Erythrocyte aging as a mechanism of anemia and a biomarker of device thrombosis in continuous-flow left ventricular assist devices.

BACKGROUND: Blood trauma caused by continuous-flow left ventricular assist devices (CF-LVADs) has been associated with device thrombosis and anemia. Accurate in vivo quantification of erythrocyte turnover and its contribution to CF-LVAD complications have yet to be elucidated. METHODS: We investigated the age (lifespan) of circulating erythrocytes in subjects with CF-LVAD. Erythrocyte lifespan is a quantitative indicator of in vivo erythrocyte turnover that can be accurately derived from measurement of the exhaled carbon monoxide (CO) level. Sixty non-smoking subjects were prospectively enrolled: 25 had a CF-LVAD without thrombosis; 10 had a CF-LVAD with thrombosis; and 25 were normal controls. End-tidal breath CO levels were measured and used to calculate erythrocyte lifespan. RESULTS: The mean erythrocyte lifespan was significantly shorter in CF-LVAD subjects with (29.7 ± 14.9 days) compared to those without (65.0 ± 17.3 days) device thrombosis (p < 0.0001). The lifespans in these 2 groups were significantly shorter compared with normal controls (96.0 ± 24.9 days, both p < 0.0001). A receiver operator curve demonstrated high sensitivity-specificity for use of erythrocyte lifespan to detect device thrombosis (AUC = 0.94). In addition, all CF-LVAD subjects had low hemoglobin (11.8 ± 2.0 g/dl), and their anemia was normochromic normocytic with elevated mean reticulocyte counts. Erythrocyte lifespan correlated significantly with mean corpuscular hemoglobin concentration (r = 0.56, p = 0.0005) and red cell distribution width (r = -0.65, p < 0.001), but not with reticulocyte count (r = 0.27, p = 0.32). CONCLUSIONS: Erythrocyte lifespan is substantially reduced in subjects with a CF-LVAD, which was more pronounced in the presence of device thrombosis. The etiology of anemia in CF-LVAD was primarily due to accelerated erythrocyte aging. Further studies are needed to determine whether erythrocyte lifespan could provide a practical means of detecting subtle pre-clinical thrombosis.

Triggers, inhibitors, mechanisms, and significance of eryptosis: the suicidal erythrocyte death.

Suicidal erythrocyte death or eryptosis is characterized by erythrocyte shrinkage, cell membrane blebbing, and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include Ca(2+) entry, ceramide formation, stimulation of caspases, calpain activation, energy depletion, oxidative stress, and dysregulation of several kinases. Eryptosis is triggered by a wide variety of xenobiotics. It is inhibited by several xenobiotics and endogenous molecules including NO and erythropoietin. The susceptibility of erythrocytes to eryptosis increases with erythrocyte age. Phosphatidylserine exposing erythrocytes adhere to the vascular wall by binding to endothelial CXC-Motiv-Chemokin-16/Scavenger-receptor for phosphatidylserine and oxidized low density lipoprotein (CXCL16). Phosphatidylserine exposing erythrocytes are further engulfed by phagocytosing cells and are thus rapidly cleared from circulating blood. Eryptosis eliminates infected or defective erythrocytes thus counteracting parasitemia in malaria and preventing detrimental hemolysis of defective cells. Excessive eryptosis, however, may lead to anemia and may interfere with microcirculation. Enhanced eryptosis contributes to the pathophysiology of several clinical disorders including metabolic syndrome and diabetes, malignancy, cardiac and renal insufficiency, hemolytic uremic syndrome, sepsis, mycoplasma infection, malaria, iron deficiency, sickle cell anemia, thalassemia, glucose 6-phosphate dehydrogenase deficiency, and Wilson's disease. Facilitating or inhibiting eryptosis may be a therapeutic option in those disorders.

Blood storage duration and morbidity and mortality in children undergoing cardiac surgery. A retrospective analysis.

