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Significance of chondroitin sulfate isomers in the synovial fluid of osteoarthritis patients
J Orthop Sci (2002) 7:232–237
Significance of chondroitin sulfate isomers in the synovial fluid of osteoarthritis patients Shinji Uesaka, Yoshihito Nakayama, Kiyoshi Yoshihara, and Hiromoto Ito Department of Orthopaedic Surgery, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8602, Japan
Abstract To test whether chondroitin sulfate (CS) isomers in synovial fluid are related to the radiographic severity of osteoarthritis (OA) and the age and sex, we investigated the concentrations of chondroitin 6-sulfate (C6S) and chondroitin 4-sulfate (C4S) in patients with OA of the knee joint and agematched healthy controls. Synovial fluid was obtained from 133 patients with OA of the knee and 27 volunteers (controls). Twenty-seven patients were radiographically classified as grade 1, 59 as grade 2, 34 as grade 3, and 13 as grade 4 according to a modified version of the Kellgren and Lawrence scale. Compared with findings in the controls, values for the concentrations of C6S and C4S, and the C6 : C4S ratio, were significantly higher in the grade 1 patients, whereas in grade 3 and 4 patients, the C6S concentration and the C6 : C4S ratio were lower. There was an obvious sex difference, and all values for the women were significantly lower than those for the men in both the control and the patient groups. In the control group, multiple regression analysis showed a moderate inverse correlation between age and all three of the above biochemical variables, and sex was moderately associated with the C6S concentration and the C6S : C4S ratio. In the patient group, age and sex showed a poor correlation with any of the three variables. However, the radiographic severity of OA showed a relatively strong inverse correlation with the C6S concentration and the C6S : C4S ratio, and a weak inverse correlation with the C4S concentration. Key words Chondroitin sulfate · Synovial fluid · Biochemical analysis · Normal value
Introduction As well as playing an important role in the lubrication and nutrition of the joint,8,10 chondroitin sulfate (CS) is the major sulfated glycosaminoglycan (GAG) in the matrix of joint tissues. In both normal and disease conditions,14 it is continuously released into the synovial fluid, where its concentration and sulfation pattern reflect the joint tissue metabolism.5–7,9,20 Most chondroitin 6-sulfate (C6S) in synovial fluid has been thought to be derived from articular cartilage. Chondroitin 4-sulfate (C4S) is distributed widely, not only in articular cartilage but also in the synovium, ligaments, and menisci.15,16,23 Measurements of GAG levels have been performed in patients with various joint diseases.4,13 Recently, much attention has been focused on the role of CS isomers in synovial fluid as a marker of joint disease.2,23 Changes in C6S and C4S concentrations and in the C6S : C4S ratio have been considered to reflect the processes of osteoarthritis (OA) and rheumatoid arthritis (RA).21–24,26 However, because these joint diseases occur commonly in the older population, especially in older women, it is important that the changes in CS isomers associated with joint pathology are identified and distinguished from normal, agerelated events. To clarify whether CS isomers in synovial fluid are related to the radiographic severity of OA, and the age and sex, we investigated the concentrations of C6S and C4S in OA patients and in healthy controls.
Patients and methods Offprint requests to: S. Uesaka, Department of Orthopaedic Surgery, Nippon Medical School, Second Hospital, 1-396 Kosugi-cho, Nakahara-ku, Kawasaki, Kanagawa 211-8533, Japan Received: June 11, 2001 / Accepted: October 22, 2001
Synovial fluid was obtained from the knee joints of 133 patients with OA of the knee (37 men and 96 women; mean age, 66.5 years; range, 41 to 90 years). The mean time interval from the beginning of osteoarthritic
S. Uesaka et al.: CS isomers of synovial fluid in OA
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Table 1. Data on osteoarthritis (OA) patients and control subjects Number of subjects
Control OA patients Grade 1 Grade 2 Grade 3 Grade 4
Total
Men
Women
27 133 27 59 34 13
13 37 8 19 7 3
14 96 19 40 27 10
Age (years) 66.7 66.5 53.3 64.8 76.2 76.5
⫾ ⫾ ⫾ ⫾ ⫾ ⫾
12.3 12.0 7.5 9.5 7.4 9.7
Table 2. Modified Kellgren and Lawrence scale11
Grade Grade Grade Grade Grade
0 1 2 3 4
Osteophytes
Joint space
⫺ ⫹ ⫹ ⫹ ⫹
6 mm or more 6 mm or more 3 to less than 6 mm Less than 3 mm Loss of joint space
symptoms to the examination in this study was 35.5 months (range, 1 to 324 months). Most of the patients had shown a pattern of deterioration and remission for many years. Patients who were suspected of having RA or who had a history of steroid therapy were excluded from this study. Data on the subjects studied are shown in Table 1. Posteroanterior radiographs were made with the subjects in the standing position with the knee at 20° flexion. The axial dimension of the clear space in the center of both the medial and lateral components was measured to evaluate narrowing of the joint space quantitatively. The radiographic severity of OA was classified into five grades according to a modified version of the Kellgren and Lawrence scale (knee version)11: grade 0, normal; grade 1, osteophytes and joint space of 6 mm or more; grade 2, osteophytes and joint space of 3 to less than 6 mm; grade 3, osteophytes and joint space of less than 3 mm; grade 4, osteophytes and loss of joint space (Table 2). Twenty-seven patients were evaluated as grade 1, 59 as grade 2, 34 as grade 3, and 13 as grade 4. As a control study, we examined synovial fluid from healthy elderly volunteers who had no history of knee symptoms or injuries and who were radiologically evaluated as normal. We obtained the approval of the Research Ethics Committee of Nippon Medical School for the aspiration of synovial fluid from the knee joint. Sufficient volumes of synovial fluid (0.2 ml) for assay were obtained from 27 volunteers. The mean age of these 27 volunteers (13 men, 14 women) was 66.7 years (range, 43 to 86 years). There was no significant difference in age between the OA patients and these
