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Praca oryginalna Original paper
The measurement of creatine kinase (CK) activity is one of the biochemical parameters determined in the blood plasma. Assessment of this parameter finds its practical application in the fitness of athletic horses (10, 11).
CK (EC 2.7.3.2) is a muscle specific enzyme which takes part in energy metabolism during muscle contr-action. During exercise, permeability of the muscle cell membrane alternates in such a way that proteins such as CK can leave the inside of a muscle cell through this membrane and are liberated into the bloodstream (10). It is a well known fact that the effect of exercise on plasma CK activity depends on the intensity and duration of the exercise as well as the age, sex, race and fitness of the horses studied (10, 11). Yet the reason for this transient increase of the muscle cell membrane permeability for CK after exercise is not yet fully understood. As a result, the normal rise in CK activity in response to exercise has not been established up till now. However, it is known that the blood plasma CK activity reaches peak values within a few hours of an effort episode and is quickly remo-ved from the bloodstream (T1/2 amounts about 2 h).
Muscular disorders called tying-up, muscle stiffness or rhabdomyolysis (RER) bring about an increased level of plasma CK activity. Due to this fact, CK determination during exercise tests can also be used as a diagnostic tool in the identification of some muscular disorders (10, 30, 31).
Recent studies indicate that a high plasma glucose level is positively correlated with plasma CK activity. Zeyner et al. (34) noticed that basal plasma glucose and CK values showed a high positive correlation. Moreover, a low-starch and high-fat diet decreased plasma glucose concentration as well as CK activity in horses (34). Van Weyenberg et al. (32) have also stated that the decrease in plasma glucose level after L-carnitine supplementation resulted in lower CK activity in healthy ponies.
On the other hand, it is also well known that hyper-glycemia can induce muscle damage in several ways (3, 7, 20, 25, 29). A high increase of glucose concen-tration can generate the formation of reactive oxygen species (ROS). This, in turn, induces peroxidation pro-cesses resulting in disturbances of cellular membrane structure, its depolarization, and loss of cellular
mem-Correlation of plasma creatine kinase activity and
glucose level in exercised Purebred Arabian horses
WITOLD KÊDZIERSKI
Department of Animal Biochemistry and Physiology, Faculty of Veterinary Medicine, University of Life Sciences, Akademicka 12, 20-033 Lublin, Poland
Kêdzierski W.
Correlation of plasma creatine kinase activity and glucose level in exercised Purebred Arabian horses
Summary
This study tests the hypothesis found in professional literature that increased plasma creatine kinase (CK) activity in horses is a direct result of a rise in the plasma glucose level.
Participants were 24 three-year-old Purebred Arabian mares and 67 three to six-year-old Purebred Arabian stallions. From the total of 91 horses, 29 of the horses were studied only once and 62 were studied two, three or four times. As a result, the total number of studied exercise tests amounted to 176. All horses were being tested whilst undergoing race training. On the days of the study, the horses took part in a conventional training session. Three blood samples from the jugular vein were collected from each horse. These samples were taken while 1) at rest, 2) after the end of training session and 3) thirty min. after the end of effort. In the obtained plasma samples, glucose level and CK activity were determined using spectrophotometric diagnostic kits.
The exercise regime which the studied horses had undertaken evoked a statistically significant increase in both means of the analyzed parameters. An analysis of results using a coefficient correlation indicated that there was no correlation between the changes in plasma glucose level and CK activity. The present study does not seem to support the hypothesis of increased plasma CK activity in horses being the result of a rise in the plasma glucose level.
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brane integrity (28, 35). An increase in blood glucose concentration in horses can be the result of high--intensity exercise (9, 11, 22). It is possible that an exercise-induced rise in the circulating glucose con-centration in horses influences the CK liberation from the muscle cells.
The aim of this study was to test the hypothesis, that changes in the plasma glucose level correlate positi-vely with CK activity in exercised horses.
Material and methods
Horses. A total of 91 clinically healthy young Purebred Arabian horses were studied. All horses were in race training and they competed in official races. The group in this study consisted of: 24 three-year-old mares and 67 three to six-year-old stallions. From the total of 91 horses, 29 horses were studied only once and 62 were studied two, three or four times. As a result, the total number of studied exercise tests amounted to 176.
