Shortened Erythrocyte Lifespan Research Articles

Novel indicator of microvascular complications in patients with type 2 diabetes mellitus and shortened erythrocyte lifespan: a multicenter cross-sectional analysis

IntroductionIn this study, we assessed whether the ratio of glucose management index (GMI) to glycated albumin (GA) was linked to microvascular complications in patients with type 2 diabetes mellitus (T2DM) who also possessed a shortened erythrocyte lifespan.MethodsThis study encompassed individuals from the Tianjin Diabetic Retinopathy Screening Cohort who completed continuous glucose monitoring and had an erythrocyte lifespan of under 90 days. Differences in GMI/GA were compared between the T2DM patients with or without microvascular complications, including diabetic kidney disease (DKD) and diabetic retinopathy (DR). The relationship between GMI/GA and microvascular complications (DKD and/or DR) was assessed by dividing GMI/GA into three groups based on tertiles.ResultsOur study comprised 140 participants with T2DM (62 men and 78 women, with a median age of 67 years) with a median DM duration of 9.68 years, a mean glycated hemoglobin A1c (HbA1c) value of 7.10%, and a median GA value of 16.10%. As expected, the lower GMI/GA group exhibited higher HbA1c and GA (P < 0.001) with similar mean glucose levels (P = 0.099). GMI/GA values were significantly higher in participants without microvascular complications than in those with microvascular complications, including DKD and/or DR (P < 0.05). After adjusting for confounders, the lowest GMI/GA group (T1) had a 3.601-fold increased risk of microvascular complications (95% CI, 1.364–9.508, P = 0.010) and a 3.830-fold increased risk of DKD, specifically (95% CI, 1.364–12.222, P = 0.023) relative to the highest group (T3).ConclusionGMI/GA serves as a novel risk indicator for microvascular complications in T2DM, independent of HbA1c.

A-100 Clinical Utility of Measuring Creatine Concentration in Erythrocyte

Abstract Background Shortened erythrocyte lifespan characterizes hemolytic anemia and impacts HbA1c values. Recently, erythrocyte creatine concentration (EC) measurement emerged as a new method to assess erythrocyte lifespan. However, literature reports conflicting findings regarding the association between erythrocyte lifespan and EC concentration. In our study, we noted low EC concentrations in renal diseases, typically linked to short erythrocyte lifespan, while iron deficiency anemia exhibited high EC concentrations. This contradicts established relationships, prompting a reevaluation of EC concentration assessment. The aim is to evaluate the clinical utility of EC concentration measurements by correlating them with various clinical parameters, including hematopoietic status. Methods EC concentrations were measured in 522 subjects (252 females, 270 males) aged 19 to 95 at Kawasaki Medical School Hospital. Medical records and laboratory test results from around one month before and after blood collection were used to create a database. Box-and-whisker plots illustrated EC concentration distribution for each disease, and scatter plots compared EC concentrations with laboratory parameters. Results EC concentrations were lower in renal diseases like Diabetes and Chronic Kidney Disease but higher in iron deficiency anemia (Fig. 1). Moreover, EC concentrations rose as hemoglobin concentrations declined, unaffected by CKD grade (Fig. 2). Conclusions While conventionally, high EC concentrations imply a short erythrocyte lifespan, our study noted the opposite trend. EC concentrations might not indicate erythrocyte lifespan but instead reflect hemoglobin synthesis. Investigating EC concentration's relationship with factors like iron and vitamin B12 involved in hemoglobin synthesis may unveil new clinical applications.

Erythroferrone exacerbates iron overload and ineffective extramedullary erythropoiesis in a mouse model of β-thalassemia.

