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Rheumatology Advance Access originally published online on September 12, 2007
Rheumatology 2007 46(11):1652-1656; doi:10.1093/rheumatology/kem220
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© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Serum keratan sulfate is a promising marker of early articular cartilage breakdown

S. Wakitani1, M. Nawata2, A. Kawaguchi1, T. Okabe3, K. Takaoka1, T. Tsuchiya4, R. Nakaoka4, H. Masuda5 and K. Miyazaki5

1Department of Orthopaedic Surgery, Osaka City University Graduate School of Medicine, 2Department of Orthopaedic Surgery, Marunouchi Hospital, 3Department of Orthopaedic Surgery, Shinshu University School of Medicine, 4Division of Medical Devices, National Institute of Health Sciences, 5Department of Pharmaceuticals information, Seikagaku Corporation.

Correspondence to: Shigeyuki Wakitani, MD, Department of Orthopaedic Surgery, Osaka City University Graduate School of Medicine, 1-4-3 Asahi, Abeno-ku, Osaka 545-8585, Japan. E-mail: wakitani{at}med.osaka-cu.ac.jp


   Abstract
Top
 Abstract
Introduction
 Patients and methods
 Results
 Discussion
 References
 
Objectives. To find serum markers that may serve as indices for an early diagnosis of degeneration or damage of the articular cartilage.

Methods. Twenty-four healthy volunteers, 19 individuals with knee trauma (KT) and 31 with knee osteoarthritis (OA) were evaluated. KT patients were divided into a group (n = 5) with an injury <2 months old (recent KT) and a group (n = 14) with that >2 months old (old KT). Articular cartilage damage was assessed using either arthroscopy or direct observation. Serum concentrations of hyaluronic acid (HA), cartilage proteoglycan aggrecan turnover epitope (CS846) and cartilage oligomeric protein (COMP) were measured using enzyme-linked immunosorbent assay kits and those of keratan sulfate (KS) and chondroitin-6-sulfate (C6S) using high-performance liquid chromatography.

Results. Serum KS in the recent KT group (2095 ± 594 ng/ml) was significantly higher than that in the old KT group (1373 ± 418 ng/ml; P = 0.021), and serum COMP in the recent KT group (1572 ± 182 ng/ml) showed a tendency that was higher than that in the old KT group (1350 ± 250 ng/ml; P = 0.079).

Serum KS in OA patients with Kellgren and Lawrence (KL) grades 0 and I (1456 ± 334 ng/ml) showed a tendency that was higher than that in OA patients with KL grades II, III and IV (1248 ± 220 ng/ml; P = 0.084).

Conclusions. The serum concentration of KS correlated with the damage of the articular cartilage and it was significantly increased even at an early stage after the injury.

KEY WORDS: Keratan sulfate, Glycosaminoglycan, Cartilage oligomeric protein, Cartilage injury, Osteoarthritis, Serum marker


   Introduction
Top
 Abstract
 Introduction
Patients and methods
 Results
 Discussion
 References
 
The prevalence of patients with articular cartilage defects among patients with symptomatic knees requiring arthroscopy has been reported as 5–20% [1–3]; when left untreated, osteoarthritic changes are observed on X-rays taken after 10–20 yrs [4, 5]. Thus, articular cartilage injury is considered a cause of osteoarthritis (OA). Even if there is no articular cartilage injury, degeneration of the articular cartilage is considered to begin in humans at a young age, and articular cartilage changes, such as changes in colour and fibrillation, can occur. Injury or early-stage alterations of the articular cartilage in OA cannot be detected using X-ray examination. Magnetic resonance imaging (MRI) can detect articular cartilage defects and cartilaginous quality changes to some extent, but this technique is not sensitive enough to detect early OA changes and is expensive to be used as a routine examination. Serum markers, on the other hand, are suitable as screening tests, and only patients with high values of serum markers should be subjected to MRI or arthroscopy to detect articular cartilage degeneration. If it were possible to detect OA or articular cartilage damage at an early stage, patients could be educated to prevent the progression of OA. Moreover, it would be useful to monitor the natural course of articular cartilage damage or repair after, for instance, autologous chondrocytes implantation, whose effectiveness is still controversial because there is no method to effectively evaluate cartilage repair.

