A lipidomic study of sphingolipid species in human synovial fluid

s / Osteoarthritis and Cartilage 22 (2014) S57–S489 S425 donors. Many of these proteins are cartilage specific proteoglycans such as hyaluronan and proteoglycan link protein 1, aggrecan core protein or lumican as well as some proteins with a well-known role in the pathogenesis of OA like COMP or MMP3. On the other hand, 21 proteins exhibited a significantly reduced abundance in OA patients when compared to controls. Interestingly, we detected several proteins which belong to the tenascin protein family, like tenascin-X, which accelerates collagen fibril formation. We also found WISP2 decreased at day 14, suggesting a lower activity of the Wnt signaling pathway in OA cells. Conclusions: The identification and quantification of these secreted proteins enhance our knowledge on the extracellular regulation of the chondrogenesis and allow the identification of extracellular markers of this process. Moreover, the lower expression of some of them in OA patients (like tenascin-X or WISP2), suggest their putative usefulness for the molecular monitorization of the chondrogenesis in cell therapybased approaches for cartilage repair. 766 A LIPIDOMIC STUDY OF SPHINGOLIPID SPECIES IN HUMAN SYNOVIAL FLUID M.K. Kosinska y, G. Liebisch z, G. Lochnit y, J. Wilhelm y, H. Klein y, U. Kaesser x, G. Lasczkowski y, M. Rickert y, G. Schmitz z, J. Steinmeyer y. yUniv. of Giessen, Giessen, Germany; zUniv. Hosp. Regensburg, Regensburg, Germany; x Internistisches Praxiszentrum am Krankenhaus Balserische Stiftung, Giessen, Germany Purpose: Articular synovial fluid (SF) is a complex mixture of components either derived from plasma or locally synthesized by synovial tissues, which are involved in nutrition, communication, shock absorption, and lubrication. Alterations in its composition can be associated with increased friction, leading to articular cartilage damage, or with changes in inflammatory status of synovial joints. We already quantified 130 phospholipid species in human osteoarthritis (OA) and rheumatoid arthritis (RA) SF. The presented lipidomic investigation aims to quantify for the first time, the composition of sphingolipids [sphingomyelins (SM), ceramides (Cer), hexosylceramides (HexCer) and dihexosylceramides (Hex2Cer) species] in the SF of knee joints from unaffected controls, patients with early (eOA) and late (lOA) stages of OA and from RA patients, and to further elucidate whether the concentrations of individual lipid species are associated with the stage of OA disease. Methods: According to predefined inclusion and exclusion criteria patients were included in the study. The use of human SF for this study was approved by the local ethics committee of our university, and all participants provided written informed consent. Lipids were extracted from SF without cells and cellular debris from 9 postmortem donors (control), 18 RA, and 30 OA patients. OA group was subcategorized as early (n 1⁄4 17) and late (n 1⁄4 13) using Outerbridge score. RA patients were classified according to the criteria of the American College of Rheumatology. Lipids were quantified using electrospray ionization tandem mass spectrometry directly or coupled with hydrophilic interaction liquid chromatography. The quantitative values of all lipid species were corrected by the method described by Kraus et al. (2002). The Kruskal-Wallis test adjusted with a false discovery rate (FDR) of 10% and the Wilcoxon rank sum test were applied to determine statistically significant differences. P values < 0.05 were considered to be statistically significant. Results: We provide a novel, detailed overview of sphingolipid species in human SF. The analytical set-up used in the present study allowed us to quantitate 35 different sphingolipid species. We identified 19 SM species, based on the length and saturation of the attached fatty acids. SM 34:1 was the predominant species among SMs. In addition, versus control SF, all species were elevated in eOA, lOA and RA SF. Remarkably, all SM species rose approximately 2-fold in SF from eOA to lOA and approximately 1.5-fold in SF from eOA to RA. However, we found no significant different levels of SM species in lOA compared with RA SF. Also, 6 Cer, 5 HexCer and 5 Hex2Cer were identified in human SF. Cer d18:0/24 was the predominant species. Moreover, most Cer species contained saturated fatty acids in all cohorts. Compared to control SF, the levels of most Cer species were 4.5-fold higher in lOA and 5.8-fold in RA SF. Moreover, all Cer species rose approximately 1.7-fold in SF from eOA to RA, and nearly 1.3-fold in SF from lOA to RA. Conclusions: This lipidomic investigation present for the first time a detailed overview of sphingolipid species in human SF. Compared to control SF the concentrations of sphingolipid species such as SM and Cer were found to be elevated in eOA, lOA and RA SF. Since, disease and stage-dependent differences in the composition and concentrations of lipid species might result not only in an impaired lubrication but also in an altered inflammatory status of joints, sphingolipids appear to be associated with the pathogenesis of OA and RA. Our study lays the foundation for addressing specific questions regarding the biosynthesis and function of sphingolipid species in SF. 767 IDENTIFICATION OF AUTOANTIBODIES IN SERUM FROM OSTEOARTHRITIS PATIENTS USING MICROARRAYS L. Lourido y, J. Fernandez-Tajes y, V. Calamia y, C. Fernandez y, B. Rocha y, P. Fern andez-Puente y, J. Mateos y, M. Fuentes z, F.J. Blanco y, C. Ruiz-Romero y. yRheumatology Div., Proteomics Group-ProteoRed/ ISCIII, INIBIC-CHUAC, A Coru~ na, Spain; zCentro de Investigaci on del C ancer/IBMCC (USAL/CIC), IBSAL, Departamento de Med., Unidad de Prote omica Servicio Gen. de Citometria, Universidad de Salamanca,