BACKGROUND: Blood transfusion is frequently required in children undergoing cardiac surgery and is associated with altered postoperative outcome. This may be due to alterations in red blood cell properties related to the storage process. OBJECTIVE: To evaluate the effect of blood storage duration on postoperative morbidity and mortality in children undergoing cardiac surgery. DESIGN: A retrospective review of a paediatric cardiac surgery database. SETTING: Department of Anaesthesiology, Queen Fabiola Children's University Hospital, Brussels, Belgium. PARTICIPANTS: Children transfused with one or two units of blood in the perioperative period. INTERVENTIONS: None. MAIN OUTCOME MEASURES: Storage duration was used to allocate children to the Group 'Young' or the Group 'Old' (cut-off = 7 days). The primary endpoint was a composite based on the incidence of hospital mortality and/or the incidence of at least one organ failure. RESULTS: From 1014 children in the database, 570 were included in the final analysis. One hundred and eighteen patients were included in the Group 'Young' [median (interquartile range, IQR) storage duration 6 (5 to 7) days] and 452 in the Group 'Old' [storage duration 14 (11 to 19) days]. No difference was found in mortality, length of ICU stay, mechanical ventilation duration, postoperative infection and major organ dysfunction. Duration of storage used as a continuous variable did not influence the incidence of the composite endpoint when evaluated by univariate or multivariate logistic regression analyses. CONCLUSION: Red blood cell storage duration did not influence postoperative morbidity and mortality in paediatric cardiac surgery patients transfused with one or two units of blood.

Transfusion-related biologic effects and free hemoglobin, heme, and iron.

BACKGROUND: Red blood cell (RBC) transfusion is common in intensive care unit (ICU) patients and is associated with complications that appear related to the duration of blood storage. We hypothesize that hemolysis of stored RBCs results in increases in the availability of non-heme-bound iron, which inhibits macrophage activation. STUDY DESIGN AND METHODS: RBCs were sampled at multiple time points to evaluate hemolysis and iron release. Activation of THP-1 monocytic cells was assessed in the presence of plasma from aged RBCs. Age of transfused blood in our pediatric intensive care unit (PICU) from 2001 to 2006 was analyzed to assess relevance to our patient population. RESULTS: Hemolysis increased significantly during storage time as demonstrated by increases in free heme and hemoglobin. While there was a trend toward elevated levels of non-heme-bound iron, this was not significant (p = 0.07). THP-1 cell activation was inhibited by exposures to both plasma and a ferric compound; the effect of plasma on macrophage activation was not reversed by the iron chelator desferroxamine. Thirty-one percent of our PICU patients received blood older than 2 weeks. CONCLUSION: Hemolysis products increased significantly over time in our stored RBCs. Ferric compounds and plasma from stored blood inhibit THP-1 cell activation. Plasma inhibition does not appear to be due primarily to increased iron. Further studies are needed to define the inhibitory effect of stored blood plasma on macrophage function. Complications related to blood storage are relevant to our PICU patients.

Calpain-1 knockout reveals broad effects on erythrocyte deformability and physiology.

Pharmacological inhibitors of cysteine proteases have provided useful insights into the regulation of calpain activity in erythrocytes. However, the precise biological function of calpain activity in erythrocytes remains poorly understood. Erythrocytes express calpain-1, an isoform regulated by calpastatin, the endogenous inhibitor of calpains. In the present study, we investigated the function of calpain-1 in mature erythrocytes using our calpain-1-null [KO (knockout)] mouse model. The calpain-1 gene deletion results in improved erythrocyte deformability without any measurable effect on erythrocyte lifespan in vivo. The calcium-induced sphero-echinocyte shape transition is compromised in the KO erythrocytes. Erythrocyte membrane proteins ankyrin, band 3, protein 4.1R, adducin and dematin are degraded in the calcium-loaded normal erythrocytes but not in the KO erythrocytes. In contrast, the integrity of spectrin and its state of phosphorylation are not affected in the calcium-loaded erythrocytes of either genotype. To assess the functional consequences of attenuated cytoskeletal remodelling in the KO erythrocytes, the activity of major membrane transporters was measured. The activity of the K+-Cl- co-transporter and the Gardos channel was significantly reduced in the KO erythrocytes. Similarly, the basal activity of the calcium pump was reduced in the absence of calmodulin in the KO erythrocyte membrane. Interestingly, the calmodulin-stimulated calcium pump activity was significantly elevated in the KO erythrocytes, implying a wider range of pump regulation by calcium and calmodulin. Taken together, and with the atomic force microscopy of the skeletal network, the results of the present study provide the first evidence for the physiological function of calpain-1 in erythrocytes with therapeutic implications for calcium imbalance pathologies such as sickle cell disease.

