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

Stacey L Oke DVM, MSc1,2, Mark B. Hurtig DVM, MVSc3, Robert A. Keates PhD4, Jen R. Wright BSc5, and John H. Lumsden DVM, MSc6
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  • 1 Department of Clinical Studies, University of Guelph, Guelph, ON, Canada N1G 2W1.
  • | 2 Present address is 20-7 Forest Hill Dr, Guelph, ON, Canada N1G 2E1.
  • | 3 Department of Clinical Studies, University of Guelph, Guelph, ON, Canada N1G 2W1.
  • | 4 Department of Chemistry and Biochemistry, College of Physical and Engineering Science, University of Guelph, Guelph, ON, Canada N1G 2W1.
  • | 5 Department of Clinical Studies, University of Guelph, Guelph, ON, Canada N1G 2W1.
  • | 6 Department of Pathobiology, University of Guelph, Guelph, ON, Canada N1G 2W1.

Abstract

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 SF. 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. (Am J Vet Res 2003;64:900–906)

Abstract

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 SF. 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. (Am J Vet Res 2003;64:900–906)