Clinical and gene mutation characteristics of patients with hereditary ellipsocytosis: nine cases report and literature review / 中华血液学杂志

Objective: To report gene mutations in nine patients with hereditary elliptocytosis (HE) and analyze the characteristics of pathogenic gene mutations in HE. Methods: The clinical and gene mutations of nine patients clinically diagnosed with HE at Institute of Hematology & Blood Diseases Hospital from June 2018 to February 2022 were reported and verified by next-generation sequencing to analyze the relationship between gene mutations and clinical phenotypes. Results: Erythrocyte membrane protein gene mutations were detected among nine patients with HE, including six with SPTA1 mutation, one with SPTB mutation, one with EPB41 mutation, and one with chromosome 20 copy deletion. A total of 11 gene mutation sites were involved, including 6 known mutations and 5 novel mutations. The five novel mutations included SPTA1: c.1247A>C (p. K416T) in exon 9, c.1891delG (p. A631fs*17) in exon 15, E6-E12 Del; SPTB: c.154C>T (p. R52W) ; and EPB41: c.1636A>G (p. I546V) . Three of the six patients with the SPTA1 mutation were SPTA1 exon 9 mutation. Conclusion: SPTA1 is the most common mutant gene in patients with HE.

Enzymatic cleavage of cell surface proteins of pig and cow erythrocytes and its effect on concanavalin-mediated agglutinability.

Study was carried out to understand and compare architecture of the proteins of erythrocyte cell surface of some mammals viz., Homo sapiens (human), Sus scorfa domestica (pig) and Bos taurus domestica (cow). In this study, we investigated the action of proteinases viz., trypsin and chymotrypsin and neuraminidase on the erythrocyte surface proteins and erythrocyte agglutination tendency with a lectin (concanavalin A). The electrophoretic pattern of membrane proteins and glycophorins (analyzed by SDS-PAGE and visualized by Coomassie brilliant blue and periodic acid-schiff stains, respectively) and concanavalin A (Con A) agglutinability revealed that: (i) There were variations in the number and molecular weights of glycophorins in human, pig and cow, (ii) trypsin action on pig and cow erythrocyte membrane proteins was similar, unlike human, (iii) glycophorins degradation by trypsin and chymotrypsin was not similar in pig, as compared to that of human and cow, (iv) erythrocytes agglutination with Con A was significantly different due to differences in membrane composition and alterations in the surface proteins after enzyme treatment, (v) a direct correlation was found between degradation of glycophorins and Con A agglutinability, and (vi) removal of erythrocyte surface sialic acids by neuraminidase specifically indicated an increase in Con A agglutinability of pig and cow erythrocytes, similar to human.

Solubilization of human erythrocyte membranes by ASB detergents

Understanding the membrane solubilization process and finding effective solubilizing agents are crucial challenges in biochemical research. Here we report results on the interaction of the novel linear alkylamido propyl dimethyl amino propanosulfonate detergents, ASB-14 and ASB-16, with human erythrocyte membranes. An estimation of the critical micelle concentration of these zwitterionic detergents (ASB-14 = 100 µM and ASB-16 = 10 µM) was obtained using electron paramagnetic resonance. The amount of proteins and cholesterol solubilized from erythrocytes by these detergents was then determined. The hemolytic activities of the ASB detergents were assayed and the detergent/lipid molar ratios for the onset of hemolysis (Re sat) and total lysis (Re sol) were calculated, allowing the determination of the membrane binding constants (Kb). ASB-14 presented lower membrane affinity (Kb = 7050 M-1) than ASB-16 (Kb = 15610 M-1). The amount of proteins and cholesterol solubilized by both ASB detergents was higher while Re sat values (0.22 and 0.08 detergent/lipid for ASB-14 and ASB-16, respectively) were smaller than those observed with the classic detergents CHAPS and Triton X-100. These results reveal that, besides their well-known use as membrane protein solubilizers to enhance the resolution of two dimensional electrophoresis/mass spectrometry, ASB-14 and ASB-16 are strong hemolytic agents. We propose that the physicochemical properties of ASB detergents determine their membrane disruption efficiency and can help to explain the improvement in the solubilization of membrane proteins, as reported in the literature.

