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Resting concentrations of cardiac troponin I in fit horses and effect of racing
Journal of Veterinary Cardiology (2008) 10, 105e109
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Resting concentrations of cardiac troponin I in fit horses and effect of racing* ¨ggstro ¨m, DVM, PhD Katarina Nostell, DVM, PhD*, Jens Ha Department of Clinical Sciences, Faculty of Veterinary Medicine, Swedish University of Agricultural Sciences, Box 7054, S-750 07 Uppsala, Sweden Received 22 February 2008; received in revised form 29 September 2008; accepted 1 October 2008
KEYWORDS Cardiac troponin I; Horse; Race
Abstract Objectives: To determine normal resting values for cardiac troponin I (cTnI) in healthy Standardbred, Thoroughbred and Warmblood horses and investigate if racing has an influence on cTnI concentrations. Background: Measuring cTnI concentrations in plasma is the gold standard for detecting myocardial injury in humans. Cardiac troponin I is highly conserved between species and has gained interest as a marker for cardiac injury in horses. Increased levels of cTnI have been reported in association with endurance and short-term strenuous exercise on a treadmill in horses. However, the effect of true racing conditions has not yet been reported. Animals, materials and methods: Blood samples for analysis of cTnI concentrations in plasma were collected from 67 Standardbred racehorses, 34 Thoroughbred racehorses and 35 Warmblood dressage horses at rest. Blood samples were also collected prior to and after racing in 22 Standardbred racehorses and 6 Thoroughbred racehorses. Results: All horses except one had resting plasma cTnI concentrations <0.022 mg/L. Mild increases in cTnI concentrations were seen in some horses 1e2 h after the race (1/17 Standardbreds and 2/6 Thoroughbreds) as well as 10e14 h after the race (4/ 21 Standardbreds and 1/6 Thoroughbreds). Conclusions: Resting cTnI concentrations in horses are low but mildly elevated cTnI concentrations may be detected in some horses 1e14 h after racing. These findings could be of importance when evaluating horses with suspected cardiac disease that recently have performed hard exercise. ª 2008 Elsevier B.V. All rights reserved.
*
Funding for this study was provided by the Swedish Foundation for Equine Research, Sweden. * Corresponding author. E-mail address: [email protected] (K. Nostell).
1760-2734/$ - see front matter ª 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jvc.2008.10.001
106
Introduction Myocardial injury is difficult to detect and diagnose in horses. The traditional methods that have been used to detect suspected myocardial injury are evaluation of electrocardiography (ECG) and echocardiography. These methods have limitations and therefore attempts have been made to find more sensitive markers for myocardial injury in the horse and biomarkers have provided new options to detect cardiac injury in different species. Increased concentrations of isoenzymes of creatine kinase (CK-MB) and lactate dehydrogenase (LDH-1 and LDH-2) have been used clinically as markers for myocardial injury in the horse. These enzymes have limitations due to the occurrence of cross-reactions with skeletal muscle and a short half-life in serum.1e6 In recent years cardiac troponin I (cTnI), a protein involved in the interaction between actin and myosin during contraction, has gained interest as a clinical marker for myocardial injury in different species. cTnI is known to leak out from injured myocardial cells and has been shown to be a sensitive and specific marker for myocardial injury in humans and dogs.2,7e10 In humans, cardiac troponin I is mainly used for diagnosing myocardial infarction but is also elevated in other conditions such as myocarditis and sepsis.11e13 In healthy humans and dogs, plasma concentrations of cTnI are generally low and usually increase between 3 and 8 h after initial myocardial injury, and in humans the test reaches reliable diagnostic sensitivity by 12e16 h.9,14e16 The concentration of cTnI in serum returns to normal within 5e12 days after an insult and the degree of increase in cTnI concentration is correlated to the degree of injury.8,14,17,18 Measuring cTnI concentrations in serum or plasma is therefore of value to determine the prognosis after myocardial injury in humans and dogs. The amino acid sequence for equine cTnI has recently been characterised and evidence supports the use of commercial human assay systems to measure equine cardiac troponin I.19 The improved sensitivity, specificity, and the fact that the concentration of cTnI shows a long persistence in the circulation in humans make cTnI an interesting marker for cardiac injury in horses. Case reports on horses with increased cTnI concentrations and documented myocardial disease have been published.20,21 Normal resting values for cardiac troponin I in horses have been reported and are generally very low, although cTnI values cannot be compared between assays.21e23 It could be expected that horses, depending on their use and
K. Nostell, J. Ha ¨ggstro ¨m exercise level and time of sampling, vary in concentrations of cTnI. Increased concentrations of cTnI have been reported in horses in association with endurance competition as well as after shortterm maximal exercise on a treadmill for 2000e 2400 m.24,25 However, the effect of short-term high intensity exercise during true race conditions, such as a trotting race, has not yet been reported. Knowledge about what effect a race has on cTnI concentrations is of importance when evaluating horses with suspicion of cardiac disease that have recently raced or performed maximal exercise. The aims of the present study were to study cTnI concentrations in plasma in healthy fit horses of different breeds and to see if racing influences cTnI concentrations.
