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    Figure 1—

    Scatterplots of CTX-II concentration in samples of SF (A and B) and serum (C and D) and calculated values of SF:serum CTX-II ratio (E and F) for carpal joints (A, C, and E) and MCP joints (B, D, and F) in 40 Thoroughbred racehorses without joint injury before and after 5 to 6 months of race training (pre-[Pre] and postexercise [Post] samples, respectively) and in 38 Thoroughbred racehorses with OCI (1 joint assessed/horse). For each group in each panel, the short horizontal solid line represents the mean value; the horizontal dashed lines represent concentrations or ratios for which there was predictive value for discrimination of samples from OCI-affected horses from the pre- or postexercise samples. *†‡Mean values in the bracketed groups differ significantly (P < 0.001, P < 0.01, and P < 0.05, respectively).

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Effect of exercise and osteochondral injury on synovial fluid and serum concentrations of carboxy-terminal telopeptide fragments of type II collagen in racehorses

Orlaith B. ClearyDepartment of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL 32610.

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Troy N. TrumbleDepartment of Veterinary Population Medicine, College of Veterinary Medicine, University of Minnesota, Saint Paul, MN 55108.

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Kelly A. MerrittDepartment of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL 32610.

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Murray P. BrownDepartment of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL 32610.

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Abstract

Objective—To investigate the effects of exercise and osteochondral injury on concentrations of carboxy-terminal telopeptide fragments of type II collagen (CTX-II) in synovial fluid (SF) and serum of Thoroughbred racehorses and to compare findings with radiographic and arthroscopic scores of joint injury severity.

Animals—78 Thoroughbreds with (n = 38) and without (40) osteochondral injury.

Procedures—Serum and metacarpophalangeal or carpal joint SF samples were collected from noninjured horses before and at the end of 5 to 6 months of race training (pre- and postexercise samples, respectively) and from horses with osteochondral injury (1 joint assessed/horse). Synovial fluid and serum CTX-II concentrations were determined by use of an ELISA. Radiographic and arthroscopic scores of joint injury severity were determined for the injured horses.

Results—The CTX-II concentrations in SF and SF:serum CTX-II ratio were significantly higher for horses with joint injuries, compared with pre- and postexercise findings in noninjured horses. Serum CTX-II concentrations in postexercise and injured-horse samples were significantly lower than values in pre-exercise samples. On the basis of serum and SF CTX-II concentrations and SF:serum CTX-II ratio, 64% to 93% of serum and SF samples were correctly classified into their appropriate group (pre-exercise, postexercise, or injured-joint samples). In horses with joint injuries, arthroscopic scores were positively correlated with radiographic scores, but neither score correlated with SF or serum CTX-II concentration.

Conclusions and Clinical Relevance—Results suggested that serum and SF CTX-II concentrations and SF:serum CTX-II ratio may be used to detect cartilage degradation in horses with joint injury.

Abstract

Objective—To investigate the effects of exercise and osteochondral injury on concentrations of carboxy-terminal telopeptide fragments of type II collagen (CTX-II) in synovial fluid (SF) and serum of Thoroughbred racehorses and to compare findings with radiographic and arthroscopic scores of joint injury severity.

Animals—78 Thoroughbreds with (n = 38) and without (40) osteochondral injury.

Procedures—Serum and metacarpophalangeal or carpal joint SF samples were collected from noninjured horses before and at the end of 5 to 6 months of race training (pre- and postexercise samples, respectively) and from horses with osteochondral injury (1 joint assessed/horse). Synovial fluid and serum CTX-II concentrations were determined by use of an ELISA. Radiographic and arthroscopic scores of joint injury severity were determined for the injured horses.

Results—The CTX-II concentrations in SF and SF:serum CTX-II ratio were significantly higher for horses with joint injuries, compared with pre- and postexercise findings in noninjured horses. Serum CTX-II concentrations in postexercise and injured-horse samples were significantly lower than values in pre-exercise samples. On the basis of serum and SF CTX-II concentrations and SF:serum CTX-II ratio, 64% to 93% of serum and SF samples were correctly classified into their appropriate group (pre-exercise, postexercise, or injured-joint samples). In horses with joint injuries, arthroscopic scores were positively correlated with radiographic scores, but neither score correlated with SF or serum CTX-II concentration.

