DEAN B. COWIE AND RICHARD B. ROBERTS 5 



shows thai despite the variables introduced (cell volume, temperature, and 

 sulfate concentration) the water space determined by the radioactivity of 

 the supernatant fluid, is correlated with the volume of cells in each tube and 

 is independent of the other variables. 



There are several limitations on such a determination. When small volumes 

 of cells are used, the loss of radioactivity from the original medium is small 

 and the method is inaccurate. Even more important, however, is the fact that 

 this method measures the total uptake of materials by the cell. If metabolic 

 uptake occurs in the experiment, an erroneous estimate of water space will 

 be made since such bound material is not distinguished from that retained 

 passively in the water space of the cell. Since in the experiments shown in 

 table 3 the highest specitic radioactivity was used with the largest volume of 

 cells, any metabolic uptake would be emphasized. Therefore, the results shown 

 in table 3 indicate that little metabolic incorporation occurred. In addition, 

 the uniformity of the results obtained over the wide range of sulfur concen- 

 tration rules out any large amount of metabolic binding. Any sulfur meta- 

 bolically bound can, moreover, be detected by measuring the radioactivity 

 contained in the cells. For example, the pellets of cells in the experiment of 

 table 3 were rinsed with 20 ml of NaCl and then resuspended in 20 ml of 

 XaCl solution. After centrifuging, aliquots of the washed cells were measured. 

 Less than 1% of the sulfur initially taken up by the cells was retained showing 

 that these resting cells did not bind the sulfur. 



Measurement of the rinse solution obtained from this experiment showed 

 that the radioactivity lost in the rinse corresponded to a volume of approxi- 

 mately 0.1 ml of the original medium. The three rinse values obtained were 

 0.1, 0.08, and 0.09 ml. If the rinse procedure were effective in removing trapped 

 intercellular medium from the pellets, a correlation with cell volume would 

 be expected. Since no correlation was observed, it is concluded that the radio- 

 activity of the rinsing solution is due to residual medium adhering to the walls 

 of the centrifuge tube or to the surface of the pellet. 



Intercellular Fluid Volume. What has been defined as 'water space' might 

 be considered to be nothing more than medium trapped in the interstices 

 between the packed cells. This intercellular volume has been measured, using 

 Fe** and I''" labeled proteins as the tracer materials. Fe^^ was used to label 

 ferric-beta-i-globulinate (30) and F'" was used to label serum albumin. Cells 

 were suspended in media containing these tracer compounds of high molecular 

 weight and immediately centrifuged at 14,000 g. After centrifuging, the super- 

 natant was decanted, and the centrifuge tube and pellet were not rinsed; the 

 radioactivity retained in the tube corresponded to a water space of 20%. In 

 a parallel experiment rinsing removed half of this retained radioactivity. The 

 residual radioactivity thus corresponded to a water space of 10%. As the 

 previous experiments showed that the rinse loss corresponded mainly to the 