Animals, materials and methods Resting concentrations of cTnI were measured in 67 Standardbred racehorses, 34 Thoroughbred racehorses and 35 Warmblood horses (Study 1). The Standardbred trotters (27 mares, 21 geldings, 19 stallions) were between 3 and 10 years old, the Thoroughbreds between 2 and 9 years old (15 mares, 16 geldings, 3 stallions) and the Warmbloods between 6 and 24 years old (7 mares, 20 geldings and 8 stallions). In addition to this, 22 Standardbred racehorses (9 mares, 4 geldings, 9 stallions) between 3 and 8 years old and 6 Thoroughbred racehorses (3 geldings and 3 mares) between 3 and 6 years old, were included in a separate study investigating the effect of racing on cTnI concentrations (Study 2). Four of the Standardbreds were also used in the first part of the study. All racehorses included in the study were fit racehorses in professional training and all Warmbloods were dressage horses that trained and competed at a high national level (FEI Prix St. George and above). Some horses (2/67 Standardbreds, 2/34 Thoroughbreds and 3/35 Warmbloods) had low intensity right sided systolic murmurs (1e2/6) on auscultation of the heart. None of the horses included in the study showed signs of disease on clinical examination or had a history of poor performance. The study was sanctioned by the Ethical Committee for Animal Experiments, Uppsala, Sweden. All resting blood samples in study 1 were collected at rest in the box at the training campus. In the 28 horses enrolled in study 2 blood samples were collected 2 days prior to the race at rest in the box at the training campus, within 2 h after the race and the day after the race (10e14 h post-race).
Cardiac troponin I in fit horses Blood samples were drawn by vacutainer technique from the jugular vein and collected in heparinised tubes. Samples were centrifuged and plasma separated within 30 min after collection and all samples were kept cool until frozen at 20 C (within 3 h after collection). The samples were kept frozen at 20 C until analysed within 6 months after collection. Repeated analysis of a sample with a cTnI concentration of 42 mg/L was conducted after 6 months and the concentration was then 47 mg/L (11% increase). This suggests that little degradation occurs after a storage period of 6 months. Plasma concentrations of cTnI were measured with an enzyme immunoassaya with an analytical sensitivity of <0.022 mg/L. The assay was evaluated for use in equine samples by macerating horse heart muscle and performing a dilution curve which showed an F value of <0.0001. To evaluate the ability of the assay to detect horses with increased cTnI concentrations, blood samples were also collected from three horses (horses A, B and C) with suspicion of primary myocardial disease. These horses all had signs of myocardial disease on clinical examination such as ventricular extra systoles (horse A), tachycardia at rest and extra systoles (horse B), and ventricular tachycardia and atrial fibrillation (horse C). Increased cTnI concentrations were observed in these horses (horse A: 1.3 mg/L, Horse B: 0.40 mg/L and Horse C: 42 mg/L). Two of the horses were necropsied; horse A was diagnosed with cardiomyopathy with acute and chronic myocardial necrosis and horse B with acute myocardial necrosis.
Results 97% (65/67) of the Standardbreds, 94% (32/34) of the Thoroughbred racehorses and 100% (35/35) of the Warmblood dressage horses had resting cTnI concentrations <0.022 mg/L. Four horses (2/67 Standardbreds and 2/34 Thoroughbreds) had resting cTnI concentrations that exceeded 0.022 mg/L (Standardbreds: 0.023 and 0.025 mg/L, Thoroughbreds: 0.043 mg/L and 0.025 mg/L), none of these four horses had murmurs on auscultation. In the horses tested after racing, a complete set of cardiac troponin I values were obtained in 15 of the 22 Standardbreds and all of the Thoroughbreds. At rest 90% (19/21) of the Standardbreds and 83% (5/6) of the Thoroughbreds had cTnI concentrations below the detection level of 0.022 mg/L. Three horses (two Standardbreds and one Thoroughbred) showed pre-race concentrations of cTnI a Architect CI8200, Abbot Laboratories, Diagnostic Division, Abbott Park, IL.
107 that exceeded 0.022 mg/L. Mild increases in cTnI concentrations were found in some horses (5/22 Standardbreds and 3/6 Thoroughbreds) both 1e2 h post-race and 10e14 h post-race (Table 1).