Physiologically aged red blood cells undergo erythrophagocytosis in vivo but not in vitro.

BACKGROUND: The lifespan of red blood cells is terminated when macrophages remove senescent red blood cells by erythrophagocytosis. This puts macrophages at the center of systemic iron recycling in addition to their functions in tissue remodeling and innate immunity. Thus far, erythrophagocytosis has been studied by evaluating phagocytosis of erythrocytes that were damaged to mimic senescence. These studies have demonstrated that acquisition of some specific individual senescence markers can trigger erythrophagocytosis by macrophages, but we hypothesized that the mechanism of erythrophagocytosis of such damaged erythrocytes might differ from erythrophagocytosis of physiologically aged erythrocytes. DESIGN AND METHODS: To test this hypothesis we generated an erythrocyte population highly enriched in senescent erythrocytes by a hypertransfusion procedure in mice. Various erythrocyte-aging signals were analyzed and erythrophagocytosis was evaluated in vivo and in vitro. RESULTS: The large cohort of senescent erythrocytes from hypertransfused mice carried numerous aging signals identical to those of senescent erythrocytes from control mice. Phagocytosis of fluorescently-labeled erythrocytes from hypertransfused mice injected into untreated mice was much higher than phagocytosis of labeled erythrocytes from control mice. However, neither erythrocytes from hypertransfused mice, nor those from control mice were phagocytosed in vitro by primary macrophage cultures, even though these cultures were able to phagocytose oxidatively damaged erythrocytes. CONCLUSIONS: The large senescent erythrocyte population found in hypertransfused mice mimics physiologically aged erythrocytes. For effective erythrophagocytosis of these senescent erythrocytes, macrophages depend on some features of the intact phagocytosing tissue for support.

Oxidative stress due to aluminum exposure induces eryptosis which is prevented by erythropoietin.

The widespread use of aluminum (Al) provides easy exposure of humans to the metal and its accumulation remains a potential problem. In vivo and in vitro assays have associated Al overload with anemia. To better understand the mechanisms by which Al affects human erythrocytes, morphological and biochemical changes were analyzed after long-term treatment using an in vitro model. The appearance of erythrocytes with abnormal shapes suggested metal interaction with cell surface, supported by the fact that high amounts of Al attached to cell membrane. Long-term incubation of human erythrocytes with Al induced signs of premature erythrocyte death (eryptosis), such as phosphatidylserine externalization, increased intracellular calcium, and band 3 degradation. Signs of oxidative stress, such as significant increase in reactive oxygen species in parallel with decrease in the amount of reduced glutathione, were also observed. These oxidative effects were completely prevented by the antioxidant N-acetylcysteine. Interestingly, erythrocytes were also protected from the prooxidative action of Al by the presence of erythropoietin (EPO). In conclusion, results provide evidence that chronic Al exposure may lead to biochemical and morphological alterations similar to those shown in eryptosis induced by oxidant compounds in human erythrocytes. The antieryptotic effect of EPO may contribute to enhance the knowledge of its physiological role on erythroid cells. Irrespective of the antioxidant mechanism, this property of EPO, shown in this model of Al exposure, let us suggest potential benefits by EPO treatment of patients with anemia associated to altered redox environment.

