I. A Study of Passive Potassium Efflux From Human Red Blood Cells Using Ion Specific Electrodes. II. Quantitation of Human Red Blood Cell Fixation by Glutaraldehyde

Citation

Morel, François M. M. (1972) I. A Study of Passive Potassium Efflux From Human Red Blood Cells Using Ion Specific Electrodes. II. Quantitation of Human Red Blood Cell Fixation by Glutaraldehyde. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/WGFY-R705. https://resolver.caltech.edu/CaltechETD:etd-12172004-160548

Abstract

I. Study of Passive Potassium Efflux From Human Red Blood Cells Using Ion Specific Electrodes. It is shown that, by using ion specific electrodes, the small potassium leakage induced by ouabain in human erythrocytes can be measured continuously and precisely. Upon addition of isotonic sucrose solution to a suspension of red cells in physiological saline the passive (ouabain-insensitive) potassium efflux is directly proportional to the chloride ratio. (i. e., an exponential function of the total membrane potential). The same result is obtained upon addition of hypertonic sucrose solution, suggesting that neither osmolarity nor intracellular concentrations have any influence on the passive potassium efflux. The independence of the potassium efflux and osmolarity can be verified by addition of glucose to the cell suspension. Glucose penetrates the red cells leaving the intracellular volume,and thus the membrane potential as well as the intracellular concentrations, unchanged. Adding water or hypertonic sodium chloride solution to red blood cell suspensions shows that the potassium efflux increases slightly in more concentrated salt solutions. Inasmuch as this effect can be interpreted as a pure ionic strength effect, the experiments corroborate the hypertonic sucrose solution experiments in demonstrating no dependence of the potassium efflux upon intracellular concentrations. The results of this investigation as well as other studies (LaCelle and Rothstein 1966, Donlon and Rothstein 1969, Cotterrell and Whittam 1971) show that the passive permeability of the human red blood cell to potassium depends uniquely on the membrane potential near physiological conditions, while it depends on parameters such as pH or concentrations for values of the membrane potential over 40mV. This suggests that two different mechanisms of transport might be involved: one would control the permeability under normal conditions; the other would represent a leak through the route normally used by anions and become important only under extreme conditions. II. Quantitation of Human Red Blood Cell Fixation by Glutaraldehyde. The uptake of glutaraldehyde by human red blood cells has been measured as a function of time by a freezing point osmometer. The rate of attachment of glutaraldehyde to the cell proteins is high over the first hour, declining to zero over a period of a few days. The number of glutaraldehyde molecules cross-linking with each hemoglobin molecule is of the order of 200, in reasonable agreement with the calculated number of attachment sites. The cell membrane is immediately highly permeable to glutaraldehyde. Selective permeability to ions is lost during fixation. Ionic equilibrium is obtained only after a few hours. An optimum fixation technique for shape preservation is suggested.

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