hypoglycin-a and Plant-Poisoning

Equine atypical myopathy in Europe results from hypoglycin A (HGA) exposure through the ingestion of samaras or seedlings of the sycamore maple tree. This pilot study aimed at better defining sources of HGA intoxication in spring. Samaras fallen on the ground and then seedlings were collected at two-week intervals from sycamore, Norway, and field maple trees over the spring 2016. In early April, rainwater from wet seedlings collected after a rainy night was harvested to be analysed. Mid-May, samaras of the box elder, common ash, and inflorescences of sycamore maples were collected on the tree. Quantification of HGA in samples was performed using high performance thin layer chromatography. Hypoglycin A was detected in all samples from sycamore including rainwater but tested negative for Norway, field maples. The samaras of the box elder found in the present study area did not contain a seed within their husk and thus tested negative. From the maximum HGA concentrations found, it may be extrapolated that at some periods and locations, about 20 g of samaras, 50 seedlings, 150 g of inforescences or 2 liters of water that has been in contact with seedlings would contain the maximum tolerated dose per day for a horse.

Topics: Animal Feed; Animals; Europe; Horse Diseases; Horses; Hypoglycins; Pilot Projects; Plant Poisoning; Seasons

Intestinal absorption of hypoglycin A (HGA) and its metabolism are considered major prerequisites for atypical myopathy (AM). The increasing incidence and the high mortality rate of AM urgently necessitate new therapeutic and/or preventative approaches.. To identify a substance for oral administration capable of binding HGA in the intestinal lumen and effectively reducing the intestinal absorption of the toxin.. Experimental in vitro study.. Substances commonly used in equine practice (activated charcoal composition, di-tri-octahedral smectite, mineral oil and activated charcoal) were tested for their binding capacity for HGA using an in vitro incubation method. The substance most effective in binding HGA was subsequently tested for its potential to reduce intestinal HGA absorption. Jejunal tissues of 6 horses were incubated in Ussing chambers to determine mucosal uptake, tissue accumulation, and serosal release of HGA in the presence and absence of the target substance. Potential intestinal metabolism in methylenecyclopropyl acetic acid (MCPA)-conjugates was investigated by analysing their concentrations in samples from the Ussing chambers.. Activated charcoal composition and activated charcoal were identified as potent HGA binding substances with dose and pH dependent binding capacity. There was no evidence of intestinal HGA metabolism.. Binding capacity of adsorbents was tested in vitro using aqueous solutions, and in vivo factors such as transit time and composition of intestinal content, may affect adsorption capacity after oral administration.. For the first time, this study identifies substances capable of reducing HGA intestinal absorption. This might have major implications as a preventive measure in cograzers of AM affected horses but also in horses at an early stage of intoxication.

Topics: Acer; Adsorption; Animals; Antidotes; Charcoal; Drug Combinations; Horse Diseases; Horses; Hypoglycins; Kaolin; Plant Bark; Plant Poisoning; Quercus; Rhabdomyolysis; Seeds; Silicates; Silicon Dioxide

Hypoglycin A (HGA) toxicity, following ingestion of material from certain plants, is linked to an acquired multiple acyl-CoA dehydrogenase deficiency known as atypical myopathy, a commonly fatal form of equine rhabdomyolysis seen worldwide. Whilst some plants are known to contain this toxin, little is known about its function or the mechanisms that lead to varied HGA concentrations between plants. Consequently, reliable tools to detect this amino acid in plant samples are needed. Analytical methods for HGA detection have previously been validated for the food industry, however, these techniques rely on chemical derivatisation to obtain accurate results at low HGA concentrations. In this work, we describe and validate a novel method, without need for chemical derivatisation (accuracy = 84-94%; precision = 3-16%; reproducibility = 3-6%; mean linear range R2 = 0.999). The current limit of quantitation for HGA in plant material was halved (from 1μg/g in previous studies) to 0.5μg/g. The method was tested in Acer pseudoplatanus material and other tree and plant species. We confirm that A. pseudoplatanus is most likely the only source of HGA in trees found within European pastures.