β-thalassemia is characterized by chronic hepcidin suppression and iron overload, even in patients who have not undergone transfusion. The HbbTh3/+ (Th3/+) mouse model of nontransfusion-dependent β-thalassemia (NTDBT) partially recapitulates the human phenotype but lacks chronic hepcidin suppression, progressive iron accumulation into adulthood, or the interindividual variation of the rate of iron loading observed in patients. Erythroferrone (ERFE) is an erythroid regulator that suppresses hepcidin during increased erythropoiesis. ERFE concentrations in the sera of patients with NTDBT correlate negatively with hepcidin levels but vary over a broad range, possibly explaining the variability of iron overload in patients. To analyze the effect of high ERFE concentrations on hepcidin and iron overload in NTDBT, we crossed Th3/+ mice with erythroid ERFE-overexpressing transgenic mice. Th3/ERFE-transgenic mice suffered high perinatal mortality, but embryos at E18.5 showed similar viability, appearance, and anemia effects as Th3/+ mice. Compared with Th3/+ littermates, adult Th3/ERFE mice had similarly severe anemia but manifested greater suppression of serum hepcidin and increased iron accumulation in the liver, kidney, and spleen. The Th3/ERFE mice had much higher concentrations of serum ERFE than either parental strain, a finding attributable to both a higher number of erythroblasts and higher production of ERFE by each erythroblast.Th3/+ and Th3/ERFE mice had similar red blood cell count and shortened erythrocyte lifespan, but Th3/ERFE mice had an increased number of erythroid precursors in their larger spleens, indicative of aggravated ineffective extramedullary erythropoiesis. Thus, high ERFE concentrations increase the severity of nontransfusional iron overload and ineffective erythropoiesis in thalassemic mice but do not substantially affect anemia or hemolysis.

Morphological Types of Anemia Associated with Chronic Renal Diseases

Introduction: Anemia develops early in the course of renal disease and worsens as kidney function declines; a typical consequence of CKD is caused by a disruption in erythropoietin production as well as caused by iron and vitamin shortages, blood loss, shortened erythrocyte life span, chronic inflammation, and the uremic environment. Aim: Our study aimed to identify and classify different types of anemia that complicated patient with chronic kidney disease. Methods: 40 patient were included in this study, 32 males (80%) and 8 females (20%), the age range was 40-70 for male and 48-68 for female .Depending on clinical sign, symptoms and laboratory diagnostic test All those patient are diagnosed previously as a cases of chronic renal failure by clinicians in our city and develop anemia during the course of diseases, so clinicians referred patients to our laboratory to find the cause of anemia. Depending’s on hematological laboratory data, peripheral blood smear examination from these 40 patients, which showed different types of anemia. Results: the most common types of anemia was Normochromic anemia (62.5 %) which develop in 25 patients followed by hypochromic microcytic anemia (8 %) which develop in 8 patients, and followed by macrocytic anemia (12.5 %) which develop in 5 patients and the less common type of anemia was hemolytic anemia (5 %) which was founded in 2 patients.. Conclusion: All types of anaemia can be detected in-patient with different types of renal diseases and it’s important to used different type of laboratory test to diagnosed type of anemia to treat it.

Hemoglobin A1c and Fructosamine Evaluated in Patients with Type 2 Diabetes Receiving Peritoneal Dialysis Using Long-Term Continuous Glucose Monitoring

Introduction: Shortened erythrocyte life span and erythropoietin-stimulating agents may affect hemoglobin A_1c (HbA_1c) levels in patients receiving peritoneal dialysis (PD). We compared HbA_1c with interstitial glucose measured by continuous glucose monitoring (CGM) in patients with type 2 diabetes receiving PD. Methods: Fourteen days of CGM (Ipro2, Medtronic) were performed in 23 patients with type 2 diabetes receiving PD and in 23 controls with type 2 diabetes and an estimated glomerular filtration rate over 60 mL/min/1.73 m². Patients were matched on gender and age (±5 years). HbA_1c (mmol/mol), its derived estimate of mean plasma glucose (eMPG_A1c) (mmol/L), and fructosamine (µmol/L) were measured at the end of the CGM period and compared with the mean sensor glucose (mmol/L) from CGM. Results: In the PD group, mean sensor glucose was 0.98 (95% con­fidence interval (CI): 0.43–1.54) mmol/L higher than the eMPG_A1c compared with the control group (p = 0.002) where glucose levels were nearly identical (−0.05 (95% CI: −0.35–0.25) mmol/L). A significant association was found between fructosamine and mean sensor glucose using linear regression with no difference between slopes (p = 0.89) or y-intercepts (p = 0.28). Discussion/Conclusion: HbA_1c underestimates mean plasma glucose levels in patients with type 2 diabetes receiving PD. However, the clinical significance of this finding is undetermined. Fructosamine seems to more accurately reflect glycemic status. CGM or fructosamine could complement HbA_1c to increase the accuracy of glycemic monitoring in the PD population.

