Hypoglycin Research Articles

A Zebrafish Embryo Model to Screen Potential Therapeutic Compounds in Sapindaceae Poisoning

Hypoglycin A (HGA) and methylenecyclopropylglycine (MCPrG) are protoxins produced by Sapindaceae plants, particularly Acer pseudoplatanus, and are responsible for causing atypical myopathy (AM) in equids. These protoxins metabolise into toxic compounds, such as methylenecyclopropylacetyl-CoA (MCPA-CoA), which alters energy metabolism and induces severe rhabdomyolysis. Currently, no specific treatment exists for this poisoning, in vitro models fail to reproduce HGA’s toxic effects on equine primary myoblasts, and mammalian models are impractical for large-scale drug screening. This study aimed to develop a zebrafish embryo model for screening therapeutic compounds against AM. Zebrafish embryos were exposed to various concentrations of HGA, MCPrG, and methylenecyclopropylacetate (MCPA) for 72 h. MCPrG did not induce toxicity, while HGA and MCPA showed median lethal concentration (LC50) values of 1.7 µM and 1 µM after 72 h, respectively. The highest levels of the conjugated metabolite MCPA–carnitine were detected 24 h after HGA exposure, and the acylcarnitines profile was highly increased 48 h post-exposure. Isovaleryl-/2- methylbutyrylcarnitine levels notably rose after 24 h, suggesting potential exposition biomarkers. Glycine and carnitine effectively reduced mortality, whereas riboflavin showed no protective effect. These findings suggest that the zebrafish embryo represents a valuable model for identifying therapeutic compounds for Sapindaceae poisoning.

Large-scale study of blood markers in equine atypical myopathy reveals subclinical poisoning and advances in diagnostic and prognostic criteria

Equine atypical myopathy (AM) is a severe rhabdomyolysis syndrome primarily caused by hypoglycin A (HGA) and methylenecyclopropylglycine protoxins. This study aimed to refine diagnostic and prognostic criteria for AM while exploring apparently healthy cograzers. Blood samples from 263 horses, including AM cases (n= 95), cograzers (n= 73), colic horses (n= 19), and controls (n= 76), were analyzed for HGA, its toxic metabolite, and acylcarnitines profile. Diseased horses exhibited alterations in acylcarnitines that strongly distinguished them from controls and colic horses. Regression analyses identified distinct acylcarnitines profiles among groups, with cograzers showing intermediate alterations. Age and gelding status emerged as protective factors against AM. Furthermore, serum acylcarnitines profiling was valuable in predicting AM survival, with isovaleryl-/2-methylbutyrylcarnitine (i.e., C5 acylcarnitine) showing promise as both a diagnostic and prognostic marker. Subclinical alterations in cograzers underscore a novel aspect: the presence of subclinical cases of AM.

Hypoglycin A in Acer genus plants

Hypoglycin A (HGA) is an amino acid occuring in the Sapindaceae family. Ingestion of certain Acer genus plants belonging to this family has been connected with atypical myopathy (AM) or seasonal pasture myopathy (SPM). To date, all cases of AM/SPM have been associated with sycamore (Acer pseudoplatanus) and boxelder maple (Acer negundo). The aim of this work was to determine and compare HGA in sycamore, boxelder and silver maple (Acer saccharinum), the trees known for HGA content, whose occurence is quite common in the Czech Republic. In sycamore and boxelder maple the effect of location, weather condition and sampling season was evaluated.The other aim was screening for presence of HGA in 12 other species of Acer genus which are grown as ornamental trees in Europe. The determination of HGA was conducted using ultra – high performance liquid chromatography – tandem mass spectrometry (LC/MS). HGA was detected in all samples of sycamore, boxelder and silver maple except for eight leave samples of boxelder maple. In the case of sycamore maple, the highest concentrations of HGA (median) were found in seedlings (770 mg/kg) followed by samaras (130 mg/kg) and by leaves (48 mg/kg) and inflorescences (24 mg/kg). In boxelder maple, significantly higher concentrations of HGA (median) were found in seedlings (550 mg/kg) compared with samaras (45 mg/kg), leaves (14 mg/kg) and inflorescences (24 mg/kg). According to the results the seedlings could pose a significant risk of poisoning, although other factors such as accessibility and palatability of other parts, especially samaras, should be considered. No significant differences of HGA concentrations in silver maple (56 mg/kg) were found between samaras, leaves and inflorescences. HGA was also identified in sugar maple (Acer saccharum), Japanese maple (Acer palmatum), trident maple (Acer buergerianum), paperbark maple (Acer griseum) and Himalayan maple (Acer oblongum). Although silver maple and other ornamental maples have not been reported to cause AM/SPM, the possibility of intoxication in animals can not be excluded.

