Direct in vivo biotinylation of erythrocytes as an assay for red cell survival studies

Direct in vivo labeling of erythrocytes with biotin is shown as a method for estimation of red cell survival as well as of enrichment of young or aged erythrocytes. Two succinimide esters (biotin-N-hydroxysuccinimide ester [BNHS], caproylamidobiotin-N-hydroxysuccinimide ester [C-BNHS] were used for biotin labeling of erythrocytes. With improved syntheses, pure BNHS (mp, 212°–214° C) and the spacered intermediate for C-BNHS, 6-(biotinylamide) hexanoate (mp, 225°–226° C) were obtained in an overall yield of 86%; the yield of C-BNHS (mp, 167°–169° C) was 68%. When three doses of 1 mg C-BNHS are injected intravenously into mice at 24-h intervals, all the red cells are biotin labeled. The rate of red cell production as well as the life span of red cells can be measured without any effect on erythropoiesis or damage by red cells in vitro. The survival curve seems to be linear, with 2.5%–3.3% disappearance of biotin-labeled red cells daily. In mice, in vivo biotin labeling avoids damaging red cells by in vitro procedures and does not influence the steady state of erythropoiesis by hypertransfusion. Therefore, in vivo biotin labeling is a very useful method for determining red cell survival time in small animals.