pyrimidinones and isouramil

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

In spite of its positive repercussions on nutrition and environment, faba bean still remains an underutilized crop due to the presence of some undesired compounds. The pyrimidine glycosides vicine and convicine are precursors of the aglycones divicine and isouramil, the main factors of favism, a genetic condition which may lead to severe hemolysis after faba bean ingestion. The reduction of vicine and convicine has been targeted in several studies but little is known about their degradation. In this study, the hydrolysis kinetics of vicine and convicine and their derivatives during fermentation with L. plantarum DPPMAB24W was investigated. In particular, a specific HPLC method coupled to ESI-MS and MS/MS analysis, including the evaluation procedure of the results, was set up as the analytical approach to monitor the compounds. The degradation of the pyrimidine glycosides in the fermented flour was complete after 48 h of incubation and the aglycone derivatives could not be detected in any of the samples. The toxicity of the fermented faba bean was established through ex-vivo assays on human blood, confirming the experimental findings. Results indicate that mild and cost effective bioprocessing techniques can be applied to detoxify faba bean also for industrial applications.

Topics: Barbiturates; Biotransformation; Fermentation; Flour; Food Technology; Glucosides; Humans; Hydrogen-Ion Concentration; Hydrolysis; Lactobacillus plantarum; Pyrimidinones; Spectrometry, Mass, Electrospray Ionization; Uridine; Vicia faba

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

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

Dialuric Acid, the reduced form of the beta-cell toxin alloxan, and the related fava bean derivatives divicine and isouramil, autoxidize rapidly in neutral solution by a radical mechanism. GSH promotes redox cycling of each compound, with concomitant GSH oxidation and H2O2 production. With superoxide dismutase present, there is a lag period in which little oxidation occurs, followed by rapid oxidation. GSH extends this lag and decreases the subsequent rate of oxidation, so that with superoxide dismutase and a sufficient excess of GSH, coupled oxidation of GSH and each pyrimidine is almost completely suppressed. This mechanism may be a means whereby GSH in combination with superoxide dismutase protects against the cytotoxic effects of these reactive pyrimidines. Superoxide dismutase may also protect cells against oxidative stress in other situations where GSH acts as a radical scavenger, and we propose that the concerted action of GSH and superoxide dismutase constitutes an important antioxidant defence.

Topics: Antioxidants; Barbiturates; Chemical Phenomena; Chemistry; Glutathione; Oxidation-Reduction; Pyrimidinones; Superoxide Dismutase

The toxicity of dialuric acid to pancreatic beta cells, and the haemolytic action of divicine and isouramil involve auto-oxidation and redox cycling reactions. Divicine and isouramil are produced on hydrolysis of the fava bean glycosides, vicine and convicine. The mechanism of auto-oxidation of the three compounds as well as the acid hydrolysis product of vicine (provisionally assigned the structure 2-amino-4,5,6-trihydroxypyrimidine) has been studied. All four pyrimidines auto-oxidized rapidly at neutral pH, generating H2O2 by an O2-dependent chain mechanism. Superoxide dismutase inhibited the initial oxidation, but inhibition was transitory, and after a lag period rapid oxidation occurred. The lag period varied with pH, temperature and pyrimidine concentration, and was much shorter for isouramil and divicine than for dialuric acid and acid-hydrolysed vicine. The initial rate of dialuric acid oxidation was greater and the acceleration less pronounced than with the other pyrimidines. A mechanism common to all four pyrimidines has been shown by kinetic analysis to account for nearly all the observations in the presence and absence of superoxide dismutase. Autocatalysis in the latter case is attributed mainly to the reactions reduced pyrimidine + oxidized pyrimidine in equilibrium 2 pyrimidine radical pyrimidine radical + O2----oxidized pyrimidine + O2- Rate constants for these and other reactions are reported. At pH 7.4 and 37 degrees the lag period before 100 microM acid-hydrolysed vicine underwent rapid oxidation was approx. 15 min. Isouramil and divicine at an equivalent concentration gave lags of less than 1 min, which became less at higher concentrations. Thus intracellular superoxide dismutase should provide only transitory protection against the oxidation products of dialuric acid, divicine or isouramil. Prolonged protection should only be achieved if accumulation of oxidized pyrimidine is also prevented.

Topics: Alloxan; Barbiturates; In Vitro Techniques; Kinetics; Oxidation-Reduction; Pyrimidinones; Superoxides

The effects of GSH on the autoxidation of the fava bean pyrimidine aglycones, divicine and isouramil, and on acid-hydrolyzed vicine (provisional identification 2-amino-4,5,6-trihydroxypyrimidine) have been studied. GSH alone promoted redox cycling of each compound, with concomitant GSH oxidation and H2O2 production. In the presence of superoxide dismutase, there is a lag period during which little pyrimidine oxidation occurs, followed by a period of accelerated oxidation. With the three pyrimidines, increasing concentrations of GSH extended this lag period and progressively decreased subsequent rates of both pyrimidine oxidation and O2 uptake. No GSH oxidation or O2 uptake occurred during the lag. These results show that the combination of GSH and superoxide dismutase is able to inhibit redox cycling of the pyrimidines. With a 10-fold excess of GSH over isouramil or acid-hydrolyzed vicine (20-fold with divicine) this coupled oxidation of GSH and the pyrimidine is almost completely suppressed. This mechanism may be a means whereby GSH in combination with superoxide dismutase protects against the cytotoxic effects of these reactive pyrimidines.

Topics: Barbiturates; Catalase; Glucosides; Glutathione; Hydrogen Peroxide; Hydrogen-Ion Concentration; Kinetics; Oxidation-Reduction; Oxygen Consumption; Pyrimidinones; Superoxide Dismutase

Release of iron from ferritin by the polyhydroxypyrimidines, dialuric acid, isouramil, divicine, and acid-hydrolyzed vicine, was measured. Iron was released at fast initial rates which gradually declined to zero in 10 min. All the compounds were better reductants for ferritin-iron under nitrogen than in air. The effects of superoxide dismutase, catalase, and glutathione on both initial rates and total iron released over 30 min in air were determined. Major effects were inhibition by superoxide dismutase for divicine and isouramil and enhancement for dialuric acid and acid-hydrolyzed vicine. Glutathione promoted increased iron release that was further enhanced by superoxide dismutase. These increases were particularly striking over the longer time period. Catalase, in all cases, gave modest enhancement. Enhanced iron release correlated with inhibition of pyrimidine oxidation. The results indicate that the reduced form of each pyrimidine releases ferritin iron directly, and the effects of the antioxidants are mainly to maintain or regenerate the reduced pyrimidines. A combination of each pyrimidine and ferritin caused peroxidation of phopholipid liposomes, above that seen with the pyrimidines and adventitious iron. Glutathione, superoxide dismutase, and catalase modulated lipid peroxidation in a way consistent with their effects being mainly on ferritin-iron release. On the basis of our findings, we propose that the release and subsequent reactions of ferritin-iron may contribute to the toxicity of these compounds. Although glutathione and superoxide dismutase together efficiently inhibit redox cycling and H2O2 production from the pyrimidines, this combination maximized iron release from ferritin and ferritin-dependent lipid peroxidation.

Topics: Barbiturates; Catalase; Ferritins; Glucosides; Glutathione; Iron; Kinetics; Lipid Peroxidation; Liposomes; Oxidation-Reduction; Oxygen Consumption; Pyrimidines; Pyrimidinones; Superoxide Dismutase

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

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

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