Assessment of three variations of the 1,9-dimethylmethylene blue assay for measurement of sulfated glycosaminoglycan concentrations in equine synovial fluid

OBJECTIVE: To determine whether 3 variations of the 1,9-dimethylmethylene blue (DMMB) assay yield comparable results when measuring sulfated glycosaminoglycan (sGAG) concentrations in equine synovial fluid (SF). SAMPLE POPULATION: 25 samples of SF collected from affected joints of 13 horses and 13 samples of SF collected from nonaffected (control) joints of 4 horses. PROCEDURE: Sulfated glycosaminoglycan concentrations were measured by the direct spectrophotometric (ie, Farndale), microplate, and indirect DMMB assays in samples of SF collected from normal and affected joints and in samples digested with nucleases, papain, and hyaluronidase. RESULTS: All 3 assays reacted similarly to standard solutions of sGAGs and digestion of SF samples with nucleases, papain, and hyaluronidase. Nucleic acids were not important interfering substances, and papain and hyaluronidase could not be used interchangeably to digest SE All 3 assays proved to have satisfactory precision (SD < 10%), but each DMMB assay resulted in significantly different measures of sGAG in equine SF. CONCLUSIONS AND CLINICAL RELEVANCE: Samples of SF should be digested with papain or hyaluronidase prior to measurement via DMMB assay. Researchers currently are unable to compare clinical information when variations of the DMMB assay are used, because each DMMB assay yields substantially different sGAG concentrations in SF. Of the 3 assays examined here, we recommend use of the direct spectrophotometric DMMB assay.     

Quantitative microanalysis of equine synovial fluid glycosaminoglycan concentration

An alcian blue precipitation method for quantifying the hyaluronic acid (HA) and sulphated glycosaminoglycan concentration (SGAG) in solutions containing both compounds was assessed. The assay was found to be rapid and reliable in solutions containing 0 to 200 mg of HA/dl and 50 to 1,000 micrograms of SGAG/dl, and was not affected by the presence of protein, hemoglobin, or methemoglobin in concentrations normally found in synovial fluid. The HA and SGAG concentrations in intercarpal synovial fluid from 13 clinically normal and 11 arthritic horses were evaluated. A relationship was not found between the concentration of HA and SGAG and any other synovial fluid variable. The SGAG concentration was found to be markedly high in several of the synovial fluid samples from arthritic horses, but did not correlate with the degree of articular cartilage erosion.     

Assessment of glycosaminoglycan concentration in equine synovial fluid as a marker of joint disease.

A modification of a colorimetric assay was used to determine synovial fluid total and individual sulphated-glycosaminoglycan concentration in various clinical presentations of joint disease in horses. Concentrations of synovial fluid and serum sulphated-glycosaminoglycan (GAG) were measured by the 1,9-dimethylmethylene blue (DMMB) dye assay in normal horses (n = 49), horses with acute (n = 26) or chronic (n = 27) joint disease (defined by clinical, radiographic, and clinicopathological parameters), and horses with cartilaginous lesions at diagnostic arthroscopy, but with normal radiographs and synovial fluid (n = 9). Horses with acute joint disease were subdivided into moderate acute (n = 21) and severe acute (n = 5) joint disease on the basis of synovial fluid analysis and clinical examination. Horses with chronic joint disease were subdivided into mild chronic (n = 9), moderate chronic (n = 10), and severe chronic (n = 8) joint disease on the basis of synovial fluid analysis, clinical examination, and radiographic findings. The concentrations of chondroitin sulphate (CS) and keratan sulphate (KS) were analyzed in each sample following sequential enzymatic digestion of the sample with chondroitinase or keratanase. In addition, the concentration of hyaluronate (HA) in each sample was determined by a colorimetric assay following digestion of the sample with microbial hyaluronidase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inflammatory mediators in equine synovial fluid

Enzyme immunoassay for prostaglandin E₂ (PGE₂), and radioimmunoassays for prostaglandin F₂α (PGF₂α, 6-keto-PGF₁α, and leukotriene B₄ (LTB₄) were performed on synovial fluid from normal middle carpal joints of 10 horses, and from 30 middle carpal or antebrachiocarpal joints of horses affected by degenerative joint disease and chip fractures to compare the concentrations of inflammatory mediators. Significantly greater concentrations of PGE₂ were detected in fluid from affected than from control joints, but there were no significant differences in the mean concentrations of PGF₂α, 6-keto-PGF₁α, and LTB₄.     