Topics: Animals; Chromatography, Liquid; Horse Diseases; Horses; Hypoglycins; Muscular Diseases; Phytochemicals; Plant Poisoning; Reproducibility of Results; Sensitivity and Specificity; Tandem Mass Spectrometry

Hypoglycin A (HGA) and methylenecyclopropylglycine (MCPG) are naturally-occurring amino acids known to cause hypoglycemia and encephalopathy. Exposure to one or both toxins through the ingestion of common soapberry (Sapindaceae) fruits are documented in illness outbreaks throughout the world. Jamaican Vomiting Sickness (JVS) and seasonal pasture myopathy (SPM, horses) are linked to HGA exposure from unripe ackee fruit and box elder seeds, respectively. Acute toxic encephalopathy is linked to HGA and MCPG exposures from litchi fruit. HGA and MCPG are found in several fruits within the soapberry family and are known to cause severe hypoglycemia, seizures, and death. HGA has been directly quantified in horse blood in SPM cases and in human gastric juice in JVS cases. This work presents a new diagnostic assay capable of simultaneous quantification of HGA and MCPG in human plasma, and it can be used to detect patients with toxicity from soapberry fruits. The assay presented herein is the first quantitative method for MCPG in blood matrices.

Topics: Chromatography, High Pressure Liquid; Cyclopropanes; Glycine; Humans; Hypoglycins; Limit of Detection; Linear Models; Plant Poisoning; Reproducibility of Results; Sapindaceae; Tandem Mass Spectrometry

Atypical myopathy (AM) in horses is caused by the plant toxin hypoglycin A, which in Europe typically is found in the sycamore maple tree (Acer pseudoplatanus). Owners are concerned about whether their horses are in danger if they graze near maple trees.. To measure hypoglycin A in the most common maple tree species in the Netherlands, and to determine whether concentration of toxin is a predictor of AM in horses.. A total of 278 samples of maple tree leaves, sprouts, and seeds were classified by species. Mean concentrations of hypoglycin A were compared for the type of sample, the season and the occurrence of AM in the pasture (non-AM versus AM). Statistical analysis was performed using generalized a linear model (SPPS22).. Almost all Acer pseudoplatanus samples contained hypoglycin A, with concentrations differing significantly among sources (P < .001). Concentrations were significantly higher in seeds from the AM group than in seeds from the non-AM group (856 ± 677 and 456 ± 358 mg/kg, respectively; P = .039). In sprouts and leaves this was not the case. Acer platanoides and Acer campestre samples did not contain detectable concentrations of hypoglycin A.. Acer platanoides and campestre seem to be safe around paddocks and pastures, whereas almost all Acer pseudoplatanus samples contained hypoglycin A. In all AM cases, Acer pseudoplatanus was found. Despite significantly higher concentration of hypoglycin A in seeds of pastures where AM has occurred, individual prediction of AM cannot be made by measuring these concentrations because of the high standard deviation.

Topics: Acer; Animals; Horse Diseases; Horses; Hypoglycins; Muscular Diseases; Netherlands; Plant Leaves; Plant Poisoning; Seeds

L-α-amino-methylenecyclopropyl propionic acid (Hypoglycin A, HGA) has been found to be the toxic compound in fruits of the Sapindaceae family causing acute intoxication when ingested as food or feed. Clinical symptoms are consistent with acquired multiple acyl-CoA dehydrogenase deficiency (MADD). Ultra performance liquid chromatography-tandem mass spectrometry was used to measure HGA after butylation. Sample volumes were 10μL for serum and 20μL for urine. Internal standard for HGA was d3-leucine, samples were plotted on a 7-point linear calibration curve. Coefficients of variation were <15% at 0.01μmol HGA/L and ≤4.1% at 10μmol/L. R(2) values for linearity were ≥0.995. In order to quantify non-metabolized HGA together with some of its metabolites plus a spectrum of acyl glycines and acyl carnitines typical for acquired MADD in one single analysis HGA measurement was integrated into a method which we previously developed for metabolites of HGA and acyl conjugates. The new method is suitable for biochemical diagnosis of Ackee fruit poisoning or atypical myopathy in horses and for forensic purposes in cases of suspected HGA poisoning.

