hypoglycin-a and Muscular-Diseases

Topics: Acyl-CoA Dehydrogenases; Animals; Carnitine; Creatine Kinase; Cyclopropanes; Fatty Acids; Glycine; Horse Diseases; Horses; Hypoglycins; Male; Muscular Diseases

Hypoglycin A (HGA) and methylenecyclopropylglycine (MCPrG) from seeds/seedlings of Sycamore maple (SM, Acer pseudoplatanus) causes atypical myopathy (AM) in horses. AM was not known to occur in wild ruminants until several fatalities in milus (Elaphurus davidianus) following the ingestion of HGA in SM seeds. However, a role for MCPrG has not previously been evaluated.. To test the hypothesis that MCPrG is also a major factor in AM in milus, three milus (M1, M2, M3) from the Zoo Dresden (aged 7-11 years, 2 females and 1 male, in good nutritional condition) that developed AM were studied.. Serum, urine and methanol extracts from the liver, kidney, rumen digesta and faeces were analysed by ultrahigh-performance liquid chromatography-tandem mass spectrometry for HGA, MCPrG and for conjugates of carnitine (C) and glycine (G): Methylenecyclopropylacetyl (MCPA)-G, MCPA-C, Methylenecyclopropylformyl (MCPF)-G, MCPF-C, butyryl-C and isobutyryl-C.. HGA in serum was high (M2 480 nmol/L; M3 460 nmol/L), but MCPrG was not. HGA and MCPrG were found in rumen and faeces extracts, and MCPrG was also identified in the liver. Metabolites of HGA and MCPrG were high in serum, urine and liver, but not in the rumen or faeces.. This study shows that MCPrG is involved in the pathophysiology of AM in milus. The metabolism of MCPrG is considered to be faster because, after ingestion, the specific metabolites appear highly concentrated in the serum. The high toxin concentration in the liver suggests that a possible transfer into products for human consumption may pose a risk.

Topics: Acer; Animals; Cyclopropanes; Deer; Female; Gastrointestinal Contents; Glycine; Hypoglycins; Male; Muscular Diseases; Rumen; Serum; Urine

Atypical myopathy (AM) is a fatal disease in horses presumably caused by hypoglycine A (HGA) from ingested maple seeds and its active metabolite methylene cyclopropyl acetic acid (MCPA). The aim of this study was the development and validation of a rapid and simple assay for HGA and MCPA-carnitine in horse serum and its application to authentic samples. Identification and quantification were carried out by ultra high performance liquid chromatography-high resolution tandem mass spectrometry (UHPLC-HRMS/MS) with full-scan/data-dependent MS/MS. Chromatographic separation was performed by isocratic elution on a hydrophilic interaction liquid chromatography (HILIC) column (100 x 2.1 mm, 1.7 μm). Serum samples (250 μL) were worked up by protein precipitation. The method was validated according to international guidelines with respect to selectivity, linearity, accuracy, precision, matrix effects, and recovery. The calibration range was from 100 to 2000 ng/mL for HGA and from 10 to 1000 ng/mL for MCPA-carnitine. HGA and MCPA-carnitine showed acceptable accuracy and precision (bias -3.0% to 1.1%; RSD 9.2% to 12.4%). The limit of quantification (LOQ) was defined as the lowest calibrator and well below the lowest published serum concentrations in affected horses. Matrix effects ranged from -79% to +20% (RSD 4.2% to 14.4%), recoveries from 17.9% to 21.1% (RSD 2.3% to 10.8 %) for low and high quality control samples, respectively. Applicability was tested in 10 authentic AM cases. In all specimens, relevant amounts of HGA and MCPA-carnitine were found (570-2000 ng/mL; ~8.5-150 ng/mL, respectively). The developed assay allows reliable identification and quantification of HGA and MCPA-carnitine in horse serum and will be helpful to further study the association between HGA/MCPA and AM.

Topics: Animals; Carnitine; Chromatography, High Pressure Liquid; Cyclopropanes; Horse Diseases; Horses; Hypoglycins; Limit of Detection; Muscular Diseases; Tandem Mass Spectrometry

