COIL PLANET CENTRIFUGATION, AN OBSERVATION ON SEEPAGE OF CYTOSOL SUBSTANCES FROM ERYTHROCYTES UNDER CONTINUOUSLY DECREASING OSMOTIC PRESSURE*
Coil planet centrifugation proviedes us with a new measure particularly suitable for observation of sequential alteration of the membrane of erythrocytes when they are exposed to a stress produced by a rectilinearly decreasing osmotic pressure of salt solution at a constant speed. The osmotic gradient is prepared in a long slender polyethylene coiled tube (“ coil”) which serves as a centrifuge tube. Venous blood was taken from 65 normal adult subjects, Anticlot ET was added to prevent coagulation, and a 10 μｌ aliquot of blood was applied to a“ coil”, sealed and centrifuged. The erythrocytes migrated from the higher to the lower osmolar side of the coil, and hemolyzed at the sites where they could no longer comply with the osmotic stress. The coil (3 m) was cut into 15 pieces of equal length and their contents were analyzed for the chemical components leaked out of erythrocytes through the injured membrane: potassium ion (MW 39), reduced glutathione (MW 307), hemoglobin (MW 64,500), glutathione reductase (MW 115,000). Erythrocyte ghosts were also counted. The results of this experiment disclosed a sequence of release of components from erythrocytes, K+ (the fastest), GSH, hemoglobin, and GSSG-R (the slowest), in the order listed, and this was followed by the appearance of ghosts. This release sequence suggested that the smaller is the intraerythrocytic substance in molecular size the faster leaks the relevant substance from the erythrocytes.