Red Blood Cell Metabolism Research Articles

Irradiation Causes Alterations of Polyamine, Purine, and Sulfur Metabolism in Red Blood Cells and Multiple Organs.

Investigating the metabolic effects of radiation is critical to understand the impact of radiotherapy, space travel, and exposure to environmental radiation. In patients undergoing hemopoietic stem cell transplantation, iron overload is a common risk factor for poor outcomes. However, no studies have interrogated the multiorgan effects of these treatments concurrently. Herein, we use a model that recapitulates transfusional iron overload, a condition often observed in chronically transfused patients. We applied an omics approach to investigate the impact of both the iron load and irradiation on the host metabolome. The results revealed dose-dependent effects of irradiation in the red blood cells, plasma, spleen, and liver energy and redox metabolism. Increases in polyamines and purine salvage metabolites were observed in organs with high oxygen consumption including the heart, kidneys, and brain. Irradiation also impacted the metabolism of the duodenum, colon, and stool, suggesting a potential effect on the microbiome. Iron infusion affected the response to radiation in the organs and blood, especially in erythrocyte polyamines and spleen antioxidant metabolism, and affected glucose, methionine, and glutathione systems and tryptophan metabolism in the liver, stool, and the brain. Together, the results suggest that radiation impacts metabolism on a multiorgan level with a significant interaction of the host iron status.

New approaches to the typology and prediction of the effectiveness of therapy for late-life depression

To analyze clinical parameters and effectiveness of therapy in depressed patients of late age with different activity of enzymes of energy, glutamate metabolism and antioxidant glutathione system in platelets and red blood cells. The study included 53 hospitalized patients (41 women and 12 men), aged 60 to 86 years (median - 66 years), with a depressive episode of mild to marked severity within the framework of recurrent depressive disorder, bipolar affective disorder or a single depressive episode according to ICD-10. The patients were examined using clinical, psychometric, biochemical and statistical methods. Psychometric assessment of patients' condition was performed before the start of therapy and on the 28th day of treatment using HAMD-17 and HARS. Blood sampling was carried out to determine the activity of cytochrome c-oxidase (COX), glutathione reductase (GR), glutathione S-transferase (GST) and glutamate dehydrogenase (GDH). Cluster analysis revealed 3 clusters (Cl.1, Cl.2, Cl.3), which differed in biochemical indicators. In Cl.1 (n=24, 45.2%), there was a decrease in the activity of COX (p<0.05) and a decrease of GR activity in red blood cells and GST activity in platelets (p<0.001). Patients of Cl.2 (n=11, 20.8%) had increased COX activity (p<0.001) and decreased GDH activity (p<0.001). In patients of Cl.3 (n=18, 34%) the enzyme activity was similar to that in the controls. Later age at disease onset was observed in Cl.1 compared to Cl.2 (51 years [40; 60.5] and 32 years [19; 59], p=0.052). Compared with Cl.2 and Cl.3, patients of Cl.1 significantly more often (p<0.05) had apathetic depressions (41.7%), while complex depressions were observed almost 2 times less often than in Cl.2 (50% and 91.9%, p<0.05). The effectiveness and tolerability of psychopharmacotherapy was higher in Cl.3. There is a relationship between the nature of changes in metabolic parameters and differences in the phenomenology and course of late-age depression. The results of the study open up new directions in the field of predicting the effectiveness of therapy and the development of personalized therapeutic approaches to improve the effectiveness and safety of treatment of depressed elderly patients.

Burden of Congenital Haemolytic Anaemias among the Rural Paediatric Population of Central India

Introduction: Childhood anaemia may be caused due to many factors, including malnutrition, chronic infections, deficiency of iron and vitamins, parasitic infections. On haematological work up the most common finding is microcytic hypochromic anaemia. Inherited defects in Red Blood Cell (RBC) and haemoglobin structure and metabolism result in congenital haemolytic anaemia. About 4.2% of all paediatric microcytic anaemias are diagnosed with haemoglobinopathies. Thalessaemia and other haemoglobinopathies have a large representation in the Indian subcontinent. A study of the extent of these hereditary anaemias will help in formulating a targeted approach of spreading awareness and educating couples in target population, who are planning conception. Aim: To comprehend the incidence of congenital haemolytic anaemias in the paediatric population of central India, and to evaluate their clinico-haematological status. Materials and Methods: This cross-sectional observational study was conducted in the Paediatric Anaemia Clinic at Chirayu Medical College and Hospital (teaching, tertiary care hospital), Bhopal, Madhya Pradesh, India, from January 2021 to December 2021. All relevant details regarding clinical history, consanguinity, family history of the patients were recorded. Investigations done included-complete blood count, reticulocyte count, sickling test, Naked Eye Single Tube Red Cell Osmotic Fragility Test (NESTROFT) and High Performance Liquid Chromatography (HPLC). All the relevant data collected was entered into Microsoft Excel and frequency, distribution, standard deviation were analysed and calculated. Results: Amongst the total 1080 cases of microcytic hypochromic anaemia in the Paediatric Anaemia Clinic, there were 55 cases of congenital haemolytic anaemias. Of these 55 cases of congenital haemolytic anaemias, there were 41 (74.54%) cases of thalassaemia (four major and 37 minor thalassaemia), 11 (19.9%) cases of sickle cell anaemia, 1 (1.81%) case of Hereditary Persistence of Foetal Haemoglobin (HPFH) and 2 (3.63%) cases of Haemoglobin J variant (HbJ). The haemoglobin values were the lowest in the β-thalassaemia major patients, followed by sickle cell disease. Foetal Hb (HbF) was highest in β-thalassaemia major at and the HPFH case had foetal haemoglobin of 35%. Conclusion: In the current study, 5.09% of the paediatric population presenting to the clinic were diagnosed with congenital haemolytic anaemias, with major representation of β-thalassaemia, followed by sickle cell trait. This lifelong burden of haemolytic anaemias on the children and their families may be reduced by active counseling and awareness programs.