Conclusions and Clinical Relevance—Results suggested that serum and SF CTX-II concentrations and SF:serum CTX-II ratio may be used to detect cartilage degradation in horses with joint injury.

Articular cartilage is composed principally of type II collagen and aggrecan.1,2 Type II collagen is the most abundant protein of articular cartilage across species and is relatively specific for this tissue.1 A combination of cytokines and growth factors stimulates the release and activation of matrix metalloproteinases, aggrecanases, and cathepsins from cartilage and synovial tissue.3–6 These proteases play an important role in the etiopathogenesis of osteoarthritis by causing cleavage of type II collagen, which leads to degradation and release of fragments into SF.7–9 However, during repetitive loading activities such as athletic training, protease activity is increased in an attempt to maintain homeostasis. Type II collagen degradation is increased in human endurance athletes10 and race-trained horses,11 compared with findings in nonathletic individuals, and in horses in response to treadmill exercise.12 Therefore, it is important to differentiate type II collagen degradation caused by normal metabolic responses to exercise from those associated with pathological conditions such as osteoarthritis. Early detection of type II collagen degradation may allow timely intervention and prevention of irreversible articular changes.

An ELISA has been developed to measure the Glu-Lys-Gly-Pro-Asp-Pro (EKGPDP) epitope of the carboxy-terminal telopeptide fragments formed during the degradation of type II collagen.13 This assay has been used to examine type II collagen degradation in explant media14 and rat serum15–18 as well as in human and rat SF7,15,16 and urine.13,14 Concentrations of CTX-II in these body fluids correlate well with histologic,15,18–20 radiographic,21–24 arthroscopic,25 and magnetic resonance imaging26–29 assessments of severity and progression of osteoarthritis. Measurements of CTX-II concentrations have been used extensively in studies30–32 evaluating the effects of disease-modifying osteoarthritis drugs on progression of osteoarthritis. On the basis of those reports as well as the conservation of the telopeptide region of type II collagen across species,33 it appears that the CTX-II assay may have potential for similar application in equine athletes.

The effects of exercise and OCI on SF and serum biomarker concentrations in horses have been investigated.12,34–37 However, to our knowledge, no previous studies have used CTX-II assessments to evaluate the effects of exercise or OCI on degradation of type II collagen in horses. The purpose of the study reported here was to identify differences in CTX-II concentrations in SF and serum samples obtained from horses with OCI and noninjured horses before and after a period of training (pre- and postexercise samples, respectively); evaluate the predictive values of SF and serum CTX-II concentrations and SF:serum CTX-II ratio to differentiate among groups of experimental samples (samples from horses with OCI and pre- and postexercise samples); assess use of discriminant analysis to classify samples into pre-exercise, postexercise, or OCI groups on the basis of the SF and serum CTX-II concentrations and the SF:serum CTX-II ratio; and compare CTX-II concentrations in SF and serum of joint-injured horses with radiographic and arthroscopic scores of joint injury severity. On the basis of observations in a previous study36 in which serum and SF BAP activities were evaluated in horses with OCI, we hypothesized that exercise and OCI would increase SF CTX-II concentrations and decrease serum CTX-II concentrations and that, in this regard, OCI would have a greater effect than exercise.

Materials and Methods

Horses and experimental design—Serum and SF samples were obtained from 2 groups of Thoroughbred racehorses. The first group comprised 40 untrained horses (age range, 14 to 21 months) that were purchased from yearling sales. All horses were considered clinically normal and without MCP or carpal joint injury on the basis of clinical and radiographic examination findings. Synovial fluid samples were obtained from 20 MCP joints and 20 carpal joints (10 RC joints and 10 MC joints) prior to the horses undergoing race training (designated as pre-exercise samples). One sample was collected from 1 joint in each horse. A pre-exercise blood sample (6 mL) was collected from each horse. All 40 horses then underwent a similar race-training program. Briefly, all horses were trotted to the racetrack, galloped at speeds of 32 km/h (1 furlong/22 s) for 1.5 miles, and walked back to their stalls 6 d/wk. After 2 months, horses were also introduced to high-speed exercise (known as breezing) 1 d/wk at speeds of 53 km/h (1 furlong/≤ 14 s) for 1 to 2 furlongs, followed by 1 day of rest. At the end of 5 to 6 months of training, another blood sample was collected from each horse and an SF sample was collected from the joint that had been selected to provide the pre-exercise sample; these samples were designated as postexercise samples.

