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Serum and Synovial Fluid Concentrations of Bovine Chondrocalcin in Dogs
The Veterinary Journal 2002, 163, 211±213 doi:10.1053/tvjl.2001.0645, available online at http://www.idealibrary.com on
Short Communication Serum and Synovial Fluid Concentrations of Bovine Chondrocalcin in Dogs Y. YAMAYA*, H. TAKEUCHIy, K. NAGAOKAy, T. KOSAKAy and S. TANAKAy *
Laboratory of Comprehensive Veterinary Clinical Studies and y Laboratory of Veterinary Surgery, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-8510, Japan
KEYWORDS: Chondrocalcin; dogs; knee; serum; synovial fluid. Bone and joint diseases in small animals are mainly diagnosed through gait analysis, palpation and radiography. However, joint and other diseases are difficult to discriminate at the early stage because joint damage may only involve articular cartilage. Many X-ray examinations may be required and the cost of diagnosis may increase if magnetic resonance imaging (MRI) or computed tomography (CT) are used. To reduce or resolve these problems in dogs, biochemical markers may be useful (Innes et al., 1999). Chondrocalcin is a carboxy-terminal propeptide of type II procollagen that is released in synovial fluid when articular cartilage is synthesized and repaired (Van der Rest et al., 1986). In addition, concentrations of chondrocalcin are high in the growth plates of bovine, rat and human joints (Kujawa et al., 1989; Poole et al., 1984a; Poole et al., 1984b). Levels of synovial fluid chondrocalcin are increased in human osteoarthritis, rheumatoid and traumatic arthritis (Kobayashi et al., 1998; Kobayashi et al., 1997; Mansson et al., 1995). Bovine chondrocalcin (B-CC) can now be quantified using a commercial enzyme immunoassay for human chondrocalcin. This assay has been used to study type II collagen synthesis in human articular cartilage (Shinmei et al., 1993). Moreover, chondrocalcin also Correspondence to: Yoshiki Yamaya, Laboratory of Comprehensive Veterinary Clinical Studies, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252±8510, Japan. Fax: 81 466 84 3900; E-mail: [email protected]
circulates in serum (Mansson et al., 1995; Nelson et al., 1998). In the present study, we examined levels of serum and synovial B-CC with aging to determine whether or not serum and synovial B-CC levels increase in dogs with a ruptured cranial cruciate ligament (RCCL). Twenty-eight clinically healthy dogs, consisting of 20 beagles, five mongrels, two Siberian huskies and one Akita (14 males and 14 females), were examined and grouped as non-lame dogs. A general physical examination determined that these animals had no lameness or pain in any joint. Lame dogs included four mongrels, one Doberman, one Labrador retriever and one Siberian husky (five males and two females). The animals were diagnosed and treated for hind limb lameness at the Animal Medical Center, Nihon University. The cause of lameness was diagnosed during surgery, and it was revealed that all dogs had unilateral RCCL. The range of ages and body weight were six months to 13 years and 7±35 kg (non-lame dogs) and three to 13 years and 4±33 kg (RCCL dogs), respectively. Blood was sampled through the intermediate cephalic vein to obtain serum from both groups of dogs. In non-lame dogs, synovial fluid was aspirated from the right or left knee joint under sedation with medetomidine hydrochloride or general anaesthesia with isoflurane. In RCCL dogs, synovial fluid was aspirated from the affected knee joint just before the start of surgery under general anaesthesia with isoflurane. Cells and tissue debris were removed from samples of blood and synovial
1090-0233/02/$ ± see front matter # 2002 Elsevier Science Ltd. All rights reserved.
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THE VETERINARY JOURNAL, 163, 2
fluid anti-coagulated with EDTA by centrifugation at 10 000 rpm for 20 min. Samples were then stored at ÿ80 C. Serum and synovial B-CC levels were measured using a chondrocalcin assay kit (Teijin). Figure 1 shows the changes in serum and synovial B-CC concentrations with aging in non-lame dogs.
Fig. 1. Changes in bovine chondrocalcin concentration against aging in non-lame dogs. Closed circles show concentrations in serum and open circles in synovial fluid.
Fig. 2. Serum (black box) and synovial fluid (white box) chondrocalcin in sound and lame dogs, over 2-year-old. Bars are SEM.
