Measurement of glycosaminoglycans in canine synovial fluid and its correlation with the cause of secondary osteoarthritis , age and body weight

Glycosaminoglycans are natural components of healthy joint cartilage and they also appear in healthy synovial fluid. An increased amount of glycosaminoglycans in synovial fluid is believed to be a marker of secondary osteoarthritis, regardless of its primary cause. The aim of our study was to define the relationship between glycosaminoglycans in the synovial fluid and joint disorders, age, and body weight. The samples of synovial fluid were obtained from dogs suffering from secondary secondary osteoarthritis (n = 35) and from control dogs (n = 18); control dogs had normal body weight. The results were compared among joints of dogs with secondary osteoarthritis divided into groups according to the criteria mentioned above and control dogs. Glycosaminoglycan concentrations in synovial fluid were measured using dimethylmethylene blue assay. The lowest mean value of glycosaminoglycans in synovial fluid was measured in the control group. Significantly higher glycosaminoglycan content (P < 0.05) was found in synovial fluid isolated from obese dogs compared to control dogs. Furthermore, we observed an agerelated trend, in which the highest mean values were reached either in old dogs or pups. Despite the absence of significant differences in glycosaminoglycan values among dogs suffering from various types of secondary secondary osteoarthritis, the highest mean values were measured in fragmented coronoid processus group. Our data suggest that abnormally increased body weight has an impact on glycosaminoglycan concentration in synovial fluid which may imply faster degradation and turnover of joint cartilage. Such observation has not yet been published in veterinary medicine.

Glycosaminoglycans (GAG) and proteoglycans form the amorphous part of cartilage matrix, contributing to 35% of total cartilage weight.These hydrophilic molecules help counteracting mechanical pressure, thus maintaining the space structure (Kempson et al. 1970); they regulate solution penetration into cartilage (Maroudas et al. 1980) and play an important role in differentiation and regeneration of cartilage tissue (Linsenmayer and Toole 1977).
Chondroitin-6-sulphate and chondroitin-4-sulphate represent the main group of GAG and are constituent parts of aggregated proteoglycans.Another GAG representative is keratan sulphate or dermatan sulphate (Uebelhart 2008).
Regarding physiological condition, GAG or chondroitin-6-sulphate a chondroitin-4sulphate are found both in cartilage matrix and synovial fluid (SF) as they enter SF within normal cartilage turnover.Their concentration is elevated if the matrix is being degraded faster as in the case of certain pathological condition, e.g.osteoarthritis (OA).The OA development, pathophysiology, diagnostics and treatment are well reviewed by Renberg (2005).Primary OA is rarely seen in dogs; on the other hand, the secondary OA is quite common since 20% of dogs older than 1 year suffer from secondary OA (Johnston 1997).
Changes in GAG concentration are thought to be a reliable marker of cartilage degradation level.An important factor that is necessary to objectify when comparing GAG concentration in SF is the chronicity of OA process.In early OA stages, the GAG amount may increase dramatically; however, GAG values drop slowly and approach nearly normal values in later OA stages (Innes et al. 1998).GAG can also be estimated in serum because they are filtrated through synovial membrane.Importantly, serum values may give false positive results as the half-time of GAG clearance in OA joints differs from healthy joints (Myers et al. 1996).Moreover, some authors believe GAG serum concentration is neither prognostic nor diagnostics marker of OA (Arican et al. 1994).
The physiological turnover of cartilage and its abnormal degradation during OA share some similarities.Metalloproteinases are primarily responsible for extracellular matrix degradation; also, they release proteoglycans from the complex with hyaluronic acid.Subsequently, free proteoglycans undergo proteolysis and their fragments of various size containing chondroitin sulphate, keratan sulphate, interglobular domains etc. enter SF and are further proceeded by synovial cells or removed by lymphatic vessels from the joint space.The majority of fragments enters the blood circulation and then is removed by the liver or kidneys.
The aim of our study was to prove the dependence of synovial GAG concentration on the primary cause of secondary OA as well as on the age and body weight of studied dogs.

