Senescent erythrocytes: isolation of in vivo aged cells and their biochemical characteristics.

Abstract

Rabbit erythrocytes were covalently labeled with biotin and then infused into the donor animial. Up to 60 days after infusion, the biotinylated cells were selectively isolated by their affinity for avidin. These aged erythrocytes, which are within 10 days of death, were analyzed for several biochemical parameters. The 2,3-bisphosphoglycerate and glutathione levels of these cells were constant with age; however, the adenosine 5'-triphosphate concentrations increased approximately 75% as the cells approached the end of their life span. The activities of several glycolytic enzymes did not change with age.The mechanisms of erythrocyte senescence are poorly understood (1). Clearly, erythrocytes have a well-defined life span; however, the study of the aging processes in these cells has been hindered by the inability to definitively isolate senescent erythrocytes. A myriad of publications have reported age-related differences in various biochemical parameters of erythrocytes (for a review, see ref. 1). However, the majority of these are based on the separation of cells over density gradients with the underlying assumption that the most dense fractions represent the old cells, an assumption that has been questioned (2,3).Two methods that are independent of the physical separation techniques outlined above have been reported for the isolation of senescent erythroctyes. Allison and Burn (4) transfused blood group 0 erythrocytes into normal blood group A individuals. At subsequent time points, the 0 cells were recovered after agglutination of the host A erythrocytes. Unfortunately, the technique was hindered by technical difficulties. More recently, Ganzoni et al. (5) Preparation of Young Erythrocyte Cohorts. In some experiments, young erythrocyte populations were used for biotinylation and replacement into donor rabbits. The advantage of using a young cohort of cells is that a larger percentage of the cells will still be circulating 50-60 days after replacement and therefore will allow recovery of greater numbers of senescent cells. Two procedures were used to produce young cell populations-phenylhydrazine treatment and repeated phlebotomy. Phenylhydrazine hydrochloride (25 mg) was subcutaneously injected into a rabbit on days 0, 2, and 3. On day 13, 50 ml of blood was collected in heparin for biotinylation. The reticulocyte count for these preparations averaged more than 50% on day 8. For the repeated phlebotomy procedure, 50-60 ml of blood was drawn from the rabbit on day 0, 2, and 4. The sample for biotinylation was taken on day 14. To protect against iron deficiency during repeated phlebotomy, each rabbit received 50 mg of iron-dextran intramuscularly on days -7, -5, -3, 0, 2, and 4. Preparation and Infusion of Biotinylated Erythrocytes.Rabbit erythrocytes were washed, labeled with ['4C]cyanate, treated with NHS-biotin, and infused into the donor rabbit exactly as described (7). The average level of biotinylation was approximately 25,000 biotin molecules per erythrocyte.Avidin-Coated Polystyrene Beads. The av...

Homing of annexin-labeled stem cells to apoptotic cells

“…1 B, D), showing the safeguard of the physiological membrane protein turnover. Consistently with this last observation, several investigators reported that the biotinylation reagents do not seem cytotoxic in in vitro and in vivo experimental models [18,20,21].…”

Measurement of Red Cell Lifespan and Aging

“…Since a large number of RBC are counted this analysis can be continued for essentially the entire RBC lifespan. In animal studies with in vivo biotinylation, this technique has been used to determine cell age-related changes in membrane immunoglobulin [43,46], aminophospholipid translocase (flippase) activity [47], enzyme activities [40], and many other properties [38,39,42] In human studies with ex vivo biotinylation, measured variables include HbF-containing RBC [19,20,22], RBC with external phosphatidylserine [21], and the time for disappearance of reticulocytes [19].…”

“…In animal studies it is possible to label all the RBC in vivo with biotin with an intravenous injection of sulfo-NHS-biotin [37][38][39][40][41][42][43]. At subsequent times the biotin-negative RBC are all younger than the time since administration of the labeling reagent, and the biotin-positive RBC are all older than that time.…”

Erythrocyte Aging: Physical and Chemical Membrane Changes

“…For obvious reasons, the majority of studies have con cerned the human red blood cell and I will refer to this cell unless stated otherwise. nylation [18,19]. In the hypertransfusion scheme, only two steps are shown but the procedure must be repeated several times every couple of days to sup press erythropoiesis in the recipients.…”