Erythrocyte Enzyme Research Articles

Sphingolipid Catabolism and Glycerophospholipid Levels Are Altered in Erythrocytes and Plasma from Multiple Sclerosis Patients.

Multiple sclerosis (MS) is an autoimmune, inflammatory, degenerative disease of the central nervous system. Changes in lipid metabolism have been suggested to play important roles in MS pathophysiology and progression. In this work we analyzed the lipid composition and sphingolipid-catabolizing enzymes in erythrocytes and plasma from MS patients and healthy controls. We observed reduction of sphingomyelin (SM) and elevation of its products—ceramide (CER) and shingosine (SPH). These changes were supported by the detected up-regulation of the activity of acid sphingomyelinase (ASM) in MS plasma and alkaline ceramidase (ALCER) in erythrocytes from MS patients. In addition, Western blot analysis showed elevated expression of ASM, but not of ALCER. We also compared the ratios between saturated (SAT), unsaturated (UNSAT) and polyunsaturated fatty acids and suggest, based on the significant differences observed for this ratio, that the UNSAT/SAT values could serve as a marker distinguishing erythrocytes and plasma of MS from controls. In conclusion, the application of lipid analysis in the medical practice would contribute to definition of more precise diagnosis, analysis of disease progression, and evaluation of therapeutic strategies. Based on the molecular changes of blood lipids in neurodegenerative pathologies, including MS, clinical lipidomic analytical approaches could become a promising contemporary tool for personalized medicine.

Whole genome sequences discriminate hereditary hemorrhagic telangiectasia phenotypes by non-HHT deleterious DNA variation

AbstractThe abnormal vascular structures of hereditary hemorrhagic telangiectasia (HHT) often cause severe anemia due to recurrent hemorrhage, but HHT causal genes do not predict the severity of hematological complications. We tested for chance inheritance and clinical associations of rare deleterious variants in which loss-of-function causes bleeding or hemolytic disorders in the general population. In double-blinded analyses, all 104 patients with HHT from a single reference center recruited to the 100 000 Genomes Project were categorized on new MALO (more/as-expected/less/opposite) sub-phenotype severity scales, and whole genome sequencing data were tested for high impact variants in 75 HHT-independent genes encoding coagulation factors, or platelet, hemoglobin, erythrocyte enzyme, and erythrocyte membrane constituents. Rare variants (all gnomAD allele frequencies <0.003) were identified in 56 (75%) of these 75 HHT-unrelated genes. Deleteriousness assignments by Combined Annotation Dependent Depletion (CADD) scores >15 were supported by gene-level mutation significance cutoff scores. CADD >15 variants were identified in 38/104 (36.5%) patients with HHT, found for 1 in 10 patients within platelet genes; 1 in 8 within coagulation genes; and 1 in 4 within erythrocyte hemolytic genes. In blinded analyses, patients with greater hemorrhagic severity that had been attributed solely to HHT vessels had more CADD-deleterious variants in platelet (Spearman ρ = 0.25; P = .008) and coagulation (Spearman ρ = 0.21; P = .024) genes. However, the HHT cohort had 60% fewer deleterious variants in platelet and coagulation genes than expected (Mann-Whitney test P = .021). In conclusion, patients with HHT commonly have rare variants in genes of relevance to their phenotype, offering new therapeutic targets and opportunities for informed, personalized medicine strategies.

Integrative Physiological Responses to Acute Dehydration in the Rufous-Collared Sparrow: Metabolic, Enzymatic, and Oxidative Traits

Predictions indicate that birds worldwide will be affected by global warming and extreme climatic events which is especially relevant for passerines because the diurnal habits, small body size, and high mass-adjusted metabolic rates of this group make it particularly susceptible to increases in temperature and aridity. Some bird species respond to conditions that stress osmoregulation by increasing their rates of energy expenditure, nevertheless, the effect of dehydration on metabolic rates in birds has produced contrasting results. It also remains unknown whether hydration state may cause shifts in tissue-specific metabolic rates or modify tissue oxidative status. We used the rufous-collared sparrow (Zonotrichia capensis), to experimentally test the effect of dehydration on metabolic enzymes in erythrocytes, tissue oxidative status, basal metabolic rate (BMR), and total evaporative water loss. We found a significant increase in mass-adjusted BMR in water restricted (WR) birds compared to control birds (CT). Activity of cytochrome-c-oxidase (COX) in red blood cells (RBCs) was also significantly higher in the WR group relative to the CT group and this activity was positively correlated with mass-adjusted BMR. We found a moderate effect of water restriction on membrane damage of skeletal muscle. In a second set of individuals subjected to the same experimental conditions, lean mass and total water were tightly correlated and decreased by 10 and 12%, respectively, in birds in the WR group relative to the CT group. Decreases in total water and lean mass leads to an increase in mass-adjusted BMR in WR Z. capensis, suggesting that birds may simultaneously increase protein catabolism and production of metabolic water through oxidation. The significant positive relationship between BMR and COX in RBCs is a finding that requires additional research to determine whether erythrocyte metabolism is affected by dehydration per se and or it more generally reflects rates of energy expenditure in birds.

