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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 Discipline: Biophysics Degree Granting Institution: University of Illinois at Urbana-Champaign Degree: Ph.D. Genre: Dissertation 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 Type: Text Description: 129 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1993. URI: http://hdl.handle.net/2142/72407 Other Identifier(s): (UMI)AAI9329063 Date Available in IDEALS: 2014-12-17 Date Deposited: 1993
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