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Levels of chondroitin sulfate isomers in synovial fluid of patients with hip osteoarthritis
J250 Orthop Sci (1999) 4:250–254
H. Yamada et al.: Chondroitin sulfate in osteoarthritis
Levels of chondroitin sulfate isomers in synovial fluid of patients with hip osteoarthritis Harumoto Yamada1, Satoshi Miyauchi2, Hiraku Hotta1, Mitsuhiro Morita1, Yasuo Yoshihara1, Toshiyuki Kikuchi1, and Kyosuke Fujikawa1 1 2
Department of Orthopaedic Surgery, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama 359, Japan Tokyo Research Institute, Seikagaku Corporation, 3-1253 Tateno, Higashi-Yamato, Tokyo, Japan
Abstract: We determined the levels of chondroitin sulfate (CS) isomers in the synovial fluid of patients with hip osteoarthritis (OA) to investigate their significance as markers reflecting extracellular matrix metabolism in joint tissues. A cross-sectional study of synovial fluid aspirated from 50 hip joints of 50 patients with OA was performed. Concentrations of chondroitin-4-sulfate (C4S) and chondroitin-6-sulfate (C6S) were determined by high-performance liquid chromatography. Levels of each marker molecule were investigated in relation to age and radiological stage of the disease. In all disease stages, the dominant CS isomer in synovial fluid was C6S. There was a significant negative correlation between levels of C6S and age. A significant inverse correlation was also observed between the ratio of C6S to C4S and age. Results of analysis of covariance in which age was covariate showed that the ratio of C6S to C4S in advanced and terminal stage OA was lower than that in early stage OA. The present results indicate that the ratio of CS isomers in synovial fluid in hip OA varies with the severity of disease. These molecules in synovial fluid may serve as a useful marker reflecting extracellular matrix metabolism in OA. Key words: chondroitin sulfate, synovial fluid, osteoarthritis of the hip joint, joint marker, cartilage
Introduction Osteoarthritis (OA) is primarily a disorder of articular cartilage, caused by mechanical stress, combined with
Offprint requests to: H. Yamada, Department of Orthopaedic Surgery, Fujita Health University, Second Hospital, 3-6-10 Otohbashi, Nakagawa-ku, Nagoya 4548509, Japan Received for publication on May 13, 1998; accepted on Dec. 17, 1998
biochemical changes in the cartilage matrix and inadequate cartilage and bone repair processes.13 Secondary synovitis resulting from the breakdown products of cartilage and bone matrix is likely to modify the symptoms of OA. In spite of the rapid progress in imaging technologies for diagnosis of arthritic diseases, it is still extremely difficult to recognize subtle changes in joint components, including articular cartilage.7 This is especially true for the hip joint, where even the resolution of magnetic resonance imaging (MRI) may not be sufficient to detect wear and focal defects of the articular cartilage. Assessment of joint pathology is essential for the correct diagnosis and subsequent treatment of OA. Recently, several joint markers reflecting the metabolism of different components of joint tissue have been identified.11,20 As OA is not a single disease entity but has diverse pathogenesis, markers which reflect the different aspects of OA have been investigated. Fragments of cartilage extracellular matrix components are important markers which reflect both normal turnover and catabolism of cartilage tissue. Since the major glycosaminoglycans (GAGs) in adult human articular cartilage are chondroitin-6-sulfate (C6S) and keratan sulfate, synovial fluid concentrations of these GAGs in pathological conditions have been investigated as catabolic markers during cartilage degradation.2,3 Regenerated fibrocartilage seen in OA joints and synovium have been shown to synthesize chondroitin-4-sulfate (C4S).6,12 The detection of chondroitin sulfate (CS) in synovial fluid has been mainly performed with cellulose acetate membrane electrophoresis or ion-exchange choromatography.2,16 The precise analysis of synovial fluid CS has been difficult using these conventional methods. High-performance liquid chromatography (HPLC), a recently developed analytical technique, provides a more sensitive method to measure CS levels in synovial fluid.18 The significance of GAGs as joint markers has
H. Yamada et al.: Chondroitin sulfate in osteoarthritis
been investigated using knee synovial fluid from patients with various arthritic diseases.18,19 However, there is at present very little data on GAG levels in the synovial fluid in other joints, including the hip joint.15 Since the volume of synovial fluid in the joint cavity and amount of articular cartilage and synovium differs significantly in each joint, the production and clearance of joint marker molecules may also be different. We report the results of a cross-sectional study of synovial fluid levels of C6S and C4S in patients with hip OA, and we examine the significance of these molecules as markers reflecting extracellular matrix metabolism in OA.
