Effect of exercise on serum concentration of cartilage oligomeric matrix protein in Thoroughbreds

Ibrahim E. Helal Department of Veterinary Surgery, Kagoshima University, 21-24 Korimoto 1-chome, Kagoshima 890-0065, Japan.
Department of Veterinary Surgery and Radiology, Suez Canal University, Ismailia, Egypt.

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Kazuhiro Misumi Department of Veterinary Surgery, Kagoshima University, 21-24 Korimoto 1-chome, Kagoshima 890-0065, Japan.

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Oki Tateno Miyazaki Training Farm, Japan Racing Association, 2347 Ohhara Hanagashima-cho, Miyazaki 880-0036, Japan.

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Takeshi Kodama Miyazaki Training Farm, Japan Racing Association, 2347 Ohhara Hanagashima-cho, Miyazaki 880-0036, Japan.

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Mutsuki Ishimaru Miyazaki Training Farm, Japan Racing Association, 2347 Ohhara Hanagashima-cho, Miyazaki 880-0036, Japan.

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Junya Yamamoto Miyazaki Training Farm, Japan Racing Association, 2347 Ohhara Hanagashima-cho, Miyazaki 880-0036, Japan.

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Daiske Miyakoshi Department of Veterinary Surgery, Kagoshima University, 21-24 Korimoto 1-chome, Kagoshima 890-0065, Japan.

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Makoto Fujiki Department of Veterinary Surgery, Kagoshima University, 21-24 Korimoto 1-chome, Kagoshima 890-0065, Japan.

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Abstract

Objective—To evaluate changes in serum cartilage oligomeric matrix protein (COMP) concentrations in response to exercise in horses.

Animals—15 horses in experiment 1 and 27 horses in experiment 2.

Procedures—In experiment 1, 15 Thoroughbreds free of orthopedic disease underwent a standardized exercise protocol. Running velocity and heart rate (HR) were recorded, and blood samples were collected immediately before (baseline) and 1, 5, and 24 hours after a single episode of exercise. In experiment 2, 27 horses underwent 9 stages of a training program in which each stage consisted of 4 to 8 consecutive daily workouts followed by a rest day. Blood samples were collected immediately before the first and final daily workouts in each stage. Serum COMP concentrations were measured via inhibition ELISA with a monoclonal antibody (14G4) against equine COMP.

Results—In experiment 1, mean serum COMP concentration was significantly higher than baseline 1 and 5 hours after exercise and returned to baseline concentrations 24 hours after exercise. Mean serum baseline COMP concentration increased as the velocity of running at maximum HR and at an HR of 200 beats/min increased, being significantly higher during the third and fourth exercise tests than during the first. In experiment 2, mean baseline COMP concentration at the final workout of each stage was significantly higher than that at the first workout, beginning with stage 3.

Conclusions and Clinical Relevance—Serum COMP concentrations changed significantly in response to exercise. Exercise may enhance movement of COMP into the circulation as well as change the basal turnover rate of COMP.

Abstract

Objective—To evaluate changes in serum cartilage oligomeric matrix protein (COMP) concentrations in response to exercise in horses.

Animals—15 horses in experiment 1 and 27 horses in experiment 2.

Procedures—In experiment 1, 15 Thoroughbreds free of orthopedic disease underwent a standardized exercise protocol. Running velocity and heart rate (HR) were recorded, and blood samples were collected immediately before (baseline) and 1, 5, and 24 hours after a single episode of exercise. In experiment 2, 27 horses underwent 9 stages of a training program in which each stage consisted of 4 to 8 consecutive daily workouts followed by a rest day. Blood samples were collected immediately before the first and final daily workouts in each stage. Serum COMP concentrations were measured via inhibition ELISA with a monoclonal antibody (14G4) against equine COMP.

Results—In experiment 1, mean serum COMP concentration was significantly higher than baseline 1 and 5 hours after exercise and returned to baseline concentrations 24 hours after exercise. Mean serum baseline COMP concentration increased as the velocity of running at maximum HR and at an HR of 200 beats/min increased, being significantly higher during the third and fourth exercise tests than during the first. In experiment 2, mean baseline COMP concentration at the final workout of each stage was significantly higher than that at the first workout, beginning with stage 3.

