The effects of the extracellular manganese concentration and variation of the interpulse delay time in the CPMG sequence on water exchange time across erythrocyte membranes

There has been broad disagreement in the literature regarding the dependence of water exchange times (T_e) across erythrocyte membranes studied by the ¹H-NMR Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence on extracellular Mn²⁺ concentration. While some workers saw no change in T_e with Mn²⁺, others reported a 35-50% decrease in T_e with this extracellular paramagnetic relaxation agent. We present ¹H-NMR evidence that a 30-50% change in T_e can be produced by interdependence of the interpulse delay time of the CPMG pulse sequence and the external Mn²⁺ concentration. Such a large dependency is interpreted in terms of the diffusional effect as a major source. However, it is shown experimentally that if a large number of refocusing π pulses are used, the observed transverse relaxation times are unaffected by Mn²⁺. Under these conditions excellent agreement of T_e obtained in our study (13.0 ± 0.64 ms (N = 36) at 21°C) and that of 12.8 ± 3.6 ms at 20-23°C reported by the radiotracer method was found. Our findings suggest new and important implications for evaluating the previous reports of the ¹H-NMR CPMG method concerning the [Mn²⁺] effect in the decrease of T_e, and provide conditions where studies of water transport across erythrocyte membranes using this magnetic resonance method can be used with confidence.