Objective. Membrane phospholipid species contribute to boundary lubrication that is provided by synovial fluid (SF). Altered levels of lubricants can be associated with increased friction, leading to articular cartilage damage. This study was undertaken to determine whether the composition of phospholipid species is altered in diseases of human knee joints.Methods. The study was performed using SF from unaffected controls and patients with early osteoarthritis (OA), late OA, or rheumatoid arthritis (RA). Lipids were extracted from cell-and vesicle-free SF from 9 control donors postmortem and from 17 patients with early OA, 13 patients with late OA, and 18 patients with RA. Phospholipid species were quantified by electrospray ionization tandem mass spectrometry.Results. We conducted lipidomic analysis to provide the first detailed overview of phospholipid species in human SF. We identified 130 lipid species belonging to 8 lipid classes (phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine, plasmalogens, phosphatidylserine, phosphatidylglycerol, sphingomyelin, and ceramide). Compared to SF from controls, SF from patients with early OA and those with late OA had higher levels of most phospholipid species. Moreover, the concentrations of 64 and 27 phospholipids differed between RA and early OA SF and between RA and late OA SF, respectively. Also, the levels of 66 phospholipid species were altered in early OA versus late OA.Conclusion. Our data indicate disease-and stagedependent differences in the relative composition and levels of phospholipid species in human SF. Such alterations might affect articular joint lubrication. Because certain phospholipids scavenge reactive oxygen species (ROS) and are pro-or antiinflammatory, any altered phospholipid level might influence ROS-scavenging activity of SF and the inflammatory status of joints. Thus, phospholipids may be associated with the pathogenesis of OA.

BackgroundHyaluronic acid (HA), lubricin, and phospholipid species (PLs) contribute independently or together to the boundary lubrication of articular joints that is provided by synovial fluid (SF). Our study is the first reporting quantitative data about the molecular weight (MW) forms of HA, lubricin, and PLs in SF from cohorts of healthy donors, patients with early (eOA)- or late (lOA)-stage osteoarthritis (OA), and patients with active rheumatoid arthritis (RA).MethodsWe used human SF from unaffected controls, eOA, lOA, and RA. HA and lubricin levels were measured by enzyme-linked immunosorbent assay. PLs was quantified by electrospray ionization tandem mass spectrometry. Fatty acids (FAs) were analyzed by gas chromatography, coupled with mass spectrometry. The MW distribution of HA was determined by agarose gel electrophoresis.ResultsCompared with control SF, the concentrations of HA and lubricin were lower in OA and RA SF, whereas those of PLs were higher in OA and RA SF. Moreover, the MW distribution of HA shifted toward the lower ranges in OA and RA SF. We noted distinct alterations between cohorts in the relative distribution of PLs and the degree of FA saturation and chain lengths of FAs.ConclusionsThe levels, composition, and MW distribution of all currently known lubricants in SF—HA, lubricin, PLs—vary with joint disease and stage of OA. Our study is the first delivering a comprehensive view about all joint lubricants during health and widespread joint diseases. Thus, we provide the framework to develop new optimal compounded lubricants to reduce joint destruction.

Articular synovial fluid (SF) is a complex mixture of components that regulate nutrition, communication, shock absorption, and lubrication. Alterations in its composition can be pathogenic. This lipidomic investigation aims to quantify the composition of sphingolipids (sphingomyelins, ceramides, and hexosyl- and dihexosylceramides) and minor glycerophospholipid species, including (lyso)phosphatidic acid, (lyso)phosphatidylglycerol, and bis(monoacylglycero)phosphate species, in the SF of knee joints from unaffected controls and from patients with early (eOA) and late (lOA) stages of osteoarthritis (OA), and rheumatoid arthritis (RA).SF without cells and cellular debris from 9 postmortem donors (control), 18 RA, 17 eOA, and 13 lOA patients were extracted to measure lipid species using electrospray ionization tandem mass spectrometry - directly or coupled with hydrophilic interaction liquid chromatography.We provide a novel, detailed overview of sphingolipid and minor glycerophospholipid species in human SF. A total of 41, 48, and 50 lipid species were significantly increased in eOA, lOA, and RA SF, respectively when compared with normal SF.The level of 21 lipid species differed in eOA SF versus SF from lOA, an observation that can be used to develop biomarkers. Sphingolipids can alter synovial inflammation and the repair responses of damaged joints. Thus, our lipidomic study provides the foundation for studying the biosynthesis and function of lipid species in health and most prevalent joint diseases.

IntroductionLysosomal cathepsins have been reported to contribute to Osteoarthritis (OA) pathophysiology due to their increase in pro-inflammatory conditions. Given the causal role of cathepsins in OA, monitoring their specific activity could provide means for assessing OA severity. To this end, we herein sought to assess a cathepsin activity-based probe (ABP), GB123, in vitro and in vivo.MethodsProtein levels and activity of cathepsins B and S were monitored by immunoblot analysis and GB123 labeling in cultured primary chondrocytes and conditioned media, following stimuli with tumor necrosis factor alpha (TNFα) and/or Interleukin 1 beta (IL-1β). Similarly, cathepsin activity was examined in sections of intact cartilage (IC) and degraded cartilage (DC) regions of OA. Finally, synovial fluid (SF) and serum from donors with no signs of diseases, early OA, late OA and rheumatoid arthritis (RA) patients were analyzed with GB123 to detect distinct activity levels of cathepsin B and S.ResultsCathepsin activity in cell lysates, conditioned media explants and DC sections showed enhanced enzymatic activity of cathepsins B and S. Further histological analysis revealed that cathepsin activity was found higher in superficial zones of DC than in IC. Examining serum and SF revealed that cathepsin B is significantly elevated with OA severity in serum and SF, yet levels of cathepsin S are more correlated with synovitis and RA.ConclusionsBased on our data, cathepsin activity monitored by ABPs correlated well with OA severity and joint inflammation, directing towards a novel etiological target for OA, which possesses significant translational potential in developing means for non-invasive detection of early signs of OA.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-015-0586-5) contains supplementary material, which is available to authorized users.

Our lipidomic analysis demonstrates that SF in the canine OA model closely mimics the early osteoarthritic changes that occur in humans. Further, the canine SF lipidome often reflects normal human lipid metabolism.