pyrimidinones and Favism

Topics: Erythrocytes; Favism; Free Radicals; Glucosephosphate Dehydrogenase Deficiency; Glucosides; Humans; Pyrimidinones; Vicia faba

The faba bean is one of the earliest domesticated crops, with both economic and environmental benefits. Like most legumes, faba beans are high in protein, and can be used to contribute to a balanced diet, or as a meat substitute. However, they also produce the anti-nutritional compounds, vicine and convicine (v-c), that when enzymatically degraded into reactive aglycones can potentially lead to hemolytic anemia or favism. Current methods of analysis use LC-UV, but are only suitable at high concentrations, and thus lack the selectivity and sensitivity to accurately quantitate the low-v-c genotypes currently being developed. We have developed and fully validated a rapid high-throughput LC-MS method for the analysis of v-c in faba beans by optimizing the extraction protocol and assessing the method of linearity, limit of detection, limit of quantitation, accuracy, precision and matrix effects. This method uses 10-times less starting material; removes the use of buffers, acids and organic chemicals; and improves precision and accuracy when compared to current methods.

Topics: Favism; Glucosides; Pyrimidinones; Uridine; 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 aglycones of vicine and convicine, divicine and isouramil, are the causative agents of favism and, therefore, should be analysed along with vicine and convicine in research seeking to eliminate them. This study investigated the stability of the aglycones produced by hydrolysis with β-glucosidase. Reversed-phase, high-performance liquid chromatography (HPLC) with UV detection was shown to be able to observe both aglycone formation and further reactions in isolated fractions and extract made from faba bean and in faba bean suspension. Divicine and isouramil were unstable and degraded almost completely in extract in 60min and completely in fractions in 120min at a pH of 5 at 37°C. Adding sodium ascorbate delayed degradation of divicine. Divicine was more stable at 20°C than at 37°C. Being able to show formation and degradation of the aglycones, the proposed method allows monitoring of the vicine and convicine detoxification process.

Topics: Barbiturates; beta-Glucosidase; Favism; Glucosides; Hydrolysis; Pyrimidinones; Uridine; Vicia faba

Favism is an acute anemic crisis that can occur in susceptible individuals who ingest fava beans. The fava bean pyrimidine aglycone divicine has been identified as a hemotoxic constituent; however, its mechanism of toxicity remains unknown. We have shown recently that divicine can induce a favic-like response in rats and that divicine is directly toxic to rat red cells. In the present study, we have examined the effect of hemotoxic concentrations of divicine on rat erythrocyte sulfhydryl status, hexose monophosphate (HMP) shunt activity, morphology, and membrane skeletal proteins. In vitro exposure of rat red cells to divicine markedly stimulated HMP shunt activity and resulted in depletion of reduced glutathione with concomitant formation of glutathione-protein mixed-disulfides. Examination of divicine-treated red cells by scanning electron microscopy revealed transformation of the cells to an extreme echinocytic morphology. SDS-PAGE and immunoblotting analysis of the membrane skeletal proteins indicated that hemotoxicity was associated with the apparent loss of skeletal protein bands 2.1, 3, and 4.2, and the appearance of membrane-bound hemoglobin. Treatment of divicine-damaged red cells with dithiothreitol reversed the protein changes, which indicated that the observed alterations were due primarily to the formation of disulfide-linked hemoglobin-skeletal protein adducts. The data suggest that oxidative modification of hemoglobin and membrane skeletal proteins by divicine may be key events in the mechanism underlying favism.

Topics: Animals; Electrophoresis, Polyacrylamide Gel; Erythrocytes; Favism; Male; Membrane Proteins; Microscopy, Electron, Scanning; Pentose Phosphate Pathway; Pyrimidinones; Rats; Rats, Sprague-Dawley; Sulfhydryl Compounds

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

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

Changes in the concentrations of vicine, convicine and L-DOPA in two cultivars of Vicia faba L. seeds in different stages of pod development were determined by high performance liquid chromatography (HPLC). The vicine and convicine content was highest in fresh green cotyledons (moisture content about 80%) and gradually declined until a constant level was reached when seed dry matter percentage was around 40%. A similar pattern of variation in glucoside concentration was observed for the seed coat. The pods contained neither vicine nor convicine but they were particularly rich in L-DOPA. These compounds were not homogeneously distributed in the seeds.