During the experiment, the horses were kept and trained in a local training centre or at the racecourse (Sluzewiec, Warsaw, Poland). All horses were housed in the racecourse stables in one-horse boxes. They were fed individually with a diet consisting of typical race horse fodder in the amounts suitable for the individual energy demands of a given horse. The horses housed in the local training centre received oats, hay and carrots, supplemented with a mineral-vitamin premix. The horses trained at the race-course were fed with bruised oats and a high-energy blend. All the animals were fed three times per day and had unlimited access to a salt lick and fresh water.
Prior to the start of the observations, the horses had been conditioned through a standard training program. The type of training, as well as its intensity, were consistent with the principles of the trainers preparing the horses for races.
Training program. Generally, two different types of exercises were used in the training program and as studied tests. At the beginning of each training season, the horses started with endurance exercises. The horses walked and trotted in field conditions for about one hour, and cantered to cover a distance of approximately 500 m at a speed of 6 m/s. This type of activity was performed three times a week. The distance and speed were gradually increased as the individual horses showed improvement. The number of studied endurance-exercise tests amounted to 72.
After about three months of endurance type exercises, the horses started regular race training. The daily training routine consisted of a warm up, a gallop with a mean speed of 8-12 m/s to cover a distance of 800-1200 m on a race--track, and a cooling down on a horse walker. Race training took place five days a week. The number of studied race training sessions amounted to 104.
Exercise test protocol. On the days of the study, the horses which performed the endurance-training session took part in the exercise test consisting of a 5 min. warm up with a walk, a 30-60 min. trot and gallop on the ground track at a mean speed of 6.0 m/s, and a 10 min. slow trot as the cool down. The horses involved in race training, performed an exercise test consisting of a 10 min. warm up trot, galloping to cover the distance of 800-1200 m at an approximate speed of 9-13 m/s, and a 5 min. trot. In general, the time and intensity of gallops were appropriate for the age and fitness level of the trained horses. No symptoms of painful muscle contractions or stiffness were observed after effort. Blood sampling and laboratory analysis. Three blood samples were collected from the jugular vein of each horse via venipuncture. The three samples were taken as follows: at rest, immediately after the training session, and 30 min. after the end of effort. The samples were collected into lithium heparin tubes and centrifuged within 30 min. for plasma separation. The plasma samples were stored at 4°C. They were then analyzed within 8 h to assess the glucose level and within 72 h to evaluate the CK activity using spectrophotometric diagnostic kits (Cormay, Poland, and Dr Lange, Germany, respectively). The obtained values were expressed as mmol/l and U/l, respectively.
Statistical analysis. The results were presented as means ± standard deviations (SD). Comparisons between results obtained for consecutive groups of horses were analyzed using the Tukey test (ANOVA). The statistical significance was accepted at the level of p £ 0.05. The coefficient correlation was also calculated to compare the changes in plasma glucose concentrations and CK activity.
