Extra- and intracellular unstirred layer effects in measurements of CO2 diffusion across membranes--a novel approach applied to the mass spectrometric 18O technique for red blood cells.

We have developed an experimental approach that allows us to quantify unstirred layers around cells suspended in stirred solutions. This technique is applicable to all types of transport measurements and was applied here to the (18)O technique used to measure CO(2) permeability of red cells (PCO2). We measure PCO2 in well-stirred red cell (RBC) suspensions of various viscosities adjusted by adding different amounts of 60 kDa dextran. Plotting 1/PCO2 vs. viscosity nu gives a linear relation, which can be extrapolated to nu=0. Theoretical hydrodynamics predicts that extracellular unstirred layers vanish at zero viscosity when stirring is maintained, and thus this extrapolation gives us an estimate of the PCO2 free from extracellular unstirred layer artifacts. The extrapolated value is found to be 0.16 cm s(-1) instead of the experimental value in saline of 0.12 cm s(-1) (+30%). This effect corresponds to an unstirred layer thickness of 0.5 microm. In addition, we present a theoretical approach modelling the actual geometrical and physico-chemical conditions of (18)O exchange in our experiments. It confirms the role of an extracellular unstirred layer in the determination of PCO2. Also, it allows us to quantify the contribution of the so-called intracellular unstirred layer, which results from the fact that in these transport measurements--as in all such measurements in general--the intracellular space is not stirred. The apparent thickness of this intracellular unstirred layer is about 1/4-1/3 of the maximal intracellular diffusion distance, and correction for it results in a true PCO2 of the RBC membrane of 0.20 cm s(-1). Thus, the order of magnitude of this is PCO2 unaltered compared to our previous reports. Discussion of the available evidence in the light of these results confirms that CO(2) channels exist in red cell and other membranes, and that PCO2 of red cell membranes in the absence of these channels is quite low.