The serum B-CC concentration was 21.6 4.7 ng/dL (mean SEM) up to the age of one-year, when it decreased to below 0.7 0.1 ng/dL. The synovial B-CC concentration was 61.6 2.1 ng/dL (mean SEM) up to one-year, decreasing to 13.0 2.8 ng/dL among one- to two-year-old dogs, and to 2.2 0.2 ng/dL thereafter. Serum and synovial B-CC concentrations did not significantly differ between males and females. In addition, serum and synovial B-CC concentrations in RCCL dogs were compared with those in eleven age-matched, non-lame dogs (over two-years of age). Serum and synovial B-CC concentrations were 0.8 0.2 ng/dL and 2.2 0.2 ng/dL in the non-lame dogs and 2.1 0.7 ng/dL and 9.0 1.7 ng/dL in the RCCL dogs, respectively (means SEM; Fig. 2). In RCCL dogs, the concentration of serum B-CC was slightly higher (P 0.118; by Mann±Whitney U test) and that of synovial B-CC increased significantly (P 0.004) compared with non-lame dogs over two-years-old. The concentration of serum B-CC increases in children between infancy and 14-years of age (Carey et al., 1997). The present study found that serum and synovial B-CC similarly changed in dogs but serum B-CC levels were high only in those under one-year of age and synovial B-CC levels were high in dogs under two-years-old. We speculated that the period of joint formation is completely different in dogs, where the joint may be formed more rapidly than in humans. Serum B-CC levels in humans are higher in osteoarthritis whereas synovial B-CC levels are similar in rheumatoid arthritis and osteoarthritis (Mansson et al., 1995; Nelson et al., 1998). These results suggest that serum B-CC reflects whole body cartilage metabolism in osteoarthritis where few joints are ordinarily involved compared with rheumatoid arthritis (Nelson et al., 1998). Comparing serum and synovial B-CC concentrations may be useful in attempting to understand whether the turnover of articular cartilage occurs systematically or regionally. Our data showed that the synovial B-CC concentration was high (1.9 ng/dL±25.5 ng/dL), whereas serum B-CC levels were close to zero (0 ng/dL±1.1 ng/dL) at one- to two-years of age. Therefore, it may be that the active turnover of articular cartilage settles down at two-years of age and in canine knee joints may be later than in other joints. In addition, our data showed that the serum B-CC concentration tended to be higher in RCCL dogs. Thus, whereas we diagnosed that RCCL was unilateral at that time, some of the RCCL dogs may in fact have had bilateral RCCL and/or other joint diseases.
CHONDROCALCIN IN DOGS
In humans, the synovial B-CC concentration correlates with cartilage damage in knees with anterior cruciate ligament deficiencies and osteoarthritis (Kobayashi et al., 1998). In dogs, procollagen accumulates in degenerative cartilage at the early stage of osteoarthritis (Burton-Wurster et al., 1982; Miller & Lust, 1979). The present study found that synovial B-CC levels were elevated in RCCL dogs, indicating that the articular cartilage had been damaged at a relative early stage in RCCL, since the dogs underwent surgery within one week of injury. Early diagnosis and treatment of RCCL is therefore required to reduce cartilage damage. REFERENCES BURTON-WURSTER, N., HUI-CHOU, C. S., GREISEN, H. A. & LUST, G. (1982). Reduced deposition of collagen in the degenerated articular cartilage of dogs with degenerative joint disease. Biochimica et Biophysica Acta 718, 74±84. CAREY, D. E., ALINI, M., IONESCU, M., HYAMS, J. S., ROWE, J. C., ROSENBERG, L. C. & POOLE, A. R. (1997). Serum content of the C-propeptide of the cartilage molecule type II collagen in children. Clinical and Experimental Rheumatology 15, 325±8. INNES, J. F., SHARIF, M. & BARR, A. R. (1999). Changes in concentrations of biochemical markers of osteoarthritis following surgical repair of ruptured cranial cruciate ligaments in dogs. American Journal of Veterinary Research 60, 1164±8. KOBAYASHI, T., TANAKA, O., YOSHIHARA, Y., SHIMMEI, M. & SAMURA, A. (1998). Synovial fluid concentrations of the C-propeptide of type II collagen correlate with body mass index in primary knee osteoarthritis. Orthopedics 21, 773±6. KOBAYASHI, T., YOSHIHARA, Y., SAMURA, A., YAMADA, H., SHINMEI, M., ROOS, H. & LOHMANDER, L. S. (1997). Synovial fluid concentrations of the C-propeptide of type II collagen correlate with body mass index in
213