Tissue Specific Distribution and Activation of Sapindaceae Toxins in Horses Suffering from Atypical Myopathy.

Equine atypical myopathy is caused by hypoglycin A (HGA) and methylenecyclopropylglycine (MCPrG), the known protoxins of sycamore maple (Acer pseudoplatanus). Various tissues from five atypical myopathy cases were analyzed but only HGA was found. Whether deamination of MCPrG has already occurred in the intestine as the first stage of metabolization has not been investigated. Activation of the protoxins to methylenecyclopropylacetyl (MCPA)-CoA and methylenecyclopropylformyl (MCPF)-CoA, respectively, occurred mainly in the skeletal muscles, as evidenced by very high concentrations of MCPA-carnitine and MCPF-carnitine in this tissue. Inhibition of the acyl-CoA dehydrogenases of short- and medium-chain as well as branched-chain fatty acids by the toxins led to a strong increase in the corresponding acylcarnitines, again preferentially in skeletal muscles. An accumulation of the long-chain acylcarnitines beyond the level of the control samples could not be detected in the tissues. As a high amount of HGA was always found unmetabolized in the organs, we speculate that targeting the interruption of further metabolization might be a way to stop the progression of intoxication. Inhibition of the mitochondrial branched-chain amino acid aminotransferase, i.e., the first enzyme responsible for the activation of sycamore maple protoxins, could be a therapeutic approach.

Detection of Hypoglycin A and MCPrG Metabolites in the Milk and Urine of Pasture Dairy Cows after Intake of Sycamore Seedlings.

Hypoglycin A (HGA), methylenecyclopropylglycine (MCPrG), hypoglycin B (HGB), and γ-glutamyl-α-(methylenecyclopropyl) glycine (γ-glutamyl-MCPrG) are secondary plant metabolites occurring in sycamore maple (Acer pseudoplatanus) as well as several other Sapindaceae (e.g., Blighia sapida). By interfering with energy metabolism, they may cause severe intoxication in humans and other species. However, to date, there is not enough data available concerning the intake, metabolism, or excretion of sycamore maple toxins in dairy cows. In May 2022, five cows were observed over four days, when they had first access to a pasture with two sycamore maples. Grazing of their seedlings that grew numerously in between the pasture plants was monitored by direct observation. Milk samples were drawn both from individual cows and from the bulk tank. Spontaneous urine samples were collected from all cows on day 3 after access to the pasture. Seedlings (100 g) were sampled on the pasture and analyzed, together with milk and urine samples, for sycamore toxins and their metabolites using liquid chromatography-tandem mass spectrometry and liquid chromatography-high-resolution mass spectrometry. Cows ingested sycamore seedlings while grazing. Values of HGA in milk were below the limit of quantification. However, metabolites of HGA and MCPrG were detected in individual milk samples already at the end of the first day of grazing. Urine samples of all five cows showed higher concentrations of conjugated HGA and MCPrG metabolites than in milk. Observations suggest that dairy cows may have a low susceptibility toward sycamore maple toxins. However, whether this could be attributed to foregut fermenting species in general requires further elucidation.

A sensitive LC–MS/MS method for the quantification of the plant toxins hypoglycin A and methylenecyclopropylglycine and their metabolites in cow’s milk and urine and application to farm milk samples from Germany

Hypoglycin A (HGA) and its homologue methylenecyclopropylglycine (MCPrG) are present in ackee and lychee as well as seeds, leaves, and seedlings of some maple (Acer) species. They are toxic to some animal species and humans. The determination of HGA, MCPrG, and their glycine and carnitine metabolites in blood and urine is a useful tool for screening for potential exposure to these toxins. In addition, HGA, MCPrG, and/or their metabolites have been detected in milk. In this work, simple and sensitive ultra-performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) methods without derivatization were developed and validated for the quantification of HGA, MCPrG, and their metabolites in cow’s milk and urine. An extraction procedure from milk samples has been developed, whereas a dilute-and-shoot approach was implemented for urine samples. For quantification, the MS/MS analysis was performed in multiple reaction monitoring mode. The methods were validated according to the European Union guidelines using blank raw milk and urine as matrices. The limit of quantification presented here for HGA in milk (1.12 µg/L) is noticeably lower than the lowest published limit of detection (9 µg/L). Acceptable values for recovery (89–106% and 85–104% in milk and urine, respectively) and precision (≤ 20%) were obtained for all the quality control levels. The stability of HGA and MCPrG in frozen milk over a period of 40 weeks has been demonstrated. The method was applied to 68 milk samples from 35 commercial dairy farms and showed the absence of any quantifiable amounts of HGA, MCPrG, and their metabolites.Graphical