Disease duration (months) 35.5 5.1 24.2 54.4 100.4
⫺ ⫾ 54.4 ⫾ 8.2 ⫾ 47.0 ⫾ 52.3 ⫾ 76.8
controls. All subjects, patients and volunteers, gave their informed consent for all procedures. Synovial fluid samples were collected in sterile plastic tubes and centrifuged at 10 000 g for 15 min at 4°C to remove cells and joint debris. The supernatants were stored at ⫺80°C until biochemical assay was performed. The unsaturated disaccharides derived from CS and hyaluronic acid (HA) were measured by high-performance liquid chromatography (HPLC).23,25 Chondroitinase ABC, 50 µl (5 units/ml), 10 mM sodium acetate buffer, 80 µl (pH 8.0), and distilled water, 70 µl, were added to a tenfold-diluted synovial fluid aliquot of 200 µl. The mixture was incubated at 37°C for 2 h, and was then ultrafiltered, with the Ultrafree C3GC system (molecular size cutoff 10 000; Japan Millipore, Tokyo, Japan). To completely reduce HA to the disaccharide, 30 µl of HAase (0.5 units/ml) and 30 µl of 100 mM sodium acetate buffer (pH 6.0) were added to 300 µl of the ultrafiltrate, and the mixture was incubated at 37°C for 2 h. After ultrafiltration, the mixture was analyzed by HPLC. The unsaturated disaccharides in each sample were eluted with a gradient of 0–100 mM sodium sulfate for 60 min, at a flow rate of 0.5 ml/min. The eluant from the column was mixed with 100 mM sodium tetraborate buffer (pH 9.0) containing 10 mg/ml of 2cyanoacetamide, at a flow rate of 0.5 ml/min, and the mixture was passed through a polyetheretherketone reaction coil (0.8 mm inner dimension ⫻10 m) set in a dry reaction bath at a temperature of 137°C. The effluent was monitored with a spectrofluorometer set at an excitation wavelength of 331 nm and an emission wavelength of 383 nm. We determined the reproducibility of the biochemical estimations. The coefficients of variance of the assay were 4.4% for C6S and 7.1% for C4S. Biochemical evaluations included determinations of the concentrations of C6S and C4S, and calculation of the C6S : C4S ratio. Statistical analysis was performed by analysis of variance, the χ2 test, and multiple regression analysis, and P⬍0.05 was accepted as the minimum value indicating significance.
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S. Uesaka et al.: CS isomers of synovial fluid in OA
Table 3. Synovial fluid concentrations of the unsaturated disaccharides derived from chondroitin sulfate and hyaluronic acid in osteoarthritis patients and control subjects Grade 1 (n ⫽ 27) Volume (ml) C6S (nmol/ml)a C4S (nmol/ml)b C6S : C4Sc HA (mg/ml)d
9.9 ⫾ 119.1 ⫾ 22.8 ⫾ 5.1 ⫾ 1.8 ⫾
6.3 66.1 8.3 1.2 0.6
Grade 2 (n ⫽ 59)
Grade 3 (n ⫽ 34)
Grade 4 (n ⫽ 13)
OA total (n ⫽ 133)
Control (n ⫽ 27)
11.1 ⫾ 9.1 72.8 ⫾ 42.4 17.2 ⫾ 6.7 4.1 ⫾ 1.0 1.9 ⫾ 0.5
9.2 ⫾ 9.9 56.2 ⫾ 22.2 16.8 ⫾ 5.5 3.3 ⫾ 0.8 1.8 ⫾ 0.7
8.7 ⫾ 9.5 44.9 ⫾ 13.3 17.0 ⫾ 3.9 2.6 ⫾ 0.4 2.1 ⫾ 0.5
10.1 ⫾ 8.8 75.2 ⫾ 48.7 18.2 ⫾ 6.9 4.0 ⫾ 1.2 1.9 ⫾ 0.6
0.6 ⫾ 0.5 76.9 ⫾ 37.4 16.6 ⫾ 4.6 4.5 ⫾ 1.1 2.8 ⫾ 0.6
Values are means ⫾ SD a P ⬍ 0.001 grade 1 vs control; P ⬍ 0.05 grade 3 and 4 vs control b P ⬍ 0.001 grade 1 vs control c P ⬍ 0.05 grade 1 vs control; P ⬍ 0.0001 grade 3 and 4 vs control d P ⬍ 0.0001 grade 1, 2, 3, and 4 vs control
Fig. 1. Chondroitin sulfate (CS) concentrations and chondroitin 6-sulfate (C6S) : chondroitin 4-sulfate (C4S) ratio in synovial fluid. The mean C6S concentration in the patients with grade 1 osteoarthritis was significantly higher than that in the controls (**P ⬍ 0.001), whereas that in the grade 3 and 4 patients was lower (*P ⬍ 0.05). The mean C4S concentration in the grade 1 patients was also significantly higher than that in
the controls (**P ⬍ 0.001), but no difference was observed between the patients with grade 2 or above and the controls. The mean C6S : C4S ratio in the grade 1 patients was significantly higher (*P ⬍ 0.05) than that in controls, but, in contrast, that in the grade 3 and 4 patients was lower (***P ⬍ 0.0001). Horizontal lines indicate means. See text and Table 2 for definitions of grade of osteoarthritis
Results
(P ⬍ 0.05) (Fig. 1). The mean C4S concentration in the grade 1 patients was also significantly higher than that in the controls (P ⬍ 0.001), but no difference was observed between the patients with grade 2 or above and the controls. The mean C6S : C4S ratio in the grade 1 patients was significantly higher than that in the controls (P ⬍ 0.05), but, in contrast, that in the grade 3 and 4 patients was lower (P ⬍ 0.0001). The mean HA concentration in all OA grades was significantly lower than that in the controls (P ⬍ 0.0001), but no differences were observed between any OA grades. There was an