Prevalence of Glucose-6-phosphate Dehydrogenase Deficiency in Aligarh, India: A Pilot Study

Introduction: Deficiency of glucose-6-phosphate dehydrogenase (G6PD) enzyme in erythrocytes is among the prevalent X-linked recessive genetic disorders and affects approximately 400 million people across the globe. It causes neonatal hyperbilirubinemia, also hemolysis eventually leading to hemolytic anemia when exposed to oxidative stress. Purpose: The purpose of the study was to find out the prevalence of G6PD deficiency in Aligarh (Western Uttar Pradesh) India. Materials and Methods: The data and samples were collected from a total of 106 individuals. All the required information were recorded in a standardized questionnaire, blood was taken with their prior consent. Blood typing test and fluorescent spot test (FST) were performed, spots were arranged accordingly and observed. Results: Out of total 106 individuals surveyed, 10% of them were found to be deficient for G6PD enzyme, 5% were intermediate, and 85% were normal. Significant association was found for G6PD deficiency and gender (P &lt; 0.05) of the individuals while non-significant association were reported for G6PD deficiency and consanguinity (P &lt; 0.8) and for G6PD deficiency and blood groups (P &lt; 0.9). Conclusion: FST is a good method for G6PD screening and detecting the deficiency in fields. This pilot study highlights the prevalence of G6PD enzymopathy in Aligarh and will help government programs of this area to prevent and control the genetic disorder

Класична галактоземія: особливості діагностики та лікування

Актуальність. Галактоземія І типу, або класична галактоземія (КГ), — найбільш тяжка форма спадкових порушень обміну вуглеводів, зокрема галактози. Галактоземія не включена до програми масового неонатального скринінгу в Україні. КГ характеризується неспецифічністю дебютних симптомів, супроводжується високими показниками смертності у неонатальному періоді. Актуальними є питання ранньої діагностики та лікування КГ за відсутності програми масового неонатального скринінгу в Україні. Мета. Аналіз клініко-лабораторних, молекулярно-генетичних особливостей КГ; визначення комплексу клінічних симптомів щодо формування селективних груп пацієнтів для призначення дієтотерапії та проведення лабораторної діагностики; розробка поетапної схеми ефективної ранньої діагностики та лікування пацієнтів при КГ за відсутності масового неонатального скринінгу з метою запобігання інвалідизації хворих, зниження показника дитячої смертності. Матеріали та методи. Впродовж 2014–2017 рр. у Центрі орфанних захворювань обстежено 317 пацієнтів із діагнозом «гепатит невизначеного генезу». Вік хворих — від 3 днів до 16 років. Хлопчиків було 145 (45 %), дівчаток — 172 (55 %). У всіх пацієнтів відзначена полісиндромна клінічна картина з переважним ураженням печінки. В усіх пацієнтів визначали активність ферменту GАLT в еритроцитах периферичної крові, проводили ДНК-дослідження з метою виявлення мажорних мутацій у гені GАLT. Всі хворі були обстежені на наявність вірусних гепатитів. За результатами біохімічного дослідження визначали рівень печінкових трансаміназ, лужної фосфатази, білірубіну, показники згортання крові. Також проводили тандемну мас-спектрометрію з метою виявлення порушень обміну амінокислот, органічних кислот і мітохондріального бета-окислення жирних кислот. У 5 пацієнтів за наявності припущення про глікогеноз проведене кількісне визначення глікогену у біоптаті печінки. Результати. Діагноз КГ підтверджений у 15 (4,7 %) з 317 пацієнтів: у 9 хлопчиків і 6 дівчаток віком від 14 діб до 15 років. У 13 (86,7 %) дітей клінічний діагноз КГ встановлений у середньому через 47 ± 15 діб життя. У 2 пацієнтів КГ діагностована у віці 2 і 15 років. У всіх пацієнтів активність ферменту GALT в еритроцитах була знижена і становила 0,08–5,8 Од/г Hb, у середньому 3,3 ± 1,6 Од/г Hb (в нормі &gt; 10 Од/г Hb). Молекулярно-генетичний аналіз проведений у 14 (93,3 %) пацієнтів. У 5 хворих виявлений гомозиготний генотип за Q188R, в одного — за K285N, у 6 — гетерозиготний генотип за двома мажорними мутаціями (Q188R/K285N), в одного пацієнта виявлений генотип був р.Y209S/р.K285N, ще в одного — виявлена мутація р.K285N тільки в одній алелі. Отже, у 14 пацієнтів досліджені 28 алелей у гені GALT, ідентифіковані 27 мутацій: Q188R — у 57 % спостережень, K285N — у 36 %, р.Y209S — у 3 % (виявлена в одного пацієнта в одній алелі). В цілому мутації Q188R і K285N виявляли з частотою 93 %, що свідчить про високий рівень їх мажорності в українській популяції. Жовтяниця виявлена у 93 % хворих, гепатомегалія — у 80 %, диспептичний синдром — у 87 %. Катаракта була діагностована у 6 (40 %) пацієнтів, в усіх спостереженнях поєднана з неврологічними симптомами. Ураження нервової системи (спастичний тетрапарез, дистонія, тремор, когнітивна недостатність, дизартрія, затримка мовленнєвого розвитку) відзначені у 7 (21,8 %) хворих при КГ. Померли 4 (26,7 %) з 15 пацієнтів віком від 20 днів до 2,5 місяця. За даними клініко-генеалогічного аналізу у родинах хворих на КГ виявлено часті випадки ранньої смерті дітей (віком до 14 тижнів) унаслідок печінкової недостатності. У неонатальному періоді померли 4 дітей із трьох родин з обтяженим анамнезом. У цілому в родинах наших пацієнтів померли 8 (42,1 %) із 19 дітей з аналогічними клінічними симптомами. Лікування всім пацієнтам призначали з суттєвим запізненням, у середньому на 44-й день. Запропоновано схему діагностично-лікувальних заходів у пацієнтів селективної групи. Висновки. КГ — загрозливе для життя захворювання, що характеризується ураженням клітин печінки, диспептичним синдромом, сепсисом і спричиняє високий рівень смертності немовлят у неонатальному періоді. В цілому мутації Q188R і K285N виявлені у 93 % спостережень, що свідчить про високий рівень їх мажорності в українській популяції. Всі немовлята з жовтяницею, зумовленою гіпербілірубінемією, за наявності гепатомегалії, диспепсії, порушень згортання крові, високого рівня трансаміназ мають бути обстежені щодо КГ і негайно переведені на безлактозну дієту до отримання остаточних результатів лабораторної діагностики.

