Evaluation of the health risks related to the presence of cyanogenic glycosides in foods other than raw apricot kernels
2019; Wiley; Volume: 17; Issue: 4 Linguagem: Inglês
10.2903/j.efsa.2019.5662
ISSN1831-4732
AutoresDieter Schrenk, Margherita Bignami, Laurent Bodin, James Kevin Chipman, Jesús del Mazo, Bettina Grasl‐Kraupp, Christer Högstrand, L.A.P. Hoogenboom, Jean‐Charles Leblanc, Carlo Nebbia, Elsa Nielsen, Evangelia Ntzani, Annette Petersen, Salomon Sand, Christiane Vleminckx, Heather Wallace, Diane Benford, Leon Brimer, Francesca Romana Mancini, Manfred Metzler, Barbara Viviani, Andrea Altieri, Davide Arcella, Hans Steinkellner, Tanja Schwerdtle,
Tópico(s)Cassava research and cyanide
ResumoEFSA JournalVolume 17, Issue 4 e05662 Scientific OpinionOpen Access Evaluation of the health risks related to the presence of cyanogenic glycosides in foods other than raw apricot kernels EFSA Panel on Contaminants in the Food Chain (CONTAM), EFSA Panel on Contaminants in the Food Chain (CONTAM)Search for more papers by this authorDieter Schrenk, Dieter SchrenkSearch for more papers by this authorMargherita Bignami, Margherita BignamiSearch for more papers by this authorLaurent Bodin, Laurent BodinSearch for more papers by this authorJames Kevin Chipman, James Kevin ChipmanSearch for more papers by this authorJesús del Mazo, Jesús del MazoSearch for more papers by this authorBettina Grasl-Kraupp, Bettina Grasl-KrauppSearch for more papers by this authorChrister Hogstrand, Christer HogstrandSearch for more papers by this authorLaurentius (Ron) Hoogenboom, Laurentius (Ron) HoogenboomSearch for more papers by this authorJean-Charles Leblanc, Jean-Charles LeblancSearch for more papers by this authorCarlo Stefano Nebbia, Carlo Stefano NebbiaSearch for more papers by this authorElsa Nielsen, Elsa NielsenSearch for more papers by this authorEvangelia Ntzani, Evangelia NtzaniSearch for more papers by this authorAnnette Petersen, Annette PetersenSearch for more papers by this authorSalomon Sand, Salomon SandSearch for more papers by this authorChristiane Vleminckx, Christiane VleminckxSearch for more papers by this authorHeather Wallace, Heather WallaceSearch for more papers by this authorDiane Benford, Diane BenfordSearch for more papers by this authorLeon Brimer, Leon BrimerSearch for more papers by this authorFrancesca Romana Mancini, Francesca Romana ManciniSearch for more papers by this authorManfred Metzler, Manfred MetzlerSearch for more papers by this authorBarbara Viviani, Barbara VivianiSearch for more papers by this authorAndrea Altieri, Andrea AltieriSearch for more papers by this authorDavide Arcella, Davide ArcellaSearch for more papers by this authorHans Steinkellner, Hans SteinkellnerSearch for more papers by this authorTanja Schwerdtle, Tanja SchwerdtleSearch for more papers by this author EFSA Panel on Contaminants in the Food Chain (CONTAM), EFSA Panel on Contaminants in the Food Chain (CONTAM)Search for more papers by this authorDieter Schrenk, Dieter SchrenkSearch for more papers by this authorMargherita Bignami, Margherita BignamiSearch for more papers by this authorLaurent Bodin, Laurent BodinSearch for more papers by this authorJames Kevin Chipman, James Kevin ChipmanSearch for more papers by this authorJesús del Mazo, Jesús del MazoSearch for more papers by this authorBettina Grasl-Kraupp, Bettina Grasl-KrauppSearch for more papers by this authorChrister Hogstrand, Christer HogstrandSearch for more papers by this authorLaurentius (Ron) Hoogenboom, Laurentius (Ron) HoogenboomSearch for more papers by this authorJean-Charles Leblanc, Jean-Charles LeblancSearch for more papers by this authorCarlo Stefano Nebbia, Carlo Stefano NebbiaSearch for more papers by this authorElsa Nielsen, Elsa NielsenSearch for more papers by this authorEvangelia Ntzani, Evangelia NtzaniSearch for more papers by this authorAnnette Petersen, Annette PetersenSearch for more papers by this authorSalomon Sand, Salomon SandSearch for more papers by this authorChristiane Vleminckx, Christiane VleminckxSearch for more papers by this authorHeather Wallace, Heather WallaceSearch for more papers by this authorDiane Benford, Diane BenfordSearch for more papers by this authorLeon Brimer, Leon BrimerSearch for more papers by this authorFrancesca