Presented in part as an abstract at the 2011 American College of Internal Medicine Forum in Denver, CO
Corresponding author: B. Sponseller, Dr. med. vet., Dipl. ABVP (Equine), Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Iowa State University, Ames, IA 50011; e-mail: email@example.com .
Seasonal pasture myopathy (SPM) is a highly fatal form of nonexertional rhabdomyolysis that occurs in pastured horses in the United States during autumn or spring. In Europe, a similar condition, atypical myopathy (AM), is common. Recently, a defect of lipid metabolism, multiple acyl-CoA dehydrogenase deficiency (MADD), has been identified in horses with AM.
To determine if SPM in the United States is caused by MADD.
Six horses diagnosed with SPM based on history, clinical signs, and serum creatine kinase activity, or postmortem findings.
Retrospective descriptive study. Submissions to the Neuromuscular Diagnostic Laboratory at the University of Minnesota were reviewed between April 2009 and January 2010 to identify cases of SPM. Inclusion criteria were pastured, presenting with acute nonexertional rhabdomyolysis, and serum, urine, or muscle samples available for analysis. Horses were evaluated for MADD by urine organic acids, serum acylcarnitines, muscle carnitine, or histopathology.
Six horses had clinical signs and, where performed (4/6 horses), postmortem findings consistent with SPM. Affected muscle (4/4) showed degeneration with intramyofiber lipid accumulation, decreased free carnitine concentration, and increased carnitine esters. Serum acylcarnitine profiles (3/3) showed increases in short- and medium-chain acylcarnitines and urinary organic acid profiles (3/3) revealed increased ethylmalonic and methylsuccinic acid levels, and glycine conjugates, consistent with equine MADD.
Conclusions and Clinical Importance
Similar to AM, the biochemical defect causing SPM is MADD, which causes defective muscular lipid metabolism and excessive myofiber lipid content. Diagnosis can be made by assessing serum acylcarnitine and urine organic acid profiles.
Seasonal pasture myopathy (SPM), first described in the United States in 2006, is a highly fatal form of nonexertional rhabdomyolysis of pastured horses during fall or spring. Muscular weakness, recumbency, and myoglobinuria are the predominant clinical signs and may be accompanied by dysphagia, esophageal obstruction, or colic-like signs. SPM can be difficult to differentiate from other forms of acute, severe rhabdomyolysis, or other causes of acute death and is likely underrecognized in the United States. The cause of SPM is unknown, and diagnosis is based on clinical findings and histopathologic evidence of severe myonecrosis with lipid accumulation mainly in postural and respiratory muscles. In Europe, the seasonal occurrence of myopathy in grazing horses is a well-recognized entity with the first reported cases occurring in Great Britain in 1939. In 1984, the term “atypical myoglobinuria” was coined by investigators of a Scottish outbreak, although in recent years the disease has been more commonly referred to as “atypical myopathy” (AM). Over the past decades, the emerging nature of this disease has become evident with sporadic, occasionally large outbreaks reported from a number of European countries.[4-6] Numerous similarities exist between SPM and AM, including clinical and clinicopathological findings, seasonal occurrence (autumn and spring) in association with inclement weather, high case fatality rate (71–100%), and the type and distribution of myopathic lesions, with Zenker's degeneration and lipid accumulation predominantly in postural, respiratory, and cardiac muscles.[1, 3]
Recent metabolic screening of lipid metabolism in horses with AM suggested a deficiency of multiple mitochondrial dehydrogenases that use flavine adenine dinucleotide (FAD) as cofactor, as seen in human MADD. Enzymes impaired in MADD include dehydrogenases that catalyze β-oxidation of short-, medium-, and long-chain fatty acids, and dehydrogenases involved in amino acid and choline metabolism.[8, 9] Disruption of enzymatic pathways in MADD leads to accumulation of enzyme-specific substrates (acyl-CoAs) that can be eliminated as acylcarnitines or acylglycines (glycine conjugates). Hence, the presence of specific acylcarnitines in serum and glycine conjugates in urine directly reflects the intramitochondrial accumulation of corresponding acyl-CoA esters. In addition, accumulating substrates may undergo alternate metabolic pathways including peroxisomal β-oxidation or microsomal ω-oxidation, resulting in a variety of disease-specific secondary metabolites. Consequently, patients with MADD display characteristic urine organic acid[11, 12] and blood acylcarnitine profiles.[9, 13, 14] Horses affected with AM have MADD specific increases in urine organic acids (ethylmalonic, methylsuccinic, and lactic acid) and glycine conjugates ([iso]valeryl-, butyryl- and hexanoylglycine). In addition, short- (C2–C5) and medium-chain (C6, C8, C10) acylcarnitines were increased in urine and plasma of horses with AM, consistent with MADD.