Co-Occurrence of Hypoglycin A and Hypoglycin B in Sycamore and Box Elder Maple Proved by LC-MS/MS and LC-HR-MS.

Hypoglycin A (HGA) and methylenecyclpropylglycine (MCPrG) are formed by some maple trees (Acer species) and have been associated with incidences of atypical myopathy among horses in pastures. In this work, a simple and sensitive ultra-performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) method without derivatization was developed for the quantification of HGA and MCPrG in maple samples and validated according to EU guidelines. The LOQ presented here for HGA (16.4 µg/kg) is considerably lower than the lowest published LOQ (500 µg/kg). This method confirms that sycamore and box elder maple contain considerable amounts of HGA and MCPrG. In addition, the presence of the dipeptides hypoglycin B and γ-glutamyl-MCPrG in these two maple species is shown using high-resolution MS. This is the first report on the presence of these dipeptides in maple since 1973. The presence of HGB and γ-glutamyl-MCPrG could change the way we understand animal intoxication following the ingestion of maple.

Acer pseudoplatanus: A Potential Risk of Poisoning for Several Herbivore Species

Acer pseudoplatanus is a worldwide-distributed tree which contains toxins, among them hypoglycin A (HGA). This toxin is known to be responsible for poisoning in various species, including humans, equids, Père David’s deer and two-humped camels. We hypothesized that any herbivore pasturing with A. pseudoplatanus in their vicinity may be at risk for HGA poisoning. To test this hypothesis, we surveyed the HGA exposure from A. pseudoplatanus in species not yet described as being at risk. Animals in zoological parks were the major focus, as they are at high probability to be exposed to A. pseudoplatanus in enclosures. We also searched for a toxic metabolite of HGA (i.e., methylenecyclopropylacetyl-carnitine; MCPA-carnitine) in blood and an alteration of the acylcarnitines profile in HGA-positive animals to document the potential risk of declaring clinical signs. We describe for the first instance cases of HGA poisoning in Bovidae. Two gnus (Connochaetes taurinus taurinus) exposed to A. pseudoplatanus in their enclosure presented severe clinical signs, serum HGA and MCPA-carnitine and a marked modification of the acylcarnitines profile. In this study, even though all herbivores were exposed to A. pseudoplatanus, proximal fermenters species seemed less susceptible to HGA poisoning. Therefore, a ruminal transformation of HGA is hypothesized. Additionally, we suggest a gradual alteration of the fatty acid metabolism in case of HGA poisoning and thus the existence of subclinical cases.

Atypical myopathy in 2 Bactrian camels.

Atypical myopathy (AM) is an acute seasonal rhabdomyolysis seen primarily in equids, caused by the ingestion of sycamore maple samaras containing hypoglycin A (HGA) and methylenecyclopropyl-glycine (MCPG). Toxic metabolites inhibit acyl-CoA dehydrogenases and enoyl-CoA hydratases, causing selective hyaline degeneration of type I muscle fibers. Two zoo-kept Bactrian camels (Camelus bactrianus) with a fatal course of AM had sudden onset of muscle pain and weakness, recumbency, and dysphagia, accompanied by increased serum creatine kinase activity and detection in serum of HGA, MCPG, and metabolites. Medical treatment was ineffective. At postmortem examination, sycamore maple tree material was found within the first gastric compartment of the 2-y-old gelding. Although musculature was macroscopically normal, histologically, monophasic hyaline degeneration was marked within type I fibers of intercostal and hypoglossal muscles of the gelding, and in neck, tongue, and masticatory muscles of the cow. The ingestion of sycamore maple material can cause AM in Bactrian camels, and trees of the Sapindaceae family should be avoided in enclosures.

