Reduced Erythrocyte Lifespan Research Articles

Testosterone and Erythrocyte Lifespan.

Endogenous and exogenous androgens increase circulating erythrocytes and hemoglobin but their effects on erythrocyte lifespan is not known. To investigate androgen effects on immature and mature erythrocyte lifespan in humans and mice using novel non-radioactive minimally invasive methods. Human erythrocyte lifespan was estimated using alveolar carbon monoxide concentration and blood hemoglobin in Levitt's formula in hypogonadal or transgender men before and up to 18 weeks after commencing testosterone (T) treatment. Erythrocyte lifespan was estimated in androgen receptor (AR) knockout and wild-type mice after T or dihydrotestosterone (DHT) treatment of intact females or orchidectomized males using in vivo biotin labelling of erythrocyte surface epitopes for reticulocytes (Ter119+CD71+) and two markers of erythrocytes (CD45-, Ter119+CD71-) monitoring their blood disappearance rate by flow cytometry. Before treatment, hypogonadal and transgender men had marked reduction in erythrocyte lifespan compared with controls. T treatment increased erythrocyte lifespan at 6 weeks but returned to pre-treatment levels at 18 weeks while serum T and blood hemoglobin were increased by T treatment remaining elevated at 18-weeks. In mice T and DHT treatment had higher erythrocyte (but not reticulocyte) lifespan but neither orchidectomy nor AR inactivation significantly influenced erythrocyte or reticulocyte lifespan. We conclude that hypogonadal men have reduced erythrocyte lifespan and acute androgen-induced increase in circulating erythrocyte lifespan may contribute to the well-known erythropoietic effects of androgens, but longer-term effects require further investigation to determine how much they contribute to androgen-induced increases in circulating hemoglobin.

High-volume endurance exercise training stimulates hematopoiesis by increasing ACE NH2-terminal activity.

One of the health benefits of endurance exercise training (ET) is the stimulation of hematopoiesis. However, the mechanisms underlying ET-induced hematopoietic adaptations are understudied. N-Acetyl-Seryl-Aspartyl-Lysyl-Proline (Ac-SDKP) inhibits proliferation of early hematopoietic progenitor cells. The angiotensin I-converting enzyme (ACE) NH2-terminal promotes hematopoiesis by inhibiting the anti-hematopoietic effect of Ac-SDKP. Here we demonstrate for the first time the role of ACE NH2-terminal in ET-induced hematopoietic adaptations. Wistar rats were subjected to 10 weeks of moderate-(T1) and high-(T2) volume swimming-training. Although both protocols induced classical ET-associated adaptations, only T2 increased plasma ACE NH2-domain activity (by 40%, P=0.0003) and reduced Ac-SDKP levels (by 50%, P<0.0001). T2 increased the number of hematopoietic stem cells (HSCs; ∼200%, P=0.0008), early erythroid progenitor colonies (∼300%, P<0.0001) and reticulocytes (∼500%, P=0.0007), and reduced erythrocyte lifespan (∼50%, P=0.022). Following, Wistar rats were subjected to T2 or T2 combined with ACE NH2-terminal inhibition (captopril (Cap) treatment: 10 mg.kg-1.day-1). T2 combined with ACE NH2-terminal inhibition prevented Ac-SDKP decrease and attenuated ET-induced hematopoietic adaptations. Altogether, our findings show that ET-induced hematopoiesis was at least partially associated with increased ACE NH2-terminal activity and reduction in the hematopoietic inhibitor Ac-SDKP.

Reduced erythrocyte lifespan measured by chromium-51 in patients with type 2 diabetes undergoing long-term hemodialysis.