We hypothesized that SPM in the United States is characterized by MADD similar to AM in Europe and that this metabolic defect would be identified by assessing lipid staining of skeletal muscle, urine organic acids, and serum acylcarnitine profiles.
Materials and Methods
Submissions from the Neuromuscular Diagnostic Laboratory at the University of Minnesota were reviewed between April 2009 and January 2010 to identify possible cases of SPM. Horses were selected for inclusion in this study if they were unexercised horses on pasture that presented with clinical signs of muscle pain, weakness, and recumbency combined with high serum creatine kinase (CK) activity or postmortem findings consistent with rhabdomyolysis; and serum, urine, or muscle samples were available for analysis. Attending veterinarians were contacted to obtain medical records and State Diagnostic Laboratories provided gross postmortem results.
Serum and urine samples available from horses 2, 5, and 6, collected and frozen at initial presentation, were sent to the Baylor Institute of Metabolic Disease in Dallas, Texas, for determination of urine organic acid and serum acylcarnitine concentrations analyzed by gas chromatography-mass spectrometry (GC-MS) and tandem mass spectrometry (MS-MS), respectively. Urine samples from 5 healthy adult Quarter Horse mares and serum samples from 35 healthy adult horses (Quarter Horses, Warmbloods, and Thoroughbreds) were used to define normal values for urine organic acids and serum acylcarnitines. The upper limit of the reference range was calculated as mean + 2 SD for C-2-carnitine and mean + 3 SD for other acylcarnitines and urine organic acids.
Skeletal muscle samples collected at necropsy from horses 1, 3, 4, and 6 were frozen in isopentane chilled in liquid nitrogen within 2–4 hours (horses 1, 3, and 6) or 12–24 hours (horse 4) after death. Cryosections were stained with hematoxylin and eosin (H&E), periodic acid-Schiff's (PAS), amylase PAS, and Oil red O. Snap frozen muscle specimens available for horses 1, 3, 4, and 6 and 2 healthy control horses were submitted to the Comparative Neuromuscular Laboratory in La Jolla, California, for measurement of total, free, and esterified carnitine by MS-MS.
In April and May of 2009, 2 Belgian draft horses from the same pasture in Iowa (horses 1 and 2) and a Quarter Horse from Minnesota (horse 3) were diagnosed with SPM. In October and November of 2009, an Appaloosa from Minnesota (horse 4), a Paint horse from Wisconsin (horse 5), and a Missouri Fox Trotter from Minnesota (horse 6) were diagnosed with SPM. The horses ranged in age from 2 to 16 years and included 1 mare, 4 geldings, and a stallion.
All horses were housed on pasture (2–10 acres) for 24 hours a day when clinical signs of SPM developed. Trees were present on or around all pastures and horses had access to dead wood and leaves. Pastures were sloped and the grassland was sparse. Five horses received no supplementary feed, 1 horse received hay. All had access to a salt or trace mineral block and all were on a routine vaccination and deworming program. Horses were used for pleasure riding 2–3 times per week (n = 3) or driving 1–2 times per month (n = 1). One horse had not been ridden but received some ground work, and another horse was taken to horse shows and used for gaming.
Five horses presented with muscular weakness and stiffness of less than 2-day duration. Additional signs included myoglobinuria (n = 5), prolonged periods of recumbency (n = 4), colic-like signs (n = 4), esophageal obstruction (n = 3), dysphagia (n = 2), dyspnea (n = 2), muscle trembling (n = 2), and profuse sweating (n = 1). Further, horse 2 exhibited excessive weight shifting of all limbs and inability to eat from the ground, presumably because of cervical pain. Three horses (horses 1, 5, and 6) progressed to persistent recumbency within 2–3 days of onset of clinical signs and were euthanized. One horse (horse 3) succumbed after developing severe dyspnea despite tracheostomy along with signs of head pressing and seizures. One horse (horse 4) was found dead on pasture after 2 days of pelvic limb stiffness, reluctance to walk, inappetance, and low volume diarrhea. Horse 2 had a complete recovery and was discharged after 7 days of hospitalization.