The other group of horses comprised 38 horses (age range, 2 to 7 years) that were undergoing arthroscopic surgery for removal of osteochondral fragments attributable to training or racing injuries. Synovial fluid samples were obtained from 16 MCP joints and 22 carpal joints (10 RC joints and 12 MC joints). One sample was collected from 1 joint in each horse. Fragments were removed from dorsal articular borders of the third carpal, RC, and intermediate carpal bones; the distal portion of the radius; and dorsoproximal portion of the first phalanx. As determined via radiographic and arthroscopic examinations, some of the horses in the OCI group had osteochondral fragments in more than 1 joint. Other joints were injured in 7 of 16 horses from which MCP joint SF was collected; 5 had injuries in the contralateral MCP joint, and 2 had injuries in the contralateral MC or RC joint. Among the 22 horses from which carpal joint SF was collected, 10 had injuries in the contralateral MC or RC joint. Synovial fluid samples were not collected for analysis from these additional injured joints. A blood sample (6 mL) was also collected from each horse.

Sample collection—For all horses, blood samples were collected from a jugular vein via needle venipuncture. Blood was allowed to clot, and samples were centrifuged and serum was decanted. Synovial fluid samples were collected at the same time as blood sample collection via aseptic needle arthrocentesis. Samples were centrifuged and decanted. All serum and SF samples were stored at −80°C until assayed. All sample collections were approved by the University of Florida Institutional Animal Care and Use Committee.

Procedure for the CTX-II immunoassay—Samples of serum and SF were appropriately diluted (1:2 or 1:4), and serum and SF CTX-II concentrations were measured by use of a sandwich ELISA that was based on a monoclonal antibody against the EKGPDP linear 6-amino acid epitope of CTX-II.a This ELISA is based on the binding of 2 identical monoclonal antibodies to cross-linked fragments of type II collagen.13 It has been validated for use in equine serum and SF samples.b,c

Arthroscopic and radiographic scores—Arthroscopic images and videos and surgical reports for horses with OCI were reviewed, and 11 categories were graded by use of a scoring system36 developed by the authors. A total arthroscopic score of 0 to 37 was assigned to the evaluated joint in each horse with OCI. The total arthroscopic score included 5 categories of inflammation (graded 0 through 3), fragment size (graded 0 through 3), fragment number (graded 0 through 3), and 4 categories of degenerative cartilage changes related to the fragments (graded 0 through 4). Each grade scale represented changes in ascending order of severity, with 0 representing none. Similarly, available radiographic views of the evaluated joints in horses with OCI were reviewed and 10 categories of radiographic changes were graded by use of a previously described scoring system.36 A total radiographic score from 0 to 30 was assigned to the evaluated joint in each horse with OCI. The total radiographic scores included scores (graded 0 through 3) for joint space, subchondral bone sclerosis, subchondral bone lucency, soft tissue swelling, and sizes and numbers of osteophytes, enthesophytes, and fragments. Each grade scale represented changes in ascending order of severity, with 0 representing none. Consensus arthroscopic and radiographic scores were determined by 2 of the investigators working together (TNT and MPB). The investigators were unaware of the radiographic or arthroscopic appearance of a joint when evaluating the other images.