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.

Band 3 ectopic expression in colorectal cancer induces an increase in erythrocyte membrane-bound IgG and may cause immune-related anemia.

Autoimmune hemolytic anemia (AIHA) is a rare comorbidity in colorectal cancer (CRC) and has an unknown etiology. Previously, we described an AIHA case secondary to CRC with ectopic band 3 expression. Herein, we investigated ectopic band 3 expression and erythrocyte membrane-bound IgG in a CRC cohort. Between September 2016 and August 2018, 50 patients with CRC and 26 healthy controls were enrolled in the present study. The expression of band 3 and SLC4A1 mRNA was observed in 97% of CRC surgical specimens. Although clinical AIHA was not observed in any patient with CRC, a direct antiglobulin test was positive in 10 of the patients in the CRC group (p = 0.01). Flow cytometry revealed significantly increased erythrocyte membrane-bound IgG among patients with CRC compared to healthy controls (mean ± standard deviation; 38.8 ± 4.7 vs. 29.9 ± 15.6, p = 0.012). Normocytic anemia was observed, including in cases negative for fecal occult blood, suggesting a shortened erythrocyte life-span due to increased membrane-bound IgG. Immunoprecipitation revealed increased anti-band 3 autoantibodies in patients' sera. Mouse experiments recapitulated this phenomenon. We also confirmed that band 3 expression is controlled by 5'AMP-activated protein kinase under hypoxic conditions. These findings increase our understanding of the etiology of cancer-related anemia.

Transglutaminase diseases: from biochemistry to the bedside.

In humans, 9 members of the transglutaminase (TG) family have been identified, of which 8 [factor XIII (FXIII)A and TG1-TG7] catalyze post-translational protein-modifying reactions, and 1 does not (protein 4.2). The TG enzymatic activities considered in our discussion of human disease include deamidation of glutamine (Gln) residues, amine incorporation into Gln residues, and protein crosslinking. Except for TG7, which remains poorly studied, all individual TG members have been correlated with disparate human diseases that arise from either TG function or lack of function. Loss of TG function is associated with numerous orphan diseases that affect a relatively small number of individuals: loss of FXIIIa (transamidase-activated form) crosslinking leads to defects in blood coagulation in FXIII deficiency; loss of TG1 and TG5 cross linking leads to defects in epidermal cornification in lamellar ichthyosis and acral peeling skin syndrome, respectively; loss of TG3 crosslinking in hair-cuticle formation leads to uncombable hair syndrome; the predicted loss of TG6 crosslinking leads to spinocerebellar ataxia-35; and loss of the structural erythrocyte membrane protein, protein 4.2, leads to hereditary spherocytosis type 5. The enzymatic activity of TG2 is involved in the exacerbation of celiac disease and in at least 1 case of hemoglobinopathy, characterized by shortened erythrocyte lifespan. TGs are also autoantigens in a number of immune diseases, resulting in the production of autoantibodies against FXIIIa in acquired FXIII deficiency, TG2 in celiac disease, TG3 in dermatitis herpetiformis, TG4 in autoimmume polyglandular syndrome type 1, and TG6 in gluten axonal neuropathy and gluten ataxia. Much still remains to be learned and confirmed with respect to disease mechanisms, particularly with respect to TG-related immune diseases, in which development of isozyme-specific inhibitors may be useful for treatment.-Lorand, L., Iismaa, S. E. Transglutaminase diseases: from biochemistry to the bedside.