From 2004 until 2016, 21 Père David's deer (Elaphurus davidianus) have died for unknown reason at Zoo Duisburg. These deer, also known as milu, have succumbed from a myopathy that occurred seasonally in autumn and in spring. The clinical signs shown by the animals closely resembles those of a disease called equine atypical myopathy (EAM), which is formerly known in horses. The cause for EAM in Europe was found in the ingestion of hypoglycin A, contained in samaras and seedlings of the sycamore maple tree (Acer pseudoplatanus). To test the hypothesis that the mortality of milus was caused by ingestion of hypoglycin A, 79 sera from all zoos and wildlife parks that have kept milus in Germany and Austria, including 19 diseased and 60 healthy animals, were used. Selected biochemical values and additionally hypoglycin A, methylenecyclopropyl acetic acid-carnitine (MCPA-carnitine), and acylcarnitines, which have been found in horses suffering from EAM, were determined. The results showed greater values of serum activities of creatine kinase (P < 0.001) and aspartate aminotransferase (P < 0.001) in diseased milus comparing to healthy ones confirming a myopathy in affected animals. Moreover, hypoglycin A and MCPA-carnitine were found in the blood of Père David's deer and thus, hypoglycin A intoxication was considered to be a potential cause for the myopathies by ingestion of sycamore maple samaras that were present in the enclosure of the affected animals. Hypoglycin A values were greater in diseased animals (P < 0.01) as well as MCPA-carnitine levels (P < 0.05). Additionally, affected milus showed greater C5-OH-carnitine (P < 0.01) and C6-carnitine (P < 0.001) values. Until now hypoglycin A intoxication was only known in the family of Equidae, in humans, and in laboratory rats, and it has not been previously described in other zoological families. Comparing to horses, ruminants do have a different digestive tract and it will need further investigation to find out if several factors are involved to trigger an outbreak in ruminants.

Topics: Acer; Animal Husbandry; Animals; Austria; Carnitine; Deer; Eating; Female; Germany; Hypoglycins; Male; Muscular Diseases; Seasons

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

Evidence suggest there is a link between equine atypical myopathy (EAM) and ingestion of sycamore maple tree seeds.. To further evaluate the hypothesis that the ingestion of hypoglycin A (HGA) containing sycamore maple tree seeds causes acquired multiple acyl-CoA dehydrogenase deficiency and might be associated with the clinical and pathological signs of EAM.. Case report.. Necropsy and histopathology, using hematoxylin and eosin and Sudan III stains, were performed on a 2.5-year-old mare that died following the development of clinical signs of progressive muscle stiffness and recumbency. Prior to death, the animal ingested sycamore maple tree seeds (Acer pseudoplatanus). Detection of metabolites in blood and urine obtained post mortem was performed by rapid ultra-performance liquid chromatography-tandem mass spectrometry. Data from this case were compared with 3 geldings with no clinical history of myopathy.. Macroscopic examination revealed fragments of maple tree seeds in the stomach and severe myopathy of several muscle groups including Mm. intercostales, deltoidei and trapezii. Histologically, the affected muscles showed severe, acute rhabdomyolysis with extensive accumulation of finely dispersed fat droplets in the cytoplasm of degenerated skeletal muscle cells not present in controls. Urine and serum concentrations of several acyl carnitines and acyl glycines were increased, and both contained metabolites of HGA, a toxic amino acid present in sycamore maple tree seeds.. The study supports the hypothesis that ingestion of HGA-containing maple tree seeds may cause EAM due to acquired multiple acyl-CoA dehydrogenase deficiency.

Topics: Acer; Animals; Fatal Outcome; Female; Horse Diseases; Horses; Hypoglycins; Muscular Diseases; Seeds

Ingestion of sycamore seeds (Acer pseudoplatanus) is the likely source of hypoglycin A in atypical myopathy (AM) but ingestion of seedlings in spring might also contribute to intoxication.. To test for hypoglycin A in seeds and seedlings collected on pastures where AM cases were reported and compare its concentration in serum of affected and healthy horses.. Field investigation of clinical cases.. Whenever present, samaras (the winged nuts that each contain one seed) and/or seedlings were collected from pastures of 8 AM cases and 5 unaffected horses from different premises. Two AM cases were each co-grazing with an apparently healthy horse. Acylcarnitines and hypoglycin A were quantified in blood samples of all horses involved in the study.. Hypoglycin A was detected in serum of AM (5.47 ± 1.60 μmol/l) but not in healthy controls pasturing where A. pseudoplatanus trees were not present. However, hypoglycin A was detected at high concentrations (7.98 μmol/l) in serum of a clinically healthy horse grazing a pasture with seedlings and samaras and also in the 2 healthy horses co-grazing with AM cases (0.43 ± 0.59 μmol/l). Hypoglycin A was detected in all samples of seeds and spring seedlings of A. pseudoplatanus.. Atypical myopathy can be associated with the ingestion of sycamore samaras and also ingestion of seedlings. Hypoglycin A can be detected in the blood of horses with no detectable clinical signs at pasture in which there is A. pseudoplatanus. Determination of hypoglycin A concentration in blood is useful for screening for exposure in suspected cases of AM.