The biochemical results are summarized in Table 3. There was little difference between the OA patients overall and the controls with respect to any of the values for the concentrations of C6S and C4S and the C6 : C4S ratio studied. However, when the variables in each OA grade were compared with those for the controls, the mean C6S concentration in the grade 1 patients was significantly higher than that in the controls (P ⬍ 0.001), whereas that in the grade 3 and 4 patients was lower
S. Uesaka et al.: CS isomers of synovial fluid in OA
235
Table 4. Sex differences in synovial fluid concentrations of the unsaturated chondroitin sulfate disaccharides
OA Men (n ⫽ 37) Women (n ⫽ 96) P value Control Men (n ⫽ 13) Women (n ⫽ 14) P value
C6S (nmol/ml)
C4S (nmol/ml)
C6S : C4S
90.0 ⫾ 56.6 69.5 ⫾ 44.3 0.03
20.1 ⫾ 7.7 17.4 ⫾ 6.5 0.04
4.4 ⫾ 1.3 3.8 ⫾ 1.1 0.02
97.4 ⫾ 42.7 57.9 ⫾ 17.5 0.004
18.3 ⫾ 5.7 15.0 ⫾ 2.5 0.05
5.2 ⫾ 1.1 3.8 ⫾ 0.8 0.0006
Table 5. Relationship between synovial fluid concentrations of the unsaturated chondroitin sulfate disaccharides and age, sex, and radiographic severity of OA on multiple regression analysis Partial correlation
OA (n ⫽ 133) Age Sex Severity of OA Control (n ⫽ 27) Age Sex
C6S
C4S
C6S : C4S
0.019 0.154 ⫺0.458
0.076 0.154 ⫺0.291
⫺0.079 0.159 ⫺0.547
⫺0.490 0.339
⫺0.445 0.192
⫺0.423 0.446
obvious sex difference, and all of the variables for the women were significantly lower than those for the men, in both the control group and the patient group (Table 4). As for the correlation of the three biochemical variables (i.e., C6S and C4S concentrations and the C6 : C4 ratio) with age, sex, and severity of OA on multiple regression analysis, in the controls, age showed a moderate inverse correlation with all three of the variables (r ⫽ ⫺0.490 for C6S concentration, r ⫽ ⫺0.445 for C4S concentration, and r ⫽ ⫺0.423 for the C6S : C4S ratio), and sex was moderately associated with the C6S concentration and the C6S : C4S ratio (r ⫽ 0.339 and r ⫽ 0.446, respectively). On the other hand, in the patients, age and sex showed a poor correlation with any of the variables. The severity of OA showed a relatively strong inverse correlation with the C6S concentration and the C6S : C4S ratio, and a weak inverse correlation with the C4S concentration (r ⫽ ⫺0.458 for C6S concentration, r ⫽ ⫺0.291 for C4S concentration, and r ⫽ ⫺0.547 for C6S : C4S ratio) (Table 5). These results suggests that the changes in CS concentrations and sulfation pattern in OA patients are influenced more by the severity of OA than by age
and sex. The measurement of CS isomers in synovial fluid may provide information that will be useful in the early detection of OA changes and in the assessment of the progression of OA.
Discussion In recent studies of OA using HPLC, C6S and C4S concentrations and the C6S : C4S ratio have been reported to range from 52.3 to 92.5 nmol/ml, 15.0 to 28.2 nmol/ml, and 2.81 to 3.81, respectively.21,23,24,26 The results described here are similar to these reported results. Comparison of these three variables between the OA patients overall and the controls showed little difference in any of the variables. However, previous studies, using capillary zone electrophoresis, have yielded conflicting results. Lewis et al.12 reported that the C6S concentrations and C6S : C4S ratios in 45 OA patients (mean age, almost 70 years) were significantly lower than those in 13 control subjects (mean age, 34 years). In marked contrast, there was no difference in C4S concentrations between the two groups. Sharif et al.22 reported that 28 OA patients (mean age, 68.7 years) showed higher C4S concentrations and lower C6S : C4S ratios than 24 controls (mean age, 39.9 years). Our review of these two studies revealed some problems: no grade 1 patients were included in either study, and, in both studies, the control subjects were younger than the patients. The reasons for the discrepancy between our results and these previously reported results are unclear, but they may arise from differences in assay methods, OA grades, and the ages of the control subjects. In studies of synovial fluid markers, most investigators have treated OA patients as a homogeneous group. Little information has been provided about the changes in CS isomers in the different stages of OA. The present study revealed that the changes in CS concentrations and sulfation patterns were closely related to the radiographic severity of OA. Early OA is characterized by an increase in all three variables, whereas advanced OA is characterized by a decrease in the C6S concentration and the C6S : C4S ratio. As for the mechanism of changes in CS concentrations and sulfation pattern in OA, it has previously been postulated that the changes may result from differences in joint tissue turnover or the cartilage mass remaining in the joint. Recently, Bayliss et al.3 reported some interesting findings concerning the zonal distribution of CS isomers in normal cartilage: C6S concentration increased from the midzones of the tissue toward the articular surface, and the highest concentration of C6S was found in the upper quartile of the tissue. These findings suggest that articular cartilage wear in the early
236