GWAS of Osmotic Hemolysis in 12,352 Healthy Blood Donors Identifies Red Cell Genetic Variants Associated with Steady State Hemolysis in Patients with Sickle Cell Disease

Background: The evolutionary pressure of endemic malaria has produced highly variable genes encoding erythrocyte proteins and enzymes, many of which modulate cellular structure and function. We hypothesized that a high throughput screen of red blood cell susceptibility to osmotic hemolysis, coupled with a GWAS study, would identify rare and common genetic variants associated with cellular structure and function during red blood cell storage and in hemolytic diseases like sickle cell and malaria.Methods: The REDS III RED Blood Cell Omics (RBC-Omics) study enrolled 13,403 successful blood donors at 4 major hubs. Two labs were used for red blood cell phenotyping of leukoreduced RBC unit-derived samples stored for ~42 days under blood bank conditions. Red blood cell osmotic fragility was assayed in 12,352 donors, including 1,483 African American, 1,477 Asian and 960 Hispanic donors. DNA was isolated and genotyped using a custom Affymetrix Axiom TM-Array, which contained approximately 875,000 SNPs enriched for blood and transfusion related polymorphisms.Results: Genome wide association analysis in the complete RBC-omics data set revealed 18 genes/loci to be associated with osmotic hemolysis at a genome wide significance threshold of P<5x10-8. The genes included candidates with clear internal validity such as HBB (specifically rs344 the HbS variant) and promotor regions of HBA2 (thalassemic variants), as well as SNPs in logical candidates, such as Ankyrin, Spectrin, and Aquaporin 1, mutations in which are responsible for spherocytosis and red cell membrane water transport defects, among others. To test the potential relevance of these finding to human disease, we evaluated the top 100 regions in the RBC-Omics studies in the 852 pediatric and adult patients with sickle cell disease enrolled in the PUSH and Walk-PHASST screening studies with genome SNP data, and analyzed the association of the SNPs with the intensity of steady state hemolysis, as measured by a principle component analysis of reticulocyte count, bilirubin, lactate dehydrogenase and aspartate amino transferase. After multiple testing correction, numerous genes were associated with the intensity of hemolysis in both studies, including SNPs in red cell metabolism enzymes genes (HK1, TKTL1 MTOR signaling), ion channels (PIEZO1), as well as regulatory gene (ZNF573) and less obvious genes (FLN, SBF2, GAB3, and TMEM116).Conclusion: An analysis of the genetic variability underlying normal human red cell susceptibility to osmotic stress hemolysis identified new candidate genes that are also associated with the severity of red blood cell hemolysis during the disease stress of human SCD. We hypothesize that many of these candidate SNPs may modulate the severity of red cell hemolysis in other diseases like malaria and potential outcomes after routine red blood cell storage and transfusion. These studies highlight the discovery of a number of new metabolic and ion/water transport variants that are associated with the severity of steady state hemolysis and under disease stress. DisclosuresMast:Novo Nordisk: Research Funding. Gordeuk:Emmaus Life Sciences: Consultancy.

