ARTHRITIS & RHEUMATISM Volume 38 Number 1, January 1995, pp 78-81 0 1995, American College of Rheumatology 78

CORRELATION BETWEEN SYNOVIAL FLUID MARKERS OF CARTILAGE AND BONE TURNOVER AND SCINTIGRAPHIC SCAN

ABNORMALITIES IN OSTEOARTHRITIS OF THE KNEE

MOHAMMED SHARIF, EMMANUEL GEORGE, and PAUL A. DIEPPE

Objective. To test the hypothesis that scinti- graphic evidence of bone activity will correlate with biochemical evidence of increased matrix turnover in osteoarthritis (OA).

Methods. Keratan sulfate epitope (5D4), chon- droitin sulfate epitope (3B3), and osteocalcin (OC) were measured in synovial fluid (SF) from 35 patients with knee OA, within 1 month of bone scan.

Results. SF OC levels correlated with 5D4 levels (r = 0.32, P = 0.047) and with abnormalities on scintigraphic scan. Mean OC levels were 47% higher (P = 0.016) in patients with severely abnormal findings on scans compared with levels in patients with mildly abnormal scan findings. No significant association of 5D4 or 3B3 levels and perfusion- or late (bone)-phase scintigraphic abnormalities was found.

Conclusion. These data support the hypothesis that there is an association between late-phase bone scan abnormalities and SF biochemical markers of bone turnover in OA.

The standard clinical and radiographic methods used to assess osteoarthritis (OA) are only able to provide crude and insensitive measures of outcome, such as pain, functional impairment, and those ana- tomic changes which are severe enough to be seen on a plain radiograph. However, modern technology is providing exciting new information about the disorder. Advanced imaging techniques such as magnetic reso-

Supported by the Arthritis and Rheumatism Council. Mohammed Sharif, PhD, Emmanuel George, PhD, Paul A.

Dieppe, MD: University of Bristol Rheumatology Unit, Bristol Royal Infirmary, Bristol, UK.

Address reprint requests to Mohammed Sharif, PhD, Rheu- matology Unit, University of Bristol Department of Medicine, Bristol Royal Infirmary, Bristol BS2 8HW, UK.

Submitted for publication December 9, 1993; accepted in revised form July 27, 1994.

nance imaging and scintigraphy (l), and new biochem- ical assays which reflect the turnover of connective tissues (2) are both being used to investigate the disease processes and consequent joint damage seen in OA.

Scintigraphy , using technetium-labeled diphos- phonates, is widely used to detect areas of abnormal bone turnover in a variety of conditions, including arthritis (1). In OA, it is recognized that findings on bone scans are often abnormal, but that there is a discrepancy between scintigraphic and radiographic evidence of disease, suggesting that the bone scan may reflect the activity of the disease process, rather than its morphologic consequences (3,4). Furthermore, it has been shown that scan results are highly predictive of subsequent radiographic progression of both hand and knee OA (5,6).

The OA process involves an alteration in the normal balance of synthesis and degradation of matrix molecules in the articular cartilage and subchondral bone. This results in the release into body fluids of numerous products of this abnormal matrix synthesis and degradation, the so-called “biochemical (or skel- etal) markers” of the OA process (7). Assays of these marker molecules in the synovial fluid (SF) of an affected joint may provide direct information about the process occurring in that joint. Antibodies have been developed that can measure products of aggrecan degradation, such as keratan sulfate (KS), or synthe- sis, such as “neoepitopes” seen on chondroitin sulfate (CS) side chains (8,9). Osteocalcin (OC), which re- flects osteoblastic activity, can also be detected in the SF, and raised levels may be indicative of increased activity in subchondral bone (10,ll).

The present study examined the hypothesis that abnormal activity seen on bone scans of knee joints with OA will correlate with findings on SF assays of putative biochemical markers of disease activity.

SCINTIGRAPHIC ABNORMALITIES AND BONE TURNOVER IN OA 79

PATIENTS AND METHODS

Local ethical committee approval was obtained for the study. Patients were recruited for the study from a hospital-based rheumatology clinic. Entry criteria included current, use-related pain in the knee, with a clinically evident effusion, radiographic evidence of knee OA includ- ing both osteophytes and joint space narrowing, and no other rheumatologic diagnosis.