Patients and methods Patients Needle puncture through the joint capsule was performed at 117 hip surgeries for 117 OA patients (77 total hip arthroplasties and 40 pelvic osteotomies). Fifty samples of hip synovial fluids could be aspirated from 50 patients; 67 patients were excluded, since no significant synovial fluid could be aspirated from the joint cavity. Diagnosis of hip OA was made according to the criteria recommended by the American College of Rheumatology.1 Forty-two patients (84%) were diagnosed with secondary hip OA after developmental acetabular dysplasia. OA patients were further subdivided into four groups (pre-, early, advanced, and terminal stage disease) based on the radiological staging system provided by the Japanese Orthopaedic Association.17 General characteristics of the patients are shown in Table 1. Patients with advanced and terminal stage OA were significantly older than those with early stage OA (P , 0.05). Thirty-nine patients had previously been treated with various nonsteroidal antiinflammatory drugs (NSAIDs) for the relief of hip pain. No patients had received prior treatment of intraarticular injection of steroids or high-molecular weight hyaluronic acid. Table 1. General characteristics of the patients in the present study Stagea PreEarly Advanced Terminal Total
Number of patients 0 17 6 27 50
Age (years)b 36.9 52.2 62.4 54.7
— 6 13.2 6 12.0* 6 11.2* 6 11.9
Female/male — 17/0 5/1 25/2 47/3
* P , 0.05 vs early stage a Stage of hip deformity was assessed by the radiological findings according to the criteria of the Japanese Orthopaedic Association17 b Values are means 6 SD
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Preparation of samples Synovial fluid was collected into sterile tubes without additives and centrifuged at 10 000 rpm for 20 min at 4°C to remove cells and tissue debris. The supernatants were stored in aliquots at 280°C for later assay. Measurement of CS isomers Levels of C4S and C6S were determined by HPLC as previously described.18 Briefly, synovial fluid samples were diluted tenfold with distilled water and digested with chondroitinase ABC (Seikagaku, Tokyo, Japan) and chondroitinase AC-II (Seikagaku) before analysis to reduce the GAGs to disaccharides, which were then analyzed by HPLC, using a 7.6 3 150 mm column packed with propylamine-bound silica gel (YMC gel PA-120; YMC, Kyoto, Japan), combined with fluorometry according to the method of Toyoda et al.21 The effluent was monitored with a spectrofluorometer (Model 920-FP, Japan Spectroscopic, Tokyo, Japan) set at an excitation wavelength of 331 nm and emission wavelength of 383 nm. Statistical analysis Correlations were sought using the Spearman’s rank correlation coefficient. Analysis of covariance (ANCOVA), in which age was the covariate, was performed to determine the relation between marker levels and disease stage. Differences between other groups were analyzed by Mann-Whitney U-test for unpaired variables.
Results Synovial fluid levels of GAGs in patients with hip OA The mean values (6SD) of the concentrations of C6S and C4S in the synovial fluids are shown in Table 2. When the amounts of the two CS isomers in each dis-
Table 2. Concentrations of chondroitin 6 sulfate (C6S) and chondroitin 4 sulfate (C4S) in synovial fluid of patients in each disease stage Stage Early Advanced Terminal
C6S
C4S
114.3 6 59.4 84.6 6 30.0 88.5 6 41.7
32.6 6 11.7 35.5 6 11.3 45.6 6 19.4
Values are means 6 SD nmoles/ml for unsaturated disaccharides derived from C6S and C4S No significant differences were observed between glycosaminoglycan (GAG) concentrations in each disease stage, using analysis of covariance (ANCOVA)
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Fig. 1. Correlation between level of chondroitin 6 sulfate (C6S) in hip synovial fluid and age. Diluted synovial fluid samples were digested with chondroitinase ABC and chondroitinase AC-II before analysis to reduce the glycosaminoglycans to disaccharides. Unsaturated dissaccharides derived from chondroitin sulfates in the synovial fluid were then analyzed by high performance liquid chromatography (HPLC) combined with fluorometry, as described in Materials and Methods. Levels of C6S were expressed as concentrations (nmoles/ml) of unsaturated disaccharides derived from C6S. A significant negative correlation was observed (Spearman’s rank correlation coefficient, 20.334; P , 0.05)
H. Yamada et al.: Chondroitin sulfate in osteoarthritis