Results and discussion
Means ± SD for measured parameters are showed in tab. 1. The endurance exercise test induced a sta-tistically significant decrease in plasma glucose level when determined just after the end of effort as com-pared to the values obtained at rest. A 30 min. rest Tab. 1. Plasma glucose level and CK activity determined in Purebred Arabian horses during the exercise tests (means ± SD)
Explanation: a b c means in rows with different superscripts differ at p £ 0.05
s e s r o h f o p u o r G n ]l /l o m m [ e s o c u l G CK[U/]l t s e r t A Aotffeerxtheerciesned 30emndino.faetfefforttrhe Atrest Aotffeerxthereceisned 30emndino.faetfeffortrthe t s e t e c n a r u d n E 172 6.65±8.84a 5.94±0.54b 6.15±0.70b 139±37.5a 231±174b 260±280b t s e t d e e p S 104 6.00±0.60a 7.19±1.72b 6.50±1.38c 162±104a 235±178b 243±351b s e s r o h ll A 176 6.27±0.78a 6.68±1.50b 6.36±1.16a 152±84.0a 233±176b 250±323b A p u o r g b u S 154 6.24±0.71a 6.76±1.54b 6.45±1.20ab 147±44.4a 187±57.8b 188±61.7b B p u o r g b u S 112 6.05±0.85a 6.06±0.87b 5.45±0.64ab 141±77.8a 354±193b 459±818b C p u o r g b u S 110 6.86±1.25a 6.46±1.12b 5.86±0.62ab 156±58.6a 691±213b 553±182b
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allowed for the plasma glucose concentra-tion to return to its resting level. After the post-exercise decrease, the level of plasma glucose continued to fall into the normal range (15). The results of other studies indicate that a decrease in plasma glucose level is characteristic for endurance type of exercises (12, 24). In the studied horses, the race exercise test caused a transient, significant increase in the plasma glucose level measured just after the end of effort, in comparison to the values obtained at rest. During the subsequent 30 min. rest, the level of this parameter decreased, but it was still statistically higher than at rest. An increase in plasma glucose level influenced by short-term, intensive exercise was also found to take place in other studies (11, 22). Generally, the plasma glucose level reflects the considerable speeding up of glycogeno-lysis in the liver. Such an action happens e.g. under the influence of a raised naline level in the blood (11). Blood adre-naline concentration in horses correlates positively with intensity of exercise (26).
In both types of exercise tests, plasma CK activity determined just after effort and after a 30 min. rest were significantly higher than the resting values. The increase in plasma CK activity varied between individual horses. In extreme cases, the increase in plasma CK activity reached values five-times higher
than those obtained at rest. However, any clinical epi-sodes of muscle stiffness or RER were carefully stated. On the basis of the obtained results, the total number of horses studied was divided into three subgroups marked A, B and C. Subgroup A consisted of horses in which plasma CK activity after exercise increased but the increased level was not more than double the resting values. Cases featuring plasma CK increase that were more than double the resting value, but not higher than threefold were placed in subgroup B. Only 10 cases had displayed a rise in CK activity exceeding the threefold increase on the resting value. These cases were additionally separated into subgroup C. The most important post-exercise increase in plasma glucose level was observed in subgroup A, whereas in subgroup C, the value of this parameter tended to decrease (tab. 1). This observation suggests that there does not exist a relationship between the increase in plasma glucose level and CK activity in exercised horses. Moreover, no correlation was found between the changes in plasma glucose level and CK activity in the studied horses (tab. 2). The coefficient correla-tion was statistically insignificant in all the analyzed groups of horses, except subgroup C (tab. 2). In this last subgroup there was a statistically significant nega-tive correlation between the analyzed parameters. All
the facts mentioned above indicate that the post-exer-cise increase in plasma CK activity in studied horses occurred regardless of the level of plasma glucose.