Topics: Animal Feed; Animals; Chromatography, High Pressure Liquid; Esterification; Horse Diseases; Horses; Hypoglycins; Multiple Acyl Coenzyme A Dehydrogenase Deficiency; Muscular Diseases; Plant Poisoning; Sapindaceae; Tandem Mass Spectrometry

Hypoglycin A (HGA) is the toxic principle in ackee (Blighia sapida Koenig), a nutritious and readily available fruit which is a staple of the Jamaican working-class and rural population. The aril of the unripe fruit has high concentrations of HGA, the cause of Jamaican vomiting sickness, which is very often fatal. HGA is also present in the samara of several species of maple (Acer spp.) which are suspected to cause seasonal pasture myopathy in North America and equine atypical myopathy in Europe, often fatal for horses. The aim of this study was to develop a method for quantifying HGA in blood that would be sensitive enough to provide toxicological evidence of ackee or maple poisoning. Analysis was carried out using solid-phase extraction (HILIC cartridges), dansyl derivatization and UHPLC-HRMS/MS detection. The method was validated in whole blood with a detection limit of 0.35 μg/L (range: 0.8-500 μg/L). This is the first method applicable in forensic toxicology for quantifying HGA in whole blood. HGA was quantified in two serum samples from horses suffering from atypical myopathy. The concentrations were 446.9 and 87.8 μg/L. HGA was also quantified in dried arils of unripe ackee fruit (Suriname) and seeds of sycamore maple (Acer pseudoplatanus L.) (France). The concentrations were 7.2 and 0.74 mg/g respectively.

Topics: Animals; Chromatography, High Pressure Liquid; Horses; Hypoglycins; Limit of Detection; Linear Models; Plant Poisoning; Reproducibility of Results; Tandem Mass Spectrometry

Hypoglycin A (HGA) in seeds of Acer spp. is suspected to cause seasonal pasture myopathy in North America and equine atypical myopathy (AM) in Europe, fatal diseases in horses on pasture. In previous studies, this suspicion was substantiated by the correlation of seed HGA content with the concentrations of toxic metabolites in urine and serum (MCPA-conjugates) of affected horses. However, seed sampling was conducted after rather than during an outbreak of the disease. The aim of this study was to further confirm the causality between HGA occurrence and disease outbreak by seed sampling during an outbreak and the determination of i) HGA in seeds and of ii) HGA and MCPA-conjugates in urine and serum of diseased horses. Furthermore, cograzing healthy horses, which were present on AM affected pastures, were also investigated. AM-pastures in Germany were visited to identify seeds of Acer pseudoplatanus and serum (n = 8) as well as urine (n = 6) from a total of 16 diseased horses were analyzed for amino acid composition by LC-ESI-MS/MS, with a special focus on the content of HGA. Additionally, the content of its toxic metabolite was measured in its conjugated form in body fluids (UPLC-MS/MS). The seeds contained 1.7-319.8 μg HGA/g seed. The content of HGA in serum of affected horses ranged from 387.8-8493.8 μg/L (controls < 10 μg/L), and in urine from 143.8-926.4 μg/L (controls < 10 μg/L), respectively. Healthy cograzing horses on AM-pastures showed higher serum (108.8 ± 83.76 μg/L) and urine concentrations (26.9 ± 7.39 μg/L) compared to control horses, but lower concentrations compared to diseased horses. The range of MCPA-carnitine and creatinine concentrations found in diseased horses in serum and urine were 0.17-0.65 mmol/L (controls < 0.01), and 0.34-2.05 μmol/mmoL (controls < 0.001), respectively. MCPA-glycine levels in urine of cograzing horses were higher compared to controls. Thus, the causal link between HGA intoxication and disease outbreak could be further substantiated, and the early detection of HGA in cograzing horses, which are clinically normal, might be a promising step in prophylaxis.

Topics: Acer; Animals; Carnitine; Disease Outbreaks; Horse Diseases; Horses; Hypoglycins; Muscular Diseases; Plant Poisoning; Plants, Toxic; Seeds; Tandem Mass Spectrometry

