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Equine veterinary journal2020; 53(3); 558-568; doi: 10.1111/evj.13303

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

Abstract: 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. Objective: To develop a rapid LCMS method for detection of serum and tissue HGA and MCPA that enables expedited diagnosis for horses with AM. Methods: Analytical test validation. Methods: 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. Results: 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. Conclusions: 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. Conclusions: 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.
© 2020 The Authors. Equine Veterinary Journal published by John Wiley & Sons Ltd on behalf of EVJ Ltd.
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Publication Date: 2020-08-12 PubMed ID: 32525217DOI: 10.1111/evj.13303Google Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

This study describes the creation and optimization of a quick, cost-effective method, using liquid chromatography-mass spectrometry, to diagnose Atypical Myopathy in horses by detecting hypoglycin A (HGA) and its toxic metabolite, methylenecyclopropylacetic acid (MCPA) in horse serum and muscle.

Study Objectives

This research aims to:

  • Facilitate a faster diagnosis of Atypical Myopathy in horses.
  • Provide a reliable and cost-effective alternative by eliminating the need for a derivatising step in the analytical process.

Research Methods

To reach their conclusions, the researchers:

  • Validated their analytical test according to industry standards, using precision, accuracy, linearity, reproducibility, and stability in analyte-spiked samples as their criteria.
  • Built their method around 9-calibration points and 3 different validation concentrations in both serum and muscle tissue.

Research Findings

The findings from the testing portion of this research include:

  • Excellent linearity of their test.
  • The accuracy of the test was quite good for detecting both analytes (93%-108%).
  • The precision did not exceed 10% coefficient of variation, and reproducibility met the requirements of the Horwitz equation.
  • The test’s ability to detect the toxins remained unaffected by variables such as storage temperatures.

Conclusion and Potential Applications

The research concludes that:

  • The simplified, validated detection method for HGA and MCPA-carnitine in equine serum and muscle significantly increases our ability to rapidly diagnose Atypical myopathy.
  • The serum-based test may lead to better toxin exposure risk assessments of other horses grazing near an affected horse, and for examining horses with undiagnosed myopathies.
  • The tissue detection test might help confirm post-mortem cases and determine toxin distribution, metabolism, and clearance across different tissues.

Therefore, this new approach has the potential to substantially optimize the diagnosis process of Atypical Myopathy in horses.

Cite This Article

APA
González-Medina S, Hyde C, Lovera I, Piercy RJ. (2020). Detection of hypoglycin A and MCPA-carnitine in equine serum and muscle tissue: Optimisation and validation of a LC-MS-based method without derivatisation. Equine Vet J, 53(3), 558-568. https://doi.org/10.1111/evj.13303

Publication

Equine veterinary journal
ISSN: 2042-3306
NlmUniqueID: 0173320
Country: United States
Language: English
Volume: 53
Issue: 3
Pages: 558-568

Researcher Affiliations

González-Medina, Sonia
  • Comparative Neuromuscular Diseases Laboratory, The Royal Veterinary College, London, UK.
Hyde, Carolyne
  • Bio-Analysis Centre, Royal College Street, London, UK.
Lovera, Imogen
  • Bio-Analysis Centre, Royal College Street, London, UK.
Piercy, Richard J
  • Comparative Neuromuscular Diseases Laboratory, The Royal Veterinary College, London, UK.

MeSH Terms

  • Animals
  • Carnitine
  • Chromatography, Liquid / veterinary
  • Cyclopropanes
  • Horse Diseases / diagnosis
  • Horses
  • Hypoglycins
  • Muscles
  • Reproducibility of Results
  • Tandem Mass Spectrometry / veterinary

Grant Funding

  • SPrj032 / Horserace Betting Levy Board
  • G6015 / The Horse Trust

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Citations

This article has been cited 3 times.
  1. François AC, Taminiau B, Renaud B, Gonza-Quito IE, Massey C, Hyde C, Piercy RJ, Douny C, Scippo ML, Daube G, Gustin P, Delcenserie V, Votion DM. In Vitro Investigation of Equine Gut Microbiota Alterations During Hypoglycin A Exposure. Animals (Basel) 2025 Nov 19;15(22).
    doi: 10.3390/ani15223343pubmed: 41302050google scholar: lookup
  2. Bochnia M, Ziegler J, Glatter M, Zeyner A. Hypoglycin A in Cow's Milk-A Pilot Study. Toxins (Basel) 2021 May 26;13(6).
    doi: 10.3390/toxins13060381pubmed: 34073628google scholar: lookup
  3. González-Medina S, Bevin W, Alzola-Domingo R, Chang YM, Piercy RJ. Hypoglycin A absorption in sheep without concurrent clinical or biochemical evidence of disease. J Vet Intern Med 2021 Mar;35(2):1170-1176.
    doi: 10.1111/jvim.16077pubmed: 33675130google scholar: lookup
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