Sialidase (neuraminidase; acylneuraminyl hydrolase; EC 3.2.1.18)treated erythrocytes obtained from different species are susceptible to rapid elimination from the circulation and are sequestered in the liver and spleen. The present studies were concerned with the mechanism of this clearance and how it may relate to the normal physiological process of removing senescent erythrocytes from the circulation. The Other investigators have shown (5-8) that the sialic acid content of circulating erythrocytes varies with the age of the erythrocyte. In older erythrocytes sialic acid content is 10-15% less than in younger erythrocytes (6).Autologous transfusion of sialidase-treated erythrocytes results in their rapid disappearance from the circulation. This is true in several different species, such as rats (9, 10), rabbits (4, 10-13), dogs (4), goats (4), and human beings (14). Removal of only 10% of the total sialic acid present on the surface of erythrocytes is sufficient to eliminate these sialidase-treated erythrocytes from the circulation (4). Moreover, the 51Cr label used to tag the sialidase-treated erythrocytes accumulates in the liver (9, 10) and spleep (9).The present studies were concerned with the process by which the sialidase-treated erythrocytes are sequestered in the liver and sp1een. These studies were initiated with the concept that the physiological removal of senescent erythrocytes from the circulation may be analogous to the removal of sialidasetreated erythrocytes. The results of this study demonstrate that sialidase-treated erythrocytes preferentially adhere to Kupffer cells in the liver or to mononuclear cells in the spleen. These investigations correlate well with the histological findings of Schauer (personal communication). MATERIALS AND METHODSMaterials. Sialidase was prepared from Vibrio cholerae as described (3). Clostridium perfringens sialidase was obtained commercially from Sigma Biochemicals (Type VI, lot SOC-8010) and Cl. histolytwum collagenase was purchased from Enzymatic Treatment of Erythrocytes. Blood was collected in EDTA (1.4 mg/ml) from 200-to 250-g male rats (SpragueDawley strain) maintained on standard laboratory chow and tap water ad lib. After anesthetic induction with ethyl ether, the blood was withdrawn by a sterile cardiac puncture. The erythrocytes were treated with sialidase as described (4). The erythrocytes were washed twice in 0.9% saline (pH 7.0) and the buffy coat was removed. After a final wash with 0.83% saline in 10 mM CaCl2, the erythrocytes were incubated at 370 for 30 min with 0.2 unit* of sialidase per ml of packed cells or incubated for 30 min at 370 in CaCl2/saline only. The erythrocytes were then washed three times in saline before further study, and the supernatants were assayed for free sialic acid by the quantitative thiobarbituric acid method (15). The erythrocytes were prepared before the liver or spleen cells because they were stable at 40 for at least 3 hr.Isolation of Rat Liver Hepatocytes and Kupffer Cells. The method is based on that of ...

Previous studies have shown that sialidasetreated mammalian erythrocytes were rapidly eliminated from circulation. In contrast, chicken asialoerythrocytes remained fully viable. This investigation was undertaken to ascertain the reason for this difference in behavior as well as to determine the extent of the similarity of the physiological mechanism for the elimination from circulation of asialoglycoproteins and mammalian asialoerythrocytes. To that end, erythrocytes from dogs, rabbits, and chickens were each subjected to the action of galactose oxidase (D-galactose:oxygen 6-oxidoreductase; EC 1.1.3.9) both before and after sialidase (acylneuraminyl hydrolase; EC 3.2.1.18) treatment. The viability of the autologously transfused erythrocytes in circulation was monitored by Na251CrO4 labeling. Galactose oxidase had no deleterious effect on the viability of either dog or chicken erythrocytes, nor did it restore the viability of dog or rabbit asialoerythrocytes. On the other hand, desialated chicken erythrocytes, which were fully viable, were rendered nonviable upon treatment with galactose oxidase. It may be concluded therefore that (a) the physiological mechanism of elimination of mammalian asialoerythrocytes from circulation is not the same as that for plasma asialoglycoproteins and (b) the treatment of chicken asialoerythrocytes with galactose oxidase results in the oxidation at carbons 6 of the galactosyl-or N-acetylgalactosaminyl residues, thereby rendering the erythrocytes nonviable.