The results found in other studies also indicate that the changes in plasma glucose level and CK activity occurred independently in exercised horses. For exam-ple, endurance rides involved a very high increase in plasma CK activity (8, 33) and a decrease in plasma glucose level (12, 24). On the other hand, short-term, intense bouts of exercise led to an important rise in plasma glucose concentration (11, 22). However, the increase in plasma CK activity after this kind of exercise, although important (2, 5, 11), was still less expressed than plasma CK activity after endurance rides (8, 33). This means that a prolonged period of exercise resulted in a decreased plasma glucose level and a simultaneous increase in CK activity (18, 27). Moreover, in horses showing the RER symptoms, plasma CK activity determined after exercise was not associated with the plasma glucose level (4, 16, 17, 19, 23). Therefore, it cannot be stated that changes in plasma glucose level correspond with plasma CK activity in exercised horses. Furthermore, a positive correlation between plasma CK and glucose values was found only in horses studied at rest (32, 34) whereas the present study concerns the exercised horses. Explanations: Ä the difference between values obtained after the end of exer-cise and at rest; Ä the difference between values obtained 30 min. after the end of effort and at rest; values in bold are statistically significant
Tab. 2. Values of coefficient correlation for compared plasma glucose and CK levels r e t e m a r a P CKeandtferofthe e s i c r e x e D KC . n i m 0 3 K C d n e e h t r e tf a tr o ff e f o D K'C 2 7 = n ,t s e t e c n a r u d n E e s i c r e x e f o d n e e h t r e tf a e s o c u l G 0.058 0.082 0.022 0.034 Dglucose 0.255 0.302 0.178 0.200 4 0 1 = n ,t s e t d e e p S e s i c r e x e f o d n e e h t r e tf a e s o c u l G 0.072 0.047 0.129 0.135 Dglucose 0.065 0.045 0.078 0.076 6 7 1 = n , s e s r o h ll A e s i c r e x e f o d n e e h t r e tf a e s o c u l G 0.054 0.070 0.108 0.122 Dglucose 0.082 0.130 0.095 0.123 4 5 1 = n , A p u o r g b u S e s i c r e x e f o d n e e h t r e tf a e s o c u l G 0.105 0.044 0.117 0.066 Dglucose 0.131 0.020 0.148 0.052 2 1 = n , B p u o r g b u S e s i c r e x e f o d n e e h t r e tf a e s o c u l G 0.230 0.194 0.301 0.303 Dglucose 0.143 0.160 0.073 0.070 0 1 = n , C p u o r g b u S e s i c r e x e f o d n e e h t r e tf a e s o c u l G 0.101 0.022 0.636 0.656 Dglucose 0.220 0.245 0.465 0.507
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Generally, the rise in plasma CK activity is the result of the increased muscle cell membrane permeability. Such cell membrane permeability may result from the influence of oxidative stress that occurs during exer-cise (5, 8). Oxidative stress leads to muscle damage by increased protein and lipid oxidation (4, 6, 13, 33). It is also known that high glucose concentration induces oxidative stress and inhibition of enzyme membrane activity, indicating uncoupling of oxidative phosphorylation in mitochondria (28). The study on cell cultures indicates that mitochondrial CK plays a key role as an antioxidant preventive against oxida-tive stress, reducing ROS production, particularly in high glucose concentration (21). In this light, a proba-ble reason for the increased post-exercise plasma CK activity in the studied horses can be found in muscle damage resulting from the influence of oxidative stress, rather than being caused by a high glucose level. The differences in plasma CK activity stated in the indivi-dual horses may result from different antioxidant status, which depend e.g. on horse performance and intensity of exercise (1, 14).
In conclusion, the hypothesis that plasma CK activity is dependent on plasma glucose level was not supported in this study. The post-exercise increase in plasma CK activity in healthy horses cannot be reco-gnized as the result of transient hyperglycemia.
References
1.Avellini L., Chiaradia C., Gaiti A.: Effect of exercise training, selenium and vitamin E on some free radical scavangers in horses (Equus caballus). Comp. Biochem. Physiol. B Biochem. Mol. Biol. 1999, 123, 147-154.
2.Balogh N., Gaál T., Ribiczeyné P. S., Petri A.: Biochemical and antioxidant changes in plasma and erythrocytes of pentathlon horses before and after exercise. Vet. Clin. Pathol. 2001, 30, 214-218.
3.Bjarnegard N., Arnqvist H. J., Lindstrom T., Jonasson L., Jonsson A., Lanne T.: Long-term hyperglycaemia impairs vascular smooth muscle cell function in women with type 1 diabetes mellitus. Diab. Vasc. Dis. Res. 2009, 6, 25-31. 4.Borgia L. A., Valberg S. J., McCue M. E., Pagan J. D., Roe C. R.: Effect of
dietary fats with odd or even numbers of carbon atoms on metabolic response and muscle damage with exercise in Quarter horse-type horses with type 1 poly-saccharide storage myopathy. Am. J. Vet. Res. 2010, 71, 326-336.
5.Chiaradia E., Avellini L., Rueca F., Spaterna A., Porciello F., Antonioni M. T., Gaiti A.: Physical exercise, oxidative stress and muscle damage in racehorses. Comp. Biochem. Physiol. B 1998, 119, 833-836.
6.Ducharme N. G., Fortier L. A., Kraus M. S., Hobo S., Mohammed H. O., McHugh M. P., Hackett R. P., Soderholm L. V., Mitchell L. M.: Effect of a tart cherry juice blend on exercise-induced muscle damage in horses. Am. J. Vet. Res. 2009, 70, 758-763.
