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Linamarin

Linamarin is a cyanogenic with the molecular formula C₁₀H₁₇NO₆, primarily occurring in the leaves and roots of (Manihot esculenta), as well as in lima beans (Phaseolus lunatus) and (Linum usitatissimum). It serves as a defense compound against herbivores and pathogens by releasing (HCN) upon tissue damage and enzymatic hydrolysis. In , linamarin constitutes over 80% of the total cyanogenic glycosides, with concentrations highest in leaves (up to 5 g/kg fresh weight) and lower in roots (approximately 20-fold less). Chemically, linamarin is the β-D-glucopyranoside of , biosynthesized from the through a pathway involving enzymes and UDP-glucosyltransferases. The compound is hydrolyzed by the enzyme linamarase, present in the same plants, to yield glucose, acetone, and HCN, the latter being acutely toxic to humans and animals at concentrations as low as 0.5–3.5 mg/kg body weight. Improper processing of , a for over 800 million people in tropical regions, can lead to linamarin-related , manifesting as (a paralytic disorder) or chronic neuropathy. Efforts to mitigate toxicity include breeding low-linamarin varieties and traditional processing methods like and drying to degrade the glycoside.

Chemical properties

Molecular structure

Linamarin is a cyanogenic characterized by the molecular formula \ce{C10H17NO6} and a molecular weight of 247.25 g/mol. This compound serves as the primary cyanogen in (Manihot esculenta). Structurally, linamarin is the β-D-glucopyranoside of , systematically named 2-hydroxy-2-methylpropanenitrile β-D-glucopyranoside. The aglycone portion consists of a central carbon atom bonded to two methyl groups, a cyano group (\ce{-C#N}), and an oxygen atom that forms the glycosidic linkage. This oxygen is part of what was originally a hydroxyl group in the . The glucose moiety is a β-D-glucopyranose ring, attached at its anomeric carbon (C1) to the aglycone's oxygen via a β-glycosidic bond, with the standard chair conformation and hydroxyl groups at C2, C3, C4, and C6. In a typical structural representation, the molecule can be depicted as: \ce{(CH3)2C(OC6H11O5)C#N} where \ce{OC6H11O5} denotes the β-D-glucopyranosyloxy group, emphasizing the ether-like connection between the sugar and the cyanohydrin-derived aglycone. Linamarin shares structural similarities with other cyanogenic glucosides, such as lotaustralin, which is the β-D-glucopyranoside of 2-hydroxy-2-methylbutanenitrile (derived from methyl ethyl ketone cyanohydrin). The key difference lies in the aglycone: lotaustralin features an ethyl group replacing one methyl group of linamarin, resulting in the formula \ce{C11H19NO6} and a slightly larger molecular framework, while maintaining the same β-glycosidic linkage to the glucose unit. This homology underscores their common biosynthetic origins from amino acids like valine for linamarin and isoleucine for lotaustralin.

Physical and chemical properties

Linamarin appears as a to off-white crystalline solid and imparts a bitter taste, which is a key sensory characteristic associated with its presence in like . It exhibits high solubility in polar solvents, including (freely soluble), , , and DMSO, but shows low solubility in non-polar solvents such as , , , and . The compound has a of 142–143 °C and is non-volatile at standard conditions. Linamarin displays optical activity, with a [\alpha]_D of -29° at 18 °C (c = 1 in ). It remains stable at neutral but is sensitive to acidic conditions, where it undergoes and , ultimately releasing (HCN).