Fig. 2. Correlation between ratio of C6S to C4S in hip synovial fluid and age. Unsaturated disaccharides derived from chondroitin sulfates in the synovial fluid by sequential digestion with chondroitinase ABC and chondroitinase AC-II were analyzed using HPLC, as described in Materials and Methods. The ratio of C6S to C4S was expressed as the molar ratio of unsaturated disaccharides derived from C6S and C4S. A significant negative correlation was observed (Spearman’s rank correlation coefficient, 20.657; P , 0.0001)
ease stage were compared, C6S was predominant in each group. Results of ANCOVA in which age was the covariate showed that there were no significant differences in the levels of C6S and C4S at each disease stage. Levels of C4S showed no significant correlation with age (data not shown). A weak but statistically significant negative correlation was observed between the level of C6S and age (r 5 20.334; P , 0.05) (Fig. 1). No significant differences in the levels of the two CS isomers were observed between primary and secondary hip OA. Treatment with NSAIDs had no significant effect on the levels of the two CS isomers (data not shown). Relationship between ratio of C6S to C4S and age/disease stage The molar ratio of the two CS isomers was investigated relative to age and disease stage. A significant inverse correlation was observed between the ratio of C6S to C4S and age (r 5 20.657; P , 0.0001) (Fig. 2). The ratios of C6S to C4S in patients with each disease stage are shown in Fig. 3. The mean value of the ratio of C6S to C4S at each stage was 3.38 6 0.82 (early), 2.37 6 0.17 (advanced), and 1.86 6 0.41 (terminal). When the ratio of C6S to C4S was compared with the radiological as-
Fig. 3. Relationship of the ratio of C6S to C4S in hip synovial fluid and the radiological disease stage. The ratio of C6S to C4S is shown as the molar ratio of unsaturated disaccharides derived from C6S and C4S. The ratio of C6S to C4S in advanced and terminal stage osteoarthritis (OA) was significantly lower than that in early stage OA (analyzed by ANCOVA). Values are presented as means and SD. * P , 0.05; ** P , 0.01
H. Yamada et al.: Chondroitin sulfate in osteoarthritis
sessment of hip OA using ANCOVA in which age was the covariate, the ratio in advanced and terminal stage OA was significantly lower than that in early stage OA (Fig. 3) (P , 0.05 for advanced stage OA, P , 0.01 for terminal stage OA). No significant differences in the ratio of the two CS isomers were observed between primary and secondary hip OA. Treatment with NSAIDs had no significant effect on the ratio of the two CS isomers (data not shown).
Discussion In the present study, levels of the unsaturated disaccharide isomers of C6S and C4S in hip synovial fluid were measured using HPLC combined with fluorometry. This assay method has previously been shown to be extremely sensitive and useful for the detection of these CS isomers in the knee synovial fluid of patients with various arthritic diseases, including OA.18 The present experimental results showed that the ratio of these two CS isomers varied significantly with the disease severity of hip OA. Remarkable progress has been made recently in research of the mechanism of cartilage destruction in arthritic diseases. The degradation of aggrecan and release of its fragments from cartilage in OA is believed to be mainly mediated by proteolytic enzymes.14 Aggrecan molecules are synthesized by chondrocytes and the cleaved fragments are liberated into the synovial fluid after enzymatic degradation. Thus, the levels of aggrecan-related fragments in synovial fluid may reflect both the synthesis and degradation of aggrecan in cartilage. Various methods have been developed to analyze the levels of aggrecan fragments in synovial fluid. The concentration of sulfated GAGs and proteoglycan fragments in knee synovial fluid was measured spectrophotometrically, using dimethylmethylene blue and a radioimmunoassay, respectively.4 These studies demonstrated an increase of aggrecanrelated fragments in arthritic diseases, including OA. Dahlberg et al.5 measured the concentration of aggrecan fragments in the synovial fluid of patients with knee OA, using an enzyme-linked immunosorbent assay (EIA), and demonstrated that the levels of aggrecan fragments decreased with advancing disease severity. Campion et al.3 have shown an inverse correlation between radiological cartilage loss and levels of antigenic keratan sulfate in osteoarthritic synovial fluid measured by EIA. In the present study, HPLC was used to measure the level of unsaturated disaccharide isomers of C6S in the synovial fluid of patients with hip OA. This measurement reflects the levels of aggrecan-related fragments present in the synovial fluid. The present assay method cannot provide information about the mole-
