pyrimidinones and Hemolysis

Topics: Adult; Child; Child, Preschool; Erythrocytes; Female; Glucosephosphate Dehydrogenase Deficiency; Glucosides; Hemolysis; Humans; Male; Pyrimidinones; Seeds; Vicia faba

Faba bean (Vicia faba) vicine and convicine (V-C) aglycones (divicine and isouramil respectively) provoke an acute hemolytic anemia called favism in individuals with a glucose-6-phosphate dehydrogenase (G6PD) enzyme defect in their red blood cells. Geneticists/plant breeders are working with faba bean to decrease V-C levels to improve public acceptance of this high-protein pulse crop. Here, we present a fast and simple ex vivo in vitro bioassay for V-C toxicity testing of faba bean or faba bean food products.. We have shown that 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU)-treated (i.e., sensitized) normal red blood cells, like G6PD-defective blood, displayed (i) continuous glutathione (GSH) depletion with no regeneration as incubation time and the dose of aglycones increased, (ii) progressive accumulation of denatured hemoglobin products into high molecular weight (HMW) proteins with increased aglycone dose, (iii) both band 3 membrane proteins and hemichromes, in HMW protein aggregates. We have also demonstrated that sensitized red blood cells can effectively differentiate various levels of toxicity among faba bean varieties through the two hemolysis biomarkers: GSH depletion and HMW clumping.. BCNU-sensitized red blood cells provide an ideal model for favism blood, to assess and compare the toxicity of faba bean varieties and their food products. © 2018 Society of Chemical Industry.

Topics: Biological Assay; Erythrocytes; Favism; Glucosephosphate Dehydrogenase; Glucosides; Hemolysis; Humans; Pyrimidinones; Uridine; Vicia faba

The aim of this study is to identify a plasticizer that is effective in the suppression of the autohemolysis of the stored blood and can be used to replace di(2-ethylhexyl) phthalate (DEHP) in blood containers. The results of hemolysis test using mannitol-adenine-phosphate/red cell concentrates (MAP/RCC) spiked with plasticizers included phthalate, phthalate-like, trimeliate, citrate, and adipate derivatives revealed that di-isononyl-cyclohexane-1,2-dicarboxylate (Hexamoll(®) DINCH), di(2-ethylhexyl)-1,2,3,6-tetrahydro-phthalate (DOTP), and diisodecyl phthalate (DIDP) exhibited a hemolysis suppression effect almost equal to that of DEHP, but not other plasticizers. This finding suggested that the presence of 2 carboxy-ester groups at the ortho position on a 6-membered ring of carbon atoms may be required to exhibit such an effect. The hemolytic ratios of MAP/RCC-soaked polyvinyl chloride (PVC) sheets containing DEHP or different amounts of DINCH or DOTP were reduced to 10.9%, 9.2-12.4%, and 5.2-7.8%, respectively (MAP/RCC alone, 28.2%) after 10 weeks of incubation. The amount of plasticizer eluted from the PVC sheet was 53.1, 26.1-36.5, and 78.4-150 µg/mL for DEHP, DINCH, and DOTP, respectively. PVC sheets spiked with DIDP did not suppress the hemolysis induced by MAP/RCC because of low leachability (4.8-6.0 µg/mL). These results suggested that a specific structure of the plasticizer and the concentrations of least more than ∼10 µg/mL were required to suppress hemolysis due to MAP/RCC.

Topics: Adenine; Benzoates; Blood Preservation; Citrates; Cyclohexanecarboxylic Acids; Depression, Chemical; Dicarboxylic Acids; Diethylhexyl Phthalate; Gas Chromatography-Mass Spectrometry; Glucose; Hemolysis; Heparin; Humans; Inosine Nucleotides; Mannitol; Oxazoles; Plasticizers; Polyvinyl Chloride; Pyrimidinones; Structure-Activity Relationship

Several thieno[2,3-d]pyrimidinediones have been synthesized and examined for antibacterial activity against a range of gram-positive and gram-negative pathogens. Two compounds displayed potent activity (2-16 mg/L) against multi-drug resistant gram-positive organisms, including methicillin resistant, vancomycin-intermediate, vancomycin-resistant Staphylococcus aureus (MRSA, VISA, VRSA) and vancomycin-resistant enterococci (VRE). Only one of these agents possessed moderate activity (16-32 mg/L) against gram-negative strains. An examination of the cytotoxicity of these agents revealed that they displayed low toxicity (40-50 mg/L) against mammalian cells and very low hemolytic activity (2-7%). Taken together, these studies suggest that thieno[2,3-d]pyrimidinediones are interesting scaffolds for the development of novel gram-positive antibacterial agents.