Romana Mancini, Francesca Romana ManciniSearch for more papers by this authorManfred Metzler, Manfred MetzlerSearch for more papers by this authorBarbara Viviani, Barbara VivianiSearch for more papers by this authorAndrea Altieri, Andrea AltieriSearch for more papers by this authorDavide Arcella, Davide ArcellaSearch for more papers by this authorHans Steinkellner, Hans SteinkellnerSearch for more papers by this authorTanja Schwerdtle, Tanja SchwerdtleSearch for more papers by this author First published: 11 April 2019 https://doi.org/10.2903/j.efsa.2019.5662Citations: 7 Correspondence: contam@efsa.europa.eu Requestor: European Commission Question number: EFSA-Q-2016-00802 Panel members: Margherita Bignami, Laurent Bodin, James Kevin Chipman, Jesús del Mazo, Bettina Grasl-Kraupp, Christer Hogstrand, Laurentius (Ron) Hoogenboom, Jean-Charles Leblanc, Carlo Stefano Nebbia, Elsa Nielsen, Evangelia Ntzani, Annette Petersen, Salomon Sand, Dieter Schrenk, Tanja Schwerdtle, Christiane Vleminckx and Heather Wallace. Acknowledgements: The Panel wishes to thank the hearing expert Klaus Abraham for the support provided to this scientific output. Adopted: 18 March 2019 Reproduction of the images listed below is prohibited and permission must be sought directly from the copyright holder: Figure C1: © Abraham K, Buhrke T, Lampen A, 2015. This publication is linked to the following EFSA Supporting Publications article: http://onlinelibrary.wiley.com/doi/10.2903/sp.efsa.2019.EN-1601/full AboutSectionsPDF ToolsExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Abstract In 2016, the EFSA Panel on Contaminants in the Food Chain (CONTAM) published a scientific opinion on the acute health risks related to the presence of cyanogenic glycosides (CNGs) in raw apricot kernels in which an acute reference dose (ARfD) of 20 μg/kg body weight (bw) was established for cyanide (CN). In the present opinion, the CONTAM Panel concluded that this ARfD is applicable for acute effects of CN regardless the dietary source. To account for differences in cyanide bioavailability after ingestion of certain food items, specific factors were used. Estimated mean acute dietary exposures to cyanide from foods containing CNGs did not exceed the ARfD in any age group. At the 95th percentile, the ARfD was exceeded up to about 2.5-fold in some surveys for children and adolescent age groups. The main contributors to exposures were biscuits, juice or nectar and pastries and cakes that could potentially contain CNGs. Taking into account the conservatism in the exposure assessment and in derivation of the ARfD, it is unlikely that this estimated exceedance would result in adverse effects. The limited data from animal and human studies do not allow the derivation of a chronic health-based guidance value (HBGV) for cyanide, and thus, chronic risks could not be assessed. Summary Following a request from the European Commission, the European Food Safety Authority (EFSA) Panel on Contaminants in the Food Chain (CONTAM Panel) evaluated the risks to human health related to the presence of cyanogenic glycosides (CNGs) in foods other than raw apricot kernels. Previous assessments from the EFSA, in particular the opinion on acute health risks related to the presence of CNGs in raw apricot kernels and products derived from raw apricot kernels (2016), and assessments from other international and national scientific bodies have been used as a starting point for the evaluation together with publications identified in a targeted literature search. EFSA guidance documents and general principles for risk assessment have been applied for hazard and exposure assessment in this opinion. CNGs contain chemically bound cyanide and are present in foods such as almonds, linseed or cassava. When the plant cells are damaged, by for example grinding or chewing, CNGs and their degrading enzymes are brought into contact and cyanide is released. Cyanide is readily absorbed from the gastrointestinal tract and rapidly distributed to all organs. Peak concentrations of cyanide in blood and tissue depend on the amount of CNGs in the food consumed and the rate of release of cyanide which in turn depends on the presence and activity of the degrading enzymes. Peak blood cyanide concentration (assessed