Host vascular endothelial growth factor is trophic for Plasmodium falciparum-infected red blood cells.

Plasmodium falciparum, the protozoan parasite responsible for severe malaria infection, undergoes a complex life cycle. Infected red blood cells (iRBC) sequester in host cerebral microvessels, which underlies the pathology of cerebral malaria. Using immunohistochemistry on post mortem brain samples, we demonstrated positive staining for vascular endothelial growth factor (VEGF) on iRBC. Confocal microscopy of cultured iRBC revealed accumulation of VEGF within the parasitophorous vacuole, expression of host VEGF-receptor 1 and activated VEGF-receptor 2 on the surface of iRBC, but no accumulation of VEGF receptors within the iRBC. Addition of VEGF to parasite cultures had a trophic effect on parasite growth and also partially rescued growth of drug treated parasites. Both these effects were abrogated when parasites were grown in serum-free medium, suggesting a requirement for soluble VEGF receptor. We conclude that P. falciparum iRBC can bind host VEGF-R on the erythrocyte membrane and accumulate host VEGF within the parasitophorous vacuole, which may have a trophic effect on parasite growth.

Erythrocyte membrane, plasma and atherosclerotic plaque lipid pattern in coronary heart disease / Perfil lipídico de membrana de eritrocito, plasma y placa ateromatosa en la enfermedad coronaria

The objective was to analyze the lipid composition of the atherosclerotic plaque (AP), plasma and erythrocyte membrane (EM) in patients with advanced coronary heart disease (CHD). AP were obtained through endarterectomy in 18 patients. Ten normolipemic healthy subjects were selected to obtain the normal lipid pattern profile. Total lipids of AP and EM were determined by HPTLC, and the fatty acid profile from AP, EM and plasma using TLC-FID. The relative amount of the lipid species analyzed in AP was in line with the data in the literature [phospholipids: 23.5 mol% more or less 3.5; total cholesterol 68.9 mol% more or less 7.9; triglyceride 7.6 mol% more or less 3.4]. Plasma and EM from CHD patients compared to controls, showed a decrease in polyunsaturated fatty acids and an increase in saturated fatty acids leading to a decrease in the unsaturation index (plasma: 1.67 more or less 0.06 vs. 1.28 more or less 0.03, P less than 0.05; EM: 2.28 more or less 0.04 vs. 1.25 more or less 0.010, P less than 0.05) and an enhancement in the saturated/unsaturated ratio (plasma: 0.35 more or less 0.02 vs. 0.52 more or less 0.02, P less than 0.05; EM: 0.45 more or less 0.01 vs. 0.83 more or less 0.04, P less than 0.05). These data are consistent with an essential fatty acid deficiency. Total cholesterol was increased in the CHD's EM (32.3 more or less 0.8 vs. 40.6 more or less 2.5, P less than 0.05) with a decrease in phospholipid percentage (67.7 more or less 0.7 vs. 59.4 more or less 2.6, P less than 0.05) indicating an alteration in membrane fluidity. These findings suggest changes in EM lipids in CHD patients in spite of different pathological conditions such as age, smoking status and diabetes. The analysis of the lipid composition of EM could provide a useful tool to monitor the evolution of the CHD.