Profiling and Bioinformatics Analysis Revealing Differential Circular RNA Expression about Storage Lesion Regulatory in Stored Red Blood Cells

Introduction: Circular RNA (circRNA) plays an important role in regulating metabolism of red blood cells (RBCs) and their storage lesions, but the study of how circRNA expression changes in stored RBCs has rarely been conducted. Methods: The expression change of circRNA was systemically evaluated via high-throughput sequencing on healthy RBCs on day 0, 20, and 40. And then we confirmed the reliability of the high-throughput sequencing analysis by RT-qPCR characterization on selected circRNAs. A higher parental gene enrichment was used to explore circRNA function in pathways. In addition, we deciphered a dysregulated circRNA-related ceRNAs network, and identified three circRNA-miRNA-mRNA regulatory axes related to storage lesion. Results: We identified 2,586 known and 6,216 putative novel circRNAs, more than 100 circRNAs expression levels were shifted, and the number of downregulated circRNAs was greater with longer storage time. Furthermore, a higher parental gene enrichment related to circRNA was found in pathways, including cAMP signaling pathway, ubiquitin-mediated proteolysis, apoptosis, adhesion, MAPK signaling pathway, cystine methionine metabolism, RNA degradation, RNA transport, TGF-β, and actin regulatory pathway. hsa_circ_0007127-miR-513a-5p-SMAD4, hsa_circ_0000033-miR-19a-3p-VAMP3, and hsa_circ_0005546-miR-4720-CCND3 regulatory axes related to storage lesion was found. Conclusions: Through investigation in circRNAs profile and circRNA-miRNA-mRNA interactions, this study provides insights on stored RBC circRNA expression changes, which closely relate to the storage lesion of RBCs and their physiological functions.

Adenine Nucleotide Metabolites in Uremic Erythrocytes as Metabolic Markers of Chronic Kidney Disease in Children.

Chronic kidney disease (CKD) is associated with multifaceted pathophysiological lesions including metabolic pathways in red blood cells (RBC). The aim of the study was to determine the concentration of adenine nucleotide metabolites, i.e., nicotinamide adenine dinucleotide (NAD)-oxidized form, nicotinamide adenine dinucleotide hydrate (NADH)-reduced form, nicotinic acid mononucleotide (NAMN), β-nicotinamide mononucleotide (NMN), nicotinic acid adenine dinucleotide (NAAD), nicotinic acid (NA) and nicotinamide (NAM) in RBC and to determine a relationship between NAD metabolites and CKD progression. Forty-eight CKD children and 33 age-matched controls were examined. Patients were divided into groups depending on the CKD stages (Group II-stage II, Group III- stage III, Group IV- stage IV and Group RRT children on dialysis). To determine the above-mentioned metabolites concentrations in RBC liquid chromatography-mass spectrometry was used. Results: the only difference between the groups was shown concerning NAD in RBC, although the values did not differ significantly from controls. The lowest NAD values were found in Group II (188.6 ± 124.49 nmol/mL, the highest in group IV (324.94 ± 63.06 nmol/mL. Between Groups II and IV, as well as III and IV, the differences were statistically significant (p < 0.032, p < 0.046 respectively). Conclusions. CKD children do not have evident abnormalities of RBC metabolism with respect to adenine nucleotide metabolites. The significant differences in erythrocyte NAD concentrations between CKD stages may suggest the activation of adaptive defense mechanisms aimed at erythrocyte metabolic stabilization. It seems that the implementation of RRT has a positive impact on RBC NAD metabolism, but further research performed on a larger population is needed to confirm it.