Discussion The results from this study show that horses generally have low cTnI concentrations at rest but that intense short-term exercise, such as a trotting race or a Thoroughbred race, induces mild increases in cTnI concentrations in some horses. This is in agreement with a previous study on fit performance horses where a tendency for increased plasma cTnI concentrations was seen in all horses 3e6 h after they had performed shortterm high intensity exercise for a distance of 2000e2400 m on a treadmill.25 This study also reported that some individuals still had cTnI concentrations that were above the limit of detection at 12 and 24 h post-exercise. In the present study, it is possible that more horses would have shown increases in cTnI concentrations if blood samples had been collected more frequently after the race. However, this was not possible for practical reasons. Another limitation in the present study is the inability of the test to reliably measure cTnI concentrations below 0.022 mg/L. In humans new ultra sensitive cTnI assays are able to identify myocardial damage, at concentrations well below this (0.006e0.04 mg/L).26
Table 1 Horses with plasma cTnI concentrations that were above the analytical sensitivity limit for one of the three sampling periods. Age Gender Breed
cTnI concentration (mg/L) Pre-race
8 6 4 5 5 8 6 5 3
S M S G S M M G M
SB SB SB SB SB SB TB TB TB
0.025a <0.022 <0.022 <0.022 <0.022 0.023a <0.022 <0.022 0.025a
1e2 10e14 h Post-race Post-race 0.029a <0.022 <0.022 <0.022 <0.022 <0.022 0.028a <0.022 0.027a
<0.022 0.025a 0.023a 0.023a 0.035a <0.022 <0.022 0.023a <0.022
S: stallion; M: mare; G: gelding; SB: Standardbred; TB: Thoroughbred. In addition to the reported results, 16 Standardbred horses (9 with complete profiles) and 3 Thoroughbred horses (all with complete profiles) had cTnI concentrations <0.022 mg/L at each of their testing intervals. a result above the analytical sensitivity of the test.
108 The reasons behind the increase found in cTnI concentrations after hard exercise are not clear. cTnI is reported to have a very high specificity for myocardium and it is therefore unlikely that these increases are related to cross-reactivity with skeletal muscle.10,19 The method used in this study also has a low reported cross-reactivity (0.07%) with skeletal muscle in humans. In the study by Durando et al., none of the horses with increased cTnI after exercise had signs of cardiac disease on ECG or echocardiography. Increased concentrations of cTnI have also been reported in connection with endurance exercise in horses and humans without signs of cardiac disease.24,25,27,28 The low grade right sided systolic murmurs heard in some horses in the present study were likely due to tricuspid insufficiency. This is a frequent finding in performance horses which is rarely associated with clinically significant changes in the valves.29 As only a thorough clinical examination was performed on the horses in the present study, it could be speculated that some of the horses might have mild cardiac disease causing the increases seen in cTnI after exercise. However, this is regarded unlikely as all horses were in professional training with experienced trainers and raced on regular basis with no suspicion or history of impaired performance. It is therefore likely that the increases seen in plasma cTnI in some horses are related to exercise. One theory is that a mild hypoxia during exercise could cause a change in the permeability of the myocardium causing a leakage of macromolecules into the blood. This is supported by the fact that a previous study in rats has shown that short periods of hypoxia can induce release of cTnI without cell death.30 The fact that some horses had small increases in cTnI concentrations could to a certain degree be normal as the interday variability of cardiac troponin I in fit Standardbreds and Thoroughbreds has not been evaluated. They could also be attributed to the kind of exercise the horses had performed the previous day. It is not likely that these differences were attributed to a difference in fitness level as a previous study that compared concentrations of cTnI in race trained Thoroughbreds and Thoroughbreds on pasture found no difference between groups.22 The exercise induced increases in cTnI were mild and not comparable to the concentrations found in the horses with signs of cardiac disease. The increased concentrations of cTnI together with the clinical symptoms and findings on ECG, echocardiography and the findings on necropsy indicate that the assay used in this study is able to detect horses with cardiac disease. Previous
K. Nostell, J. Ha ¨ggstro ¨m studies on humans have shown a correlation between the degree of myocardial infarction size (indicating myocardial damage) and the release of cTnI.17 Although myocardial infarcts are seldom seen in horses, there is reason to believe that an association between the severity of myocardial damage and the degree of increase in cTnI also exists in horses. It is therefore likely that the horse with the highest plasma cTnI concentrations (horse C) in this study also had the most generalised myocardial injury. Unfortunately the horse was not necropsied so this claim could not be confirmed. The individual differences in cTnI concentrations are also influenced by the time lapse between myocardial damage and sampling. In humans, cTnI concentrations have been shown to increase in other conditions that are primarily extra-cardiac such as sepsis and kidney failure.13,31,32 One theory is that these elevations might reflect toxic or inflammatory damage to the heart as opposed to ischemic injury. Elevated cTnI concentrations and evidence of myocarditis have also been reported in patients with bacterial enteritis and in septic foals.33,34,35
Conclusions and clinical implications Resting cTnI concentrations in horses are low, but can increase slightly above the baseline level in some horses 11e14 h after racing. A conclusion whether these elevated cTnI concentrations should be considered as normal for horses that have recently raced or are a reflection of myocardial disease cannot be drawn form the results of this study. However, it seems unlikely that the horses in the present study had cardiac disease since they were performing well. It is also important to remember that cTnI numbers may vary between assays and values can never be directly compared from one assay to another. Mildly elevated concentrations of cTnI alone will unlikely lead to a definitive diagnosis, but together with the clinical presentation and findings on ECG and echocardiography it can become an important marker for myocardial disease in horses with impaired performance. Marked elevations of cTnI alone could be considered as a strong indication of myocardial disease. Further studies are needed on a larger population of horses with signs of cardiac disease to establish the correlation between elevated concentrations of cTnI and pathological findings. Likewise, further studies are needed to establish ‘normal’ post-exercise/post-race cTnI reference ranges in horses and to evaluate cTnI levels in poorly performing horses.