A complete blood count (CBC) was performed in 5 horses. Neutrophilia (n = 4; range 7,332–14,410 cells/μL; reference range 2,100–6,700 cells/μL), lymphopenia (n = 3; range 530–1,010 cells/μL; reference range 1,300–4,500 cells/μL), or both were common hematologic abnormalities. Biochemistry profiles (n = 5) revealed hyperglycemia (range 146–358 mg/dL; reference range 80–113 mg/dL), hypocalcemia (range 7.5–10.1 mg/dL; reference range 10.6–12.8 mg/dL), and marked increases in CK (range 47,700–813,933 IU/L; reference range 100–575 IU/L) and AST activities (2,143–18,043 IU/L; reference range 100–465 IU/L). Alkaline phosphatase activity (264–538 IU/L; reference range 50–220 IU/L) was increased in 3 of 4 horses measured. Serum bicarbonate concentrations were low (19.2–22.5 mEq/L; reference range 24.5–33.5 mEq/L) and the anion gap high (19–27; reference range 7–14) in 4 horses measured. Creatinine was mildly increased in 1 horse (2.5 mg/dL; reference range 1.0–2.1 mg/dL). Serum selenium was measured in 1 horse (horse 2) and was low (76 ppb; reference range 140–250 ppb); serum vitamin E level in the same horse was within normal limits. Blood lactate levels (1.8–8.24 mmol/L; reference range 0.37–1.43 mmol/L) measured in horses 2 and 5, and peritoneal fluid lactate levels (6.37–14.3 mmol/L; reference range 0.22–0.98 mmol/L) measured in horses 1 and 5, were increased.
Treatment included intravenous administration of fluids and flunixin-meglumine (n = 5), dimethyl sulfoxide (n = 4), continuous rate infusion (CRI) of lidocaine, detomidine, butorphanol, or ketamine for pain control (n = 2), acepromazine (n = 2), methocarbamol (n = 1), fentanyl patches (n = 1), systemic antimicrobials (n = 1), and intranasal oxygen insufflation (n = 1). In addition, horse 2, the only survivor, received antioxidants (vitamin E/selenium and vitamin C) and a single dose of dexamethasone.
Necropsy results available for horses 1, 3, 4, and 6 revealed areas of pallor in numerous skeletal muscles (n = 4) and myocardium (n = 2), presence of dark brown urine in the urinary bladder (n = 4), and tissue edema (n = 3). Other findings included pallor of kidneys (n = 2) and liver (n = 1), mucosal lesions in the squamous (n = 2) or glandular (n = 1) portion of the stomach, and diffuse reddening and mucosal congestion of the distal portion of the large colon (n = 1). Diffuse, dark red semicircular foci in the lung parenchyma and bronchi filled with coagulated blood were evident in 1 horse. The latter also had dark purple discoloration and swelling of the epiglottis, vocal folds, and arytenoid cartilages.
Gastric contents of horses 1 and 3 were tested for ionophores and results were negative. Hepatic vitamin E concentration was decreased in horse 1 (2 ppm; reference range 4.3–7.1 ppm) and normal in horse 3.
Extensive, acute, multifocal myocyte degeneration with hypereosinophilia and fragmentation was present in respiratory muscles (intercostals and diaphragm) and limb muscles (including deep gluteal, semitendinosus, psoas minor/major, and longissimus) of all (n = 4) horses and in the myocardium of 3 horses. Similar changes were found in the tongue (n = 2), masseter muscle (n = 1), and cervical muscle (n = 1). Paucity of cellular infiltrates and lack of signs of myocyte regeneration were common features.
Fresh frozen samples of diaphragm, iliopsoas, and gluteal muscles (horses 3, 4, and 6), postural (horse 3), semimembranosus (horses 4 and 6), and quadriceps (horse 1) muscle showed a moderate to marked number of myofibers with Zenker's necrosis, absent to mild degree of macrophage infiltration, and normal to decreased periodic acid-Schiff's staining for glycogen. Excessive lipid storage was found in all samples in numerous myofibers using the Oil Red O stain for neutral triglycerides (Fig 1).
Other histopathologic findings included vacuolation of hepatocytes (n = 4), severe, acute, bilateral renal tubular necrosis with intratubular myoglobin casts (n = 3), bilateral diffuse renal congestion (n = 1), subacute (n = 2) to chronic (n = 1) gastric ulceration, and hemorrhage in the colonic mucosa and pulmonary alveoli/bronchi (n = 1).
Skeletal muscle from horses (n = 4) with SPM showed a marked decrease in free carnitine and increased carnitine esters with an increased ester to free ratio compared with healthy control horses (Table 1).