Genetic Modifiers of Fetal Haemoglobin in Sickle Cell Disease.

Fetal haemoglobin (HbF) levels have a clinically beneficial effect on sickle cell disease (SCD). Patients with SCD demonstrate extreme variability in HbF levels (1-30%), a large part of which is likely genetically determined. The main genetic modifier loci for HbF persistence, HBS1L-MYB, BCL11A and the β-globin gene cluster in adults also act in SCD patients. Their effects are, however, modified significantly by a disease pathology that includes a drastically shortened erythrocyte lifespan with an enhanced survival of those red blood cells that carry HbF (F cells). We propose a model of how HbF modifier genes and disease pathology interact to shape HbF levels measured in patients. We review current knowledge on the action of these loci in SCD, their genetic architecture, and their putative functional components. At each locus, one strong candidate for a causative, functional DNA change has been proposed: Xmn1-HBG2 at the β-globin cluster, rs1427407 at BCL11A and the 3bp deletion rs66650371 at HBS1L-MYB. These, however, explain only part of the impact of these loci and additional variants are yet to be identified. Further progress in understanding the genetic control of HbF levels requires that confounding factors inherent in SCD, such as ethnic complexity, the role of F cells and the influence of drugs, are suitably addressed. This will depend on international collaboration and on large, well-characterised patient cohorts with genome-wide single-nucleotide polymorphism or sequence data.

Effects of Packed Red Cell Transfusion on Blood Glucose Concentrations in Beta Thalassemia Major (BTM) Ashort Presentations of Personal Experience

The most accurate method with which to evaluate altered glucose metabolism in patients with TM is still controversial. Even if the annual oral glucose tolerance test (OGTT) by the age of 10 years is the recommended method, a diagnosis of ’normal’ glucose tolerance during OGTT does not exclude abnormal postprandial glucose levels at home . There is now evidence that the OGTT method, evaluating fasting and 2-h post load glucose, may miss episodes of hyperglycaemia . Furthermore, the credibility of Hb A1c has been questioned because the hemoglobin composition of patients’ erythrocytes are considerably modified, due to regular and frequent transfusions. The results may be falsely increased or decreased depending on the proximity to transfusion, shortened erythrocyte lifespan and the assay used . It has been demonstrated recently that the continuous glucose monitoring system (CGMS) is a useful and valid tool in defining glucose metabolism in children and adults affected by TM with early glucose derangements . Indeed, the CGMS allows monitoring of glycaemic profiles throughout a period of 72 h for a total of 288 glycaemic registrations per day. It identifies glycaemic excursions and constitutes a valid device to understand the 24-h glycaemic trend and profiles. Rimondiet al. investigated the value of using CGMS in six TM patients with abnormal glucose homeostasis after an oral glucose tolerance test (OGTT) . Two-hour OGTT glucose values and CGMS fluctuations were classified as normal if < 7.8 mmol/l, impaired if 7.8 to 11.1 mmol/l, diabetic if > 11.1 mmol/l. The TM patients spent from 1 to 23% of the time with a blood glucose level from 7.8 to 11.1 mmol/l. we evaluate three patients with Beta Thalassemai major using CGMSPatient 1 A 15 year old male with TM presented with nocturia. His FBG was 5.6 mmol/L and OGTT showed a BG level at 2hrs of 8.5 mmol/l. His CGMS showed a diabetic range of BG after dinner and overnight. Based on this tracing, a basal insulin (Glargine) was prescribed at night. A satisfactory response was recorded by CGMS .Patient 2 A 14 year old girl with TM with no symptoms related to glycemic abnormalities. Her FBG was 4.9 mmol/L and an OGTT showed a BG level at 2 hrs of 6.9 mmol/L (IGT). CGMS tracing showed prolonged persistent hyperglycemia after lunch suggesting a need for prandial insulin to cover her carbohydrate load. Insulin aspart before lunch properly controlled her glycemia . Patient 3 A 13 year old boy with TM had a normal FBG (4.5 mmol/L) and OGTT (2h BG =7.6 mmo/l). Applying CGMS monitoring showed IGT state. Furthermore, the effect of packed red cell transfusion showed marked reduction of his BG before meals and overnight. This is the first case to document the rapid beneficial effect of blood transfusion on glycemia in patiens with Beta thalssemia Major Advances in TM care have led to improved survival and quality of life in patients with TM; however, many chronic endocrine diseases have emerged as a result of potential endocrine complications by routine screening and a high index of suspicion is imperative for patients with TM to receive timely treatment.Our results demonstrate that the CGMS is a useful method to detect the variability of glucose fluctuations and offers the opportunity for better assessment of glucose homeostasis in TM patients. Proper and early iron chelation or the use of intensive iron chelation in those with high iron load the new oral chelators have been shown to decrease or reversethese glycemic abnormalities.In addition, optimization of insulin therapy with the help of CGMS appears to be clinically useful and costeffective approach. DisclosuresNo relevant conflicts of interest to declare.