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cular weight of the CS isomers. However, this assay method is extremely sensitive for the detection of total aggrecan fragments. Multiple etiologic factors have been shown to be associated with the development of OA. Eighty-four percent of the patients who entered the present study were diagnosed with secondary hip OA after congenital acetabular dysplasia. Congenital acetabular dysplasia in Japanese occurs predominantly in females, and 94% of the subjects in this study were women. These characteristics may suggest that the patients with hip OA in the present study have a relatively uniform background as regards the etiopathogenesis of this disease. No significant correlation was observed between levels of C6S and the severity of hip OA assessed by radiological findings. These data were different from those compiled from previous reports on knee OA. This discrepancy between results for hip and knee OA may be explained, in part, by differences in the assay of aggrecan fragments and assessments of radiological findings, and, mainly, by differences in the anatomical structure of the joints. For example, clearance and volume flux may differ markedly in the hip and knee joints.10 Furthermore, it has been shown that aggrecan fragments are easily retained in hip cartilage, while being lost quickly from knee cartilage into the synovial fluid.8 Although normal human articular cartilage contains only minor amounts of C4S, it is well known that regenerated osteoarthritic cartilage and chondro-osseous spurs may produce singnificant amounts of C4S.12 C4S is produced by synovial tissue and may also be derived from plasma by filtration through the synovium.9 C4S is thus considered as a marker that reflects both changes in cartilage metabolism and synovial inflammation. Shinmei et al.19 have indicated that the ratio of C6S to C4S is significantly higher in primary knee OA and traumatic arthritis than in rheumatoid arthritis, and this ratio is a useful diagnostic marker reflecting the matrix metabolism in joint tissues. In the present study, the ratio of these two CS isomers in the synovial fluid of patients with hip OA was shown to be significantly dependent on the disease severity of OA. In the present cross-sectional study, patients with each disease stage varied significantly in age. Since the disease severity and age were closely related, ANCOVA was used to control for possible confounding between these two variables. The effect of disease stage on the ratio of the two CS isomers remained significant on ANCOVA in which age was the covariate. We propose that this ratio is more useful as a marker reflecting the metabolism of cartilage extracellular matrix than the concentration of C6S alone, since the ratio of these two CS isomers correlates significantly with the disease severity of hip OA. It cannot be concluded from the present experiment alone whether these CS isomers are
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useful for determining the prognosis of hip OA. Further studies will also be needed to more fully explore the clinical significance of these molecules for the differential diagnosis of OA from other arthritic disease. Acknowledgments. This study was partly supported by a grant from the Hip Joint Foundation of Japan. The authors are deeply grateful to the late Professor Masayuki Shinmei, of the National Defense Medical College, for his encouragement in the initiation of this study.
References 1. Altman R, Alarcón G, Appelrouth D, et al. The American College of Rheumatology criteria for the classification and reporting of osteoarthritis of the hip. Arthritis Rheum 1991;34:505–14. 2. Barker SA, Hawkins CF, Hewins M. Mucopolysaccharides in synovial fluid. Detection of chondroitin sulphate. Ann Rheum Dis 1966;25:209–13. 3. Campion GV, McCrae F, Schnitzer TJ, et al. Levels of keratan sulfate in the serum and synovial fluid of patients with osteoarthritis of the knee. Arthritis Rheum 1991;34:1254–9. 4. Carroll G. Measurement of sulphated glycosaminoglycans and proteoglycan fragments in arthritic synovial fluid. Ann Rheum Dis 1989;48:17–24. 5. Dahlberg L, Ryd L, Heinegård D, et al. Proteoglycan fragments in joint fluid. Influence of arthrosis and inflammation. Acta Orthop Scand 1992;63:417–23. 6. Hamerman D, Smith C, Keiser HD, et al. Glycosaminoglycans produced by human synovial cell cultures. Collagen Rel Res 1982;2:313–29. 7. Hodgson RJ, Carpenter TA, Hall LD. Magnetic resonance imaging of osteoarthritis. In: Kuettner KE, Schleyerbach R, Peyron JG, et al, editors. Articular cartilage and osteoarthritis. New York: Raven, 1992:629–42. 8. Inerot S, Heinegård D, Olsson S-E, et al. Proteoglycan alterations during developing experimental osteoarthritis in a novel hip joint model. J Orthop Res 1991;9:658–73.