Table 1. Skeletal muscle carnitine concentrations (nmol/mg protein) in horses with SPM (n = 4) and healthy control horses (n = 2)
Mean ± SD
18.08 ± 7.14
16.3 ± 2.4
3.03 ± 0.99
13.55 ± 1.48
15.05 ± 6.98
2.75 ± 0.92
Ester/free carnitine ratio
5.41 ± 3.57
0.2 ± 0
The urinary organic acid and glycine conjugate profile measured in 3 horses revealed markedly increased levels of ethylmalonic acid, methylsuccinic acid, lactic acid, adipic acid, butyrylglycine, isovalerylglycine, and hexanoylglycine consistent with MADD (Table 2).[7, 11] Glutaric acid was increased in horses 5 and 6 but was normal in horse 2, the only survivor. The serum acylcarnitine profile in horses 5 and 6 resembled that of MADD, with a generalized increase in short- (C2–C5), medium- (C6–C12), and long-chain (C14–C20) acylcarnitines (Table 3). C2, C4, C5, and C6 acylcarnitines showed the most prominent increases. In contrast, the only surviving horse (horse 2) had only mild increases in acylcarnitines C5 through C10 and minor increases in C12 and C16.
Table 2. Organic acid and glycine conjugate concentrations (mmol/mol creatinine) in urine from horses (n = 3) with seasonal pasture myopathy
Mean ± SD
303.67 ± 78.31
54.67 ± 5.69
471.33 ± 302.58
30.67 ± 16.29
61.67 ± 62.52
4,164.00 ± 2,391.61
177.00 ± 66.12
181.33 ± 133.98
Table 3. Serum acylcarnitines (μmol/L) from horses (n = 2) with fatal SPM
This study documents that U.S. horses affected with SPM have a metabolic profile very similar to AM. The characteristic abnormalities in the serum and urine of affected horses are pathognomonic for MADD and provide a means to accurately diagnose this emerging disease. Further, comparison of the epidemiology of SPM and AM in the midwestern United States and Europe may provide key insights into the cause of this acquired myopathy.
The history, seasonal occurrence, clinical signs, and clinical or postmortem pathology for the 6 pastured horses were consistent with SPM and AM.[1, 3] A lipid storage myopathy was evident in fresh samples from the 4 fatal cases with necropsies by neutral lipid accumulation in myofibers of postural and respiratory muscles in Oil red O stains. Lipid storage in skeletal muscle is a hallmark of horses affected with SPM/AM[1, 3] and the extent of lipid storage is severe. Less than 6% of healthy horses exhibit a degree of lipid accumulation in myofibers independent of dietary fat content and very few horses with exertional rhabdomyolysis show evidence of abnormal lipid accumulation in skeletal muscle fibers. Excessive lipid storage alone, however, is not diagnostic of MADD as there may be other conditions within the category of nonexertional rhabdomyolysis that have excessive lipid storage. Thus, in this study the most specific diagnosis of MADD was made in 3 SPM horses based on the urinary excretion pattern of organic acids (ethylmalonic, methylsuccinic, and adipic acid) and glycine conjugates (butyryl-, isovaleryl- and hexanoylglycine) and the increase in predominantly short- and medium-chain serum acylcarnitines. Mild increases in long-chain acylcarnitines were evident in serum of 2 horses with fatal SPM in this report, but not in serum of the surviving horse collected during the acute phase of the disease. Long-chain fatty acids may undergo compensative β-oxidation in peroxisomes. However, when this compensation fails, increased amounts of long-chain acylcarnitines may be found in serum, reflecting a more serious clinical course of the disease. Urinary excretion of glutaric acid is a hallmark of human MADD, also known as glutaric aciduria type II, and was present in 2 horses with SPM in this study, but not in healthy control horses. This is in contrast to a study by Westermann et al, who documented the presence of glutaric acid in healthy control horses and speculated that glutarate may be a normal constituent of horse urine. The absence of glutaric aciduria in horse 2, the only survivor, may be explained by a milder course of the disease and is consistent with findings in mild forms of human MADD. Increased urinary excretion of lactic acid is an unspecific finding in equine MADD and can be found in horses affected with other types of acute myopathies.