by serial measurements of cyanide in whole-blood after ingestion) can be used as a reliable biomarker for acute cyanide exposure. In a human bioavailability study, mean peak concentrations of cyanide in blood were different after consumption cassava root, linseed and persipan, indicating a fast and practically complete release of cyanide after chewing of bitter almonds and cassava roots but not with linseed and persipan. In experimental animals, acute toxicity of cyanide and CNGs is characterised by dyspnoea, ataxia, arrhythmia, convulsions, loss of consciousness, decreased respiration and death. Upon repeated dose exposure to cyanide, histopathological alterations in the thyroid, kidney, liver and central nervous system (CNS), and changes in epididymis cauda weights, sometimes paralleled with clinical signs have been reported, but the findings are not consistent between different studies. With the CNGs linamarin and amygdalin, alterations in haematology and clinical chemistry parameters and histopathological alterations were seen. With gari (a cassava product for direct human consumption) and cassava, behavioural changes have been observed. There are indications of developmental effects in hamsters exposed to CNGs or cassava and in rats exposed to potassium cyanide (KCN), which were often observed in the presence of maternal toxicity. Cyanide is not genotoxic. No information is available on the genotoxicity of CNGs. The acute lethal oral dose of cyanide in humans is reported to be between 0.5 and 3.5 mg/kg body weight (bw). The toxic threshold value for cyanide in blood is considered to be between 0.5 mg/L (ca. 20 μM) and 1.0 mg/L (ca. 40 μM), the lethal threshold value ranges between 2.5 mg/L (ca. 100 μM) and 3.0 mg/L (ca. 120 μM). Signs of acute cyanide poisoning in humans include headache, vertigo, agitation, respiratory depression, metabolic acidosis, confusion, coma, convulsions and death. Poisoning cases, some fatal, have resulted from ingestion of amygdalin preparations, bitter almonds and cassava. Several neurological disorders and other diseases have been associated with chronic exposure to cyanide in populations where cassava constitutes the main source of calories. The primary mode of action for acute toxicity of cyanide is the inhibition of oxidative phosphorylation leading to anaerobic energy production. Due to the high oxygen and energy demand, brain and heart are particularly sensitive to cyanide which can result in hypoxia, metabolic acidosis and impairment of vital functions. The role of cyanide in neurological impairment upon long-term consumption of foods containing CNGs has not been elucidated. The CONTAM Panel concluded that there are no data indicating that the acute reference dose (ARfD) for cyanide of 20 μg/kg bw, established in 2016, should be revised and that it is applicable for acute effects of cyanide regardless of the dietary source. For exposure to cyanide from foods other than raw apricot kernels, bitter almonds and cassava roots, this ARfD is likely to be over-conservative because of the lower bioavailability of cyanide from these foods, but establishment of different ARfDs for different types of food is not appropriate. However, to account for the differences in cyanide bioavailability after ingestion of certain food items, for cassava and cassava derived products and for almonds a factor of 1, for linseed a factor of 3 and for marzipan/persipan, a factor of 12 was calculated based on results from a human bioavailability study. Occurrence data on these foods were divided by the respective factors for inclusion in the exposure assessment. For all other food items, no data on bioavailability were available, and a factor of 1 was used as a default worst-case value assuming complete cyanide bioavailability. The limited data from animal and human studies do not allow the derivation of a chronic health-based guidance value (HBGV) for cyanide (CN). A total of 2,586 analytical results on total cyanide in foods were available in the EFSA database (of which about 89% came from Germany and of which 46% were left-censored) to estimate acute and chronic dietary exposure. Highest occurrence values were reported in bitter almonds (mean concentration 1,437 mg/kg) and in linseed (mean concentration 