Animals
Our study included 36 privately owned dogs treated at the Small Animal Clinic of the University of Veterinary and Pharmaceutical Sciences Brno.Thirty-one of these dogs (35 joints) suffered from joint disease and 5 (18 joints) animals were control dogs that were euthanized for disorders not affecting joints (Table 1).These control animals had healthy weight and their age ranged from 6 to 12.5 years.The diagnosis of OA and its underlying cause was based on history, clinical examination, radiographic assessment and perioperative findings.
Dogs with joint disease were divided into several groups based on their disease, age, and body weight.Based on disease status, dogs were divided into four groups: control dogs (n = 5), dogs with rupture of CCL (RCCL) (n = 18), dogs with FCP (n = 7) and dogs with other causes of OA (OCOA), such as patellar luxation, OCD or hip OA (n = 6).Age clustering was the following: pups (up to 6 months for small breeds, 9 months for medium breeds and 12 months for large breeds) (n = 6), young dogs (age 6-12 months for small breeds, 9-18 months for medium breeds and 12-30 months for large breeds) (n = 8), and old dogs (9 years or older for small breeds, 8 years or older for medium breeds and 6 years or older for large breeds) (n = 4).The breed category (small, medium, and large breed) is used accordingly in Table 1.Animals exceeding Fédération Cynologique Internationale (FCI) standard for given breed by 10-19% were considered overweight (n = 4), dogs surpassing standards by 20% or more were rated as obese (n = 5).Overweight and obese dogs were pooled into one group.Obese dogs were also evaluated separately.

Sample collection
Synovial fluid samples were collected from all dogs by aseptic arthrocentesis and concentrations of GAG were examined.After assessing the sample volume, heparin solution (50 ml) was added and the sample was diluted sevenfold with phosphate-buffered saline (PBS), pH 7.0.The sample was then centrifuged at 200 g for 10 min at room temperature.The supernatant fluid was stored at -80 °C until assayed.

Dimethylmethylene blue assay for GAG concentration in SF
We used a modified colorimetric method that was based on the dimethylmethylene blue (DMMB) assay published by Arican et al. (1994).Briefly, samples of SF supernatant were sonicated (20 pulses q 0.5s, 80% power, Branson Sonifer 150, Branson Corp., Danbury, CT, USA), transfered to a 96-well plate and incubated with N-acetylcysteine and papain at 65 °C for 2 h.After digestion, iodoacetic acid, sodium chloride and DMMB were added and sample absorbance at 540 nm was determined by ELISA-reader iMS READER MF (LABSYSTEMS, Helsinki, Finland).Shark cartilage (BioChemika Fluka, Germany) chondroitin-6-sulphate solution was used to construct a standard curve.If not indicated otherwise, all chemicals were purchased from Sigma-Aldrich (Prague, Czech Republic).

Statistical analyses
The normality of data distribution was tested first.The criterion of normality was not fulfilled hence the data were evaluated by the non-parametric Kruskal-Wallis test.The P values lower than 0.05 were considered significant.

Results
The GAG concentrations were measured in SF samples obtained from 53 joints (36 dogs) of which 18 joints were used as control samples.The results were compared among dog groups divided according to given criteria (disease status, age, body weight) and control dogs.The GAG found in SF together with other variables characterizing the dogs are shown in Table 1.
The GAG concentrations in SF compared among groups of dogs with different disease status are shown in Fig. 1.The mean values of GAG concentrations in SF were following: 1.14 ± 0.58 mg/ml, 1.43 ± 1.088 mg/ml, 1.81 ± 1.51 mg/ml, and 2.87 ± 2.43 mg/ml in the control group, RCCL, OCOA, and FCP groups, respectively.
The GAG concentrations in SF compared among age groups of dogs are presented in Fig. 2. The mean values of GAG concentrations in SF were following: 1.14 ± 0.58 mg/ml, 1.22 ± 0.51 mg/ml, 1.44 ± 1.42 mg/ml, 2.50 ± 2.59 mg/ml, and 2.71 ± 1.79 mg/ml in the control group, the group of young animals, adult dogs, pups, and old dogs, respectively.
The GAG concentrations in SF compared among body weight groups are presented in Fig. 3.The mean values of GAG concentrations in SF were following: 1.14 ± 0.58 mg/ml, 1.80 ± 0.73 mg/ml, 1.80 ± 0.58 mg/ml, and 2.85 ± 1.80 mg/ml in the control group, group of dogs with healthy weight, overweight group including obese dogs, and obese group, respectively.The mean value of GAG concentration in SF measured in the obese group was significantly higher compared to control dogs (P < 0.05).