Aautoimmune Hemolytic Anemia: Pattern of Clinical Presentation and Management Outcome — a Nine Year Observation from a Tertiary Care Hospital in Saudi Arabia

Introduction: Autoimmune hemolytic anemia (AIHA) is characterized by production of auto-antibodies that bind to antigen on the erythrocyte surface that leads to its premature destruction and removal from the circulation by macrophages. Occurrence of AIHA in children and adolescents is rare, exact incidence unknown. Objective of this retrospective study was to assess clinical and laboratory characteristics, and management outcome of patients diagnosed with AIHA seen at Pediatric Hematology/Oncology department of King Faisal specialist Hospital and Research Center ,Riyadh from 2005 to 2014. Method: Retrospective chart review of patients (1-14 yrs.) diagnosed with AIHA was conducted. Children were excluded if they have congenital hemolytic anemia, such as hemoglobinopathies, congenital membranopathy and erythrocyte enzyme deficiencies. Result: Thirty-one patients identified, 19(61%) female and 12(39%) male with a median age at diagnosis of 6 years (0.1-12.4). The direct Coombs test was positive in 23 (74%) patients. Underlying diseases observed as cause of AIHA in 14 (45%) patients: majority secondary to viral infections followed by severe combined immunodeficiency syndrome post bone marrow transplant GVHD complication, while systemic lupus erythematosus, antiphospholipid antibody syndrome, autoimmune hepatitis, hypereosinophilic syndrome, Hyper IgE syndrome, post-liver transplant, diabetes was observed in individual cases. Remaining 17 (55%) patients diagnosed as primary AIHA .Corticosteroids and/or immunoglobulin was given as first line of treatment to 64.5% (20) patients while 35.5% (11) required Rituximab and/or splenectomy as second line of treatment, of which 73%(8) were diagnosed as primary AIHA. Five-year overall survival (Kaplan-Meir) rate of 89% was observed with twenty-seven alive patients showing partial or complete response ,while four patients died due to underlying diseases.Conclusion: Our results demonstrates patients responding to first line of treatment, however serious cases required management through second line of treatment. Further, a regional multicenter observational study will enable better understanding of differences in disease presentation and treatment outcomes. DisclosuresNo relevant conflicts of interest to declare.

Antioxidant Status, and Blood Zinc and Copper Concentrations in Dogs with Uncomplicated Babesiosis due to Babesia Canis Infections.

IntroductionThe aim of the study was to demonstrate a link between uncomplicated Babesia canis infection in dogs and blood concentrations of zinc and copper and erythrocytic antioxidant defence – activities of glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT).Material and MethodsThe study was based on 15 naturally occurring cases of canine babesiosis with anorexia, pyrexia, depression, pale mucous membrane, splenomegaly and dark red urine. Microscopic examination of Giemsa-stained peripheral blood smears and the results of PCR confirmed B. canis infection. Seven apparently healthy dogs brought in for either a check-up or vaccination were used for comparison.ResultsThe levels of the erythrocytic antioxidant enzymes - SOD and CAT - were significantly higher in the infected dogs than in cytologically negative dogs. The levels of blood micronutrients were significantly lower in the infected dogs (0.478 μg of zinc per mL vs 1.241 μg/mL and 0.722 μg of copper per mL vs 1.392 μg/mL).ConclusionOxidative stress can be posited as one of the mechanisms leading to anaemia in dogs with babesiosis, and therefore antioxidant biomarker and copper and zinc concentrations could be used as indicators of disease severity and prognostic markers.

Hereditary Red Blood Cell Enzymopathies

Red cell metabolic disturbances result in hemolysis, which leads to a significant shortening of the erythrocyte life span. The most common enzyme deficiencies are glucose 6-phosphate dehydrogenase (G6PD) in the antioxidant pathway, pyruvate kinase in the anaerobic glycolysis pathway, and pyrimidine 5’ nucleotidase (P5’N) in the nucleotide metabolism. While the X chromosome inherits G6PD and phosphoglycerate kinase deficiencies, other enzymopathies show autosomal recessive inheritance. Although the causes of hereditary hemolytic disorders are diverse, clinical, laboratory findings and complications overlap. A history of neonatal jaundice requiring phototherapy and exchange transfusion is quite usual. Mild to severe anemia may be accompanied with episodic or constant hemolysis associated with icterus, hyperbilirubinemia, growth retardation, gallstones, splenomegaly, and a variable degree of iron overload. Erythrocyte enzyme disorders should be suspected in patients with severe hemolytic episodes, or chronic hemolysis, after excluding hemoglobinopathies, membranopathies, and immune-mediated hemolysis.