Topics: Chromatography, High Pressure Liquid; Fabaceae; Favism; Glucosides; Levodopa; Plants, Medicinal; Pyrimidinones; Seeds; Toxins, Biological; Uridine

Topics: Fabaceae; Favism; Glucosides; Humans; Plants, Medicinal; Pyrimidinones; Toxins, Biological

Favism is an acute hemolytic anemia triggered by ingestion of fava beans in genetically susceptible subjects with severe deficiency of glucose-6-phosphate dehydrogenase (G6PD) activity. Erythrocytes from 10 favic patients had constantly and markedly increased calcium levels, as compared with values detected in 4 asymptomatic G6PD-deficient controls. Correspondingly, the calcium permeability of erythrocytes, estimated as the fraction of intracellular calcium exchangeable with externally added 45Ca2+, was invariably enhanced in favism and returned to normal patterns after several months from the acute hemolytic crisis. In favic patients, the levels of erythrocyte calcium ATPase activities showed wide variability, ranging from 2.0-12.9 mumol Pi/ml RBC/h, while control values in asymptomatic G6PD-deficient subjects were 10.62 +/- 2.03 mumol Pi/ml RBC/h. Analysis of the calcium ATPase in situ in erythrocyte membranes from favic patients showed the same molecular mass of 134 kD as observed in the control subjects. Exposure of G6PD-deficient erythrocytes in vitro to autoxidizing divicine, a pyrimidine aglycone strongly implicated in the pathogenesis of favism which leads to late accumulation of intracellular calcium, caused: (i) a marked inactivation of calcium ATPase, without changes in the molecular mass of 134 kD; and (ii) the concomitant loss of spectrin, band 3 and band 4.1, all known substrates of the calcium activated procalpain-calpain proteolytic system. Thus, the increased intraerythrocytic calcium apparently results in the degradation of calcium ATPase observed in some favic patients. It is proposed that both enhanced calcium permeability and a calcium-stimulated degradation of the calcium pump are the mechanisms responsible for the perturbation of erythrocyte calcium homeostasis in favism.

Topics: Calcimycin; Calcium; Calcium-Transporting ATPases; Calpain; Erythrocytes; Favism; Glucosephosphate Dehydrogenase; Homeostasis; Humans; Male; Oxidation-Reduction; Pyrimidinones

The toxic glycosides vicine and convicine which are present in fababeans have been implicated in favism, an anaemic disease of humans. Vicine and convicine concentrations are reduced by growth of Lactobacillus plantarum on fababean suspensions. The glycosides are eliminated from the fababean substrate by the growth of the filamentous fungus Fusarium graminearum. Incubation of fababean suspension with concentrated culture filtrate of Aspergillus oryzae, induced for extracellular beta-glucosidase production, results in complete degradation of the glycosides. This study suggests a potential use of micro-organisms or microbial enzymes for detoxification of fababeans.

Topics: Aspergillus oryzae; beta-Glucosidase; Electrophoresis; Fabaceae; Favism; Fusarium; Glucosides; Humans; Hydrolysis; Lactobacillus; Plants, Medicinal; Pyrimidinones; Uridine

The response of faba bean 'Vicia faba' (Giza 2 variety) towards soaking conditions differed greatly since the absorbed quantities of water (either by the whole or the decorticated forms) are a function of their chemical constituents. On the other hand, 28.45% of the total vicine (vicine & convicine) present in the whole faba bean samples was extracted after soaking for 72 h at room temperature. Subsequently, other soaking mediums, i.e., 0.5% sodium carbonate and/or 1% acetic acid were used in an attempt to increase the level of vicine elimination. Percentage removal of total vicine in whole faba bean was higher in the acidic (61.31%) than the alkaline (38.40%) medium under the conditions tested, i.e., at room temperature for 72 hours. The rates of vicine + convicine elimination in decorticated faba bean for the acidic acid and alkaline soaking media were 78.46 and 79.13%, respectively. The solubility ratio of total vicine relative to soaking solutions (H2O:Na2CO3:Acetic acid) was 1:1.35:2.16 in the whole broad bean and 1:2.41:2.39 in the decorticated samples. The residual amounts of total vicine (78.33% and 77.27%) present after stewing under normal and under pressure cooking conditions could be expected to be decreased to 30.33% for the former and 29.92% for the later after 72 h of soaking. Regression analysis was used to estimate the theoretical zero point of vicine elimination from faba bean through soaking in 1% acetic acid.