Shp2 and Pten have antagonistic roles in myeloproliferation but cooperate to promote erythropoiesis in mammals

Previous data suggested a negative role of phosphatase and tensin homolog (Pten) and a positive function of SH2-containing tyrosine phosphatase (Shp2)/Ptpn11 in myelopoiesis and leukemogenesis. Herein we demonstrate that ablating Shp2 indeed suppressed the myeloproliferative effect of Pten loss, indicating directly opposing functions between pathways regulated by these two enzymes. Surprisingly, the Shp2 and Pten double-knockout mice suffered lethal anemia, a phenotype that reveals previously unappreciated cooperative roles of Pten and Shp2 in erythropoiesis. The lethal anemia was caused collectively by skewed progenitor differentiation and shortened erythrocyte lifespan. Consistently, treatment of Pten-deficient mice with a specific Shp2 inhibitor suppressed myeloproliferative neoplasm while causing anemia. These results identify concerted actions of Pten and Shp2 in promoting erythropoiesis, while acting antagonistically in myeloproliferative neoplasm development. This study illustrates cell type-specific signal cross-talk in blood cell lineages, and will guide better design of pharmaceuticals for leukemia and other types of cancer in the era of precision medicine.

PRKAA1/AMPKα1 is required for autophagy-dependent mitochondrial clearance during erythrocyte maturation

AMP-activated protein kinase α1 knockout (prkaa1−/−) mice manifest splenomegaly and anemia. The underlying molecular mechanisms, however, remain to be established. In this study, we tested the hypothesis that defective autophagy-dependent mitochondrial clearance in prkaa1−/− mice exacerbates oxidative stress, thereby enhancing erythrocyte destruction. The levels of ULK1 phosphorylation, autophagical flux, mitochondrial contents, and reactive oxygen species (ROS) were examined in human erythroleukemia cell line, K562 cells, as well as prkaa1−/− mouse embryonic fibroblasts and erythrocytes. Deletion of Prkaa1 resulted in the inhibition of ULK1 phosphorylation at Ser555, prevented the formation of ULK1 and BECN1- PtdIns3K complexes, and reduced autophagy capacity. The suppression of autophagy was associated with enhanced damaged mitochondrial accumulation and ROS production. Compared with wild-type (WT) mice, prkaa1−/− mice exhibited a shortened erythrocyte life span, hemolytic destruction of erythrocytes, splenomegaly, and anemia, all of which were alleviated by the administration of either rapamycin to activate autophagy or Mito-tempol, a mitochondria-targeted antioxidant, to scavenge mitochondrial ROS. Furthermore, transplantation of WT bone marrow into prkaa1−/− mice restored mitochondrial removal, reduced intracellular ROS levels, and normalized hematologic parameters and spleen size. Conversely, transplantation of prkaa1 −/− bone marrow into WT mice recapitulated the prkaa1−/− mouse phenotypes. We conclude that PRKAA1-dependent autophagy-mediated clearance of damaged mitochondria is required for erythrocyte maturation and homeostasis.

