Development of EPR Methods for Simultaneous Measurements of Intra- and Extra-Cellular Oxygen Concentration in Vitro
Hu, Huping
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https://hdl.handle.net/2142/72407
Description
Title
Development of EPR Methods for Simultaneous Measurements of Intra- and Extra-Cellular Oxygen Concentration in Vitro
Author(s)
Hu, Huping
Issue Date
1993
Doctoral Committee Chair(s)
Swartz, Harold M.
Department of Study
Center for Biophysics and Computational Biology
Discipline
Biophysics
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Animal Physiology
Biophysics, Medical
Abstract
In this thesis, the electron paramagnetic resonance (EPR) methods for simultaneous measurement of intra- and extracellular concentration of oxygen ( (O$\sb2$)) in viable cells have been developed.
In the presence of Fe(CN)$\sb6\sp{-3}$ in the medium, intracellular (O$\sb2$) is measured by a neutral $\sp $N-nitroxide and extracellular (O$\sb2$) is measured by a negatively charged $\sp $N, since charged species do not enter cells and the EPR spectrum of a $\sp $N-nitroxide does not overlap with that of a $\sp $N-nitroxide. The method is based in part on the minimal broadening of negatively charged nitroxides by Fe(CN)$\sb6\sp{-3}$ and the very effective broadening of neutral nitroxides by the same paramagnetic ions. Results with this method confirm the existence of gradients in (O$\sb2$) between the extracellular and intracellular compartment in CHO cells and M5076 tumor cells, even without stimulation of cellular resiration by carbonyl cyanide m-chlorophenylhydrazone (CCCP). The nature of the barrier that needs to be involved to account for the experimental results raises some significant questions.
Due to the potential toxic effect of Fe(CN)$\sb6\sp{-3}$ to cells after long time exposure and the difficulties to extend the measurement of intracellular (O$\sb2$) to in vivo in the presence of Fe(CN)$\sb6\sp{-3},$ a new method without the use of Fe(CN)$\sb6\sp{-3}$ was also studied. The study has been focused on the feasibility of developing nitroxides that localize selectively in the intracellular compartment so that the intracellular (O$\sb2$) could be measured without the use of Fe(CN)$\sb6\sp{-3}$ and extracellular (O$\sb2$) could be measured simultaneously by a charged $\sp $N-nitroxide. It is based on the use of readily hydrolysed ester linkages, such that the nitroxides become converted intracellularly to ionic derivatives that do not cross cell membranes readily. Up to 120-fold increased concentrations of intracellular nitroxides and their one electron reduction product, the hydroxylamines, were obtained. The EPR spectra of the intracellular nitroxides and their partitions into water and CHCl$\sb3$ were consistent with their conversion to the ionic species. Further studies indicate that these nitroxides have the properties needed for their use as probes of intracellular (O$\sb2$) and that it should be feasible to synthesize nitroxides that will be even more effective for this purpose.
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