H. Yamada et al.: Chondroitin sulfate in osteoarthritis 9. Jessen TE, Faarvang KL, Ploug M. Carbohydrate as covalent crosslink in human inter-α-trypsin inhibitor: a novel plasma protein structure. FEBS Lett 1988;230:195–200. 10. Levick JR. Synovial fluid: Determinations of volume turnover and material concentration. In: Kuettner KE, Schleyerbach R, Peyron JG, et al, editors. Articular cartilage and osteoarthritis. New York: Raven, 1992:529–41. 11. Lohmander LS. Articular cartilage and osteoarthrosis. The role of molecular markers to monitor breakdown, repair and disease. J Anat 1994;184:477–92. 12. Mankin HJ, Lippiello L. The glycosaminoglycans of normal and arthritic cartilage. J Clin Invest 1971;50:1712–9. 13. Mankin HJ, Brandt KD. Biochemistry and metabolism of articular cartilage in osteoarthritis. In: Moskowitz RW, Howell DS, Goldberg VM, et al, editors. Osteoarthritis, diagnosis and medical/surgical management. Philadelphia: WB Saunders, 1984:109– 54. 14. Nagase H, Woessner JF Jr. Role of endogenous proteinases in the degradation of cartilage matrix. In: Woessner JF Jr, Howell DS, editors. Joint cartilage degradation. New York: Marcel Dekker, 1993:159–85. 15. Saari H, Santavirta S, Nordstrom D, et al. Hyaluronate in total hip replacement. J Rheumatol 1993;20:87–90. 16. Seppäla P, Lehtonen A, Karkkäinen J, et al. The presence of chondroitin sulphate in the synovial fluid in rheumatoid arthritis. Clin Chim Acta 1967;16:115–9. 17. Shima Y, Ueno R. Comparison of the therapies for osteoarthritis of the hip joint. Evaluation for the clinical result and long term result. Nippon Seikeigekagakkai Zasshi (J Jpn Orthop Assoc) 1971;45:813–33 (in Japanese). 18. Shinmei M, Miyauchi S, Machida A, et al. Quantitation of chondroitin 4-sulfate and chondroitin 6-sulfate in pathologic joint fluid. Arthritis Rheum 1992;35:1304–8. 19. Shinmei M, Inamori Y, Yoshihara Y, et al. The potential of cartilage markers in joint fluid for drug evaluation. In: Kuettner KE, Schleyerbach R, Peyron JG, et al, editors. Articular cartilage and osteoarthritis. New York: Raven, 1992:597–609. 20. Thonar EJ-MA, Shinmei M, Lohmander LS. Body fluid markers of cartilage changes in osteoarthritis. Rheum Dis Clin North Am 1993;19:635–57. 21. Toyoda H, Shinomiya K, Yamanashi S, et al. Microdetermination of unsaturated disaccharides produced from chondroitin sulfates in rabbit plasma by high performance liquid chromatography with fluorometric detection. Anal Sci 1988;4:381–4.
H. Yamada et al.: Chondroitin sulfate in osteoarthritis
Levels of chondroitin sulfate isomers in synovial fluid of patients with hip osteoarthritis Harumoto Yamada1, Satoshi Miyauchi2, Hiraku Hotta1, Mitsuhiro Morita1, Yasuo Yoshihara1, Toshiyuki Kikuchi1, and Kyosuke Fujikawa1 1 2
Department of Orthopaedic Surgery, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama 359, Japan Tokyo Research Institute, Seikagaku Corporation, 3-1253 Tateno, Higashi-Yamato, Tokyo, Japan
Abstract: We determined the levels of chondroitin sulfate (CS) isomers in the synovial fluid of patients with hip osteoarthritis (OA) to investigate their significance as markers reflecting extracellular matrix metabolism in joint tissues. A cross-sectional study of synovial fluid aspirated from 50 hip joints of 50 patients with OA was performed. Concentrations of chondroitin-4-sulfate (C4S) and chondroitin-6-sulfate (C6S) were determined by high-performance liquid chromatography. Levels of each marker molecule were investigated in relation to age and radiological stage of the disease. In all disease stages, the dominant CS isomer in synovial fluid was C6S. There was a significant negative correlation between levels of C6S and age. A significant inverse correlation was also observed between the ratio of C6S to C4S and age. Results of analysis of covariance in which age was covariate showed that the ratio of C6S to C4S in advanced and terminal stage OA was lower than that in early stage OA. The present results indicate that the ratio of CS isomers in synovial fluid in hip OA varies with the severity of disease. These molecules in synovial fluid may serve as a useful marker reflecting extracellular matrix metabolism in OA. Key words: chondroitin sulfate, synovial fluid, osteoarthritis of the hip joint, joint marker, cartilage
Introduction Osteoarthritis (OA) is primarily a disorder of articular cartilage, caused by mechanical stress, combined with
Offprint requests to: H. Yamada, Department of Orthopaedic Surgery, Fujita Health University, Second Hospital, 3-6-10 Otohbashi, Nakagawa-ku, Nagoya 4548509, Japan Received for publication on May 13, 1998; accepted on Dec. 17, 1998