A reduced erythrocyte lifespan potentially explains the low hemoglobin A1c values found in hemodialysis patients. However, data supporting this notion in patients with type 2 diabetes is unclear. We evaluated the erythrocyte lifespan in patients with type 2 diabetes undergoing long-term hemodialysis and investigated potential predictors of erythrocyte lifespan. Long-term hemodialysis patients with type 2 diabetes and type 2 diabetes patients without nephropathy (estimated glomerular filtration rate > 60 mL/min/1.73 m2 ) were included. The erythrocyte lifespan was measured using chromium-51 (51 Cr)-labeled erythrocytes. Blood radiotracer activity was measured six to nine times over a period of 3-5 weeks to determine the erythrocyte lifespan of each patient. Biochemical markers were obtained five times over 16 weeks and associated with the erythrocyte lifespan. Type 2 diabetes patients undergoing hemodialysis (N = 13) had a significantly shorter median erythrocyte lifespan of 49.7 (interquartile range [IQR] = 44.1-58.6) days compared with 64.2 (IQR = 62.6-83.5) days in the control group (N = 10) (P ˂ 0.001) with a difference between medians of 14.5 (95% confidence interval = 8.1-38.8) days. In the hemodialysis group, no association could be detected between the erythrocyte lifespan and markers of hemolysis, level of inflammation, or urea. A reduced erythrocyte lifespan was detected in type 2 diabetes patients undergoing long-term hemodialysis. This may contribute to the reduced hemoglobin A1c values observed in the type 2 diabetic hemodialysis population. An association could not be detected between the erythrocyte lifespan and biochemical markers of hemolysis or inflammation.

Congenital Hemolytic Anemias: Is There a Role for the Immune System?

Congenital hemolytic anemias (CHAs) are a heterogeneous group of rare hereditary conditions including defects of erythrocyte membrane proteins, red cell enzymes, and disorders due to defective erythropoiesis. They are characterized by variable degree of anemia, chronic extravascular hemolysis, reduced erythrocyte life span, splenomegaly, jaundice, biliary lithiasis, and iron overload. Although few data are reported on the role of the immune system in CHAs, several immune-mediated mechanisms may be involved in the pathogenesis of these rare diseases. We reported in ~60% of patients with hereditary spherocytosis (HS), the presence of naturally-occurring autoantibodies (NAbs) directed against different membrane proteins (α- and β-spectrin, band 3, and dematin). Positive HS subjects showed a more hemolytic pattern and NAbs were more evident in aged erythrocytes. The latter is in line with the function of NAbs in the opsonization of damaged/senescent erythrocytes and their consequent removal in the spleen. Splenectomy, usually performed to reduce erythrocyte catheresis and improve Hb levels, has different efficacy in various CHAs. Median Hb increase is 3 g/dL in HS, 1.6–1.8 g/dL in pyruvate kinase deficiency (PKD), and 1 g/dL in congenital dyserythropoietic anemias (CDA) type II. Consistently with clinical severity, splenectomy is performed in 20% of HS, 45% of CDAII, and in 60% of PKD patients. Importantly, sepsis and thrombotic events have been registered, particularly in PKD with a frequency of ~7% for both. Furthermore, we analyzed the role of pro-inflammatory cytokines and found that interleukin 10 and interferon γ, and to a lesser extent interleukin 6, were increased in all CHAs compared with controls. Moreover, CDAII and enzymatic defects showed increased tumor necrosis factor-α and reduced interleukin 17. Finally, we reported that iron overload occurred in 31% of patients with membrane defects, in ~60% of CDAII cases, and in up to 82% of PKD patients (defined by MRI liver iron concentration >4 mg Fe/gdw). Hepcidin was slightly increased in CHAs compared with controls and positively correlated with ferritin and with the inflammatory cytokines interleukin 6 and interferon γ. Overall the results suggest the existence of a vicious circle between chronic hemolysis, inflammatory response, bone marrow dyserythropoiesis, and iron overload.

