Metabolism of vicine and convicine in rat tissues: absorption and excretion patterns and sites of hydrolysis

The objectives of this study were to determine whether dietary vicine and convicine were absorbed by rat tissue, and to determine their excretion patterns and/or sites of degradation. Orally administered vicine and convicine were excreted in relatively low amounts via the kidney and faeces. However, no vicine or convicine was detected in the blood, liver, kidney or muscle tissue of rats which had been fed these compounds. In-vitro studies demonstrated that vicine and convicine were not hydrolysed in liver, kidney, muscle, caecal wall or intestinal wall homogenates. In contrast, digesta samples from the large intestine and caeca were able to rapidly hydrolyse these compounds, with the concomitant formation of new compounds. Digesta from the stomach and small intestine promoted the slow hydrolysis of these compounds, as did fresh faecal samples. These results would suggest that vicine and convicine are absorbed by the rat in only limited amounts, are not hydrolysed by rat tissues, and are rapidly cleared from tissue via the kidney. The bulk of the dietary vicine and convicine are hydrolysed in the large intestine and caecum.     

Hydrolysis of the toxic constituents (vicine and convicine) in fababean (Vicia faba L.) food preparations following treatment with β-glucosidase

Several experiments were carried out to determine whether the concentrations of vicine and convicine in fababean food preparations were reduced following heat and β-glucosidase treatment. Heating fababeans to relatively high temperatures was not effective in reducing the concentration of these glycosides. Treatment of cooked fababeans (fababean paste) with ground raw almond powder, which is a rich source of β-glucosidase (EC 3.2.1.21) activity, resulted in the hydrolysis of vicine and convicine. The degree of hydrolysis ranged from partial to complete and was dependent on the duration of the treatment period (1 to 24 h), the temperature at which treatment was carried out (1 to 30°C), the pH (6.4 to 4.6) as adjusted by the addition of lemon juice and the amount of ground almonds added to the fababean paste. For example, 88 to 89% of the glycosides were hydrolysed when 1 g of fababean paste was mixed with 0.1 g of almond powder and 0.1 ml of lemon juice and incubated at 30°C for 3 h. This study demonstrated that the concentration of vicine and convicine and their corresponding aglycones can be greatly reduced in fababean food preparations by enzymic hydrolysis.     

The estimation of vicine and convicine in fababeans (Vicia faba L.) and isolated fababean proteins

A specific method of analysis for each of the pyrimidine glucosides, vicine and convicine, is described. This involves extraction by ethanol, evaporation, separation on t.l.c. and quantitative ultraviolet spectrophotometry. Using this method mature dehulled fababeans were shown to contain on average 0.73% vicine and 0.30% convicine on a dry matter basis. No significant variation was found between varieties. The glycosides seem to be associated to the protein bodies in the raw bean, and are absent from the hull. The total content of vicine + convicine in isolated fababean proteins ranged from 0.07 to 2.0%. The highest content was found in a protein fraction produced by air-classification of dry milled fababeans. The lowest content was found in proteins isolated from a watery extract either by precipitation with acetic acid or by diafiltration. Protein isolates produced by direct ultrafiltration have contents of about 0.60%. The ratio vicine:convicine was found to be relatively constant in the raw beans (average 2.5), but varied from 1.4 to 3.0 in the protein isolates, thus indicating a different reaction of vicine and convicine to conditions during the processing of the fababean. It is the opinion of the authors that the etiology of favism is still controversial as far as vicine and convicine are concerned.     

Removal of favism-inducing factors vicine and convicine and the associated effects on the protein content and digestibility of fababeans (Vicia faba L)†