Cardiac troponin I in fit horses
References 1. Apple F. Tissue specificity of cardiac troponin I, cardiac troponin T and creatine kinase-MB. Clin Chim Acta 1999; 284:151e9. 2. Adams 3rd JE, Bodor GS, Davila-Roman VG, Delmez JA, Apple FS, Ladenson JH, Jaffe AS. Cardiac troponin I. A marker with high specificity for cardiac injury. Circulation 1993;88:101e6. 3. Evans GO. Biochemical assessment of cardiac function and damage in animal species. A review of the current approach of the academic, governmental and industrial institutions represented by the Animal Clinical Chemistry Association. J Appl Toxicol 1991;11:15e21. 4. Lee TH, Goldman L. Serum enzyme assays in the diagnosis of acute myocardial infarction. Recommendations based on a quantitative analysis. Ann Intern Med 1986;105:221e33. 5. Nakai K, Nakai K, Nagane Y, Obara W, Sato M, Ohi K, Matsumoto N, Takanashi N, Itoh C. Serum levels of cardiac troponin I and other marker proteins in patients with chronic renal failure. Clin Exp Nephrol 2004;8:43e7. 6. Argiroudis SA, Kent JE, Blackmore DJ. Observations on the isoenzymes of creatine kinase in equine serum and tissues. Equine Vet J 1982;14:317e21. 7. Mair J, Genser N, Morandell D, Maier J, Mair P, Lechleitner P, Calzolari C, Larue C, Ambach E, Dienstl F, Pau B, Puschendorf B. Cardiac troponin I in the diagnosis of myocardial injury and infarction. Clin Chim Acta 1996;245:19e38. 8. Ricchiuti V, Sharkey SW, Murakami MM, Voss EM, Apple FS. Cardiac troponin I and T alterations in dog hearts with myocardial infarction: correlation with infarct size. Am J Clin Pathol 1998;110:241e7. 9. Schober KE, Kirbach B, Oechtering G. Noninvasive assessment of myocardial cell injury in dogs with suspected cardiac contusion. J Vet Cardiol 1999;1:17e25. 10. O’Brien PJ, Landt Y, Ladenson JH. Differential reactivity of cardiac and skeletal muscle from various species in a cardiac troponin I immunoassay. Clin Chem 1997;43:2333e8. 11. Smith SC, Ladenson JH, Mason JW, Jaffe AS. Elevations of cardiac troponin I associated with myocarditis. Experimental and clinical correlates. Circulation 1997;95:163e8. 12. Greaves K, Oxford JS, Price CP, Clarke GH, Crake T. The prevalence of myocarditis and skeletal muscle injury during acute viral infection in adults: measurement of cardiac troponins I and T in 152 patients with acute influenza infection. Arch Intern Med 2003;163:165e8. 13. Ammann P, Fehr T, Minder EI, Gunter C, Bertel O. Elevation of troponin I in sepsis and septic shock. Intensive Care Med 2001;27:965e9. 14. Alexander RW, Pratt CM, Ryan TJ, et al. Diagnosis and management of patients with acute myocardial infarction. 10th ed. New York: McGraw-Hill; 2001. p. 1275e359. 15. Fromm RE, Roberts R. Sensitivity and specificity of new serum markers for mild cardionecrosis. Curr Probl Cardiol 2001;26:241e84. 16. Sleeper MM, Clifford CA, Laster LL. Cardiac troponin I in the normal dog and cat. J Vet Intern Med 2001;15:501e3. 17. Mair J, Wagner I, Morass B, Fridrich L, Lechleitner P, Dienstl F, Calzolari C, Larue C, Puschendorf B. Cardiac troponin I release correlates with myocardial infarction size. Eur J Clin Chem Clin Biochem 1995;33:869e72.