P1004REDUCED ERYTHROCYTE LIFE SPAN IN PATIENTS WITH TYPE 2 DIABETES UNDERGOING LONG-TERM HAEMODIALYSIS

Abstract Background and Aims Anaemia in long-term haemodialysis (HD) is caused by reduced erythropoietin levels but shortened erythrocyte life span is thought to contribute. However, reduced erythrocyte life span has never been documented in patients with type 2 diabetes (T2D) undergoing long-term HD. The aim of the study was to establish if the erythrocyte life span is decreased in in patients with type 2 diabetes (T2D) undergoing long-term HD and to investigate predictors of reduced erythrocyte life span. Method Patients with T2D undergoing long-term HD and patients with T2D without nephropathy (control group) (eGFR above 60ml/min) were included. Erythrocyte life span was measured using Chromium-51 (51Cr) labelled erythrocytes. Blood radiotracer activity was measured 6-8 times over 3-5 weeks to calculate the erythrocyte half-life for each patient. Markers for haemolysis, blood pressure and time spend in dialysis were obtained 5 times over 16 weeks and correlated to the erythrocyte half-life. Results For patients with T2D undergoing HD (n=13), the median half-life was 34.5 days (30.6 - 40.7) compared with 44.6 days (43.5 - 58.0) in the control group (n=10) corresponding to a 23 % difference (p=0.0003). From the half-life, the median erythrocyte life span was estimated to 49.7 days (44.1-58.6) and 64.2 days (62.6-83.5), respectively. For patients with T2D undergoing HD no correlation was found between the erythrocyte half-life and markers of haemolysis, blood pressure or time spend in dialysis. Conclusion The half-life of erythrocytes was reduced by 23 % in patients with T2D undergoing long-term HD compared with a control group. This reflects that erythrocyte life span is reduced in these patients. No specific markers of haemolysis could be correlated to the findings.

Bacterial neuraminidase-mediated erythrocyte desialylation provokes cell surface aminophospholipid exposure.

Surface desialylation is associated with erythrocyte aging and mediates phagocytic recognition and clearance of senescent erythrocytes. Neuraminidases, a family of glycohydrolytic enzymes, cleave the glycosidic linkages between sialic acid and mucopolysaccharides and have previously been implicated in erythrocyte dysfunction associated with sepsis. Erythrocytes in septic patients further display a phenotype of accelerated eryptosis characterized by membrane phospholipid scrambling resulting in phosphatidylserine (PS) externalization. Herein, we examined the impact of artificial erythrocyte desialylation on eryptosis. Using flow cytometry and/or fluorescence microscopy, we analyzed desialylation patterns and eryptotic alterations in erythrocytes exposed to Clostridium perfringens-derived neuraminidase. Exogenous bacterial neuraminidase significantly augmented membrane PS exposure and cytosolic Ca2+ levels in a dose- and time-dependent manner. Neuraminidase treatment significantly reduced fluorescence-tagged agglutinin binding, an effect temporally preceding the increase in PS externalization. Neuraminidase-induced PS exposure was significantly curtailed by pretreatment with the pan-sialidase inhibitor N-acetyl-2,3-dehydro-2-deoxyneuraminic acid. Neuraminidase treatment further induced hemolysis but did not significantly impact erythrocyte volume, ceramide abundance, or the generation of reactive oxygen species. Collectively, our data reveal that alteration of erythrocyte sialylation status by bacterial neuraminidase favors eryptotic cell death, an effect potentially contributing to reduced erythrocyte lifespan and anemia in sepsis.

Mice Deficient in the Putative Phospholipid Flippase ATP11C Exhibit Altered Erythrocyte Shape, Anemia, and Reduced Erythrocyte Life Span*♦

Transmembrane lipid transporters are believed to establish and maintain phospholipid asymmetry in biological membranes; however, little is known about the in vivo function of the specific transporters involved. Here, we report that developing erythrocytes from mice lacking the putative phosphatidylserine flippase ATP11C showed a lower rate of PS translocation in vitro compared with erythrocytes from wild-type littermates. Furthermore, the mutant mice had an elevated percentage of phosphatidylserine-exposing mature erythrocytes in the periphery. Although erythrocyte development in ATP11C-deficient mice was normal, the mature erythrocytes had an abnormal shape (stomatocytosis), and the life span of mature erythrocytes was shortened relative to that in control littermates, resulting in anemia in the mutant mice. Thus, our findings uncover an essential role for ATP11C in erythrocyte morphology and survival and provide a new candidate for the rare inherited blood disorder stomatocytosis with uncompensated anemia.

