An improved non-radioisotopic method for measuring ouabain-sensitive Na + efflux from erythrocytes

Abstract

Ouabain-sensitive Nat efflux (Na+, KC ATPase activity) from erythrocytes is most commonly measured by monitoring the efflux of radioactive sodium from previously loaded cells [l-3), a technique which requires considerable in vitro manipulation of the erythrocytes and the handling of radioisotopes. Cumberbatch and Morgan [4] have proposed a method based on the assumption that the sodium fluxes of red cells in whole blood are in equilibrium. In their method, which does not require the use of labeled sodium, ouabain is added to the whole blood to block the ouabain-sensitive Na+ efflux. Na+ influx is not blocked; therefore, the intracellular Na+ continues to increase. Cumberbatch and Morgan reason that the rate of increase in intra~llular Na+ is equal. to the ouabain-sensitive Na+ efflux, an assumption that is valid only if other Na+ efflux pathways are insignificant. We describe a simple method for measuring ouabain-sensitive Na+ efflux which does not use radioactivity and which does not rely on assumptions about other Na+ transport mechanisms. Sodium efflux via the Naf pump is determined from the difference in efflux of intracellular Na+ into two media, one containing Kf to maximize pump activity, the other containing ouabain to inhibit pump activity.