Statistical analysis—Statistical analysis was performed by use of personal computer–based statistical software.d Normality plots of the data were assessed. Analysis of box plots identified possible outliers, and extreme Studentized deviate tests were then performed to determine whether the value was > 2 SDs or < 2 SDs from the mean. If the value was > 2 SDs from the mean, it was considered an outlier and was eliminated from further analysis. Differences in CTX-II concentrations between pre- and postexercise samples were evaluated by use of a paired t test. Differences in CTX-II concentrations between pre- or postexercise samples and samples from horses with OCI were determined by use of an unpaired t test. Differences in radiographic and arthroscopic scores between the carpal and MCP joints were determined by use of an unpaired t test. To determine whether the CTX-II assay could discriminate among the groups of horses (ie, samples from horses with OCI and pre- and postexercise samples), sensitivity, specificity, positive and negative predictive values, and likelihood ratios for SF CTX-II concentration, serum CTX-II concentration, and SF:serum CTX-II ratio were determined by use of a Fisher exact test. Correlations (age, sex, limb, joint, radiographic score, arthroscopic score, serum CTX-II concentration, SF CTX-II concentration, and SF: serum CTX-II ratio) were determined by use of Spearman rank correlation. Discriminant analysis was used to classify samples into the appropriate grouping (pre-exercise, postexercise, or OCI) on the basis of the serum and SF CTX-II concentrations as well as the ratio of those 2 variables. A quadratic discriminant function was computed from a random sampling of the carpal and MCP joints with prior probabilities proportional to the population sizes. To estimate how well the model could discriminate each joint into its respective grouping, a subset validation was performed on a random sampling of carpal and MCP joints that were not used to determine the function. A value of P < 0.05 was considered significant for all analyses.

Results

Concentrations of CTX-II were significantly higher in SF samples from horses with OCI of carpal (P < 0.001) and MCP joints (P < 0.01), compared with concentrations in pre- and postexercise SF samples (Figure 1). No significant difference in SF CTX-II concentration was detected between pre- and postexercise samples for either joint. Carpal joints with SF CTX-II concentrations > 300 pg/mL were 11 times as likely to be associated with OCI as were carpal joints with concentrations ≤ 300 pg/mL. Metacarpophalangeal joints with SF CTX-II concentrations > 200 pg/mL were 12 times as likely to be associated with OCI as were MCP joints with concentrations ≤ 200 pg/mL (Table 1).

Figure 1—
Figure 1—

Scatterplots of CTX-II concentration in samples of SF (A and B) and serum (C and D) and calculated values of SF:serum CTX-II ratio (E and F) for carpal joints (A, C, and E) and MCP joints (B, D, and F) in 40 Thoroughbred racehorses without joint injury before and after 5 to 6 months of race training (pre-[Pre] and postexercise [Post] samples, respectively) and in 38 Thoroughbred racehorses with OCI (1 joint assessed/horse). For each group in each panel, the short horizontal solid line represents the mean value; the horizontal dashed lines represent concentrations or ratios for which there was predictive value for discrimination of samples from OCI-affected horses from the pre- or postexercise samples. *†‡Mean values in the bracketed groups differ significantly (P < 0.001, P < 0.01, and P < 0.05, respectively).

Citation: American Journal of Veterinary Research 71, 1; 10.2460/ajvr.71.1.33

Table 1—

Sensitivity, specificity, positive and negative predictive values, and likelihood ratio for CTX-II concentrations in SF and serum and the calculated SF:serum CTX-II ratio with regard to discrimination between samples from OCI-affected horses and pre- or postexercise samples.

SampleSensitivity(%)Specificity (%)Positive predictive value (%)Negative predictive value(%)Likelihood ratio
Carpal joint SF (> 300 pg/mL)7194868611
MCP joint SF (> 200 pg/mL)6295808812
Serum (≤ 65 pg/mL)5595868012
SF:serum ratio (≥ 2.5)6997908821

Calculations were based on data from samples collected from carpal or MCP joints in 40 Thoroughbred racehorses without joint injury before and at the end of 5 to 6 months of race training (pre- and postexercise samples, respectively) and in 38 Thoroughbred racehorses with OCI (1 joint assessed/horse). Values in parentheses represent concentrations or ratios for which there was predictive value for discrimination of samples from OCI-affected horses from the pre- or postexercise samples.

Serum CTX-II concentrations were significantly lower in postexercise samples obtained from noninjured horses that underwent SF sample collection from carpal and MCP joints (P < 0.001 and P < 0.01, respectively), samples from horses with OCI of carpal joints (P < 0.001), and samples from horses with OCI of MCP joints (P < 0.001), compared with values in pre-exercise samples (Figure 1). There was no significant difference in CTX-II concentrations between postexercise serum samples and injured-horse serum samples. Serum samples with CTX-II concentrations ≤ 65 pg/mL were 12 times as likely to be associated with OCI as were serum samples with concentrations > 65 pg/mL (Table 1). Horses with OCI of only 1 carpal joint had higher mean ± SD serum CTX-II concentration (80.45 ± 47.35 pg/mL) than horses with OCI of multiple carpal joints (64.51 ± 31.66 pg/mL), but the difference was not significant (P = 0.39). Similarly, horses with OCI of only 1 MCP joint had higher mean serum CTX-II concentration (67.47 ± 29.46 pg/mL) than horses with OCI of multiple MCP joints (59.86 ± 43.67 pg/mL), but the difference was not significant (P = 0.72).