Urea as a measure of dilution of equine synovial fluid

This paper tests the hypothesis that serum and synovial urea concentrations are similar and that urea concentration can be used as an accurate marker for synovial fluid dilution in normal equine joints. Serum and synovial fluid urea concentrations were compared in 42 horses and were equivalent for individual horses (P<0.0001). Mean +/- s.e. serum concentration was 6.1+/-0.552 mmol/l and synovial concentration 6.0+/-0.459 mmol/l. The normal range for synovial urea concentration was determined as 2.5-7.7 mmol/l. The synovial urea concentration from different synovial structures in individual horses were compared and were equivalent (P = 0.002). Known dilutions of synovial fluid with saline were made. The actual and expected synovial urea concentrations were compared and were equivalent (P<0.001). An accurate method of calculating synovial fluid dilution has been determined.     

A hemolytic assay for the measurement of equine complement

A hemolytic assay was developed for the measurement of functional equine complement activity. The assay utilizes antibody sensitized chicken erythrocytes as the target cell and was specific for classical pathway (antibody dependent) complement activity. The assay was found to be reproducible and more sensitive than previous reports using other species of target cells. Total serum complement (CH50) values were determined for five mares and their foals and followed over a period of 3 months.     

Development of an enzyme-linked immunosorbent assay for specific equine neutrophil myeloperoxidase measurement in blood.

Equine inflammatory disease is accompanied by a neutrophil activation resulting in the release of granulocytic enzyme myeloperoxidase (MPO). To measure MPO in horse plasma as marker of neutrophil activation, the authors purified equine neutrophil MPO and developed a specific enzyme immunoassay using 2 specific polyclonal antibodies obtained from rabbit (primary antibody) and guinea pig (secondary antibody). The sandwich complex "primary antibody-MPO-secondary antibody" was detected using a goat anti-guinea pig immunoglobulin antibody conjugated to alkaline phosphatase. The enzyme-linked immunosorbent assay (ELISA) showed good precision and accuracy, with intra- and interassay coefficients of variation below 10% for MPO concentrations ranging from 0.78 to 50 ng/ml. A stable plasma MPO value, unaffected by time elapsed between blood collection and centrifugation, was obtained with plasma from EDTA anticoagulated blood. The mean MPO value measured in 38 healthy horses was 181.80 +/- 64.74 ng/ml. In 20 horses suffering from obstruction of the large or small intestine, MPO concentrations measured at the time of arrival at the intensive care unit were significantly higher than mean normal value, ranging from 477.88 to 2,748.13 ng/ml. Work is in progress to apply this MPO ELISA technique to other biological fluids and other equine diseases.     

马骨关节炎模型关节液中COMP含量变化的研究

为探索马骨关节炎病变程度与关节液中软骨代谢标志物软骨寡聚基质蛋白(COMP)水平的关系,试验选用13匹蒙古马,随机分为试验组(n=8)和对照组(n=5)试验组左侧腕关节内注入2 mL两性霉素B(25 mg/mL体重),建立马骨关节炎模型;对照组左侧腕关节注射2 mL生理盐,水。每周采集关节液1次,用ELISA法检测马关节液中COMP的含量。结果显示,试验组关节液COMP浓度在注射两性霉素B后第14天开始显著升高(15.54μg/L±1.46μg/L),与对照组(11.29μg/L±0.73μg/L)比较差异极显著(P0.01);试验组COMP浓度保持增高的趋势直至第63天试验结束;第21天试验组马匹开始出现不同程度跛行。试验结果表明,关节液内COMP浓度升高比马跛行出现得早,推测COMP可以作为马骨关节炎(OA)早期诊断的生物标记物。     

Concentration and degree of polymerization of hyaluronate in equine synovial fluid