biochemical changes in the cartilage matrix and inadequate cartilage and bone repair processes.13 Secondary synovitis resulting from the breakdown products of cartilage and bone matrix is likely to modify the symptoms of OA. In spite of the rapid progress in imaging technologies for diagnosis of arthritic diseases, it is still extremely difficult to recognize subtle changes in joint components, including articular cartilage.7 This is especially true for the hip joint, where even the resolution of magnetic resonance imaging (MRI) may not be sufficient to detect wear and focal defects of the articular cartilage. Assessment of joint pathology is essential for the correct diagnosis and subsequent treatment of OA. Recently, several joint markers reflecting the metabolism of different components of joint tissue have been identified.11,20 As OA is not a single disease entity but has diverse pathogenesis, markers which reflect the different aspects of OA have been investigated. Fragments of cartilage extracellular matrix components are important markers which reflect both normal turnover and catabolism of cartilage tissue. Since the major glycosaminoglycans (GAGs) in adult human articular cartilage are chondroitin-6-sulfate (C6S) and keratan sulfate, synovial fluid concentrations of these GAGs in pathological conditions have been investigated as catabolic markers during cartilage degradation.2,3 Regenerated fibrocartilage seen in OA joints and synovium have been shown to synthesize chondroitin-4-sulfate (C4S).6,12 The detection of chondroitin sulfate (CS) in synovial fluid has been mainly performed with cellulose acetate membrane electrophoresis or ion-exchange choromatography.2,16 The precise analysis of synovial fluid CS has been difficult using these conventional methods. High-performance liquid chromatography (HPLC), a recently developed analytical technique, provides a more sensitive method to measure CS levels in synovial fluid.18 The significance of GAGs as joint markers has
H. Yamada et al.: Chondroitin sulfate in osteoarthritis
been investigated using knee synovial fluid from patients with various arthritic diseases.18,19 However, there is at present very little data on GAG levels in the synovial fluid in other joints, including the hip joint.15 Since the volume of synovial fluid in the joint cavity and amount of articular cartilage and synovium differs significantly in each joint, the production and clearance of joint marker molecules may also be different. We report the results of a cross-sectional study of synovial fluid levels of C6S and C4S in patients with hip OA, and we examine the significance of these molecules as markers reflecting extracellular matrix metabolism in OA.
Patients and methods Patients Needle puncture through the joint capsule was performed at 117 hip surgeries for 117 OA patients (77 total hip arthroplasties and 40 pelvic osteotomies). Fifty samples of hip synovial fluids could be aspirated from 50 patients; 67 patients were excluded, since no significant synovial fluid could be aspirated from the joint cavity. Diagnosis of hip OA was made according to the criteria recommended by the American College of Rheumatology.1 Forty-two patients (84%) were diagnosed with secondary hip OA after developmental acetabular dysplasia. OA patients were further subdivided into four groups (pre-, early, advanced, and terminal stage disease) based on the radiological staging system provided by the Japanese Orthopaedic Association.17 General characteristics of the patients are shown in Table 1. Patients with advanced and terminal stage OA were significantly older than those with early stage OA (P , 0.05). Thirty-nine patients had previously been treated with various nonsteroidal antiinflammatory drugs (NSAIDs) for the relief of hip pain. No patients had received prior treatment of intraarticular injection of steroids or high-molecular weight hyaluronic acid. Table 1. General characteristics of the patients in the present study Stagea PreEarly Advanced Terminal Total
Number of patients 0 17 6 27 50
Age (years)b 36.9 52.2 62.4 54.7
— 6 13.2 6 12.0* 6 11.2* 6 11.9
Female/male — 17/0 5/1 25/2 47/3
* P , 0.05 vs early stage a Stage of hip deformity was assessed by the radiological findings according to the criteria of the Japanese Orthopaedic Association17 b Values are means 6 SD
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Preparation of samples Synovial fluid was collected into sterile tubes without additives and centrifuged at 10 000 rpm for 20 min at 4°C to remove cells and tissue debris. The supernatants were stored in aliquots at 280°C for later assay. Measurement of CS isomers Levels of C4S and C6S were determined by HPLC as previously described.18 Briefly, synovial fluid samples were diluted tenfold with distilled water and digested with chondroitinase ABC (Seikagaku, Tokyo, Japan) and chondroitinase AC-II (Seikagaku) before analysis to reduce the GAGs to disaccharides, which were then analyzed by HPLC, using a 7.6 3 150 mm column packed with propylamine-bound silica gel (YMC gel PA-120; YMC, Kyoto, Japan), combined with fluorometry according to the method of Toyoda et al.21 The effluent was monitored with a spectrofluorometer (Model 920-FP, Japan Spectroscopic, Tokyo, Japan) set at an excitation wavelength of 331 nm and emission wavelength of 383 nm. Statistical analysis Correlations were sought using the Spearman’s rank correlation coefficient. Analysis of covariance (ANCOVA), in which age was the covariate, was performed to determine the relation between marker levels and disease stage. Differences between other groups were analyzed by Mann-Whitney U-test for unpaired variables.
