Hypertonic cryohemolysis of human red blood cells

Hypertonic cryohemolysis of human erythrocytes is caused by incubation of the cells in hypertonic medium at a temperature of 20--50 degrees C (stage 1), with subsequent cooling to 0 degrees C (stage 2). In 0.86 M sucrose hemolysis increases, with increasing stage 1 temperature, whereas in 1 M NaCl cryohemolysis has a temperature optimum at a stage 1 temperature of about 30 degrees C. Cryohmeolysis is inhibited by preceding ATP depletion of the cells and by preincubation of the cells in hypertonic medium at 0 degrees C. In general, anesthetics inhibit cryohemolysis strongly. Only in 1 M NaCl at stage 1 temperatures in the range of 40--50 degrees C is cryohemolysis stimulated by these drugs, if present during the entire incubation period. This effect is abolished, however, when the anesthetic is added after prior incubation of the cells at 40--50 degrees C in 1 M NaCl. Ghost-bound ANS fluorescence indicates complicated conformation changes in the membrane structure during the various experimental stages leading to cryohemolysis. Some of the experimental results can be considered as examples of molecular hysteresis, thus indicating several different metastable structures of the membrane, under various experimental conditions. The described results support the working hypothesis of Green and Jung that the experimental procedure results in membrane protein damage, preventing normal adaptation of the membrane during cooling.