In addition to its well-known physicochemical properties, hyaluronate (HA) has recently been shown to have important biological and pathophysiologic regulatory effects on granulocytes, monocytes, fibroblasts, and endothelial cells, as well as on the healing of wounds and various joint disorders. Many of these effects depend on or are reflected in the concentration and degree of polymerization of HA. Therefore, high-performance liquid chromatography with size-exclusion column was used to characterize the concentration and degree of polymerization of HA in equine synovial fluid (SF). The mean (+/- SD) HA concentration was 0.47 +/- 0.19 mg/ml and there was no difference between control joints and those with positive response to local anesthetic administration (0.61 +/- 0.20 mg/ml vs 0.42 +/- 0.17 mg/ml), suggesting that in horses with acute traumatic synovitis causing lameness, HA concentration in SF cannot be used as a marker for the condition. High-performance liquid chromatograms disclosed considerable variation between horses in the degree of polymerization reflected in the peak area to height ratio (mean +/- SD, 3.207 +/- 0.447; range, 2.229 to 3.915), indicating differences in local synthesis, degradation, or mobilization into lymph of SF HA. In addition, the correlation between SF HA concentration and degree of polymerization was 0.760 (P less than 0.01; linear regression analysis), suggesting that HA concentration and chain length are independently regulated.     

Development of a novel in vitro equine anthelmintic assay

An in vitro assay involving the use of a horse strongyle (Strongylus edentatus) and the micromotility meter has been developed to test for equine anthelmintic activity. Three commercially available equine anthelmintics (dichlorvos, ivermectin, and pyrantel pamoate) and an investigational drug (p-toluoyl chloride phenylhydrazone) were evaluated in this assay at four concentrations. After a 24-h incubation, greater than or equal to 10 micrograms/ml of all four drug treatments significantly (P less than or equal to 0.05) reduced the motility of ensheathed L-3 S. edentatus larvae, thereby indicating anthelmintic activity. Pyrantel pamoate also reduced motility at 1 microgram/ml, while the hydrazone significantly increased movement at this level. At 0.1 microgram/ml, none of the treatments significantly reduced motility; one treatment (dichlorvos) significantly increased larval motility. Incubation for 48 h resulted in significant activity (reduction in motility) at greater than or equal to 1 microgram/ml with two drugs (ivermectin, pyrantel pamoate); dichlorvos and the hydrazone reduced motility at greater than or equal to 10 micrograms/ml. None of the treatments significantly reduced motility at the lowest concentration (0.1 microgram/ml); however, at 48 h, two treatments (dichlorvos, hydrazone) significantly increased motility at the lowest concentration (0.1 microgram/ml). The in vitro S. edentatus motility assay proved to be sensitive, accurate and rapid. This assay system should be a valuable addition to tests used to identify potential equine anthelmintics, monitor helminth resistance to drugs, and perhaps define the kinetics and mode of action for drugs.     

Increased cartilage oligomeric matrix protein concentrations in equine digital flexor tendon sheath synovial fluid predicts intrathecal tendon damage

Objectives: To evaluate digital flexor tendon sheath (DFTS) synovial fluid cartilage oligomeric matrix protein (COMP) concentrations as a molecular marker for intrathecal pathology. Study Design: Case control study. Animals: Horses (n=46) with DFTS tenosynovitis; 23 fresh cadaver horses. Methods: DFTS synovial fluid samples were collected from clinical cases with noninfected DFTS tenosynovitis and from control DFTS. Clinical and surgical findings were recorded, and dissection of control limbs was performed to confirm the DFTS to be grossly normal. Synovial fluid COMP was quantified using a homologous competitive inhibition ELISA. Results: Abnormalities were identified tenoscopically: intrathecal tendon/ligament tearing was identified in 37 cases and 9 had other lesions. In control horses, synovial fluid COMP was higher in younger horses. Clinical cases with intrathecal tendon/ligament tearing had higher synovial fluid COMP than either clinical cases with other lesions, or controls. In horses ≥5 years old, the sensitivity and specificity of the assay was high for diagnosing intrathecal tendon/ligament tearing. Conclusions: COMP concentrations in DFTS synovial fluid were significantly greater than those in normal horses with noninfected tenosynovitis caused by intrathecal tendon/ligament tearing, but not by other lesions.     

Ethylenediaminetetraacetic acid (EDTA) anticoagulant affects the refractometric measurement of total protein concentration in equine synovial fluid