Ingestion of fababeans is associated with precipitation of the haemolytic disease favism in certain glucose-6-phosphate dehydrogenase-deficient humans. Hence their incorporation into weaning foods has not yet been practised. Few, if any, attempts to detoxify fababeans have so far been made. The present investigation has evaluated the effects of a series of treatments designed for vicine and convicine extraction and hydrolysis followed by oxidation of their pyrimidine moieties (divicine and isouramil). The effects of seed germination and oxidative treatment by hydrogen peroxide have also been examined. Extracts of treated samples were assayed for residual vicine and convicine. Toxicity was monitored by the changes in vitro of reduced glutathione (GSH) of red blood cells (RBCs) from Sprague Dawley rats deliberately made deficient in glutathione reductase activity to mimic favism-susceptible human RBCs. Treatments of whole cotyledons resulted in recovery of 59–93% vicine and 50–70% convicine originally present in the seeds. Treatments of fababean powders, however, were capable of lowering the vicine content by 94–100% and convicine content by 100%. Germinated seeds showed a drop in vicine content of 86% and their hydrogen peroxide treatment 91–93%. Convicine was totally absent in germinated and oxidised seeds. The results of toxicity assays were concomitant with vicine and convicine analyses. Protein content of original fababean powders was well recovered (up to 94.00%) and its digestibility was almost complete (99.34%). © 1999 Society of Chemical Industry     

Effects of intraperitoneally injected vicine and convicine on the rat: Induction of favism-like signs

Rats were injected intraperitoneally with single or several successive doses of vicine or convicine. Single injections of these compounds at different concentrations caused increased respiration rates, generalised cyanosis, abdominal convolutions and, after several hours, death which appeared to be caused by asphyxiation. The tissues of the dead animals were engorged with dark brown blood and the large intestine and caecum contained entrapped gases and watery digesta and faecal matter. A second study demonstrated that injected vicine and convicine were cleared from the intraperitoneal cavity over several hours via the kidney and large intestine and that they were cleaved in the digesta of the large intestine and colon to divicine and isouramil. In a third study, rats were injected daily for 10 days with vicine or convicine. There were increases in spleen weight and blood monocytes and neutrophil counts and decreases in liver weight, blood glutathione and glucose concentrations, and lymphocyte counts. Blood from rats pretreated in vivo with convicine was shown to have an altered ultraviolet absorbance pattern. A similar pattern developed in vitro only in the presence of the aglycones of vicine (divicine) or convicine (isouramil) but not in the presence of the compounds themselves. The results of these studies demonstrate that vicine and convicine when injected intraperitoneally into the rat are converted to their aglycones which cause signs similar in many respects to those observed in the human metabolic disease, favism.     

Development of a simple procedure for the complete extraction of vicine and convicine from fababeans (Vicia faba? L.)

Egyptian foods prepared from fababeans contain variable and relatively high concentrations of vicine. Vicine and convicine can be readily extracted from whole or dehulled fababeans. The rate and completeness of extraction depends upon size of beans, the presence or absence of testa, the pH, temperature and volume of the aqueous extracting solution, the duration of the extraction procedure and the frequency with which the extracting solution is changed. In a typical experiment vicine and convicine were almost completely removed from whole or dehulled fababeans when they were extracted at 20 or 40°C with 1% glacial acetic acid (0.17M) or water over a 72 h period with changes in the extracting media every 24 h. This appears to be the most practical procedure for extraction. The low pH of the acid solution prevents bacterial fermentation that occurs when water is used. Water at temperatures greater than 70°C is a highly effective extracting agent. Extraction of fababeans with acetic acid does not markedly affect the concentration of protein (N × 5.7), fat or fibre. Ash content, however, was reduced by a factor of 2 and the total dry matter content of the extracted beans was reduced by 16–17% in whole beans and 20–21% in dehulled beans.     

A modified procedure for the purification of vicine and convicine from fababeans (Vicia faba L.)

A procedure was developed for the simultaneous isolation of pure vicine [2,6-diamino-4,5-dihydroxypyrimidine 5-(β-D glucopyranoside)] and convicine [2,4,5-trihydroxy-6-aminopyrimidine 5-(β-D glucopyranoside)] from dehulled fababeans. The procedure involved the extraction of the glycosides with an ethanol-water (70:30, by vol.) solution, concentration of the extract to a minimal volume and adjustment of the pH of the aqueous extract to 7.2. Mixed vicine and convicine crystals, which were formed over a period of from 1 to 4 days, were harvested by centrifuging and washed with water and ethanol. Vicine was readily obtained from the mixed crystals by extraction at a low pH and recrystallised by adjustment of the pH to neutrality. Convicine, which is relatively insoluble in an acidic medium, was solubilised in an alkaline solution and recrystallised at a neutral pH.     