Natural occurrence

In plants

Linamarin is a cyanogenic primarily found in various plant species, serving as a key component in their arsenal. It constitutes up to 80% of the cyanogenic glycosides in (Manihot esculenta), a in tropical regions, where it accumulates in both roots and leaves. Other major sources include lima beans (Phaseolus lunatus) and (Linum usitatissimum), with linamarin present in their seeds and vegetative tissues. Concentrations of linamarin vary significantly across plant tissues and developmental stages, often expressed as hydrogen cyanide (HCN) potential. In , levels are highest in young leaves (up to 5 g/kg fresh weight) and peels, while mature roots typically exhibit lower concentrations, around 10 to 50 mg/kg in sweet varieties and up to 400 mg/kg in bitter ones. leaves contain significantly higher linamarin than roots (up to 20-fold more), with overall HCN levels in foliage reaching 200 to 5,000 mg/kg fresh weight. Linamarin also occurs in other economically important plants, such as (various species) where taxiphyllin predominates. In lima beans, seed concentrations equate to 144 to 167 mg HCN/kg, while flax seeds have a total cyanogenic potential averaging 192 mg HCN/kg, with linamarin as a minor component. can reach up to 1,000 mg HCN/kg in fresh material, highlighting their prevalence in edible wild plants. Evolutionarily, linamarin functions as a defense compound in , deterring herbivores and pathogens by releasing toxic upon tissue damage. This role is evident across families like () and (lima beans), where its structure allows safe storage in vacuoles until is triggered. Agriculturally, linamarin levels in exhibit substantial varietal differences, influencing and practices. Sweet cultivars, with low cyanogenic potential (under 50 mg HCN/kg), are preferred for direct consumption, whereas bitter varieties (15 to 400 mg/kg or higher) require but offer to pests. These differences guide breeding programs to balance yield, toxicity, and resilience in tropical farming systems.

In other organisms

Linamarin, a cyanogenic primarily associated with defense, has also been identified in certain , where it serves similar protective functions against predators. In various species of , including and moths, linamarin is present either through or from host plants. For instance, larvae of burnet moths (Zygaena filipendulae) in the family both synthesize linamarin endogenously using enzymes such as P450s (CYP405A2 and CYP332A3) and UDP-glucosyltransferase (UGT33A1), and sequester it intact from cyanogenic host plants in the family, storing it in and tissues for release as (HCN) upon predation. In butterfly species within the superfamily , such as those in the Heliconiinae subfamily (e.g., ), linamarin and its homolog lotaustralin are biosynthesized from and , respectively, across all life stages, independent of dietary sources. These compounds accumulate in wing tissues and , contributing to by deterring , , and through HCN emission when tissues are damaged; additionally, adult males transfer significant quantities (up to 30% of body mass) of linamarin as nuptial gifts during mating, signaling mate quality. occurs in some Papilionidae larvae feeding on cyanogenic plants, where linamarin is absorbed intact from the diet and translocated to defensive structures, enhancing survival without endogenous production. Although millipedes in orders like exhibit cyanogenesis via defensive secretions containing HCN precursors, linamarin specifically has not been confirmed in these organisms; instead, they typically produce aromatic cyanogenic compounds such as mandelonitrile glucosides. In vertebrates and microbes, linamarin is not endogenously produced but can be transiently present in trace amounts in animals like through dietary intake of cyanogenic (e.g., ), where it is rapidly hydrolyzed by to HCN, preventing significant tissue accumulation. Endophytic bacteria in cassava roots, such as species, interact with linamarin primarily through enzymatic degradation via linamarase, aiding in cyanide detoxification rather than harboring the compound. Rare reports of cyanogenic glucosides in exist, but linamarin has not been quantified in species like Spirulina platensis.

Biosynthesis

Biosynthetic pathway

The biosynthetic pathway of linamarin in cyanogenic plants initiates with the amino acid L- as the primary precursor. This pathway follows a linear sequence characteristic of cyanogenic formation, involving sequential oxidation, dehydration, and steps to derive the β-glucosylated α-hydroxynitrile structure. In model systems like (Manihot esculenta), valine serves as the starting substrate for linamarin production, distinguishing it from the parallel pathway yielding lotaustralin from . The initial transformation converts to the intermediate 2-methylpropanal through N- followed by . This then undergoes further modification to , involving an , additional , and C-, primarily facilitated by P450-mediated oxidation and processes. The final step entails the addition of a glucose moiety to the acetone via UDP-glucose-dependent glucosylation, yielding linamarin as the end product. These steps ensure the incorporation of from the precursor into the cyanohydrin aglycone, with the overall pathway compartmentalized within cells to mitigate premature . Linamarin accumulates in the vacuoles of cells, where and occur separately from catabolic enzymes, preventing accidental and release during normal growth. This vacuolar sequestration is a key for safe containment in tissues of producing such as .

