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

This research focuses on comparing three variations of the 1,9-dimethylmethylene blue (DMMB) assay and how they perform in measuring sulfated glycosaminoglycan (sGAG) concentrations in equine synovial fluid. The study shows that all three DMMB assays react similarly, but produce significantly different measures of sGAG, suggesting the need for standardization in clinical comparisons.

Methods

• The researchers studied 25 samples of synovial fluid (SF) collected from the affected joints of 13 horses, and 13 samples from the non-affected joints of 4 horses. They used these samples to test the performance of three variations of the DMMB assay.
• sGAG concentrations were measured using the direct spectrophotometric (i.e., Farndale), microplate, and indirect DMMB assays.
• The researchers treated some of the SF samples with substances like nucleases, papain, and hyaluronidase. This was done to gauge the response of the assays to standard solutions of sGAGs, and to these digestion agents.

Results

• The results of the study showed that all three DMMB assays behaved similarly to sGAG standard solutions and to SF samples that were digested with nucleases, papain, and hyaluronidase.
• The study demonstrated that nucleic acids were not a significant factor of interference, and that papain and hyaluronidase could not be used interchangeably for digesting SF.
• While each DMMB assay demonstrated satisfactory precision (with a standard deviation of less than 10%), they each resulted in significantly different measurements of sGAG concentrations in the equine SF.

Conclusions

• The study concludes that to measure sGAG concentrations in SF using the DMMB assay, SF samples should first be digested with either papain or hyaluronidase.
• The study also highlights that the lack of uniformity between the DMMB assay variations makes it impossible to compare clinical data when different versions of the DMMB assay are used, since they yield distinctly different sGAG concentrations in SF.
• From the three assays examined, the study recommends the use of the direct spectrophotometric DMMB assay for the most accurate results.