Topics: Acer; Animals; Horse Diseases; Horses; Hypoglycins; Muscular Diseases; Seedlings; Seeds

During April and May 2014 four horses aged between 5 months and 9 years, located in the Canterbury, Marlborough and Southland regions, presented with a variety of clinical signs including recumbency, stiffness, lethargy, dehydration, depression, and myoglobinuria suggestive of acute muscle damage. Two horses were subjected to euthanasia and two recovered. In all cases seeds of sycamore maple (Acer pseudoplatanus) or box elder (A. negundo) were present in the area where the horse had been grazing.. The samaras (seeds) of some Acer spp. may contain hypoglycin A, that has been associated with cases of atypical myopathy in Europe and North America. To determine if hypoglycin A is present in the samaras of Acer spp. in New Zealand, samples were collected from trees throughout the country that were associated with historical and/or current cases of atypical myopathy, and analysed for hypoglycin A. Serum samples from the four cases and four unaffected horses were analysed for the presence of hypoglycin A, profiles of acylcarnitines (the definitive diagnosis for atypical myopathy) and activities of creatine kinase and aspartate aminotransferase.Markedly elevated serum activities of creatine kinase and aspartate aminotransferase, and increased concentrations of selected acylcarnitines were found in the case horses. Hypoglycin A was detected in the serum of those horses but not in the healthy controls. Hypoglycin A was detected in 10/15 samples of samaras from sycamore maple and box elder from throughout New Zealand.. Cases of atypical myopathy were diagnosed on properties where samaras containing hypoglycin A were also found.. Sycamore and box elder trees in New Zealand are a source of hypoglycin A associated with the development of atypical myopathy. If pastured horses present with clinical and biochemical signs of severe muscle damage then the environment should be checked for the presence of these trees. Horses should be prevented from grazing samaras from Acer spp. in the autumn.

Topics: Acer; Animals; Horse Diseases; Horses; Hypoglycins; Male; Muscular Diseases; New Zealand; Plants, Toxic; Seeds

Topics: Acer; Animals; Horse Diseases; Horses; Hypoglycins; Muscle, Skeletal; Muscular Diseases

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

Equine atypical myopathy in Europe is a fatal rhabdomyolysis syndrome that results from the ingestion of hypoglycin A contained in seeds and seedlings of Acer pseudoplatanus (sycamore maple). Acylcarnitine concentrations in serum and muscle OXPHOS capacity were determined in 15 atypical myopathy cases. All but one acylcarnitine were out of reference range and mitochondrial respiratory capacity was severely decreased up to 49% as compared to 10 healthy controls. The hallmark of atypical myopathy thus consists of a severe alteration in the energy metabolism including a severe impairment in muscle mitochondrial respiration that could contribute to its high death rate.

Topics: Acer; Animal Feed; Animals; Carnitine; Europe; Female; Horse Diseases; Horses; Hypoglycins; Male; Mitochondrial Diseases; Muscles; Muscular Diseases; Oxidative Phosphorylation

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

Topics: Animals; Horse Diseases; Horses; Hypoglycins; Muscular Diseases; Sentinel Surveillance

Hypoglycin A has been recently identified has the causal agent of atypical myopathy (AM) in horses. Its identification and quantification in equine's biological fluids is thus a major concern to confirm maple poisoning and to provide insight into the poorly understood mechanism of hypoglycin A intoxication.. Quantification of hypoglycin A has been achieved with the aTRAQ kit for amino acid analysis of physiological fluids (AB Sciex). Acquisition method on mass spectrometer has been updated to record the hypoglycin A specific MRM transition.. Outlined accuracy profiles demonstrated very reliable data. A good linearity was observed from 0.09 to 50μmol/L and precision was very good with coefficient of variation below 8%. Fifty-five samples collected from 25 confirmed AM horses revealed significant hypoglycin A concentrations, while toxin was not found in serum of 8 control animals.. The described aTRAQ variant method has been analytically and clinically validated. The reliability of our approach is thus demonstrated into the workup of atypical myopathy.

Topics: Animals; Horse Diseases; Horses; Hypoglycins; Isotope Labeling; Linear Models; Mass Spectrometry; Muscular Diseases; Reproducibility of Results; Sensitivity and Specificity

Topics: Animal Husbandry; Animals; Horse Diseases; Horses; Humans; Hypoglycins; Muscular Diseases; Seasons; Seeds; Societies, Medical; Trees; United Kingdom; Veterinary Medicine

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