Factors affecting the release of haemoglobin and enzymes from human erythrocytes

The efflux of lactate dehydrogenase and haemoglobin from human erythrocytes during prolonged incubation at 37 degrees was significantly reduced by ATP, ADP, AMP, UTP, creatine phosphate, or phosphoenolpyruvate and to a lesser extent by fructose, glucose 6-phosphate or fructose 6-phosphate, but not by glucose. Iodoacetate, however, markedly increased the loss of haemoglobin and slightly increased that of lactate dehydrogenase. Phospholipase C greatly accelerated the relase of haemoglobin, lactate dehydrogenase, pyruvate kinase, hexokinase, glucose 6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase from human erythrocytes, but this effect was also reduced in the presence of ATP or ADP. The loss of lactate dehydrogenase, malate dehydrogenase, and pyruvate kinase from the cells treated with phospholipase C increased as their ATP content fell. In a series of experiments in which the action of phospholipase C was stopped by the subsequent addition of trypsin, ATP and ADP (1 mmol/l) significantly reduced the efflux of haemoglobin, but AMP had no such effect. The results are consistent with the conclusion from our previous work that enzyme leakage is related to diminution in the energy content of the cells. The protective action of AMP on cells not treated with phospholipase C, however, differs from earlier findings with rat lymphocytes and it is suggested that in red cells it might be converted into ATP or that it has a direct effect on the permeability of the cell membrane.