Red blood cell storage is associated with length of stay and renal complications after cardiac surgery.

BACKGROUND: The association of red blood cell (RBC) storage on morbidity outcome after cardiac surgery is debated. We sought to clarify the association of the age of transfused blood on outcome in patients undergoing cardiac surgery. STUDY DESIGN AND METHODS: Data were drawn from a prospective, observational cohort study of morbidity outcome in patients undergoing cardiac surgery. Blood transfusion data were obtained retrospectively via the Trust blood bank electronic records. Old blood was defined as more than 14 days old. The primary outcome measure was postoperative length of stay (PLOS). Secondary outcome measures included renal failure and morbidity as defined within the postoperative morbidity survey. RESULTS: A total of 176 (39.6%) of 444 participants received a blood transfusion. Patients transfused with new blood had a reduced PLOS compared with patients receiving exclusively old or any old blood (old blood ± new blood; 7 days vs. 8 days, p = 0.04 and vs. 10 days, p = 0.002, respectively). In patients who only had 1 unit transfused, PLOS was longer in those receiving only old blood compared with those receiving only new blood (8 days vs. 6 days, p = 0.02) with a 3.8-fold risk of longer stay. Compared with patients receiving exclusively new blood, patients receiving any old blood had a higher incidence of new renal complications (65.7% vs. 43.9%, p = 0.008). Each 1-day increase in storage was associated with a 7% increase in risk of new renal complications. CONCLUSION: Our data support previous suggestions of an association between transfusion of older RBCs and poorer outcome in cardiac surgery patients. Randomized controlled trials are required to determine the true causal nature of any such association.

Peroxyl-induced oxidative stress in aging erythrocytes of rat.

This study aims at determining the possible changes in intracellular calcium (Ca (i) (2+) ), plasma membrane calcium ATPase (PMCA) activity and phosphatidylserine (PS) along with glutathione (GSH) level in response to an oxidant challenge in vitro. Erythrocytes were isolated on Percoll and incubated with 2, 2'azobis (2-aminopropane) hydrochloride (AAPH) as well as with vitamin C preceding AAPH incubation. Membrane integrity in terms of hemolysis was negatively related to acetylcholine esterase (AChE) activity with the extent of reduction under OS being higher in the old erythrocyte than in the young. A divergent pattern was seen towards lower PMCA and higher (Ca (i) (2+) ) in the young and old cells. However, the PMCA activity in the stressed young cell was high when pre-treated with vitamin C. PS externalization in the young under OS is perhaps analogous to normal aging, with vitamin C preventing premature death. These findings suggest that young erythrocytes may benefit from vitamin C in therapies addressed towards the mechanisms underlying the reduced effects of OS.

Duration of red blood cell storage and outcomes in pediatric cardiac surgery: an association found for pump prime blood.

INTRODUCTION: Cardiac surgery using cardiopulmonary bypass in newborns, infants and small children often requires intraoperative red blood cell transfusions to prime the circuit and oxygenator and to replace blood lost during surgery. The purpose of this study was to investigate the influence of red blood cell storage time prior to transfusion on postoperative morbidity in pediatric cardiac operations. METHODS: One hundred ninety-two consecutive children aged five years or less who underwent cardiac operations using cardiopulmonary bypass and who received red blood cells for priming the cardiopulmonary bypass circuit comprised the blood-prime group. Forty-seven patients receiving red blood cell transfusions after cardiopulmonary bypass were separately analyzed. Patients in the blood-prime group were divided into two groups based on the duration of storage of the red blood cells they received. The newer blood group included patients who received only red blood cells stored for less than or equal to four days and the older blood group included patients who received red blood cells stored for more than four days. RESULTS: Patients in the newer blood group had a significantly lower rate of pulmonary complications (3.5% versus 14.4%; P = 0.011) as well as a lower rate of acute renal failure (0.8% versus 5.2%; P = 0.154) than patients in the older blood group. Major complications (calculated as a composite score based on pulmonary, neurological, and gastroenterological complications, sepsis and acute renal failure) were found in 6.9% of the patients receiving newer blood and 17.1% of the patients receiving older blood (P = 0.027). After adjusting for other possible confounding variables, red blood cell storage time remained an independent predictor of major morbidity. The same association was not found for patients receiving red blood cell transfusions after cardiopulmonary bypass. CONCLUSIONS: The storage time of the red blood cells used for priming the cardiopulmonary bypass circuit in cardiac operations on newborns and young infants is an independent risk factor for major postoperative morbidity. Pulmonary complications, acute renal failure, and infections are the main complications associated with increased red blood cell storage time.