Topics: Animals; Anti-Bacterial Agents; Bacteria; Hemolysis; Mice; NIH 3T3 Cells; Pyrimidinones; Sheep

Lipid peroxidation and the accompanying translocation of phosphatidylserine (PS) from the inner to the outer leaflet of the lipid bilayer have recently been identified as key components of a signaling pathway for phagocytosis of apoptotic cells by macrophages. Drug-induced hemolytic anemia has long been known to be caused by an accelerated uptake of damaged (but intact) erythrocytes by macrophages in the spleen, and this process has been associated with enhanced formation of reactive oxygen species (ROS). However, the role of lipid peroxidation in hemolytic injury has remained unclear, and the effect of hemolytic agents on the distribution of PS in the erythrocyte membrane is unknown. The present studies were undertaken to determine whether lipid peroxidation and PS translocation could be detected in rat and human erythrocytes by three types of direct-acting hemolytic agents--dapsone hydroxylamine, divicine hydroquinone, and phenylhydrazine. 2',7'-Dichlorodihydrofluorescein diacetate was employed as a probe for intracellular ROS formation; lipid peroxidation was assessed by GC/MS analysis of F2-isoprostanes; and PS externalization was measured by annexin V labeling and the prothrombinase assay. The data confirmed that all three hemolytic agents generate ROS within erythrocytes under hemolytic conditions; however, no evidence for lipid peroxidation or PS translocation was detected. Instead, ROS production by these hemolytic agents was associated with extensive binding of oxidized and denatured hemoglobin to the membrane cytoskeleton. The data suggest that the transmembrane signal for macrophage recognition of hemolytic injury may be derived from oxidative alterations to erythrocyte proteins rather than to membrane lipids.

Topics: Animals; Dapsone; Dose-Response Relationship, Drug; Erythrocytes; Hemoglobins; Hemolysis; Humans; In Vitro Techniques; Lipid Peroxidation; Lipids; Phenylhydrazines; Phosphatidylserines; Proteins; Pyrimidinones; Rats; Reactive Oxygen Species

Incubation of cells and tissues with saponin makes the lipid bilayer permeable to macromolecules. Ghosts (membrane preparations) of saponin-lysed erythrocytes do not reseal, thus indicating an irreversible damage of the lipid bilayer. We investigated the influence of disturbance of the lipid bilayer on membrane proteins by comparing ghosts of saponin-lysed erythrocytes with ghosts of cells lysed in hypotonic buffer. Transmission electron microscopy revealed destruction of the lipid bilayer and emergence of multilamellar buds in saponin-lysed ghosts. Freeze-fracture electron microscopy showed regions with crystalline lipids and an increase in particle-free areas on fracture faces. The number of protein sulfhydryl groups and the binding of hemoglobin were diminished in saponin-lysed ghosts. A Scatchard plot of hemoglobin binding revealed the decrease of high affinity binding sites. All these results indicate an aggregation of band 3 protein also demonstrated by laser scanning microscopy after incubation of cells labelled with eosin-5-maleimide with sublytic concentration of saponin. Hemolysis with saponin also affected the interaction between transmembrane proteins and the cytoskeleton. Dissociation of peripheral membrane proteins by incubation of ghosts in low salt buffer or by blocking sulfhydryl groups was increased and the association of spectrin with spectrin-depleted vesicles was decreased. The increased incorporation of the fluorescent probe Merocyanine 540 into saponin-lysed ghosts and the increased relative fluorescence quantum yield confirmed the perturbation of the lipid bilayer and the changed interaction between membrane lipids and intrinsic membrane proteins. Our results suggest that permeabilization of the lipid bilayer with saponin to admit the access of antibodies to the cytoplasmic surface of cells can aggregate transmembrane proteins and affect the immunocytochemical localization of associated proteins of the cytoskeleton.

Topics: Anion Exchange Protein 1, Erythrocyte; Binding Sites; Cell Membrane Permeability; Erythrocyte Membrane; Erythrocytes; Freeze Fracturing; Hemoglobins; Hemolysis; Humans; Lipid Bilayers; Membrane Proteins; Pyrimidinones; Saponins; Spectrin

Favism is an acute hemolytic anemia known to occur in susceptible individuals who ingest fava beans. Susceptibility to favism is conferred by a genetic deficiency in erythrocytic glucose-6-phosphate dehydrogenase (G6PD) activity. Although the fava bean pyrimidine aglycones, divicine and isouramil, have been implicated in the onset of favism in humans, the lack of a well-defined experimental animal model for favism has hampered progress in elucidating the mechanism underlying hemotoxicity. We have examined whether a favic-like response could be provoked in G6PD-normal rats treated with synthetic divicine. Intraperitoneal administration of divicine to rats preloaded with 51Cr-tagged erythrocytes resulted in a severe, dose-dependent decrease in blood radioactivity (TD50 approximately 0.5 mmol/kg) within 24 h. The increased rate of removal of blood radioactivity was accompanied by a rapid decline in reduced glutathione levels in the blood, decreased hematocrits, marked hemoglobinuria, splenic enlargement, and reticulocytosis. In vitro exposure of 51Cr-tagged red cells to divicine before their re-administration to isologous rats also resulted in a sharp, concentration-dependent decrease in erythrocyte survival in vivo (TC50 approximately 1.5 mM), and these divicine-damaged red cells were removed from the circulation by the spleen. These data demonstrate that a favic response can be induced in G6PD-normal rats treated with divicine, and that hemolytic activity can be reproduced in isolated red cells under conditions that will allow a direct examination of the mechanism underlying this hemotoxicity.

