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  • Author or Editor: Stacey L. Oke x
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in Journal of the American Veterinary Medical Association

Abstract

Objective—To develop a new 1,9-dimethylmethylene blue (DMMB) assay for measurement of sulfated glycosaminoglycan (sGAG) concentrations in equine synovial fluid (SF) by use of membrane technology and to compare the assay's ability to measure sGAG concentrations with that of 2 other established DMMB assays.

Sample Population—25 samples of SF collected from affected joints of 14 horses and 13 samples of SF collected from nonaffected (control) joints of 4 horses.

Procedure—A solid-phase DMMB assay was developed to measure sGAG concentrations in SF. Results for the assay were then compared with results obtained by use of the direct spectrophotometric method (ie, Farndale method) and microplate DMMB assay.

Results—The solid-phase assay and direct spectrophotometric assay measured the same sGAG concentrations in identical equine SF, but those concentrations differed significantly from results obtained by use of the microplate DMMB assay. All other aspects of the solidphase DMMB assay were comparable to both the direct spectrophotometric and microplate DMMB assays.

Conclusions and Clinical Relevance—The new solid-phase assay can be used interchangeably with the direct spectrophotometric method to measure sGAG concentrations in equine SF samples, but it cannot be interchanged with the microplate DMMB assay. Results can be rapidly obtained with the solidphase assay. Also, the solid-phase assay can detect nanogram quantities of sGAGs in SF, circumvent the problem of premature precipitation of sGAG-dye complexes, and provide quantitative or qualitative results. The solid-phase assay may replace other DMMB assays for measuring sGAG concentrations in SF obtained from horses. ( Am J Vet Res 2003; 64:894–899)

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in American Journal of Veterinary Research

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)

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in American Journal of Veterinary Research