Key enzymes involved

The of linamarin in (Manihot esculenta) involves several key enzymes that catalyze specific steps in the conversion of to the cyanogenic . The monooxygenases CYP79D1 and CYP79D2 initiate the pathway by performing the N-hydroxylation of , forming the corresponding as the first committed step. These isoenzymes exhibit similar catalytic properties, with CYP79D1 and CYP79D2 showing overlapping substrate specificity for and , leading to linamarin and lotaustralin production, respectively. Subsequent steps are mediated by the enzyme , which converts the intermediate through dehydration and C-hydroxylation to yield , the aglycone of linamarin. This enzyme, present as paralogs in the genome, colocalizes transcriptionally with CYP79D1 and CYP79D2, supporting coordinated expression during . The final is catalyzed by UDP-glucosyltransferases of the UGT85K family, such as UGT85K4 and UGT85K5, which add the β-D-glucose moiety to , forming linamarin. These enzymes co-express with the upstream P450s in relevant plant tissues, ensuring efficient pathway flux. The genes encoding these enzymes are clustered in the cassava genome on , forming a gene-dense region that includes CYP79D2, two CYP71E paralogs, and two UGT85K genes, which facilitates their coordinated regulation and evolutionary conservation. This genomic organization aids in identifying and manipulating the pathway for trait improvement. Functional studies using (RNAi) to silence CYP79D1 and CYP79D2 have demonstrated significant reductions in linamarin levels, with up to 94% decrease in leaves and 99% in when targeted via leaf-specific promoters, confirming their rate-limiting roles without major impacts on . Similar knockdown approaches highlight the pathway's plasticity, as root-specific silencing has minimal effects on tuber linamarin, indicating primary in leaves followed by .

Metabolism

Enzymatic hydrolysis

The enzymatic of linamarin is catalyzed primarily by linamarase, a classified under 3.2.1.21, which specifically cleaves the β-glycosidic bond between the glucose moiety and the aglycone in the cyanogenic glycoside. This reaction proceeds as follows: \text{Linamarin} + \text{H}_2\text{O} \rightarrow \text{glucose} + \text{acetone cyanohydrin} Linamarase exhibits optimal activity at an acidic pH range of 5.5–6.0 and is predominantly localized in the cell walls of tissues, such as leaves and roots. In intact plants, linamarin and linamarase are compartmentalized separately—linamarin is stored in the vacuoles of mesophyll and epidermal cells, while linamarase resides in the apoplastic space (cell walls)—preventing spontaneous and maintaining plant until tissue disruption occurs. Cassava varieties differ in linamarase expression levels, with high-linamarase cultivars, often associated with lower overall potential, facilitating accelerated enzymatic breakdown of linamarin during post-harvest processing steps like or , thereby enhancing efficiency.

Cyanide release

The release in linamarin occurs following the enzymatic of linamarin to its aglycone, , as the final step leading to (HCN) production. serves as the key intermediate substrate, which decomposes to yield acetone and HCN. This process can proceed through both non-enzymatic and enzymatic pathways, with the choice depending on environmental conditions and organism-specific factors. Non-enzymatic breakdown involves the spontaneous dissociation of in s, particularly under neutral or alkaline conditions. At values greater than 5, the undergoes , accelerating with increasing due to the instability of the C-CN bond in basic environments. For instance, in a 0.1% at 6.8, the is approximately 8 minutes, shortening further at higher levels such as 7.4, where decomposition occurs within minutes. This spontaneous release of HCN is temperature-dependent as well, with rates increasing above 35°C, making it relevant in both plant tissues and during . In certain species, such as (Manihot esculenta), enzymatic facilitation by α-hydroxynitrile lyase (HNL) significantly accelerates release from . HNL catalyzes the cleavage of the into acetone and HCN, exhibiting optimal activity at acidic around 5.0, with a Michaelis constant (Km) of 0.93 mM and maximum velocity (Vmax) of 11.5 μmol HCN per mg protein per hour. This is particularly active in cassava leaves but notably absent in roots, leading to reliance on spontaneous in root s and potential accumulation of the toxic intermediate if processing is inadequate. The dependence of HNL contrasts with spontaneous breakdown, allowing compartmentalized cyanogenesis in plant cells where vacuolar favors linamarin storage and cytoplasmic conditions promote rapid HCN release upon tissue disruption.