Results Synovial fluid levels of GAGs in patients with hip OA The mean values (6SD) of the concentrations of C6S and C4S in the synovial fluids are shown in Table 2. When the amounts of the two CS isomers in each dis-
Table 2. Concentrations of chondroitin 6 sulfate (C6S) and chondroitin 4 sulfate (C4S) in synovial fluid of patients in each disease stage Stage Early Advanced Terminal
C6S
C4S
114.3 6 59.4 84.6 6 30.0 88.5 6 41.7
32.6 6 11.7 35.5 6 11.3 45.6 6 19.4
Values are means 6 SD nmoles/ml for unsaturated disaccharides derived from C6S and C4S No significant differences were observed between glycosaminoglycan (GAG) concentrations in each disease stage, using analysis of covariance (ANCOVA)
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Fig. 1. Correlation between level of chondroitin 6 sulfate (C6S) in hip synovial fluid and age. Diluted synovial fluid samples were digested with chondroitinase ABC and chondroitinase AC-II before analysis to reduce the glycosaminoglycans to disaccharides. Unsaturated dissaccharides derived from chondroitin sulfates in the synovial fluid were then analyzed by high performance liquid chromatography (HPLC) combined with fluorometry, as described in Materials and Methods. Levels of C6S were expressed as concentrations (nmoles/ml) of unsaturated disaccharides derived from C6S. A significant negative correlation was observed (Spearman’s rank correlation coefficient, 20.334; P , 0.05)
H. Yamada et al.: Chondroitin sulfate in osteoarthritis
Fig. 2. Correlation between ratio of C6S to C4S in hip synovial fluid and age. Unsaturated disaccharides derived from chondroitin sulfates in the synovial fluid by sequential digestion with chondroitinase ABC and chondroitinase AC-II were analyzed using HPLC, as described in Materials and Methods. The ratio of C6S to C4S was expressed as the molar ratio of unsaturated disaccharides derived from C6S and C4S. A significant negative correlation was observed (Spearman’s rank correlation coefficient, 20.657; P , 0.0001)
ease stage were compared, C6S was predominant in each group. Results of ANCOVA in which age was the covariate showed that there were no significant differences in the levels of C6S and C4S at each disease stage. Levels of C4S showed no significant correlation with age (data not shown). A weak but statistically significant negative correlation was observed between the level of C6S and age (r 5 20.334; P , 0.05) (Fig. 1). No significant differences in the levels of the two CS isomers were observed between primary and secondary hip OA. Treatment with NSAIDs had no significant effect on the levels of the two CS isomers (data not shown). Relationship between ratio of C6S to C4S and age/disease stage The molar ratio of the two CS isomers was investigated relative to age and disease stage. A significant inverse correlation was observed between the ratio of C6S to C4S and age (r 5 20.657; P , 0.0001) (Fig. 2). The ratios of C6S to C4S in patients with each disease stage are shown in Fig. 3. The mean value of the ratio of C6S to C4S at each stage was 3.38 6 0.82 (early), 2.37 6 0.17 (advanced), and 1.86 6 0.41 (terminal). When the ratio of C6S to C4S was compared with the radiological as-
Fig. 3. Relationship of the ratio of C6S to C4S in hip synovial fluid and the radiological disease stage. The ratio of C6S to C4S is shown as the molar ratio of unsaturated disaccharides derived from C6S and C4S. The ratio of C6S to C4S in advanced and terminal stage osteoarthritis (OA) was significantly lower than that in early stage OA (analyzed by ANCOVA). Values are presented as means and SD. * P , 0.05; ** P , 0.01
H. Yamada et al.: Chondroitin sulfate in osteoarthritis
sessment of hip OA using ANCOVA in which age was the covariate, the ratio in advanced and terminal stage OA was significantly lower than that in early stage OA (Fig. 3) (P , 0.05 for advanced stage OA, P , 0.01 for terminal stage OA). No significant differences in the ratio of the two CS isomers were observed between primary and secondary hip OA. Treatment with NSAIDs had no significant effect on the ratio of the two CS isomers (data not shown).