Safety and Efficacy of Mitapivat (AG-348), an Oral Activator of Pyruvate Kinase R, in Subjects with Sickle Cell Disease: A Phase 2, Open-Label Study (ESTIMATE)

BackgroundSickle cell disease (SCD) is one of the most common and devastating inherited blood disorders characterized by a single nucleotide mutation in the beta-globin chain leading to the production of mutant hemoglobin S (HbS). HbS polymerizes upon deoxygenation causing red blood cells (RBC) to sickle which results in extremely painful episodes of vaso-occlusive crisis (VOC), severe hemolytic anemia, chronic multiorgan failure and a reduced life span. An important metabolic feature directly associated with RBC sickling is increased intracellular levels of the glycolytic intermediate 2,3-diphosphyglycerate (2,3-DPG) which promotes deoxygenation by lowering the oxygen affinity of hemoglobin (Hb). Pyruvate kinase R (PKR) is a key enzyme in RBC metabolism, generating adenosine triphosphate (ATP) to maintain energy homeostasis, membrane integrity and deformability, and modulates 2,3-DPG levels. Mitapivat (AG-348) is an oral, small molecule allosteric activator of PKR and shows promise as an anti-sickling agent in addition to its effect in PK deficiency and thalassemia. The safety and efficacy of mitapivat in subjects with SCD was evaluated in the dose finding period of this ongoing phase 2 study.MethodsThe ESTIMATE study is a phase 2, open-label study in which subjects ≥16 years with SCD (HbSS, HbS/β0, HbS/β+) with a baseline hemoglobin >6.1 g/dL and ≤11.1 g/dL, no chronic transfusion and adequate organ function were eligible. In the 8-week Dose Finding Period, initial dosing of mitapivat was 20 mg twice daily (BID). Subjects received a maximum of two sequential dose escalations of mitapivat (i.e. from 20 mg BID to 50 mg BID and 100 mg BID) depending on safety. The primary endpoints were safety, evaluated by frequency and severity of adverse events (AEs), and efficacy of mitapivat on RBC sickling. RBC sickling was evaluated by change in Point of Sickling (PoS), the pO2 at which sickling occurs as measured by oxygen gradient ektacytrometry on the Lorrca (RR Mechatronics). Secondary endpoints included changes in hematological parameters, levels of 2,3-DPG and ATP, Hb-oxygen affinity (p50) and surrogate markers of organ damage and mortality. Subjects who safely tolerated mitapivat and showed evidence of clinical improvement, were eligible to continue a 52-week follow-up period (Fixed Dose Extension Period).ResultsSix subjects have been enrolled as of September 2020 and completed the Dose Finding Period. All had homozygous HbSS except one patient who had HbS/β0-thalassemia. Baseline characteristics were: median age of 36 years (range 20-59 years), 4 (66.7%) were female and 5 (83.3%) were on stable-dose hydroxyurea. All subjects received dose escalation to a maximum dose of 100 mg BID. No serious adverse events (SAEs) occurred. Adverse events (AEs) were mild and often transient, with the most common treatment emergent AEs: transaminase increase (n=3 [50.0%], Grade 1), gastrointestinal disorders including dyspepsia, diarrhea and abdominal discomfort (n=3 [50.0%], Grade 1) and headache (n=2 [33.3%], Grade 1). One VOC occurred without hospital admission and did not require dose reduction or discontinuation. Table 1 summarizes the anti-sickling effect as well as the hematological and biochemical response to mitapivat treatment. Sickling occurred at lower pO2 levels in all 6 patients during the Dose Finding Period reflected by a significant decrease in treatment week 8 mean PoS compared to baseline. 5/6 subjects (83.3%) achieved a Hb increase of ≥1 g/dL during this period, which was accompanied by a decrease in hemolytic markers. Consistent with activation of PKR, 2,3-DPG levels decreased and ATP levels increased. Additional results including biomarker data will be presented.ConclusionMitapivat demonstrated an adequate safety profile during the 8-week Dose Finding Period in patients with SCD. The data show promising efficacy in terms of a decrease in the pO2 at which RBCs start to sickle, as well as increase in Hb from baseline and a concomitant decrease in markers reflecting hemolysis. The observed changes in 2,3-DPG and ATP levels are consistent with the proposed mechanism of the drug. The study is ongoing and further data including follow-up data, patient-reported outcomes, PKR activity and thermostability will be reported at a later stage. [Display omitted] Disclosuresvan Dijk: Agios Pharmaceuticals: Research Funding; Axcella Health: Research Funding. Rab: Axcella Health: Research Funding; Agios Pharmaceuticals: Research Funding. Rijneveld: Servier: Research Funding; Amgen: Research Funding. Nur: Celgene: Speakers Bureau; Novartis: Research Funding, Speakers Bureau; Roche: Speakers Bureau. Schutgens: CSL Behring: Research Funding; Novo Nordisk: Research Funding; OctaPharma: Research Funding; Pfizer: Research Funding; Shire/Takeda: Research Funding; Bayer: Research Funding. Wijk: Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Axcella health: Research Funding; Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding. Van Beers: Pfizer: Research Funding; Novartis: Research Funding; Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; RR Mechatronics: Research Funding.

Red Blood Cell Metabolism in Pyruvate Kinase Deficient Patients.

