Keratan Sulfate Concentration Research Articles

Mucopolysaccharidosis IVA: Correlation between genotype, phenotype and keratan sulfate levels

Mucopolysaccharidosis IVA (MPS IVA) is caused by deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS), leading to systemic skeletal dysplasia because of excessive storage of keratan sulfate (KS) in chondrocytes. In an effort to determine a precise prognosis and personalized treatment, we aim to characterize clinical, biochemical, and molecular findings in MPS IVA patients, and to seek correlations between genotype, phenotype, and blood and urine KS levels. Mutation screening of GALNS gene was performed in 55 MPS IVA patients (severe: 36, attenuated: 13, undefined: 6) by genomic PCR followed by direct sequence analysis. Plasma and urine KS levels were measured by ELISA method. Genotype/phenotype/KS correlations were assessed when data were available. Fifty-three different mutations including 19 novel ones (41 missense, 2 nonsense, 4 small deletions, 1 insertion, and 5 splice-site) were identified in 55 patients and accounted for 93.6% of the analyzed mutant alleles. Thirty-nine mutations were associated with a severe phenotype and ten mutations with an attenuated one. Blood and urine KS concentrations in MPS IVA patients were age-dependent and markedly higher than those in age-matched normal controls. Plasma and urine KS levels in MPS IVA patients with the severe phenotype were higher than in those with an attenuated form.This study provides evidence for extensive allelic heterogeneity of MPS IVA. Accumulation of mutations as well as clinical descriptions and KS levels allows us to predict clinical severity more precisely and should be used for evaluation of responses to potential treatment options.

Validation of keratan sulfate level in mucopolysaccharidosis type IVA by liquid chromatography–tandem mass spectrometry

Mucopolysaccharidosis type IVA (MPS IVA, Morquio A disease), a progressive lysosomal storage disease, causes skeletal chondrodysplasia through excessive storage of keratan sulfate (KS). KS is synthesized mainly in cartilage and released to the circulation. The excess storage of KS disrupts cartilage, consequently releasing more KS into circulation, which is a critical biomarker for MPS IVA. Thus, assessment of KS level provides a potential screening strategy and determines clinical course and efficacy of therapies. We have recently developed a tandem mass spectrometry liquid chromatography [LC/MS/MS] method to assay KS levels in blood. Forty-nine blood specimens from patients with MPS IVA [severe (n = 33), attenuated (n = 11) and undefined (n = 5)] were analyzed for comparison of blood KS concentration with that of healthy subjects and for correlation with clinical severity. Plasma samples were digested by keratanase II to obtain disaccharides of KS. Digested samples were assayed by LC/MS/MS. We found that blood KS levels (0.4-26 µg/ml) in MPS IVA patients were significantly higher than those in age-matched controls (0.67-4.6 µg/ml; P < 0.0001). It was found that blood KS level varied with age and clinical severity in the patients. Blood KS levels in MPS IVA peaked between 2 years and 5 years of age (mean 11.4 µg/ml). Blood KS levels in severe MPS IVA (mean 7.3 µg/ml) were higher than in the attenuated form (mean 2.1 µg/ml) (P = 0.012). We also found elevated blood KS levels in other types of MPS. These findings indicate that the new KS assay for blood is suitable for early diagnosis and longitudinal assessment of disease severity in MPS IVA.

Periodic knee injections of BMP‐7 delay cartilage degeneration induced by excessive running in rats

Strenuous running of rats enhances mechanical stress on the knee, thereby inducing degeneration of articular cartilage. Bone morphogenetic protein-7 (BMP-7) has an inhibitory effect on cartilage degeneration, suggesting its usefulness for human osteoarthritis patients. However, its mode of administration should be investigated. We examined whether weekly knee injections of BMP-7 delayed the progression of cartilage degeneration. Wistar rats were forced to run 30 km in 6 weeks on a rodent treadmill, and BMP-7 was injected weekly into the knee. Macroscopically and histologically, this strenuous running regimen induced cartilage degeneration. Weekly injections of 250 ng BMP-7 delayed the progression of cartilage degeneration. Immunohistochemically, in the control knee, type II collagen expression decreased, while BMP-7 expression in chondrocytes slightly increased. Interestingly, weekly injection of BMP-7 increased BMP-7 expression even 9 days after the final injection. Disulfate disaccharide keratan sulfate in serum transiently increased in the control group, while it remained at a low level in the BMP-7 group. Weekly BMP-7 injection increased BMP-7 expression in chondrocytes and its effect seemed to last more than 7 days. The effect of BMP-7 could be monitored by serum keratan sulfate concentration. Periodical injections of BMP-7 delayed progression of cartilage degeneration induced by excessive running in rats.