primary knee osteoarthritis. Annals of the Rheumatic Diseases 56, 500±3. KUJAWA, M. J., WEITZHANDLER, M., POOLE, A. R., ROSENBERG, L. & CAPLAN, A. I. (1989). Association of the C-propeptide of type II collagen with mineralization of embryonic chick long bone and sternal development. Connective Tissue Research 23, 179±99. MANSSON, B., CAREY, D., ALINI, M., IONESCU, M., ROSENBERG, L. C., POOLE, A. R., HEINEGARD, D. & SAXNE, T. (1995). Cartilage and bone metabolism in rheumatoid arthritis. Differences between rapid and slow progression of disease identified by serum markers of cartilage metabolism. Journal of Clinical Investigation 95, 1071±7. MILLER, D. R. & LUST, G. (1979). Accumulation of procollagen in the degenerative articular cartilage of dogs with osteoarthritis. Biochimica et Biophysica Acta 583, 218±31. NELSON, F., DAHLBERG, L., LAVERTY, S., REINER, A., PIDOUX, I., IONESCU, M., FRASER, G. L., BROOKS, E., TANZER, M., ROSENBERG, L. C., DIEPPE, P. & POOLE, A. R. (1998). Evidence for altered synthesis of type II collagen in patients with osteoarthritis. Journal of Clinical Investigation 102, 2115±25. POOLE, A. R., PIDOUX, I., REINER, A., CHOI, H. & ROSENBERG, L. C. (1984). The association of a newly discovered protein, called chondrocalcin, with cartilage calcification. Acta Biologica Hungarica 35, 143±9. POOLE, A. R., PIDOUX, I., REINER, A., CHOI, H. & ROSENBERG, L. C. (1984). Association of an extracellular protein (chondrocalcin) with the calcification of cartilage in endochondral bone formation. Journal of Cell Biology 98, 54±65. SHINMEI, M., ITO, K., MATSUYAMA, S., YOSHIHARA, Y. & MATSUZAWA, K. (1993). Joint fluid carboxy-terminal type II procollagen peptide as a marker of cartilage collagen biosynthesis. Osteoarthritis and Cartilage 1, 121±8. VAN DER REST, M., ROSENBERG, L. C., OLSEN, B. R. & POOLE, A. R. (1986). Chondrocalcin is identical with the C-propeptide of type II procollagen. Biochemical Journal 237, 923±5. (Accepted for publication 19 August 2001)
Short Communication Serum and Synovial Fluid Concentrations of Bovine Chondrocalcin in Dogs Y. YAMAYA*, H. TAKEUCHIy, K. NAGAOKAy, T. KOSAKAy and S. TANAKAy *
Laboratory of Comprehensive Veterinary Clinical Studies and y Laboratory of Veterinary Surgery, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-8510, Japan
KEYWORDS: Chondrocalcin; dogs; knee; serum; synovial fluid. Bone and joint diseases in small animals are mainly diagnosed through gait analysis, palpation and radiography. However, joint and other diseases are difficult to discriminate at the early stage because joint damage may only involve articular cartilage. Many X-ray examinations may be required and the cost of diagnosis may increase if magnetic resonance imaging (MRI) or computed tomography (CT) are used. To reduce or resolve these problems in dogs, biochemical markers may be useful (Innes et al., 1999). Chondrocalcin is a carboxy-terminal propeptide of type II procollagen that is released in synovial fluid when articular cartilage is synthesized and repaired (Van der Rest et al., 1986). In addition, concentrations of chondrocalcin are high in the growth plates of bovine, rat and human joints (Kujawa et al., 1989; Poole et al., 1984a; Poole et al., 1984b). Levels of synovial fluid chondrocalcin are increased in human osteoarthritis, rheumatoid and traumatic arthritis (Kobayashi et al., 1998; Kobayashi et al., 1997; Mansson et al., 1995). Bovine chondrocalcin (B-CC) can now be quantified using a commercial enzyme immunoassay for human chondrocalcin. This assay has been used to study type II collagen synthesis in human articular cartilage (Shinmei et al., 1993). Moreover, chondrocalcin also Correspondence to: Yoshiki Yamaya, Laboratory of Comprehensive Veterinary Clinical Studies, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252±8510, Japan. Fax: 81 466 84 3900; E-mail: [email protected]
circulates in serum (Mansson et al., 1995; Nelson et al., 1998). In the present study, we examined levels of serum and synovial B-CC with aging to determine whether or not serum and synovial B-CC levels increase in dogs with a ruptured cranial cruciate ligament (RCCL). Twenty-eight clinically healthy dogs, consisting of 20 beagles, five mongrels, two Siberian huskies and one Akita (14 males and 14 females), were examined and grouped as non-lame dogs. A general physical examination determined that these animals had no lameness or pain in any joint. Lame dogs included four mongrels, one Doberman, one Labrador retriever and one Siberian husky (five males and two females). The animals were diagnosed and treated for hind limb lameness at the Animal Medical Center, Nihon University. The cause of lameness was diagnosed during surgery, and it was revealed that all dogs had unilateral RCCL. The range of ages and body weight were six months to 13 years and 7±35 kg (non-lame dogs) and three to 13 years and 4±33 kg (RCCL dogs), respectively. Blood was sampled through the intermediate cephalic vein to obtain serum from both groups of dogs. In non-lame dogs, synovial fluid was aspirated from the right or left knee joint under sedation with medetomidine hydrochloride or general anaesthesia with isoflurane. In RCCL dogs, synovial fluid was aspirated from the affected knee joint just before the start of surgery under general anaesthesia with isoflurane. Cells and tissue debris were removed from samples of blood and synovial
1090-0233/02/$ ± see front matter # 2002 Elsevier Science Ltd. All rights reserved.