Anemia, Shortened Erythrocyte Lifespan and Stomatocytosis In a Flippase Mutant Mouse Strain

BackgroundMammalian erythrocytes are anucleate biconcave discs with marked flexibility and deformability, features necessary for efficient function. The cell membrane consists of a lipid bilayer containing proteins and an asymmetric distribution of phospholipids (PL). Phosphatidylcholine and sphingomyelin are predominantly concentrated in the outer layer and phosphatidylserine (PS) and phosphatidylethanolamine are mainly confined to the inner layer of the erythrocyte membrane. Maintenance of PL asymmetry is essential for erythrocyte survival and function as increased externalization of PS results in adherence of erythrocytes to vascular endothelium, activation of plasma blood clotting factors and premature removal from the circulation. Flippases, floppases and scramblases are the enzymes responsible for the establishment and maintenance of PL distribution.A mouse deficient in the flippase ATP11C was generated through ENU mutagenesis and was found to have reduced hemoglobin in comparison to wild type (WT) littermates. This study was performed to characterize the etiology of anemia in these mice. ResultsATP11C-deficient mice had significant reductions in erythrocyte numbers and hematocrit compared to WT but higher MCH and MCV. Reticulocyte numbers were comparable to WT as were serum iron parameters. Bone marrow and splenic erythropoiesis was normal in ATP11C mutant mice, however, erythrocyte lifespan was reduced by 35%. A marked increase in the frequency of PS+ erythrocytes was demonstrated in ATP11C mutant mice and was shown to increase with erythrocyte age. Bone marrow and splenic erythroblasts from ATP11C-deficient animals displayed a lower rate of PS translocation in vitro compared to WT confirming defective flippase activity.Erythrocytes and late-stage splenic erythroblasts in ATP11C mutants were significantly larger than WT based on flow cytometry. SEM revealed the majority of mutant erythrocytes displayed stomatocyte-like morphology, as confirmed on peripheral blood smears. The osmotic fragility of the ATP11C-deficient erythrocytes was comparable to WT erythrocytes as was Na+ and K+ homeostasis. DiscussionThese studies reveal the important role of flippases in maintaining normal erythrocyte function through the maintenance of the asymmetric distribution of PS within the membrane. Perturbation of this process, as seen in the ATP11C-deficient mice, results in (i) increased exposure of PS on the erythrocyte outer membrane, (ii) increased size of late-stage erythroblasts and erythrocytes with stomatocyte formation (iii) reduced erythrocyte lifespan and (iv) normochromic anemia. This study, therefore, reveals a new mechanism for stomatocytosis and raises the question of whether mutations in ATP11C may serve as a previously unclassified cause of anemia in humans. Disclosures:No relevant conflicts of interest to declare.

Ribavirin-Induced Anemia in Hepatitis C Virus Patients Undergoing Combination Therapy