Serum and synovial fluid concentrations of keratan sulfate and hyaluronan in dogs with induced stifle joint osteoarthritis following cranial cruciate ligament transection

To examine longitudinal changes in serum and synovial fluid concentrations of keratan sulfate (KS) and hyaluronan (HA) after cranial cruciate ligament (CCL) transection in dogs. 12 clinically normal adult mixed-breed dogs. Following CCL transection in the right stifle joint, KS and HA concentrations were determined in serum and neat (undiluted) synovial fluid prior to and 1, 2, 3, and 12 months after surgery. Postsurgical dilution of synovial fluid was corrected by use of urea as a passive marker. Synovial fluid KS and HA concentrations decreased at 1, 2, and 3 months after surgery in operated stifle joints, compared with baseline values. Synovial fluid KS concentration decreased in unoperated stifle joints at 1 month. A decrease in synovial fluid KS concentration was found in operated stifle joints, compared with unoperated stifle joints, at 2 and 3 months, and a decrease in synovial fluid HA concentrations was also found in operated stifle joints, compared with unoperated stifle joints, at 1, 2, and 3 months. Serum KS concentrations increased from baseline values at 3 months after surgery. Hyaluronan concentrations in operated stifle joints were lower than baseline values at 1, 2, and 3 months. Urea-adjusted synovial fluid concentrations revealed that dilution did not account for the decline in biomarker concentrations. The initial decrease and subsequent increase in synovial fluid concentrations of HA and KS may be caused by an acute inflammatory response to surgical intervention that negatively affects cartilage metabolism or an increase in production of immature proteoglycans.

The Effects of Growth and Disease in Serum Keratan Sulfate Concentration in Dogs

The purpose of this study is to investigate keratan sulfate (KS) concentration in the serum of puppies and the effects of age, body weight, breed and diseases. Serum samples from six neonatal dogs (4 Beagles, 2 Labrador Retrievers), and from 127 adult dogs with various diseases were collected at a Teaching Animal Hospital. Canine serum KS concentration was measured by inhibition enzyme-linked immunosorbent assay (ELISA). Samples from puppies were evaluated for growth-related changes, and samples from patients were evaluated for age, body weight, breed and disease-related changes. Serum KS concentration was high in puppies from birth to 4 months of age. KS values started to decrease from 4 months to 9 months of age, and then gradually reached to the plateau. Though in the small sample, mean KS concentration in a Labrador Retriever was higher than in Beagles during the first 10 months. The values of serum KS showed body weight-related increase within retrievers among teaching hospital population and there was significant increase in body weight-related change. Cartilage metabolism is high in canine immature joint and that activity continues for 5 months, and that higher in Labrador Retrievers rather than in Beagles. There was no effect from other factors, including age, body weight, breed and disease in all patients. Serum KS concentration of Retrievers is higher than Beagles, and that value increased with gain of body weight. We suggest that Retriever have higher cartilage metabolism with growth or ageing.

Comparative assessment of bone among wild-type, restricted ovulator and out-of-production hens

1. The aim of this study was to assess bone characteristics in restricted ovulator (RO) hens. These hens generally are unable to ovulate due to a point mutation in the oocyte VLDL receptor gene whose protein product mediates the uptake of yolk precursors. Because these hens do not have the cyclic calcium (Ca) metabolism associated with egg formation, they could be a useful model for studying bone metabolism. 2. RO hens had greater humerus, femur and tibia ash concentrations than wild-type (WT) and out-of-production (OP) hens. Bone mineral content and density obtained with dual-energy X-ray absorptiometry (DXA) were highly correlated with the results of conventional bone assays. 3. Gross and histological examination of the femurs confirmed the presence of extremely dense medullary bone deposition in the RO hens. However, the composition of non-collagenous protein extracts of medullary bone was similar for the two genotypes. 4. Analysis of medullary bone extracts for glycosaminoglycans (GAG) confirmed the presence of large amounts of keratan sulphate (KS) in the matrix of medullary bone. 5. Plasma Ca, total GAG and KS concentrations of RO hens were markedly higher than WT and OP hens. The changes in plasma calcium and keratan sulphate are probably a reflection of elevated Ca-binding yolk precursor molecules and intensive medullary bone formation in response to increased plasma oestrogen observed by others in RO hens.