In 1985, Thonar et al. [6] measured serum keratan sulfate (KS) using an enzyme-linked immunosorbent assay (ELISA) by anti-KS antibody (1/20/5-D-4), and suggested its usefulness as a marker of OA. However, the correlation was weak and it did not correlate with X-ray grading [7]. Many researchers have tried to detect the metabolic products of articular cartilage components (proteoglycan, type II collagen and non-collagenous proteins) in joint fluid or blood and thereby a marker of OA [8–11]. As reported by Okumura et al. [12], early OA articular cartilage destruction begins with a loss of glycosaminoglycans (GAGs) from articular cartilage surfaces, followed by collagenolysis. Thus, the first event in OA or articular cartilage damage is the release of GAGs, which play an important role in maintaining articular cartilage function. Consequently, early markers of articular cartilage damage or OA change might be among GAG metabolic products. We selected KS, chondroitin 6 sulfate (C6S), cartilage proteoglycan aggrecan turnover epitope (CS846) and hyaluronan (HA) as candidate markers, and cartilage oligomeric protein (COMP), which is not a component of GAGs but has been reported to be a marker of OA [9]. These components have been reported to correlate with OA, to some extent, but not with cartilage damage caused by degradation and/or injury. These metabolic products can be measured in joint fluid, serum and urine, but we measured them in serum because it is easy to collect.

We measured KS using high-performance liquid chromatography (HPLC), which has been reported to be more accurate than ELISA [13]; C6S using HPLC, and CS846, HA and COMP using ELISA. We measured these markers in healthy volunteers and in patients with knee trauma (KT) or OA, who were subjected to knee surgery and whose articular cartilage was optically assessed (by arthroscopy or direct observation). We examined the correlation of these markers with the articular cartilage assessment to evaluate their usefulness as markers of early articular cartilage breakdown caused by degeneration and/or injury but that showed no change by X-ray examination.


   Patients and methods
Top
 Abstract
 Introduction
 Patients and methods
Results
 Discussion
 References
 
This study was approved by the institutional Review Board of Marunouchi Hospital and was conducted in accordance with the Helsinki Declaration of 1975, revised in 1983. Written informed consent was obtained from the healthy volunteers and patients prior to their participation in the study.

Blood collection from healthy volunteers
Ten men and 14 women (23–52 yrs old) volunteered to participate in the study. The volunteers were healthy with no gross obesity, inferior limb malalignment, history of knee injury or knee disorders. Sera were collected and stored at –80°C.

Patients with KT or knee OA
Nineteen KT patients (11 men and 8 women; 20–54 yrs old) and 31 patients with knee OA (11 men and 20 women; 40–80 yrs old) who were diagnosed to undergo knee surgery participated in the study. X-rays of knee and lumbar spine were available for all the patients. Sera samples were collected before surgery and stored at –80°C. The condition of the knee articular cartilage was observed at the time of surgery either arthroscopically or by direct observation. Among the 19 KT patients, two had meniscal injuries, 12 had ligament injuries and five had both meniscal and ligament injuries. KT patients were divided into a group of 5 patients with injuries <2 months old (recent KT) and a group of 14 patients with injuries >2 months old (old KT). Among the 31 OA patients, eight underwent total knee replacement, one underwent a high tibial osteotomy and 22 underwent arthroscopic debridement.

Assessment of articular cartilage surfaces by X-ray and visual inspection
X-ray images were assessed using the Kellgren and Lawrence (KL) grading scale [14]. All the KT patients were KL grade 0. Seven of the OA patients were KL grade 0, seven were KL grade I, four were KL grade II, six were KL grade III and seven were KL grade IV. Articular cartilage damage was assessed using the Societé Francaise d’ Arthroscopie (SFA) scaling system [15]. In brief, the degree of articular cartilage damage was estimated from 0 to IV according to the SFA grading scale. The width of the damaged area was evaluated as a percentage of the damaged area in the medial and medial femoro-tibial and patello-femoral areas, separately. The SFA score was then calculated using a coefficient. The SFA score represents not only the degree of articular cartilage surface damage, but also the width of the damaged area.