7.Dyntar D., Eppenberger-Eberhardt M., Maedler K., Pruschy M., Eppenberger H. M., Spinas G. A., Donath M. Y.: Glucose and palmitic acid induce degenera-tion of myofibrils and modulate apoptosis in rat adult cardiomyocytes. Diabetes 2001, 50, 2105-2113.
8.Frankiewicz-Jóko A., Szarska E.: Anti-oxidant level to exercise in the blood of endurance horses. Biol. Sport 2000, 17, 217-227.
9.Gordon M. E., McKeever K. H., Betros C. L., Manso Filho H. C.: Exercise--induced alternations in plasma concentrations of ghrelin, adiponectin, leptin, glucose, insulin, and cortisol in horses. Vet. J. 2007, 173, 532-540.
10.Harris P. A., Marlin D. J., Gray J.: Plasma aspartate aminotransferase and creatine kinase activities in Thoroughbred racehorses in relation to age, sex, exercise and training. Vet. J. 1998, 155, 295-304.
11.Kêdzierski W., Bergero D.: Comparison of plasma biochemical parameters in Thoroughbred and Purebred Arabian horses during the same-intensity exercise. Polish J. Vet. Sci. 2006, 9, 233-238.
12.Kêdzierski W., Podolak M.: Wp³yw treningu koni rasy arabskiej na poziom parametrów biochemicznych zwi¹zanych z gospodark¹ wêglowodanowo-lipi-dow¹. Medycyna Wet. 2002, 58, 788-791.
13.Kinnunen S., Hyyppä S., Lappalainen J., Oksala N., Venojärvi M., Nakao C., Hänninen O., Sen C. K., Atalay M.: Exercise-induced oxidative stress and muscle stress protein responses in trotters. Eur. J. Appl. Physiol. 2005, 93, 496--501.
14.Korhonen P. A. S., Lilius E. M., Hyyppa S., Räsänen L. A., Pösö A. R.: Produc-tion of reactive oxygen species in neutrophils after repeated bouts of exercise In Standardbred trotters. J. Vet. Med. A. 2000, 47, 565-573.
15.Krumrych W.: Wskaniki laboratoryjne krwi koni wartoci referencyjne i in-terpretacja. Pañstwowy Instytut Weterynaryjny, Pu³awy 2003.
16.MacLeay J. M., Valberg S. J., Pagan J. D., De La Corte F. D., Roberts J., Billstrom J., McGinnity J., Kaese H.: Effect of diet on Thoroughbred horses with recurrent rhabdomyolysis performing a standardised exercise test. Equine Vet. J. Suppl. 30, 1999, 458-462.
17.MacLeay J. M., Valberg S. J., Pagan J. D., Xue J. L., De La Corte F. D., Roberts J.: Effect of ration and exercise on plasma creatine kinase activity and lactate concentration in Thoroughbred horses with recurrent exertional rhabdomyolyysis. Am. J. Vet. Res. 2000, 61, 1390-1395.
18.Marlin D. J., Fenn K., Smith N., Deaton C. D., Roberts C. A., Harris P. A., Dunster C., Kelly F. J.: Changes in circulatory antioxidant status in horses. J. Nutr. 2002, 132, 1622S-1627S.
19.McKenzie E. C., Valberg S. J., Godden S. M., Pagan J. D., MacLeay J. M., Geor R. J., Carlson G. P.: Effect of dietary starch, fat, and bicarbonate content on exercise responses and serum creatine kinase activity in equine recurrent exertional rhabdomyolysis. J. Vet. Intern. Med. 2003, 17, 693-701.
20.Meugnier E., Faraj M., Rome S., Beauregard G., Michaut A., Pelloux V., Chiasson J. L., Laville M., Clement K., Vidal H., Rabasa-Lhoret R.: Acute hyperglycemia induces a global downregulation of gene expression in adipose tissue and skeletal muscle of healthy subjects. Diabetes 2007, 56, 992-999. 21.Meyer L. E., Machado L. B., Santiago A. P., da-Silva W. S., De Felice F. G.,
Holub O., Oliveira M. F., Galina A.: Mitochondrial creatine kinase activity prevents reactive oxygen species generation: antioxidant role of mitochondrial kinase-dependent ADP re-cycling activity. J. Biol. Chem. 2006, 281, 37361--37371.