Quantitative analysis of mechanisms that govern red blood cell age structure and dynamics during anaemia.

Mathematical modelling has proven an important tool in elucidating and quantifying mechanisms that govern the age structure and population dynamics of red blood cells (RBCs). Here we synthesise ideas from previous experimental data and the mathematical modelling literature with new data in order to test hypotheses and generate new predictions about these mechanisms. The result is a set of competing hypotheses about three intrinsic mechanisms: the feedback from circulating RBC concentration to production rate of immature RBCs (reticulocytes) in bone marrow, the release of reticulocytes from bone marrow into the circulation, and their subsequent ageing and clearance. In addition we examine two mechanisms specific to our experimental system: the effect of phenylhydrazine (PHZ) and blood sampling on RBC dynamics. We performed a set of experiments to quantify the dynamics of reticulocyte proportion, RBC concentration, and erythropoietin concentration in PHZ-induced anaemic mice. By quantifying experimental error we are able to fit and assess each hypothesis against our data and recover parameter estimates using Markov chain Monte Carlo based Bayesian inference. We find that, under normal conditions, about 3% of reticulocytes are released early from bone marrow and upon maturation all cells are released immediately. In the circulation, RBCs undergo random clearance but have a maximum lifespan of about 50 days. Under anaemic conditions reticulocyte production rate is linearly correlated with the difference between normal and anaemic RBC concentrations, and their release rate is exponentially correlated with the same. PHZ appears to age rather than kill RBCs, and younger RBCs are affected more than older RBCs. Blood sampling caused short aperiodic spikes in the proportion of reticulocytes which appear to have a different developmental pathway than normal reticulocytes. We also provide evidence of large diurnal oscillations in serum erythropoietin levels during anaemia.

Separación de poblaciones de glóbulos rojos senescentes por gradientes preformados de Percoll / Separation of populations of senescent red blood cells by Percoll performed gradients

Los mecanismos que determinan la senescencia de los eritrocitos han sido extensamente estudiados, sin embargo, no se han logrado conclusiones definitivas debido a la ausencia de una técnica que permita el aislamiento de grupos etáreos bien definidos. Los métodos más comúnmente empleados se basan en el aumento de densidad de los eritrocitos durante el envejecimiento. En este trabajo desarrollamos una técnica para la separación de glóbulos rojos de distintas edades empleando gradientes preformados de Percoll, un polímero sintético con propiedades fisicoquímicas adecuadas para trabajar con células vivas. En las suspensiones eritrocitarias obtenidas se realizaron determinaciones hematológicas, actividades de enzimas antioxidantes y el ensayo de eritrofagocitosis. Los valores de los parámetros hematológicos evaluados fueron significativamente mayores en las suspensiones de glóbulos rojos jóvenes. Las actividades enzimáticas mostraron una disminución de la capacidad antioxidante en las poblaciones de eritrocitos senescentes. Este proceso favorecería la interacción de los hematíes envejecidos con las células fagocíticas, demostrada median­te el ensayo de eritrofagocitosis. Los resultados obtenidos indican que el método de gradientes de Percoll permite una adecuada separación de las suspensiones eritrocitarias de distintas edades, con una eficiencia comparable a la observada en la técnica de centrifugación diferencial considerada de referencia.