Topics: Animals; Chromium; Chromium Radioisotopes; Dose-Response Relationship, Drug; Erythrocytes; Fabaceae; Favism; Glucosephosphate Dehydrogenase; Glutathione; Hemoglobins; Hemolysis; Lethal Dose 50; Male; Plants, Medicinal; Plants, Toxic; Pyrimidinones; Rats; Rats, Sprague-Dawley

Incubation of glutathione (GSH) depleted mouse erythrocytes with the oxidants phenylhydrazine, acrolein, divicine and isouramil resulted in the release of free iron and in lipid peroxidation and hemolysis. The addition of the flavonoid quercetin, which chelates iron and penetrates erythrocytes, resulted in remarkable protection against lipid peroxidation and hemolysis. The protection seems to be due to intracellular chelation of iron, since a semi-stoichiometric ratio between released iron and the amount of quercetin necessary to prevent lipid peroxidation and hemolysis was found. Incubation of GSH depleted human erythrocytes with divicine and isouramil did not induce lipid peroxidation and hemolysis in spite of a substantial release of iron. However, divicine and isouramil produced alterations of membrane proteins, such as spectrin and band 3, as well as formation of senescent cell antigen. The addition of quercetin prevented these alterations.

Topics: Acrolein; Animals; Barbiturates; Chelating Agents; Erythrocyte Membrane; Erythrocytes; Flavonoids; Glutathione; Hemolysis; Humans; Iron; Iron Chelating Agents; Kinetics; Lipid Peroxidation; Male; Malondialdehyde; Methemoglobin; Mice; Pyrimidinones; Quercetin

The interaction of the red cell membrane with merocyanine 540 or protoporphyrin led to four phenomena, most probably interrelated. (i) The morphology changed from the normal discoid to an echinocytic form. This morphological change persisted when followed over a period of 24 h. (ii) Simultaneously, cell deformability was decreased, as revealed by viscosity measurements and a cell-filtration technique. (iii) Both drugs caused swelling of the erythrocytes in isotonic medium, due to a very-short-term increased permeability of the membrane, also for larger molecules such as lactose. The pathway of this temporary leak seems to be unrelated to the Na+/K+ -ATPase, the K+/Cl- and the Na+/K+/Cl- cotransport systems, the Ca2+-activated Gardos pathway, the oxidation/deformation-activated leak pathway and the so-called residual transport route. Despite the morphological changes, K+-leakage induced by mechanical stress was not increased. (iv) During osmotic swelling, the critical hemolytic volume was found to be increased in the presence of either merocyanine 540 or protoporphyrin. The increase critical volume protected erythrocytes against osmotic hemolysis.

Topics: Antiviral Agents; Cell Membrane Permeability; Chemical Phenomena; Chemistry, Physical; Erythrocyte Deformability; Erythrocyte Membrane; Erythrocytes; Hemolysis; Humans; Osmosis; Phospholipids; Photosensitizing Agents; Protoporphyrins; Pyrimidinones; Viscosity

The endemic occurrence of favism in certain Mediterranean regions provided an investigative opportunity for testing in vivo the validity of claims as to the role of catalase in protecting human erythrocytes against peroxidative injury. Reduced activity of catalase was found in the erythrocytes of six boys who were deficient in erythrocytic glucose-6-phosphate dehydrogenase (G6PD) and who were studied while suffering hemolysis after ingesting fava beans. Activity of catalase was further reduced when their red blood cells were incubated with aminotriazole. In contrast, minimal reduction of catalase activity was found, both with and without incubation with aminotriazole, in erythrocytes of a G6PD-deficient boy who had ingested fava beans 7 days earlier and in erythrocytes of seven G6PD-deficient men with a past history of favism. These results confirmed earlier studies in vitro indicating that catalase is a major disposer of hydrogen peroxide in human erythrocytes and, like the glutathione peroxidase/reductase pathway, is dependent on the availability of reduced nicotinamide adenine dinucleotide phosphate (NADPH). The effect of divicine on purified catalase and on the catalase of intact G6PD-deficient erythrocytes was similar to the previously demonstrated effect on catalase of a known system for generating hydrogen peroxide. This effect of divicine strengthens earlier arguments that divicine is the toxic peroxidative component of fava beans.