Conclusions and Clinical Relevance—Serum COMP concentrations changed significantly in response to exercise. Exercise may enhance movement of COMP into the circulation as well as change the basal turnover rate of COMP.

To help investigators understand the disease processes of osteoarthritis, biomarkers such as cytokines, catabolic enzymes, and macromolecules of joint extracellular matrix have been evaluated. Biomarkers yield information regarding cartilage or bone destruction and remodeling associated with infiltrative, proliferative, and malfunctioning cells and may be indicators of extracellular matrix injury resulting from abnormal physical forces.1,2 Studies1,3-7 of these markers in synovial fluid have yielded much information pertaining to the pathologic changes associated with osteoarthritis in injured joints, whereas analysis of serum markers is less often considered to be informative of developing osteoarthritis. The most useful serum markers of osteoarthritis would be molecules that originate in the joint but move efficiently from joint fluid into blood. Limitations associated with use of serum biomarkers include poor specificity of degraded joint components for joint disease and restricted movement of molecules from joints into the circulation.

Equine cartilage oligomeric matrix protein was first purified from tendon tissue8 and has been analyzed by use of certain antibodies found in digital sheath synovial fluid and serum in horses with tendon injury.9 Findings from the latter study9 suggest that analysis of COMP in serum may yield information pertaining to cartilage turnover but not tendon health because tendonitis does not induce substantial changes in serum COMP concentration. Results of other studies1,3 indicate that analysis of COMP fragmentation in synovial fluid, in combination with ELISA measurements of COMP in synovial fluid, would be useful for monitoring early development of joint disease in horses. Evaluation of urinary COMP concentration in racehorses with joint disorders has confirmed that COMP molecules move from synovial fluid into blood and, subsequently, into urine.10

Using mAb 12C4, which has high affinity for less degraded or intact equine COMP, investigators found that COMP concentrations in synovial fluid from joints with osteoarthritis were lower than concentrations in healthy joints and that the numbers of COMP bands on immunoblots of osteoarthritic synovial fluid were decreased.1 When mAb 14G4, which recognizes both small (ie, more degraded) and intact fragments of equine COMP, was used, COMP concentration was significantly higher in synovial fluid from osteoarthritic joints, compared with synovial fluid from healthy joints, and more fragmented COMP was seen in immunoblots of synovial fluid from osteoarthritic joints.3 We speculated that mAb 12C4 would be more useful for ELISA monitoring of increased COMP turnover (ie, synthesis) than COMP degradation in diseased equine joints, whereas mAb 14G4 would be valuable for identifying and quantifying COMP fragmentation. In an earlier prospective study11 of COMP as a serum marker of osteoarthritis, we detected a significant correlation between serum and synovial fluid COMP concentrations by use of ELISA and mAb 12C4, but it remains to be determined whether osteoarthritis can be diagnosed in horses on the basis of serum COMP concentration. The difficulty in using these antibodies in serum COMP assays for diagnosing osteoarthritis in horses may be the result of limited detection of COMP degradation at different stages of osteoarthritis lesions as well as severity and factors unrelated to the disease process that affect the release of COMP into synovial fluid and, subsequently, into the blood. Cartilage oligomeric matrix protein may undergo less degradation in lymphatics and blood because high serum activity of proteases such as MMPs, which cleave the molecule in the serum of horses with osteoarthritis, has not been detected. Similarly, detection of degraded COMP in healthy liver tissue has not been reported.

Another putative factor contributing to development of osteoarthritis is mechanical loading of joint cartilage and synovium during exercise. Normal mechanical loading during exercise has effects on matrix molecule turnover in cartilage and on transfer of molecular fragments to the lymphatics. Neidhart et al12 reported a transient but significant increase in serum COMP concentration in human runners during and after a marathon, suggesting a correlation between serum COMP concentration and joint inflammation in association with strenuous exercise. In 1 study,13 equine cartilage explants cultured in postexercise synovial fluid had enhanced glycosaminoglycan synthesis and diminished release, compared with explants cultured in preexercise synovial fluid collected after a period of stall rest. Application of mechanical load consistent with a physiologic degree of strain to healthy cartilage explants promotes synthesis of proteoglycan and protein, whereas synthesis is decreased following application of hyperphysiologic degrees of stress.14 In another in vitro study,15 cyclic compression resulted in an imbalance of MMPs and their inhibitors, induced catabolic events in cartilage, or both.