Background: Pyruvate kinase deficiency (PKD) is the most frequent congenital enzymatic defect of glycolysis, and one of the most common causes of hereditary non spherocytic hemolytic anemia. Therapeutic interventions are limited, in part because of the incomplete understanding of the molecular mechanisms that compensate for the metabolic defect.Methods: Mass spectrometry-based metabolomics analyses were performed on red blood cells (RBCs) from healthy controls (n=10) and PKD patients (n=5).Results: In PKD patients, decreases in late glycolysis were accompanied by accumulation of pentose phosphate pathway (PPP) metabolites, as a function of oxidant stress to purines (increased breakdown and deamination). Markers of oxidant stress included increased levels of sulfur-containing compounds (methionine and taurine), polyamines (spermidine and spermine). Markers of hypoxia such as succinate, sphingosine 1-phosphate (S1P), and hypoxanthine were all elevated in PKD subjects. Membrane lipid oxidation and remodeling was observed in RBCs from PKD patients, as determined by increases in the levels of free (poly-/highly-unsaturated) fatty acids and acyl-carnitines.Conclusion: In conclusion, in the present study, we provide the first overview of RBC metabolism in patients with PKD. Though limited in scope, the study addresses the need for basic science to investigate pathologies targeting underrepresented minorities (Amish population in this study), with the ultimate goal to target treatments to health disparities.

Proteinuric chronic kidney disease is associated with altered red blood cell lifespan, deformability and metabolism

Anemia is a common complication of chronic kidney disease, affecting the quality of life of patients. Among various factors, such as iron and erythropoietin deficiency, reduced red blood cell (RBC) lifespan has been implicated in the pathogenesis of anemia. However, mechanistic data on invivo RBC dysfunction in kidney disease are lacking. Herein, we describe the development of chronic kidney disease-associated anemia in mice with proteinuric kidney disease resulting from either administration of doxorubicin or an inducible podocin deficiency. In both experimental models, anemia manifested at day 10 and progressed at day 30 despite increased circulating erythropoietin levels and erythropoiesis in the bone marrow and spleen. Circulating RBCs in both mouse models displayed altered morphology and diminished osmotic-sensitive deformability together with increased phosphatidylserine externalization on the outer plasma membrane, a hallmark of RBC death. Fluorescence-labelling of RBCs at day 20 of mice with doxorubicin-induced kidney disease revealed premature clearance from the circulation. Metabolomic analyses of RBCs from both mouse models demonstrated temporal changes in redox recycling pathways and Lands' cycle, a membrane lipid remodeling process. Anemic patients with proteinuric kidney disease had an increased proportion of circulating phosphatidylserine-positive RBCs. Thus, our observations suggest that reduced RBC lifespan, mediated by altered RBC metabolism, reduced RBC deformability, and enhanced cell death contribute to the development of anemia in proteinuric kidney disease.

Red Blood Cell Metabolism in Patients with Propionic Acidemia

Propionic acidemia (PA) is a rare autosomal recessive disorder with an estimated incidence of 1:100,000 live births in the general population. Due in part to an insufficient understanding of the disease’s pathophysiology, PA is often associated with complications, and in severe cases can cause coma and death. Despite its association with hematologic disorders, PA’s effect on red blood cell metabolism has not been described. Mass spectrometry-based metabolomics analyses were performed on RBCs from healthy controls (n = 10) and PKD patients (n = 3). PA was associated with a significant decrease in the steady state level of glycolytic products and the apparent activation of the PPP. The PA samples showed decreases in succinate and increases in the downstream dicarboxylates of the TCA cycle. BCAAs were lowered in the PA samples and C3 carnitine, a direct metabolite of propionic acid, was increased. Trends in the markers of oxidative stress including hypoxanthine, allantoate and spermidine were the opposite of those associated with elevated ROS burden. The alteration of short chain fatty acids, the accumulation of some medium chain and long chain fatty acids, and decreased markers of lipid peroxidation in the PA samples contrasted with previous research. Despite limitations from a small cohort, this study provides the first investigation of RBC metabolism in PA, paving the way for targeted investigations of the critical pathways found to be dysregulated in the context of this disease.

Metabolic profile of irradiated whole blood by chemical isotope-labeling liquid chromatography-mass spectrometry

Irradiated blood is a new type of blood product used to prevent transfusion-associated graft-versus-host disease. However, the effects of irradiation on the metabolism of plasma, red blood cells (RBCs), and peripheral blood mononuclear cells (PBMCs) are largely unknown. We developed a workflow for testing metabolic changes in whole blood to determine the impact of irradiation by chemical isotope labeling liquid chromatography-mass spectrometry (CIL LC–MS). Blood parameters, PBMC proliferation and apoptosis were examined before and after irradiation. Next, the amine/phenol metabolites in the blood components were assayed by 12C- and13C-dansylation labeling LC–MS. We identified 1654, 1730, and 1666 peak pairs in plasma, RBCs, and PBMCs, respectively. We screened out 367, 177, and 219 significant metabolites in plasma, RBCs, and PBMCs, respectively, by principle component analyses, volcano plots, and Venn plots. Metabolic pathway analyses showed that irradiation modulated taurine and hypotaurine metabolism in plasma and purine metabolism in RBCs and PBMCs. Changes in potential biomarkers, including an increase in hypoxanthine level and a decrease in adenine level, may be related to the dysfunction of DNA synthesis in PBMCs. The decreased AMP level in RBCs may interfere with RBC storage lesions. Our research provides a more comprehensive perspective on blood metabolism associated with irradiation.