The SF:serum CTX-II ratio was significantly higher in horses with OCI of carpal joints, compared with the ratio for horses before (pre-exercise; P < 0.001) and after (postexercise; P < 0.001) training (Figure 1). The SF:serum CTX-II ratio was also significantly higher in horses with OCI of MCP joints, compared with findings in pre- (P < 0.01) and postexercise (P < 0.05) samples. In the noninjured horses in which carpal joint SF was analyzed, the postexercise SF:serum CTX-II ratio was significantly (P = 0.03) higher than the pre-exercise value. However, in the noninjured horses in which MCP joint SF was analyzed, there was no significant (P = 0.14) difference between pre- and postexercise SF: serum CTX-II ratios. Horses with a SF:serum CTX-II ratio ≥ 2.5 were 21 times as likely to have OCI of a joint as horses with a ratio < 2.5 (Table 1).

For horses with OCI of carpal joints, SF and serum CTX-II concentrations and SF:serum CTX-II ratio all made a significant (P < 0.01) contribution to the discriminant analysis model. Overall, serum CTX-II concentration was the best discriminator, followed by SF: serum CTX-II ratio, and then SF CTX-II concentration. After accounting for collinearity, serum CTX-II concentration was the best discriminator for the pre- and postexercise samples, whereas the SF:serum CTX-II ratio was the best discriminator for samples from horses with OCI. The discriminant model was developed on the basis of 31 randomly selected samples and overall correctly classified 81% of samples into their source groups (P < 0.01; Table 2). To verify the predictive nature of this model, an additional subset of 14 samples that was not used in model creation was evaluated and the model correctly classified 93% of those samples into their source groups.

Table 2—

Determination (3 × 3 table) of the predictive ability of serum CTX-II concentration, carpal and MCP joint SF CTX-II concentration, and SF:serum CTX-II ratio (assessed in Thoroughbred racehorses without joint injury before and at the end of 5 to 6 months of race training [pre- and postexercise samples, respectively] and in Thoroughbred racehorses with OCI [1 joint assessed/horse]) for use in classifying samples into the appropriate source group (pre-exercise [Pre] samples, postexercise [Post] samples, or samples from OCI-affected horses [OCI]).

JointFunctionsamples categoryPredicted grouping determined via discriminant analysis
PrePostOCITotalPercentage of samples correctly classified
CarpalModel development* (n = 31)Pre9101080.6
Post09211
OCI03710
Model validationt (n = 14)Pre510692.9
 Post0202
 OCI0066
MCPModel development* (n = 27)Pre9301274.1
Post1809
OCI0336
Model validation (n = 14)Pre330664.3
 Post0404
 OCI1124

Values in parentheses represent the number of samples used in the analysis.

A quadratic discriminant function was computed from a random sampling of the carpal and MCP joints (model development).

As a test of the model (model validation), an additional random subset of samples that were not used for determination of the original function (model development) was used to estimate how well the model could discriminate each sample into its respective source group.

For horses with OCI of MCP joints, SF and serum CTX-II concentrations and SF:serum CTX-II ratio all made a significant (P < 0.05) contribution to the discriminant analysis model. Overall, serum CTX-II concentration was the best discriminator, followed by SF: serum CTX-II ratio, and then SF CTX-II concentration. After accounting for collinearity, serum CTX-II concentration was the best discriminator for the pre- and postexercise samples, whereas SF CTX-II concentration was the best discriminator for samples from horses with OCI. The discriminant model was developed on the basis of 27 randomly selected samples and overall correctly classified 74% of samples into their source groups (P < 0.05; Table 2). To verify the predictive nature of this model, an additional subset of 14 samples that was not used in model creation was evaluated and the model correctly classified 64% of those samples into their source groups.

