the techniques of electron spin resonance (ESR) to measure intracellular oxygen concentrations.
The method requires a membrane permeant nitroxide spin label and a membrane impermeant paramagnetic broadening agent. When added to a cell suspension, the nitroxide distributes throughout the suspension, but the broadening agent remains extracellular. Spin exchange between the paramagnetic ion and the nitroxide broadens the ESR signal from extracellular nitroxide, allowing the selective visualization of the signal from intracellular label. Molecular oxygen, being paramagnetic, broadens ESR spectral lines via Heisenberg spin exchange. The oxygen-induced line broadening is proportional to the oxygen concentration and can be calibrated as a function of that concentration. The measurement of the ESR linewidth of nitroxides located within actively respiring cells was shown to allow the routine measurement and study of intracellular oxygen concentrations without affecting cell viability.
We have developed an analogous methodology to measure extracellular oxygen concentrations. This method employs a membrane impermeant nitroxide and no added broadening agent. Since the nitroxide remains extracellular, its ESR spectrum reflects the extracellular oxygen concentration.
Using these ESR methodologies, we studied the intracellular and extracellular oxygen concentrations in suspensions of actively respiring mammalian cells. In the absence of respiratory stimulation, the measured values of intracellular and extracellular oxygen concentrations did not differ to a statistically significant level. When oxygen utilization was maximally stimulated by uncoupling electron transport, however, the measured extracellular concentration exceeds the intracellular value by approximately 20 $\mu$M. These experimental results were shown to be consistent with diffusion theory.
The technique was also applied to the study of nitroxide reduction as a function of oxygen concentration. The rate of nitroxide reduction increases markedly as the concentration in the system is decreased. These data suggest that nitroxides might serve well as contrast agents for nuclear magnetic resonance imaging, yielding enhanced images of hypoxic regions.
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