The mechanisms that determine the senescence of the erythrocytes have been extensively studied; however. definitive conclusions have not been achieved mainly because of the lack of a technique that allows the isolation of well-defined etarian groups. The methods most commonly used for separating erythrocytes from different ages are based on the increase in density that these cells present during their aging. In the present work we have developed a technique for obtaining red blood cells from different ages using Percoll preformed gradients, a synthetic polymer with adequate physic-chemic properties to work with lives cells. In the erythrocytes suspensions we have made hematological determinations. activities of antioxidants enzymes and the essay of erythrophagocytosis. The values of the hematological parameters were significantly higher in the suspensions of young red blood cells. In the measurements of the enzymatic activity we observed a decrease of the antioxidant capacity in the populations of senescent erythrocytes. This process would promote the interaction between the old erythrocytes and the phagocyte cells, demonstrated by the erythrophagocytosis essay. The results obtained indicate that the method Percoll density gradients allows an appropriate separation of the erythrocytes suspensions of different ages with a comparable efficiency to that observed in the technique differential centrifugation, considered as reference.

Clinical relevance of anemia and transfusion iron overload in myelodysplastic syndromes.

Most patients with myelodysplastic syndrome eventually become dependent on regular red cell transfusions. This dependency has a negative impact on clinical outcome, primarily because it may be associated with more severe marrow failure. In addition, however, transfusion dependency may involve clinical consequences of chronic anemia and iron overload. Although transfusion iron is primarily taken up by the reticuloendothelial cells, the metal is later redistributed to parenchymal cells. This redistribution is modulated by several factors, including the degree of ineffective erythropoiesis through its suppressive effect on hepcidin production. Body iron status is routinely assessed by serum ferritin and transferrin saturation, but there is a need of reliable tools for locating iron accumulation in patients. Magnetic resonance imaging T2* provides a non-invasive method for detecting and quantifying both liver and myocardial iron overload. Clinical consequences of parenchymal iron overload have been reported not only in thalassemia major, but also in patients with myelodysplastic syndrome. Transfusion-dependent patients with isolated erythroid dysplasia and low risk of leukemic evolution are more likely to develop parenchymal iron overload and its toxicity, and therefore may benefit from chelation therapy. There may also be a benefit of chelation therapy in patients with transfusion iron overload undergoing allogeneic stem cell transplantation. Deferoxamine and deferasirox are currently available for treatment of transfusion iron overload in patients with myelodysplastic syndrome.

Blood transfusion promotes cancer progression: a critical role for aged erythrocytes.

BACKGROUND: In cancer patients, allogeneic blood transfusion is associated with poorer prognosis, but the independent effect of the transfusion is controversial. Moreover, mediating mechanisms underlying the alleged cancer-promoting effects of blood transfusion are unknown, including the involvement of donors' leukocytes, erythrocytes, and soluble factors. METHOD: Two syngeneic tumor models were used in Fischer 344 rats, the MADB106 mammary adenocarcinoma and the CRNK-16 leukemia. Outcomes included host ability to clear circulating cancer cells, and host survival rates. The independent impact of blood transfusion was assessed, and potential deleterious characteristics of the transfusion were studied, including blood storage duration; the role of erythrocytes, leukocyte, and soluble factors; and the kinetics of the effects. RESULTS: Blood transfusion was found to be an independent and significant risk factor for cancer progression in both models, causing up to a fourfold increase in lung tumor retention and doubling mortality rates. Blood storage time was the critical determinant of these deleterious effects, regardless of whether the transfused blood was allogeneic or autogenic. Surprisingly, aged erythrocytes (9 days and older), rather than leukocytes or soluble factors, mediated the effects, which occurred in both operated and nonoperated animals. The effects of erythrocytes transfusion in the MADB106 model emerged immediately and dissipated within 24 h. CONCLUSIONS: In rats, transfusion of fresh blood is less harmful than transfusion of stored blood in the context of progressing malignancies. Further studies should address mediating mechanisms through which erythrocytes' storage duration can impact the rate of complications while treating malignant diseases and potentially other pathologies.

