SURFACE CHARGE OF OLD, TRANSFORMED, AND EXPERIMENTALLY DETERIORATED ERYTHROCYTES a

Twenty-three years ago we conducted experiments with our late Professor, Aharon Katzir, on thrombogenesis, using the positively charged poly-L-lysine for the agglutination of red blood cells (RBC).' Doing our electron microscopical studies on agglutinated cell membranes, we observed for the first time an ultrastructural difference between RBC membranes of the two extremes in age of the cells.' The electron microscopical evidence for a structural difference between young and old human RBC, which was demonstrated also in domestic animal^,^ induced us to look for other biophysical differences between young and old cells. Thus the difference between young and old RBC and their mobility in an electric field was reported in 1961.4 Later a difference in agglutinability between young and old RBC by poly-L-lysine related to their surface charge differences was r e p ~ r t e d . ~ These results have since been confirmed by several a u t h o r ~ ~ ~ and questioned recently by None of the investigators has questioned the decrease in sialic acid contents on the membrane of old cells as compared with young ones. We have recently submitted these results to a further rigorous test by using the apparatus for measuring cell electrophoresis designed and used by one of the who claimed that there is no difference in charge density between young and old RBC; we employed the same system with a single modification. We have replaced the direct observation of the moving cells through the microscope eyepiece and the hand on the stopwatch, by continuous cinematographic recording with enumerated frames on the film, thus eliminating the possibility of human error. The histograms (FIGS. 1 and 2) depict the electric mobility distribution of unseparated RBC (FIG. 1) and those separated into extreme age groups of "youngest" and "oldest" cells (FIG. 2). The earlier data and later confirmations of the fact that old RBC are less negatively charged led us to believe that the diminished charge plays a role in the elimination of old erythrocytes from the circulation. We have often suggested that the diminished amount of sialic acid on the surface of the RBC reveals new antigenic sites that could be "recognized by the m a c r ~ p h a g e s . ~ ~ ' ~ We looked for a different experimental model in which the macrophages identify and eliminate modified "undesirable" self cells" and therefore approached new methods for the evaluation of the negative charge on cell surface. First, we have used the col-