Topics: Catalase; Child; Child, Preschool; Enzyme Inhibitors; Erythrocytes; Favism; Hemolysis; Humans; Hydrogen Peroxide; Male; NADP; Oxidative Stress; Pyrimidinones

Some differences were established between mopyridone-sensitive (MCU-s) wild strain and mopyridone-resistant (MCU-r) mutant progenies of influenza virus A/Hong Kong/1/68 (H3N2). The virions of MCU-r mutant had a lower buoyant density in linear sucrose gradient as compared to those of MCU-s strain, and an increased ability of aggregation as well. HA content (HAU/micrograms protein) in the purified virions of MCU-r mutant was twice lower as compared to MCU-s strain. The surface glycoproteins of MCU-r mutant were solubilized by octylglucoside faster than those of MCU-s strain. No differences were found between MCU-r strain and MCU-s mutant-induced red blood cell lysis at acid pH, and mopyridone did not influence this phenomenon. MCU-r mutant showed a lower thermostability as compared with MCU-s strain, but similar UV-inactivation curves for both viruses were observed. The quantity of the purified HA-NA complex and M1 protein incorporated into multilamellar liposomes was greater in the case of MCU-s mutant. Electron microscopy examination of liposomes which contained M1 protein from MCU-r mutant manifested pleomorphism with unusual gigantic forms tending to aggregate, whereas MCU-s M1 protein-containing liposomes were uniform and did not form aggregates. No morphological differences were found between the two viruses in HA-NA complex containing liposomes. These data indicate changes in the protein-lipid interactions in MCU-r mutant virions. Amino acid analysis of M1 protein revealed significantly lower content of asparagine, glutamine and serine, and a higher one oof hisitidine in MCU-r mutant as compared to MCU-s wild strain.

Topics: Amino Acids; Animals; Cell Line; Chick Embryo; Drug Resistance, Microbial; Glucosides; Hemolysis; Humans; Influenza A virus; Influenza A Virus, H3N2 Subtype; Liposomes; Mutation; Pyrimidinones; Ultraviolet Rays; Viral Matrix Proteins; Viral Proteins; Virion

Mouse erythrocytes were incubated with oxidizing agents, phenylhydrazine, divicine and isouramil. With all the oxidants a rapid release of iron in a desferrioxamine (DFO)-chelatable form was seen and it was accompanied by methaemoglobin formation. If the erythrocytes were depleted of GSH by a short preincubation with diethyl maleate, the release of iron was accompanied by lipid peroxidation and, subsequently, haemolysis. GSH depletion by itself did not induce iron release, methaemoglobin formation, lipid peroxidation or haemolysis. Rather, the fate of the cell in which iron is released depended on the intracellular availability of GSH. In addition, iron release was higher in depleted cells than in native ones, suggesting a role for GSH in preventing iron release when oxidative stress is imposed by the oxidants. Iron release preceded lipid peroxidation. The latter was prevented when the erythrocytes were preloaded with DFO in such a way (preincubation with 10 mM-DFO) that the intracellular concentration was equivalent to that of the released iron, but not when the intracellular DFO was lower (preincubation with 0.1 mM-DFO). Extracellular DFO did not affect lipid peroxidation and haemolysis, suggesting again that the observed events occur intracellularly (intracellular chelation of released iron). The relevance of iron release from iron complexes in the mechanisms of cellular damage induced by oxidative stress is discussed.

Topics: Animals; Barbiturates; Deferoxamine; Erythrocyte Membrane; Glutathione; Hemolysis; Iron; Lipid Peroxidation; Male; Methemoglobin; Mice; Oxidants; Phenylhydrazines; Pyrimidinones

In the present study we have assayed the effect of divicine in G6PD-deficient red blood cells in the presence of deferoxamine (iron-chelating drug) and NaN3 (inhibitor of catalase). The effect of divicine has been compared to oxidative stress by H2O2; haemolysis is regarded as an index of cellular toxicity. In addition, we have tested antioxidant enzymatic systems. No significant change in antioxidant enzymatic systems was found in RBCs from subjects with G6PD deficiency when compared to the control group, either in oxidative haemolysis by divicine or by H2O2; a significant decrease in oxidative haemolysis by H2O2 was observed in the presence of deferoxamine, whereas no change was found in oxidative haemolysis by divicine. The replacement of incubation medium by homologous plasma or the supplementation with bovine serum albumin resulted in a marked decrease of percentage of haemolysis by divicine.