192.1 mg/kg). No occurrence data were available in the database for cassava and products derived thereof. Estimated acute exposures to cyanide originating from foods containing CNGs across 43 different dietary surveys and all age groups ranged from 0.0 to 13.5 μg/kg bw per day (mean, minimum lower bound (LB) to mean maximum upper bound (UB)) and 0.0–51.7 μg/kg bw per day (95th percentile (P95), minimum LB to maximum UB). Estimated chronic exposures to cyanide originating from foods containing CNGs across 38 different dietary surveys and all age groups ranged from 0.0 to 13.5 μg/kg bw per day (mean, minimum LB to maximum UB) and from 0.6 to 34.5 μg/kg bw per day (P95, minimum LB to maximum UB). The highest acute and chronic exposures were estimated for 'Infants', 'Toddlers' and 'Other children' and the main contributors to acute and chronic exposure to cyanide in all age groups were 'Biscuits (cookies)', 'Juice or nectar from fruits' and 'Pastries and cakes'. Estimated mean dietary acute exposures did not exceed the ARfD of 20 μg CN/kg bw in any age group. At the P95, the ARfD was exceeded by up to about 2.5-fold in some consumption surveys for 'Infants', 'Toddlers', 'Other children' and the adolescent age groups. The CONTAM Panel notes that these are likely overestimations, in particular because of the assumptions made regarding full cyanide bioavailability from foods other than bitter almonds, cassava roots, linseed, persipan and marzipan. A chronic exposure assessment has also been carried out, although there are insufficient data to characterise potential risks of chronic exposure to cyanide in a European population. In addition, exposure 'back-calculations' have been carried out to estimate the amount of certain food items that can be ingested without exceeding the ARfD. This was done for raw cassava root, gari, cassava flour, ground linseed and bitter almonds as well as for food items for which an EU maximum level (ML) for cyanide has been established. The bioavailability factors applied for the exposure assessment have also been applied for these calculations. Depending on the body weight, consumption of 1.3–14.7 g ground linseed containing a high concentration of 407 mg CN/kg could reach the ARfD, the corresponding values for consumption of raw cassava root containing a high concentration of 235 mg CN/kg, being 0.7–8.5 g. If gari or cassava flour containing the respective Codex Alimentarius Commission (Codex) MLs of 2 mg total CN/kg and 10 mg total CN/kg, respectively, are consumed, the ARfD is reached with consumption of 87–1,000 g gari and with 17–200 g cassava flour. Consumption of 0.1–1.4 g bitter almonds (1,477 mg CN/kg) reaches the ARfD. This corresponds to an amount of less than half a small kernel in 'Toddlers' and of 1 large kernel in 'Adults'. If marzipan or persipan containing the respective EU maximum limit (ML) of 50 mg CN/kg are consumed, the ARfD is reached with 42–480 g. Consumption of 35–400 g canned stone fruits containing the respective EU ML of 5 mg total cyanide/kg leads to an exposure equivalent to the ARfD. If stone fruit marc spirits and stone fruit spirits contain the EU ML of 35 mg total cyanide/kg, the ARfD is reached by consumption of 26–57 g, depending on the body weight of the individual. The overall uncertainty incurred with the present assessment is considered as high. It is more likely to overestimate than to underestimate the risk. Validated methods for the quantification of CNGs and total cyanide and investigations on the variation of hydrolytic enzymes are needed in different foods. The variation of hydrolytic enzymes in food crops and the potential to identify cultivars of crops with relatively low content of CNG or of hydrolytic enzymes need to be investigated. More occurrence data for cyanide in raw and processed foods and consumption data for CNG containing foods are also needed. Human toxicokinetics of CNGs and released cyanide after ingestion of food items containing CNGs need to be studied further. More information is needed on the presence of hydrolytic activity in processed foods. More data are needed to evaluate the potential of cyanide and food items that contain CNGs to cause chronic effects. 