212
THE VETERINARY JOURNAL, 163, 2
fluid anti-coagulated with EDTA by centrifugation at 10 000 rpm for 20 min. Samples were then stored at ÿ80 C. Serum and synovial B-CC levels were measured using a chondrocalcin assay kit (Teijin). Figure 1 shows the changes in serum and synovial B-CC concentrations with aging in non-lame dogs.
Fig. 1. Changes in bovine chondrocalcin concentration against aging in non-lame dogs. Closed circles show concentrations in serum and open circles in synovial fluid.
Fig. 2. Serum (black box) and synovial fluid (white box) chondrocalcin in sound and lame dogs, over 2-year-old. Bars are SEM.
The serum B-CC concentration was 21.6 4.7 ng/dL (mean SEM) up to the age of one-year, when it decreased to below 0.7 0.1 ng/dL. The synovial B-CC concentration was 61.6 2.1 ng/dL (mean SEM) up to one-year, decreasing to 13.0 2.8 ng/dL among one- to two-year-old dogs, and to 2.2 0.2 ng/dL thereafter. Serum and synovial B-CC concentrations did not significantly differ between males and females. In addition, serum and synovial B-CC concentrations in RCCL dogs were compared with those in eleven age-matched, non-lame dogs (over two-years of age). Serum and synovial B-CC concentrations were 0.8 0.2 ng/dL and 2.2 0.2 ng/dL in the non-lame dogs and 2.1 0.7 ng/dL and 9.0 1.7 ng/dL in the RCCL dogs, respectively (means SEM; Fig. 2). In RCCL dogs, the concentration of serum B-CC was slightly higher (P 0.118; by Mann±Whitney U test) and that of synovial B-CC increased significantly (P 0.004) compared with non-lame dogs over two-years-old. The concentration of serum B-CC increases in children between infancy and 14-years of age (Carey et al., 1997). The present study found that serum and synovial B-CC similarly changed in dogs but serum B-CC levels were high only in those under one-year of age and synovial B-CC levels were high in dogs under two-years-old. We speculated that the period of joint formation is completely different in dogs, where the joint may be formed more rapidly than in humans. Serum B-CC levels in humans are higher in osteoarthritis whereas synovial B-CC levels are similar in rheumatoid arthritis and osteoarthritis (Mansson et al., 1995; Nelson et al., 1998). These results suggest that serum B-CC reflects whole body cartilage metabolism in osteoarthritis where few joints are ordinarily involved compared with rheumatoid arthritis (Nelson et al., 1998). Comparing serum and synovial B-CC concentrations may be useful in attempting to understand whether the turnover of articular cartilage occurs systematically or regionally. Our data showed that the synovial B-CC concentration was high (1.9 ng/dL±25.5 ng/dL), whereas serum B-CC levels were close to zero (0 ng/dL±1.1 ng/dL) at one- to two-years of age. Therefore, it may be that the active turnover of articular cartilage settles down at two-years of age and in canine knee joints may be later than in other joints. In addition, our data showed that the serum B-CC concentration tended to be higher in RCCL dogs. Thus, whereas we diagnosed that RCCL was unilateral at that time, some of the RCCL dogs may in fact have had bilateral RCCL and/or other joint diseases.