Blood pro-oxidant/antioxidant balance in young men with class II obesity after 20 sessions of whole body cryostimulation: a preliminary study

ABSTRACT Objectives: In obesity, there is a shift in the pro-oxidative-antioxidant balance towards the oxidationreactions. However, it has been shown that in people with normal body composition, after a series of whole-body cryotherapy (WBC), the balance shifts in the opposite direction. Design: The aim of the study was to assess the impact of 20 WBC treatments on blood pro-oxidative-antioxidant balance. Interventions: Study included 14 obese (BMI > 35) and 10 non-obese volunteers. Methods: The total antioxidative (TAS/TAC) and pro-oxidative status (TOS/TOC) in serum and activity of antioxidant enzymes in erythrocytes were determined before the first and 2 hours after the last cryostimulation. Results: In the obese group, a significantly higher level of TOS/TOC, and its significant decrease after the WBC series, was observed. Cryotherapy had no influence on TAS/TAC level which was similar in both groups. Changes in activity of antioxidant enzymes were multidirectional. An increase in CAT activity in the obese group was observed. OSI, both before and after a series of treatments, was significantly higher in obese subjects. Conclusions: A beneficial effect on the level of TOS/TOC and CAT activity was indicated, but the proposed number of treatments for patients with class II obesity turned out to be insufficient. Trial registration: Australian New Zealand Clinical Trials Registry identifier: ACTRN12619000524190.

Clinical, metabolic, and molecular genetic characterization of hereditary methemoglobinemia caused by cytochrome b5 reductase deficiency in 30 dogs

Genotype–phenotype correlations of humans and dogs with hereditary methemoglobinemia are not yet well characterized. We determined total hemoglobin and methemoglobin (MetHb) concentrations, cytochrome b5 reductase (CYB5R) enzyme activities, genotypes, and clinical signs in 30 dogs with persistent cyanosis without cardiopulmonary disease. Erythrocytic CYB5R enzyme activities were low in all dogs assayed. Owner-reported quality of life ranged from subclinical to occasional exertional syncope. Two previously reported and two novel CYB5R3 missense variants were identified among the methemoglobinemic cohort and were predicted to impair enzyme function. Two variants were recurrent: a homozygous Ile194Leu substitution was found in Pomeranians and other small dogs, and a homozygous Arg219Pro change occurred predominately in pit bull terriers. The other two variants were Thr202Ala and Gly76Ser substitutions in single dogs. Of the two common CYB5R3 genotypes, Arg219Pro was associated with a more severe metabolic phenotype. We conclude that CYB5R3 deficiency is the predominate cause of canine hereditary methemoglobinemia. Although this finding is unlikely to alter the clinical approach to hereditary methemoglobinemia in dogs, it demonstrates the possibility of how genotype–phenotype cohort analysis might facilitate precision medicine in the future in veterinary medicine.

Host Porphobilinogen Deaminase Deficiency Confers Malaria Resistance in Plasmodium chabaudi but Not in Plasmodium berghei or Plasmodium falciparum During Intraerythrocytic Growth.

An important component in host resistance to malaria infection are inherited mutations that give rise to abnormalities and deficiencies in erythrocyte proteins and enzymes. Understanding how such mutations confer protection against the disease may be useful for developing new treatment strategies. A mouse ENU-induced mutagenesis screen for novel malaria resistance-conferring mutations identified a novel non-sense mutation in the gene encoding porphobilinogen deaminase (PBGD) in mice, denoted here as PbgdMRI58155. Heterozygote PbgdMRI58155 mice exhibited ~50% reduction in cellular PBGD activity in both mature erythrocytes and reticulocytes, although enzyme activity was ~10 times higher in reticulocytes than erythrocytes. When challenged with blood-stage P. chabaudi, which preferentially infects erythrocytes, heterozygote mice showed a modest but significant resistance to infection, including reduced parasite growth. A series of assays conducted to investigate the mechanism of resistance indicated that mutant erythrocyte invasion by P. chabaudi was normal, but that following intraerythrocytic establishment a significantly greater proportions of parasites died and therefore, affected their ability to propagate. The Plasmodium resistance phenotype was not recapitulated in Pbgd-deficient mice infected with P. berghei, which prefers reticulocytes, or when P. falciparum was cultured in erythrocytes from patients with acute intermittent porphyria (AIP), which had modest (20–50%) reduced levels of PBGD. Furthermore, the growth of Pbgd-null P. falciparum and Pbgd-null P. berghei parasites, which grew at the same rate as their wild-type counterparts in normal cells, were not affected by the PBGD-deficient background of the AIP erythrocytes or Pbgd-deficient mice. Our results confirm the dispensability of parasite PBGD for P. berghei infection and intraerythrocytic growth of P. falciparum, but for the first time identify a requirement for host erythrocyte PBGD by P. chabaudi during in vivo blood stage infection.