Hypoglycin A in Cow's Milk-A Pilot Study.

Hypoglycin A (HGA) originating from soapberry fruits (litchi, and ackee) seeds or seedlings from the sycamore maple (SM) tree (related to Sapindaceae) may cause Jamaican vomiting sickness in humans and atypical myopathy in horses and ruminants. A possible transfer into dairy cow’s milk cannot be ruled out since the literature has revealed HGA in the milk of mares and in the offal of captured deer following HGA intoxication. From a study, carried out for another purpose, bulk raw milk samples from four randomly selected dairy farms were available. The cows were pastured in the daytime. A sycamore maple tree was found on the pasture of farm No. 1 only. Bulk milk from the individual tank or milk filling station was sampled in parallels and analyzed for HGA by LC-ESI-MS/MS. Measurable concentrations of HGA occurred only in milk from farm No. 1 and amounted to 120 and 489 nmol/L. Despite low and very variable HGA concentrations, the results indicate that the ingested toxin, once eaten, is transferred into the milk. However, it is unknown how much HGA the individual cow ingested during grazing and what amount was transferred into the bulk milk samples. As a prerequisite for a possible future safety assessment, carry-over studies are needed. Furthermore, the toxins’ stability during milk processing should also be investigated as well.

Hypoglycin A absorption in sheep without concurrent clinical or biochemical evidence of disease.

BackgroundHypoglycin A (HGA) intoxication after ingestion of Acer spp. tree material has never been confirmed in domesticated ruminants despite their similar grazing habitats.ObjectivesTo investigate whether sheep have low HGA bioavailability caused by rumen HGA breakdown.AnimalsStomach and rumen fluid samples from 5 adult horses and 5 adult sheep respectively. Residual serum samples from 30 ewes and lambs.MethodsExperimental and retrospective cohort study. Hypoglycin A concentration was quantified in horse gastric and sheep ruminal samples after in vitro incubation with Acer pseudoplatanus seeds. Serum samples from grazing sheep (n = 20) and nursing lambs (n = 10) obtained before and after their release onto pastures with and without Sycamore seedlings were analyzed for HGA and methylenecyclopropyl‐acetic acid carnitine, and serum biochemistry.ResultsNeither ovine rumen nor equine gastric fluid affected HGA content in samples incubated for up to 2 hours. Despite HGA's detection in serum from sheep (n = 13/15; median, 23.71 ng/mL; range, 5.62‐126.4 ng/mL) grazing contaminated pastures and in their nursing lambs (n = 2/5; median, 12.5 ng/mL; range, 8.82‐15.67 ng/mL), there was no apparent clinical or subclinical disease.Conclusions and Clinical ImportanceAny reduced sensitivity to HGA intoxication in sheep seems unrelated to ruminal degradation. Serum HGA concentrations in sheep were similar to those of subclinically affected atypical myopathy horses. Any reduced sensitivity of sheep to HGA might be related to greater metabolic resistance rather than selective grazing habits or lower bioavailability. Hypoglycin A was found in nursing lambs, suggesting that HGA is excreted in milk.

Comparison of Fecal Microbiota of Horses Suffering from Atypical Myopathy and Healthy Co-Grazers.

Simple SummaryEquine atypical myopathy is a muscular disease caused by a plant intoxication, which seems to affect only certain horses sharing a pasture, and the role of the intestinal bacteria (microbiota) in this selective impairment is still poorly understood. The aim of this study was to describe and compare fecal microbiota of horses suffering from atypical myopathy and healthy co-grazers. We concluded that fecal microbiota of horses suffering from atypical myopathy is different from their co-grazers and changes are more severe in horses that do not survive the disease. Understanding those changes may help in developing therapeutic and/or preventive strategies for horses at risk for atypical myopathy.Equine atypical myopathy (AM) is caused by hypoglycin A (HGA) and methylenecyclopropylglycine (MCPG) intoxication resulting from the ingestion of seeds or seedlings of some Acer tree species. Interestingly, not all horses pasturing in the same toxic environment develop signs of the disease. In other species, it has been shown that the intestinal microbiota has an impact on digestion, metabolism, immune stimulation and protection from disease. The objective of this study was to characterize and compare fecal microbiota of horses suffering from AM and healthy co-grazers. Furthermore, potential differences in fecal microbiota regarding the outcome of diseased animals were assessed. This prospective observational study included 59 horses with AM (29 survivors and 30 non-survivors) referred to three Belgian equine hospitals and 26 clinically healthy co-grazers simultaneously sharing contaminated pastures during spring and autumn outbreak periods. Fresh fecal samples (rectal or within 30 min of defecation) were obtained from all horses and bacterial taxonomy profiling obtained by 16S amplicon sequencing was used to identify differentially distributed bacterial taxa between AM-affected horses and healthy co-grazers. Fecal microbial diversity and evenness were significantly (p < 0.001) higher in AM-affected horses as compared with their non-affected co-grazers. The relative abundance of families Ruminococcaceae, Christensenellaceae and Akkermansiaceae were higher (p ≤ 0.001) whereas those of the Lachnospiraceae (p = 0.0053), Bacteroidales (p < 0.0001) and Clostridiales (p = 0.0402) were lower in horses with AM, especially in those with a poor prognosis. While significant shifts were observed, it is still unclear whether they result from the disease or might be involved in the onset of disease pathogenesis.

