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

Abstract

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 degrees-214 degrees C) and the spacered intermediate for C-BNHS, 6-(biotinylamide) hexanoate (mp, 225 degrees-226 degrees C) were obtained in an overall yield of 86%; the yield of C-BNHS (mp, 167 degrees-169 degrees 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.

A novel approach for in vivo measurement of mouse red cell redox status

“…Expression of roGFP2 had no effect on RBC survival as the survival curves are identical for both the GFP positive and GFP negative RBC ( Figure 1B) and agree with studies performed by our group using nontransgenic mice on the same strain (B6/J) background (data not shown). [17][18][19] We compared complete blood counts of transgenic and nontransgenic mice and found no significant differences in RBC indices, WBC parameters or platelet count (supplemental Table 1, available on the Blood Web site; see Supplemental Materials link at the top of the online article), indicating that expression of roGFP2 does not impact RBC survival or development.…”

“…20 In vitro incubation with PHZ resulted in a dose-dependent increase of R405/488 that was reversed by administration of 10mM DTT (Figure 2A-B). We next probed for redox changes accompanying normal RBC aging using an in vivo biotin labeling method 17,21 ( Figure 2C). R405/488 of the labeled cells (increasing in age) was followed over time and compared with that of unlabeled, GFP positive RBC in control mice.…”

“…17,18 Blood samples (5 L) were taken and analyzed by flow cytometry after staining with streptavidin Cy-Chrom (BD Pharmingen) for determination of cell age and roGFP2 ratio. All animal protocols were approved by the TSRI IACUC.…”

“…Expression of roGFP2 had no effect on RBC survival as the survival curves are identical for both the GFP positive and GFP negative RBC ( Figure 1B) and agree with studies performed by our group using nontransgenic mice on the same strain (B6/J) background (data not shown). [17][18][19] We compared complete blood counts of transgenic and nontransgenic mice and found no significant differences in RBC indices, WBC parameters or platelet count (supplemental Table 1, available on the Blood Web site; see Supplemental Materials link at the top of the online article), indicating that expression of roGFP2 does not impact RBC survival or development.We performed in vitro flow cytometry experiments using roGFP2-expressing RBCs to characterize the response and dynamic fluorescence range of the redox sensor. When transgenic RBCs are incubated with t-butyl hydroperoxide (tBOOH), fluorescence intensity excited at 405 nm (emitted at 520 nm) increases while fluorescence intensity excited at 488nm (emitted at 520 nm) decreases (supplemental Figure 1).…”

Red blood cell dysfunction in septic glucose-6-phosphate dehydrogenase-deficient mice

“…This discrepancy is most likely the result of the fact that the current study used 10-to 12-wk-old animals to allow erythrocytes of the entire age spectrum to appear in the circulation (the life span of erythrocytes is 40 days in mice; Ref. 23). Younger mice also have elevated erythrocyte counts compared with older animals.…”