pyrimidinones and convicine

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 (

Topics: Deep Learning; Genotype; Glucosides; Polymorphism, Single Nucleotide; Pyrimidinones; Regulatory Sequences, Nucleic Acid; Seeds; Synteny; Transcription Factors; Uridine; Vicia faba

Faba bean (Vicia faba L.) provides environmental and health benefits; however, the presence of the pyrimidine glycosides vicine and convicine (v-c) in its seeds limits consumption. Low v-c genotypes have been introduced, but the convicine levels in these genotypes have not been quantified. To improve detection, the polar nature of v-c was exploited by implementing hydrophilic interaction liquid chromatography (HILIC). A sample preparation method using a two-step extraction was developed for use with UV and/or tandem mass spectrometry (SRM) detection. The HILIC-UV method was suitable for over three orders of magnitude, covering the range of v-c concentrations in faba bean seeds across all genotypes tested. The linear range of HILIC-SRM was slightly less (∼3 orders of magnitude), but improved sensitivity and selectivity make it more suitable for quantifying low v-c samples. The analysis of 13 genotypes suggests that v-c concentrations in faba bean seeds may be independent quantitative traits.

Topics: Chromatography, Liquid; Glucosides; Hydrophobic and Hydrophilic Interactions; Pyrimidinones; Seeds; Uridine; Vicia faba

Although faba bean provides environmental and health benefits, vicine and convicine (v-c) limit its use as a source of vegetable protein. Crop improvement efforts to minimize v-c concentration require low-cost, rapid screening methods to distinguish between high and low v-c genotypes to accelerate development of new cultivars and to detect out-crossing events. To assist crop breeders, we developed a unique and rapid screening method that uses a 60 s instrumental analysis step to accurately distinguish between high and low v-c genotypes. The method involves flow injection analysis (FIA) coupled with tandem mass spectrometry (i.e., selective reaction monitoring, SRM). Using seeds with known v-c levels as calibrants, measured v-c levels were comparable with liquid chromatography (LC)-SRM results and the method was used to screen 370 faba bean genotypes. Widespread use of FIA-SRM will accelerate breeding of low v-c faba bean, thereby alleviating concerns about anti-nutritional effects of v-c in this crop.

Topics: Chromatography, Liquid; Flow Injection Analysis; Glucosides; High-Throughput Screening Assays; Pyrimidinones; Seeds; Tandem Mass Spectrometry; 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 faba bean (Vicia faba L.) is a potential source of proteins for poultry, mainly for laying hens whose protein requirements are lower than those of other birds such as growing broilers and turkeys. However, this feedstuff contains anti-nutritional factors, that is, vicine (V) and convicine (C) that are already known to reduce laying hen performance. The aim of the experiment reported here was to evaluate the effects of a wide range of dietary V and C concentrations in laying hens. Two trials were performed with laying hens fed diets including 20% or 25% of faba bean genotypes highly contrasting in V+C content. In Trial 1, faba beans from two tannin-containing cultivars, but with high or low V+C content were dehulled in order to eliminate the tannin effect. In addition to the contrasting levels of V+C in the two cultivars, two intermediate levels of V+C were obtained by mixing the two cultivars (70/30 and 30/70). In Trial 2, two isogenic zero-tannin faba bean genotypes with high or low V+C content were used. In both trials, a classical corn-soybean diet was also offered to control hens. Each experimental diet was given to 48 laying hens for 140 (Trial 1) or 89 (Trial 2) days. Laying performance and egg quality were measured. The redox sensitivity of red blood cells (RBCs) was assessed by measuring hemolysis and reduced glutathione (GSH) concentration in these cells. Egg weight was significantly reduced by the diets containing the highest concentrations of V+C (P<0.0001) in Trial 1 and slightly reduced (P<0.10) in Trial 2, but only weak linear relationships between egg weight and dietary V+C concentration were established. No negative effect of V+C level was observed for egg quality parameters. In contrast, certain parameters (i.e. Haugh units, yolk color) were improved by feeding low V+C diets (P<0.05). Hemolysis of RBCs was higher in hens fed high V+C diets. A decrease in GSH concentration in RBCs of hens fed the highest levels of V+C was observed. Faba bean genotypes with low concentrations of V+C can therefore be used in laying hen diets up to 25% without any detrimental effects on performance levels or egg characteristics, without any risk of hemolysis of RBCs.

Topics: Animal Feed; Animals; Chickens; Diet; Erythrocytes; Female; Genotype; Glucosides; Glycine max; Ovulation; Ovum; Pyrimidinones; Tannins; 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

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 antinutritional factors (ANFs) present in Vicia spp. seeds are a major constraint to the wider utilization of these crops as grain legumes. In the case of faba bean (Vicia faba L.), a breeding priority is the absence vicine and convicine (v-c); responsible for favism in humans and for the reduced animal performance or low egg production in laying hens. The discovery of a spontaneous mutant allele named vc-, which induces a 10-20 fold reduction of v-c contents, may facilitate the process. However, the high cost and difficulty of the chemical detection of v-c seriously restricts the advances in breeding-selection. To identify random amplified polymorphic DNA (RAPD) markers linked to this gene, we have analysed an F(2 )population derived from a cross between a line with high v-c content (Vf6) and the vc- genotype (line 1268). Quantification of v-c was done by spectrophotometry on the parents and the F(2 )population (n = 136). By using bulked segregant analysis (BSA), two RAPD markers linked in coupling and repulsion phase to the allele vc- were identified and further converted into sequence characterized amplified regions (SCARs). Amplification of SCARS was more consistent, although the initial polymorphism between pools was lost. To recover the polymorphisms several approaches were explored. Restriction digestion with HhaI (for SCAR SCH01(620)) and RsaI (for SCAR SCAB12(850)) revealed clear differences between the parental lines. The simultaneous use of the two cleavage amplified polymorphism (CAP) markers will allow the correct fingerprinting of faba bean plants and can be efficiently used in breeding selection to track the introgression of the vc- allele to develop cultivars with low v-c content and improved nutritional value.