A superficial network of fenestrated synovial microvessels filters fluid slowly into the joint cavity when intra-articular pressure is low, such as when a joint is in extension; joint fluid exits in the spaces between synoviocytes when synovial fluid pressure is high, such as during joint flexion.16,17 Dynamic forms of exercise such as walking and cycling increase synovial blood flow in effusive joints in humans with arthropathies18 and increase intra-articular pressure in healthy and rheumatoid joints.19

Before COMP analysis can be used clinically for evaluating osteoarthritis in racehorses, it is necessary to validate the extent to which exercise affects its release. The purpose of the present study was to determine changes in serum COMP concentrations before and after a standardized exercise test conducted during different stages of developing athletic ability for 4 months and changes in serum concentrations at the onset and end of each of 9 contiguous stages of training in young Thoroughbred racehorses. We hypothesized that exercise would enhance differences in serum COMP concentration between clinically normal racehorses and horses with joint disease.

Materials and Methods

Experiment 1—In experiment 1, 15 horses (8 females and 7 males) with a mean ± SD age of 20 ± 1 months were studied from December through April. During this time, horses were trained continuously on a schedule of 6 consecutive days of exercise followed by 1 day of rest. Horses were determined to be free of orthopedic disease on the basis of radiographic examination and ultrasonographic evaluation of tendons throughout the experimental period. Horses were fitted with a saddle containing telemetry instrumentsa for global positioning and electrocardiography. The standardized exercise protocols were conducted every 40 days from December 8th through April 8th. Before commencing the protocol on test days, horses had a warm-up period consisting of a 500-m walk. The exercise protocol consisted of trotting for 200 m, cantering for 1,200 to 1,500 m, trotting for 200 m, and walking for 400 m. From running velocity and heart rates obtained during the exercise, maximum heart rate (HRmax, beats/min), velocity at maximum heart rate (VHRmax, m/min), and velocity at a heart rate of 200 beats/min (V200, m/min) were calculated. Blood samples for COMP measurement were collected immediately before warm-up (baseline) and 1, 5, and 24 hours after completion of the exercise.

Experiment 2—Twenty-seven Thoroughbreds (14 females and 13 males) with a mean ± SD age of 22 ± 1 months were subjected to 9 stages of a training program in which each stage consisted of 4 to 8 consecutive daily workouts followed by a day of rest. The 9 stages of the training program took place in 60 days. Horses were determined to be free of orthopedic disease by means of radiographic examination and ultrasonographic tendon imaging before beginning the study and underwent additional diagnostic imaging if clinical signs of lameness or joint swelling developed during the training program. Each daily workout consisted of walking for 800 m, trotting for 1,400 m, cantering or galloping for 2,400 m, and walking for 1,500 to 2,000 m. Running speed (m/s) during the canter-gallop stage was increased as the training stage advanced; maximum speed was 10 m/s in stages 1 and 2 (mild exercise stage), 13.3 m/s in stages 3 to 5 (moderate stage), and 15.4 m/s in stages 6 to 9 (intense exercise stage. Blood samples were collected immediately before the first (baseline) and final daily workouts at each stage. Blood samples were collected from a jugular vein. Clotted samples were centrifuged, and sera were stored at −70°C until the COMP assays were performed.