1 Introduction 1.1 Background and Terms of Reference as provided by the requestor 1.1.1 Background On 1 March 2016, the Panel on Contaminants in the Food Chain (CONTAM) adopted the scientific opinion on acute health risks related to the presence of cyanogenic glycosides in raw apricot kernels and products derived from raw apricot kernels.1 The CONTAM Panel established an ARfD for cyanide of 0.02 mg/kg bw (20 μg/kg bw) for use in assessing the risks associated with the presence of cyanogenic glycosides in apricot kernels. Cyanogenic glycosides are also present in other food such as linseed and cassava. Furthermore, maximum levels for hydrocyanic acid are established in nougat, marzipan or its substitutes or similar products (50 mg/kg) canned stone fruits (5 mg/kg) and alcoholic beverages (35 mg/kg) by Regulation (EC) No 1334/20082 and 7 g of hydrocyanic acid per hectolitre of 100% vol. alcohol in stone fruit spirits and fruit marc spirit, established by Regulation (EC) No 110/20083. In the scientific literature there is evidence that this acute reference dose is applicable to unprocessed foods with cyanogenic glycosides also containing intact plant β-glucosidase. It is mentioned that for some foods the approach may be overly conservative due to the delayed and/or incomplete release of cyanide from the cyanogenic glycosides depending on many factors, as was demonstrated for linseed. In case of missing or inactivated β-glucosidase, the hazard potential would be much lower.4 Furthermore, in the scientific opinion of the Scientific Panel on Food Additives, Flavourings, Processing Aids and Materials in Contact with Food (AFC) on hydrocyanic acid in flavourings and other food ingredients with flavouring properties,5 adopted on 7 October 2004 the following is concluded 'Cassava flour is used as a staple food mainly outside Europe; a consumption of 200 g/person would lead to an estimated intake level of 30 μg HCN/kg bw for a 60 kg adult. In accordance with the JECFA view such an intake would not be associated with acute toxicity. The highest level of HCN found in retail marzipan paste is 20 mg HCN/kg. Assuming on one sitting a person of 60 kg consumes 100 g marzipan containing such a level, that intake would be equivalent to 2 mg HCN or to 0.03 mg/kg bw'. It is appropriate to consider the need to take regulatory measures as regards the presence of cyanogenic glycosides in foods which are not yet regulated at EU level and to assess the appropriateness of existing maximum levels for hydrocyanic acid in food to provide a high level of human health protection. Therefore, it is appropriate that EFSA assesses the applicability of the Acute Reference Dose (ARfD) for cyanogenic glycosides in raw apricot kernels to other food in which cyanogenic glycosides are present. In case it is concluded that the ARfD for cyanogenic glycosides in raw apricot kernels is not applicable to other foods in which cyanogenic glycosides are present, EFSA is requested to assess the human health risks of the presence of cyanogenic glycosides in foods other than raw apricot kernels. 1.1.2 Terms of Reference In accordance with Art. 29 (1) of Regulation (EC) No 178/2002, the European Commission asks the European Food Safety Authority for a scientific opinion on the human health risks related to the presence of hydrocyanic acid in foods other than raw apricot kernels and products derived from apricot kernels (ground, milled, cracked, chopped). In particular, the scientific opinion should inter alia comprise: Evaluation of the applicability of the ARfD established for cyanogenic glycosides in raw apricot kernels for other foods in which cyanogenic glycosides are present. Evaluation of the relevance of chronic effects related to the human dietary exposure to cyanogenic glycosides. Estimation of acute and (if relevant) chronic dietary exposure of the EU population, including consumption patterns of specific (vulnerable) groups of the population. 