Electron-spin resonance oximetry in cellular systems and in vivo
Glockner, James Frederick
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https://hdl.handle.net/2142/23806
Description
Title
Electron-spin resonance oximetry in cellular systems and in vivo
Author(s)
Glockner, James Frederick
Issue Date
1991
Doctoral Committee Chair(s)
Swartz, Harold M.
Department of Study
Biophysics, General
Discipline
Biophysics, General
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biophysics, General
Language
eng
Abstract
In this thesis, techniques of electron spin resonance (ESR) were developed and applied to measurement of oxygen concentrations in cellular suspensions as well as in vivo systems.
ESR oximetry was used to measure extracellular and intracellular oxygen concentrations in suspensions of CHO cells. No difference was noted between the extracellular and intracellular values of cells in their baseline state. However, when cellular respiration was stimulated with an uncoupler of oxidative phosphorylation, significant oxygen gradients developed, and these gradients increased as the cellular oxygen consumption rate increased. Attempts to fit these data to standard models of oxygen diffusion and consumption were largely unsuccessful, even when oxygen diffusion barriers at the plasma membrane were invoked.
A technique for measuring the concentration of extracellular oxygen using liposome-encapsulated nitroxides was developed, and it is shown that this method yields results which are similar to those obtained using other well established ESR oximetric techniques. The liposome system was used to measure extracellular oxygen in environments where free nitroxides are rapidly reduced, and it was then applied to measure intracellular and extracellular oxygen concentrations simultaneously in the same sample. The simultaneous technique was applied to suspensions of rapidly respiring myoblast cells, and significant oxygen gradients are demonstrated in unstimulated cells.
In vivo oxygen concentration measurements can be performed with a low frequency (1.1 GHz) ESR spectrometer and surface probe. Liposomes containing the oxygen-sensitive nitroxide d-Cat$\sb1$ were injected into mouse skeletal muscle, and changes in the local oxygen tension were measured as the fraction of inspired oxygen was varied.
Finally, two novel ESR oxygen probes, fusinite and lithium phthaloryanine, were characterized and then applied to the measurement of in vivo skeletal muscle oxygen tensions. Again, the fraction of inspired oxygen was varied, and changes in tissue pO$\sb2$ noted. The results of the three in vivo methods were compared, and issues of accuracy, calibration, and oxygen sensitivity discussed.
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