Hypoglycin A Content in Blood and Urine Discriminates Horses with Atypical Myopathy from Clinically Normal Horses Grazing on the Same Pasture

<div><p>Hypoglycin A (HGA) in seeds of <i>Acer spp</i>. 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 <i>Acer pseudoplatanus</i> 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.</p></div

Specific characteristics of AM-affected and cograzing horses dedicated to the individual AM-pasture.

<p>TN = total number of horses on individual pasture; ¹ in hrs/d, *additional food stuffs (hay, concentrate); AH = affected horses, CG = cograzing horses: the denomination is made up of the letter of pasture (A-L) and the number of affected or cograzing horses on this pasture; WB = Warmblood, GRP = German Riding Pony, HF = haflinger; g = gelding, m = male, s = stallion; **survived, affect date 17^th Nov 2013, ***not available.</p

Hypoglycin A concentration in seeds of <i>Acer pseudoplatanus</i> collected from affected pastures detected by LC-ESI-MS/MS analysis.

<p>pasture B,C,E,F sampled in 2013; pasture G,H,J,K,L sampled in 2014.</p

Pasture B.

<p>Numerous seeds found on pasture where the closest sycamore maple tree was 50 meters away from the pasture.</p