A mouse model of anemia of inflammation: complex pathogenesis with partial dependence on hepcidin

AbstractAnemia is a common complication of infections and inflammatory diseases, but the few mouse models of this condition are not well characterized. We analyzed in detail the pathogenesis of anemia induced by an injection of heat-killed Brucella abortus and examined the contribution of hepcidin by comparing wild-type (WT) to iron-depleted hepcidin-1 knockout (Hamp-KO) mice. B abortus–treated WT mice developed severe anemia with a hemoglobin nadir at 14 days and partial recovery by 28 days. After an early increase in inflammatory markers and hepcidin, WT mice manifested hypoferremia, despite iron accumulation in the liver. Erythropoiesis was suppressed between days 1 and 7, and erythrocyte destruction was increased as evidenced by schistocytes on blood smears and shortened red blood cell lifespan. Erythropoietic recovery began after 14 days but was iron restricted. In B abortus–treated Hamp-KO compared with WT mice, anemia was milder, not iron restricted, and had a faster recovery. Similarly to severe human anemia of inflammation, the B abortus model shows multifactorial pathogenesis of inflammatory anemia including iron restriction from increased hepcidin, transient suppression of erythropoiesis, and shortened erythrocyte lifespan. Ablation of hepcidin relieves iron restriction and improves the anemia.

L-carnosine alters some hemorheologic and lipid peroxidation parameters in nephrectomized rats

BackgroundChronic kidney disease (CKD) is a major health problem worldwide. Oxidative stress is one of the mediators of this disease. Systemic complications of oxidative stress are involved in the pathogenesis of hypertension, endothelial dysfunction, shortened erythrocyte lifespan, deformability, and nitric oxide (NO) dysfunction. L-carnosine is known as an antioxidant. In this study, our aim was to investigate the effect of carnosine on hemorheologic and cardiovascular parameters in CKD-induced rats.Material/MethodsWe used 4-month-old male Sprague-Dawley rats divided into 4 groups of 6 rats each. Three days after subtotal nephrectomy and sham operations, the surviving rats were divided into the 4 groups; 1) Sham (S), 2) Sham+Carnosine (S-C), 3) Subtotal nephrectomy (Nx), and 4) Subtotal nephrectomy + Carnosine (N-C). Carnosine was injected intraperitoneally (i.p.) (50 mg/kg) for 15 days. The control group received the same volume of physiological saline.ResultsIn CKD rats, malondialdehyde (MDA) levels were increased, and NO and RBC deformability were decreased compared to Sham. Carnosine treatment decreased MDA levels, improved RBC (red blood cell) ability to deform, and increased NO levels. However, carnosine did not affect blood pressure levels in these rats.ConclusionsWe found that carnosine has beneficial effects on CKD in terms of lipid peroxidation and RBC deformability. Carnosine may have a healing effect in microcirculation level, but may not have any effect on systemic blood pressure in CKD-induced rats.

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.

Unreliable oral glucose tolerance test and haemoglobin A1C in beta thalassaemia major – a case for continuous glucose monitoring?

Unreliable oral glucose tolerance test and haemoglobin A1C in beta thalassaemia major – a case for continuous glucose monitoring?