Grazing Mares on Pasture with Sycamore Maples: A Potential Threat to Suckling Foals and Food Safety through Milk Contamination.

Simple SummaryEquine atypical myopathy is seasonal poisoning resulting from the ingestion of seeds and seedlings of the sycamore maple that contains toxins. Literature mentions several cases of intoxication among gravid mares and in unweaned foals. The objective of this study was to determine whether the toxins responsible for atypical myopathy could pass to the foal via suckling. Four mares that were pasturing with sycamore in the vicinity were milked. Analysis revealed the presence of toxins in milk. This unprecedented observation could partially explain cases of unweaned foals suffering from atypical myopathy. However, a transplacental transfer of the toxin cannot be excluded for newborn cases. Besides being a source of contamination for offspring, milk contamination by toxins from fruits of trees of the Sapindaceae family might constitute a potential risk for food safety regarding other species’ raw milk or dairy products.Equine atypical myopathy (AM) is seasonal intoxication resulting from the ingestion of seeds and seedlings of the sycamore maple (Acer pseudoplatanus) that contain toxins, among them, hypoglycin A (HGA). Literature mentions several cases of AM among gravid mares and in unweaned foals. The objective of this study was to determine whether HGA and/or its metabolite are present in milk from grazing mares exposed to sycamore maple trees as confirmed by detection of HGA and its metabolite in their blood. Four mare/foal couples were included in the study. Both HGA and its metabolite were detectable in all but one of the milk samples. To our knowledge, this is the first study describing transfer of HGA to the milk. This unprecedented observation could partially explain cases of unweaned foals suffering from AM. However, a transplacental transfer of the toxin cannot be excluded for newborn foals. Besides being a source of contamination for offspring, milk contamination by toxins from fruits of trees of the Sapindaceae family might constitute a potential risk for food safety regarding other species’ raw milk or dairy products.

Detection of hypoglycin A and MCPA‐carnitine in equine serum and muscle tissue: Optimisation and validation of a LC‐MS–based method without derivatisation

Measurement of hypoglycin A (HGA) and its toxic metabolite, methylenecyclopropylacetic acid (MCPA), in equine serum confirms a diagnosis of atypical myopathy (AM), a pasture-associated toxic rhabdomyolysis with high mortality linked to the ingestion of Acer trees plant material. Supportive diagnostic tests include plasma acyl-carnitine profiling and urine organic acid testing, but these are not specific for AM. Previously reported HGA and MCPA analytical techniques used liquid chromatography-mass spectrometry (LC-MS) with a derivatising step, but the latter prolongs testing and increases costs. To develop a rapid LCMS method for detection of serum and tissue HGA and MCPA that enables expedited diagnosis for horses with AM. Analytical test validation. Validation parameters to industry standards using as criteria precision, accuracy, linearity, reproducibility and stability in analyte-spiked samples were calculated on 9-calibration points and 3 different validation concentrations in both serum and muscle tissue. The test was successfully validated for the detection of HGA and MCPA-carnitine in equine serum and muscle. Test linearity was excellent (r2 =.999), accuracy was very good for both analytes (93%-108%), precision did not exceed 10% coefficient of variation and reproducibility met the requirements of the Horwitz equation. Stability was unaffected by storage at a range of temperatures. The spectrum of the tested analytes was limited to only two relevant analytes in favour of a quick and easy analysis. Linearity of the muscle method was not evaluated as calibration curves were not produced in this matrix. We report an optimised, simplified and validated method for detection of HGA and MCPA-carnitine in equine serum and muscle suitable for rapid diagnosis of suspected AM cases. The serum-based test should also enable risk assessment of toxin exposure in cograzing horses and assessment of horses with undiagnosed myopathies, while the tissue detection test should help to confirm cases post-mortem and to determine toxin distribution, metabolism and clearance across different tissues.

