Osmotic equilibria in human erythrocytes studied by immersion refractometry

Abstract

New data are presented on volume-osmotic pressure relationships at equilibrium in human erythrocytes. The inadequacy of previous theoretical treatments of the subject is pointed out and a new theoretical approach is presented. The refractive index of the erythrocyte gives a direct measure of its solid concentration, and the volume is estimated as the reciprocal of concentration. The erythrocyte volume was found to be linearly related to the reciprocal of the osmotic pressure over a range from 300 m-osm. down to a lower limit of between 130 and 200 m-osm. (depending on the pH of the immersion medium). The gradient of the regression line was not, however, that expected from the simple Boyle-van't Hoff law. The isotonic water content of the erythrocyte derived from the gradient on the basis of the simple Boyle-van't Hoff Law was found to be less than the actual water content obtained by direct refractometry, i.e. the value of Ponder's $\germ{R}$ was found to average 0.95. Below a point between 130 and 200 m-osm. it was found that the erythrocyte volume did not increase in proportion to the fall of osmotic pressure. This was attributed to incipient haemolysis. The isotonic haemoglobin concentration of erythrocytes was found to vary from 38.5% at pH 6.9 to 30.3% at pH 5.6 in the same subject. The thermodynamic basis of the Boyle-van't Hoff law is examined. It is explained that a cell will obey the simple Boyle-van't Hoff law only if the osmotic coefficients of all fractions of the cell solute do not change significantly within the range of intracellular concentrations produced in osmotic experiments. From the fact that the osmotic coefficient of haemoglobin changes rapidly with concentration, it is concluded that the erythrocyte will not obey the simple Boyle-van't Hoff law; swelling in hypotonic solutions will be less than that predicted from the law. The degree of the discrepancy from the Boyle-van't Hoff law is calculated from known osmotic coefficients of haemoglobin, and is shown to result in an expected value of Ponder's $\germ{R}$ less than 1.0, and ranging from 0.925 to 0.975 depending on the corpuscular haemoglobin concentration. The observed values of $\germ{R}$ in different experiments are compared with the expected values and agreement is found within the limits of error of the experimental method.