Point-of-Care Measurements of HbA1c: Simplicity Does Not Mean Laxity With Controls

Point-of-care HbA1c measurements (POC-A1Cs) have been adopted by many diabetes clinics to improve the quality of care provided to their patients (1). Herein, we show that reliability of this approach might be questioned. POC-A1Cs routinely used in the ambulatory section of our diabetes clinic was evaluated on 100 diabetic patients (type 1, n = 58; type 2, n = 42) attending the clinic from 1 October 2011 to 30 November 2011. Patients with abnormal hemoglobin traits or shortened erythrocyte life span were excluded. Blood-capillary samples were analyzed by POC-A1C (DCA Vantage; Siemens Medical Solutions Diagnostics, Cergy-Pontoise, France) and venous EDTA-anticoagulated blood specimens by the central laboratory high-performance liquid chromatography measurement (Tosoh HLC-723 GHb G8; BioSciences, Lyon, France). Both methods were certified (NGSP/Diabetes Control and Complications Trial [DCCT] and International Federation of Clinical …

Shortened Erythrocyte Life Span and Increased Oxidative Stress In Erythroid Precursors Are Consistent with Normocytic Anemia In Mice with Chronic Inflammation

AbstractAbstract 3205The anemia of inflammation is a common co-morbid condition associated with various infections, autoimmune disorders, and other chronic disease states. In general, hemoglobin levels are mildly to moderately decreased. In vitro studies have demonstrated that pro-inflammatory cytokines can inhibit erythropoiesis at various stages of development, but many of these studies are limited to the analysis of early developmental stages and primarily assess proliferation as opposed to maturation or survival. Sufficient data from in vivo studies and whole animal models are lacking. We characterized the anemia in 8–10 week old C57BL/6 mice with turpentine oil-induced sterile abscess. We found that following 3 weeks of sterile abscess, the mice had a normocytic, normochromic anemia.Using in vivo biotin-labeling of peripheral blood, we found the coefficient describing erythrocyte life span significantly decreased from -0.044 ± 0.002 SE in control mice to -0.048 ± 0.002 SE in mice with sterile abscess (p = 0.04). In further support of the mechanism of increased erythrocyte turnover, we found that splenic macrophages isolated from mice with abscess significantly increased expression of Heme-regulated gene 1 (Hrg-1) 2.5 fold, (p < .001) and significantly increased expression of Ferroportin (Fpn) 2.76 fold (p = 0.003).Oxidative stress can lead to increased erythrocyte turnover and can inhibit erythroid maturation. To determine whether erythrocytes from mice with sterile abscess had impaired capacity for survival, we assessed staining of peripheral blood and erythroid precursors with chloromethyl dichlorodihydrofluorescein diacetate, acetyl ester (DCF). DCF fluoresces upon oxidation. We found that mean DCF fluorescence intensity (MFI) in peripheral blood increased from 3.2 ± 1.0 in control mice to 5.5 ± 0.6 in mice with abscess (p=0.004). Similarly, we found a greater percentage of bone marrow derived erythroid precursors from mice with abscess had high DCF staining (16.2 ± 3.9%) when compared to control mice (6.7 ± 1.9%, p=0.003). In conclusion, these data support the hypothesis that increased oxidative stress in erythroid precursors of mice with sterile abscess results in increased erythrocyte turnover and normocytic, normochromic anemia. Future studies will investigate whether pro-inflammatory cytokines inhibit the expression of key erythrocyte anti-oxidant enzymes or whether reactive oxygen species from neighboring granulocytes damage erythroid precursors in the bone marrow. Disclosures:No relevant conflicts of interest to declare.

Plasminogen Deficiency Impairs Erythropoietic Recovery Capacity In Male C57BL/6J Mice through Testosterone Defect