Effect of genotype and environment on vicine and convicine levels in fababeans (Vicia faba minor)

The concentrations of vicine and convicine (1.94 and 0.83%, dry basis, respectively) in fababeans were highest in young seeds and decreased rapidly with maturity. By 21-28 August 1978 the concentrations had levelled off and the rank order of four cultivars, which differed widely in days required to mature, remained unchanged for the rest of the growing season. Analysis of variance on 36 fababean cultivars grown at different locations and years in Saskatchewan showed that genotype and environment had highly significant (P=0.01) effects on the glucoside level of fababean samples. Although lower in concentration (mean=0.31%), convicine showed greater variability among cultivars than vicine (mean = 0.66%). The glucoside contents of 242 diverse cultivars grown at Saskatoon ranged from 0.44 to 0.82% for vicine and 0.13 to 0.64% for convicine with the lowest cultivar containing 0.62% total glucoside. Individual seeds of a cultivar differed greatly in glucoside content and could become another source of variability for plant breeding and selection of low-glucoside cultivars. Hulls were relatively free of glucosides but, despite variations in hull content, it was unnecessary to dehull samples before analysis.     

Purification,identification and quantification of an egg-weight-depressing factor (vicine) in fababeans (Vicia faba L.)

An egg-weight-depressing factor in fababeans was isolated in pure form from a white precipitate which had been shown in a previous study, to contain a concentrated form of this factor. The analytically pure compound was obtained in colourless, needle crystals. Its melting point was 242-244°C, it contained 17.4% nitrogen but did not react with ninhydrin. The spectra had adsorption maxima at 274, 276 and 269 nm in acidic, neutral, and alkaline solutions, respectively. These and other properties were identical to those of vicine [2,6-diamino-4,5-dihydroxy pyrimidine,5-(β-D-glucopyrano-side)] a compound known to be present in fababeans. Vicine and convicine [2,4,5-trihydroxy, 6-aminopyrimidine,5-(β-D-glucopyranoside)]; compounds having similar chemical and probably physiological activity, were resolved on a cation-exchange column and were detected by monitoring at 280 nm. Quantitative results obtained with the procedure was shown to be similar to those obtained with two other procedures. This method, however, was superior to other methods as it gave individual values for both vicine and convicine rather than a composite value; sample preparation was simple and the method could readily be adapted to automatic analysis.     

The Distribution of Pyrimidinone Glucosides in Developing Seedlings of Vicia faba and Vicia narbonensis

The distribution of the pyrimidinone glucopyranosides vicine and convicine was investigated in resting seed and axenic developing seedlings up to 17 days of age in Vicia faba cv Troy, a low seed pyrimidinone glucopyranoside mutant (MTG5) derived from Troy, and a related species V narbonensis. The lower amounts of total vicine and convicine in resting seed of MTG5 were due primarily to a reduction in cotyledon pyrimidinone glucopyranosides, with the amount in the embryo axis much less affected. Distribution within developing seedlings revealed consistent patterns. A pulse of vicine appeared in both species in stems and roots at day 4 to day 7, then declined. Amounts of convicine rose initially then declined in seedling stems of both species, whereas in roots the rise in convicine continued throughout the period. The rise in convicine in the roots of V narbonensis could not have been accounted for by conversion from reserves of vicine. The low pyrimidinone glucopyranoside mutant MTG5 had less vicine initially in stems and roots than both Troy and V. narbonensis but by 17 days this had risen close to the levels of Troy and exceeded those of V narbonensis. By flowering, amounts of vicine and convicine in glass-house grown plants were similar in MTG5 and Troy. The levels of both compounds in the roots of MTG5 exceeded those of the sown seed. Consequently, both V faba and V narbonensis have the capacity to synthesise vicine and convicine in young plants. A mutation in a gene controlling levels of pyrimidinone glucopyranosides in the developing seed of V faba does not suppress the biosynthetic ability of young plants.     

The concentration of vicine and convicine in Vicia faba and some related species and their distribution within mature seeds

Mature seeds from a range of species from the Vicieae tribe were analysed for vicine and convicine content. Quantifiable amounts of vicine were found in all the Vicia species analysed but neither glucopyranoside was detected in Pisum or Lathyrus. The concentrations of both vicine and convicine were similar for both Vicia faba and V bithynica but in all the other Vicia species glucopyranoside concentrations were significantly lower. Seeds from three faba bean lines and one each from V narbonensis and V bithynica were dissected into their constituent seed parts and these parts analysed. Only trace quantities were found in the seed coat but concentrations of vicine ranging from 39 g kg^?1 to 81 g kg^?1 were detected in the radicle samples. The implications of these results in relation to the role of these compounds in the plant are discussed.     