The current standard of care for hepatitis C virus (HCV) infection – combination therapy with pegylated interferon and ribavirin – elicits sustained responses in only ∼50% of the patients treated. No alternatives exist for patients who do not respond to combination therapy. Addition of ribavirin substantially improves response rates to interferon and lowers relapse rates following the cessation of therapy, suggesting that increasing ribavirin exposure may further improve treatment response. A key limitation, however, is the toxic side-effect of ribavirin, hemolytic anemia, which often necessitates a reduction of ribavirin dosage and compromises treatment response. Maximizing treatment response thus requires striking a balance between the antiviral and hemolytic activities of ribavirin. Current models of viral kinetics describe the enhancement of treatment response due to ribavirin. Ribavirin-induced anemia, however, remains poorly understood and precludes rational optimization of combination therapy. Here, we develop a new mathematical model of the population dynamics of erythrocytes that quantitatively describes ribavirin-induced anemia in HCV patients. Based on the assumption that ribavirin accumulation decreases erythrocyte lifespan in a dose-dependent manner, model predictions capture several independent experimental observations of the accumulation of ribavirin in erythrocytes and the resulting decline of hemoglobin in HCV patients undergoing combination therapy, estimate the reduced erythrocyte lifespan during therapy, and describe inter-patient variations in the severity of ribavirin-induced anemia. Further, model predictions estimate the threshold ribavirin exposure beyond which anemia becomes intolerable and suggest guidelines for the usage of growth hormones, such as erythropoietin, that stimulate erythrocyte production and avert the reduction of ribavirin dosage, thereby improving treatment response. Our model thus facilitates, in conjunction with models of viral kinetics, the rational identification of treatment protocols that maximize treatment response while curtailing side effects.

Simultaneous Loss of Mouse Dematin and beta-Adducin Results in Severe Erythrocyte Fragility and Hemolytic Anemia.

The mechanical strength and stability of the erythrocyte membrane are regulated by a network of proteins that participate in both horizontal and vertical interactions. The actin-containing junctional complexes, located at the tail ends of spectrin molecules, serve as the critical regulatory nodes for the maintenance of membrane stability. Dematin and beta-adducin, two actin-binding proteins of the junctional complex, are known to play essential roles in the regulation of erythrocyte shape and membrane stability, as revealed recently by the development of mouse knockout models. Here, we show that simultaneous loss of functional dematin (headpiece domain deletion) and beta-adducin results in severe fragility and abnormal shape of erythrocytes, despite the presence of major skeletal proteins. Adducin/Dematin Double Knockout (ADKO) mice are viable and can be distinguished at birth by their pallor with pronounced spleomegaly and regenerative hematopoiesis. Hematological evaluations show a reduction of erythrocytes, reduced hematocrit and hemoglobin, and a ~52% increase in the number of reticulocytes. The presence of a variety of misshapen and fragmented erythrocytes in the ADKO mice correlates with increased osmotic fragility and reduced erythrocyte life span in vivo. Despite an apparently normal composition of ghosts and skeletal proteins, the retention of spectrin in the ADKO erythrocyte plasma membrane is significantly compromised. Atomic force microscopy (AFM) revealed similar volume parameters in the four genotypes examined, but an increased grain size, and a decreased filament number in the ADKO erythrocyte membrane. In addition, highly aggregated, disassembled, and irregular features were visualized by AFM in the ADKO erythrocyte membrane. Staining of filamentous actin provided further evidence for the existence of large protein aggregates in the ADKO erythrocyte membrane. Together, these results demonstrate a crucial function of dematin and beta-adducin in the maintenance of erythrocyte shape and membrane stability, and more importantly, suggest the existence of an alternate mechanism for the linkage of junctional complexes to the plasma membrane.

HDL cholesterol levels are an important factor for determining the lifespan of erythrocytes

