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Evaluation of hyaluronidase activity in equine and bovine sera and equine synovial fluid samples by use of enzyme zymography

Jennifer M. WilliamsDepartments of Anatomy, Physiology, and Cell Biology, School of Veterinary Medicine, University of California, Davis, CA 95616.

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Jessica M. StokesDepartments of Anatomy, Physiology, and Cell Biology, School of Veterinary Medicine, University of California, Davis, CA 95616.

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Melinda H. MacDonaldDepartment of Surgical and Radiological Sciences, and Cell Biology, School of Veterinary Medicine, University of California, Davis, CA 95616.

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Hilary P. BentonDepartments of Anatomy, Physiology, and Cell Biology, School of Veterinary Medicine, University of California, Davis, CA 95616.

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Abstract

Objective—To investigate the activities of hyaluronidases in equine sera and synovial fluid samples and sera from fetal and adult bovids and evaluate the extent to which the degradation of hyaluronan is influenced by chondrocytes.

Sample Population—Commercial and noncommercial samples of equine (n = 6) and bovine (6) sera and 16 synovial fluid samples from horses.

Procedure—Hyaluronidase activities in sera and synovial fluid samples were assessed via enzyme zymography (performed at pH 4, 5, 6, or 7). Chondrocytes were isolated from equine cartilage and cultured with or without hyaluronan (1 mg/mL); the degradation of hyaluronan was assessed via agarose gel electrophoresis.

Results—Hyaluronidase activity was detected in equine sera and synovial fluid samples at pH 4, but not at pH 7, and in bovine sera at both pH values. In all samples at pH 4, a major band of activity (molecular weight, approx 60 kd) and some additional higher molecular weight bands were detected; high- and low-molecular-weight activities were detected in bovine sera at pH 7. Hyaluronan in tissue culture medium with or without fetal calf serum was degraded in the presence, but not the absence, of equine chondrocytes.

Conclusions and Clinical Relevance—Hyaluronidase activity was detected in equine sera and synovial fluid at pH 4 and in bovine sera at pH 4 and 7. Primary chondrocytes in monolayer culture can degrade exogenous hyaluronan. Modulating native hyaluronidase activity may offer a new approach to improve the quantity and quality of hyaluronan in articular joints. ( Am J Vet Res 2005;66:984–990)

Abstract

Objective—To investigate the activities of hyaluronidases in equine sera and synovial fluid samples and sera from fetal and adult bovids and evaluate the extent to which the degradation of hyaluronan is influenced by chondrocytes.

Sample Population—Commercial and noncommercial samples of equine (n = 6) and bovine (6) sera and 16 synovial fluid samples from horses.

Procedure—Hyaluronidase activities in sera and synovial fluid samples were assessed via enzyme zymography (performed at pH 4, 5, 6, or 7). Chondrocytes were isolated from equine cartilage and cultured with or without hyaluronan (1 mg/mL); the degradation of hyaluronan was assessed via agarose gel electrophoresis.

Results—Hyaluronidase activity was detected in equine sera and synovial fluid samples at pH 4, but not at pH 7, and in bovine sera at both pH values. In all samples at pH 4, a major band of activity (molecular weight, approx 60 kd) and some additional higher molecular weight bands were detected; high- and low-molecular-weight activities were detected in bovine sera at pH 7. Hyaluronan in tissue culture medium with or without fetal calf serum was degraded in the presence, but not the absence, of equine chondrocytes.

Conclusions and Clinical Relevance—Hyaluronidase activity was detected in equine sera and synovial fluid at pH 4 and in bovine sera at pH 4 and 7. Primary chondrocytes in monolayer culture can degrade exogenous hyaluronan. Modulating native hyaluronidase activity may offer a new approach to improve the quantity and quality of hyaluronan in articular joints. ( Am J Vet Res 2005;66:984–990)