stage of OA may be reflected as an increase in the C6S concentration. When the articular surface wears to some degree with advancing OA stage, the amount of C6S released into the joint declines sharply, resulting in marked decreases in the C6S concentration and the C6S : C4S ratio. With respect to cartilage turnover in early OA, Adams and Brandt1 reported that both the synthesis and the degradation of proteoglycans in dog articular cartilage were activated in early OA, and Ratcliffe et al.18 also found that the 3-B-3 epitope, as an anabolic marker of C6S, was elevated 33 to 35 folds in the synovial fluid of dogs with early OA. Clinically, Rizkalla et al.19 reported that proteoglycan synthesis was increased in the early stage of cartilage degradation. Thus, our findings that the C6S concentration and the C6S : C4S ratio in synovial fluid were increased in early OA may reflect not only an increase in cartilage degradation but also an acceleration of proteoglycan synthesis activity. With respect to demographic variables, the present study showed that, in the controls, all three of the biochemical variables showed a decreasing tendency with increasing age. In an analysis of human articular cartilage, Bayliss et al.3 found that the C6S content in the full depth of cartilage of the femoral condyle increased with age up to 20 years, whereas the C4S content decreased immediately after birth, and continued to decrease until age 20 years. During subsequent aging (20–85 years), there was little change in the overall content of C6S and C4S. Platt et al.17 demonstrated that the C6S : C4S ratio in equine articular cartilage in the metacarpophalangeal joints decreased with advancing age. The reasons why age-related changes in synovial fluid CS do or do not parallel those in articular cartilage may be explained, in part, by topographical and zonal variations of sulfation in cartilage, and different CS composition in joint tissues as the source of synovial fluid. Interestingly, in the present study, in the patient group, age showed a poor correlation with any of the biochemical variables, whereas the severity of OA showed an inverse correlation with all three variables, with the correlation being relatively strong for the C6S concentration and the C6S : C4S ratio. Thus, the changes in CS concentrations and sulfation pattern in OA patients were not influenced by age as much as those in controls, and these changes may be, in part, disease-related. As for sex differences, Sharif et al.22 reported that women showed higher C4S concentrations and lower C6S : C4S ratios than men. In our results, all three variables studied were lower in women than in men in both the control and OA groups, indicating that degenerative changes of articular cartilage may proceed more rapidly in women than in men during the normal
S. Uesaka et al.: CS isomers of synovial fluid in OA
aging process. In fact, the susceptibility of women to cartilage damage is reflected in the difference in the ratio of women to men between the control and OA groups. The ratio was high in the OA group (2.6 : 1), compared with that in the control group (1.1 : 1). Moreover, the incidence of severe OA (grade 3 or 4) was higher in the female patients (38.5%) than in the male patients (27.0%). In summary, CS concentrations and sulfation pattern in synovial fluid were shown to be closely related to the radiographic severity of OA. Compared with values in controls, the values for the C6S and C4S concentrations and the C6S : C4S ratio were increased in the early stage of OA, and the C6S concentration and the C6S : C4S ratio decreased in the advanced stage. Age and sex showed moderate correlations with all three of the biochemical variables in the control group, but showed a poor correlation in the patient group.
References 1. Adams ME, Brandt KD. Hypertrophic repair of canine articular cartilage in osteoarthritis after anterior cruciate ligament transection. J Rheumatol 1991;18:428–35. 2. Asari A, Miyauchi S, Sekiguchi T, et al. Hyaluronan, cartilage destruction and hydrarthrosis in traumatic arthritis. Osteoarthritis Cartilage 1994;2:79–89. 3. Bayliss MT, Osborne D, Woodhouse S, et al. Sulfation of chondroitin sulfate in human articular cartilage. J Biol Chem 1999;274:15892–900. 4. Belcher C, Yaqub R, Fawthrop F, et al. Synovial fluid chondroitin and keratan sulphate epitopes, glycosaminoglycans, and hyaluronan in arthritic and normal knees. Ann Rheum Dis 1997;56:299– 307. 5. Brown MP, West LA, Merritt KA, et al. Changes in sulfation patterns of chondroitin sulfate in equine articular cartilage and synovial fluid in response to aging and osteoarthritis. Am J Vet Res 1998;59:786–91. 6. Caterson B, Mahmoodian F, Sorrell JM, et al. Modulation of native chondroitin sulphate structure in tissue development and in disease. Cell Sci 1990;97:411–7. 7. Caterson B, Hughes CE, Roughley P, et al. Anabolic and catabolic markers of proteoglycan metabolism in osteoarthritis. Acta Orthop Scand 1995;66:121–4. 8. Hardingham TE, Fosang AJ. Proteoglycans: many forms and many functions. FASEB J 1992;6:861–70. 9. Hardingham T. Changes in chondroitin sulphate structure induced by joint disease. Acta Orthop Scand 1995;66:107–10. 10. Hascall VC, Heinegård DK, Wight TN. Proteoglycans. Metabolism and pathology. In: Hay ED, editor. Cell biology of extracellular matrix. 2nd ed. New York: Plenum; 1991. p. 149– 75. 11. Kellgren JH, Lawrence JS. Radiological assessment of osteoarthrosis. Ann Rheum Dis 1957;16:494–502. 12. Lewis S, Crossman M, Flannelly J, et al. Chondroitin sulphation patterns in synovial fluid in osteoarthritis subsets. Ann Rheum Dis 1999;58:441–5. 13. Lohmander LS, Dahlberg L, Ryd L, et al. Increased levels of proteoglycan fragments in knee joint fluid after injury. Arthritis Rheum 1989;32:1434–42. 14. Lohmander LS. Markers of cartilage metabolism in arthrosis: a review. Acta Orthop Scand 1991;62:623–32.