Characterization and kinetics of gastric emptying of peptides derived from milk proteins in the preruminant calf.

The gastric emptying kinetics of peptides derived from milk protein were studied in vivo in preruminant calves by collecting and characterizing the whole effluent leaving the stomach for 12 h after ingestion of crude skim milk. Peptides were isolated by reversed-phase HPLC and identified. Particular attention was paid to biologically active peptides and to peptides that could be precursors of biologically active sequences. A gastrin inhibitor, the caseinomacropeptide, was emptied from the stomach only during the first 0.5 h of digestion and rapidly hydrolysed. Precursors of immunostimulatory peptides from alpha s1- and beta-caseins were emptied throughout digestion in the gastric effluent. A precursor of beta-casomorphins (peptide 58-93 of beta-casein) was emptied from the stomach 3.5 h after the meal when it was taken on an empty stomach. From this precursor, peptides that may be resistant to hydrolysis by intestinal peptidase were obtained after in vitro hydrolysis by pancreatic enzymes. A phosphopeptide (fragment 110-142 of alpha s1-casein) was also found in digesta after a few hours of digestion. When the meal was not taken on an empty stomach, these peptides were emptied in the first digesta at a low concentration. The potential activity of these peptides is discussed. The results support the hypothesis that active sequences could still be present in the gut after the action of pancreatic enzymes.     

Content and distribution of vicine,convicine and <Emphasis Type="SmallCaps">l</Emphasis>-DOPA during germination and seedling growth of two <Emphasis Type="Italic">Vicia faba</Emphasis> L. varieties

This work examined the content and distribution of the pyrimidine glucosides vicine and convicine, and the nonproteic amino acid l-DOPA in cotyledons and embryo axes along the germination and seedling growth of Vicia faba L. vars. Alameda and Brocal. The behaviour of these compounds was similar in both varieties. Vicine and convicine, implicated in favism disease, slowly declined in cotyledons. In embryo axis, vicine levels were sharply reduced and convicine amount was slightly increased as assay progressed. Total pyrimidine glucosides remained unchanged in the whole plant during the study. l-DOPA only appeared in the embryo axis of V. faba seeds and the highest content was observed in Brocal variety at 6 DAI (days after imbibition). The information provided in this study could be valuable for a possible role of embryo axis, rich in l-DOPA, for the treatment of Parkinson’s disease and also as a choice ingredient for functional foods or as nutraceutical.     

Extraction of favism‐inducing agents from whole seeds of faba bean (Vicia faba L var major)

The objective of the present study was to establish a suitable procedure by which favism‐inducing glycosides vicine and convicine could be completely removed from whole seeds of faba beans without distorting their shape or reducing their nutritive value. Detoxification procedures carried out were extraction by water or, 10 g l^?1 acid solutions (acetic, citric and phosphoric acid) using stepwise or continuous soaking procedures. Three cultivars of the beans were used in the study. In the stepwise soaking procedures, the seeds were soaked in either water or acid solution at 40 °C for different periods while in the continuous flow techniques, the solutions were constantly passed through a seed reservoir under different time–temperature–flow rate conditions. Extraction of vicine and convicine increased with increasing temperature and period of soaking and/or continuous flow of soaking solutions. Neither stepwise soaking nor autoclaving could effectively remove the favism factors from the whole seeds. The best results were obtained with continuous flow soaking in tap water, giving vicine‐ and convicine‐free seeds. The protein contents of treated seeds were only slightly affected and their physical structure remained intact. Thus, it was concluded that the latter procedure was the method of choice for detoxification of dry whole seeds. Copyright © 2005 Society of Chemical Industry     

The enzymic deacylation of p-nitrophenyl esters and phosphatidylcholine in vicia faba minor

Two enzymic deacylating activities have been detected in crude extracts of small fababeans—the hydrolysis of p-nitrophenyl fatty acyl esters and that of phosphatidylcholine. Kinetic studies demonstrated differences between the two activities with respect to substrate inhibition and to the effects of pH, Triton X-100, calcium chloride and mercuric chloride. The sequential activity of phospholipases A₁ and A₂, and lysophospholipase, in fababeans is postulated, and some similarity is indicated between the hydrolysis of p-nitrophenyl palmitate and fababean alkaline lipase. The significance of results from this study is discussed in relation to earlier work on lipid-degrading enzymes in fababeans.     