COMP assay—Serum COMP concentration was analyzed via inhibition ELISA by use of an mAb 14G4 against equine COMP. Purification of equine COMP from cartilage and the inhibition ELISA used to quantify COMP in serum were performed according to modifications of previous protocols.1 In brief, 50 μL of purified horse COMP antigen in a coating buffer (20mM sodium carbonate, 20mM sodium bicarbonate, and 0.002% sodium azide [pH, 10]) was placed into each well (end concentration, 5 μg/mL) and incubated for 2 hours at approximately 24°C and for approximately 12 hours at 4°C. Seventy microliters of diluted standard solutions (range, 13.5 to 0.01 μg/mL) and serum (final dilution, 1:20) was mixed with the same volume of mAb 14G4 (final dilution, 1:100,000) in PBS solution containing 0.05% Tween 20 and was incubated for approximately 12 hours at 4°C. Coated wells were washed with PBS solution, blocked, and incubated with 100 μL/well of the COMP-antibody mixture for 1 hour at approximately at 24°C and for 1 hour at 4°C. The primary antibody binding to COMP on the plate was detected by use of goat anti-mouse IgG conjugated to alkaline phosphatase and addition of an alkaline phosphatase chromophore. After 1 hour, chromophore production was stopped by addition of 2M H2 CO3(30 μL/well) and absorbance was read at 405 nm.

Statistical analysis—Data were reported as mean ± SD. In the first experiment, HRmax, VHRmax, V200, and baseline COMP concentrations were compared among the 4 exercise tests with factorial ANOVA. Significance of changes detected in serum COMP concentrations after exercise was determined by repeated-measures ANOVA. Relationships between COMP data and exercise variables were evaluated by use of regression analysis. In the second experiment, the significance of differences in baseline COMP concentrations between the first and final daily workouts at each stage or between the final and first workouts after 1 day of rest was also analyzed by use of repeated-measures ANOVA. In both experiments, the Scheffé method was used for simultaneous multiple comparisons. For all comparisons, values of P < 0.05 were considered significant.

Results

Experiment 1—Changes in heart-related physical variables of horses during the 4 exercise tests were summarized (Table 1). Maximum heart rate increased in response to increased work during the exercise tests, with HRmax in February and April significantly (P = 0.01) higher than that in December. Accordingly, VHRmax and V200, indices of the horses' physical fitness, also increased, with values for both variables significantly (P < 0.05) higher in April than in December.

Table 1—

Mean ± SD running velocities and heart rates in 15 Thoroughbreds during a standardized exercise protocol that was conducted every 40 days during a 4-month training period.

Exercise testMaximum speed (m/s)HRmax (beats/min)VHRmax (m/min)V200 (m/min)
First (Dec)8.6 ± 0.5184.4 ± 11.9692.3 ± 65.4608.0 ± 52.5
Second (Jan)9.2 ± 0.5193.2 ± 10.4682.8 ± 57.1600.1 ± 55.1
Third (Feb)12.1 ± 1.1*210.9 ± 12.0*714.1 ± 50.6630.9 ± 55.8
Fourth (Apr)13.1 ± 1.3*219.2 ± 8.5*760.4 ± 50.7668.7 ± 56.2

Values are significantly (P = 0.01) different from those obtained in the first exercise test.

Values are significantly (P < 0.05) different from those obtained in the first exercise test.

HRmax = Maximum heart rate. VHRmax = Velocity of running at maximum heart rate. V200 = Velocity of running at a heart rate of 200 beats/min.

Serum COMP concentrations after exercise were summarized (Figure 1). In December and January, serum COMP concentrations after 5 hours of exercise were significantly (P = 0.01) higher than baseline values. In February and April, serum COMP concentrations after 1 and 5 hours of exercise were significantly (P = 0.01) higher than baseline values. During all 4 exercise tests, the high COMP concentrations measured 1 or 5 hours after exercise returned to preexercise baseline values 24 hours later.

Figure 1—
Figure 1—

Mean ± SD serum COMP concentrations before and after standardized exercise tests in 15 Thoroughbreds free of lameness or clinically evident joint disease. Tests were conducted every 40 days from December through April. Notice that baseline concentrations for the third and fourth tests (in February and April; asterisks) were significantly (P = 0.01) higher than those for the first test (in December). Also notice that COMP concentrations increased significantly (P = 0.01) from baseline values after exercise (daggers) and returned to baseline values within 24 hours. B = Baseline (preexercise) concentration.