The specific PKC-\u03b1 inhibitor chelerythrine blunts costunolide-induced eryptosis

Costunolide, a natural sesquiterpene lactone, has multiple pharmacological activities such as neuroprotection or induction of apoptosis and eryptosis. However, the effects of costunolide on pro-survival factors and enzymes in human erythrocytes, e.g. glutathione and glucose-6-phosphate dehydrogenase (G6PDH) respectively, have not been studied yet. Our aim was to determine the mechanisms underlying costunolide-induced eryptosis and to reverse this process. Phosphatidylserine exposure was estimated from annexin-V-binding, cell volume from forward scatter in flow cytometry, and intracellular glutathione [GSH]i from high performance liquid chromatography. The oxidized status of intracellular glutathione and enzyme activities were measured by spectrophotometry. Treatment of erythrocytes with costunolide dose-dependently enhanced the percentage of annexin-V-binding cells, decreased the cell volume, depleted [GSH]i and completely inhibited G6PDH activity. The effects of costunolide on annexin-V-binding and cell volume were significantly reversed by pre-treatment of erythrocytes with the specific PKC-α inhibitor chelerythrine. The latter, however, had no effect on costunolide-induced GSH depletion. Costunolide induces eryptosis, depletes [GSH]i and inactivates G6PDH activity. Furthermore, our study reveals an inhibitory effect of chelerythrine on costunolide-induced eryptosis, indicating a relationship between costunolide and PKC-α. In addition, chelerythrine acts independently of the GSH depletion. Understanding the mechanisms of G6PDH inhibition accompanied by GSH depletion should be useful for development of anti-malarial therapeutic strategies or for synthetic lethality-based approaches to escalate oxidative stress in cancer cells for their sensitization to chemotherapy and radiotherapy.

Inhibition Effect of Eosin Y on Carbonic Anhydrase (CA) I and II Isoenzymes Purified from Human Erythrocytes

All cells produce carbon dioxide (CO2), which is released as a result of metabolism and must be removed from the body. A large part of this CO2 is converted to bicarbonate by the carbonic anhydrase (CA) enzyme in erythrocytes and is discarded from the body. So, CA has a vital role in red blood cells. In addition to, CA involved in many other pathological and physiological processes and it was determined that the inhibitors of CA were effective in the treatment and diagnosis of many diseases particularly glaucoma. Considering the importance of the CA's inhibitors, in this study it was intended to research the inhibition effects of Eosin Y on CA I and CA II isoenzymes activity purified from human erythrocytes. Eosin Y is a dye molecule commonly used in histological and medical applications. For this purpose, firstly CA I and CA II isoenzymes were purified from human erythrocytes by using Sepharose-4B-L-tyrosine-sulfanilamide affinity chromatography. Then the inhibitory effect of Eosin Y on the activity of these human erythrocyte CA I (hCA I) and CA II (hCA II) isoenzymes was investigated. It was determined that hCA I and hCA II were inhibited by Eosin Y in the millimolar range. IC50 values were found to be 3.78 mM for hCA I and 2.04 mM for hCA II and Ki values were determined as 9.65±0.968 mM and 7.52±2.88 mM for hCA I and hCA II, respectively. In conclusion, it is hoped that the results obtained in this study may be beneficial in the development of new CA inhibitors which may be drug candidates.

Smokeless tobacco induced biophysical and biochemical alterations in the plasma, erythrocytes, and platelets of panmasala users: Subsequent biological effects

Aim & backgroundSmokeless tobacco (SLT) products are extensively consumed throughout the world including India. These products act as the primary addictive agents, due to the presence of nicotine among other tobacco products to humans and animals and its quitting is difficult. Higher the exposure of SLT products more is the toxic effects and alterations in erythrocytes and platelets. ObjectivesThe products of smokeless tobacco could cause increase in the concentrations of oxidants (free radicals), decrease the activities antioxidant enzymes, activate the process of programmed cell death through enhanced expression of inducible nitric oxide synthase. Smokeless tobacco products represent a major modifiable risk factor for the development of redox imbalance through the enhanced production of reactive oxygen species and diminished activities of antioxidant enzymes in plasma, bio-membranes of erythrocytes, and platelets and induction of apoptosis in the blood. Materials and methodsThe protein expression of inducible nitric oxide synthase (iNOS) was studied by western blot and gene expression of apoptotic proteins, tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) was evaluated by RT-PCR technique. Membrane fluidity of erythrocytes and platelets was studied by the fluorescence method. ResultsThe results of the present study revealed that significantly elevated levels of iNOS enzyme in plasma, erythrocyte, and platelet membranes of panmasala users. We found that gene expression levels of Bcl2, Bax, IL-6, caspase proteins (Caspase 8, Caspase 10, and Caspase 12) are greater and decreased levels of TNF-α with no significant change in blood of smokeless tobacco users in comparison with normal controls. In addition, there were substantial significantly higher in concentrations of nicotine, cotinine, and epinephrine in the plasma of panmasala users than non-tobacco users. Panmasala can be caused a significant increase in nitroxidative stress marker (LPO, NO, and ONOO−) values and significant decrease in the levels of antioxidant enzymes in erythrocytes and platelets. ConclusionOn the basis of the present study results, it may be concluded that the chronic use of panmasala than any smokeless tobacco products may be a contributory risk factor or may give conclusive idea and has been associated with the expansion of the development of structural and functional alterations in the erythrocyte and platelet membranes induced oxidative damage and apoptosis, possibly further enhanced by nicotine and tobacco-specific N-nitrosamines. SLT exposure had implicated a threat and enormous implications on public health and is required to prove that may not be viewed as a safe alternative to any tobacco products.

