2H2O quadrupolar splitting used to measure water exchange in erythrocytes

The 2H NMR resonance from HDO (D=2H) in human red blood cells (RBCs) suspended in gelatin that was held stretched in a special apparatus was distinct from the two signals that were symmetrically arranged on either side of it, which were assigned to extracellular HDO. The large extracellular splitting is due to the interaction of the electric quadrupole moment of the 2H nuclei with the electric field gradient tensor of the stretched, partially aligned gelatin. Lack of resolved splitting of the intracellular resonance indicated greatly diminished or absent ordering of the HDO inside RBCs. The separate resonances enabled the application of a saturation transfer method to estimate the rate constants of transmembrane exchange of water in RBCs. However both the theory and the practical applications needed modifications because even in the absence of RBCs the HDO resonances were maximally suppressed when the saturating radio-frequency radiation was applied exactly at the central frequency between the two resonances of the quadrupolar HDO doublet. More statistically robust estimates of the exchange rate constants were obtained by applying two-dimensional exchange spectroscopy (2D EXSY), with back-transformation analysis. A monotonic dependence of the estimates of the efflux rate constants on the mixing time, tmix, used in the 2D EXSY experiment were seen. Extrapolation to tmix=0, gave an estimate of the efflux rate constant at 15 degrees C of 31.5+/-2.2 s(-1) while at 25 degrees C it was approximately 50 s(-1). These values are close to, but less than, those estimated by an NMR relaxation-enhancement method that uses Mn2+ doping of the extracellular medium. The basis for this difference is thought to include the high viscosity of the extracellular gel. At the abstract level of quantum mechanics we have used the quadrupolar Hamiltonian to provide chemical shift separation between signals from spin populations across cell membranes; this is the first time, to our knowledge, that this has been achieved.