Citation: American Journal of Veterinary Research 68, 2; 10.2460/ajvr.68.2.134

Baseline concentrations of COMP in the February (24.4 ± 5.6 μg/mL) and April (25.9 ± 5.0 μg/mL) tests were also significantly (P = 0.01) higher than in December (15.5 ± 4.3 μg/mL). Variations in serum COMP concentration after exercise did not change significantly (Table 2). The increased baseline concentration of COMP was not significantly correlated with increases in VHRmax or V200 (correlation coefficients, 0.22 and 0.24, respectively).

Table 2—

Mean ± SD differences in serum concentration of COMP (μg/mL; calculated as the postexercise concentration minus baseline concentration) in the same 15 horses as in Figure 1 and Table 1. Negative values indicate that the serum concentration of COMP after exercise was less than the preexercise (baseline) concentration.

Exercise testAfter 1 hourAfter 5 hoursAfter 24 hours
First (Dec)1.1 ± 0.71.6 ± 1.70.1 ± 1.4
Second (Jan)1.0 ± 0.91.5 ± 1.40.1 ± 1.8
Third (Feb)1.4 ± 0.81.7 ± 1.0−0.4 ± 1.5
Fourth (Apr)1.5 ± 1.01.5 ± 1.5−0.5 ± 1.5

Experiment 2—Baseline serum COMP concentrations remained in the range of 10 to 20 μg/mL during the exercise period. When the line graph of serum COMP concentration was overlaid on a stair-step graph representing maximum running speeds during daily workouts (Figure 2), the mean baseline COMP concentration had an oscillatory pattern between stages 3 and 9; measurements obtained just before the final daily workouts were significantly (P = 0.01) higher than those obtained before the first daily workouts at each stage. The increased baseline concentration at the final workout decreased significantly (P = 0.01) after 1 day of rest (when measurements were conducted before the first workout in the next stage) at stages 4, 5, 7, 8, and 9. Serum COMP concentration in 1 horse increased markedly after stage 6. At that time, the horse had swelling and heat around both tibiotarsal joints and all 4 metacarpophalangeal and metatarsophalangeal joints (Figure 3).

Figure 2—
Figure 2—

Association between serum COMP concentration before daily episodes of exercise (circles) and maximum running speed during those episodes (stairstep graph) in various stages of a 9-stage training program in 27 Thoroughbreds. Each stage consisted of 4 to 8 consecutive days of an exercise protocol followed by a day of rest, and intensity of exercise (mild, moderate, and intense) increased with increasing stage number. Notice that COMP concentrations immediately prior to the final daily exercise in each stage were significantly (P = 0.01) higher (asterisks) than those before the first daily exercise at each stage. Also notice that the higher concentrations at the final daily exercise decreased significantly (P = 0.01) after 1 day of rest (daggers; those measurements were obtained before the first daily exercise of the next stage) in stages 4, 5, 7, 8, and 9.

Citation: American Journal of Veterinary Research 68, 2; 10.2460/ajvr.68.2.134

Figure 3—
Figure 3—

Individual serum COMP concentrations during the 9-stage training protocol in the same 27 horses as in Figure 2. Notice that most concentrations remained between 10 and 20 μg/mL, but the concentration in 1 horse that developed joint effusion (solid line) was substantially higher than in the others (dashed lines) during and after stage 6, when exercise intensity was intense.

Citation: American Journal of Veterinary Research 68, 2; 10.2460/ajvr.68.2.134

Discussion

Previous studies investigating exercise-related changes in serum concentration of COMP and other joint-derived biomarkers have yielded conflicting results regarding changes in concentrations in humans and other animals. In 1 study20 in which the effect of exercise on serum keratan sulfate concentration was investigated in 2-year-old horses, serum concentration peaked immediately after exercise, whereas 1, 5, 9, and 24 hours after exercise, keratan sulfate concentration was the same as before exercise. In the present study, keratan sulfate concentration returned to preexercise values at 1 hour, whereas serum COMP concentration took 24 hours to return to preexercise values. Delayed clearance of COMP from the circulation may be a result of delayed fragmentation or elimination of the macromolecules in synovial fluid. Consistent with those results, in a controlled study12 of marathon runners, serum COMP concentrations increased significantly during a run (31 and 42 km) and decreased 24 and 48 hours after exercise. In a study21 of physically active adults, serum concentrations of COMP increased by 9.7% immediately after 30 minutes of walking exercise, then decreased. In that study, a second increase in serum COMP concentration was detected 5.5 hours after the walking exercise.