Pyruvate Kinase Deficiency Cases Mimicking Congenital Dyserythropoietic Anemia

Introduction Pyruvate kinase (PK) deficiency is the most common glycolytic pathway defect that leads to hereditary hemolytic anemia with autosomal recessive inheritance pattern, which may present with severe hemolysis in the neonatal period, frequent blood transfusion requirement during infancy or with milder course of anemia. The prevalence reported among the Caucasians is 51 per million. Herein we present two cases of PK deficiency who were clinically and morphologically mimicking congenital dyserythropoietic anemia (CDA) at presentation and were diagnosed molecularly. Case 1 A three-year-old male patient was admitted with jaundice. Personal history revealed that he was born prematurely from a non-consanguineous family, and he did not need phototherapy at neonatal period. By the third month, he received first erythrocyte transfusion; then he needed transfusions every month. Strikingly, transfusion requirements increased in febrile episodes. Physical examination at admission revealed jaundice and splenomegaly. Complete blood count showed hemoglobin (Hb) 7.2 g/dL, mean corpuscular volume (MCV) 86.1 fL, mean corpuscular hemoglobin (MCH) 27.3 pg, mean corpuscular hemoglobin concentration (MCHC) 31.7 pg, and reticulocytes 12.3%. A blood film examination showed macrocytosis, hypochromia, anisocytosis, and rare spherocytes. Hemoglobin electrophoresis was normal, and direct Coombs' test was negative. The PK enzyme level was ignored since the patient was regularly transfused. Bone marrow aspiration showed erythroid hyperactivity and severe megaloblastic changes with double and multi-nucleated erythroid precursors suggestive for CDA. However, no genetic mutation detected concerning CDA (C15orf41, F8, F9, KLF1, CDAN1, VWF, SEC23B, GATA1, G6IBA genes). After negative molecular testing for CDA, sequencing of the PK-LR gene revealed the presence of a homozygous c.1151C&gt; T mutation. Case 2 A three-month-old male patient presented with pallor and jaundice. Personal history revealed that he was born at term from a non-consanguineous family, and he required phototherapy and exchange transfusion due to icterus and anemia at the neonatal period. He needed erythrocyte transfusions during follow-up. Hemogram revealed Hb 6.7 g/dL, MCV 81.2 fL, MCH 25.2 pg, MCHC 31.1 pg, and reticulocytes 2.6%. Blood film examination showed polychromasia, rare spherocytes, and acanthocytes. Direct Coombs' test and Parvovirus B19 PCR analysis were negative. Bone marrow aspiration showed erythroid hyperactivity along with double and multi-nucleated erythroid precursors, which suggested CDA. However, no genetic mutation detected concerning CDA (SEC23B, CDAN1). The PK enzyme level was normal, which was 2 ½ months after the last transfusion. Finally, the molecular analysis of the PK-LR gene revealed the presence of a novel homozygous c.880G&gt;A mutation. The mutation predicted as possibly damaging by in Silico analysis. Discussion The diagnostic approach to transfusion-dependent hereditary hemolytic anemia could be challenging related to false normal erythrocyte enzyme studies and osmotic fragility tests in previously transfused patients. It was reported that PK deficiency cases with inappropriately low reticulocytes might mimic CDA, which was discerned in cases where NGS performed for CDA but revealed PK-LR mutation. PK deficiency may lead to ineffective erythropoiesis due to erythrocyte ATP depletion, as well as CDA-like morphological findings (Cases 1 and 2) and low reticulocyte counts (Case 2). We suggest that in cases where CDA is suspected, PK deficiency should be considered in the differential diagnosis. Furthermore, instead of the PK enzyme level in patients with regular transfusions, PK-LR genetic analysis would be appropriate. Disclosures No relevant conflicts of interest to declare.

TGA/Chemometric Test Is Able to Detect the Presence of a Rare Hemoglobin Variant Hb Bibba.

