Hemolysis of sheep erythrocytes with the cell membrane of liquid paraffin-induced guinea pig macrophages.

As reported previously, the lysate of liquid paraffin-induced guinea pig peritoneal macrophages contains a hemolytic factor which is composed of two components: the soluble (S) and membrane-bound (M) components. To investigate the mechanism whereby the factor hemolysis sheep erythrocytes, an attempt was made to identify the S and M components. The fractionation of the cytosol of macrophages by DEAE-cellulose chromatography and the failure of the lysate from L-ascorbate-depleted macrophages to lyse erythrocytes demonstrated that the S component was L-ascorbate. In addition, L-ascorbate was found to be replaced by NADPH, a substrate of the membrane-bound NADPH oxidase, showing that L-ascorbate acts as a donor of active oxygen. When L-ascorbate was combined with the phospholipids isolated from the membrane fraction by extraction with chloroform-methanol and thin layer chromatography, it became able to lyse erythrocytes. The results so far obtained indicate that the hemolysis by the macrophage lysate is dependent on the formation of peroxidized phospholipids in the membrane fraction with certain active oxygen species produced either from L-ascorbate or by the NADPH oxidase.