AbstractAbstract 3212Plasminogen (Plg), a classical fibrinolytic factor, is essential for maintaining homeostasis of blood coagulation, and has important roles in wound healing as well as pathophysiology of atherosclerosis, cancer and Alzheimer's disease. Recently, we have shown that the activation of the Plg fibrinolytic pathway is critical for hematopoietic regeneration from stem cells after myelosuppression. Here, we investigated the role of Plg in regulating erythropoiesis.Male and female C57BL/6J mice with normal or homozygously disrupted Plg genes were used under steady state conditions and in a model of acute erythropoietic stress selectively induced by intravenous injection of the hemolytic drug phenylhydrazine hydrochloride. Orbital vein blood, bone marrow and spleen were obtained to determine erythropoietic parameters. In addition, testosterone concentrations were measured in murine serum.Normochromic normocytic anemia was found in Plg-/- mice under steady state conditions. Microscopic evaluation of blood smears revealed the presence of altered erythrocytes, such as acanthocytes, echinocytes (burr cells) and to lesser extent schistocytes. In addition, flow cytometry of intravenously injected autologous erythrocytes, labeled with a fluorescent probe, showed significantly shortened erythrocyte lifespan, suggestive of hemolysis as a cause of the anemia. In concordance, there was an increase in plasma erythropoietin with augmented reticulocyte counts, erythroid burst forming units in bone marrow, and finally erythroid progenitors in spleens, which were significantly enlarged, suggestive of extramedullar erythropoiesis. Interestingly, the intensity of compensatory erythropoiesis was higher in Plg-/- female mice, resulting in lesser hematocrit decrease when compared to that observed in Plg-/- male mice. In addition, similar gender differences could be observed in a model of acute erythropoietic stress. Concretely, acute hemolytic anemia induced by intravenous injection of phenylhydrazine hydrochloride resulted in a significant erythrocyte recovery delay in Plg-/- male mice that, in contrast, was not as profound in Plg-/- female mice. To understand the reason for the observed gender differences, a competitive enzyme immunoassay was employed to measure serum concentrations of testosterone. As a result, a significant decrease in serum testosterone levels was detected in Plg-/- male mice.In summary, we found that Plg plays important role in maintaining normal lifespan of erythrocytes by preventing their mechanical alteration by microangiopathy from abnormal fibrin deposition. In addition, given that testosterone is well known to regulate erythropoiesis, through increasing erythropoietin levels as well as having non-erythropoietin stimulating effects, our present results suggest that Plg might also play roles in regulating erythropoiesis through being important for normal testosterone production. Disclosures:No relevant conflicts of interest to declare.

Association between Markers of Glycemic Control, Cardiovascular Complications and Survival in Type 2 Diabetic Patients with End-Stage Renal Disease

The influence of glycemic control on cardiovascular (CV) complications or survival is not clear in diabetic patients with end-stage renal disease (ESRD). Although glycohemoglobin (HbA1c) is widely used as a marker of hyperglycemia in these patients, it may be unreliable because of shortened erythrocyte lifespan. Glycated albumin (GA) is an alternative marker. We investigated the relation between these markers and development of CV complications or survival in diabetic ESRD patients. We obtained three variables as markers of glycemic control: 1) mean HbA1c levels during 1-year after initiation of dialysis (HbA1c1), 2) mean HbA1c levels during 3 months from August to October 2002 (HbA1c2), 3) GA on October 2002 (GA2) from 78 type 2 diabetic patients on chronic hemodialysis. We examined the influence of these variables on survival or development of CV diseases using the multivariate Cox proportional-hazards models until September 2006. The 3-year survival rate was 73%. A total of 27 patients died, 15 from CV diseases. A total of 23 CV diseases developed in 20 patients. Neither HbA1c1 nor HbA1c2 was associated with all-cause mortality, CV mortality or development of CV diseases. GA2 was also not associated with mortality. However, the higher GA2 group (GA > or = 23.0%) had a significantly higher rate of development of CV diseases than the lower GA2 group (GA < 23.0%) (log-rank test p=0.03). The higher GA2 group was significantly associated with development of CV diseases relative to the lower GA2 group (hazard ratio 3.25, p=0.04). Neither HbA1c levels nor GA levels, at initiation of dialysis or on chronic dialysis, predicted mortality in diabetic ESRD patients. However, poor glycemic control as reflected by higher GA levels may be associated with the development of CV diseases. More studies are needed to clarify the beneficial effect of glycemic control in these patients.