S. Uesaka et al.: CS isomers of synovial fluid in OA 15. Mourão PAS. Distribution of chondroitin 4-sulfate and chondroitin 6-sulfate in human articular and growth cartilage. Arthritis Rheum 1988;31:1028–33. 16. Plaas AHK, West LA, Wong-Palms S, et al. Glycosaminoglycan sulfation in human osteoarthritis. J Biol Chem 1998;273:12642–9. 17. Platt D, Bird JLE, Bayliss MT. Ageing of equine articular cartilage: structure and composition of aggrecan and decorin. Equine Vet J 1998;30:43–52. 18. Ratcliffe A, Shurety W, Caterson B. The quantitation of a native chondroitin sulfate epitope in synovial fluid lavages and articular cartilage from canine experimental osteoarthritis and disuse atrophy. Arthritis Rheum 1993;36:543–51. 19. Rizkalla G, Reiner A, Bogoch E, et al. Studies of the articular cartilage proteoglycan aggrecan in health and osteoarthritis. J Clin Invest 1992;90:2268–77. 20. Sandy JD, Flannery CR, Neame PJ, et al. The structure of aggrecan fragments in human synovial fluid. J Clin Invest 1992;89: 1512–6. 21. Sato M, Kasai C, Takemura M, et al. Analysis of synovial fluid in patients with osteoarthritis and rheumatoid arthritis.
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Chubuseisaishi (Cent Jpn J Orthop Surg Traumatol) 1995;38:119– 20 (in Japanese). Sharif M, Osborne DJ, Meadows K, et al. The relevance of chondroitin and keratan sulphate markers in normal and arthritic synovial fluid. Br J Rheumatol 1996;35:951–7. 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. Shiota E, Maekawa M, Ohtani M. Analysis of synovial fluid components of hydrarthrosis in long-term hemodialysis patients. J Orthop Sci 1999;4:171–5. 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. Ueyoshi A, Kiura Y. Quantitation of synovial fluid in rheumatoid arthritis and osteoarthritis. Analysis of chondroitin sulfate content. Kansetsunogeka (Joint Surg) 1997;24:124–8 (in Japanese).
Significance of chondroitin sulfate isomers in the synovial fluid of osteoarthritis patients Shinji Uesaka, Yoshihito Nakayama, Kiyoshi Yoshihara, and Hiromoto Ito Department of Orthopaedic Surgery, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8602, Japan
Abstract To test whether chondroitin sulfate (CS) isomers in synovial fluid are related to the radiographic severity of osteoarthritis (OA) and the age and sex, we investigated the concentrations of chondroitin 6-sulfate (C6S) and chondroitin 4-sulfate (C4S) in patients with OA of the knee joint and agematched healthy controls. Synovial fluid was obtained from 133 patients with OA of the knee and 27 volunteers (controls). Twenty-seven patients were radiographically classified as grade 1, 59 as grade 2, 34 as grade 3, and 13 as grade 4 according to a modified version of the Kellgren and Lawrence scale. Compared with findings in the controls, values for the concentrations of C6S and C4S, and the C6 : C4S ratio, were significantly higher in the grade 1 patients, whereas in grade 3 and 4 patients, the C6S concentration and the C6 : C4S ratio were lower. There was an obvious sex difference, and all values for the women were significantly lower than those for the men in both the control and the patient groups. In the control group, multiple regression analysis showed a moderate inverse correlation between age and all three of the above biochemical variables, and sex was moderately associated with the C6S concentration and the C6S : C4S ratio. In the patient group, age and sex showed a poor correlation with any of the three variables. However, the radiographic severity of OA showed a relatively strong inverse correlation with the C6S concentration and the C6S : C4S ratio, and a weak inverse correlation with the C4S concentration. Key words Chondroitin sulfate · Synovial fluid · Biochemical analysis · Normal value
Introduction As well as playing an important role in the lubrication and nutrition of the joint,8,10 chondroitin sulfate (CS) is the major sulfated glycosaminoglycan (GAG) in the matrix of joint tissues. In both normal and disease conditions,14 it is continuously released into the synovial fluid, where its concentration and sulfation pattern reflect the joint tissue metabolism.5–7,9,20 Most chondroitin 6-sulfate (C6S) in synovial fluid has been thought to be derived from articular cartilage. Chondroitin 4-sulfate (C4S) is distributed widely, not only in articular cartilage but also in the synovium, ligaments, and menisci.15,16,23 Measurements of GAG levels have been performed in patients with various joint diseases.4,13 Recently, much attention has been focused on the role of CS isomers in synovial fluid as a marker of joint disease.2,23 Changes in C6S and C4S concentrations and in the C6S : C4S ratio have been considered to reflect the processes of osteoarthritis (OA) and rheumatoid arthritis (RA).21–24,26 However, because these joint diseases occur commonly in the older population, especially in older women, it is important that the changes in CS isomers associated with joint pathology are identified and distinguished from normal, agerelated events. To clarify whether CS isomers in synovial fluid are related to the radiographic severity of OA, and the age and sex, we investigated the concentrations of C6S and C4S in OA patients and in healthy controls.