Detection of toxic methylenecyclopropylglycine and hypoglycin A in litchi aril of three Chinese cultivars

As a subtropical fruit with high commercial values, litchi is also a source of methylenecyclcopropylglycine (MCPG) and hypoglycin A (HGA), which could cause hypoglycemia and fatal encephalopathy in human. In this work, a quantitative method was developed well to detect MCPG and HGA present in litchi aril of different cultivars. Method validation was evaluated well by linearity, recovery, precision and sensitivity. Among three cultivars, ‘Feizixiao’ contained the highest toxin level with 0.60–0.83 mg kg−1 of MCPG and 10.66–14.46 mg kg−1 of HGA, followed by ‘Huaizhi’ with 0.08–0.12 mg kg−1 of MCPG and 0.63–1.54 mg kg−1 of HGA, and ‘Nuomici’ with 0.09–0.11 mg kg−1 of MCPG and 0.35–0.91 mg kg−1 of HGA. The toxin levels were highly associated with litchi cultivar and storage time. These findings can provide new knowledge to help to recommend the safe consumption of fresh litchi based on human health.

Liquid chromatography-mass spectrometry-based determination of ergocristine, ergocryptine, ergotamine, ergovaline, hypoglycin A, lolitrem B, methylene cyclopropyl acetic acid carnitine, N-acetylloline, N-formylloline, paxilline, and peramine in equine hair

Ingestion of hypoglycin A (HGA) in maple seeds or alkaloids produced by symbiotic fungi in pasture grasses is thought to be associated with various syndromes in grazing animals. This article describes analytical methods for monitoring long-term exposure to HGA, its metabolite MCPA-carnitine, as well as ergocristine, ergocryptine, ergotamine, ergovaline, lolitrem B, N-acetylloline, N-formylloline, peramine, and paxilline in equine hair.After extraction of hair samples separation was achieved using two ultra high performance liquid chromatographic systems (HILIC or RP-C18, ammonium formate:acetonitrile). A benchtop orbitrap instrument was used for high resolution tandem mass spectrometric detection. All analytes were sensitively detected with limits of detection between 1 pg/mg and 25 pg/mg. Irreproducible extraction or ubiquitous presence in horse hair precluded quantitative validation of lolitrem B/paxilline and N-acetylloline/N-formylloline, respectively. For the other analytes validation showed no interferences in blank hair. Other validation parameters were as follows: limits of quantification (LOQ), 10 to 100 pg/mg; recoveries, 18.3 to 91.0%; matrix effects, −48.2 - 24.4%; linearity, LOQ - 1000 pg/mg; accuracy, −14.9 - 6.4%, precision RSDs ≤10.7%.The method allows sensitive detection of all analytes and quantification of ergocristine, ergocryptine, ergotamine, ergovaline, HGA, MCPA-carnitine, and peramine in horse hair. Applicability was proven for N-acetylloline and N-formylloline by analyzing hair of 13 horses.

Detection of MCPG metabolites in horses with atypical myopathy.

