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A BST R A CT The hematocrit method as a technique for determining red cell volume under anisotonic conditions has been reexamined and has been shown, with appropriate corrections for trapped plasma, to provide a true measure of cell volume. Cell volume changes in response to equilibration in anisotonic media were found to be much less than those predicted for an ideal osmometer; this anomalous behavior cannot be explained by solute leakage or by the changing osmotic coefficient of hemoglobin, but is quantitatively accounted for by the hypothesis that 20 per cent of intracellular water is bound to hemoglobin and is unavailable for participation in osmotic shifts.When the chemical potential of the water surrounding a red cell is altered, the cell responds by a shift in volume. The magnitude of this shift provides important clues concerning the state of solvent and solute within the ceil. Dick and Lowenstein (1) and Hendry (2) have challenged the long standing observation (3) that a portion of the water of red cells does not appear to take part in these osmotic responses. We have therefore reexamined the hematocrit method, on which most of the earlier work had been based, as a method for measuring cell volume. Our results show that this method provides an accurate estimate of cell volume, even under anisotonic conditions, so long as suitable corrections are made for the medium trapped in the cell column. We have also made new measurements of the osmotic behavior of red cells. The change in red cell water content is less than expected on an ideal basis, an observation we have attributed to the layer of water of hydration surrounding the protein, called "bound water" by Perutz (4), rather than to changes in the osmotic coefficient of hemoglobin with protein concentration as suggested by Maizels and McConaghey (5) and others. I. EVALUATION OF THE HEMATOCRIT METHODIn order to evaluate the hematocrit method, it is necessary to compare the results obtained by this technique with volumes determined by an independent procedure. Isotope dilution has been chosen for this purpose, a method which depends only on volume measurements of the total suspension and the medium and therefore is not influenced by the means of separating 79

A new technique to determine the rate of water passage through the membrane of the human erythrocyte under an osmotic gradient has been developed. It utilizes a rapid mixing apparatus of the Hartridge-Roughton type whichpermits measurements at short intervals after the reaction has begun. This is coupled with a light-scattering device of new design which permits the determination of very small changes in volume of the cells without disturbing them. With this technique it was possible to measure the change in volume of freshly drawn human erythrocytes after about 50, 100, 155, and 215 msec. of exposure to anisotonic media. The experimental curves were compared with theoretical curves derived from accepted equations for the process and a permeability coei~cient of 0.23 ~ 0.03 (cm.4/osm., sec.) was obtained.The present study has been undertaken to measure the rate of water entrance into human red cells under an osmotic pressure gradient. This rate has been measured previously by Jacobs (1) for red cells of the ox and man using the technique of hemolysis time measurement. Recently, however, Jacobs (2) has criticized these earlier measurements on the grounds that delays in rupture of the cell lead to permeability constant values that are too low. The present measurements have been made by a different method, which depends on volume changes smaller than those which produce hemolysis. A modification of the flow method of Hartridge and Roughton (3) permits observation of cell size at intervals of 47, 99, 155, and 216 milliseconds after the cells have been exposed to a variety of anisotonic media; the cell volume in the flowing solution is measured by the intensity of 90 ° scattered light, a modification of the method used by 0rskov (4) and Parpart (5). Equations developed by Jacobs (6) have been used to determine the rate of water entrance into human red cells from the measured cell swelling curves. EquipmentWhole blood was mixed with one of four salt solutions in a mixing chamber as shown schematically in Fig. 1. Gas (5 per cent COr-95 per cent air) under a pressure of