Knee radiographs included the standing anteroposte- rior (AP) view, obtained with the leg in full extension, and the lateral supine view, with 30" of flexion. These were read by a single observer (PAD) who was blinded to the biochem- ical and scintigraphic data. The information recorded in- cluded the Kellgren and Lawrence grading of knee OA (12). Scintigraphy was performed on the same day as or within 1 month of SF aspiration and radiography. AP scans were obtained within 5 minutes of intravenous injection of 600 mBq of 99mtechnetium-labeled hydroxymethylene diphos- phonate (the perfusion phase), and both AP and lateral scans were obtained 3 hours later (the late, or bone, phase) (3). All scans were read by a single observer (PAD) who was blinded to the radiographic and biochemical data. Abnormal isotope retention was graded as present or absent in both the perfusion and the bone phase, and the scan results were reported as normal (no uptake in either phase), abnormal (uptake in the bone phase only), or severely abnormal (uptake in both the perfusion and the bone phase). In addition, the localization and pattern of any uptake on the late-phase scans was recorded. The degree of intraobserver error in both radio- graphic and scintigraphic readings has been reported previ- ously (6).

SF was aspirated from the suprapatellar pouch and examined within 2 hours. The fluid was centrifuged at 2,OOOg for 10 minutes to remove cells and debris, aliquoted, and stored at -70°C. Aliquots were thawed only once immedi- ately prior to use, and all assays were carried out in duplicate, without any pretreatment of fluids. Total levels of glycosaminoglycan (GAG) were measured by the method of Farndale et a1 (13), using dimethylene blue and whale CS to standardize the technique. The KS epitope was measured with a mouse IgG monoclonal antibody (5D4) which recog- nizes a sulfated epitope at the nonreducing end of the KS chain (8), using the inhibition enzyme-linked immunosorbent assay developed by Thonar et a1 (14). The CS epitope was measured using a mouse IgM monoclonal antibody (3B3) which recognizes a sulfated epitope (9), as described previ- ously (15). The levels of both 5D4 and 3B3 epitopes were expressed as ratios of total GAG, to account for variations in the volume of SF aspirated. OC levels were measured with a radioimmunoassay kit (purchased from Biogenesis Ltd., Poole, England) which utilizes an IgG human monoclonal antibody to OC. Intra- and interassay variations were calcu- lated for all assays of SF biochemical markers.

Analysis of variance was used to compare mean levels of markers with scan categories. Student's t-test was used to investigate associations between cartilage markers and scintigraphic patterns in different knee compartments.

RESULTS

Twenty-three women and 12 men, with a mean age of 67 years (range 42-82 years) and a mean disease duration of 16 years (range 1-39 years) were recruited for the study. All patients had activity-related pain in the index knee at the time of the study. The Kellgren and Lawrence score in that knee was grade 2 in 6 patients, grade 3 in 19, and grade 4 in 10. Scintigraphy results were normal in 3 index knees, abnormal (late- phase retention only) in 18, and severely abnormal (late and perfusion phases) in 14. Late-phase localiza- tion of uptake was to the medial compartment in 26, the lateral compartment in 12, and the patellofemoral joint in 8 (15 knees had abnormal uptake in more than 1 compartment on the late-phase image).

The intra- and interassay coefficients of varia- tion were <7% and <12%, respectively, for the GAG assay, <3% and <4% for the KS assay, <7% and <13% for the CS assay, and <6% and 4 % for the OC assay. The concentrations of the different epitopes varied greatly among the 35 SF samples. The mean 2 SD values were total GAG 71.5 k 30.8 pg/ml (range 26.2-172), KS (5D4) 10.44 t 5.84 pg/ml (range 1.96- 27.97), CS (3B3) 1.29 ? 0.78 pg/ml (range 0.32-3.13), and OC 5.8 t 2.6 ng/ml (range 1.9-12.8). There was a weak but significant positive correlation between OC and 5D4 levels (r = 0.32, P = 0.047), but no significant correlation between other assay results.