Determination of the serum markers
Keratan sulfate was determined by HPLC after digestion with keratanase II (Seikagaku Corporation, Tokyo, Japan) according to the method of Tomatsu et al. [13] Each serum sample (0.2 ml) was treated with a protease (actinase E: Kaken Pharmaceutical Co. Ltd., Tokyo) and the negatively charged substance containing KS was fractionated by Q sepharose and digested by keratanase II. The KS-derived ß-galactosyl-(1-4)-6-O-sulfo-N-acetylglucosamine (m-ks) and ß-6-O-sulfo-galactosyl-(1-4)-6-O-sulfo-N-acetylglucosamine (d-ks) were contained in the solution that was digested by the enzyme and were measured using HPLC. Standard KS derived from bovine cornea (Seikagaku) was used to measure KS under identical conditions; and the quantity of KS in each serum sample was calculated as the sum of m-ks and d-ks. To determine C6S concentration, 0.2 ml of each serum sample was first treated with chondroitinase ABC (Seikagaku). The quantity of unsaturated disaccharide contained in the digested fluid was determined and C6S was detected by HPLC [16]. For the determination of CS846, COMP and HA, the Aggrecan Chondroitin Sulfate 846 Epitope ELISA Kit (IBEX Technologies, Inc., Montreal, Quebec, Canada), Human COMP ELISA Kit (Kamiya Biomedical Company, Seattle, WA, USA) and Hyaluronan Assay Kit (Seikagaku Corporation) were used respectively.

Statistical analysis
To determine the statistical significance of inter-group differences, Steel's multiple comparison test for patient group vs control and Wilcoxon rank-sum test for inter-patient group were conducted, and the P-level was set at <0.05.


   Results
Top
 Abstract
 Introduction
 Patients and methods
 Results
Discussion
 References
 
Arthroscopic findings in KT patients and serum concentrations of KS, C6S, CS846, HA and COMP
All KT patients had articular cartilage damage. Their cartilaginous damage scores (SFA) for the recent KT group and the old KT group were 1.2 ± 0.7 and 3.8 ± 3.9, respectively. On X-ray examination, no changes were noted in the knee or intervertebral joints (Fig. 1).


Figure 1
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  		FIG. 1. Representative radiographs of the knee and lumbar spine and photograph of the articular cartilage in the knee of a 36-yr-old man with traumatic arthropathy. The X-ray grade, SFA score and serum KS concentration were normal, 0.7 and 2537 ng/ml, respectively. The serum KS concentration was high, although damage of the cartilage was minimal.

 
The serum concentrations of KS, C6S, CS846, HA and COMP in KT patients are shown in Table 1. KS, C6S, CS846 and COMP were significantly higher in the recent KT group (P = 0.001, P = 0.047, P = 0.022 and P = 0.001, respectively), and KS and COMP higher in the old KT group (both P < 0.001) than in controls.


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  		TABLE 1. Serum concentration of markers of cartilage degeneration or damage in patients with knee trauma

 
X-ray and arthroscopic examination of OA patients and serum concentrations of KS, C6S, CS846, HA and COMP
The SFA scores of OA patients distributed by their KL grade are presented in Table 2. The SFA score increased in relation with increased KL grade. Even in patients with KL grade 0 OA, degeneration or damage of the articular cartilage surface was observed by direct optical methods. In such patients, no X-ray findings were detected in the knee nor in the intervertebral discs (Fig. 2).


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  		TABLE 2. Serum concentration of markers of cartilage degeneration or damage in OA patients

 

Figure 2
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  		FIG. 2. Representative radiographs of the knee and lumbar spine and photograph of the articular cartilage in the knee of a 66-yr-old woman with KL grade 0 OA. The SFA score and serum KS concentration were 1.7 and 2145 ng/ml, respectively. The serum KS concentration was high, although the patient had early-stage OA.

 
Serum concentrations of KS were significantly higher in most OA stages (KL grade 0: P = 0.004, I: P < 0.001, III: P = 0.004 and IV: P = 0.008) and serum COMP were significantly higher in all OA stages (KL grade 0: P = 0.004, I: P = 0.002, II: P = 0.008, III: P = 0.002 and IV: P < 0.001) than in controls. C6S and HA in patients with KL grade IV were significantly higher than in controls (both P < 0.001).