Development and Testing of New Screening Method for Keratan Sulfate in Mucopolysaccharidosis IVA

Mucopolysaccharidosis IVA (MPS IVA), a progressive lysosomal storage disease, causes skeletal dysplasia through excessive storage of keratan sulfate (KS). We developed an ELISA-sandwich assay that used a MAb specific to KS. Forty-five blood and 59 urine specimens from MPS IVA patients (ages 1-65 y) were analyzed to determine whether KS concentration is a suitable marker for early diagnosis and longitudinal assessment of disease severity. Blood specimens were obtained from patients categorized as phenotypically severe (n = 36) and milder (n = 9). Urine specimens were also analyzed from patients categorized as severe (n = 56) and milder (n = 12), respectively. Blood KS levels (101-1525 ng/mL) in MPS IVA patients were two to eight times higher than those in age-matched controls (15-323 ng/mL). It was found that blood KS level varied with age and clinical severity. Blood KS levels in both MPS IVA and controls peaked between 5 and 10 y of age (mean, 776 versus 234 ng/mL, respectively). Blood levels in severe MPS IVA were 1.5 times higher than in the milder form. In contrast to blood, urine KS levels in both MPS IVA and controls peaked between 1 and 5 y (15.3 versus 0.26 mg/g creatinine), and thereafter declined with age. Urine KS level also varied with age and clinical severity, and the severe MPS IVA phenotype was associated with 6.7 times greater urine KS excretion than the milder one. These findings indicate that the new assay for blood or urine KS may be suitable for early diagnosis and longitudinal assessment of disease severity in MPS IVA.

Serum level of cartilage oligomeric matrix protein (COMP) in equine osteoarthritis.

This study was designed to assay and compare cartilage oligomeric matrix protein (COMP) in horse sera, in samples from normal and joint diseased horses, and to investigate the relationships between COMP in sera and synovial fluids (SF) with keratan sulphate (KS) data. Sera from 38 horses free of any joint pathology (controls) and from horses with aseptic joint disease (AJD horses, n = 40) were assayed for COMP and KS concentrations. Of the 78 horses in the study, 53 were also assayed for COMP and KS concentrations in SF. COMP and KS were measured by inhibition ELISA, using monoclonal antibodies 12C4 and 5D4, respectively. The COMP concentration in sera from AJD horses (mean +/- s.d. 10.7 +/- 7.4 microg/ml) was significantly (P<0.02) lower than in control sera (14.8 +/- 7.8 microg/ml). The joint disease sera also had significantly lower (P<0.01) KS levels (180.5 +/- 61.8 ng/ml) than controls (237.1 +/- 116.1 ng/ml). A significant correlation (r = 0.52, n = 53, P<0.001) was seen between serum and SF in COMP levels; no such relationship was seen in KS levels. It is possible that serum COMP concentration could be a more specific marker of equine joint disease than any other described to date.

Serum concentrations of keratan sulfate, osteocalcin, and pyridinoline crosslinks after oral administration of glucosamine to standardbred horses during race training.

To determine the effects of orally administered glucosamine on concentrations of markers of bone and cartilage metabolism in Standardbred horses during race training. Twenty 16- to 20-month-old Standardbreds beginning race training. Horses were randomly assigned to 2 groups. One group received glucosamine hydrochloride (4 g, PO, q 12 h), and the second (control) group received glucose (4 g, PO, q 12 h). Serum samples were obtained prior to onset of the study (baseline) and at regular intervals for 48 weeks for determination of concentrations of keratan sulfate (KS), osteocalcin (OC), and pyridinoline crosslinks (PYD). Osteocalcin concentrations changed significantly with time; mean serum concentrations were significantly higher than baseline values for samples obtained at 24 to 48 weeks after onset of the study. Although a significant effect of time was observed for mean concentration of KS, concentrations did not differ significantly from baseline values at any time during the study when groups were analyzed separately. However, pooled analysis revealed significant increases of mean serum KS concentration at weeks 24 and 30. Significant changes in serum PYD concentrations were not detected. Oral administration of glucosamine did not significantly affect serum concentrations of any of the markers. Increased serum OC in clinically normal Standardbreds during race training may reflect bone formation that accompanies adaptive remodeling of the appendicular skeleton. For these experimental conditions, glucosamine did not appear to exert a detectable influence on serum concentrations of these 3 markers of connective tissue metabolism.