Patients and methods Offprint requests to: S. Uesaka, Department of Orthopaedic Surgery, Nippon Medical School, Second Hospital, 1-396 Kosugi-cho, Nakahara-ku, Kawasaki, Kanagawa 211-8533, Japan Received: June 11, 2001 / Accepted: October 22, 2001
Synovial fluid was obtained from the knee joints of 133 patients with OA of the knee (37 men and 96 women; mean age, 66.5 years; range, 41 to 90 years). The mean time interval from the beginning of osteoarthritic
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Table 1. Data on osteoarthritis (OA) patients and control subjects Number of subjects
Control OA patients Grade 1 Grade 2 Grade 3 Grade 4
Total
Men
Women
27 133 27 59 34 13
13 37 8 19 7 3
14 96 19 40 27 10
Age (years) 66.7 66.5 53.3 64.8 76.2 76.5
⫾ ⫾ ⫾ ⫾ ⫾ ⫾
12.3 12.0 7.5 9.5 7.4 9.7
Table 2. Modified Kellgren and Lawrence scale11
Grade Grade Grade Grade Grade
0 1 2 3 4
Osteophytes
Joint space
⫺ ⫹ ⫹ ⫹ ⫹
6 mm or more 6 mm or more 3 to less than 6 mm Less than 3 mm Loss of joint space
symptoms to the examination in this study was 35.5 months (range, 1 to 324 months). Most of the patients had shown a pattern of deterioration and remission for many years. Patients who were suspected of having RA or who had a history of steroid therapy were excluded from this study. Data on the subjects studied are shown in Table 1. Posteroanterior radiographs were made with the subjects in the standing position with the knee at 20° flexion. The axial dimension of the clear space in the center of both the medial and lateral components was measured to evaluate narrowing of the joint space quantitatively. The radiographic severity of OA was classified into five grades according to a modified version of the Kellgren and Lawrence scale (knee version)11: grade 0, normal; grade 1, osteophytes and joint space of 6 mm or more; grade 2, osteophytes and joint space of 3 to less than 6 mm; grade 3, osteophytes and joint space of less than 3 mm; grade 4, osteophytes and loss of joint space (Table 2). Twenty-seven patients were evaluated as grade 1, 59 as grade 2, 34 as grade 3, and 13 as grade 4. As a control study, we examined synovial fluid from healthy elderly volunteers who had no history of knee symptoms or injuries and who were radiologically evaluated as normal. We obtained the approval of the Research Ethics Committee of Nippon Medical School for the aspiration of synovial fluid from the knee joint. Sufficient volumes of synovial fluid (0.2 ml) for assay were obtained from 27 volunteers. The mean age of these 27 volunteers (13 men, 14 women) was 66.7 years (range, 43 to 86 years). There was no significant difference in age between the OA patients and these
Disease duration (months) 35.5 5.1 24.2 54.4 100.4
⫺ ⫾ 54.4 ⫾ 8.2 ⫾ 47.0 ⫾ 52.3 ⫾ 76.8
controls. All subjects, patients and volunteers, gave their informed consent for all procedures. Synovial fluid samples were collected in sterile plastic tubes and centrifuged at 10 000 g for 15 min at 4°C to remove cells and joint debris. The supernatants were stored at ⫺80°C until biochemical assay was performed. The unsaturated disaccharides derived from CS and hyaluronic acid (HA) were measured by high-performance liquid chromatography (HPLC).23,25 Chondroitinase ABC, 50 µl (5 units/ml), 10 mM sodium acetate buffer, 80 µl (pH 8.0), and distilled water, 70 µl, were added to a tenfold-diluted synovial fluid aliquot of 200 µl. The mixture was incubated at 37°C for 2 h, and was then ultrafiltered, with the Ultrafree C3GC system (molecular size cutoff 10 000; Japan Millipore, Tokyo, Japan). To completely reduce HA to the disaccharide, 30 µl of HAase (0.5 units/ml) and 30 µl of 100 mM sodium acetate buffer (pH 6.0) were added to 300 µl of the ultrafiltrate, and the mixture was incubated at 37°C for 2 h. After ultrafiltration, the mixture was analyzed by HPLC. The unsaturated disaccharides in each sample were eluted with a gradient of 0–100 mM sodium sulfate for 60 min, at a flow rate of 0.5 ml/min. The eluant from the column was mixed with 100 mM sodium tetraborate buffer (pH 9.0) containing 10 mg/ml of 2cyanoacetamide, at a flow rate of 0.5 ml/min, and the mixture was passed through a polyetheretherketone reaction coil (0.8 mm inner dimension ⫻10 m) set in a dry reaction bath at a temperature of 137°C. The effluent was monitored with a spectrofluorometer set at an excitation wavelength of 331 nm and an emission wavelength of 383 nm. We determined the reproducibility of the biochemical estimations. The coefficients of variance of the assay were 4.4% for C6S and 7.1% for C4S. Biochemical evaluations included determinations of the concentrations of C6S and C4S, and calculation of the C6S : C4S ratio. Statistical analysis was performed by analysis of variance, the χ2 test, and multiple regression analysis, and P⬍0.05 was accepted as the minimum value indicating significance.