Among horses with OCI (age range, 2 to 7 years), serum concentrations were negatively correlated with age (r = −0.51; P < 0.001), indicating that younger horses had higher serum CTX-II concentrations than older horses. In addition, total arthroscopic scores were positively correlated (r = 0.50; P < 0.001) with total radiographic scores in horses with OCI. No significant correlations were identified between either total arthroscopic or radiographic scores and SF CTX-II concentration, serum CTX-II concentration, or SF:serum CTX-II ratio. Mean ± SD total radiographic scores in horses with OCI of carpal joints (8.77 ± 3.29) and horses with OCI of MCP joints (8.06 ± 4.63) were not significantly (P = 0.58) different. Mean arthroscopic score was higher for horses with OCI of carpal joints (15.21 ± 6.08), compared with the finding in horses with OCI of MCP joints (12.40 ± 4.21), but the difference was not significant (P = 0.14).

Discussion

Results of the present study indicated that SF CTX-II concentrations in samples collected from OCI-affected MCP or carpal joints in horses were significantly higher than values in similar samples collected from noninjured horses before (pre-exercise) and after (postexercise) training. This increase in SF CTX-II concentration in association with OCI suggests that increased type II collagen degradation occurs within a joint following that type of injury. The 2-fold increase in CTX-II concentration in SF samples obtained from horses with OCI, compared with concentrations in pre- and postexercise SF samples, was similar to findings of a previous human study7 in which SF CTX-II concentrations in individuals with various types of knee joint injury were ≥ 3 times as great as concentrations in joints of healthy persons.

In our study, exercise appeared to have no effect on SF CTX-II concentrations in MCP or carpal joints. However, increases in several SF biomarkers in response to treadmill exercise and early-stage osteoarthritis in horses have been reported.12 In that study, 16 horses were exercised on a treadmill for approximately 3 months with a 2-week rest period after 21 days. At 21 days, all horses underwent arthroscopy of both MC joints to ensure that the joints were normal (exercise control group; n = 8) or to create an osteochondral fragment in 1 joint (osteoarthritis-affected group; 8). Of the 8 SF biomarkers analyzed, concentrations of 5 increased in the exercise control horses by day 21, compared with prestudy baseline values. In the exercise control horses, concentrations of all 8 SF biomarkers were increased from baseline values at some time point after day 21, but it is unclear whether this was attributable to exercise or the arthroscopic examination. In contrast, the noninjured racehorses used in our study underwent training on a track surface for 5 to 6 months with no more than 1 day of rest/wk. No arthroscopic surgery was performed on these horses during the course of the study. If samples had been collected for analysis after 3 months of racetrack exercise, it is possible that findings may have been similar to those of that previous study. However, after 5 to 6 months of training, the SF CTX-II concentrations did not differ from pre-exercise values. The disparity in SF CTX-II concentrations between the 2 studies may be a consequence of duration and type of activity (treadmill exercise vs racetrack training).

In the present study, serum CTX-II concentrations were lower in postexercise samples and OCI-affected joint samples, compared with concentrations in preexercise samples. This pattern is similar to changes in serum BAP activity detected in horses with OCI, compared with findings in healthy horses.36 Horses in our study that had OCIs in multiple joints had lower serum CTX-II concentrations than horses that had OCI of only 1 joint. Even though this difference was not significant, it highlights the fact that cumulative joint damage from multiple sources can decrease serum CTX-II concentration. To the authors' knowledge, studies in horses or humans to assess changes in serum CTX-II concentrations in association with joint damage have not been performed. However, in rats with collagen-induced arthritis, serum CTX-II concentrations substantially increased and were predictive of articular cartilage damage.16 In contrast to the extent of joint damage in the horses of our study, the experimentally induced arthritis in those rats was more severe in a shorter period, which likely is the reason for the difference in findings. In horses with experimentally induced osteoarthritis, serum concentrations of 8 biomarkers increased in response to exercise and in response to OCI.12 In fact, in that study, measurements of 6 of 8 serum biomarkers could be used to distinguish between exercised horses and horses with OCI. We have no definitive explanation as to why CTX-II concentrations in postexercise serum samples and serum samples from horses with OCI were lower than the pre-exercise value in our study. The clearance of CTX-II from SF to serum may be affected by both exercise and injury. In addition, it is also possible that continued degradation of collagen fragments in the SF alters the structure of fragments that are cleared into the lymphatic system or blood-stream. This may result in a lack of assay recognition of CTX-II once the fragment reaches systemic circulation because of the aggregation or deletion of amino acids in the target sequence.