Viscous Food Matrix Influences Absorption and Excretion but Not Metabolism of Blackcurrant Anthocyanins in Rats

ABSTRACT:  The aim of the present study was to investigate the effect of a simultaneous intake of food and anthocyanins (ACNs) on ACN absorption, metabolism, and excretion. Blackcurrant ACNs (BcACNs) were dissolved in water with or without the addition of oatmeal and orally administered to rats, providing approximately 250 mg total ACNs per kilogram BW. Blood, urine, digesta, and tissue samples of the stomach, jejunum, and colon were subsequently collected at 0.25, 0.5, 1, 2, 3, 7, and 24 h. Identification and quantification of ACNs were carried out by Reversed phase-high-performance liquid chromatography (RP-HPLC) and liquid chromatography-mass spectrometry (LC-MS). Four major ACNs were present in the blackcurrant extract: delphinidin 3- O -glucoside, delphinidin 3- O -rutinoside, cyanidin 3- O -glucoside, and cyanidin 3- O -rutinoside. In plasma, the 4 ACNs of blackcurrant were identified and quantified. The time to reach maximal total ACN plasma concentration ( C _max BcACN/water = 0.37 ± 0.07 μmol/L; C _max BcACN/oatmeal = 0.20 ± 0.05 μmol/L) occurred faster after BcACN/water ( t _max= 0.25 h), than after BcACN/oatmeal administration ( t _max= 1.0 h). In digesta and tissue samples, the 4 original blackcurrant ACNs were detected. The relative concentration of rutinosides in the digesta increased during their passage through the gastrointestinal tract, while the glucosides decreased. Maximum ACN excretion in urine occurred later after BcACN/oatmeal than after BcACN/water administration (3 compared with 2 h). The 4 original ACNs of blackcurrant in their unchanged form, as well as several metabolites, were identified in the urine samples of both groups. The simultaneous intake of food affects ACN absorption and excretion in the urine, but not metabolism.     

Comparison of methods to determine the endogenous amino acid flow at the terminal ileum of the growing rat

The aim was to compare a recently developed peptide alimentation method and the protein-free and regression methods for determining endogenous ileal nitrogen (N) and amino acid excretion in the rat, and to apply the endogenous loss estimates to determine the true ileal digestibility of a meat and bone meal (MBM). Preliminary investigations determined the effect of the time of sampling of digesta after a meal (slaughter method) for rats given a protein-free or an enzymically hydrolysed casein (EHC) based diet, on ileal digesta and endogenous N excretion. There was a significant (P < 0.05) effect of the time of sampling on the amount of digesta collected and the endogenous N excretion for both the EHC and protein-free fed rats. The amount of digesta collected from the terminal 20 cm of ileum and the endogenous N excretion for both the EHC and protein-free fed rats were greatest and least variable at 3 h post-feeding. In the main study, endogenous ileal amino acid excretions were determined in the growing rat fed an EHC-based diet (n = 6) and with subsequent treatment of the digesta using ultrafiltration or in six rats given a protein-free diet or by extrapolation from data for 30 animals given five diets which contained graded levels of MBM as the sole protein source. For the EHC treatment, the ileal digesta precipitate plus retentate was used to determine the endogenous flows. The ultrafiltration step excludes unabsorbed dietary amino acids from the measure of endogenous loss. Chromic oxide was the reference marker in all diets. The endogenous N flows determined by the protein-free and regression methods were similar but both significantly (P < 0.01) lower than those for rats fed the EHC-based diet. The mean endogenous ileal N flows determined by the peptide alimentation method, the protein-free and regression approaches were 1866, 1103 and 1019 μg g^?1 freeze dry matter intake, respectively. The endogenous amino acid flows at the terminal ileum were lower when determined using the traditional protein-free or regression methods. The true ileal digestibility coefficients for MBM were considerably higher based on the EHC versus protein-free endogenous flows.     