Serum keratan sulphate as a cartilage metabolic marker in horses: the effect of exercise.

Keratan sulphate (KS) concentration in sera from resting horses and horses training daily on a racetrack was measured by an inhibition enzyme-linked immunosorbent assay (ELISA) using anti-equine KS antibody 1/14/16H9. For the in-training horses, serum KS concentrations in 2-year-old-horses was significantly higher than 3- or 4-year-old-horses. A higher concentration of serum KS was found in the in-training group than in the long-term resting group in 2-year-old-horses. Serum KS concentration increased remarkably immediately after training in healthy horses, and at 1, 5, 9 and 24 h after training remained at similar levels to the pre-training concentration. The results suggest that serum KS concentration could represent the situation of joint loading, induced by daily racetrack training, affecting the metabolic activities in joint cartilage.

Evaluation of the role of keratan sulphate as a molecular marker to monitor cartilage metabolism in horses.

The role of keratan sulphate (KS) as a metabolic marker of cartilage was evaluated using an in vitro model of equine articular cartilage. Articular cartilage was harvested from clinically healthy 6-month-old foals (n = 3). Chondrocytes were centrifuged and cultured as pellets. Chondrocyte pellets were stimulated by insulin-like growth factor-I alpha (IGF-I alpha) or interleukin-1 alpha (IL-1 alpha) for 2 weeks. The concentrations of sulphated glycosaminoglycans (GAG) and KS in the culture media were measured by a 1,9-dimethyl-methylene blue (DMMB) colorimetric assay and an inhibition enzyme-linked immunosorbent assay using a 1/20/5D4 antibody, respectively. The concentration of GAG was significantly increased both in the media of pellets stimulated by IGF-I alpha and in those stimulated by IL-1 alpha. KS concentration was significantly increased in those stimulated by IL-1 alpha, while no significant change was found in those stimulated by IGF-I alpha. A high correlation between GAG and KS concentrations was found in the media of pellets stimulated by IL-1 alpha (r = 0.84), but not in those stimulated by IGF-I alpha (r = 0.59). The results suggest that the concentration of KS reacting to 1/20/5D4 mirrors the GAG concentration during the stage of cartilage catabolism, but not during the cartilage anabolic stage. The KS concentration in biological fluids could therefore be a useful marker to understand further the cartilage catabolic process. It may also represent some aspects of the cartilage anabolic process.

Consideration of the role of antigenic keratan sulphate reacting to a 1/14/16H9 antibody as a molecular marker to monitor cartilage metabolism in horses.

The role of keratan sulphate (KS) as a marker of cartilage metabolism was evaluated by using an in vitro model of equine articular cartilage. Articular cartilage was harvested from clinically healthy 6-month-old foals (n=3). Chondrocytes were centrifuged and cultured as pellets. Chondrocyte pellets were stimulated by insulin-like growth factor (IGF)-Ialpha or interleukin (IL)-1alpha for 2 weeks. The sulfated glycosaminoglycans (GAG) and antigenic KS concentrations in the culture media were measured by a 1,9-dimethyl-methylene blue (DMMB) colorimetric assay and an inhibition ELISA using a 1/14/16H9 antibody, respectively. Concentration of GAG was significantly increased in the media of pellets stimulated by both IGF-Ialpha and IL-1alpha. Antigenic KS concentration was significantly increased in those stimulated by IL-1alpha, while no significant change was found in those stimulated by IGF-Ialpha. A high correlation between GAG and antigenic KS concentrations was found in the media of pellets stimulated by IL-1alpha (r=0.87), but not in those stimulated by IGF-Ialpha (r=0.43). The results suggest that the concentration of antigenic KS reacting to 1/14/16H9 mirrors the GAG concentration during the stage of cartilage catabolism, but not during the cartilage anabolic stage. The concentration of antigenic KS reacting to 1/14/16H9 antibody in biological fluids could therefore be a useful marker to further understand principally the catabolic and slightly the anabolic process of articular cartilage metabolism.

Oncostatin M induces leukocyte infiltration and cartilage proteoglycan degradation in vivo in goat joints.

