Urea exchange across the human erythrocyte membrane measured using ¹³C NMR lineshape analysis

Abstract

The ¹³C NMR spectrum of ¹³C-urea in a suspension of human red cells of reduced mean cell volume was observed to contain partially resolved resonances arising from the intra- and extracellular populations of the compound. It was shown that at 25°C and a magnetic field strength of 9.4 T, the rate of exchange of urea between the intra- and extracellular populations was such that the NMR lineshape was sensitive to a change in the rate of ¹³C-urea exchange, induced either by the addition of the urea transport inhibitor phloretin, or by the addition of ¹²C-urea. Total lineshape analysis of ^r3C NMR spectra of ¹³C-urea in red cell suspensions containing different concentrations of ¹²C-urea resulted in a weighted mean estimate for the K_m and V_max for urea equilibrium exchange from three experiments of 44 ± 18 mM and 3.1 ± 0.6 × 10⁻⁸ molcm⁻² s⁻¹, respectively (the errors denote the weighted mean standard deviations). These estimates of K_m and V_max, were significantly lower than previous values reported in the literature and determined using other techniques.