Dehydration and delayed proton equilibria of red blood cells suspended in isosmotic phosphate buffers. Implications for studies of sickled cells.

PO4 buffers isosmotic with plasma or phosphate-buffered saline solution with a substantial proportion of PO4 are often used to wash and suspend red blood cells in studies of respiratory or sickling behavior. Measurements of sequential changes in mean cell hemoglobin concentration, pH, and ion content of red blood cells suspended in 295 mOsm Na-phosphate, pH 7.4, at 23 degrees or 37 degrees C, showed (1) rapid, persistent cell dehydration (mean cell hemoglobin concentration greater than 40 gm/dl) caused initially by Cl- efflux and later by replacement of monovalent Cl- by divalent HPO=4; and (2) temporary reversal of membrane pH gradients with normalization time (30 to 120 minutes) dependent on factors controlling the rate of phosphate-chloride exchange. Sequential equilibration of red blood cells in isosmotic citrate (impermeable) followed by PO4 demonstrated the two stages of the observed shifts in PO4 alone, and red blood cells suspended in 0.15 mol/L 32PO4 at 37 degrees C showed PO4 influx consistent with pH equilibrium kinetics. Sickle trait red blood cells deoxygenated at 37 degrees C, pH 7.4, in plasma or 10 mmol/L HEPES-buffered saline solution showed only 6% to 20% sickling. In isosmotic PO4, mean cell hemoglobin concentration was 40 to 41 gm/dl with approximately 80% sickling. In phosphate-buffered saline solution containing 70 mmol/L PO4, red blood cells showed smaller, similar changes (mean cell hemoglobin concentration approximately 38 gm/dl) with a longer equilibration period and deoxygenated sickle trait cells showed 40% sickling. The altered properties of red blood cells suspended in PO4 or phosphate-buffered saline solution were neither intended nor appropriate for many studies using these media, particularly with hemoglobin S-containing red blood cells, and interpretations of reported results must be reassessed in light of these findings.