Topics: Cooking; Egypt; Fabaceae; Favism; Glucosides; Glycosides; Plants, Medicinal; Pyrimidinones; Water

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

The biochemical events that take place during acute hemolysis of G6PD-deficient subjects in favism are far from being elucidated. Evidence is here reported for a constantly and heavily disordered calcium homeostasis in the erythrocytes from seven favic patients. The abnormality, ie, a significantly impaired calcium ATPase activity and a parallel marked increase of intracellular calcium levels, was characteristic of the acute hemolytic crisis although unrelated to the attendant reticulocytosis. Concomitantly, a remarkable decrease of intracellular potassium was also observed. The mean +/- SD Ca2+-ATPase activity in the favic patients was 20.8 +/- 7.8 mumol Pi/g Hb/h compared with 37.2 +/- 8.5 in the matched controls represented by 12 healthy G6PD-deficient subjects (P less than .001). The mean +/- SD intraerythrocytic calcium content was 288 +/- 158 mumol/L of erythrocytes in the favic patients as compared with 22.0 +/- 8.2 in the G6PD-deficient controls (P less than .001). The intraerythrocytic potassium content was 76.6 +/- 19.3 mmol/L of erythrocytes in the favic patients and 106.6 +/- 8.2 in the G6PD-deficient controls (P less than .001). In vitro incubation of normal and G6PD-deficient erythrocytes with divicine, a pyrimidine aglycone present in fava beans and strongly implicated in the pathogenesis of favism, reproduces most of these events, including drop of calcium ATPase, increased intracellular calcium, and leakage of erythrocyte potassium.

Topics: Acute Disease; Calcium; Calcium-Transporting ATPases; Cell Separation; Erythrocyte Count; Erythrocytes; Favism; Homeostasis; Humans; In Vitro Techniques; Ion Channels; Male; Methemoglobin; Potassium; Pyrimidinones

Divicine (2,6-diamino-4,5-dihydroxypyrimidine), an aglycone implicated in the pathogenesis of favism, produces a remarkable and consistent inactivation of the Ca2+-ATPase activity of the erythrocyte calcium pump. The patterns of inactivation are similar in normal and glucose-6-phosphate dehydrogenase (G6PD)-deficient erythrocytes. Inactivation of Ca2+-ATPase is apparently unrelated to the cellular GSH system, to the proteolytic machinery of mature erythrocytes, and to calmodulin, and also occurs in hemoglobin-free, unsealed erythrocytes membranes at 50-100 microM concentrations of divicine. Analysis of erythrocytes that have escaped destruction during the acute hemolytic crisis of a number of favic patients revealed a dramatic elevation of erythrocyte calcium and a significant decrease of Ca2+-ATPase activity. These results support the view that divicine plays a toxic role in the pathogenesis of favism and suggest that acute electrolyte imbalances, mostly affecting calcium homeostasis, are involved in the mechanisms of erythrocyte damage and destruction in this hemolytic disease.

Topics: Calcium; Calcium-Transporting ATPases; Calmodulin; Erythrocyte Membrane; Erythrocytes; Favism; Humans; Male; Membrane Proteins; Potassium; 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

Enzymatic activity of purified or membrane-bound acetylcholine esterase was lost when incubated aerobically in the presence of both favism-inducing agent (isouramil or divicine) and copper ions. The requirement for oxygen could be substituted by hydrogen peroxide. Chelating agents provided total protection to the proteins. The suggested mechanism of enzymatic inactivation is analogous to that suggested earlier for the effects of superoxide and ascorbate, and involves the site-specific formation of hydroxyl radicals in the metal-mediated Haber-Weiss reaction. These findings may be relevant to the understanding of the pathogenesis of favism.

Topics: Ascorbic Acid; Barbiturates; Cholinesterase Inhibitors; Copper; Electron Spin Resonance Spectroscopy; Favism; Humans; In Vitro Techniques; Oxidation-Reduction; 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

Divicine (2,6-diamino-4,5-dihydroxypyrimidine) is a quinoid compound contained in high amounts in all fava bean strains. It elicits a number of metabolic and rheological modifications in G6PD-deficient RBC. Besides this, it stimulates erythrophagocytosis by mouse peritoneal macrophages and formation of crossbonded red cells. The latter are intracellularly membrane-bonded red cells, which form during hypertonic incubation and subsequent swelling. All effects observed in vitro were also observed in vivo in the early phases of fabic hemolysis. In particular, phagocytosis and crossbonding offer a basis for understanding extravasal hemolysis.

Topics: Animals; Erythrocytes; Favism; Glutathione; Humans; Macrophages; Male; Membrane Lipids; Membrane Proteins; Mice; Phagocytosis; Phospholipids; Pyrimidinones; Rheology

A new method for the quantitation of the favism-inducing agents in legumes is described. The procedure involves differential extraction of the glucosides vicine and convicine with acetic acid (25%), followed by an enzymatic hydrolysis by beta-glucosidase under anaerobic conditions. Each of the aglycone moieties, isouramil and divicine, anaerobically reduces two molecules of o-ferriphenanthroline to o-ferrophenanthroline. This reaction is readily followed spectrophotometrically at 515 nm. Using this procedure, it was found that in various strains of Vicia faba, the level of these two glucosides comprises approximately 0.5% of the wet weight of the seeds. In contrast, these glucosides could not be detected in either green peas or chick peas.