Although the biomarkers measured, type of exercise, and characteristics of joint damage in our study and in the previous investigation12 involving treadmill exercise and early-stage osteoarthritis in horses differed, the results of the 2 studies should be relatively similar. Age may have played a role because the horses used in the previous study12 were all 2 years old. The noninjured horses in our study were all approximately 2 years old, and samples were collected before and 5 to 6 months after commencement of training. However, the horses with OCI were 2 to 7 years old, and the younger affected horses had higher serum CTX-II concentrations than did the older affected horses. The higher serum CTX-II concentrations in those younger horses may be related to turnover of type II collagen in active physes, which may result in systemic release of CTX-II.38 In horses, closure of physes starts at < 3 months of age and extends as late as 3.5 years of age.39 Even though the relative amount of type II collagen released from the growth plates is probably minor at 2 to 3 years of age, compared with growth plate collagen release in neonates, this may contribute to the differences in results between studies as well as to the age-related differences within the group of OCI-affected horses in our study. Future studies are needed to determine the effect of age on serum CTX-II concentrations in horses.

To examine the relationship between serum and SF biomarker concentrations, other investigators have used a ratio of SF to serum biomarker concentrations to determine whether biomarkers were produced locally within the SF or to detect injury more effectively.36,40,41 In our study, SF:serum CTX-II concentration ratio was significantly higher in horses with OCI of carpal and MCP joints, compared with ratios in noninjured horses before and after training. This finding is similar to that of a previous study36 in which SF:serum BAP ratios were significantly higher in horses with OCI of carpal joints, compared with ratios in healthy horses. In our study, exercise appeared to increase the carpal joint SF: serum CTX-II ratio, compared with findings in horses before training. However, exercise did not increase MCP joint SF:serum CTX-II ratio, compared with findings in horses before training. This suggests that there is potential for the assessment of the SF:serum CTX-II ratio to identify an exercise effect more effectively than assessment of CTX-II concentration in either SF or serum alone.

Use of the CTX-II assay to distinguish between joints with and without OCI may be of diagnostic value. For this reason, we evaluated the ability of the assay to distinguish the pre- and postexercise samples from samples collected from the horses with OCI. To determine predictive values, cutoff values were determined for CTX-II concentrations in carpal and MCP joint SF samples, CTX-II concentration in serum samples, and SF:serum CTX-II ratio. The different cutoff values for SF CTX-II concentration in carpal and MCP joints suggest that there are some inherent differences in the mechanism and degree of injury between the 2 joints. It has been previously reported36,42–45 that there are biochemical and biomechanical differences among joints, and this should be considered in biomarker studies. Overall, for the purposes of joint injury detection, SF:serum CTX-II ratio had higher positive and negative predictive values and higher likelihood ratio than either SF CTX-II concentration or serum CTX-II concentration alone. Thus, at a cutoff for SF:serum CTX-II ratio of 2.5, there was a high probability that horses classified as positive had joint injury and that those classified as negative did not.

Discriminant analysis was also performed to determine how well the CTX-II concentrations could identify the different source groups by use of a combination of the SF CTX-II concentration, serum CTX-II concentration, and SF:serum CTX-II ratio. By combining all 3 variables, we hoped to not only determine the effect of OCI, but also determine whether the CTX-II assay could be used to discriminate pre-exercise samples from postexercise samples. The discriminant models correctly classified 64% to 93% of samples into their source group. This was similar to findings of another study46 in which discriminant analysis allowed 27 of 34 (79%) horses with carpal joint osteochondral fragments to be correctly classified as having or not having osteochondral fragmentation on the basis of 2 serum biomarker concentrations. All 3 variables (SF CTX-II concentration, serum CTX-II concentration, and SF: serum CTX-II ratio) were useful for discrimination among source groups in our model. Overall, serum CTX-II concentration was the best discriminator, likely because when examined alone, serum CTX-II concentration was most useful for distinguishing differences between the pre- and postexercise samples. In general, when misclassification occurred, it was usually because samples from OCI-affected joints were classified as postexercise samples or because pre-exercise samples were classified as postexercise samples. There was some error in this analysis because we assumed that the pre- and postexercise samples were independent of each other, when in reality they were not because they were obtained from the same horses. Even though we believe that this effect was minimal, further examination of SF CTX-II concentration, serum CTX-II concentration, and SF:serum CTX-II ratio should be performed on independent groups. Use of the discriminant analysis provided evidence of the usefulness and interaction of SF CTX-II concentration, serum CTX-II concentration, and SF:serum CTX-II ratio in biomarker studies.