Topics: Adult; Catalase; Deferoxamine; Erythrocytes; Glucosephosphate Dehydrogenase Deficiency; Glutathione Peroxidase; Glutathione Reductase; Hemolysis; Humans; Hydrogen Peroxide; In Vitro Techniques; Male; Oxidation-Reduction; Pyrimidinones

Cytotoxic T lymphocytes (CTL) release from their granules a 70 kDa protein, called PFP, perforin or cytolysin, which inserts into the target cell plasma membrane in its monomeric form. Here it polymerizes into a macromolecular complex forming pores as large as 20 nm. Although purified PFP/perforin can effectively lyze all target cells tested. CTL are refractory to lysis. The mechanism underlying the resistance of CTL is currently unknown. This study represents a search for membrane structural properties that could confer resistance to CTL against PFP/perforin-mediated lysis. The fluorescent dye merocyanine 540 was used to measure the lipid head group packing of CTL and several target cells, and 1-[4-(trimethylamine)phenyl]-6-phenylhexa-1,3,5-triene was used to estimate the fluidity of the membrane hydrocarbon region. The resistance against PFP/perforin-mediated lysis was determined by the 51Cr release assay. A comparison of the membrane rigidity with cell resistance led to the conclusion that the membrane lipid structure cannot account for the unusually high resistance of CTL. In particular, the resistant CTL line CTLL-2 has a lipid head group packing that is looser than that of Yac-1, and the sensitive target cells Jy-25 and EL-4 have membrane acyl chains that are less fluid than those of the effector CTLL-R8.

Topics: Animals; Cell Line; Cell Membrane; Diphenylhexatriene; Erythrocyte Membrane; Fatty Alcohols; Fluorescent Dyes; Hemolysis; Membrane Fluidity; Membrane Glycoproteins; Membrane Lipids; Membrane Proteins; Mice; Perforin; Pore Forming Cytotoxic Proteins; Pyrimidinones; T-Lymphocytes, Cytotoxic

Peroxidation of erythrocyte membrane lipids by hydrogen peroxide perturbs the lipid bilayer and increases phagocytosis by macrophages. This study addresses the underlying mechanism of these processes, and in particular the role of malondialdehyde, a major byproduct of lipid peroxidation. When erythrocytes were treated with hydrogen peroxide or ascorbate/iron to generate malondialdehyde, or with malondialdehyde itself, only those cells treated with hydrogen peroxide showed increased phospholipid spacing and enhanced phagocytosis. This result indicates that the alterations observed are unique to hydrogen peroxide treatment, and that malondialdehyde does not play a role in inducing these changes in surface properties. Comparison of adherence to human umbilical vein endothelial cells and phagocytosis showed that increased phagocytosis was not mirrored by enhanced adherence. This result suggests that two different signals may mediate recognition of erythrocytes by macrophages and by endothelial cells.

Topics: Cell Adhesion; Cells, Cultured; Erythrocyte Membrane; Hemolysis; Humans; Hydrogen Peroxide; Lipid Bilayers; Lipid Peroxidation; Macrophages; Malonates; Malondialdehyde; Membrane Lipids; Phagocytosis; Phospholipids; Pyrimidinones; Spectrometry, Fluorescence

The effects of 9-methyl-3-(1H-tetrazol-5-yl)-4H-pyrido[1,2-a]pyrimidin-4-one potassium salt (TBX) on type II to IV allergic reactions and immunological functions were investigated in animal models. Types II to IV allergic reactions in rodents were unaffected in vivo by TBX, even at higher doses than those capable of completely inhibiting the type I allergic reaction. However, both complement-mediated hemolysis via the classical pathway and hypotonic shock-induced hemolysis were slightly inhibited in vitro only by a high concentration of the drug (10(-4) g/ml). In the mouse system, TBX had no ability to suppress anti-hapten IgE antibody formation as well as hemagglutinin formation and to inhibit the proliferation of spleen cells induced by non-specific T and B cell mitogens. The results obtained indicate that TBX is an antiallergic drug essentially devoid of inhibitory actions on types II to IV allergic reactions and immunological functions, thus indicating that it is a specific inhibitor of type I allergic reactions.

Topics: Animals; Arthus Reaction; Cell Division; Complement System Proteins; Erythrocytes; Forssman Antigen; Guinea Pigs; Hemolysis; Histamine Antagonists; Hypersensitivity; Immunity, Cellular; Male; Mice; Mice, Inbred BALB C; Mice, Inbred ICR; Passive Cutaneous Anaphylaxis; Pyridines; Pyrimidinones; Rats; Rats, Inbred Strains; Sheep; Tuberculin Test

Topics: Animals; Erythrocyte Membrane; Erythrocytes; Heme; Hemolysis; Male; Methemoglobin; Oxidation-Reduction; Pyrimidinones; Rats; Rats, Inbred Strains; Reference Values; Vitamin E; Vitamin E Deficiency

Damaged RBC drawn from favic patients during acute hemolysis showed marked alterations in their two major proteolytic systems. Cytosolic procalpain (i.e., the proenzyme species of Ca2+-activated neutral proteinase, or calpain) had considerably lower activity than in matched RBC from asymptomatic G6PD-deficient subjects. The total RBC activity of the three acid endopeptidases that are normally membrane-bound was not reduced in favism, but its subcellular distribution was mostly cytosolic, suggesting quantitative release from membranes. Changes in procalpain activity are the result of both autoxidation of divicine and of the intracellular elevation of Ca2+ that is found in favism. Changes in acid endopeptidase activity are the consequence of perturbed Ca2+ homeostasis. Overall, the picture shows a marked impairment of the RBC proteolytic machinery that in turn may worsen cellular damage.