109 18. Cummins B, Cummins P. Cardiac specific troponin-I release in canine experimental myocardial infarction: development of a sensitive enzyme-linked immunoassay. J Mol Cell Cardiol 1987;19:999e1010. 19. Rishniw M, Simpson KW. Cloning and sequencing of equine cardiac troponin I and confirmation of its usefulness as a target analyte for commercial troponin I analyzers. J Vet Diagn Invest 2005;17:582e4. 20. Cornelisse CJ, Schott 2nd HC, Olivier NB, Mullaney TP, Koller A, Wilson DV, Derksen FJ. Concentration of cardiac troponin I in a horse with a ruptured aortic regurgitation jet lesion and ventricular tachycardia. J Am Vet Med Assoc 2000;217:231e5. 21. Schwarzwald CC, Hardy J, Buccellato M. High cardiac troponin I serum concentration in a horse with multiform ventricular tachycardia and myocardial necrosis. J Vet Intern Med 2003;17:364e8. 22. Phillips W, Giguere S, Franklin RP, Hernandez J, Adin D, Peloso JG. Cardiac troponin I in pastured and race-training Thoroughbred horses. J Vet Intern Med 2003;17:597e9. 23. Begg LM, Hoffmann KL, Begg AP. Serum and plasma cardiac troponin I concentrations in clinically normal Thoroughbreds in training in Australia. Aust Vet J 2006;84:336e7. 24. Holbrook TC, Birks EK, Sleeper MM, Durando M. Endurance exercise is associated with increased plasma cardiac troponin I in horses. Equine Vet J Suppl 2006:27e31. 25. Durando MM, Reef VB, Kline K, Birks EK. Acute effects of short duration maximal exercise on cardiac troponin I in healthy horses. Equine Comp Exerc Physiol 2006;4:217e23. 26. Apple FS. Use of centaur Tn-I ultra. Clin Chem 2008;54:723e8. 27. Rifai N, Douglas PS, O’Toole M, Rimm E, Ginsburg GS. Cardiac troponin T and I, echocardiographic [correction of electrocardiographic] wall motion analyses, and ejection fractions in athletes participating in the Hawaii Ironman Triathlon. Am J Cardiol 1999;83:1085e9. 28. Urhausen A, Scharhag J, Herrmann M, Kindermann W. Clinical significance of increased cardiac troponins T and I in participants of ultra-endurance events. Am J Cardiol 2004; 94:696e8. 29. Patteson MW, Cripps PJ. A survey of cardiac auscultatory findings in horses. Equine Vet J 1993;25:409e15. 30. Piper HM, Schwartz P, Spahr R, Hutter JF, Spieckermann PG. Early enzyme release from myocardial cells is not due to irreversible cell damage. J Mol Cell Cardiol 1984;16:385e8. 31. Wayand D, Baum H, Schatzle G, Scharf J, Neumeier D. Cardiac troponin T and I in end-stage renal failure. Clin Chem 2000;46:1345e50. 32. Roppolo LP, Fitzgerald R, Dillow J, Ziegler T, Rice M, Maisel A. A comparison of troponin T and troponin I as predictors of cardiac events in patients undergoing chronic dialysis at a Veteran’s Hospital: a pilot study. J Am Coll Cardiol 1999;34:448e54. 33. Williams P, Lainchbury J. Enteritis-associated myocarditis. Heart Lung Circ 2004;13:106e9. 34. Wanby P, Olsen B. Myocarditis in a patient with salmonella and campylobacter enteritis. Scand J Infect Dis 2001;33: 860e2. 35. Slack JA, McGuirk SM, Erb HN, Lien L, Coombs D, Semrad SD, Riseberg A, Marques F, Darien B, Fallon L, Burns P, Murakami MA, Apple FS, Peek SF. Biochemical markers of cardiac injury in normal, surviving septic, or nonsurviving septic neonatal foals. J Vet Intern Med 2005;19:577e80.