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Table 3. Synovial fluid concentrations of the unsaturated disaccharides derived from chondroitin sulfate and hyaluronic acid in osteoarthritis patients and control subjects Grade 1 (n ⫽ 27) Volume (ml) C6S (nmol/ml)a C4S (nmol/ml)b C6S : C4Sc HA (mg/ml)d
9.9 ⫾ 119.1 ⫾ 22.8 ⫾ 5.1 ⫾ 1.8 ⫾
6.3 66.1 8.3 1.2 0.6
Grade 2 (n ⫽ 59)
Grade 3 (n ⫽ 34)
Grade 4 (n ⫽ 13)
OA total (n ⫽ 133)
Control (n ⫽ 27)
11.1 ⫾ 9.1 72.8 ⫾ 42.4 17.2 ⫾ 6.7 4.1 ⫾ 1.0 1.9 ⫾ 0.5
9.2 ⫾ 9.9 56.2 ⫾ 22.2 16.8 ⫾ 5.5 3.3 ⫾ 0.8 1.8 ⫾ 0.7
8.7 ⫾ 9.5 44.9 ⫾ 13.3 17.0 ⫾ 3.9 2.6 ⫾ 0.4 2.1 ⫾ 0.5
10.1 ⫾ 8.8 75.2 ⫾ 48.7 18.2 ⫾ 6.9 4.0 ⫾ 1.2 1.9 ⫾ 0.6
0.6 ⫾ 0.5 76.9 ⫾ 37.4 16.6 ⫾ 4.6 4.5 ⫾ 1.1 2.8 ⫾ 0.6
Values are means ⫾ SD a P ⬍ 0.001 grade 1 vs control; P ⬍ 0.05 grade 3 and 4 vs control b P ⬍ 0.001 grade 1 vs control c P ⬍ 0.05 grade 1 vs control; P ⬍ 0.0001 grade 3 and 4 vs control d P ⬍ 0.0001 grade 1, 2, 3, and 4 vs control
Fig. 1. Chondroitin sulfate (CS) concentrations and chondroitin 6-sulfate (C6S) : chondroitin 4-sulfate (C4S) ratio in synovial fluid. The mean C6S concentration in the patients with grade 1 osteoarthritis was significantly higher than that in the controls (**P ⬍ 0.001), whereas that in the grade 3 and 4 patients was lower (*P ⬍ 0.05). The mean C4S concentration in the grade 1 patients was also significantly higher than that in
the controls (**P ⬍ 0.001), but no difference was observed between the patients with grade 2 or above and the controls. The mean C6S : C4S ratio in the grade 1 patients was significantly higher (*P ⬍ 0.05) than that in controls, but, in contrast, that in the grade 3 and 4 patients was lower (***P ⬍ 0.0001). Horizontal lines indicate means. See text and Table 2 for definitions of grade of osteoarthritis
Results
(P ⬍ 0.05) (Fig. 1). The mean C4S concentration in the grade 1 patients was also significantly higher than that in the controls (P ⬍ 0.001), but no difference was observed between the patients with grade 2 or above and the controls. The mean C6S : C4S ratio in the grade 1 patients was significantly higher than that in the controls (P ⬍ 0.05), but, in contrast, that in the grade 3 and 4 patients was lower (P ⬍ 0.0001). The mean HA concentration in all OA grades was significantly lower than that in the controls (P ⬍ 0.0001), but no differences were observed between any OA grades. There was an
The biochemical results are summarized in Table 3. There was little difference between the OA patients overall and the controls with respect to any of the values for the concentrations of C6S and C4S and the C6 : C4S ratio studied. However, when the variables in each OA grade were compared with those for the controls, the mean C6S concentration in the grade 1 patients was significantly higher than that in the controls (P ⬍ 0.001), whereas that in the grade 3 and 4 patients was lower
S. Uesaka et al.: CS isomers of synovial fluid in OA
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Table 4. Sex differences in synovial fluid concentrations of the unsaturated chondroitin sulfate disaccharides
OA Men (n ⫽ 37) Women (n ⫽ 96) P value Control Men (n ⫽ 13) Women (n ⫽ 14) P value
C6S (nmol/ml)
C4S (nmol/ml)
C6S : C4S
90.0 ⫾ 56.6 69.5 ⫾ 44.3 0.03
20.1 ⫾ 7.7 17.4 ⫾ 6.5 0.04
4.4 ⫾ 1.3 3.8 ⫾ 1.1 0.02
97.4 ⫾ 42.7 57.9 ⫾ 17.5 0.004
18.3 ⫾ 5.7 15.0 ⫾ 2.5 0.05
5.2 ⫾ 1.1 3.8 ⫾ 0.8 0.0006
Table 5. Relationship between synovial fluid concentrations of the unsaturated chondroitin sulfate disaccharides and age, sex, and radiographic severity of OA on multiple regression analysis Partial correlation
OA (n ⫽ 133) Age Sex Severity of OA Control (n ⫽ 27) Age Sex
C6S
C4S
C6S : C4S
0.019 0.154 ⫺0.458
0.076 0.154 ⫺0.291
⫺0.079 0.159 ⫺0.547
⫺0.490 0.339
⫺0.445 0.192
⫺0.423 0.446
obvious sex difference, and all of the variables for the women were significantly lower than those for the men, in both the control group and the patient group (Table 4). As for the correlation of the three biochemical variables (i.e., C6S and C4S concentrations and the C6 : C4 ratio) with age, sex, and severity of OA on multiple regression analysis, in the controls, age showed a moderate inverse correlation with all three of the variables (r ⫽ ⫺0.490 for C6S concentration, r ⫽ ⫺0.445 for C4S concentration, and r ⫽ ⫺0.423 for the C6S : C4S ratio), and sex was moderately associated with the C6S concentration and the C6S : C4S ratio (r ⫽ 0.339 and r ⫽ 0.446, respectively). On the other hand, in the patients, age and sex showed a poor correlation with any of the variables. The severity of OA showed a relatively strong inverse correlation with the C6S concentration and the C6S : C4S ratio, and a weak inverse correlation with the C4S concentration (r ⫽ ⫺0.458 for C6S concentration, r ⫽ ⫺0.291 for C4S concentration, and r ⫽ ⫺0.547 for C6S : C4S ratio) (Table 5). These results suggests that the changes in CS concentrations and sulfation pattern in OA patients are influenced more by the severity of OA than by age
and sex. The measurement of CS isomers in synovial fluid may provide information that will be useful in the early detection of OA changes and in the assessment of the progression of OA.