Conversely, other research on healthy active runners revealed that serum COMP concentrations were not increased 25 minutes or 2.5 hours after a run.22 Because those data were obtained from active runners who had been free of injury for a minimum of 3 years prior to commencement of the study and who had no surgical scars on knees or legs, those authors22 speculated that the higher COMP concentrations detected in the earlier study12 of marathon runners had resulted from early-stage degenerative joint disease in the experienced runners. They also speculated that the discrepancy between their results and those of the study21 involving healthy active adults may have resulted from modulation by other chemical regulators or physical factors such as lymphatic drainage.

In the first experiment in the present study, serum COMP concentrations increased 1 or 5 hours (in some instances, both) after exercise and returned to baseline concentration 24 hours later. These results were similar to those in the study12 of marathon runners.Because there was no indication that our horses had had any joint disease since birth, it seems unlikely that the high serum COMP concentrations were indicative of earlystage cartilage degradation. Therefore, our results support the speculation that the higher serum COMP concentrations detected after the run were an indication of increased catabolism and clearance in response to severe physical strain on joint structures and subsequent proteolysis, rather than a result of pathologic turnover of COMP associated with osteoarthritis.

An experimental study23 of rats subjected to running stress revealed a significant increase in MMP-3 immunoreactivity in chondrocytes, and an increase in the concentration of stromelysin-1 has been colocalized with broken collagen at the articular surface in explants subjected to mechanical loading.24 Results of earlier studies25,26 indicate that moderate exercise does not significantly affect MMP activity in synovial fluid of healthy joints, but MMP activity may be different when exercise is excessive or joints are diseased.26 Exercise may enhance proteolytic fragmentation (ie, degradation) of matrix molecules such as COMP in synovial fluid of joints with osteoarthritis, and subsequent clearance may be more rapid after exercise in horses with osteoarthritis if lymphatic drainage via the synovium is not impaired. It has been postulated that exercise also increases clearance of degraded matrix molecules from joints without altering turnover of the molecules via increased rate of passage through the synovium and increased lymphatic flow.27 Another study28 revealed that exercise increases blood flow in the synovium of healthy adult dogs, and in a study18 of humans with arthropathy, physiologic exercise of appropriate intensity, such as walking and cycling, increased the rate of synovial clearance in effusive joints. Thus, increased blood flow and clearance via the synovium could also have contributed to the increased serum COMP concentrations after exercise in horses in the present study. However, results of a clinical study29 of joint effusion indicate that high intra-articular pressure during exercise may induce synovial tissue hypoxia and that subsequent reperfusion may result in injury from superoxide radicals. It has not yet been ascertained how intense work induces pathologic changes in the joint extracellular matrix or how blood flow and clearance rate in articular soft tissues change in response to exercise.

Baseline serum COMP concentrations in the third and fourth tests (February and April) were significantly higher than those in the first test (December). It was reported by Neidhart et al11 that baseline COMP concentrations before exercise in marathon runners were significantly higher than those in healthy nonrunning volunteers. Although it is possible that the higher serum baseline concentration was a result of early-stage degradation in the joints of athletes, we speculate that the higher baseline concentration of COMP in our horses reflected increased COMP turnover rather than joint damage secondary to exercise because none of the horses had clinically important joint disease that was evident with radiography or ultrasonographic examination. In another study20 of 2-year-old horses, serum keratan sulfate concentrations were significantly higher in trained horses than in rested horses, suggesting that joint loading during daily training may affect metabolic activity in cartilage extracellular matrix. In porcine cartilage explants, COMP release increases in proportion to the magnitude of dynamic mechanical stress, whereas keratan sulfate increases in a bimodal pattern with increasing stress.14 The basal turnover rate of matrix molecules in the cartilage of young horses may change slightly in response to daily physical stress, and changeable baseline COMP concentrations could obscure changes in serum concentrations after exercise. No significant correlation was detected between the increase in baseline COMP concentration and increases in V200 and VHRmax, and no significant change in variation of serum COMP concentrations from baseline after exercise was detected.