Atypical myopathy (AM) in horses is caused by ingestion of seeds of the Acer species (Sapindaceae family). Methylenecyclopropylacetyl-CoA (MCPA-CoA), derived from hypoglycin A (HGA), is currently the only active toxin in Acer pseudoplatanus or Acer negundo seeds related to AM outbreaks. However, seeds or arils of various Sapindaceae (e.g., ackee, lychee, mamoncillo, longan fruit) also contain methylenecyclopropylglycine (MCPG), which is a structural analogue of HGA that can cause hypoglycaemic encephalopathy in humans. The active poison formed from MCPG is methylenecyclopropylformyl-CoA (MCPF-CoA). MCPF-CoA and MCPA-CoA strongly inhibit enzymes that participate in β-oxidation and energy production from fat. The aim of our study was to investigate if MCPG is involved in Acer seed poisoning in horses. MCPG, as well as glycine and carnitine conjugates (MCPF-glycine, MCPF-carnitine), were quantified using high-performance liquid chromatography-tandem mass spectrometry of serum and urine from horses that had ingested Acer pseudoplatanus seeds and developed typical AM symptoms. The results were compared to those of healthy control horses. For comparison, HGA and its glycine and carnitine derivatives were also measured. Additionally, to assess the degree of enzyme inhibition of β-oxidation, several acyl glycines and acyl carnitines were included in the analysis. In addition to HGA and the specific toxic metabolites (MCPA-carnitine and MCPA-glycine), MCPG, MCPF-glycine and MCPF-carnitine were detected in the serum and urine of affected horses. Strong inhibition of β-oxidation was demonstrated by elevated concentrations of all acyl glycines and carnitines, but the highest correlations were observed between MCPF-carnitine and isobutyryl-carnitine (r = 0.93) as well as between MCPA- (and MCPF-) glycine and valeryl-glycine with r = 0.96 (and r = 0.87). As shown here, for biochemical analysis of atypical myopathy of horses, it is necessary to take MCPG and the corresponding metabolites into consideration.

Atypical myopathy-associated hypoglycin A toxin remains in sycamore seedlings despite mowing, herbicidal spraying or storage in hay and silage.

Several pasture management strategies have been proposed to avoid hypoglycin A (HGA) intoxication in horses, but their efficacy has never been investigated. To evaluate the effect of mowing and herbicidal spraying on HGA content of sycamore seedlings and the presence of HGA in seeds and seedlings processed within haylage and silage. Experimental study. Groups of seedlings were mowed (n=6), sprayed with a dimethylamine-based (n=2) or a picolinic acid-based herbicide (n=1). Seedlings were collected before intervention, and at 48h, 1 and 2weeks after. Cut grass in the vicinity of mowed seedlings was collected pre-cutting and after 1week. Seeds and seedling (n=6) samples processed within haylage and silage were collected. HGA concentration in samples was measured using a validated LC-MS-based method. There was no significant decline in HGA content in either mowed or sprayed seedlings; indeed, mowing induced a temporary significant rise in HGA content of seedlings. HGA concentration increased significantly (albeit to low levels) in grass cut with the seedlings by 1week. HGA was still present in sycamore material after 6-8months storage within either hay or silage. Restricted number of herbicide compounds tested. Neither mowing nor herbicidal spraying reduces HGA concentration in sycamore seedlings up to 2weeks after intervention. Cross contamination is possible between grass and sycamore seedlings when mowed together. Mowing followed by collection of sycamore seedlings seems the current best option to avoid HGA toxicity in horses grazing contaminated pasture. Pastures contaminated with sycamore material should not be used to produce processed hay or silage as both seedlings and seeds present in the bales still pose a risk of intoxication.

Lychee-Associated Hypoglycaemic Encephalopathy: A New Disease of Children Described in India

Fruits of lychee tree (Litchi sinensis), extensively horticultured in India, China and many other countries, are delicious and possess many nutritious and medicinal properties. In India and other Asian countries, in lychee harvest season, outbreaks have been occurring of rapidly developing hypoglycaemia, encephalopathy, seizures and cerebral oedema in young children when they ingest lychee fruit arils in large numbers on empty stomach. It has been shown that the acute neurological illness is hypoglycaemic encephalopathy, caused by the actions of hypoglycin A (HGA) and methylenecyclopropylglycine (MCPG), the non-protein l-amino acids present in the edible arils of lychee fruits. Both HGA and MCPG phytotoxins are known to disrupt the pathways of β-fatty acid oxidation and gluconeogenesis in human body cells, result in accumulation of many undesirable metabolites of the blocked energy generating pathways, and altogether produced the often fatal hypoglycaemic illness. Here, the related work is summarized and commented upon and prospective genetical interventions in Litchi sinensis to eradicate the problem are outlined. Toxin-deficient lychee genotypes need to be developed by screening of germplasm accessions, and use of conventional and new site-directed mutagenesis technique of plant breeding. Lychee trees that produce super-toxin-rich (× 10 average toxin concentration) fruits are required to be identified and tagged to stop consumption of their fruits. New plantings must use toxin-deficient (low-toxin) lychee genotypes.

Quantification of hypoglycin A and methylenecyclopropylglycine in human plasma by HPLC-MS/MS

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.