Radiography is useful for detection of advanced articular changes, whereas arthroscopy has been viewed as the gold standard in detection of early chondropathy in osteoarthritis.47 In our study, carpal joints typically had higher total arthroscopic scores than MCP joints but, similar to findings of another equine biomarker study36 that used the same scoring systems, radiographic and arthroscopic scores for OCI-affected carpal or MCP joints were not significantly different. In the present study, there was no correlation between radiographic or arthroscopic scores and SF CTX-II concentration, serum CTX-II concentrations, or the SF:serum CTX-II ratio, which differs from findings of other equine biomarker studies36,37 in which these scoring systems have been used. In addition, the lack of correlation differs from results of osteoarthritis studies23,25,26 in people with moderate to severe and progressive osteoarthritis in which urinary CTX-II concentrations correlated with radiographic, arthroscopic, and magnetic resonance imaging changes. In our study, at least 66% of samples from horses with OCI could be distinguished from pre- and postexercise samples from clinically normal horses on the basis of SF and serum CTX-II concentrations and SF:serum CTX-II ratios, indicating that the combination of CTX-II concentrations in SF and serum and their ratio can be predictive of OCI even though CTX-II concentration in SF and serum does not correlate to radiographic and arthroscopic scores. One possible reason for this is that changes in CTX-II concentration in SF and serum reflect early joint changes that are not yet detectable via these imaging techniques.

Some factors are difficult to control when collecting clinical samples from horses with naturally occurring OCI, including chronicity of injury, exercise regimen, and medication history. Racehorses may be trained and treated in many different ways that could affect concentrations of CTX-II. For example, phenylbutazone (an NSAID that is commonly used in horses) can alter some biomarker concentrations in healthy joints after short periods of administration at recommended doses.48 Medication histories of the horses with OCI in the present study were unknown. Because of these types of factors, the number of OCI-affected horses (n = 38) may have been too low to yield sufficient statistical power to detect a correlation between SF CTX-II concentration, serum CTX-II concentration, or SF:serum CTX-II ratio and total radiographic or arthroscopic scores.

Results of our study support the use of the CTX-II assay in analyses of SF and serum samples to identify type II collagen degradation in OCI-affected horses. The assay was used to identify differences in CTX-II concentrations among samples from horses with OCI and pre- and postexercise samples from noninjured horses in training. In addition, serum and SF CTX-II concentrations as well as the ratio of those 2 variables had predictive value and discriminant ability to classify horses into their appropriate groups (pre-exercise, postexercise, or OCI). On the basis of the results of our study, measurements of SF and serum CTX-II concentrations and calculation of their ratio may be helpful in detecting cartilage degradation in horses with joint injury.

ABBREVIATIONS

BAP

Bone alkaline phosphatase

CTX-II

Carboxy-terminal telopeptide fragments of type II collagen

MC

Middle carpal

MCP

Metacarpophalangeal

OCI

Osteochondral injury

RC

Radiocarpal

SF

Synovial fluid

a.

Preclinical Cartilaps, IDS Nordic a/s, Fountain Hills, Ariz.

b.

Trumble TN, Brown MP, Merritt KA, et al. CTX II (Cartilaps) assay using equine synovial fluid (abstr). Osteoarthritis Cartilage 2006;14:S70–S71.

c.

Trumble TN, Brown MP, Merritt KA, et al. Serum pre-clinical CTX II (Cartilaps) assay validation using equine serum (abstr). Osteoarthritis Cartilage 2007;15:C78–C79.

d.

SPSS, version 15.0 for Windows, SPSS Inc, Chicago, Ill.

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Contributor Notes

Supported by the American College of Veterinary Surgeons resident-in-training grant No. 06103003.

Address correspondence to Dr. Trumble (trumb016@umn.edu).