El objetivo fue analizar la composición lipídica de las membranas de eritrocitos (ME), plasma y placas ateromatosas (PA) en pacientes con enfermedad coronaria avanzada (ECV). Las PA fueron obtenidas de endarterectomías coronarias de 18 pacientes. Fueron seleccionados 10 sujetos sanos, normolipémicos, como grupo control. Los lípidos totales de PA y ME se determinaron utilizando HPTLC, y el perfil de ácidos grasos de las PA, ME y plasma mediante TLC-FID. La cantidad relativa de las especies lipídicas obtenidas de las PA coinciden con la literatura [fosfolípidos 23.5 mol% más o menos 3.5; colesterol total 68.9 mol% más o menos 7.9; triglicéridos 7.6 mol% más o menos 3.4]. En el plasma y en las ME de los pacientes con ECV se observó, comparando con los pacientes controles, una disminución de los ácidos grasos poli-no saturados acompañado de un aumento de los ácidos grasos saturados que provocó el descenso del índice de instauración (plasma: 1.67 más o menos 0.06 vs. 1.28 más o menos 0.03, P menor que 0.05; ME: 2.28 más o menos 0.04 vs. 1.25 más o menos 0.010, P menor que 0.05) y el incremento del cociente AG saturados/insaturados (plasma: 0.35 más o menso 0.02 vs. 0.52 más o menos 0.02, P menor que 0.05; ME: 0.45 más o menos 0.01 vs. 0.83 más o menos 0.04, P menor que 0.05). Estos datos indicarían una deficiencia de ácidos grasos esenciales. Se observó una elevación en los valores de colesterol total (32.3 más o menos 0.8 vs. 40.6 más o menos 2.5, P menor que 0.05) y una disminución de los valores de fosfolípidos (67.7 más o menos 0.7 vs. 59.4 más o menos 2.6, P menor que 0.05) en las ME de los pacientes con ECV. Estos hallazgos sugieren cambios en los lípidos de las ME en los pacientes con ECV a pesar de presentar diferencias con respecto a edad, tabaquismo y diabetes. El conocimiento del perfil lipídico de las ME podría constituirse en una herramienta útil para monitorear la evolución de la enfermedad.

A unified biochemical and continuum mechanical red blood cell membrane bilayer--couple model.

We study the shapes and biochemical characteristics of human red blood cells using a unified biochemical and continuum mechanical model. In particular, we model the crenated, echinocytic shapes and show how they may shift due to changes in the pH and various amphipaths affecting the osmotic pressure by also utilizing pressure as an independent variable. In contrast to earlier works which advocate that biochemical factors may be attributable to mechanical control parameters, cytoskeletal elastic constants and effective relaxed bilayer area difference of outer plasma membrane and inner protein-based membrane skeleton, our unified model agrees well with Band 3 diffusion experimental root mean square distance data.

Erythrocyte membrane fatty acid composition in cancer patients

Essential fatty acids (EFA) have an important role in complex metabolic reactions. The metabolism of essential polyunsaturated fatty acids (PUFA) appears to be one of the critical targets in the complex metabolic stages that lead to, or are associated with cancer. The goal of our research was to analyze the erythrocyte specific types of membrane fatty acid content, level and distribution in cancer patients as compared to non-cancer patients. Changes in fatty acid composition may affect different aspects of cell structure and function, including proliferation. Analyses of RBCs membrane fatty acids were performed for 255 patients with different types of cancer (breast, prostate, liver, pancreas, colon, and lung), 2,800 non-cancer patients and 34 healthy volunteers. Our research study demonstrated a lower level of stearic acid and an increased content of oleic acid in RBC of cancer patients in comparison with control and non-cancer patients. According to the results of this investigation, the ratio of Eicosa pentaenoic acid (EPA) and Decosa hexaenoic acid (DHA) to Alpha-linolenic acid (ALA) may be useful to estimate PUFA imbalances in cancer patients. EPA and DHA acid may be recommended as supplementation and in addition to current therapy during cancer treatment

Effect of metal ions on binding of bilirubin to erythrocyte membranes.

Binding of bilirubin to different erythrocyte membranes, namely, human, buffalo, sheep and goat, pre-incubated with different concentrations of metal ions was studied. The results showed that among the different metal ions used, Ca2+ had the highest potential in increasing the amount of bound bilirubin followed by Sr2+ and Mg2+, whereas Ba2+ had the lowest potential. Treatment of these membranes with Ca2+ led to an increase in the amount of bound bilirubin in all membranes. However, human erythrocyte membranes pretreated with Ca2+, bound the highest amount of bilirubin compared to other erythrocyte membranes. Increase in bilirubin binding upon Ca2+-treatment can be ascribed to shielding effect, redistribution of phospholipids as well as increase in surface hydrophobicity induced by Ca2+.

Hypothalamic digoxin and neural regulation of blood pressure and vascular thrombosis.