Paradoxical elevated thiopurine S-methyltransferase activity after pancytopenia during azathioprine therapy: potential influence of red blood cell age.

There is an increased risk of developing bone marrow depression and infections during azathioprine therapy for inflammatory bowel disease. Patients with low or absent thiopurine S-methyltransferase (TPMT) activity have an increased risk of developing myelotoxicity. We describe a patient who developed pancytopenia combined with cytomegalovirus pneumonia after several years of azathioprine use. The bone marrow depression was probably caused by the viral infection, as all others causative factors were unlikely. Surprisingly, we observed grossly elevated TPMT activity (182 nmol/g/h) during the recovery phase, following the pancytopenic period. After complete recovery of the bone marrow suppression, TPMT activity returned to usual reference activity (43 nmol/g/h). This remarkable change in enzymatic activity of TPMT may be explained by differences in the age of red blood cells, as younger erythrocytes have a higher TPMT activity. Determination of a patient's TPMT status by phenotyping should therefore not be performed just after bone marrow depression or in cases of activated erythropoieses.

A role of phosphatidylserine externalization in clearance of erythrocytes exposed to stress but not in eliminating aging populations of erythrocyte in mice.

Age dependent changes in phosphatidylserine (PS) externalization were studied in mouse erythrocytes of different age groups (range 1-55 days) by using a newly developed double in vivo biotinylation (DIB) technique. Around 3-4% of the erythrocytes freshly released in the circulation were PS(+) but this proportion fell rapidly to 1% or less and did not increase at later time points. Blocking erythrocyte clearance from the circulation by in vivo depletion of macrophages (by treatment with clodronate loaded liposomes) for up to 7 days did not result in accumulation of PS(+) erythrocytes in the circulation indicating that the low percentage of PS(+) cells within old erythrocytes (age >40 days) was not related to the clearance of PS(+) erythrocytes by macrophages. In vitro treatment with stress inducing agents like deoxyglucose or Ca(++)/calcium ionophore resulted in a marked induction of PS externalization in mouse erythrocytes and this effect was most prominent in the youngest erythrocyte population (age <10 days). Kinetics of clearance of different age groups of stress exposed erythrocytes after intravenous infusion into recipient mice indicated that the young erythrocytes were cleared at fastest rate from the circulation as compared to erythrocytes of older age groups. Within young erythrocytes exposed to stress, PS(+) erythrocytes were preferentially cleared. Taken together our results suggest that PS externalization is unlikely to have a role in the removal of old erythrocytes from blood circulation but may have a role in the clearance of stressed and damaged young erythrocytes in blood circulation.

Vanadate-induced suicidal erythrocyte death.

Vanadium, a trace element, as vanadate (VO4(3-)) is known to interfere with a wide variety of enzymes including Ca2+ ATPase and Na+/+ ATPase. VO4(3-) is excreted mainly via the kidney. In renal insufficiency, the impaired VO4(3-) excretion leads to VO4(3-) accumulation in blood.The present study explored the effect of VO4(3-) on eryptosis, the suicidal death of erythrocytes. Eryptosis is characterized by cell shrinkage and phosphatidylserine exposure at the erythrocyte surface. Eryptotic cells are phagocytosed and thus rapidly cleared from circulating blood. Stimulators of eryptosis include an increase of the cytosolic Ca2+ concentration. Erythrocyte Ca2+ activity was estimated from Fluo-3 fluorescence, phosphatidylserine exposure from annexin V-binding, and erythrocyte volume from forward scatter in FACS analysis. Exposure of erythrocytes to VO4(3-) increased cytosolic Ca2+ concentration, enhanced the percentage of annexin V-binding erythrocytes, decreased erythrocyte forward scatter, and lowered the intracellular ATP concentration. In conclusion, VO4(3-) induces eryptosis at least partially through increase of cytosolic Ca2+ concentration, an effect presumably contributing to the development of anemia in chronic renal failure.

Iron homeostasis in chronic inflammation.

