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Title:Development of EPR Methods for Simultaneous Measurements of Intra- and Extra-Cellular Oxygen Concentration in Vitro
Author(s):Hu, Huping
Doctoral Committee Chair(s):Swartz, Harold M.
Department / Program:Center for Biophysics and Computational Biology
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(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.
Issue Date:1993
Description:129 p.
Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1993.
Other Identifier(s):(UMI)AAI9329063
Date Available in IDEALS:2014-12-17
Date Deposited:1993

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