The ability to assess osteoarthritis progression is paramount to clinicians and patients. One area of great interest in the assessment of osteoarthritis progression is the identification and validation of molecular markers (biomarkers and biochemical markers). Synovial fluid concentrations of biomarkers involved in cartilage and synovial membrane metabolism have long been considered as potentially important surrogate measures of the local disease progression. Yet, when attempting to correlate these fluid marker concentrations with osteoarthritis progression, several potential confounding factors must be considered. One important concern is that the measured synovial fluid concentration of a biomarker is dependent on the method of collection. Arthrocentesis is the standard collection method; however, the actual aspiration of the fluid can be done either neatly (ie, without dilution) or following addition of an intra-articular lavage solution. Although preaspiration lavage is a common procedure to facilitate synovial fluid removal, this method contributes substantial error to synovial fluid biomarker analyses as a result of the inability to predict sample dilution.1 Furthermore, lavage may affect joint metabolism afterward; however, data exist that suggest that this is not the case.1,2 Other factors involved in the intra-articular concentration of any biomarker include but are not limited to the following: 1) the input rate of the marker molecule, 2) the flow of water across the synovial membrane, 3) synovial membrane properties that affect the clearance rate of the biomarker, and 4) the osmotic gradient between the synovial fluid and the serum.3–5 The clinical result of many of these potential factors is joint effusion even prior to the joint lavage. Thus, the question of whether effusion or induced dilution has occurred is vital when trying to analyze the importance of synovial fluid concentrations of a biomarker.
Recently, concurrent measurement of synovial fluid urea concentrations in a sample has been reported as a highly quantifiable method of accurately correcting for the unpredictable dilution caused by inflammation or lavage in dogs with induced osteoarthritis.1 Previously, synovial fluid biomarker data on experimental dogs with osteoarthritis have been reported by use of neat (undiluted) and lavaged sample collection techniques with little discussion of the aforementioned possible dilutional effects.3,5-20 The purpose of the study reported here was to examine the longitudinal changes in unadjusted and urea-adjusted synovial fluid concentrations of KS and HA in samples obtained without lavage from dogs with induced osteoarthritis as a result of CCL transection. Keratan sulfate concentrations have been used to document proteoglycan degradation in joints, and serum KS concentrations have been advocated as a marker of osteoarthritis progression.6,21-25 Hyaluronan has also been examined in serum and synovial fluid in an effort to associate concentration changes with the progression of osteoarthritis.7,26,27 However, conflicting results of KS and HA concentrations in clinically normal and diseased dogs have been reported.8–18 A strong possibility is that some of these conflicting results may be attributable in part to a failure in accounting for dilution factors between studies.
Materials and Methods
Study design—Twelve clinically normal adult mixed-breed hounds were used in the study. The University of Georgia Animal Care and Use Care Committee approved the study. Anesthesia was induced in dogs with thiopental and maintained with isoflurane. Under sterile conditions, the CCL of the right stifle joint was transected through a small lateral parapatellar arthrotomy site. The left stifle joint was not operated. Dogs were given butorphanol (0.2 mg/kg, IM) prior to and following extubation. If lameness was observed at the time of recovery, dogs were given additional doses of butorphanol for 24 hours. Serum and synovial fluid samples for determination of KS and HA concentrations were taken prior to and 1, 2, 3, and 12 months after surgery.
Sample collection—Blood was collected from the external jugular vein. All blood samples were drawn in the early morning. Serum was removed and frozen at −70°C until analyzed. Dogs were anesthetized by IV administration of propofol. Synovial fluid was then collected via arthrocentesis. All synovial fluid samples were drawn in the midmorning. Synovial fluid was stored in aliquots at −70°C until analyzed.
Assays—Keratan sulfate (5D4 epitope) concentrations in serum and synovial fluid were measured by use of a competitive indirect ELISA, as previously described.28 Serum and synovial fluid concentrations of HA were measured with a sandwich ELISA, as previously described.29 For the KS assay, the interassay variation was 4% and the intra-assay was 3%. For the HA assay, the interassay variation was 6% and the intra-assay was 4%. Serum and synovial fluid urea concentrations were measured by use of an enzymatic in vitro assay, a chemistry analyzer,a and a urea-BUN assay kit.b Briefly, the reaction begins as urease hydrolyzes urea to form CO2 and ammonia. The ammonia then reacts with α-ketoglutarate and the reduced form of nicotinamide adenine dinucleotide in the presence of glutamate dehydrogenase to form glutamate and the oxidized form of nicotinamide adenine dinucleotide. The consumption of the reduced form of nicotinamide adenine dinucleotide is measured kinetically at a wavelength of 340 nm. Synovial fluid concentrations of HA and KS were adjusted on the basis of synovial fluid urea concentrations by use of the method previously described.1
Statistical analysis—A repeated-measures ANOVA was used to analyze serum and synovial fluid concentrations of HA and KS over time within and between stifle joints. Values of P < 0.05 were considered significant.
Results
Operated stifle joints had significantly lower synovial fluid concentrations of KS at 1, 2, and 3 months after surgery, compared with baseline values (Figure 1). This finding was consistent for urea-adjusted and unadjusted samples (Figure 2). Unoperated contralateral stifle joints had significantly lower KS concentrations at 1 month after surgery, compared with baseline values; however, the concentrations returned to preoperative values by 2 months and were maintained at that concentration for the duration of the study. Comparison of operated stifle joints to unoperated stifle joints revealed that operated stifle joints had significantly lower synovial fluid KS concentrations at 2 and 3 months after surgery. Hyaluronan concentrations in operated stifle joints significantly decreased, compared with baseline values, at 1, 2, and 3 months after surgery (Figure 3). Again, these findings were consistent for urea-adjusted and unadjusted samples (Figure 4). No significant changes were found in unoperated stifle joints over time. Comparison of operated stifle joints to unoperated stifle joints revealed that operated stifle joints had significantly lower HA concentrations at 1, 2, and 3 months after surgery.
