Characterization of the erythrocyte sodium-lithium countertransporter: limitations and assumptions of traditional and kinetic methodologies.

Abstract

The present work examined the key elements featuring in the various methods used to characterize the erythrocyte sodium-lithium countertransport. Effects of medium composition on lithium efflux were investigated in 20 subjects. Mean lithium efflux (mmol Li/l RBC.h) into a 150 mM sodium medium was significantly higher than efflux into a revised sodium-rich medium (149 mM) containing 1 mM Mg (0.335 +/- 0.100 vs. 0.298 +/- 0.085 respectively; P < 0.03). Mean lithium efflux into sodium-free media where sodium had been entirely replaced by magnesium, was significantly lower than efflux into a choline-based medium containing only 1 mM magnesium (0.088 +/- 0.027 vs. 0.109 +/- 0.034 respectively; P = 0.03). Sodium-lithium countertransport activity and the transporter's kinetic profile were measured simultaneously in 35 subjects using traditional choline-based and kinetic methodologies. There was a significant correlation between countertransport activity and maximal rate of turnover (Vmax) (r = 0.62; P < 0.001); Vmax values were consistently greater than their corresponding countertransport activities (P < 0.001). On subdividing the subject group into tertiles based on the Michaelis-Menten constant (km) values (mM), < 75, 75-150 and > 150, the slopes of the regression lines for each group diminished progressively (0.64, 0.49 and 0.23 respectively), correlations within each group remained significant (P < 0.001, P < 0.001 and P < 0.02). No significant correlation was found between km values and countertransport activity (r = 0.035; P = NS). Increasing the number of points representing sodium concentrations within the range 0-150 mM, improved the confidence in the emerging estimates of Vmax and km obtained by linear transformation. Comparison of kinetic data derived using four different analytical methods (two linear transformations, a nonlinear regression and a statistical method), showed no significant differences between the estimates yielded for either Vmax (P = 0.88. NS) or km (P = 0.92, NS). This study has highlighted the critical roles of assay conditions and derivation techniques used when measuring sodium-lithium countertransport, emphasizing the need for standardization of the methodology.