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Resting concentrations of cardiac troponin I in fit horses and effect of racing* ¨ggstro ¨m, DVM, PhD Katarina Nostell, DVM, PhD*, Jens Ha Department of Clinical Sciences, Faculty of Veterinary Medicine, Swedish University of Agricultural Sciences, Box 7054, S-750 07 Uppsala, Sweden Received 22 February 2008; received in revised form 29 September 2008; accepted 1 October 2008
KEYWORDS Cardiac troponin I; Horse; Race
Abstract Objectives: To determine normal resting values for cardiac troponin I (cTnI) in healthy Standardbred, Thoroughbred and Warmblood horses and investigate if racing has an influence on cTnI concentrations. Background: Measuring cTnI concentrations in plasma is the gold standard for detecting myocardial injury in humans. Cardiac troponin I is highly conserved between species and has gained interest as a marker for cardiac injury in horses. Increased levels of cTnI have been reported in association with endurance and short-term strenuous exercise on a treadmill in horses. However, the effect of true racing conditions has not yet been reported. Animals, materials and methods: Blood samples for analysis of cTnI concentrations in plasma were collected from 67 Standardbred racehorses, 34 Thoroughbred racehorses and 35 Warmblood dressage horses at rest. Blood samples were also collected prior to and after racing in 22 Standardbred racehorses and 6 Thoroughbred racehorses. Results: All horses except one had resting plasma cTnI concentrations <0.022 mg/L. Mild increases in cTnI concentrations were seen in some horses 1e2 h after the race (1/17 Standardbreds and 2/6 Thoroughbreds) as well as 10e14 h after the race (4/ 21 Standardbreds and 1/6 Thoroughbreds). Conclusions: Resting cTnI concentrations in horses are low but mildly elevated cTnI concentrations may be detected in some horses 1e14 h after racing. These findings could be of importance when evaluating horses with suspected cardiac disease that recently have performed hard exercise. ª 2008 Elsevier B.V. All rights reserved.
*
Funding for this study was provided by the Swedish Foundation for Equine Research, Sweden. * Corresponding author. E-mail address: [email protected] (K. Nostell).
1760-2734/$ - see front matter ª 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jvc.2008.10.001
106
Introduction Myocardial injury is difficult to detect and diagnose in horses. The traditional methods that have been used to detect suspected myocardial injury are evaluation of electrocardiography (ECG) and echocardiography. These methods have limitations and therefore attempts have been made to find more sensitive markers for myocardial injury in the horse and biomarkers have provided new options to detect cardiac injury in different species. Increased concentrations of isoenzymes of creatine kinase (CK-MB) and lactate dehydrogenase (LDH-1 and LDH-2) have been used clinically as markers for myocardial injury in the horse. These enzymes have limitations due to the occurrence of cross-reactions with skeletal muscle and a short half-life in serum.1e6 In recent years cardiac troponin I (cTnI), a protein involved in the interaction between actin and myosin during contraction, has gained interest as a clinical marker for myocardial injury in different species. cTnI is known to leak out from injured myocardial cells and has been shown to be a sensitive and specific marker for myocardial injury in humans and dogs.2,7e10 In humans, cardiac troponin I is mainly used for diagnosing myocardial infarction but is also elevated in other conditions such as myocarditis and sepsis.11e13 In healthy humans and dogs, plasma concentrations of cTnI are generally low and usually increase between 3 and 8 h after initial myocardial injury, and in humans the test reaches reliable diagnostic sensitivity by 12e16 h.9,14e16 The concentration of cTnI in serum returns to normal within 5e12 days after an insult and the degree of increase in cTnI concentration is correlated to the degree of injury.8,14,17,18 Measuring cTnI concentrations in serum or plasma is therefore of value to determine the prognosis after myocardial injury in humans and dogs. The amino acid sequence for equine cTnI has recently been characterised and evidence supports the use of commercial human assay systems to measure equine cardiac troponin I.19 The improved sensitivity, specificity, and the fact that the concentration of cTnI shows a long persistence in the circulation in humans make cTnI an interesting marker for cardiac injury in horses. Case reports on horses with increased cTnI concentrations and documented myocardial disease have been published.20,21 Normal resting values for cardiac troponin I in horses have been reported and are generally very low, although cTnI values cannot be compared between assays.21e23 It could be expected that horses, depending on their use and
K. Nostell, J. Ha ¨ggstro ¨m exercise level and time of sampling, vary in concentrations of cTnI. Increased concentrations of cTnI have been reported in horses in association with endurance competition as well as after shortterm maximal exercise on a treadmill for 2000e 2400 m.24,25 However, the effect of short-term high intensity exercise during true race conditions, such as a trotting race, has not yet been reported. Knowledge about what effect a race has on cTnI concentrations is of importance when evaluating horses with suspicion of cardiac disease that have recently raced or performed maximal exercise. The aims of the present study were to study cTnI concentrations in plasma in healthy fit horses of different breeds and to see if racing influences cTnI concentrations.