Topics: Animals; Crosses, Genetic; Genetic Markers; Glucosides; Humans; Polymorphism, Genetic; Pyrimidinones; Random Amplified Polymorphic DNA Technique; Uridine; Vicia faba

The balanced polymorphism of glucose-6-phosphate dehydrogenase deficiency (G6PD-) is believed to have evolved through the selective pressure of malarial combined with consumption of fava beans. The implicated fava bean constituents are the hydroxypyrimidine glucosides vicine and convicine, which upon hydrolysis of their beta-O-glucosidic bond, became protein pro-oxidants. In this work we show that the glucosides inhibit the growth of Plasmodium falciparum, increase the hexose-monophosphate shunt activity and the phagocytosis of malaria-infected erythrocytes. These activities are exacerbated in the presence of beta-glucosidase, implicating their pro-oxidant aglycones in the toxic effect, and are more pronounced in infected G6PD- erythrocytes. These results suggest that G6PD- infected erythrocytes are more susceptible to phagocytic cells, and that fava bean pro-oxidants are more efficiently suppressing parasite propagation in G6PD- erythrocytes, either by directly affecting parasite growth, or by means of enhanced phagocytic elimination of infected cells. The present findings could account for the relative resistance of G6PD- bearers to falciparum malaria, and establish a link between dietary habits and malaria in the selection of the G6PD- genotype.

Topics: Animals; Erythrocytes; Fabaceae; Female; Glucosephosphate Dehydrogenase; Glucosephosphate Dehydrogenase Deficiency; Glucosides; Humans; Hydrogen-Ion Concentration; Hydrolysis; Malaria, Falciparum; Male; Pentose Phosphate Pathway; Phagocytosis; Plants, Medicinal; Plasmodium falciparum; Pyrimidinones; Uridine

Uridine (C9H12N2O6) was applied as an internal standard for the determination of glucopiranosides in faba bean seeds by the HPLC method. The relative UV response factors of vaccine or convicine to uridine were determined (1.12 and 0.79, respectively). The changes in the content of vaccine and convicine during ripening of faba bean seeds were followed. It was observed that with the increase in the seed dry matter content from 0.18 kg/kg to 0.78 kg/kg, the dry matter based content of vaccine decreased from 26.225 g/kg to 0.243 g/kg, and that of convicine from 9.051 g/kg to 0.094 g/kg.

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

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

This study in the rat established the effects that a broad-spectrum and poorly absorbed antibiotic, neomycin sulfate, had on the in vitro and in vivo hydrolysis of vicine and convicine by the intestinal microflora, and on vicine- and convicine-induced depletion of blood glutathione and the resulting toxicity. The in vitro studies demonstrated that digesta from the caecum and large intestine were highly effective in hydrolysing vicine and convicine, whereas digesta from the same sections of the gastro-intestinal tract of neomycin-treated rats were much less effective (P < 0.0001). The in vivo studies showed that the total amount of vicine and convicine excreted in the urine and faeces was much greater in neomycin-treated rats compared with controls (P < 0.05), indicating the ability of neomycin to increase the amount of glycosides, particularly that of vicine, excreted in the faeces. The ability of glycosides to decrease the concentration of glutathione in blood (P < 0.05) and to increase rat mortality was greatly reduced in rats that were treated with neomycin, particularly in those treated ip with the toxin. Thus, the results demonstrated that neomycin reduced the rate at which vicine and convicine were hydrolysed in the lower section of the gastro-intestinal tract, and that neomycin treatment was associated with a reduced toxicity of the glycosides.

Topics: Administration, Oral; Analysis of Variance; Animals; Cecum; Chromatography, High Pressure Liquid; Drug Interactions; Feces; Gastrointestinal Contents; Glucosides; Glutathione; Hydrolysis; Injections, Intraperitoneal; Intestine, Large; Male; Neomycin; Pyrimidinones; Rats; Rats, Sprague-Dawley; Toxins, Biological; Uridine

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

Vicine and convicine as natural occurring toxic compounds in Vicia faba are involved in the formation of hemolytic anemia (favism). Removal of these compounds needs high technological expense. Microbiological degradation with different strains of bacteria was tried. After 3 h incubation with Streptococcus faeciens convicine was converted completely and Vicine to about 95%.

Topics: Biodegradation, Environmental; Escherichia coli; Fabaceae; Flour; Food Microbiology; Glucosides; Lactobacillus; Lactobacillus acidophilus; Plants, Medicinal; Pyrimidinones; Streptococcus; Toxins, Biological; Uridine

Topics: Animals; Glucosides; Glycosides; Hydrolysis; Intestinal Absorption; Male; Pyrimidinones; Rats; Rats, Inbred Strains; Uracil; Uridine

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

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

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

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