Inter-study and time-dependent variability of metabolite abundance in cultured red blood cells

BackgroundCultured human red blood cells (RBCs) provide a powerful ex vivo assay platform to study blood-stage malaria infection and propagation. In recent years, high-resolution metabolomic methods have quantified hundreds of metabolites from parasite-infected RBC cultures under a variety of perturbations. In this context, the corresponding control samples of the uninfected culture systems can also be used to examine the effects of these perturbations on RBC metabolism itself and their dependence on blood donors (inter-study variations).MethodsTime-course datasets from five independent studies were generated and analysed, maintaining uninfected RBCs (uRBC) at 2% haematocrit for 48 h under conditions originally designed for parasite cultures. Using identical experimental protocols, quadruplicate samples were collected at six time points, and global metabolomics were employed on the pellet fraction of the uRBC cultures. In total, ~ 500 metabolites were examined across each dataset to quantify inter-study variability in RBC metabolism, and metabolic network modelling augmented the analyses to characterize the metabolic state and fluxes of the RBCs.ResultsTo minimize inter-study variations unrelated to RBC metabolism, an internal standard metabolite (phosphatidylethanolamine C18:0/20:4) was identified with minimal variation in abundance over time and across all the samples of each dataset to normalize the data. Although the bulk of the normalized data showed a high degree of inter-study consistency, changes and variations in metabolite levels from individual donors were noted. Thus, a total of 24 metabolites were associated with significant variation in the 48-h culture time window, with the largest variations involving metabolites in glycolysis and synthesis of glutathione. Metabolic network analysis was used to identify the production of superoxide radicals in cultured RBCs as countered by the activity of glutathione oxidoreductase and synthesis of reducing equivalents via the pentose phosphate pathway. Peptide degradation occurred at a rate that is comparable with central carbon fluxes, consistent with active degradation of methaemoglobin, processes also commonly associated with storage lesions in RBCs.ConclusionsThe bulk of the data showed high inter-study consistency. The collected data, quantification of an expected abundance variation of RBC metabolites, and characterization of a subset of highly variable metabolites in the RBCs will help in identifying non-specific changes in metabolic abundances that may obscure accurate metabolomic profiling of Plasmodium falciparum and other blood-borne pathogens.

Identification of a Digenic Inheritance of Pklr and KLF1 Mutations Resulting in Hemolytic Anemia in a Neonate from a Study of Genotype-Phenotype Correlation of Globin, Pklr and KLF1 in Thai Patients with Pyruvate Kinase (PK) Deficiency

Introduction: Pyruvate kinase deficiency (PK def) is an inherited disorder that affects red blood cells metabolism and causes a wide range of clinical manifestation from fetal anemia, severe neonatal jaundice, and chronic transfusion dependent anemia to fully compensated hemolytic anemia. This condition is rarely reported in Southeast Asian population due to a lack of knowledge and recognition by clinicians and unavailability of laboratory for confirmation. The majority of patients with PK def result from mutations of the PKLR genes encoding this enzyme. Our group has described for the first time (Viprakasit V et al., Blood 2014) that mutations in trans of an erythroid specific transcription factor; KLF1 that regulate PKLR expression, could result in clinical presentation mimic that of classical PK def. Moreover, α and β globin mutations are highly prevalent in our region (up to 40% carrier rate) but their roles as a genetic modifier in PK def remain unknown. In addition, mutations in KLF1 seems to be highly prevalent in our population (14.6 in 1000, n= 2464 chr.), it is of interest to determine the epistatic role of α and β globin, PKLR and KLF1 mutations in patients with classical presentation of PK def.Objectives: To characterize α and β globin, PKLR and KLF 1 mutations and correlate these with clinical presentation and outcome(s) of them in patients with PK def and their family members.Methods: After obtaining informed consent, 9 index PK def patients with their available family members were evaluated for PKLR and KLF1 mutations by direct genomic sequencing as previously described. Hematological parameters and hemoglobin analyses were performed using standard techniques. DNA analysis for all common α and β globin mutations leading to thalassemia and hemoglobinopathies found in this region was performed using our standard protocol.Results: We successfully identified PKLR mutations in 7 out of 9 index patients as shown in Table 1. All patients were diagnosed with clinical PK def following several measurement of biochemical activity. We found 11 different mutations in 6 compound heterozygotes (5 of two nucleotide mutations and one with 1 missense and a large PKLR deletion) and 1 homozygote. One mutation (c.941T>C =p.I314T) seemed to be recurrent since it was found in two families; one homozygous and one compounded with N118S. We failed to identified PKLR and KLF1 in only one patient (PK-9) suggesting that this patient might have a novel molecular mechanism that remains to be found. We found only one family with globin abnormality (Hb E trait in PK-1) without any deleterious effect on patient's clinical severity. Moreover, most of PK patients (except one) have no mutation in KLF1 genes. Interestingly, the PK-3 patient (who is an offspring of the PK-2) was subsequently found to be double heterozygous of PKLR (p.V548fs) and KLF1 (G176RfsX179 (+7bp)) mutations. This patient was previously diagnosed with PK def at birth when he had serious hemolytic anemia (Hb 10 g/dL, haptoglobin < 10 IU/L) with hyperbilirubinemia requiring phototherapy, based on a very low PK enzyme activity (6.5 IU/g Hb, 20% of normal level) and the PK level from his mother was also low (11 IU/g Hb). Since then, he had an uneventful history for 6 years, except mild anemia from iron deficiency anemia during infant period. Physical examination showed no hepatosplenomegaly. Currently his baseline Hb levels were around 12-12.5 g/dL with normal reticulocyte count (1%) and hypochromic microcytosis. There is no globin abnormality in this family. The PK enzyme activity at 6-year-of age showed an improved PK activity (41% of normal activity) without further hemolysis. Our finding showed for the first time a possible interaction between PKLR and KLF1 mutations in man and this digenic inheritance seems to cause a more serious phenotype than each of these carriers alone. However, since the clinical presentation of our index case (PK-3) appeared to be apparent only at the early age, this might suggest the possible impact of KLF1 mutations that were more serious at the early age (similar to those of patients with two KLF1 mutations; KLF1 disease) and might be further compensated by other hitherto mechanisms during erythroid development.Conclusions: We found no effects of globin mutations on the clinical phenotypes of patients with classical PK def. However, a mutation in KLF1 could deteriorate clinical presentations even in a carrier of PKLR mutation at the early age. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The interactome of the N-terminus of band 3 regulates red blood cell metabolism and storage quality.