Comparison of the serum markers between patient groups
Since the serum concentrations of KS and COMP were higher in most stages of KT and OA than in controls, those differences between stages were compared in Fig. 3. The serum KS in the recent KT group (2095 ± 594 ng/ml) was significantly higher than that in the old KT group (1373 ± 418 ng/ml; P = 0.021) and those in OA patients with KL grades 0 and I (1456 ± 334 ng/ml) showed a tendency that was higher than that in patients with KL grades II, III and IV (1248 ± 220 ng/ml; P = 0.084). The serum concentrations of COMP in the recent KT group (1572 ± 182 ng/ml) showed a tendency that was higher than that in the old KT group (1350 ± 250 ng/ml; P = 0.079), but those in OA patients showed no difference between the patient group with KL grades 0 and I (1639 ± 434 ng/ml) and the patient group with KL grades II, III and IV (1731 ± 355 ng/ml).


Figure 3
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  		FIG. 3. Comparison of the serum markers between stage groups in KT or OA patients. KT patients were divided into a group with an injury <2 months old (recent) and a group with an injury >2 months old (old). OA patients were divided into a group with KL grades 0 and I (early) and a group with KL grades II, III and IV (advanced). Data are the mean ± S.D. *P < 0.05 (Wilcoxon rank-sum test).

 

   Discussion
Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
References
 
This study showed that the serum concentration of KS was high in patients with early-stage damage of the articular cartilage undetectable by X-ray imaging. Serum KS may be suitable as a screening test for articular cartilage damage and to monitor the natural course of articular damage or repair.

In the KT patients with recent injuries, KS was significantly higher than in those with old injuries, suggesting that serum KS might indicate the release of cartilaginous GAG in the early stage after injury in spite of moderate cartilaginous damage.

In OA patients, the serum concentrations of KS, C6S, HA and COMP were significantly higher than in healthy controls as reported previously [6, 17–19]. Among these parameters, KS was high in patients with KL grades 0 and I, indicating that KS might serve as a marker of early-stage OA. The KS concentrations in OA patients tended to decrease as the KL grade increased from 0 to IV, which reflects disappearance of the joint space. It may mean that in OA, a greater quantity of the cartilage matrix is released when the joint space has not yet narrowed. On the contrary, COMP was high in KL grade IV. This can be explained by the fact that COMP is a non-collagen protein that exists in the synovial membrane, meniscus and tendon, as well as in the cartilage and its increase is most likely related to the inflammation of various intra-articular tissues. The changes in C6S and HA were marked in KL grade IV, indicating that these are not markers of early-stage cartilage destruction.

KS is a component of proteoglycans found in the articular cartilage, intervertebral discs and corneas. Because corneas are relatively small tissues, serum KS mainly originates from articular cartilage and intervertebral discs. Thus, the serum concentration of KS is not only a marker of knee articular cartilage, but also of other joints and intervertebral discs. Therefore, before concluding that the elevated serum concentration of KS originated from damage to the knee joint articular cartilage, the possibility of spondyloarthropathy and OA in other joints must be examined. We verified that there were no X-ray changes in the lumbar spine nor symptoms caused by lumbar spinal abnormalities in KT patients (Fig. 1), although spondyloarthrotic changes existed in OA patients because most of these patients were of advanced age (Fig. 2). We verified that no OA symptoms were observed in joints other than the knee in these patients. We are planning to investigate serum KS in patients with spondyloarthropathy or intervertebral disk herniation in the future.

Serum KS is considered to reflect the normal metabolism of cartilage, and KS increases in case of mechanical injuries within a few months after injury. Budsberg et al. [20] found that serum KS increased 1–3 months after resection of the anterior cruciate ligaments of dog knees. In our report, KT patients who were evaluated within 2 months after the injury exhibited an acute release of KS. Although the SFA score of KT patients was very small, indicating that damage was confined, serum KS was high (Table 1). After the rapid release of KS ends, release from the injured surfaces continues at a relatively high rate. This phase is considered to continue for a few years to a couple of decades as in KT evaluated >2 months after the injury and in early-stage OA patients (KL grades 0 and I). The persistence of this condition leads to OA in a few decades. This phase corresponds to advanced OA (KL grades II, III, IV).

This report is the first study to show that serum KS increases early after an injury causing small articular damage and in patients with early-stage OA undetectable by X-ray imaging. As only a small volume of blood is required for the measurement of serum KS, this parameter may serve as a screening test to detect articular cartilage injury and it is expected to contribute greatly to the decision on a therapeutic strategy for the management of OA or cartilage injury.

Formula

Disclosure statement: The authors have declared no conflicts of interest.


   References
Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 

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Submitted 11 April 2007; revised version accepted 19 July 2007.
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