Equine synovial fluid protein equalization via combinatorial peptide ligand libraries

To gain further knowledge of the equine synovial fluid (SF) proteome, we propose a protocol based on the equalization of the relative concentrations of its proteins, which leads to the modification of the standard electrophoretic pattern revealing low-abundance proteins that otherwise remain undetected. Fresh SF samples were collected from ten macroscopically normal metacarpophalangeal joints of crossbred horses. The samples were processed using standard procedures as the control and via combinatorial peptide ligand libraries (CPLL) using low ionic forces (NaH2PO4 10 mM) at different pHs (4.0, 7.0, and 9.3) with 10% sodium dodecyl sulfate (SDS) and 25 mM DTT for protein resolubilization. Proteins were then separated by conventional 8% SDS-PAGE and stained with coomassie blue. After separation of the equalized proteins, there was a significant reduction in the albumin (the most abundant protein in the SF) and, at the same time, other protein bands arise that were not visible without CPLL processing. In addition, there was variation in the protein profiles at different pHs. The results suggest that protein equalization of the equine SF by CPLL could be a useful tool to better understand the articular homeostasis and/or for the detection of new biomarkers of joint pathology.

Saline-Assisted Aspirations for Collecting Synovial Fluid From Noneffused Knees: Technique and Validation

Context: To better understand why a knee develops osteoarthritis after joint trauma we need to assess the local biochemical changes. Unfortunately, it is challenging to obtain synovial fluid from a knee with no effusion. Objective: To describe the authors' protocol for aspirating synovial fluid from noneffused knees. Second, they demonstrate the validity of this method by evaluating the relationships between normalized and raw biomarker concentrations among knees with effusion (undergoing a traditional aspiration) and without effusion (requiring a saline-assisted aspiration). Design: Validation study based on secondary analyses from 2 cohort studies. Setting: Outpatient orthopedic clinic and basic-science laboratory. Participants: Participants had moderate to severe radiographic knee osteoarthritis (n = 15 with and 11 without effusion) and no osteoarthritis or effusion (n = 4). Interventions: The same orthopedic surgeon performed all synovial-fluid joint aspirations, including saline-assisted aspirations. Main Outcome Measures: The authors used multiplex enzyme-linked immunosorbent assays to determine 7 synovial-fluid biomarker concentrations. They then calculated correlations between raw and normalized (to total synovial-fluid protein content) biomarker concentrations. Results: The authors excluded 1 sample collected with a saline-assisted aspiration because it contained blood. Normalized biomarker concentrations had positive associations with raw biomarker concentrations (r = .77-99), with the exception of interleukin-13 and interleukin-1Β among knees that underwent a saline-assisted aspiration. Excluding interleukin-1Β, associations between normalized and raw biomarker concentrations were consistent between knees that had a saline-assisted or traditional aspiration. Conclusions:Saline-assisted aspiration is a valid technique for assessing the local biochemical changes in knees without effusion.