Enzymes of the small intestine of the calf: effect of dietary protein source on the activities of some enzymes in the small intestinal mucosa and digesta

The objective of this study was to examine the effect of dietary protein source on the morphology and enzyme activities in the small intestinal mucosa and digesta of preruminant calves. The control diet contained skim milk powder. Plant proteins from soy concentrate (unhydrolysed or partially hydrolysed) and potato concentrate were incorporated into experimental diets. Duodenal, jejunal and ileal mucosal biopsies and digesta were collected from calves fitted with simple cannulae and continuously infused with milk replacers into the abomasum. Biopsies were used for morphology measurements. The activity of dipeptidylpeptidase IV, aminopeptidases, lactase and alkaline phosphatase was estimated in biopsies and digesta. Incorporating plant protein had a limited effect on the intestinal mucosa. A partial villus atrophy and crypt hyperplasia were noted with the soy concentrate only. Decreases in the activity of dipeptidylpeptidase IV with the potato concentrate diet and of aminopeptidase N and lactase with the hydrolysed soy concentrate diet, were also observed in ileal biopsies. Mucosal enzymes were released in the lumen and most of them accumulated in digesta of the distal small intestine. The activity of aminopeptidase N and lactase in ileal digesta was higher in calves fed the soy and potato concentrate diets than in those fed the control diet. © 2002 Society of Chemical Industry     

Digestive lipolysis in the preruminant calf. The abomasal hydrolysis of butter oil, coconut oil, palm oil and tallow

The effect of intramolecular short-chain fatty acids on the abomasal hydrolysis of long chain acyl ester bonds has been examined using four different fats. An evaluation of abomasal infusion of feeds as a technique to avoid hydrolysis of fat by salivary lipase has been made. Only calves considered to be typically functional preruminants were used for the investigations. Five preruminant calves each received five experimental milk feeds consisting of skim milk plus 3% of either butter oil, coconut oil, palm oil or tallow. All feeds were given by mouth but the feed that contained butter oil was also introduced directly into the abomasum via a simple cannula. In all cases, digesta leaving the abomasum were continuously sampled for 12 h via a re-entrant cannula in the proximal duodenum, anterior to the entry of the bile and pancreatic ducts. Butter oil given by mouth was hydrolysed to a greater extent than the other fats. This was due not only to the ease of liberation of the short chain fatty acids, but also to an enhanced liberation of longer chain acids when short chain acids are present in the triglyceride. Butter oil introduced directly into the abomasum of truly preruminant calves was not hydrolysed, suggesting that by using this technique studies could be made in vivo of the hydrolysis of fat by pancreatic lipase alone.     

The effect of digesta sampling time and dietary protein source on ileal nitrogen digestibility for the growing rat

The effect of time that digesta were collected on the quantity and representativeness of ileal digesta and on the determination of apparent and true ileal nitrogen (N) digestibility for growing rats was investigated. Semi‐synthetic diets containing chromic oxide as an indigestible marker were given to 200 g live weight rats for 8 days as a single daily meal. The experimental treatments comprised six diets (lactalbumin, soy protein isolate, wheat gluten, fish, protein‐free (PF), enzymically hydrolysed casein (EHC)) and four ileal digesta sampling times (3, 4, 5 and 6 h after the start of feeding). On the eighth day the rats were fed and slaughtered according to treatment, and digesta were sampled from the terminal ileum. Endogenous ileal nitrogen excretion was determined using both the protein‐free and peptide alimentation methods. There was a significant (P = 0.027) effect of experimental diet averaged across sampling times for chromium recovery (CrRec). The CrRec was higher for the rats fed the PF diet than for the other diets. Responses to sampling time varied significantly (P < 0.05) among diets for ileal digesta weight (IDW), nitrogen/chromium (N/Cr) ratio, ileal digesta weight relative to dry matter intake (IDW/DMI), CrRec, N flow, apparent N digestibility and true N digestibility (determined using the protein‐free method). The optimal digesta sampling times for each of the diets were 3 h for PF, EHC, lactalbumin and fish and 4 h for soy protein isolate and wheat gluten. Consequently, 3 or 4 h after the start of feeding is recommended as the optimum ileal digesta sampling time for most purified protein sources when fed to the growing rat as a single daily meal. © 2002 Society of Chemical Industry