To evaluate the effect of intraarticular injections of recombinant human oncostatin M (rHuOSM) in the goat joint. One milliliter of endotoxin-free normal saline (vehicle) containing either 40 ng, 200 ng, or 1,000 ng of rHuOSM was injected into the right radiocarpal joints (RCJs) of 12 male angora goats, while the left RCJs were injected with an equivalent volume of vehicle alone. In subsequent studies, the right and left RCJs of 8 male angora goats were injected with 200 ng of rHuOSM, and 1 hour later, the right RCJs were injected with either 5 microg of recombinant murine leukemia inhibitory factor binding protein (rMuLBP) or 1 mg of recombinant human interleukin-1 receptor antagonist (rHuIL-1Ra) in 1 ml of vehicle, while the left RCJs received 1 ml of vehicle alone. Goat joints were examined for clinical features of inflammation, and synovial fluid (SF) was aspirated on day 0 (before injection) and at days 2 and 6 postinjection. Injections of rHuOSM stimulated dose-dependent increases in the carpal:metacarpal ratio, SF volume, and SF leukocyte numbers, and stimulated dose-dependent decreases in the cartilage proteoglycan (PG) content ex vivo and PG synthesis. No significant changes were observed in the control joints that received saline alone, or between RCJs that were injected with 200 ng rHuOSM followed by 5 microg rMuLBP and RCJs that were injected with 200 ng of rHuOSM alone, except in respect to synovial fluid keratan sulfate concentrations, where a modest statistically significant reduction was observed in the joints injected with the combination of rHuOSM and rMuLPB. In contrast, RCJs injected with 200 ng rHuOSM followed by 1 mg of rHuIL-1Ra had significantly lower SF volumes (P<0.0001) and a significantly higher rate of ex vivo PG synthesis (P<0.0001). These results indicate that rHuOSM stimulates inflammation and modulates cartilage PG metabolism in vivo. Some of the effects of rHuOSM in vivo appear to be due, in part, to elaboration of IL-1. Even at very high doses, however, the rHuIL-1Ra did not attenuate OSM-mediated cartilage PG resorption. Thus, OSM has the potential to contribute to synovitis in vivo and can stimulate cartilage PG resorption in vivo, independent of IL-1.

Measurement of synovial fluid and serum concentrations of the 846 epitope of chondroitin sulfate and of carboxy propeptides of type II procollagen for diagnosis of osteochondral fragmentation in horses

Abstract Objective To determine whether serum or synovial fluid concentrations of chondroitin sulfate epitope 846 and carboxy propeptides of type II collagen (CPII) can be used to diagnose osteochondral fragmentation (OC) in horses. Animals 38 horses with unilateral OC of the radiocarpal (n = 31) or intercarpal (33) joints and 8 clinically and radiographically normal horses. Procedures For horses with OC, serum and synovial fluid concentrations of epitope 846, CPII, and keratan sulfate (KS) were determined, along with synovial fluid WBC counts and total protein concentrations. Serum epitope 846, CPII, and KS concentrations were measured in control horses. Results Synovial fluid epitope 846 and total protein concentrations were significantly higher in the joints with OC than in unaffected joints, but CPII and KS concentrations and WBC counts were not. Synovial fluid total protein and 846 epitope concentrations were linearly related to grade of OC. Serum epitope 846 and CPII concentrations were significantly higher in horses with OC than in control horses. Discriminant analysis allowed 27 of 34 (79%) horses to be correctly classified as having or not having OC on the basis of serum epitope 846 and CPII concentrations. Conclusions Results suggest that serum and synovial fluid concentrations of epitope 846 and CPII are associated with OC. Increases in concentrations of epitope 846 and CPII suggest that increased synthesis of cartilage aggrecan and type II procollagen may be associated with OC. Clinical Relevance Measurement of serum epitope 846 and CPII concentrations may be useful in the diagnosis of OC in horses. (Am J Vet Res 1999;60:306–309)

Establishment of a monoclonal antibody (1/14/16H9) for detection of equine keratan sulfate