Discussion In recent studies of OA using HPLC, C6S and C4S concentrations and the C6S : C4S ratio have been reported to range from 52.3 to 92.5 nmol/ml, 15.0 to 28.2 nmol/ml, and 2.81 to 3.81, respectively.21,23,24,26 The results described here are similar to these reported results. Comparison of these three variables between the OA patients overall and the controls showed little difference in any of the variables. However, previous studies, using capillary zone electrophoresis, have yielded conflicting results. Lewis et al.12 reported that the C6S concentrations and C6S : C4S ratios in 45 OA patients (mean age, almost 70 years) were significantly lower than those in 13 control subjects (mean age, 34 years). In marked contrast, there was no difference in C4S concentrations between the two groups. Sharif et al.22 reported that 28 OA patients (mean age, 68.7 years) showed higher C4S concentrations and lower C6S : C4S ratios than 24 controls (mean age, 39.9 years). Our review of these two studies revealed some problems: no grade 1 patients were included in either study, and, in both studies, the control subjects were younger than the patients. The reasons for the discrepancy between our results and these previously reported results are unclear, but they may arise from differences in assay methods, OA grades, and the ages of the control subjects. In studies of synovial fluid markers, most investigators have treated OA patients as a homogeneous group. Little information has been provided about the changes in CS isomers in the different stages of OA. The present study revealed that the changes in CS concentrations and sulfation patterns were closely related to the radiographic severity of OA. Early OA is characterized by an increase in all three variables, whereas advanced OA is characterized by a decrease in the C6S concentration and the C6S : C4S ratio. As for the mechanism of changes in CS concentrations and sulfation pattern in OA, it has previously been postulated that the changes may result from differences in joint tissue turnover or the cartilage mass remaining in the joint. Recently, Bayliss et al.3 reported some interesting findings concerning the zonal distribution of CS isomers in normal cartilage: C6S concentration increased from the midzones of the tissue toward the articular surface, and the highest concentration of C6S was found in the upper quartile of the tissue. These findings suggest that articular cartilage wear in the early
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stage of OA may be reflected as an increase in the C6S concentration. When the articular surface wears to some degree with advancing OA stage, the amount of C6S released into the joint declines sharply, resulting in marked decreases in the C6S concentration and the C6S : C4S ratio. With respect to cartilage turnover in early OA, Adams and Brandt1 reported that both the synthesis and the degradation of proteoglycans in dog articular cartilage were activated in early OA, and Ratcliffe et al.18 also found that the 3-B-3 epitope, as an anabolic marker of C6S, was elevated 33 to 35 folds in the synovial fluid of dogs with early OA. Clinically, Rizkalla et al.19 reported that proteoglycan synthesis was increased in the early stage of cartilage degradation. Thus, our findings that the C6S concentration and the C6S : C4S ratio in synovial fluid were increased in early OA may reflect not only an increase in cartilage degradation but also an acceleration of proteoglycan synthesis activity. With respect to demographic variables, the present study showed that, in the controls, all three of the biochemical variables showed a decreasing tendency with increasing age. In an analysis of human articular cartilage, Bayliss et al.3 found that the C6S content in the full depth of cartilage of the femoral condyle increased with age up to 20 years, whereas the C4S content decreased immediately after birth, and continued to decrease until age 20 years. During subsequent aging (20–85 years), there was little change in the overall content of C6S and C4S. Platt et al.17 demonstrated that the C6S : C4S ratio in equine articular cartilage in the metacarpophalangeal joints decreased with advancing age. The reasons why age-related changes in synovial fluid CS do or do not parallel those in articular cartilage may be explained, in part, by topographical and zonal variations of sulfation in cartilage, and different CS composition in joint tissues as the source of synovial fluid. Interestingly, in the present study, in the patient group, age showed a poor correlation with any of the biochemical variables, whereas the severity of OA showed an inverse correlation with all three variables, with the correlation being relatively strong for the C6S concentration and the C6S : C4S ratio. Thus, the changes in CS concentrations and sulfation pattern in OA patients were not influenced by age as much as those in controls, and these changes may be, in part, disease-related. As for sex differences, Sharif et al.22 reported that women showed higher C4S concentrations and lower C6S : C4S ratios than men. In our results, all three variables studied were lower in women than in men in both the control and OA groups, indicating that degenerative changes of articular cartilage may proceed more rapidly in women than in men during the normal
S. Uesaka et al.: CS isomers of synovial fluid in OA
aging process. In fact, the susceptibility of women to cartilage damage is reflected in the difference in the ratio of women to men between the control and OA groups. The ratio was high in the OA group (2.6 : 1), compared with that in the control group (1.1 : 1). Moreover, the incidence of severe OA (grade 3 or 4) was higher in the female patients (38.5%) than in the male patients (27.0%). In summary, CS concentrations and sulfation pattern in synovial fluid were shown to be closely related to the radiographic severity of OA. Compared with values in controls, the values for the C6S and C4S concentrations and the C6S : C4S ratio were increased in the early stage of OA, and the C6S concentration and the C6S : C4S ratio decreased in the advanced stage. Age and sex showed moderate correlations with all three of the biochemical variables in the control group, but showed a poor correlation in the patient group.
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