Discussion
Secondary OA is one of the most common orthopaedic disorders in dogs.Despite the fact that secondary OA is a common result of abnormal development of joint structures (elbow or hip dysplasia, FCP, UAP etc.), the extending lifespan, increasing number of  for disorders not affecting joints, n = 18 (joints); pup -dogs with joint disease (6 months and younger for small breeds, 9 months and younger for medium breeds, and 12 months and younger for large breeds), n = 7 (joints); young -dogs with joint disease with the ages of 6 -12 months for small breeds, 9-18 months for medium breeds, and 12-30 months for large breeds, n = 9 (joints); adult -dogs with joint disease between the ages of 1-9 years for small breeds, 1.5-8 years for medium breeds, and 2.5-6 years for large breeds, n = 14 (joints); old -dogs with joint disease with the age of 9 years and older for small breeds, 8 years and older for medium breeds, and 6 years and older for large breeds), n = 5 (joints).The OA is a chronic degenerative joint disorder involving joint cartilage, underlying bone structures and the synovial membrane that interact together during degradation and reparation process (Owens and Biery 1999).The OA is considered a non-inflammatory arthropathy; its typical features are fragmentation and loss of joint cartilage, narrowing or even collapsing of joint space well seen on radiographs, increased subchondral density (sclerosis) and newly formed bone structures at the edge of joint surfaces (osteophytes) (Pedersen and Poole 1978).In this study, we focused on cartilage degradation products in SF, which are believed to be elevated during certain stages of OA.
In spite of the relatively low number of evaluated joints along with high data variability, some interesting conclusions could be drawn.The GAG concentrations in SF samples seem to be higher in dogs suffering from OA than in control dogs, regardless of their actual body weight.However, obese dogs reached a significant difference compared to control dogs.Although the finding supports the general idea of a relationship between the body weight and OA development in joints, there are no studies available specifically discussing the body weight versus GAG concentration in SF.
Also, there is a certain age influence seen as the old dogs and pups have the highest mean values and on the other hand, the lowest mean value was measured in young dogs group.A similar conclusion was made by authors evaluating the correlation of GAG concentration in SF and age in horses; the highest values were measured in a group of newborn foals and those values kept decreasing over the lifetime (van den Boom et al. 2004).Later, the same authors cast doubts on GAG measurement in early stages of cartilage damage when no obvious changes could be seen since those values were lower compared to values of horses with at least minimal visible OA changes (van den Boom et al. 2005).Negative correlations between GAG values in SF and cartilage damage stage were reported by other authors in horses, too (Fuller et al. 2001).The GAG concentration and severity of radiographic changes correlated negatively even in dogs (Innes et al. 1998) and there was no correlation between radiographic changes and GAG in human patients (Belcher et al. 1997).These indicators (severity of radiographic changes and GAG content in SF) did not correlate in our study either (data not shown).The elevated GAG and keratan sulphate concentrations were observed in the patients' SF samples during the acute process compared to the chronic disease.The authors explained this as a result of higher metabolic rate and final GAG depletion in joint cartilage content (Ratcliffe et al. 1988).The same fact was also confirmed in dogs throughout the early and late OA stages (Innes et al. 2005).
It may be considered that high GAG values are caused with a high metabolic rate in pups when anabolic processes significantly outnumber catabolic processes during the intensive phase of their growth; on the other hand, in the old dogs, catabolic processes that degrade cartilage tissue may also increase GAG in SF.Another explanation for this phenomenon can be an increased glycosylation of cartilage proteins in older animals, which leads to higher GAG concentration in SF (DeGroot et al. 2001).
The variability in age of dogs included in our study might explain the differences among groups based on primary joint disorder due to the fact that the FCP group, which achieved the absolutely highest mean value of GAG concentration in the whole study, was dominated by pups and old dogs (66% of dogs diagnosed with FCP) but the RCCL group with the lowest mean GAG value was mainly formed from adult and young dogs (81%) who had low GAG values.Nevertheless, the high FCP values in FCP group can be influenced by the fact that all samples were obtained from one compartment, the elbow joint exclusively.The GAG concentration may significantly differ among joints of a healthy animal (Fuller et al. 1996).
Finally, our study supports the widely-accepted dogma that obesity is a negative factor contributing to joint degradation process, manifested by an increased level of GAG released into samples of canine SF.Further evaluation is needed in order to answer the question how soon the obesity-dependent degradation starts and whether the process can be significantly slowed down after the individual has lost the abundant body weight.

Fig. 2 .
Fig.2.Comparison of glycosaminoglycan (GAG) mean values among age groups of dogs.Control -dogs euthanized for disorders not affecting joints, n = 18 (joints); pup -dogs with joint disease (6 months and younger for small breeds, 9 months and younger for medium breeds, and 12 months and younger for large breeds), n = 7 (joints); young -dogs with joint disease with the ages of 6 -12 months for small breeds, 9-18 months for medium breeds, and 12-30 months for large breeds, n = 9 (joints); adult -dogs with joint disease between the ages of 1-9 years for small breeds, 1.5-8 years for medium breeds, and 2.5-6 years for large breeds, n = 14 (joints); old -dogs with joint disease with the age of 9 years and older for small breeds, 8 years and older for medium breeds, and 6 years and older for large breeds), n = 5 (joints).