In experiment 2, baseline serum COMP concentration had an oscillating pattern of variation that corresponded with exercise and rest in stages 3 through 9. Baseline concentrations immediately prior to the final daily workouts were higher than those before the first daily workouts at each stage, and the increased baseline concentration at the final workout decreased after 1 day of rest, a finding that has not been reported before, to the authors' knowledge. The increase in baseline serum COMP concentration suggests that COMP released during the repeated daily works was not completely eliminated before the final workout during stages 3 to 9, when exercise intensity was moderate or intense. However, after the day of rest, residual serum COMP may have been removed from the blood compartment. Physical stimulation at appropriate levels of intensity is essential for growth and maintenance of bone and articular cartilage, but excessive mechanical loading is more destructive and less regenerative in healthy as well as diseased joints. Physiologic compressive loads (ie, corresponding to weight-bearing forces) upregulate aggrecan and collagen II expression in healthy cartilage explants, whereas osteochondrosis lesions contain higher concentrations of catabolic enzymes and have less regeneration of matrix molecules in response to mechanical forces.30 Mechanical loading of the articular surface induces increased COMP immunoreactivity at sites of cartilage fibrillation.31 In the present study, some of the horses in experiment 2 had clinical signs of joint disease such as joint swelling or heat and lameness during the training period, unlike horses in experiment 1. In most of the horses, serum COMP concentrations varied between the range of 10 and 20 μg/mL, although serum concentrations in 1 horse with joint swelling and heat increased substantially after stage 6. If exercise increases extracellular matrix catabolism in osteoarthritis lesions, COMP degradation and elimination after exercise could be used to differentiate between horses with and without osteoarthritis. Routine monitoring of serum COMP concentration during training may permit early detection of abnormalities in joint tissue metabolism and intervention before permanent damage occurs.

The present study revealed significant changes in serum COMP concentration associated with exercise in horses. Thus, when comparing serum COMP concentrations between healthy horses and those with joint disease, samples should be collected before daily exercise and the different baselines should be taken into consideration. Measurements of COMP in synovial fluid and urine are useful in distinguishing between osteoarthritic joints3 and horses with other types of joint disease.10 It is possible that high COMP concentrations in osteoarthritic synovial fluid do not contribute to increased serum concentrations. In addition to the effect of different levels of physical activity in horses, proteolytic fragmentation of COMP in osteoarthritic joint fluid or dilution of fragments in the serum pool could also obscure the increased COMP in the serum. In a recent study,10 we found that COMP concentrations in urine, which is a concentrated filtrate of plasma, may be increased with advanced osteoarthritis.

One of the limitations of our study was that early joint lesions, such as cartilage degradation and synovitis without bone deformity, could have been undetected by radiographic or ultrasonographic imaging. In addition, subclinical joint disease in the absence of lameness could also increase serum COMP concentrations in response to physical activity, although no horses in the present study had osteochondrosis before or during the study. Periodic measurement of serum COMP concentration in young racehorses in training may be useful in early diagnosis of joint disease because pathologic upregulation of matrix turnover might be exaggerated by exercise even if the lesion is clinically inapparent when a horse is at rest. It remains to be clarified to what extent COMP upregulation can be normalized and the extent to which upregulation indicates development of disease subsequent to overwork. The effects of therapeutic exercise at different intensities (eg, walking and running) and in different forms (eg, swimming and running) on COMP release and elimination warrant additional investigation.

ABBREVIATIONS

COMP

Cartilage oligomeric matrix protein

mAb

Monoclonal antibody

MMP

Matrix metalloproteinase

a.

EquiPILOT, Fidelak Ltd, Kamen, Germany.

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