In vivo aging of canine biotinylated erythrocytes: Validation and preliminary findings as a model system for the study of erythrocyte senescence

An improved system for studying red blood cell (RBC) senescence has been validated which utilizes an appropriate animal model, the dog, and avoids the controversial density gradient centrifugation methods for isolating presumably old RBC\u27s. The dog is chosen for its low random RBC loss and relatively long RBC life span, both of which are similar to man. RBC\u27s having a defined age are obtained (after the method of Suzuki and Dale) by labeling RBC with biotin, aging the biotinylated RBC\u27s in vivo, and then recovering the senescent biotinylated RBC\u27s by affinity to an avidin support. Validation of essential components demonstrated that the biotinylation system is suitable for studies in the dog. (1) Labeling. Greater than 98% of circulating RBC were biotinylated by intravenous infusion of solubilized N-Hydroxysuccinimido biotin. (2) Survival. The circulating life span (using C-14 cyanate) of biotinylated RBC\u27s was indistinguishable from non-biotinylated RBC\u27s, indicating unaltered viability. (3) Recovery. At 100-110 days post labeling, biotinylated RBC\u27s having 80-95% purity were recovered using a magnetic cell sorter (MACS, Becton Dickinson). This ensures a highly enriched population of senescent RBC\u27s for analysis. Canine biotinylated RBC\u27s aged in vivo for 104-110 days and control RBC\u27s consisting of a random age-distribution of circulating RBC\u27s were incubated with 125-I sheep anti-dog IgG. Three times as many molecules of the anti-IgG bound to senescent RBC\u27s as bound to control RBC\u27s. Furthermore, anti-biotin molecules did not contribute significantly to the increased IgG binding to old RBC\u27s since serum taken after biotinylated RBC\u27s had circulated for 113 days failed to show increased recognition of autologous, random age biotinylated RBC\u27s. Therefore, we conclude that senescence of canine RBC\u27s is characterized by opsonization with autologous IgG. Furthermore, application of the biotinylation system to the dog appears to provide an excellent model for the study of RBC senescence