Band 3 (anion exchanger 1; AE1) is the most abundant membrane protein in red blood cells, which in turn are the most abundant cells in the human body. A compelling model posits that, at high oxygen saturation, the N-terminal cytosolic domain of AE1 binds to and inhibits glycolytic enzymes, thus diverting metabolic fluxes to the pentose phosphate pathway to generate reducing equivalents. Dysfunction of this mechanism occurs during red blood cell aging or storage under blood bank conditions, suggesting a role for AE1 in the regulation of the quality of stored blood and efficacy of transfusion, a life-saving intervention for millions of recipients worldwide. Here we leveraged two murine models carrying genetic ablations of AE1 to provide mechanistic evidence of the role of this protein in the regulation of erythrocyte metabolism and storage quality. Metabolic observations in mice recapitulated those in a human subject lacking expression of AE11-11 (band 3 Neapolis), while common polymorphisms in the region coding for AE11-56 correlate with increased susceptibility to osmotic hemolysis in healthy blood donors. Through thermal proteome profiling and crosslinking proteomics, we provide a map of the red blood cell interactome, with a focus on AE11-56 and validate recombinant AE1 interactions with glyceraldehyde 3-phosphate dehydrogenase. As a proof-of-principle and to provide further mechanistic evidence of the role of AE1 in the regulation of redox homeo stasis of stored red blood cells, we show that incubation with a cell-penetrating AE11-56 peptide can rescue the metabolic defect in glutathione recycling and boost post-transfusion recovery of stored red blood cells from healthy human donors and genetically ablated mice.

The effect of N-acetyl-cysteine (NAC) on RBC oxidative damage and RBC metabolism during storage of red blood cell product in blood bank condition

Background: Oxidative damage is one of the main causes of Red blood cell (RBC) storage lesion which can reduce RBC survival during RBC storage in blood bank condition. In this study, we evaluate the effect of N-acetyl cysteine (NAC) as an anti-oxidant compound on RBC oxidative damage and RBC metabolism during storage of this product. Methods: In this experimental study, 10 bag of packed RBC were provided to the Iranian Blood Transfusion Organization's Innovation Center were randomly selected and effect of NAC was investigated on metabolism, oxidative status and hematologic variables of RBC during RBC storage. The results of this study were compared between two groups of NAC treated RBC and untreated RBC (without NAC). All of the data were analyzed with SPSS statistical program (version 22). Results: In this study, the concentration of lactate and LDH enzyme activity in the NAC treated RBC were lower than the control group (without NAC). The concentration of malondialdehyde (MDA) as a lipid peroxidation marker in the NAC treated RBC was lower increase than the untreated RBC. Also antioxidant capacity was so higher in the NAC - RBC than in the control group, especially in the 28th day of RBC storage (Pday28 &lt;0.05). Conclusion: The results of this study indicated that the use of NAC as an additive solution could decrease oxidative damage via maintaining of the RBC oxidant capacity during RBC storage. In the future NAC may be used as an additive for maintaining of the RBC survival and RBC quality during storage of RBC in blood bank condition.

Red Cell Distribution Width: A Routinely Available Biomarker with Important Clinical Implications in Patients with Atrial Fibrillation.

Red blood cell distribution width (RDW) is an inexpensive marker of anisocytosis easily available in the standard complete blood cell count. Besides its traditional use in the differential diagnosis of anemias, RDW values reflect abnormalities in erythropoiesis and red blood cell metabolism related to aging, sex, ethnicity, systemic inflammatory state, and oxidative stress. Thus, higher RDW values are common findings in several acute clinical conditions and chronic diseases. Increasing evidence suggests a prognostic role of higher RDW levels in many cardiovascular diseases. Among them, we aimed to review current literature focusing on the possible relation between RDW and atrial fibrillation (AF). Since aging, inflammation, and atrial substrate remodeling have a well-established role in AF pathogenesis, AF burden, and patient prognosis, we analyzed available data exploring the possible use of RDW in identifying patients at higher risk of AF and as a biomarker of worse outcomes for AF patients.

