Membrane structure as revealed by 

 permeability studies 



by 



HANS H. USSING 



Zpqfysiologisk Laboratorium, Kobenhavns Universitet 



It may be appropriate to take as the starting-point for the present discussion some 

 experiments performed by Hevesy, Hofer and Krogh (1935) some twenty years ago. 

 At that time Krogh was engaged in a study of the osmotic regulation of aquatic 

 animals, and when heavy water (D 2 0) became available it occurred to him that 

 isotopic water might provide a valuable tool for characterizing the osmotic properties 

 of biological membranes. In order to check the reliability of the new tool he wanted 

 to perform determinations of the permeability of a living membrane to water with 

 D 2 and, simultaneously, with the classical method of osmosis. As test material he 

 chose frogs with a rubber bag sewn onto the cloaca to collect the urine formed 

 during the experiment. The animals were submerged in tap water containing a 

 suitable concentration of heavy water. Owing to the difference of osmotic pressure 

 between the bathing solution and body fluids, water would be taken up osmotically. 

 The uptake could be measured as the increase in weight of the frog plus the rubber 

 bag. At the same time heavy water would exchange through the skin with ordinary 

 water, as indicated by a drop in the deuterium concentration of the bathing solution. 



In order to relate the exchange of heavy water to the net rate of uptake the authors 

 made the very plausible assumption that the net uptake of water is equal to the 

 difference between the amount of water diffusing in and that diffusing out. 



The unidirectional diffusion of water may be taken, as a first approximation, to be 

 proportional to the water concentration in the phase from which the diffusion takes 

 place. If the bathing solution is pure water, its concentration is 55 5 moles per litre. 

 The body fluid, however, which is some 0*2 osmolar with respect to solutes, is 

 accordingly 55 3 molar with respect to water. Thus, for every 55 5 moles diffusing 

 m > 55 '3 moles will diffuse out, resulting in a net uptake of o - 2 moles. 



The experimental results were not in agreement with these assumptions, however. 

 The net uptake of water was between three and five times higher than the theoretical 

 value calculated from the heavy-water flux and the difference in osmotic pressure 

 across the skin. The authors concluded that until more became known, diffusion of 

 heavy water could not be used to calculate rates of osmotic uptake. 



In 1944 Visscher et al. made a study of the water movements between gut and blood 

 of the dog, determining both the net water transfer and the rate of D a O diffusion. 

 Their theoretical assumptions were essentially the same as those of Hevesy et al. (I.e.), 

 except that Visscher and collaborators assumed the rate of diffusion of water to be 



33 