Topics: Barbiturates; Fabaceae; Favism; Glucosidases; Glucosides; Oxidation-Reduction; Phenanthrolines; Plants, Medicinal; Pyrimidinones; Spectrophotometry; Uracil; Uridine

Topics: Chromatography, High Pressure Liquid; Fabaceae; Favism; Glucosides; Glycosides; Levodopa; Plants, Medicinal; Pyrimidinones; Seeds; Uracil; Uridine

Isouramil and divicine are pyrimidine aglycones of two glucosides found in broad beans. They have been shown to be strong reducing agents. Their reaction with oxygen in a (gas) saturated solution, 26 degrees C, is characterized by tau 1/2 = 1 min and 3 min respectively. Hydrogen peroxide is formed in this reaction stoichiometrically (1:1). The pyrimidines lose two hydrogen and form an intermediate that is structurally analogues to alloxan. This intermediate is not stable, and in the absence of reducing agents it decomposes, possibly by ring-cleavage. In the presence of reduced glutathione the intermediate is reduced and can now react with oxygen once again. Thus, the pyrimidines cycle between the two states and the net reaction is the catalytic oxidation of glutathione by molecular oxygen; in each cycle 4 molecules of glutathione are dissipated. The possible involvement of these pyrimidines in the pathogenesis of favism may be in a similar mechanism. Red blood cells deficient in glucose-6-phosphate dehydrogenase cannot cope with such an oxidative challenge exerted by the pyrimidines. Consequently an irreversible cellular damage can take place leading to the enhanced sequestration of these red blood cells by the reticuloendothelial system.

Topics: Animals; Barbiturates; Chemical Phenomena; Chemistry; Erythrocytes; Favism; Free Radicals; Glutathione; Humans; Oxidation-Reduction; Pyrimidinones

Fava beans contain high amounts (up to 6.7 g/100 g dry weight) vicine and convicine. Their active aglycones divicine and isouramil have equivalent metabolic effects. They rapidly oxidize GSH to GSSG in normal and G6PD-deficient red cells. No regeneration of GSH occurs in deficient cells. The stoichiometry of the divicine oxidation of GSH is 1:1. Ascorbic acid is quickly oxidized by isouramil in both normal and deficient cells but regenerates only in normal cells. Isouramil oxidizes NADH at a much lesser extent than NADPH. Glycolysis is activated at the glyceraldehyde 3-phosphate dehydrogenase step. Divicine strongly stimulates hexone monophosphate shunt only in normal red cells. Divicine alone or associated with ascorbic acid has almost no effect in deficient red cells. Malonyl dialdehyde production is slight and virtually the same in normal and deficient cells treated with 5 mM isouramil. Large polypeptide aggregates are formed after 12 and 24 hours incubation with 1 mM divicine in deficient cells only. Divicine (0.25 mM) markedly decreases the filterability of deficient cells. The results are consistent with a causal role of divicine/isouramil in the genesis of the hemolytic crisis occurring in G6PD-deficient subjects after fava bean ingestion.

Topics: Ascorbic Acid; Barbiturates; Erythrocytes; Favism; Glucosephosphate Dehydrogenase Deficiency; Glucosides; Glutathione; Humans; Kinetics; Pyrimidinones; Reference Values; Species Specificity; Toxins, Biological; Uracil; Uridine

Covicine + vicine, L-DOPA-glucoside + L-DOPA and ascorbic acid were determined in different lines of Vicia faba beans throughout the biological cycle of the plant. As the seed matures the levels of convicine + vicine as well as of ascorbic acid decrease with seed maturation in all the lines examined. L-DOPA, which is lacking in cotyledons but present in the tegument, also decrease and is nearly undectable in some lines with white flowers.

Topics: Ascorbic Acid; Fabaceae; Favism; Glucosides; Hemolysin Proteins; Levodopa; Plants, Medicinal; Pyrimidinones; Seeds; Uracil; Uridine

Topics: Fabaceae; Favism; Glucosides; Glycosides; Plants, Medicinal; Plants, Toxic; Pyrimidinones; Species Specificity; Toxins, Biological

Topics: Favism; Glucosephosphate Dehydrogenase; Glucosides; Humans; In Vitro Techniques; NADP; Pyrimidinones

Topics: Favism; Glucosides; Glycosides; Humans; Pyrimidinones