Blood donor exposome and impact of common drugs on red blood cell metabolism.

Computational models based on recent maps of the RBC proteome suggest that mature erythrocytes may harbor targets for common drugs. This prediction is relevant to RBC storage in the blood bank, in which the impact of small molecule drugs or other xenometabolites deriving from dietary, iatrogenic, or environmental exposures (“exposome”) may alter erythrocyte energy and redox metabolism and, in so doing, affect red cell storage quality and posttransfusion efficacy. To test this prediction, here we provide a comprehensive characterization of the blood donor exposome, including the detection of common prescription and over-the-counter drugs in blood units donated by 250 healthy volunteers in the Recipient Epidemiology and Donor Evaluation Study III Red Blood Cell–Omics (REDS-III RBC-Omics) Study. Based on high-throughput drug screenings of 1366 FDA-approved drugs, we report that approximately 65% of the tested drugs had an impact on erythrocyte metabolism. Machine learning models built using metabolites as predictors were able to accurately predict drugs for several drug classes/targets (bisphosphonates, anticholinergics, calcium channel blockers, adrenergics, proton pump inhibitors, antimetabolites, selective serotonin reuptake inhibitors, and mTOR), suggesting that these drugs have a direct, conserved, and substantial impact on erythrocyte metabolism. As a proof of principle, here we show that the antacid ranitidine — though rarely detected in the blood donor population — has a strong effect on RBC markers of storage quality in vitro. We thus show that supplementation of blood units stored in bags with ranitidine could — through mechanisms involving sphingosine 1–phosphate–dependent modulation of erythrocyte glycolysis and/or direct binding to hemoglobin — improve erythrocyte metabolism and storage quality.

Hypochlorous acid decreases antioxidant power, inhibits plasma membrane redox system and pathways of glucose metabolism in human red blood cells.

Hypochlorous acid (HOCl) is generated at a high concentration by activated neutrophils at sites of inflammation in a myeloperoxidase catalyzed reaction. The increased and sustained production of HOCl at inflammatory sites may lead to tissue injury and this process is believed to play an important role in the progression of several diseases like chronic inflammation, atherosclerosis and some types of cancers. We have examined the effect of HOCl on human red blood cells (RBCs) under in vitro conditions. Treatment of RBC with different concentrations of HOCl (0.05-2.5mM) at 37°C resulted in decreased activities of major antioxidant enzymes while the antioxidant power of RBC was weakened, as shown by lowered metal-reducing and free radical quenching ability of HOCl treated cells. RBC plasma membrane redox system was also inhibited suggesting membrane damage. The enzymes of glucose metabolism were inhibited indicating deranged energy metabolism. Electron microscopic images showed gross morphological changes in HOCl treated RBC. These results show that HOCl causes major alterations in the cellular antioxidant defense system and inhibition of glycolytic pathways, which increase the susceptibility of RBC to oxidative damage.

Associations between red blood cells fatty acids, desaturases indices and metabolism of platelet activating factor in healthy volunteers

IntroductionPlatelet-activating-factor is an inflammatory lipid mediator. Key enzymes of its biosynthesis are CDP-choline:1-alkyl-2-acetyl-sn-glycerol-cholinephosphotransferase (PAF-CPT) and acetyl-CoA:lyso-PAF-acetyltransferases (Lyso-PAF-AT) while PAF-AH/Lp-PLA2 degrade PAF. The interplay between PAF and fatty acids metabolism was explored. Material and methodsIn a healthy population, PAF levels, its metabolic enzymes activity and RBC fatty acids were measured while desaturases indices (D) were estimated. A principal component analysis was also applied to assess patterns of RBC fatty acids. ResultsSFA were related to increased PAF biosynthesis and decreased Lp-PLA2 only in women. MUFA were inversely associated with PAF biosynthesis and positively with Lp-PLA2. Omega-6 fatty acids were positively correlated only with PAF-CPT while no significant correlations were observed with n3 fatty acids. D6 index was positively related with PAF biosynthetic enzymes and inversely with Lp-PLA2 while D9 correlated positively with Lp-PLA2. The pattern of high MUFA and low n6 was associated with reduced PAF biosynthesis and/or increased catabolism in both sexes. ConclusionThe role of fatty acids in amplifying or reducing inflammation seems to be also reflected in PAF metabolism.