In this study the TGA/Chemometric test was applied for diagnosis of a case of congenital hemolytic anemia for which the common first level diagnostic tests were not able to find the erythrocyte congenital defect. A 6 years old girl presented chronic hemolytic anemia characterized by hyperbilirubinemia, increased spleen, negative Coombs tests, normal hemoglobin values, decreased mean corpuscular volume (MCV), increased red cell distribution width (RDW), reticulocytes and lactate dehydrogenase (LDH), and altered erythrocyte morphology (ovalocytes, spherocytes, and rare schizocytes). The diagnostic protocols for differential diagnosis of hereditary hemolytic anemia were carried out by the investigation of the congenital hemolytic anemias due to defects of membrane proteins and the most common erythrocyte enzymes, but no defect was found. The TGA/Chemometric test was applied and the PLS-DA model of prediction was used to process results. The thermogravimetric profile of the patient was very distinct from those of healthy subjects and comparable with those of thalassemia patients. The classification model applied to the patient identified a chronic hemolytic anemia due to a hemoglobin defect and the molecular characterization confirmed the TGA/Chemometrics results, demonstrating the presence of a very rare hemoglobin variant Hb Bibba (α2136(H19)Leu → Proβ2). In conclusion the TGA/Chemometric test proved to be a promising tool for the screening of the hemoglobin defects, in a short time and at low cost, of this case of congenital hemolytic anemia of difficult diagnosis. This method results particularly suitable in pediatric patients as it requires small sample volumes and is able to characterize patients subjected to transfusion.

Type 2 diabetes differentially affects the substrate saturation kinetic attributes of erythrocyte hexokinase and phosphofructokinase.

The substrate kinetic parameters of hexokinase (HK) and phosphofructokinase (PFK)-the key irreversible enzymes of glycolysis-in erythrocytes from type 2 diabetic subjects were examined in comparison with control subjects. It was observed that the kinetic parameters such as Km , Vmax , Apparent Kcat , Kcat /Km , and substrate (ATP) inhibition kinetic and substrate binding characteristics are significantly altered in the diabetic group. The observed changes are suggestive of compositional changes in the subunit makeup of HK and PFK. The implication of these findings in relation to energy status of the diabetic erythrocyte and its interrelationship with loss of cell deformability are discussed here.

Oxidative Stress Status In Patients With Rheumatoid Arthritis.

Background: Rheumatoid arthritis (RA) is the most common form of inflammatory arthritis, which is an autoimmune disease characterized by chronic inflammation of synovial joints, ultimately leading to joint destructions and permanent disability. In RA oxidative stress are impaired (which caused by free radicals) might have an essential role in the etiology of RA. Objectives: The objective of this study was to determine oxidative stress by measuring malondialdehyde and enzymatic status by estimating superoxide dismutase, catalase and glutathione peroxidase in patients of RA and then comparing with healthy individuals. Setting and design:&nbsp;&nbsp; A total 42 patients with Rheumatoid Arthritis (20 females, 22 males) in the age of 30-50 years were included, as a control, 50 matched healthy volunteers (20 females and 30 males) were involve, non of the subjects smokes nor receiving any form of drugs. Subjects with any acute infections or with coexisting system disease such as coronary artery disease, hypertension or chronic renal failure were excluded. Methods: Antioxidant enzymes in erythrocyte , superoxide dismutase (SOD), glutathione peroxidase (Gpx)&nbsp; and Cayalase (CAT) was measured. Malondialdehyde (MDA) along with Copper, Zink and Iron are measured by using the serum of the patients and control group. Results: The serum level of MDL was higher in RA patients compared with the control group, the differences among both groups was statistically significant (P &lt; 0.001) Conclusion There is oxidative stress in RA patients evidenced by increased serum MDA and decreased antioxidant enzymes activity.

Phenotypic and genotypic characterization of glucose-6-phosphate dehydrogenase deficiency in Argentina. Retrospective and descriptive study.

Glucose-6-phosphate dehydrogenase deficiency is an erythrocyte enzyme disorder caused by mutations in the G6PD gene, which has an X-linked inheritance. Here we analyze the clinical and laboratory characteristics of 24 subjects with G6PD deficiency over 25 years. Their median age at diagnosis was 10.2 years (range: 0.6-56.4). No symptoms were observed in 54.2 % of patients, whereas 25 % had chronic non-spherocytic hemolytic anemia; 12.5 %, neonatal jaundice and postinfectious hemolytic anemia; and 8.3 %, acute hemolytic anemia after ingestion of fava beans. The 24 studied patients had variants that had been previously described in the bibliography. The clinical characteristics observed here were consistent with the variants found. A total of 21 women from the maternal line of affected subjects were identified as deficiency carriers using molecular biology techniques, so they received the corresponding genetic counseling.