The isoprenoid pathway produces three key metabolites--digoxin (membrane sodium-potassium ATPase inhibitor and regulator of neurotransmitter/aminoacid transport), dolichol (regulates N-glycosylation of proteins) and ubiquinone (free radical scavenger). This was assessed in patients with essential hypertension, familial hypotension, acute coronary artery disease and acute thrombotic strokes. The pathway was also assessed in patients with right hemispheric, left hemispheric and bihemispheric dominance for comparison. In patients with acute coronary artery disease, acute thrombotic stroke, essential hypertension and right hemispheric dominance, there was elevated digoxin synthesis, increased dolichol and glycoconjugate levels and low ubiquinone and high free radical levels. There was also an increase in tryptophan catabolites, reduction in tyrosine catabolites, increase in cholesterol-phospholipid ratio and a reduction in glycoconjugate level of RBC membrane in this group of patients as well as in those with right hemispheric dominance. In patients with familial hypotension and left hemispheric dominance, the patterns were reversed. The role of a dysfunctional isoprenoid pathway and endogenous digoxin in the pathogenesis of essential hypertension and familial hypotension and in thrombotic vascular disease in relation to hemispheric dominance is discussed.

Effect of pH and temperature on the binding of bilirubin to human erythrocyte membranes.

Effect of pH and temperature on the binding of bilirubin to human erythrocyte membranes was studied by incubating the membranes at different pH and temperatures and determining the bound bilirubin. At all pH values, the amount of membrane-bound bilirubin increased with the increase in bilirubin-to-albumin molar ratios (B/As), being highest at lower pH values in all cases. Further, linear increase in bound bilirubin with the increase in bilirubin concentration in the incubate was observed at a constant B/A and at all pH values. However, the slope value increased with the decrease in pH suggesting more bilirubin binding to membranes at lower pH values. Increase in bilirubin binding at lower pH can be explained on the basis of increased free bilirubin concentration as well as more conversion of bilirubin dianion to monoanion. Temperature dependence of bilirubin binding to membranes was observed within the temperature range of 7 degrees -60 degrees C, showing minimum binding at 27 degrees C and 37 degrees C which increased on either side. Increase in bilirubin binding at temperatures lower than 20 degrees C and higher than 40 degrees C can be ascribed to the change in membrane topography as well as bilirubin-albumin interaction.

Changes in the composition of erythrocyte membrane during storage of blood in di-(2-ethyl hexyl) phthalate [DEHP] plasticized poly vinyl chloride (PVC) blood storage bags.

Very little information is available on the changes in the erythrocyte membrane composition during storage of blood at 4 degrees C, particularly with respect to the glycosaminoglycans and glycoproteins. In view of this, a detailed study was carried out on the changes in the membrane proteins, glycosaminoglycans (GAG), carbohydrate components of glycoproteins, cholesterol, phospholipids and vitamin E in blood stored in glass bottles and a di-(2-ethyl hexyl) phthalate (DEHP) plasticized PVC bag (Penpol blood bag). Blood was collected in CPDA solution in glass bottles and in Penpol blood bags and kept at 4 +/- 1 degrees C. Analysis was made immediately after blood collection and after 28 and 42 days. Significant increase in the total protein in the erythrocyte membrane was observed during storage of whole blood in glass bottles and Penpol blood bag at 4 degrees C. This increase was progressively more with increase in storage time. Significant changes were also observed in GAG, carbohydrate components of glycoproteins, cholesterol, phospholipids and vitamin E in the erythrocyte membrane under these conditions. The protein:GAG ratio, protein:carbohydrate ratio, cholesterol:phospholipid ratio as well as protein:lipid ratio showed significant increase in the membrane. The extent of these changes was lower in the Penpol bag, indicating the stabilizing effect of DEHP on the erythrocyte membrane.

Surface changes of the erythrocytes membrane in alcoholics--a study of lectin binding.

Erythrocytes from 30 alcoholic patients as well as age and sex matched healthy control were examined, for hemagglutination titre, against, a purified galactose specific lectin from the latex of Pedilanthus tithymalodies. Mean hemagglutinin titre was significantly elevated (P < 0.001) in alcoholics as compared to controls. The results indicate that alcohol abuse is associated with adoptive cell surface changes on erythrocytes, measurable by its lectin binding capacity.