1.2 Interpretation of the Terms of Reference In the Terms of Reference (ToR) as provided by the European Commission, EFSA was requested to address the risks to human health related to the presence of hydrocyanic acid (hydrogen cyanide, HCN) in foods other than raw apricot kernels. The EFSA Panel on Contaminants in the Food Chain (CONTAM Panel) noted that free HCN is actually not present in food at toxicologically relevant concentrations and that any risks are related to the release of HCN from cyanogenic glycosides (CNGs) present in plant-derived food. CNGs are produced as secondary metabolites by various plant species and probably serve as a defence mechanism against herbivores, because CNGs release highly toxic HCN when hydrolysed. Hydrolytic enzymes are stored separately from CNGs in intact plants. However, when plant material is chewed or otherwise processed, hydrolytic enzymes and CNGs come in contact and HCN is formed. Because of its weak acidity, HCN always exists as a mixture of non-dissociated acid (HCN) and its dissociated form (cyanide ions, CN−) in aqueous biological fluids, the proportion of each form in the dissociation equilibrium depending on the pH of the fluid. Therefore, the term 'cyanide' (or CN) will be used throughout this opinion to inclusively represent the inorganic forms of cyanide, i.e. the undissociated HCN and the dissociated CN−. Very low levels of cyanide are also produced in the brain as neuromodulators (Cipollone and Visca, 2007). This source is negligible in terms of toxicity. The CONTAM Panel limited the assessment to plant-derived foods as in terms of CNG content, occurrence in foodstuffs and consumption, non-plant-derived foods were considered to be a negligible source of dietary cyanide. 1.3 Additional information 1.3.1 Chemistry Hydrocyanic acid (hydrogen cyanide or HCN) does virtually not occur in plants as free compound but 'hidden' in so-called CNGs, which allow the plant to store HCN without suffering from its toxicity. Cyanogenic glycosides At least 60 different CNGs have been identified in plants (Seigler, 1991). In general, CNGs contain cyanide (CN) in a chemically fixed state as a cyanohydrin (α-hydroxynitrile) which is stabilised as a β-glycoside of a monosaccharide like glucose or a disaccharide like gentiobiose (Poulton, 1990; Jones, 1998; Ballhorn, 2011). As an example, the complete chemical structures of the widely occurring glucoside linamarin and its homologous gentiobioside, linustatin are depicted in Figure 1. In intact plant cells, CNGs are stored in vacuoles and thereby separated from β-glycosidase enzymes (EC 3.2.1.21) located in plant cell walls. When plant cells are physically destroyed, e.g. by chewing or grinding, the CNGs come into contact with the β-glycosidase enzymes and are degraded with the release of HCN. In aqueous biological fluids, free HCN exists in a pH-dependent dissociation equilibrium with cyanide ions (CN−). The mixture of non-dissociated HCN and cyanide ions is termed 'cyanide' (see EFSA CONTAM Panel, 2016). Figure 1Open in figure viewerPowerPoint Chemical structures of linamarin and linustatin The chemical structures and some of the features of typical CNGs are listed in Table 1. The aglycones of some but not all of the CNGs contain chiral centres, i.e. C-atoms with four different substituents. Of particular practical importance is the fact that different amounts of CN are released from different CNGs, because of the different molecular masses. For example, 1 g of linamarin, which has a relatively low molecular mass, yields almost twice as much HCN compared to 1 g of amygdalin with a much higher molecular mass. Due to the polar glycoside group, all CNGs are solids with quite high melting points and a similar solubility, which is much higher in polar solvents like water or ethanol than in non-polar solvents such as chloroform or benzene. Table 1. Important cyanogenic glycosides (CNGs) in food plants, arranged according to maximum release of CN (calculated as HCN equivalents) Chemical structure CAS number Element formula Molecular mass CN (mg/g CNG) Examples for occurrencea 554-35-8 C10H17NO6 247.3 109.2 Cassava (Manihot esculenta Crantz) Lima beans (Phaseolus lunatus L.) 534-67-8 C11H19NO6 261.3 103.3 Cassava (Manihot esculenta Crantz) Lima beans (Phaseolus lunatus L.) 99-18-3 C14H17NO6 295.3 91.4 Bitter almonds (Prunus amygdalus var. amara Stokes) 499-20-7 C14H17NO7 311.3 86.7 Sorghum (Sorghum bicolor (L.) Moench) 21401-21-8 C14H17NO7 311.3 86.7 Bamboo (Bambusa vulgaris Schrad. and Bambusa edulis Carriere) 72229-40-4 C16H27NO11 409.4 66.0 Linseed (Linum usitatissimum L.) 72229-42-6 C17H29NO11 423.4 63.8 