The objectives of this study were to (i) determine the effect of ethylenediaminetetraacetic acid (EDTA) concentration on the refractometric measurement of the total protein (TP) concentration of equine synovial fluid and (ii) assess the effect of time and temperature on the refractometric measurement of TP concentration. Synovial fluid was retrieved aseptically from horses presenting to Liphook Equine Hospital either (a) immediately after euthanasia or (b) from those requiring arthrocentesis as part of their clinical investigation. Samples were aliquoted into commercially available (i) plain tubes (0.5 mL; control) and (ii) EDTA tubes (at different volumes to obtain serial dilutions of EDTA:synovial fluid). TP concentration was measured by two independent observers using a calibrated hand-held refractometer. Samples from a separate group of horses were stored for 24 h at 4°C and 20°C before analysis to assess the effects of time and temperature. Results were compared using repeated measures ANOVA. TP concentration was significantly higher at all EDTA concentrations compared to control samples, with the greatest effect seen with higher concentrations (P<0.0001 for samples with ≥3.17 mg EDTA/mL). Storage time or temperature did not significantly affect TP concentration. Refractometric measurement of small volumes of synovial fluid in commercially available EDTA tubes leads to artificially elevated TP concentrations. Delays in sample analysis and alterations in storage conditions did not significantly affect TP concentration within the parameters studied. The magnitude of overestimation of synovial total protein concentration could result in misclassification of suspect synovial sepsis cases where EDTA tubes are used.     

Proinflammatory cytokine activities, matrix metalloproteinase-3 activity, and sulfated glycosaminoglycan content in synovial fluid of dogs with naturally acquired cranial cruciate ligament rupture

OBJECTIVE: To measure and compare activities of interleukin-1beta (IL-1beta), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), and matrix metalloproteinase-3 (MMP-3); as well as sulfated glycosaminoglycan (S-GAG) content in synovial fluid from dogs with cranial cruciate ligament rupture (CCLR) and dogs with clinically normal stifles. To determine whether correlations exist between demographic and disease-related variables and these synovial markers. STUDY DESIGN: Prospective clinical study. ANIMALS: Dogs with CCLR (n=23) and Beagles with normal stifle joints (n=21). METHODS: Synovial fluid activities of proinflammatory cytokines (IL-1beta, IL-6, and TNF-alpha) were determined by bioassay. MMP-3 activity was measured using fluorogenic substrate. S-GAG contents were determined by dimethylmethylene blue dye-binding assay. Mann-Whitney U-test was used to compare results from CCLR joints with normal controls. Spearman's rank correlation test was used to evaluate associations between demographic and disease-related markers and synovial markers. RESULTS: Mean values for synovial markers were significantly higher in CCLR joints compared with controls. IL-1beta and MMP-3 were positively correlated with lameness duration. CONCLUSIONS: Activities of proinflammatory cytokines, MMP-3 activity and S-GAG contents were significantly elevated in synovial fluid from canine stifle joints with naturally acquired CCLR. These results indicate that there is joint inflammation and increased release of GAGs into synovial fluid, suggesting that these inflammatory changes are associated with depletion of proteoglycan from articular cartilage. CLINICAL RELEVANCE: Medical and surgical treatments designed to decrease joint inflammation and breakdown of proteoglycans may be of value in the management of CCLR in the dog.     

Inhibition of interleukin-1 activity by equine synovial fluid.

The presence, in equine synovial fluid, of inhibitors of interleukin-1 (IL-1) activity has been investigated by means of an assay involving IL-1-mediated production of PGE2 by synovial cells. Inhibitors of IL-1 alpha and IL-1 beta were identified in normal synovial fluid and synovial fluid from two horses with early joint disease. Inhibitors of IL-1 alpha were also present in synovial fluid from two horses with long-standing joint disease. However, IL-1 beta inhibitory activity was not present in fluid from the horses with more chronic joint disease. The effect appeared to be specific for IL-1, and not a direct action on PGE2 production, as synovial fluid had no effect on lipopolysaccharide-mediated PGE2 production. It is suggested that the inhibitory activity may be involved physiologically in the control of IL-1 activity in the joint, and the loss of IL-1 inhibition may be at least as important biologically as increased production of IL-1.     

The disposition of gentamicin in equine plasma, synovial fluid and lymph

Plasma (P), synovial fluid (SF) and lymph (L) concentrations of gentamicin were studied in two trials. A lymph vessel in the hindlimb was cannulated. The day after surgery (trial A), P and L samples were collected for 12 h after intravenous injection of gentamicin sulphate at 2.2 mg/kg dose rate. Approximately 48 h after surgery (trial B), the fetlock joint of the cannulated hindlimb was catheterized and P, SF and L samples collected for 12 h after a similar intravenous injection. The kinetic parameters were similar to those in other reports and did not differ between trials ( P < 0.05). The P, L and SF disposition profiles were similar. The 95% confidence interval for P & L concentrations overlapped 2–3 h after injection. Thereafter, parallelism between L and P concentrations was observed, but L concentrations were on average 60% higher than P concentrations, and elimination from L was slower than from P. The mean L/SF and P/SF ratios were 1.54 ± 0.2 and 1.25 ± 0.2, 2–4 h after injection. Gentamicin elimination from SF appeared to be slower than from L and P. Lymph cannulation is a viable technique for antibiotic disposition studies. A sample of any of the fluids 3 h after injection was representative of the others. While SF concentrations were of limited value for predicting tissue fluid (L) concentrations 3–8 after injection, P concentrations were a useful index.     