Inflammation induced anemia and resistance to erythropoietin are common features in patients with chronic kidney disease (CKD). Elevated levels of cytokines and enhanced oxidative stress, conditions associated with inflammatory states, are implicated in the development of anemia. Accumulating evidence suggests that activation of cytokine cascade and the associated acute-phase response, as it often occurs in patients with CKD, divert iron from erythropoiesis to storage sites within the reticuloendothelial system leading to functional iron deficiency and subsequently to anemia or resistance to erythropoietin. Other processes have also been shown to be involved in the pathogenesis of anemia provoked by the activated immune system including an inhibition of erythroid progenitor proliferation and differentiation, a suppression of erythropoietin production and a blunted response to erythropoietin. The present review concerns the underlying alterations in iron metabolism induced by chronic inflammation that result in anemia.

Adenosine protects against suicidal erythrocyte death.

Suicidal death of erythrocytes or eryptosis is characterized by cell shrinkage and cell membrane scrambling leading to phosphatidylserine exposure at the erythrocyte surface. The cell membrane scrambling is triggered by an increase in cytosolic Ca(2+) activity and activation of protein kinase C (PKC). Phosphatidylserine exposure fosters adherence of affected erythrocytes to the vascular wall. Thus, microcirculation in ischemic tissues may be impaired by the appearance of eryptotic erythrocytes. Ischemia leads to release of adenosine, which in most tissues leads to vasodilation and protects against cell injury. The present experiments explored whether adenosine influences mechanisms underlying eryptosis. Erythrocyte phosphatidylserine exposure was estimated from annexin V binding, cell volume from forward scatter and cytosolic Ca(2+) activity from Fluo3 fluorescence. Glucose depletion (for 24 or 48 h) significantly increased annexin binding and decreased forward scatter, effects partially reversed by adenosine. The protective effect of adenosine reached statistical significance (s.d.) at > =30 microM. Low Cl(-) solution (Cl(-) exchanged by gluconate for 24 h) similarly increased annexin binding and decreased forward scatter, effects again reversed by adenosine (s.d. at > or =10 and 30 microM, respectively). Similarly, phosphatase inhibitor okadaic acid (OA, 1 microM) and PKC activator phorbol 12-myristate-13-acetate (PMA, 3 microM) significantly enhanced annexin binding and decreased forward scatter. Adenosine significantly blunted the effects of OA and PMA on annexin V binding (s.d. at > or =30 and 10 microM, respectively) and the effect of OA on forward scatter (s.d. at > or =10 microM). In conclusion, adenosine inhibits eryptosis by a mechanism presumably effective downstream of PKC. The effect may participate in the maintenance of microcirculation in ischemic tissue.

Determinación de IgG en eritrocitos senescentes por un ensayo enzimático con consumo de antiglobulina / Determination of senescent red blood cells bound IgG using an enzyme test based an the consuption of antiglobulin

Durante el envejecimiento de los GR se acumula IgG autóloga sobre la membrana eritrocitaria. Esta IgG esta dirigida a un neo antígeno de senescencia, ubicado en la proteína banda 3. El objetivo de este trabajo fue determinar pequeñas cantidades de IgG en la membrana del GR utilizando un inmunoensayo con antiglobulina conjugada con una enzima. Las suspensiones de GRSe y GRJ fueron incubadas con anti-IgG humana conjugada con fosfatasa alcalina. Se transfirieron alícuotas a microplacas sensibilizadas con IgG humana. Se agregó el sustrato de la enzima y se midió la IgG libre. Las concentraciones de IgG unida a la membrana eritrocitaria en GRSe (13.31 x 10-4(g/(L(1.57 x 10-4) fueron significativamente mayores (p(0.0001) que los valores observados en GR (3.35 x 10-4(g/(L(1.39 x 10-4). Los resultados obtenidos indican que esta metodología constituye una herramienta útil para determinar pequeñas contidades de IgG unida a la membrana eritrocitaria.