Muscle carnitine was evaluated in 4 horses with SPM. Carnitine plays an important role in fatty acid metabolism and acts as a shuttle for long-chain fatty acids from the cytosol into mitochondria. Furthermore, carnitine buffers the mitochondrial free CoA/acyl-CoA ratio by binding activated acyl residues and liberating free CoA. When organic acids and acyl-CoAs accumulate in mitochondria, as seen in oxidation defects, the carnitine cycle can reverse leading to increased cellular excretion of acylcarnitines[17, 18] as seen in MADD. The decrease in free carnitine and increase in carnitine esters in affected muscle of horses with SPM in this study likely reflect this buffering capacity of carnitine and lend further support that SPM is caused by a defect in muscular lipid metabolism (MADD). A similar decrease in free carnitine and increase in esterified carnitine have previously been reported in muscle and serum of human patients with mitochondrial myopathy.
The pathophysiology of oxidation defects is attributed to a lack of cellular energy production that is exacerbated by direct toxic effects of accumulating metabolites on key enzymes of mitochondrial energy metabolism. Recently, decreased mitochondrial ATP production with reduced activities of all complexes of the respiratory chain was documented in skeletal muscle of horses affected with AM. Cells that utilize fatty acids as primary energy source, such as highly oxidative (type 1) myofibers, are predominantly affected.[1, 22] The latter fiber type is especially high in postural and respiratory muscles explaining the profound muscular weakness and respiratory dyspnea seen in horses with SPM. Similarly, cardiac myocytes depend largely on energy derived from oxidation of fatty acids and were affected in 2 of 4 horses that were available for necropsy in the current report. As lipid metabolism is blocked and oxygen delivery impaired by failing cardiac and respiratory muscles, metabolism becomes more reliant on anaerobic glycolysis, resulting in muscle glycogen depletion and lactic acidosis. In human MADD, hepatic glycogen stores are readily depleted and hypoglycemia is a common finding. The observed hyperglycemia in horses with SPM might reflect higher hepatic glucose mobilization from greater liver glycogen stores compared with humans.[23, 24] Conversely, hyperglycemia might reflect insulin resistance. Nevertheless, provision of glucose (IV or PO) and insulin to horses with SPM is indicated to support muscle energy production from carbohydrate metabolism and stimulate insulin-mediated lipogenesis as previously suggested in AM. Some authors advocate the use of oral chromium to enhance insulin sensitivity in horses affected with AM. Studies on the efficacy and safety of supplementation with L-carnitine in human fatty acid oxidation disorders are controversial. Some authors suggest that supplementation with carnitine may be beneficial in MADD patients with secondary carnitine deficiency caused by formation and urinary excretion of acylcarnitines. Possible adverse effects of carnitine supplementation include cardiac arrhythmias through increased concentrations of circulating acylcarnitines[26, 28] and decreased disposal of acyl moieties through inhibition of glycine conjugation. Provision of riboflavin (Vitamin B2) to horses with SPM may be of benefit considering that a significant number of human MADD patients are riboflavin responsive and equine MADD may be associated with a decreased amount of FAD. Riboflavin is a precursor of FAD, a cofactor of all acyl-CoA dehydrogenases affected in MADD, and supplementation might enhance residual enzyme activity. Furthermore, treatment with antioxidants seems prudent as fatty acid oxidation disorders can result in increased free radical production and increased vulnerability to oxidative stress.[30, 31] Increased production of reactive oxygen species might in part be mediated by inhibitory effects of accumulating ethylmalonic acid on complexes I and III of the electron transport chain.
Human inherited MADD (glutaric aciduria type II) has been associated with mutations in the electron transfer flavoprotein (ETF) gene, ETF-dehydrogenase (ETF-DH) gene, or a possible defect in the mitochondrial FAD transporter. The clinical manifestation of this autosomal recessive disease is highly variable, ranging from a fatal neonatal form to an adult onset mild lipid storage myopathy. The variety of breeds affected and histories of horses with SPM do not support a heritable basis for MADD and an acquired disorder caused by an unknown toxin is likely. Recent research on AM implicated Clostridium sordellii lethal toxin or European Tar Spot (Rhytisma acerinum) as possible culprits.
In conclusion, horses with SPM have an acquired defect in fatty acid oxidation similar to AM that is characterized by excessive lipid storage in postural and respiratory muscles and reliably diagnosed by performing serum acylcarnitine and urine organic acid profiles. The authors suggest that the term acquired equine MADD might best reflect the biochemical basis for atypical myopathy and seasonal pasture myopathy.
The authors thank Michelle Lucio for technical assistance and greatly appreciate the contributions of involved referring veterinarians (Drs Mark Jaehnig, Bill Herberg, Gretchen Geib, Jo-Anne Archambault) and pathologists (Drs Jesse Hostetter, Joseph Haynes, Anibal Armién, Arno Wünschmann).