Glycoconjugate profile in plasma and erythrocytes of gastric cancer patients.

The present study has examined the glycoconjugate profile in plasma and erythrocyte membranes of 24 adult male gastric cancer patients and an equal number of age and sex-matched controls. Protein-bound hexose, hexosamine and sialic acid were significantly increased in plasma and erythrocytes of gastric cancer patients compared to controls. Elevation of glycoconjugates in circulation is suggested to be a result of increased shedding by the tumor cells or increased synthesis by liver, due to acute phase response.

Hormonal-induced changes on lipidi composition and DPH fluorescence anisotropy of erithrocyte ghost from pre-and postmenopausal women

Fluorescence anisotropy determination were performed on hypotonically lysed red blood cells from 12 healthy, free living, premenopausal and 10 postmenopausal women fed a self-selected diet. Fatty acid composition of phospholipids and cholesterol esters, lipid/lipid and lipid/protein ratios were also determined in erythrocyte ghosts along the menstrual cycle or during hormonal replacemente therapy. The postmenopausal women were treated with transdermal 17-beta estradiol (approx. delivery rate of 50 mug/day) for a 5-week period and with a combination of percutaneous administration of estradiol and orally administered medroxyprogesterone acetate (5 mg/day) for 5 weeks more. Premenopausal women were studied in the mid-follicular and mid-luteal phases of their menstrual cycles. Membrane fluidity in cells from the luteal phase was greater than that observed in the follicular phase. After estradiol treatment, postmenopausal women exhibited a significant raise in the fluorescence anisotropy. The association with progesterone prevented the estrogen-induced increase of the anisotropy values. Estrogen administration also produced an increment in the relative content of cholesterol and membrane proteins that was reverted by progesterone addition. Changes induced by estradiol treatment in the fatty acid pattern of phospholipids and cholesterol esters were characterized by an increment in the monoenoic/saturated fatty acid ratio and linoleic relative content, with a concomitant decrease in the proportion of arachidonic and docosatetraenoic acids. These results suggest that the modification of the desaturase activities may be involved in a mechanism by which these hormonal-induced changes take place. The present study demonstrated that human erythrocyte fluidity is under hormonal control subjected through changes in the proportion and quality of the membrane lipids.

Glycosylation of hemoglobin and erythrocyte membrane proteins mediated changes in osmotic fragility of erythrocytes.

The increased levels of glycosylated hemoglobin and glycosylated erythrocyte membrane proteins in gestational diabetes could bring about alterations in osmotic fragility in erythrocytes of gestational diabetes during their first trimester. The glycosylated hemoglobin levels in erythrocytes were found to be significantly elevated with a mean of 5.42 +/- 0.77 mg/dl when compared to 3.45 +/- 0.62 mg/ dl in non diabetic pregnant control (where as in non pregnant women the value was 3.4 +/- 0.47 (n = 25)). The mean osmotic fragility (MOF) of control cells was 60.2 + 0.6 mMol/L Nacl and MOF of gestational diabetic cells was 63.93 +/- 0.6 mMol/L Nacl. The mean osmotic fragility, operationally defined as the Nacl concentration for 50% hemolysis, was found significantly higher by 3.9 + 0.01 mMol/L Nacl in gestational diabetes, than in normal cells. The total levels of glycosylated membrane proteins were increased from 50.60 +/- 8.0 in control to 69.14 + 0.47 in gestational diabetes. Therefore it is proposed that the increased levels of glycosylated Hb as well as glycosylated membrane proteins have a role in altering the membrane permeability resulting in increased osmotic fragility of erthrocytes in gestational diabetes.

Interaction of organophosphorus insecticides phosphamidon & malathion on lipid profile & acetylcholinesterase activity in human erythrocyte membrane.