Linseed (Linum usitatissimum L.) 29883-15-6 C20H27NO11 457.4 59.0 Apricot kernels (Prunus armeniaca L.) Almond kernels (Prunus amygdalus var. dulcis Stokes) a Latin names and names on authors according to 'The PlantList – a working list of all plant species' (http://www.theplantlist.org). All relevant synonyms may also be found at this list. Chiral Catoms in the aglycones (i.e. C-atoms carrying four different substituents) are labelled with the stereochemical descriptors R or S according to the Cahn–Ingold–Prelog system. The biosynthesis of CNGs, which is believed to occur in more than 3,000 plant species, follows a general scheme starting with the cytochrome P450-mediated hydroxylation of an aliphatic or aromatic amino acid (e.g. valine, isoleucine, phenylalanine, or tyrosine) to an N-hydroxyl amino acid, which is converted by oxidative decarboxylation to an oxime. Subsequent release of water yields a nitrile. Another hydroxylation then leads to an α-hydroxynitrile, which is finally stabilised by glycosylation. As an example, the biosynthesis of linamarin is depicted in Figure 2. Figure 2Open in figure viewerPowerPoint Biosynthesis of linamarin CYP: cytochrome P450; Glc: glucose; UDP-Glc: uridine diphosphoglucose; UGT: uridine diphosphoglucosyltransferase. Whereas CNGs are chemically quite stable both under acidic and alkaline conditions, the intermediate α-hydroxynitriles (cyanohydrins) are only stable in acidic media but spontaneously dissociate into the respective carbonyl compound and CN at neutral or alkaline pH (Fomunyam et al., 1985). Thus, if the glycosidic bond is hydrolysed, a process known as cyanogenesis is initiated as shown in Figure 3 for linamarin (McMahon et al., 1995). The hydrolysis of linamarin to acetone cyanohydrin and glucose is mediated by the β-glucosidase linamarase (EC 3.2.1.21). The subsequent conversion of acetone cyanohydrin to acetone and HCN proceeds spontaneously, but is much faster in the presence of the enzyme hydroxynitrile lyase (EC 4.1.2.37). Complete hydrolysis of 1 g of linamarin generates 109 mg of HCN (see Table 1). Figure 3Open in figure viewerPowerPoint Formation of HCN from linamarin Glc: glucose; HNL: hydroxynitrile lyase. The process of cyanogenesis is sometimes also called the 'cyanide bomb' (Morant et al., 2008). CNGs and their catabolic enzymes are stored in separate compartments in intact plant cells, but are brought into contact upon tissue disruption, caused, e.g. by chewing or physical processes such as maceration or freezing during food processing (Gleadow and Woodrow, 2002). The strategy of handling CNGs and their catabolic enzymes as a binary system endows plants with an effective defence against generalist herbivores. Because CNGs protect plants for herbivore attacks, they are referred to as 'phytoanticipins'. As an additional role, CNGs are believed to represent a pool of nitrogen to be used by the plant if needed (Gleadow and Møller, 2014). The hydrolysis of CNGs to release cyanide can involve various enzymes. With regard to the genuine glycosidases of the plant tissue, the activity may vary between cultivars (Iglesias et al., 2002). In addition to the plant enzymes mentioned above, β-glucosidases located in the mammalian intestinal epithelium and in colonic bacteria appear to play an important role (see Section 3.1.1 on Toxicokinetics). Hydrocyanic acid is also named hydrogen cyanide, formonitrile, methanenitrile or prussic acid, among others. It has the chemical formula HCN, the molecular mass 27.03 g/mol and the Chemical Abstracts Service (CAS) number 74-90-8. In pure form, it is a colourless liquid with a boiling point of 25.6°C and a melting point of −14°C. Its density is 0.687 g/mL and its vapour pressure is 630 mm Hg at 20°C. It is completely miscible with water or ethanol. HCN is a very weak acid with a pKa of 9.2 and a pKb of 4.8, and aqueous solutions of its alkali salts (cyanides) are therefore quite alkaline. HCN vapours have a characteristic odour like bitter almond oil, but one person out of four does not readily smell HCN (Brown and Robinette, 1967). 1.3.2 Analytical methods This chapter does not provide a full list of potential methods to quantify the concentration of CNGs, cyanohydrins a
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