Abstract Objectives To establish a sensitive and specific monoclonal antibody (MAB) against equine keratan sulfate (KS) and to develop an enzyme immunoassay for measurement of the concentration of KS in serum and synovial fluid from horses. Sample Population 18 synovial fluid and 48 serum samples were obtained from clinically normal horses and horses with arthritis. Procedure BALB/c mice were immunized with chondroitinase-ABC-digested proteoglycan monomer from equine joint cartilage, and MAB were raised, using Sp2/O cells as a fusion partner. A competitive ELISA was optimized, using one of the established MAB, and KS concentration in synovial fluid and sera from horses was measured. Results The high-titer MAB1/14/16H9, which specifically recognizes the epitope on equine KS, was identified. This antibody had no reactivity with chondroitin sulfate and core protein of proteoglycan monomers, hyaluronan, heparin, dermatan sulfate, and heparan sulfate. A competitive ELISA for determination of KS concentration was optimized, using this antibody. Precision data were obtained for the range of 10 to 160 ng/ml. The within- and between-assay coefficients of variation were 10.0 and 12.7%, respectively. Conclusions MAB 1/14/16H9 that specifically recognized equine KS was established and was used to develop an enzyme immunoassay for measurement of the concentration of KS in synovial fluid and sera from horses. It is expected that the assay system using MAB 1/14/16H9 will contribute to evaluation of cartilage metabolic activity in horses. (Am J Vet Res 1998;59:1203-1208)

Markers of cartilage and synovial metabolism in joint fluid and serum of patients with chondromalacia of the patella

Objective: To further our understanding of the pathogenesis of chondromalacia of the patella (CM), we have studied the release into knee joint fluid and serum, obtained from patients with CM, of molecules associated with the metabolism of joint cartilage matrix and synovium.Methods: Interleukin-1 α (IL-1α), interleukin-1 β (IL-1β), interleukin-6 (IL-6), stromelysin-1 (MMP-3), interstitial collagenase (MMP-1), tissues inhibitor for metalloproteinases-1 (TIMP-1), phospholipase activity A2(PLA2), hyaluronan (HA), aggrecan fragments (AGN) and antigenic keratan sulfate (KS) were quantified in knee joint lavage fluid from 96 patients with CM; KS and HA also was measured in serum. Chondromalacia was graded on a scale of I to IV according to Outerbridge (1961). The histopathology of the synovial membrane close to the patellofemoral joint was evaluated. Control samples were obtained from nine patients with knee pain presenting with arthroscopically normal knee joints.Results: The concentrations of MMP-3, MMP-1 and TIMP-1 proteins in joint lavage fluid were increased in advanced (grade IV) CM, compared with controls. Levels of MMP-1 in lavage fluid correlated with the severity of CM (r=0.38,P<0.01) and MMP-1 and MMP-3 concentrations correlated with each other (r=0.45,P<0.001). TIMP-1 was elevated in grade IV CM compared with grades II and III CM (P<0.02,P<0.01). Interleukins (IL-1α, IL-1β and IL-6) showed no significant change in CM. The lavage fluid level of PLA2increased with the severity of CM (r=0.40,P<0.001). Serum KS was higher in CM IV than in controls (P=0.05), while lavage fluid KS concentration was elevated in CM I (P=0.04). There were no differences in the lavage fluid levels of AGN and HA between the different study groups. Synovium showed slight or moderate histological signs of inflammation in 9% of CM patients.Conclusion: The changes in the release and activity of these marker molecules from serum and synovial fluid may reflect changes in the metabolism of articular cartilage and synovium in CM, that are consistent with those observed in early-stage tibiofemoral cartilage changes in OA.

Evaluation of the catabolic activity of cartilage by measurement of serum keratan sulfate concentration in foals

Abstract Objectives To determine keratan sulfate (KS) concentration in the serum of foals at the early stage of growing, and to evaluate the role of serum KS as a cartilage catabolic marker, comparing its values with the fluctuation of serum alkaline phosphatase (ALP) activity as a measurement of osteoblastic activity. Animals 12 foals with normal growth and 3 foals with joint abnormalities within 18 months after birth. Procedure Measurement of KS concentration and ALP activity in serum and radiographic and physical examinations were done. Results In all foals, serum KS concentration was high from 1 week after birth to 3 months of age, while serum ALP decreased with aging. The value started to decrease rapidly from 3 to 5 months of age, then gradually reached adult values. During the first 3 months, KS concentration in male foals was significantly higher than that in female foals. In 3 foals which had joint problems, KS concentration was higher than that in normally growing foals at 1 week, and at 1, 2, and 3 months of age. Conclusions Cartilage catabolic activity is higher in developing foals up to 3 months of age, suggesting that the immature joint at this time could be easily affected by any factor of loading. Moreover, though only 3 diseased foals were examined, higher serum KS concentration in these foals suggest that this variable might be a useful measure of joint diseases, even at an early stage of life in foals. (Am J Vet Res 1997;58:925–929)