It is hypothesised that European atypical myopathy (AM) has a similar basis as seasonal pasture myopathy in North America, which is now known to be caused by ingestion of hypoglycin A contained in seeds from the tree Acer negundo. Serum from horses with seasonal pasture myopathy contained the conjugated toxic metabolite of hypoglycin A, methylenecyclopropyl acetic acid (MCPA).. Retrospective study on archived samples.. 1) To determine whether MCPA-carnitine was present in serum of European horses confirmed to have AM; 2) to determine whether Acer negundo or related Acer species were present on AM pastures in Europe.. Concentrations of MCPA-carnitine were analysed in banked serum samples of 17 AM horses from Europe and 3 diseased controls (tetanus, neoplasia and exertional rhabdomyolysis) using tandem mass spectrometry. Atypical myopathy was diagnosed by characteristic serum acylcarnitine profiles. Pastures of 12 AM farms were visited by experienced botanists and plant species were documented.. Methylenecyclopropyl acetic acid-carnitine at high concentrations (20.39 ± 17.24 nmol/l; range 0.95-57.63 nmol/l; reference: <0.01 nmol/l) was identified in serum of AM but not disease controls (0.00 ± 0.00 nmol/l). Acer pseudoplatanus but not Acer negundo was present on all AM farms.. Atypical myopathy in Europe, like seasonal pasture myopathy in North America, is highly associated with the toxic metabolite of hypoglycin A, MCPA-carnitine. This finding coupled with the presence of a tree of which seeds are known to also contain hypoglycin A indicates that ingestion of Acer pseudoplatanus is the probable cause of AM. This finding has major implications for the prevention of AM.

Topics: Acer; Animals; Cyclopropanes; Europe; Female; Horse Diseases; Horses; Hypoglycins; Male; Muscular Diseases; Plant Poisoning; Plants, Toxic; Retrospective Studies; Seasons

We hypothesised that seasonal pasture myopathy (SPM), which closely resembles atypical myopathy (AM), was caused by ingestion of a seed-bearing plant abundant in autumn pastures.. To identify a common seed-bearing plant among autumn pastures of horses with SPM, and to determine whether the toxic amino acid hypoglycin A was present in the seeds and whether hypoglycin metabolites were present in SPM horse serum or urine.. Twelve SPM cases, 11 SPM pastures and 23 control farms were visited to identify a plant common to all SPM farms in autumn. A common seed was analysed for amino acid composition (n = 7/7) by GC-MS and its toxic metabolite (n = 4/4) identified in conjugated form in serum [tandem mass spectrometry (MS/MS)] and urine [gas chromatography (GC) MS]. Serum acylcarnitines and urine organic acid profiles (n = 7) were determined for SPM horses.. Seeds from box elder trees (Acer negundo) were present on all SPM and 61% of control pastures. Hypoglycin A, known to cause acquired multiple acyl-CoA dehydrogenase deficiency (MADD), was found in box elder seeds. Serum acylcarnitines and urine organic acid profiles in SPM horses were typical for MADD. The hypoglycin A metabolite methylenecyclopropylacetic acid (MCPA), known to be toxic in other species, was found in conjugated form in SPM horse serum and urine. Horses with SPM had longer turn-out, more overgrazed pastures, and less supplemental feeding than control horses.. For the first time, SPM has been linked to a toxin in seeds abundant on autumn pastures whose identified metabolite, MCPA, is known to cause acquired MADD, the pathological mechanism behind SPM and AM. Further research is required to determine the lethal dose of hypoglycin A in horses, as well as factors that affect annual seed burden and hypoglycin A content in Acer species in North America and Europe.

Topics: Acer; Animals; Case-Control Studies; Cyclopropanes; Data Collection; Female; Hypoglycins; Iowa; Male; Minnesota; Muscular Diseases; Plant Poisoning; Seasons; Seeds; Surveys and Questionnaires; Wisconsin

1. The lethal, hypoglycaemic and hypothermic effects of hypoglycin in fasted rats are prevented if the rats had been fed on a diet containing clofibrate (0.5% w/w). 2. Injection of hypoglycin into fasted rats maintained on a standard diet caused severe prostration, hypothermia and a massive dicarboxylic aciduria [Tanaka (1972) J. Biol. Chem. 247, 7465-7478]. 3. Rats maintained on a diet containing clofibrate appeared normal after injection of hypoglycin, but had a marked dicarboxylic aciduria which was less than that induced in rats on a normal diet. 4. After administration of hypoglycin, butyryl-CoA and decanoyl-CoA, but not palmitoyl-CoA, dehydrogenase activities were strongly inhibited (80-95%) in the livers of animals on a standard diet. 5. Clofibrate feeding decreased the inhibition of these dehydrogenases to about 40-60%. 6. It was concluded that although clofibrate protects against the toxic effects of hypoglycin, some enzyme inhibitions as indicated by dicarboxylic aciduria are only partly prevented.

Topics: Animals; Chromatography, Gas; Clofibrate; Cyclopropanes; Dicarboxylic Acids; Fatty Acid Desaturases; Hypoglycins; Male; Mitochondria, Liver; Plant Poisoning; Rats; Rats, Inbred Strains