CHONDROCALCIN IN DOGS
In humans, the synovial B-CC concentration correlates with cartilage damage in knees with anterior cruciate ligament deficiencies and osteoarthritis (Kobayashi et al., 1998). In dogs, procollagen accumulates in degenerative cartilage at the early stage of osteoarthritis (Burton-Wurster et al., 1982; Miller & Lust, 1979). The present study found that synovial B-CC levels were elevated in RCCL dogs, indicating that the articular cartilage had been damaged at a relative early stage in RCCL, since the dogs underwent surgery within one week of injury. Early diagnosis and treatment of RCCL is therefore required to reduce cartilage damage. REFERENCES BURTON-WURSTER, N., HUI-CHOU, C. S., GREISEN, H. A. & LUST, G. (1982). Reduced deposition of collagen in the degenerated articular cartilage of dogs with degenerative joint disease. Biochimica et Biophysica Acta 718, 74±84. CAREY, D. E., ALINI, M., IONESCU, M., HYAMS, J. S., ROWE, J. C., ROSENBERG, L. C. & POOLE, A. R. (1997). Serum content of the C-propeptide of the cartilage molecule type II collagen in children. Clinical and Experimental Rheumatology 15, 325±8. INNES, J. F., SHARIF, M. & BARR, A. R. (1999). Changes in concentrations of biochemical markers of osteoarthritis following surgical repair of ruptured cranial cruciate ligaments in dogs. American Journal of Veterinary Research 60, 1164±8. KOBAYASHI, T., TANAKA, O., YOSHIHARA, Y., SHIMMEI, M. & SAMURA, A. (1998). Synovial fluid concentrations of the C-propeptide of type II collagen correlate with body mass index in primary knee osteoarthritis. Orthopedics 21, 773±6. KOBAYASHI, T., YOSHIHARA, Y., SAMURA, A., YAMADA, H., SHINMEI, M., ROOS, H. & LOHMANDER, L. S. (1997). Synovial fluid concentrations of the C-propeptide of type II collagen correlate with body mass index in
213
primary knee osteoarthritis. Annals of the Rheumatic Diseases 56, 500±3. KUJAWA, M. J., WEITZHANDLER, M., POOLE, A. R., ROSENBERG, L. & CAPLAN, A. I. (1989). Association of the C-propeptide of type II collagen with mineralization of embryonic chick long bone and sternal development. Connective Tissue Research 23, 179±99. MANSSON, B., CAREY, D., ALINI, M., IONESCU, M., ROSENBERG, L. C., POOLE, A. R., HEINEGARD, D. & SAXNE, T. (1995). Cartilage and bone metabolism in rheumatoid arthritis. Differences between rapid and slow progression of disease identified by serum markers of cartilage metabolism. Journal of Clinical Investigation 95, 1071±7. MILLER, D. R. & LUST, G. (1979). Accumulation of procollagen in the degenerative articular cartilage of dogs with osteoarthritis. Biochimica et Biophysica Acta 583, 218±31. NELSON, F., DAHLBERG, L., LAVERTY, S., REINER, A., PIDOUX, I., IONESCU, M., FRASER, G. L., BROOKS, E., TANZER, M., ROSENBERG, L. C., DIEPPE, P. & POOLE, A. R. (1998). Evidence for altered synthesis of type II collagen in patients with osteoarthritis. Journal of Clinical Investigation 102, 2115±25. POOLE, A. R., PIDOUX, I., REINER, A., CHOI, H. & ROSENBERG, L. C. (1984). The association of a newly discovered protein, called chondrocalcin, with cartilage calcification. Acta Biologica Hungarica 35, 143±9. POOLE, A. R., PIDOUX, I., REINER, A., CHOI, H. & ROSENBERG, L. C. (1984). Association of an extracellular protein (chondrocalcin) with the calcification of cartilage in endochondral bone formation. Journal of Cell Biology 98, 54±65. SHINMEI, M., ITO, K., MATSUYAMA, S., YOSHIHARA, Y. & MATSUZAWA, K. (1993). Joint fluid carboxy-terminal type II procollagen peptide as a marker of cartilage collagen biosynthesis. Osteoarthritis and Cartilage 1, 121±8. VAN DER REST, M., ROSENBERG, L. C., OLSEN, B. R. & POOLE, A. R. (1986). Chondrocalcin is identical with the C-propeptide of type II procollagen. Biochemical Journal 237, 923±5. (Accepted for publication 19 August 2001)