Measurement of the distribution of anion exchange function in normal human red cells

1. The aim of the present work was to investigate cell-to-cell variation in anion exchange turnover in normal human red cells. Red cells permeabilized to protons and K⁺ dehydrate extremely rapidly by processes that are rate-limited by the induced K⁺ permeability or by anion exchange turnover. Conditions were designed to render dehydration rate-limited by anion exchange turnover. Cell-to-cell variation in anion exchange function could then be measured from the distribution of delay times required for dehydrating cells to attain resistance to haemolysis in a selected hypotonic medium. 2. Red cells were suspended at 10% haematocrit in a low-K⁺ solution and, after a brief pre-incubation with 20 μM SITS at 4°C, were warmed to 24°C, and the protonophore CCCP was added (20 μM) followed 2 min later by valinomycin (60 μM). Delay times for cells to become resistant to lysis were measured from the instant of valinomycin addition by sampling suspension aliquots into thirty volumes of 35 mM NaCl. After centrifugation the per cent lysis was estimated by measuring the haemoglobin concentration in the supernatant. Typical median delay times with this standardized method were 4-5 min. 3. The statistical parameters of the delay time distributions report the population spread in the transport function that was limiting to dehydration. In the absence of SITS and CCCP, dehydration was limited by the diffusional Cl^- permeability (P(cl)). Delay time distributions for P(cl)- and anion exchange-limited dehydration were measured in red cells from three normal donors. For both distributions, the coefficients of variation ranged between 13.0 and 15.2%, indicating a high degree of uniformity in P(cl) and anion exchange function among individual red cells.