38 



ELECTROLYTES IN BIOLOGICAL SYSTEMS 



samples as they are analyzed is due to contamination by the sea water which 

 might be in the interstitial spaces. To this end several experiments were carried 

 out to determine the extracellular space in the tissue under the conditions 

 employed in preparation of the samples for analysis. 



In one set of experiments samples were placed in sea water containing i % 

 inulin for several hours,^ then transferred to exactly loo ml of fresh sea water. 

 Immediately before transfer some of the samples were dipped very briefly 

 (i-2 sec.) into fresh sea water to remove adhering inulin solution; the other 

 samples were rinsed for 30 seconds in fresh sea water, then blotted three times 

 in absorbent tissue (same procedure used in preparation for sodium and potas- 

 sium analyses) and then transferred. 'Inulin space' was measured under these 

 conditions by determination of the inulin in the final solution a few hours after 

 transfer. At the end of an experiment the samples were removed, blotted and 

 weighed. Results of a typical experiment, indicating that the combination of 



Table i. Effects of rinsing and blotting on inulin and sucrose 'spaces' in ulva 



I- to 3-Sec. 

 Rinse, No Blot 



30-Sec. Rinse, 

 Triple Blot 



I- to 2-Sec. Rinse, 

 No Blot 



I- to 2-Sec. Rinse, 

 Triple Blot 



Inulin space 

 Sucrose space 



A . Rinsing and Blotting 

 19-21% 



20-21% 



^1% 

 c^i% 



B. Blotting 



20-22% 

 20-21% 



2-3% 

 2-3% 



the 30-second rinse plus the triple-blot are sufificient to reduce the inulin space 

 to almost zero, are presented in table lA. 



In the same manner sucrose was used as an indicator of extracellular space. 

 Representative data, which agree very well with the inulin values, are also 

 shown in table lA. 



To verify these results other experiments were designed to determine the 

 effects of rinsing procedure and the blotting technic alone on the extracellular 

 space. Samples were soaked in inulin-sea water as before and rinsed for i to 2 

 seconds in fresh sea water. One set was then transferred directly to fresh sea 

 water, while the other was first blotted according to the standard procedure. 

 Typical results are shown in table iB, where it is seen that blotting alone re- 

 duces the extracellular space from about 20% to about 2 to 3%. A similar 

 experiment was done using sucrose as the indicator of extracellular space. 



Finally sodium and potassium determinations were run on samples removed 

 from sea water under the following conditions: all samples were rinsed for 2 to 



* The time course for diffusion of inulin into the tissue indicates that equilibrium was 

 established within 1-2 hours. 