The organophosphorus insecticides phosphamidon and malathion were found to inhibit the activity of human acetylcholinesterase in vitro, in the human erythrocyte membrane. Lineweaver-Burk analysis indicated that the insecticide induced inhibition of acetylcholinesterase activity was uncompetitive in nature. The total lipid, cholesterol and phospholipid contents of erythrocyte membranes were reduced following phosphamidon and technical malathion treatment, while the level of lipid peroxidation was raised following malathion treatment.

Transport pathways in the malaria-infected erythrocyte: characterization and their use as potential targets for chemotherapy

The intraerythrocytic malarial parasite is involved in an extremely intensive anabolic activity while it resides in its metabolically quiescent host cell. The necessary fast uptake of nutrients and the discharge of waste product, are guaranteed by parasite-induced alterations of the constitutive transporters of the host cell and the production of new parallel pathways. The membrane of the host cell thus becomes permeable to phospholipids, purine bases and nucleosides, small non-electrolytes, anions and cations. When the new pathways are quantitatively unimportant, classical inhibitors of native transporters can be used to inhibit parasite growth. Several compounds were found to effectively inhibit the new pathways and consequently, parasite growth. The pathways have also been used to introduce cytotoxic agents. The parasitophorous membrane consists of channels which are highly permeable to small solutes and display no ion selectivity. Transport of some cations and anions across the parasite membrane is rapid and insensitive to classical inhibitors, and in some cases it is mediated by specific antiporters which respond to their respective inhibitors. Macromolecules have been shown to reach the parasitophorous space through a duct contiguous with the host cell membrane, and subsequently to be endocytosed at the parasite membrane. The simultaneous presence of the parasitophorous membrane channels and the duct, however, is incompatible with experimental evidences. No specific inhibitors were found as yet that would efficiently inhibit transport through the channels or the duct.

Comparative analysis of erythrocyte fatty acid composition in a sample of Mexico city children and young adults on a free diet

In the present study the influence of age on red blood cell fatty acid (RBCFA) composition was analyzed in a sample of Mexico City children and young people on a free diet, as there is scarce information about RBCFA composition in the Mexican population. Erythrocyte lipids were extracted with isopropyl alcohol and fatty acid methyl esters were prepared to be analyzed by gas liquid chromatography. The 1. to 2-year-old group showed a higher percent level of C18:0 (34.73 ñ 2.5 vs. 29.67 ñ 1.3, p<0.002) and lower of C16:1 (0.58 ñ 0.2 vs. 1.09 ñ 0.2, p<0.005), C20:4 (14.08 ñ 4.1 vs, 18.20 ñ 1.2, p<0.05) and C22:5 (2.79 ñ 1.7 vs. 7.68 ñ 0.8, p<0.001) than the 20- to 25-year-old group. Both groups showed a very low linoleic acid proportion, children 0.48 percent and young adults 0.54 percent. The unsaturated/saturated fatty acid ratio was found to be 0.55 ñ 0.2 in children and 0.91 ñ 0.1 in adults (p<0.001). These findigs indicate the presence of factors related to age that affect the fatty acid composition in the erythrocyte membrane different from diet habits in the sample analyzed. Results are compared with reports in the literature

Estudio clinico y bioquimico de 9 pacientes con eliptocitosis hereditaria / Clinical and biochemical study of 9 patients with hereditary elliptocytosis

Se estudiaron nueve pacientes (cuatro familias) con eliptocitosis herediataria (EH). Desde el punto de vista clínico, se identificaron dos tipos de presentación: siete pacientes fueron formas compensadas y dos (de 7 y 63 años) pertenecientes a distintas familias mostraron anemia, esplenomegalia, reticulocitosis y fragmentación globular. En una familia se comprobó una disminución del stock total de la proteína 4.1 en la electroforesis de proteína de membrana en gel de poliacrilamida con SDS (SDS-PAGE). Uno de sus miembros se comprobó como forma descompensada. El estudio de dímeros y tetrámeros y la digestión tríptica de espectrina fue normal en todos los casos. En la determinación de la deformabilidad en función de la osmoralidad con el ectacitómetro se halló una buena correlación entre el índice de deformabilidad (ID) y el grado de anemia. De acuerdo a los resultados de este estudio pudimos confirmar la gran heterogeneidad clínica de la EH aún en miembros de una misma familia