Animals, materials and methods Resting concentrations of cTnI were measured in 67 Standardbred racehorses, 34 Thoroughbred racehorses and 35 Warmblood horses (Study 1). The Standardbred trotters (27 mares, 21 geldings, 19 stallions) were between 3 and 10 years old, the Thoroughbreds between 2 and 9 years old (15 mares, 16 geldings, 3 stallions) and the Warmbloods between 6 and 24 years old (7 mares, 20 geldings and 8 stallions). In addition to this, 22 Standardbred racehorses (9 mares, 4 geldings, 9 stallions) between 3 and 8 years old and 6 Thoroughbred racehorses (3 geldings and 3 mares) between 3 and 6 years old, were included in a separate study investigating the effect of racing on cTnI concentrations (Study 2). Four of the Standardbreds were also used in the first part of the study. All racehorses included in the study were fit racehorses in professional training and all Warmbloods were dressage horses that trained and competed at a high national level (FEI Prix St. George and above). Some horses (2/67 Standardbreds, 2/34 Thoroughbreds and 3/35 Warmbloods) had low intensity right sided systolic murmurs (1e2/6) on auscultation of the heart. None of the horses included in the study showed signs of disease on clinical examination or had a history of poor performance. The study was sanctioned by the Ethical Committee for Animal Experiments, Uppsala, Sweden. All resting blood samples in study 1 were collected at rest in the box at the training campus. In the 28 horses enrolled in study 2 blood samples were collected 2 days prior to the race at rest in the box at the training campus, within 2 h after the race and the day after the race (10e14 h post-race).
Cardiac troponin I in fit horses Blood samples were drawn by vacutainer technique from the jugular vein and collected in heparinised tubes. Samples were centrifuged and plasma separated within 30 min after collection and all samples were kept cool until frozen at 20 C (within 3 h after collection). The samples were kept frozen at 20 C until analysed within 6 months after collection. Repeated analysis of a sample with a cTnI concentration of 42 mg/L was conducted after 6 months and the concentration was then 47 mg/L (11% increase). This suggests that little degradation occurs after a storage period of 6 months. Plasma concentrations of cTnI were measured with an enzyme immunoassaya with an analytical sensitivity of <0.022 mg/L. The assay was evaluated for use in equine samples by macerating horse heart muscle and performing a dilution curve which showed an F value of <0.0001. To evaluate the ability of the assay to detect horses with increased cTnI concentrations, blood samples were also collected from three horses (horses A, B and C) with suspicion of primary myocardial disease. These horses all had signs of myocardial disease on clinical examination such as ventricular extra systoles (horse A), tachycardia at rest and extra systoles (horse B), and ventricular tachycardia and atrial fibrillation (horse C). Increased cTnI concentrations were observed in these horses (horse A: 1.3 mg/L, Horse B: 0.40 mg/L and Horse C: 42 mg/L). Two of the horses were necropsied; horse A was diagnosed with cardiomyopathy with acute and chronic myocardial necrosis and horse B with acute myocardial necrosis.
Results 97% (65/67) of the Standardbreds, 94% (32/34) of the Thoroughbred racehorses and 100% (35/35) of the Warmblood dressage horses had resting cTnI concentrations <0.022 mg/L. Four horses (2/67 Standardbreds and 2/34 Thoroughbreds) had resting cTnI concentrations that exceeded 0.022 mg/L (Standardbreds: 0.023 and 0.025 mg/L, Thoroughbreds: 0.043 mg/L and 0.025 mg/L), none of these four horses had murmurs on auscultation. In the horses tested after racing, a complete set of cardiac troponin I values were obtained in 15 of the 22 Standardbreds and all of the Thoroughbreds. At rest 90% (19/21) of the Standardbreds and 83% (5/6) of the Thoroughbreds had cTnI concentrations below the detection level of 0.022 mg/L. Three horses (two Standardbreds and one Thoroughbred) showed pre-race concentrations of cTnI a Architect CI8200, Abbot Laboratories, Diagnostic Division, Abbott Park, IL.
107 that exceeded 0.022 mg/L. Mild increases in cTnI concentrations were found in some horses (5/22 Standardbreds and 3/6 Thoroughbreds) both 1e2 h post-race and 10e14 h post-race (Table 1).
Discussion The results from this study show that horses generally have low cTnI concentrations at rest but that intense short-term exercise, such as a trotting race or a Thoroughbred race, induces mild increases in cTnI concentrations in some horses. This is in agreement with a previous study on fit performance horses where a tendency for increased plasma cTnI concentrations was seen in all horses 3e6 h after they had performed shortterm high intensity exercise for a distance of 2000e2400 m on a treadmill.25 This study also reported that some individuals still had cTnI concentrations that were above the limit of detection at 12 and 24 h post-exercise. In the present study, it is possible that more horses would have shown increases in cTnI concentrations if blood samples had been collected more frequently after the race. However, this was not possible for practical reasons. Another limitation in the present study is the inability of the test to reliably measure cTnI concentrations below 0.022 mg/L. In humans new ultra sensitive cTnI assays are able to identify myocardial damage, at concentrations well below this (0.006e0.04 mg/L).26
Table 1 Horses with plasma cTnI concentrations that were above the analytical sensitivity limit for one of the three sampling periods. Age Gender Breed
cTnI concentration (mg/L) Pre-race
8 6 4 5 5 8 6 5 3
S M S G S M M G M
SB SB SB SB SB SB TB TB TB
0.025a <0.022 <0.022 <0.022 <0.022 0.023a <0.022 <0.022 0.025a
1e2 10e14 h Post-race Post-race 0.029a <0.022 <0.022 <0.022 <0.022 <0.022 0.028a <0.022 0.027a
<0.022 0.025a 0.023a 0.023a 0.035a <0.022 <0.022 0.023a <0.022
S: stallion; M: mare; G: gelding; SB: Standardbred; TB: Thoroughbred. In addition to the reported results, 16 Standardbred horses (9 with complete profiles) and 3 Thoroughbred horses (all with complete profiles) had cTnI concentrations <0.022 mg/L at each of their testing intervals. a result above the analytical sensitivity of the test.
