hypoglycin-a and methylenecyclopropylacetic-acid

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 (r. 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.

Topics: Animals; Carnitine; Chromatography, Liquid; Cyclopropanes; Horse Diseases; Horses; Hypoglycins; Muscles; Reproducibility of Results; Tandem Mass Spectrometry

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

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

To examine the changes in coenzyme A profile and the possible corrective effects of carnitine supplementation in the genetic disorders of mitochondrial beta-oxidation, we carried out experiments using an inhibitor of multiple acyl-CoA dehydrogenase enzymes, methylenecyclopropaneacetic acid (MCPA), in rat hepatocytes. MCPA irreversibly inhibited ketone synthesis from straight-chain fatty acids (butyrate, octanoate, palmitate) and branched-chain fatty acids (alpha-ketoisocaproate) with a parallel 70-90% reduction of hepatocyte acetyl-CoA levels. Alone, MCPA or substrates halved free CoA levels to 15% of total CoA and doubled short- and medium-chain acyl-CoA levels to 30% of total CoA. With MCPA plus substrates combined, free CoA levels were 10% of total CoA, and short- and medium-chain acyl-CoA levels were 45% of total CoA. Comparable changes in CoA profiles were found in a patient with a severe genetic defect in beta-oxidation. Neither the suppression of ketogenesis nor the alterations in CoA profiles induced by MCPA inhibition could be corrected by carnitine supplementation.

Topics: Acyl-CoA Dehydrogenases; Animals; Carnitine; Coenzyme A; Cyclopropanes; Enzyme Inhibitors; Hypoglycins; Ketones; Lipid Metabolism, Inborn Errors; Liver; Mitochondria, Liver; Rats; Rats, Sprague-Dawley; Spiro Compounds