Assessment of the effects of age and joint disease on hydroxyproline and glycosaminoglycan concentrations in synovial fluid from the metacarpophalangeal joint of horses

OBJECTIVE: To assess the effects of age and joint disease on hydroxyproline and glycosaminoglycan (GAG) concentrations in synovial fluid from the metacarpophalangeal joint of horses and evaluate the association of those concentrations with severity of osteoarthritis and general matrix metalloproteinase (MMP) activity. SAMPLE POPULATION: Synovial fluid was collected from the metacarpophalangeal joints of foals at birth (n = 10), 5-month-old foals (10), 11-month-old foals (5), and adult horses (73). PROCEDURE: Hydroxyproline and GAG concentrations were determined in synovial fluid samples. The severity of osteoarthritis in adult joints was quantified by use of a cartilage degeneration index (CDI) and assessment of general MMP-activity via a fluorogenic assay. RESULTS: Hydroxyproline and GAG concentrations in synovial fluid were highest in neonates and decreased with age. Concentrations reached a plateau in adults by 4 years and remained constant in healthy joints. In synovial fluid from osteoarthritic joints, hydroxyproline and GAG concentrations were not increased, compared with unaffected joints, but hydroxyproline were significantly correlated with the CDI and general MMP activity. There was no significant correlation between GAG concentration and CDI value or MMP activity. CONCLUSIONS AND CLINICAL RELEVANCE: Changes in hydroxyproline concentration in synovial fluid appeared to indicate damage to collagen of the articular cartilage. In joints with osteoarthritis, the lack of high GAG concentration in synovial fluid and the absence of a significant correlation between GAG concentration and CDI values or MMP activity may severely limit the usefulness of this marker for monitoring equine joint disease.     

Sandwich ELISA system for cartilage oligomeric matrix protein in equine synovial fluid and serum

Reasons for performing study: Measurement of cartilage oligomeric matrix protein (COMP) in serum has potential for diagnosis of equine osteoarthritis (OA), but clinical use is currently limited by the lack of specificity of an inhibition ELISA as well as by baseline increases due to exercise. Improved methods for ELISA with increased antigen specificity and sensitivity are therefore required for reliable measurement. Hypothesis: Measurement of the serum level of COMP by sandwich ELISA allows identification of horses with OA. Methods: New monoclonal antibodies (mAbs) were elicited against equine cartilage COMP, their epitopes were determined and a sandwich ELISA was developed. The concentrations of COMP in synovial fluid (SF; n = 100) and sera (n = 100) from OA cases were measured by sandwich ELISA as well as by inhibition ELISA and compared with concentrations in normal joints (n = 95) and horses (n = 50). Results: Immunoblots of enzymatically cleaved COMP showed that the new mAbs recognised different epitopes located on a 20 kDa fragment between K⁶³ and K²³⁸ of the EGF‐like repeats. Inhibition ELISA with any mAb detected significantly increased levels of COMP in OA SF compared with normal SF, whereas no significant difference was detected between serum levels of COMP in OA and normal horses. Conversely, sandwich ELISA with the combination of unlabelled 2A11 × biotinylated 11F10 mAbs detected a significant increase in COMP levels in both serum and SF from OA cases compared with levels in normal animals. Conclusions and potential relevance: Measurement of serum COMP with sandwich ELISA may be useful in identifying horses with OA.     

Use of polysulphated glycosaminoglycan in equine lameness

Four cases of equine lameness were treated with intra-articular polysulphated glycosaminoglycan. Two, two-year-old flat racers were treated conservatively after traumatic injury. They subsequently became lame owing to the formation of new bone and osteophytes. Two older steeplechasers were lame owing to degenerative joint disease. The four horses were treated with intra-articular injections of polysulphated glycosaminoglycans and, after three or four injections, they became clinically sound and were able to continue racing with varying degrees of success.