108 The reasons behind the increase found in cTnI concentrations after hard exercise are not clear. cTnI is reported to have a very high specificity for myocardium and it is therefore unlikely that these increases are related to cross-reactivity with skeletal muscle.10,19 The method used in this study also has a low reported cross-reactivity (0.07%) with skeletal muscle in humans. In the study by Durando et al., none of the horses with increased cTnI after exercise had signs of cardiac disease on ECG or echocardiography. Increased concentrations of cTnI have also been reported in connection with endurance exercise in horses and humans without signs of cardiac disease.24,25,27,28 The low grade right sided systolic murmurs heard in some horses in the present study were likely due to tricuspid insufficiency. This is a frequent finding in performance horses which is rarely associated with clinically significant changes in the valves.29 As only a thorough clinical examination was performed on the horses in the present study, it could be speculated that some of the horses might have mild cardiac disease causing the increases seen in cTnI after exercise. However, this is regarded unlikely as all horses were in professional training with experienced trainers and raced on regular basis with no suspicion or history of impaired performance. It is therefore likely that the increases seen in plasma cTnI in some horses are related to exercise. One theory is that a mild hypoxia during exercise could cause a change in the permeability of the myocardium causing a leakage of macromolecules into the blood. This is supported by the fact that a previous study in rats has shown that short periods of hypoxia can induce release of cTnI without cell death.30 The fact that some horses had small increases in cTnI concentrations could to a certain degree be normal as the interday variability of cardiac troponin I in fit Standardbreds and Thoroughbreds has not been evaluated. They could also be attributed to the kind of exercise the horses had performed the previous day. It is not likely that these differences were attributed to a difference in fitness level as a previous study that compared concentrations of cTnI in race trained Thoroughbreds and Thoroughbreds on pasture found no difference between groups.22 The exercise induced increases in cTnI were mild and not comparable to the concentrations found in the horses with signs of cardiac disease. The increased concentrations of cTnI together with the clinical symptoms and findings on ECG, echocardiography and the findings on necropsy indicate that the assay used in this study is able to detect horses with cardiac disease. Previous
K. Nostell, J. Ha ¨ggstro ¨m studies on humans have shown a correlation between the degree of myocardial infarction size (indicating myocardial damage) and the release of cTnI.17 Although myocardial infarcts are seldom seen in horses, there is reason to believe that an association between the severity of myocardial damage and the degree of increase in cTnI also exists in horses. It is therefore likely that the horse with the highest plasma cTnI concentrations (horse C) in this study also had the most generalised myocardial injury. Unfortunately the horse was not necropsied so this claim could not be confirmed. The individual differences in cTnI concentrations are also influenced by the time lapse between myocardial damage and sampling. In humans, cTnI concentrations have been shown to increase in other conditions that are primarily extra-cardiac such as sepsis and kidney failure.13,31,32 One theory is that these elevations might reflect toxic or inflammatory damage to the heart as opposed to ischemic injury. Elevated cTnI concentrations and evidence of myocarditis have also been reported in patients with bacterial enteritis and in septic foals.33,34,35
Conclusions and clinical implications Resting cTnI concentrations in horses are low, but can increase slightly above the baseline level in some horses 11e14 h after racing. A conclusion whether these elevated cTnI concentrations should be considered as normal for horses that have recently raced or are a reflection of myocardial disease cannot be drawn form the results of this study. However, it seems unlikely that the horses in the present study had cardiac disease since they were performing well. It is also important to remember that cTnI numbers may vary between assays and values can never be directly compared from one assay to another. Mildly elevated concentrations of cTnI alone will unlikely lead to a definitive diagnosis, but together with the clinical presentation and findings on ECG and echocardiography it can become an important marker for myocardial disease in horses with impaired performance. Marked elevations of cTnI alone could be considered as a strong indication of myocardial disease. Further studies are needed on a larger population of horses with signs of cardiac disease to establish the correlation between elevated concentrations of cTnI and pathological findings. Likewise, further studies are needed to establish ‘normal’ post-exercise/post-race cTnI reference ranges in horses and to evaluate cTnI levels in poorly performing horses.
Cardiac troponin I in fit horses
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