22.Podolak M., Kêdzierski W., Janczarek I.: Wp³yw intensywnego treningu na poziom wybranych parametrów biochemicznych krwi i liczbê têtna u koni rasy arabskiej. Medycyna Wet. 2004, 60, 403-406.
23.Ribeiro W. P., Valberg S. J., Pagan J. D., Gustavsson B. E.: The effect of varying dietary starch and fat content on serum creatine kinase activity and substrate availability in equine polysaccharide storage myopathy. J. Vet. Intern. Med. 2004, 18, 887-894.
24.Rose R. J., Hodgson D. R., Sampson D., Chan W.: Changes in plasma bioche-mistry in horses competing in a 160 km endurance ride. Aust. Vet. J. 1983, 60, 101-105.
25.Russell S. T., Rajani S., Dhadda R. S., Tisdale M. J.: Mechanism of induction of muscle protein loss by hyperglycaemia. Exp. Cell Res. 2009, 315, 16-25. 26.Snow D. H., Harris R. C., MacDonald I. A., Forster C. D., Marlin D. J.:
Effects of high-intensity exercise on plasma catecholamines In the Thorough-bred horse. Equine Vet. J. 1992, 24, 462-267.
27.Stopyra A.: Wskaniki gospodarki tlenowej i aktywnoæ wybranych enzymów surowicy koni w warunkach ekstremalnego wysi³ku. Medycyna Wet. 2002, 58, 543-548.
28.Szczepañski M., Kamianowska M., Skrzydlewska E.: Wp³yw hiperglikemii na procesy oksydacyjno-redukcyjne w komórkach ródb³onka ludzkiej ¿y³y pêpowinowej. Pol. Merkur. Lekarski 2007, 23, 246-250.
29.Thompson L. H., Kim H. T., Ma Y., Kokorina N. A., Messina J. L.: Acute, muscle-type specific insulin resistance following injury. Mol. Med. 2008, 14, 715-723.
30.Valberg S. J., MacLeay J. M., Billstrom J. A., Hower-Moritz M. A., Mickelson J. R.: Skeletal muscle metabolic response to exercise in horses with tying-up due to polysaccharide storage myopathy. Equine Vet. J. 1999, 31, 43-47. 31.Valberg S. J., Mickelson J. R., Gallant E. M., MacLeay J. M., Lentz L., de la
Corta F.: Exertional rhabdomyolysis in quarter horses and Thoroughbreds: one syndrome, multiple aetiologies. Equine Vet. J. Suppl. 1999, 30, 533-538. 32.Van Weyenberg S., Buyse J., Janssens G. P. J.: Increased plasma leptin through
L-carnitine supplementation is associated with an enhanced glucose tolerance in healthy ponies. J. Anim. Physiol. Anim. Nutr. 2009, 93, 203-208.
33.Williams C. A., Kronfeld D. S., Hess T. M., Saker K. E., Waldron J. E., Crandell K. M., Harris P. A.: Comparison of oxidative stress and antioxidant status in endurance horses in three 80-km races. Equine Comp. Exerc. Physiol. 2005, 2, 153-157.
34.Zeyner A., Hoffmeister C., Einspanier A., Gottschalk J., Lengwenat O., Illies M.: Glycaemic and insulinaemic response of quarter horses to concentrates high in fat and low in soluble carbohydrates. Equine Vet. J. Suppl. 2006, 36, 643-647. 35.Zhang W., Wang X. H., Chen S. F., Zhang G. P., Lu N., Hu R. M., Jin H. M.: Biphasic response of endothelial progenitor cell proliferation induced by high glucose and its relationship with reactive oxygen species. J. Endocrinol. 2008, 197, 463-470.
Authors address: Witold Kêdzierski PhD, Akademicka 12, 20-033 Lublin; e-mail: witold.kedzierski@up.lublin.pl
