A simplified method of determining synovial fluid chondroitin sulfate chain length

SummaryObjectiveTo determine whether dimethylmethylene blue (DMMB) analysis, when combined with agarose gel filtration chromatography (Superose 6), can be performed instead of fluorophore-assisted carbohydrate electrophoresis (FACE) to determine chondroitin sulfate (CS) chain length in synovial fluid (SF).MethodsSF was obtained from (1) normal horses after 8 weeks of rest, (2) the same horses after 9 months of treadmill training, and (3) horses with osteochondral (OC) injury from racing. SF CS concentrations and chain lengths were determined by gel chromatography and DMMB analysis and compared with previous results determined by FACE analysis on the same samples.ResultsDMMB analysis showed that SF CS peak chain length in the OC injury group increased significantly (18.7kDa) when compared to rested and exercised normal horses (15.6kDa). The assay had a positive predictive value of 71% and a negative predictive value of 75% for discriminating between normal and injured joints.ConclusionsWe report a simple and inexpensive DMMB analysis of SF CS chain length, which, when coupled with Superose 6 chromatography, discriminates between normal and post-injury joints. Similar to our previous FACE analysis results [Brown MP, Trumble TN, Plaas AHK, Sandy JD, Romano M, Hernandez J, et-al. Exercise and injury increase chondroitin sulfate chain length and decrease hyaluronan chain length in synovial fluid. Osteoarthritis Cartilage 2007;15], our DMMB results show an increase in the chain length of the CS in the SF of injured joints