Toxicity

Mechanism of action

Linamarin, a cyanogenic found in certain plants, is intrinsically non-toxic but becomes hazardous through enzymatic that releases (HCN) upon plant tissue disruption, such as during chewing or mechanical damage, serving as a against herbivores. The primary toxic effect stems from HCN, which diffuses rapidly across cellular membranes and binds to the ferric iron in , the terminal enzyme of the mitochondrial . This binding inhibits the enzyme's function, halting electron transfer to oxygen and thereby blocking , which prevents ATP production and disrupts . The inhibition of aerobic metabolism forces cells to rely on , leading to accumulation of and resulting in as a secondary effect. In humans, the dose-response for HCN indicates a ranging from approximately 0.5 to 3.5 mg/kg body weight, depending on exposure conditions and individual factors. arises from rapid HCN release during plant tissue damage, causing immediate and severe cellular , while exposure occurs through low-level dietary intake of linamarin-containing foods, leading to gradual accumulation of and its metabolites.

Health effects in humans and animals

Linamarin, a found in , poses significant health risks upon to release , particularly in regions where is a dietary staple. In humans, chronic exposure through insufficiently processed bitter has been linked to , an irreversible disorder characterized by spastic paraparesis, primarily affecting children and women in . Outbreaks of have been reported since the 1930s, with notable epidemics in the 1980s and 1990s in countries such as the of , , and , and more recently in (as of 2024), often correlating with periods of , food scarcity, and increased reliance on cyanogen-rich . continues to occur, with recent outbreaks reported in in 2020 and suspected cases in 2024, highlighting the persistent risk in protein-deficient diets reliant on . Additionally, prolonged low-level exposure from consumption contributes to tropical ataxic neuropathy (TAN), a neurodegenerative condition involving , , and bilateral deafness, observed in populations in and other tropical areas. This inhibits mitochondrial respiration, leading to damage over time. In animals, linamarin toxicity manifests as goiter in , such as and sheep, due to —a metabolite—competing with iodine for uptake in the gland, thereby disrupting thyroid hormone synthesis. This effect is exacerbated in iodine-deficient environments where byproducts are fed to animals. Acute from linamarin has also been documented in herbivores like ruminants and in consuming cyanogenic plants, resulting in rapid onset of respiratory distress, convulsions, and death from cyanide's interference with cellular oxygen utilization. Certain populations are particularly vulnerable to linamarin's effects, including those with protein deficiencies, as sulfur-containing amino acids like and are essential for via the rhodanese pathway, converting to less toxic . In protein-poor diets common in rural during famines or conflicts, this impaired heightens the risk of chronic accumulation and associated neurological disorders. Mitigation strategies significantly reduce linamarin content in cassava; for instance, traditional of grated roots can eliminate up to 95% of linamarin, while fresh chips for 25 minutes removes approximately 55%, though combining methods like soaking followed by achieves 90–97% reduction overall. These processing techniques are crucial in preventing in both human and animal consumption.

