Calcium-induced bilirubin-dependent hemolysis of human erythrocytes

Human erythrocytes, preincubated with different concentrations of calcium chloride (0.17-1.67 mM) showed hemolysis after addition of bilirubin (72 mu M). Hemolysis was observed only when cells were incubated first with calcium followed by bilirubin and not vice versa. This hemolysis was found to be dependent upon several factors such as concentration of bilirubin, time of incubation of erythrocytes with calcium and time of incubation of bilirubin with the calcium-loaded erythrocytes. Inclusion of EDTA in the incubation medium reduced the percentage hemolysis to a significant extent. Involvement of activated oxygen species in hemolytic process seems to be unlikely as inclusion of sodium azide and catalase did not prevent hemolysis. A comparison of other bivalent cations such as Ba2+, Mg2+, Mn2+ and Cu2+ with Ca2+ for their ability to hemolyse cells in presence of bilirubin shows that Ba2+ and Mg2+ are ineffective, whereas both Mn2+ and Cu2+ induce hemolysis both in the absence as well as in the presence of bilirubin. However, their mechanism of hemolysis is different from that of calcium-induced hemolysis. Formation of calcium-induced hydrophobic aggregates of phospholipid molecules in erythrocyte membrane may open the new binding sites for bilirubin on these membranes which may perturb the membrane conformation.