Osteocalcin levels were significantly associated with scan abnormalities (Figure 1). The mean ? SD concentration of OC in the SF of patients with normal, abnormal, and severely abnormal scan results was 3.0 t 0.6 ng/ml, 4.9 ? 1.9 ng/ml, and 7.8 & 3.0 ng/ml, respectively (P = 0.016 between abnormal and se- verely abnormal scan results, by unpooled t-test). There were no significant associations between scan patterns and other biochemical assay results. When SF concentrations were related to the pattern of late- phase scan abnormality, a trend toward higher KS/ GAG and CS/GAG ratios in those with abnormal uptake in the medial compartment was noted, but this did not reach statistical significance.

DISCUSSION

Scintigraphy (3,4) and SF assays of biochemical process markers (2,7) are two new techniques being used to explore the disease process in OA. Findings on scintigraphy , which is thought to reflect increased turnover of the subchondral bone, have been shown to

80 SHARIF ET AL

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SCINTIGRAPHY SCAN PATTERNS

Figure 1. Synovial fluid osteocalcin (OC) levels in osteoarthritis patients by category of results on scintigraphy scan. Horizontal bars indicate the median concentration in each group.

be predictive of subsequent radiographic change (61, and scan results therefore may reflect an important aspect of the process driving disease progression. Findings on assays of OC, and of GAG, KS, and cs epitopes, are thought to reflect turnover of bone and cartilage, respectively (7,ll). This study was designed to test the hypothesis that the results of SF-based assays of these molecules would correlate with scinti- graphic evidence of increased bone turnover, and that there would be a positive correlation between bone and cartilage turnover markers in the SF.

The patients recruited for the study had well- established knee OA, and a distribution of clinical, radiographic, and scintigraphic abnormalities that are representative of those described in previous hospital- based studies of knee OA (3,16). However, an effusion that was easy to detect clinically also had to be present for entry into the study, which may mean that these Patients were less representative than would be ideal.

The main finding reported here is a positive association between the severity of the scintigraphic

abnormalities and the SF level of OC. The grading of the scans as severely abnormal when both perfusion- and late-phase abnormalities were present is consis- tent with our previous studies, which showed that only those patients with the most scan-active joints, and the greatest areas and density of uptake on the bone phase, also had perfusion-phase abnormalities (perfusion-phase uptake without late-phase uptake has not been observed) (3). Osteocalcin is a marker of bone synthesis (1 l), and serum levels of OC have been used to assess generalized bone turnover in a number of disorders (17,18). Previous data on SF levels of OC are conflicting (10,19). Poole et a1 (19) reported higher levels in the SF than in the serum of many OA patients, indicating that at least some of the SF OC is likely to be derived from local subchondral bone. However, whether the relationship between SF OC and scan activity reported here reflects generalized or localized increase in bone turnover is unknown.

Further support for the notion that SF OC levels may in part reflect local disease activity comes from the second significant finding reported here, the association between OC and KS levels. It is well established that SF levels of KS are far higher than serum levels, and they almost certainly reflect local articular cartilage turnover (14,ZO). The data therefore provide some additional support for the concept of a linkage between the active turnover or metabolism of the subchondral bone and of the articular cartilage in the OA process. The trend toward an association of KS/GAG and CS/GAG ratios with medial compart- ment scan abnormalities, although not significant, also suggests a possible link between bone and cartilage turnover.

A scan or SF sample obtained at a single time point can provide only a partial snapshot of a disease process that evolves over many years, and probably changes during different phases of the condition. How- ever, our findings indicate that these techniques may provide valuable insights into the abnormal turnover of cartilage and bone that is central to the OA process, and be of potential value in disease assessment and the investigation of novel therapeutic approaches.

ACKNOWLEDGMENTS

We acknowledge the excellent technical assistance of Ms Kate Meadows. We are also grateful to Mr. Lee Shepstone for his help and advice with data management.

SCINTIGRAPHIC ABNORMALITIES AND BONE TURNOVER IN OA 81

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