Experimental demonstration of the effect of the unstirred water layer on the kinetic constants of the membrane transport of d-glucose in rabbit jejunum

The rate of active transport of a probe molecule into the intestinal mucosal cells is determined by the rate of movement of the solute molecule across two barriers, the unstirred water layer and the microvillus membrane of the epithelial cell. Previously a theoretical equation has been derived which described J_d, the velocity of unidirectional flux, as a function of the characteristics of the transport carrier in the membrane and of the resistance of the overlying unstirred water layer (UWL). The predictions of these equations have been tested experimentally by studying the effect of the rate of stirring of the bulk phase on the in vitro uptake of d-glucose by rabbit jejunum. These studies demonstrated that, first, alterations in the UWL have a profound effect on the magnitude of the apparent affinity constant, x K_m^*, of the active transport process. Second, at bulk phase concentrations in excess of the K_m the passive component of the experimentally determined flux rate becomes of such magnitude as to introduce significant error into the estimate of both the maximal transport rate, J_d^m, and the true K_m. Third, as a result of the UWL, the use of double-reciprocal plots to determine J_d^m and K_m leads to the overestimation of these constants. Finally, failure to account for the UWL leads to important quantitative errors describing a number of the characteristics of the transport process: these include an underestimation of the Q₁₀ and the effect of sodium ion on the active transport of glucose in the jejunum. The results confirm that the kinetic characteristics of the uptake of an actively transported molecule are a complex function of the resistance of both the UWL and the mucosal cell membrane, and this transport process can be adequately described by a newly-derived equation. It is apparent that there are serious limitations in the interpretation of much of the previously published data dealing with active transport processes in the intestine, since these studies failed to account for the effect of the UWL.