Sander Johannes, Cavalleri Jessika-M V, Terhardt Michael, Bochnia Mandy, Zeyner Annette, Zuraw Aleksandra, Sander Stefanie, Peter Michael, Janzen Nils
Screening-Labor Hannover, Germany (J Sander, Terhardt, S Sander, Peter, Janzen)Clinic for Horses, University of Veterinary Medicine Hannover, Foundation, Hannover, Germany (Cavalleri)Department of Clinical Chemistry, Hannover Medical School, Hannover, Germany (Janzen)Department of Animal Nutrition, Institute for Agricultural and Nutritional Sciences, Martin-Luther-University Halle-Wittenberg, Halle (Saale), Germany (Bochnia, Zeyner)Department of Veterinary Pathology, College of Veterinary Medicine, Freie, Universität Berlin, Berlin, Germany (Zuraw)
Screening-Labor Hannover, Germany (J Sander, Terhardt, S Sander, Peter, Janzen)Clinic for Horses, University of Veterinary Medicine Hannover, Foundation, Hannover, Germany (Cavalleri)Department of Clinical Chemistry, Hannover Medical School, Hannover, Germany (Janzen)Department of Animal Nutrition, Institute for Agricultural and Nutritional Sciences, Martin-Luther-University Halle-Wittenberg, Halle (Saale), Germany (Bochnia, Zeyner)Department of Veterinary Pathology, College of Veterinary Medicine, Freie, Universität Berlin, Berlin, Germany (Zuraw).
J Vet Diagn Invest. 2016 Mar;28(2):98-104. doi: 10.1177/1040638715624736.
Hypoglycin A (2-amino-3-(2-methylidenecyclopropyl)propanoic acid) is the plant toxin shown to cause atypical myopathy in horses. It is converted in vivo to methylenecyclopropyl acetic acid, which is transformed to a coenzyme A ester that subsequently blocks beta oxidation of fatty acids. Methylenecyclopropyl acetic acid is also conjugated with carnitine and glycine. Acute atypical myopathy may be diagnosed by quantifying the conjugates of methylenecyclopropyl acetic acid plus a selection of acyl conjugates in urine and serum. We describe a new mass spectrometric method for sample volumes of <0.5 mL. Samples were extracted with methanol containing 5 different internal standards. Extracts were analyzed by ultra-high-performance liquid chromatography-tandem mass spectrometry focusing on 11 metabolites. The total preparation time for a series of 20 samples was 100 min. Instrument run time was 14 min per sample. For the quantification of carnitine and glycine conjugates of methylenecyclopropyl acetic acid in urine, the coefficients of variation for intraday quantification were 2.9% and 3.0%, respectively. The respective values for interday were 9.3% and 8.0%. Methylenecyclopropyl acetyl carnitine was detected as high as 1.18 µmol/L in serum (median: 0.46 µmol/L) and 1.98 mmol/mol creatinine in urine (median: 0.79 mmol/mol creatinine) of diseased horses, while the glycine derivative accumulated up to 1.97 mmol/mol creatinine in urine but was undetectable in most serum samples. In serum samples from horses with atypical myopathy, the intraday coefficients of variation for C4-C8 carnitines and glycines were ≤4.5%. Measured concentrations exceeded those in healthy horses by ~10 to 1,400 times.
低血糖素A（2-氨基-3-（2-亚甲基环丙基）丙酸）是一种已证实可导致马匹发生非典型肌病的植物毒素。它在体内转化为亚甲基环丙基乙酸，后者再转化为辅酶A酯，进而阻断脂肪酸的β氧化。亚甲基环丙基乙酸还会与肉碱和甘氨酸结合。急性非典型肌病可通过定量尿液和血清中亚甲基环丙基乙酸的结合物以及一系列酰基结合物来诊断。我们描述了一种适用于样本量小于0.5 mL的新型质谱方法。样本用含有5种不同内标的甲醇进行提取。提取物通过超高效液相色谱-串联质谱进行分析，重点检测11种代谢物。一系列20个样本的总制备时间为100分钟。仪器每个样本的运行时间为14分钟。对于尿液中亚甲基环丙基乙酸的肉碱和甘氨酸结合物的定量分析，日内定量的变异系数分别为2.9%和3.0%。日间的相应值分别为9.3%和8.0%。在患病马匹的血清中检测到亚甲基环丙基乙酰肉碱高达1.18 μmol/L（中位数：0.46 μmol/L），尿液中为1.98 mmol/mol肌酐（中位数：0.79 mmol/mol肌酐），而甘氨酸衍生物在尿液中累积高达1.97 mmol/mol肌酐，但在大多数血清样本中无法检测到。在患有非典型肌病的马匹的血清样本中，C4 - C8肉碱和甘氨酸的日内变异系数≤4.5%。测得的浓度比健康马匹高出约10至1400倍。