Topics: Calcium; Calpain; Endopeptidases; Enzyme Precursors; Erythrocytes; Favism; Glucosephosphate Dehydrogenase Deficiency; Hemolysis; Humans; Male; Peptide Hydrolases; Pyrimidinones

Topics: Hemolysis; Humans; Light; Oxygen; Photochemistry; Pyrimidinones; Radiation-Sensitizing Agents; Rose Bengal; Singlet Oxygen

The effects of divicine (DV), one of its degradation products ("blue DV"), and H2O2 on normal and pre-treated rat erythrocyte (RBC) reduced glutathione (GSH) content, spontaneous hemolysis at different tonicity levels, optical absorption spectrum of the hemolysate, as well as on their morphology were investigated. The influence of experimental conditions (temperature, pre- and post-treatment incubation period, presence and absence of glucose in the medium, aerobic and anaerobic conditions and levels of DV and "blue DV") were also studied. Only DV caused a marked decrease in GSH, which is regenerated when the DV level is below 4mM, and in the absence of glucose the regenerating capacity is abolished. DV and "blue DV" not only failed to induce hemolysis but they actually increased the cells' resistance to it; especially "blue DV", which probably lacked GSH-depressing effect. DV caused changes in the absorption spectrum of the hemolysate and to some extent in that of a purified hemoglobin solution, whereas "blue DV" and H2O2 did not. DV also produced profound and long-lasting morphologic changes in the RBC.

Topics: Animals; Erythrocytes; Glutathione; Hemolysis; Hydrogen Peroxide; Male; Microscopy, Electron, Scanning; Pyrimidinones; Rats; Rats, Inbred Strains; Spectrophotometry; Time Factors

Topics: Ascorbic Acid; Carbon Monoxide; Erythrocytes; Glucosephosphate Dehydrogenase Deficiency; Hemolysis; Humans; Oxidation-Reduction; Peroxides; Plasma; Pyrimidinones; tert-Butylhydroperoxide

Bee venom phospholipase A2 and the fluorescent probe merocyanine 540 were used to examine plasma membrane phospholipid organization in the spicules released by deoxygenation and reoxygenation of sickle red cells, as well as in reversibly and irreversibly sickled erythrocytes. Digestion of phosphatidyl ethanolamine in spicules was comparable to that of phosphatidyl choline, and these structures were stained by the fluorescent probe. Both assays suggest that membrane lipid asymmetry is disrupted in spicules. The residual cells, from which the spicules were derived, retain the normal asymmetry in phospholipid distribution between the outer and inner leaflets of the plasma membrane bilayer. Comparable experiments with cell fractions enriched in irreversibly sickled cells revealed a partial enhancement of phosphatidyl ethanolamine digestion, confirming the similar experiments of Lubin et al (1981). Staining of these cells with merocyanine 540, however, did not reveal a subfraction of stainable cells, indicating that this increase in phosphatidyl ethanolamine digestion is not due to the presence of a small fraction of cells which have completely lost their membrane asymmetry.

Topics: Anemia, Sickle Cell; Erythrocyte Membrane; Fluorescent Dyes; Hemolysis; Histocytochemistry; Humans; Membrane Lipids; Phosphatidylcholines; Phosphatidylethanolamines; Phospholipases A; Phospholipases A2; Phospholipids; Pyrimidinones

Red cells of G6PD (D-glucose-6-phosphate:NADP+ 1-oxidoreductase; G6PD) deficient (Mediterranean variant) subjects were studied during a fava bean haemolytic crisis. Two representative cases are described. In Case 1, haemolysis was still going on. In more than 50% of the red cells the Hb was confined to one part of the cell, leaving the other part as transparent as a Hb-free ghost. In this part the membranes appeared tightly bonded because swelling did not peel apart the bonded membrane areas. This feature is defined as membrane cross bonding (MCB). In Case 2, haemolysis had terminated and MCB-cells were less than 1%. MCB was reproduced in vitro by incubating G6PD-deficient whole blood with 1 mM divicine for up to 10 h. Subsequent shrinkage of red cells in hypertonic plasma (400 mOsm) resulted in the rapid formation of MCB. Membrane modifications by divicine, contained in fava beans, followed by osmotic shrinkage in the kidney and/or squeezing in the microcirculation are proposed as the cause of MCB during the favic crisis. MCB reduces the effective surface area of red cells. This is a plausible cause for sequestration by the reticulo-endothelial system. Intravascular haemolysis observed in favic crisis cannot be explained by mechanical forces, but it is possible that the effective surface area is reduced by MCB to such an extent that red cells lyse osmotically.