Scavenger receptor class B, type I (SR-BI) is a multifunctional receptor that promotes the selective uptake of cholesteryl esters from high-density lipoprotein (HDL). Disruption of SR-BI in mice results in a dramatic increase in HDL cholesterol. Interestingly, mice lacking SR-BI also develop anemia, as evidenced by accumulation of reticulocytes in the circulation. The objective of the current study was to delineate the mechanism underlying development of anemia in the absence of SR-BI. Expression of important mediators of erythropoiesis, as well as key enzymes in the degradation of erythrocytes, were analyzed using real-time polymerase chain reaction in SR-BI wild-type and SR-BI knockout mice. In addition, in vivo studies were performed using biotinylated erythrocytes to determine erythrocyte survival. mRNA expression of TAL-1, GATA-1, FOG-1, erythropoietin receptor, and ferrochelatase, important mediators of erythropoiesis, was increased in spleens of SR-BI-deficient mice. In addition, the relative amount of early Ter119(high)CD71(high) -expressing erythroblasts was increased in SR-BI-deficient spleens. Interestingly, also expression of hemeoxygenase 1 and biliverdin reductase, enzymes involved in the degradation of erythrocytes, was increased. Furthermore, an elevated amount of conjugated bilirubin, the breakdown product of hemoglobin, was found in bile. Using biotinylated erythrocytes, we show that survival of erythrocytes was decreased in SR-BI-deficient mice. Thus, the observed increased erythropoiesis in the SR-BI-deficient mice is most likely a direct response to the reduced erythrocyte lifespan. Finally, we show that increased HDL cholesterol levels due to SR-BI deficiency induce erythrocyte cholesterol:phospholipid ratios, resulting in decreased deformability and increased osmotic fragility, thereby providing an explanation for the observed reduced lifespan. SR-BI is not only essential for HDL cholesterol homeostasis and atherosclerosis susceptibility, but also for maintaining normal erythrocyte lifespan.

Some aspects of erythrocyte metabolism in insulin-treated diabetic dogs

Insulin-dependent diabetes mellitus (iddm) is a common metabolic disease often complicated by a number of pathological conditions among which are haematological changes and alterations in blood cell function. Human and feline diabetes mellitus patients have been reported to be associated with oxidative stress that can lead to membrane alterations and to reduced erythrocyte life-span. Erythrocyte function in dogs affected by iddm has been investigated during insulin therapy, paying attention to antioxidant status, membrane resistance, enzyme activities and 2,3-diphosphoglycerate (2,3dpg) concentration. Thirteen diabetic and 36 healthy dogs were bled and haematology and blood chemistry assays were performed to evaluate the degree of compensation. Osmotic fragility, the activities of the enzymes glucose-6-phosphate dehydrogenase (G6PD) and pyruvate-kinase (pk) and the concentrations of reduced glutathione (gsh) and 2,3DPG were evaluated in the erythrocytes. Diabetic dogs did not differ from controls in terms of haematological parameters, except for higher numbers of platelets. Higher values of fructosamine, glucose, protein, plasma potassium and calculated osmolality were detected in the plasma from diabetic dogs. No differences were detected in osmotic fragility, gsh concentration and pk activity between the two groups but 2,3DPG concentration and G6PD activity were statistically significantly higher in the diabetic group. The results indicate minimal alterations in erythrocyte functions occur in insulin-treated diabetic dogs. This contrasts with what has been reported foriddm humans and cats.

Viral-associated haemophagocytosis with parvovirus-B19-related pancytopenia

Viral-associated haemophagocyte syndrome in response to infection with human parvovirus B19 was seen in 2 patients with hereditary spherocytosis. Depressed reticulocyte response during acute parvovirus infection is a known cause of hypoproliferative crises in patients with reduced erythrocyte lifespan; the observation of parvovirus-associated haemophagocytosis could account for the pancytopenia that may accompany human parvovirus B19 infection.

Haemolysis in patients with chronic lymphocytic leukaemia during extracorporeal irradiation of the blood.

Erythrocyte destruction was studied in six patients with chronic lymphocytic leukaemia treated with extracorporeal irradiation of the blood (ECI). Before treatment all patients were anaemic. 51Cr‐labelling showed an apparent half‐life of 14 to 19 days indicating a reduced erythrocyte life span. 59Fe‐data were suggestive of random destruction of erythrocytes in four of the six patients. During the later part of the ECI period considerable reticulocytosis developed, and increased disappearance rate of 51Cr‐label was evident after a mean cumulative erythrocyte dose between 18,500 and 34,000 rads had been reached. This indicates the occurrence of haemolysis which was most pronounced in the one patient with a slightly positive Coombs' test. The 59Fe‐curves were either unaffected by the irradiation or showed only minor effects; since labelling was performed immediately before treatment, this indicates that young erythrocytes are less radiosensitive.