Discussion In the present study, levels of the unsaturated disaccharide isomers of C6S and C4S in hip synovial fluid were measured using HPLC combined with fluorometry. This assay method has previously been shown to be extremely sensitive and useful for the detection of these CS isomers in the knee synovial fluid of patients with various arthritic diseases, including OA.18 The present experimental results showed that the ratio of these two CS isomers varied significantly with the disease severity of hip OA. Remarkable progress has been made recently in research of the mechanism of cartilage destruction in arthritic diseases. The degradation of aggrecan and release of its fragments from cartilage in OA is believed to be mainly mediated by proteolytic enzymes.14 Aggrecan molecules are synthesized by chondrocytes and the cleaved fragments are liberated into the synovial fluid after enzymatic degradation. Thus, the levels of aggrecan-related fragments in synovial fluid may reflect both the synthesis and degradation of aggrecan in cartilage. Various methods have been developed to analyze the levels of aggrecan fragments in synovial fluid. The concentration of sulfated GAGs and proteoglycan fragments in knee synovial fluid was measured spectrophotometrically, using dimethylmethylene blue and a radioimmunoassay, respectively.4 These studies demonstrated an increase of aggrecanrelated fragments in arthritic diseases, including OA. Dahlberg et al.5 measured the concentration of aggrecan fragments in the synovial fluid of patients with knee OA, using an enzyme-linked immunosorbent assay (EIA), and demonstrated that the levels of aggrecan fragments decreased with advancing disease severity. Campion et al.3 have shown an inverse correlation between radiological cartilage loss and levels of antigenic keratan sulfate in osteoarthritic synovial fluid measured by EIA. In the present study, HPLC was used to measure the level of unsaturated disaccharide isomers of C6S in the synovial fluid of patients with hip OA. This measurement reflects the levels of aggrecan-related fragments present in the synovial fluid. The present assay method cannot provide information about the mole-
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cular weight of the CS isomers. However, this assay method is extremely sensitive for the detection of total aggrecan fragments. Multiple etiologic factors have been shown to be associated with the development of OA. Eighty-four percent of the patients who entered the present study were diagnosed with secondary hip OA after congenital acetabular dysplasia. Congenital acetabular dysplasia in Japanese occurs predominantly in females, and 94% of the subjects in this study were women. These characteristics may suggest that the patients with hip OA in the present study have a relatively uniform background as regards the etiopathogenesis of this disease. No significant correlation was observed between levels of C6S and the severity of hip OA assessed by radiological findings. These data were different from those compiled from previous reports on knee OA. This discrepancy between results for hip and knee OA may be explained, in part, by differences in the assay of aggrecan fragments and assessments of radiological findings, and, mainly, by differences in the anatomical structure of the joints. For example, clearance and volume flux may differ markedly in the hip and knee joints.10 Furthermore, it has been shown that aggrecan fragments are easily retained in hip cartilage, while being lost quickly from knee cartilage into the synovial fluid.8 Although normal human articular cartilage contains only minor amounts of C4S, it is well known that regenerated osteoarthritic cartilage and chondro-osseous spurs may produce singnificant amounts of C4S.12 C4S is produced by synovial tissue and may also be derived from plasma by filtration through the synovium.9 C4S is thus considered as a marker that reflects both changes in cartilage metabolism and synovial inflammation. Shinmei et al.19 have indicated that the ratio of C6S to C4S is significantly higher in primary knee OA and traumatic arthritis than in rheumatoid arthritis, and this ratio is a useful diagnostic marker reflecting the matrix metabolism in joint tissues. In the present study, the ratio of these two CS isomers in the synovial fluid of patients with hip OA was shown to be significantly dependent on the disease severity of OA. In the present cross-sectional study, patients with each disease stage varied significantly in age. Since the disease severity and age were closely related, ANCOVA was used to control for possible confounding between these two variables. The effect of disease stage on the ratio of the two CS isomers remained significant on ANCOVA in which age was the covariate. We propose that this ratio is more useful as a marker reflecting the metabolism of cartilage extracellular matrix than the concentration of C6S alone, since the ratio of these two CS isomers correlates significantly with the disease severity of hip OA. It cannot be concluded from the present experiment alone whether these CS isomers are
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useful for determining the prognosis of hip OA. Further studies will also be needed to more fully explore the clinical significance of these molecules for the differential diagnosis of OA from other arthritic disease. Acknowledgments. This study was partly supported by a grant from the Hip Joint Foundation of Japan. The authors are deeply grateful to the late Professor Masayuki Shinmei, of the National Defense Medical College, for his encouragement in the initiation of this study.
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