Topics: Child; Child, Preschool; Erythrocyte Membrane; Erythrocytes; Favism; Glucosephosphate Dehydrogenase Deficiency; Heinz Bodies; Hemolysis; Humans; Male; Pyrimidinones

A significant inactivation of red blood cell glutathione peroxidase (25% less than the physiological value) was observed after exposure of intact erythrocytes to 2 mM divicine (an autoxidizable aminophenol from Vicia faba seeds) and 2 mM ascorbate for 3 h at 37 degrees C. Addition of catalase and conversion of Hb to the carbomonoxy derivative resulted in protection against enzyme inactivation. Oxidation of Hb was a concurrent phenomenon, and augmented the inactivating effect. In hemolysates, much stronger effects were observed at shorter times (2 h); divicine was effective also without ascorbate, and the presence of reductants (ascorbate or glutathione or NADPH) enhanced its inactivating power. Of the other antioxidant enzymes, superoxide dismutase was unaffected under the same experimental conditions. Catalase was found to be much less sensitive to the inactivation; it was almost unaffected in experiments with intact erythrocytes and specifically protected by NADPH in experiments with hemolysates. This specific damage of glutathione peroxidase, apparently involving interaction of H2O2 and HbO2, may be related to the pathogenesis of hemolysis in favism.

Topics: Adult; Carboxyhemoglobin; Erythrocytes; Favism; Glutathione Peroxidase; Hemolysis; Humans; Methemoglobin; Pyrimidinones

Favism is an acute hemolysis occurring in glucose-6-phosphate dehydrogenase (G6PD)-deficient (Mediterranean variant) individuals after intake of fava beans. Divicine (D), 2,6-diamino-4,5-dihydroxypyrimidine, is present in high amounts in the beans, and is suspected to play a role in hemolysis. Its mechanism of action was studied in a cell-free system and in G6PD (Mediterranean variant)-deficient red cells (RBC). Upon hydrolysis of the inactive beta-glucoside vicine, reduced divicine is formed. Oxygen acts as a one- or two-electron acceptor; superoxide anion and hydrogen peroxide are formed, respectively, together with the semiquinoid free-radical form of D. This free radical gives an electron spin resonance (ESR) signal, which is similar to that of the alloxan free radical. Added reduced glutathione (GSH) is rapidly oxidized with a stoichiometry of one to one, and the ESR signal is abolished. Additional GSH is oxidized by hydrogen peroxide and by a slow redox cycle which continuously regenerates oxidized D. The fast-direct and the slow-indirect oxidation result in nonstoichiometric oxidation of GSH. D added to G6PD-deficient RBC rapidly oxidizes GSH with an end point kinetics and a stoichiometry of one to one. Hydrogen peroxide and superoxide anion are scavenged in the RBC and no redox cycling is taking place. No GSH is regenerated even after long incubation periods. After the primary event, i.e., oxidation of GSH and--SH groups, a number of metabolic, rheologic, and membrane modifications, together with increased erythrophagocytosis take place in G6PD-deficient, D-treated RBC only.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Ascorbic Acid; Cell-Free System; Cytochrome c Group; Erythrocytes; Favism; Glucosephosphate Dehydrogenase Deficiency; Glutathione; Hemolysis; Humans; Hydrogen Peroxide; In Vitro Techniques; Kinetics; Male; Oxidation-Reduction; Pyrimidinones; Superoxides

Complete removal of leucocytes and platelets from erythrocytes and the development of a sensitized procedure for the assay of G6PD activity allowed the biochemical mechanisms of the Mediterranean variety of G6PD deficiency to be re-evaluated. Activity in the young erythrocytes from 9 G6PD-deficient subjects averaged 0.1% of the levels observed in the corresponding erythrocyte fraction from normal individuals: moreover, the decline of activity during aging of the G6PD-deficient erythrocytes was comparable with that observed for the normal enzyme. Mutant G6PD purified from granulocytes of a G6PD-deficient subject and entrapped within the corresponding erythrocytes was remarkably stable. Exposure of native erythrocytes to an oxidative stress (divicine plus ascorbate) resulted in a decrease of G6PD activity that was significantly more rapid and extensive in control than in G6PD-deficient cells. These results seem to exclude enhanced intracellular breakdown of the mutant protein within the circulating erythrocytes.

Topics: Ascorbic Acid; Erythrocyte Aging; Erythrocytes; Glucosephosphate Dehydrogenase; Glucosephosphate Dehydrogenase Deficiency; Hemolysis; Humans; Male; Mediterranean Islands; Mutation; Pyrimidinones

Red blood cells of favism patients with acute hemolytic crisis have markedly more superoxide dismutase (superoxide:superoxide oxidoreductase, EC 1.15.1.1) and less glutathione peroxidase (glutathione:hydrogenperoxide oxidoreductase, EC 1.11.1.9) than either normal controls, glucose-6-phosphate dehydrogenase-deficient subjects or favism patients outside hemolytic crisis. This altered value of the two enzyme activities is not due to increased reticulocyte content of blood. The electrophoretic triplet pattern of superoxide dismutase is also changed, with significant increase of the most positively charged band. Similar modifications of the two enzyme activities are observed after treatment of normal red blood cells with high concentrations of divicine and ascorbate, which are redox compounds that are contained in fava seeds. This treatment produces no hemolysis, but leads to hemolysis if the treated cells are resuspended in the homologous plasma. These results suggest a possible role of active oxygen species in the development of favism.