Mean ± SE synovial fluid KS concentrations over time in operated (solid bars) and unoperated (open bars) stifle joints of dogs. *Significant (P < 0.05) difference from baseline value.
†Significant (P < 0.05) difference from unoperated stifle joint.
Citation: American Journal of Veterinary Research 67, 3; 10.2460/ajvr.67.3.429
Mean ± SE unadjusted (solid bars) and urea-adjusted (open bars) synovial fluid KS concentrations in operated stifle joints of dogs. See Figure 1 for key.
Citation: American Journal of Veterinary Research 67, 3; 10.2460/ajvr.67.3.429
Mean ± SE synovial fluid HA concentrations over time in operated (solid bars) and unoperated (open bars) stifle joints of dogs. See Figure 1 for key.
Citation: American Journal of Veterinary Research 67, 3; 10.2460/ajvr.67.3.429
Mean ± SE unadjusted (solid bars) and urea-adjusted (open bars) synovial fluid HA concentrations in operated stifle joints of dogs. See Figure 1 for key.
Citation: American Journal of Veterinary Research 67, 3; 10.2460/ajvr.67.3.429
Serum KS concentrations did significantly increase from baseline values at 3 months after surgery. At the other time points, serum KS concentrations were increased from baseline values but these changes were not significant (Figure 5). No significant changes were found in serum HA concentrations over time, compared with baseline values (Figure 6).
Mean ± SE serum KS concentrations over time in dogs following CCL transection in the right stifle joint. See Figure 1 for key.
Citation: American Journal of Veterinary Research 67, 3; 10.2460/ajvr.67.3.429
Mean ± SE serum HA concentrations over time in dogs following CCL transection in the right stifle joint.
Citation: American Journal of Veterinary Research 67, 3; 10.2460/ajvr.67.3.429
Discussion
To our knowledge, this is the first longitudinal study to correct for an unknown dilution of the synovial fluid by use of urea as a passive marker.1 It also appears to be the first study on dogs with experimentally induced osteoarthritis of the stifle joint in which synovial fluid marker concentrations are measured in samples obtained without lavage and for a period of > 6 months. The fact that synovial fluid concentrations of HA and KS decreased following CCL transection in our study and then began to increase for unadjusted and urea-adjusted values is interesting. It is also noteworthy that no difference was found between absolute and urea-adjusted values at any time point. It appears that by 1 month after surgery, chronic effusion is in equilibrium with regard to fluid fluxes.
Our findings are consistent with synovial fluid HA concentrations in samples obtained without lavage in a 12-week evaluation of dogs with the same type of experimentally induced osteoarthritis.17 Results of our study are also consistent with those of dogs with naturally occurring CCL deficiencies in which lower synovial fluid concentrations of the 5D4 epitope of KS were found in affected stifle joints, compared with contralateral unaffected stifle joints.15 However, our results differ from those of other studies13,18 of dogs with naturally occurring CCL ruptures in which an increase in 5D4 epitope concentrations were found in synovial fluid samples collected without lavage. Our finding of low synovial fluid 5D4 epitope concentrations is also consistent with those of people with acute knee joint injuries and people with established knee joint osteoarthritis.23,30 The exact cause of this initial decrease in synovial fluid KS and HA concentration is not known. Historically, synovitis and associated increased joint volume (effusion) have been suggested as probable contributors to this decrease; however, comparing neat (undiluted) unadjusted values to urea-adjusted values, dilution appears less likely to be a valid explanation. It is possible that the KS and HA are degraded into fragments too small to be detected by the ELISA. The significant decrease in synovial fluid KS concentration found in the unoperated stifle joint suggests that, at least initially, a systemic effect on KS production occurs, which may be inflammatory in nature. The subsequent increase in concentrations of the 2 markers may confirm speculation that the effect of surgery is temporary; however, it may also indicate an increased metabolic response of the cartilage.15,25 Certainly, our findings do not provide information on other potential factors that can affect synovial fluid marker concentrations during the continuum of acute inflammation, cartilage destruction, and subsequent repair. Recent results from longitudinal studies on dogs with similarly induced osteoarthritis of the stifle joint indicate that synovial fluid concentrations of 7D4 (native chondroitin sulfate), 3B3 (degraded/abnormal chondroitin-4-sulfate neoepitope), and type II collagen collagenase-generated cleavage neoepitope (COL2long) increase following injury in samples obtained without lavage.19,20 These data were not urea-adjusted, but it does provide additional information to suggest that surgically induced inflammation does not ubiquitously decrease all cartilage metabolism markers even transiently.
Changes in absolute and urea-adjusted synovial fluid concentrations of KS in our study, in which synovial fluid samples were collected without lavage, are in direct contrast to findings in other studies in which synovial fluid samples were collected via lavage in dogs with experimentally inducted osteoarthritis of the stifle joint. In these studies, increases are seen at all time periods in a variety of markers including KS.11,12,16 These discrepancies further deepen the question as to what effect lavage may have on synovial fluid analysis results.
| KA | Keratan sulfate |
| HA | Hyaluronan |
| CCL | Cranial cruciate ligament |
Roche BMC Hitachi 912 automated chemistry analyzer, Roche Diagnostics Corp, Indianapolis, Ind.
Roche Diagnostic urea/BUN assay kit, Roche Diagnostics Corp, Indianapolis, Ind.
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