vicine and divicine

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

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

Vicine is a glucoside from broad beans (Vicia faba) that is hydrolyzed upon ingestion to the unstable aglycon divicine, the autoxidation of which has been implicated in the onset of hemolysis in favism,possibly via production of superoxide and hydrogen peroxide. The autoxidation of divicine proceeds through a series of reactions involving the formation of a radical species. In this study divicine radicals were produced either by incubation of vicine with beta-glucosidase or by boiling vicine in hydrochloric acid. On the basis of electron spin resonance spectra, it was shown that the two treatments produce different radicals. By spectral simulation the acid-produced radical was demonstrated to be a deaminodivicine. The autoxidation rates of the two radicals were determined from the disappearence of their electron spin resonance signals in the presence of air: at physiological pH the enzymatically produced divicine radical was much more stable to oxygen than the chemically produced radical. The two radicals may thus be expected to behave differently in a biological system. The repercussions of these findings could be considerable, given that most of the pharmacological and biochemical studies on vicine action have been done with the chemically produced compound, which is shown here to be an unphysiological intermediate.

Topics: Cellulases; Electron Spin Resonance Spectroscopy; Free Radicals; Glucosides; Hydrolysis; Pyrimidinones; Vicia faba

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: Absorption; Animals; Anti-Bacterial Agents; Cecum; Chickens; Glucosides; Glycosides; Half-Life; Hydrolysis; Intestines; Male; Pyrimidinones; Tissue Distribution; Uracil; Uridine

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

Topics: Chemistry, Pharmaceutical; Glucosides; Humans; Pyrimidines; Pyrimidinones