Topics: Ascorbic Acid; Electrophoresis, Polyacrylamide Gel; Erythrocyte Aging; Erythrocytes; Favism; Glutathione Peroxidase; Hemolysis; Humans; Oxidation-Reduction; Pyrimidinones; Reticulocytes; Superoxide Dismutase

Aqueous extracts of a different variety of fresh broad bean seeds obtained from a favism endemic area in Turkey, were incubated with blood from sensitive and non-sensitive (control) subjects. Red blood cells were characterized by a whole blood glutathione (GSH) and a deficiency of Glucose-6-phosphate dehydrogenase (G-6-PD) activity. As the decrease in GSH percent is taken as an index of haemolytic activity, the test results were as following: Sakiz , Milas -Region, French broad bean extracts reduced the blood GSH levels 48%, 70%, 46% and 53%, respectively, in favism sensitive subjects. Active principles which are responsible for the haemolysis ( Vicine and Convicine ) were isolated from broad beans and their effects on GSH levels of blood were 99% and 81%, respectively, in favism sensitive subjects and 33.3% and 19% in normal subjects.

Topics: Fabaceae; Favism; Glucosides; Hemolysis; Humans; In Vitro Techniques; Plant Extracts; Plants, Medicinal; Pyrimidinones; Turkey; Uracil; Uridine

Intravenous injection of divicine into mice infected with Plasmodium vinckei rapidly killed the parasites and caused haemolysis. Degenerating parasites were observed frequently inside intact circulating erythrocytes, implying that parasite death was not a passive consequence of haemolysis. Both parasite death and haemolysis were prevented by the iron chelator desferrioxamine. In vitro, divicine caused the accumulation of malonyldialdehyde and the depletion of reduced glutathione in normal mouse erythrocytes. Desferrioxamine inhibited the former event, but not the latter. These observations support the hypothesis advanced by Huheey & Martin (Experientia, 31, 1145, 1975) to explain the patchy geographical distribution of glucose-6-phosphate dehydrogenase deficiency in historic malarial areas and also suggest that desferrioxamine, a drug already in clinical use, is a potential treatment for favism and other examples of oxidative haemolysis.

Topics: Animals; Deferoxamine; Erythrocytes; Favism; Female; Glucosephosphate Dehydrogenase Deficiency; Glutathione; Hemolysis; Malaria; Male; Malondialdehyde; Mice; Mice, Inbred CBA; Microscopy, Electron; Pyrimidinones

1. Experiments were conducted to study the effects of dietary vicine (2, 6-diamino-4, 5 dihydroxy pyrimidine-5 (beta-D-glucopyranoside)) and supplemental vitamin E on the performance of laying hens and growing chicks, haemolysis of erythrocytes than birds fed on a control diet. 3. Vicine when fed to laying hens had a very dramatic effect. It depressed food consumption, egg weight, fertility and hatchability of eggs. Packed cell volume and erythrocyte haemoglobin levels and led to increased liver weights, liver glutathione levels, liver and plasma lipid levels, plasma lipid peroxide levels and erythrocyte haemolysis in vitro. Liver protein and plasma vitamin E:lipid levels were not altered. Vitamin E supplementation slightly increased egg weights, markedly improved fertility and hatchability of eggs and lowered liver weights and lipid levels but did not affect the other factors examined. 4. It is concluded that vicine which was isolated from faba beans (Vicia faba L.) has a marked influence on the metabolism of the laying hen and only a slight effect on growing chick. Vicine or its metabolites or both cause peroxidation of cellular components which result in abnormal lipid transport of synthesis or both, increased fragility of erythrocytes, and reduced fertility. These effects are overcome to varying extents by supplemental vitamin E.

Topics: Animals; Chickens; Diet; Feeding Behavior; Female; Fertility; Glucosides; Glycosides; Hemolysis; Lipid Metabolism; Liver; Organ Size; Ovulation; Pyrimidinones; Toxins, Biological; Vitamin E

The effects of azathioprine, either alone or combined with delta-(2-amino-6-hydroxy-3,4-dihydro-4-oxo-5-pyrimidinyl)-valeric acid (VUFB-9777, Damvar) on serum antibody formation was studied in mice. The immunosuppressive effect of azathioprine depended on the regimen of administration. VUFB-9777 alone produced no immunosuppressive effect but markedly enhanced that of azathioprine.

Topics: Animals; Antibody Formation; Azathioprine; Drug Interactions; Female; Hemagglutination Tests; Hemolysis; Mice; Pyrimidinones; Sheep; Time Factors