Detection and analysis

Analytical methods

Linamarin, a cyanogenic , is analyzed using a variety of techniques that leverage its for identification and quantification in plant-derived samples. These methods include chromatographic separation, enzymatic , and spectroscopic , often employing authentic standards for accuracy. Chromatographic methods, particularly (HPLC) coupled with (UV) detection or (), are widely used for the separation and quantification of linamarin due to their high resolution and specificity. In HPLC-UV approaches, linamarin is typically extracted from samples and separated on reversed-phase columns using gradients of water and with , allowing detection at wavelengths around 210 nm based on its chromophoric groups. For enhanced sensitivity and confirmation, HPLC- or LC-/ variants employ to generate characteristic ions such as m/z 270 [M+Na]⁺, enabling precise identification amid complex matrices. These techniques are favored for their ability to distinguish linamarin from structurally similar cyanogenic glycosides like lotaustralin. Enzymatic assays provide an indirect yet reliable means of detection by exploiting linamarin's structure, which facilitates -based measurement. In these procedures, samples are incubated with linamarase () to release (HCN), followed by colorimetric quantification of the cyanide using reagents like and pyridine-barbituric acid, which produce a detectable at 578 nm. This method is particularly useful for total cyanogenic potential assessments and offers simplicity for routine screening, though it requires careful control of conditions to avoid spontaneous . Spectroscopic techniques are employed for structural confirmation of linamarin, providing orthogonal validation to chromatographic data. (NMR) , including 1H and 13C NMR, reveals key signals such as the anomeric proton at δ 4.8-5.0 ppm for the glucosyl moiety and methyl groups at δ 1.5 ppm from the unit, confirming the β-D-glucopyranosyloxy . (IR) complements this by identifying functional groups, with characteristic absorptions at 3400 cm⁻¹ (O-H stretch), 2250 cm⁻¹ (C≡N stretch), and 1050 cm⁻¹ (C-O stretch), aiding in purity assessment during isolation. These methods are essential for characterizing purified linamarin but are less suited for trace-level quantification in crude samples. Calibration in these assays relies on authentic linamarin standards (CAS 554-35-8), commercially available at high purity (>98%) from suppliers, which are used to generate standard curves ensuring linearity and accuracy across concentration ranges. Sensitivity varies by method, with HPLC-MS achieving detection limits as low as 0.001-0.025 mg/kg, enabling reliable trace analysis.

Quantification in biological samples

Sample preparation for linamarin quantification in biological samples typically involves homogenizing or animal tissues in aqueous or at 80% concentration to extract the intact while minimizing enzymatic . To avoid , tissues are often processed rapidly or heated to inactivate β-glucosidase enzymes, with solvents like or used alongside techniques such as or shaking for efficient recovery. Buffers may be employed for pH control during extraction from complex matrices like , followed by (SPE) cleanup to remove interferences. In food analysis, particularly for , total cyanogens including linamarin are quantified via the alkaline method, which measures released (HCN) equivalents after . samples are homogenized in , hydrolyzed, and reacted with alkaline to form a colored complex measured spectrophotometrically at 488 nm, expressing results in mg HCN/kg. This method is widely used for screening processed products, with limits of detection around 0.05 µg/mL HCN. For plant tissues, linamarin is determined by acid to release , followed by spectrophotometric detection. Tissues are extracted, treated with 2.0 M at 100°C for 50 minutes to convert linamarin to cyanohydrins and then to under alkaline conditions, and quantified via the König reaction using and pyridine-barbituric acid at 583 nm. Recoveries range from 70–95% for added linamarin, and this approach has measured levels as low as 4 mg HCN/kg in low-cyanogen cultivars. In biological fluids, gas chromatography-mass spectrometry (GC-MS) is applied to detect metabolites following linamarin exposure. Headspace GC-MS analyzes free in blood under acidic volatilization, while (a primary ) is measured in as a , with elevated levels (e.g., 1720 µmol/L) indicating chronic exposure from cyanogenic glycosides. This method addresses the instability of free in blood, requiring prompt analysis post-exposure. The (WHO), through the Joint FAO/WHO Expert Committee on Food Additives (JECFA), sets a maximum limit of 10 mg HCN/kg for processed flour to ensure safety from . This guideline applies to products and reflects evaluations confirming no acute risks at this level. (HPLC) serves as a core confirmatory method for linamarin in these matrices after extraction.