Concentrations of keratan sulfate in plasma and synovial fluid from clinically normal horses and horses with joint disease

Objective To determine whether keratan sulfate concentrations in plasma or synovial fluid from clinically normal horses were different from concentrations in horses with joint disease and whether concentrations varied with type of joint disease. Design Case-control study. Animals 67 clinically normal horses, 10 clinically normal foals, and 160 horses with joint disease. Procedure ELISA was used to measure keratan sulfate concentrations. Results Mean plasma keratan sulfate concentration (mean ± SEM, 580 ± 124 ng/ml) in foals peaked at 10 weeks of age. Mean plasma keratan sulfate concentration in clinically normal horses was 200 ng/ml (95% confidence interval, 157 to 251 ng/ml). Horses with osteochondral (chip) fractures, other closed intraarticular fractures, inflammatory arthritis (synovitis), infectious arthritis, or osteochondrosis had significantly higher plasma keratan sulfate concentrations than did clinically normal horses, but horses with osteoarthritis did not. Breed, gender, and type of joint disease affected keratan sulfate concentration in synovial fluid. Standardbreds with chip fractures of the metacarpophalangeal/ metatarsophalangeal joints had significantly higher keratan sulfate concentrations in synovial fluid than did Thoroughbreds. Keratan sulfate concentrations in synovial fluid from osteoarthritic carpal joints were lower than concentrations in normal carpal joints and tarsocrural joints with inflammatory joint disease. Clinical Implications Keratan sulfate concentration alone was not a specific marker of joint disease but was affected by various joint diseases. (J Am Vet Med Assoc 1997;210:369–374

Glycosaminoglycans of the uterine and vaginal cul-de-sac tissue in the brushtail possum (Trichosurus vulpecula).

Live-captured feral brushtail possums (Trichosurus vulpecula) received slow-release progestagen implants for 16 days after removal of pouch young. At the time of implant removal one group was injected intramuscularly with pregnant mare serum gonadotrophin (PMSG, 2 I.U. kg-1 in 0.2 mL saline) and another group received an equivalent volume of sterile saline. Animals from each group were killed on Days 0, 3, 4, 5 or 6 after implant removal (PMSG, n = 4 per day; saline controls, n = 3 per day). The uterus and vaginal cul-de-sac were excised and tissue was analysed for glycosaminoglycans (GAGs) and keratan sulfate content. GAG concentration increased progressively over the six-day period following implant removal. Mean uterine GAG concentrations were significantly higher than those in vaginal cul-de-sac from Day 3 to Day 6 in PMSG-treated and saline-treated animals following progestagen implant removal (P < 0.05). Keratan sulfate concentrations showed a similar response--concentrations were higher in uterine tissue than in vaginal cul-de-sac tissue and PMSG-treated animals showed a greater response than did saline controls in the uterine tissue, but the same response in cul-de-sac tissue (P < 0.05 for all groups). The progressive increase in GAG concentrations of reproductive tract tissue following removal of the progesterone implant is indicative of hormonal stimulation and suggests that GAGs may have a supportive role in gamete and embryo nutrition and growth in the brushtail possum.

Demonstration of immunogenic keratan sulphate in commercial chondroitin 6-sulphate from shark cartilage. Implications for ELISA assays

The prototype monoclonal keratan sulphate (KS) antibody 5D4 that is widely used for detection of KS in tissues and biological fluids reacts strongly with commercial low grade shark cartilage chondroitin 6-sulphate. Characterization of the immunogenic material by chondroitinase ABC digestion, ELISA inhibition studies, immunoblotting and HPLC analyses confirmed the presence of substantial amounts of KS, probably as a large proteoglycan (120 kDa). Commercial and heterogenic glycosaminoglycan preparations therefore must be used with great caution in immunological analyses. On the other hand the shark cartilage chondroitin 6-sulphate is an easy accessible source of immunogenic KS that can be used as a reference standard and as coating antigen in KS-ELISAs. The concentration of immunogenic KS in synovial fluid measured with an ELISA based solely on reagents of shark cartilage chondroitin 6-sulphate correlated well ( r = 0.90) with the concentrations obtained with a traditional KS-ELISA that uses purified aggrecan as standard and coating antigen, and KS in both serum and synovial fluid could be measured with sufficient linearity.