Fluoride enhances generation of reactive oxygen and nitrogen species, oxidizes hemoglobin, lowers antioxidant power and inhibits transmembrane electron transport in isolated human red blood cells

Fluoride is a widespread environmental pollutant that at high levels exerts numerous deleterious effects on human health. The toxic effects of fluoride are a matter of serious concern since many countries have regions of endemic fluorosis. The main source of fluoride exposure for humans is intake of contaminated groundwater. Fluoride is absorbed from the gastrointestinal tract and enters the circulating blood, where the abundant red blood cells (RBC) are an early and major target of fluoride toxicity. Chronic fluoride exposure generates free radicals, reactive species which leads to redox imbalance, cytotoxicity and hematological damage. This study aimed to determine the effect of sodium fluoride (NaF) on human RBC under in vitro conditions. Isolated RBC were incubated with different concentrations of NaF (10–500 µM) for 8 h at 37 °C. Several biochemical parameters were determined in hemolysates or whole cells. Treatment of RBC with NaF enhanced the generation of reactive oxygen and nitrogen species. This increased the oxidation of hemoglobin to yield methemoglobin and oxoferrylhemoglobin, which are inactive in oxygen transport. NaF treatment increased the degradation of heme causing release of free iron from its porphyrin ring. Cellular antioxidant power was significantly decreased in NaF-treated RBC, lowering the metal reducing and free radical quenching ability of cells. The two pathways of glucose metabolism in RBC i.e. glycolysis and hexose monophosphate shunt, were inhibited. NaF also inhibited the plasma membrane redox system, and its associated ascorbate free radical reductase, to disrupt transmembrane electron transport. These results suggest that fluoride generates reactive species that cause extensive oxidative modifications in human RBC.

Metabolomic Characterization of Red Blood Cell Differentiation

Background: Erythropoiesis is the highly coordinated multi-step process by which multipotent hematopoietic stem cells differentiate into mature enucleated red blood cells (RBCs). As erythroid cells become more terminally differentiated, they undergo changes in morphology and gene expression, start synthesizing hemoglobin, commit to an irreversible loss of proliferation, and eventually expulse their nuclei and other cytoplasmic organelles. Thus, RBCs must rely on their proteome and metabolome for proper function. The RBC proteome is estimated to contain 2,800 proteins, including a variety of receptors and transporters that allow RBCs to uptake xenobiotics or endogenous metabolites as they circulate for ∼120 days. Furthermore, they are metabolically active with glycolysis, nucleotide catabolism, and glutathione metabolism as the major pathways supporting cell survival and function. However, it is unclear how the metabolome is altered during erythropoiesis, what role metabolites play in normal erythropoiesis, and if dysregulation of metabolites contributes to diseases of ineffective erythropoiesis, such as sickle cell anemia and thalassemia. Methods: Four models of erythropoiesis were used in this study. 1) Mice were treated with phenylhydrazine (Phz) to induce acute hemolysis followed by erythropoietic recovery, leading to an increase in circulating reticulocytes. 2) Mice were lethally irradiated and transplanted with wild-type or sickle cell bone marrow, leading to anemic profiles in sickle cell chimeras. 3) The mouse erythroleukemic (MEL) cell line was treated with DMSO to induce differentiation. 4) The human erythroleukemic (K562) cell line was treated with sodium butyrate to induce differentiation. For the in vivo mouse models, blood was collected from control and treated animals, and complete blood count (CBC) analysis was performed. For the in vitro cell culture models, the mRNA levels of β-globin were measured by Q-RT-PCR in control and differentiated cells, and the degree of hemoglobinization was determined visually and via staining for heme. In addition, metabolites were extracted from the collected RBCs and erythroleukemic cell lines, and a Snapshot LC/MS metabolomic platform was used to identify commonly altered metabolites. Results: We first validated our four models of erythropoiesis. Treatment with Phz decreased the number of total RBCs while increasing the RBC distribution width, indicating an increased number of reticulocytes (more immature RBCs) in circulation. Similar results were seen in the sickle cell chimeras. Treatment of MEL and K562 cells with DMSO and sodium butyrate, respectively, resulted in increased expression of β-globin, increased levels of heme, and increased red color. Then, using our Snapshot metabolomic platform, we identified global changes in RBC metabolism during erythropoiesis. Analyses of the commonly altered metabolites in the in vitro and in vivo models revealed an increase in amino acid, mitochondrial, and urea cycle metabolism during erythropoiesis. L-aspartate levels were particularly upregulated, especially in DMSO-treated MEL cells. We are now investigating the role of aspartate in the regulation of erythropoiesis. Conclusions: We defined how the metabolome was altered in multiple in vitro and in vivo models of erythropoiesis and identified global changes in RBC metabolism between the different models. Specifically, we found that L-aspartate was upregulated during RBC differentiation in all four models. Aspartate is an amino acid that plays a role in many processes in cells, including nucleotide biosynthesis, redox homeostasis, and amino acid biosynthesis. We hypothesize that aspartate metabolism is critical for RBC differentiation and that its dysregulation exacerbates disease of ineffective erythropoiesis, such as sickle cell anemia and β-thalassemia. We are currently testing its role in inducing hemoglobinization and in regulating the commitment of erythroid progenitor cells to an irreversible loss of proliferation. Overall, we believe that understanding the precise mechanisms by which cellular metabolism plays a role in proper RBC differentiation may lead to better therapies for diseases of ineffective erythropoiesis, such as sickle cell anemia and thalassemia. Disclosures No relevant conflicts of interest to declare.