A NEW SERUM BIOCHEMICAL MARKER SPECIFIC OF OXIDATIVE-INDUCED SYNOVIAL TISSUE METABOLISM IN ARTHRITIS: NITROSYLATED N-TELOPEPTIDE OF TYPE III COLLAGEN (IIINYS)

Background: Nitric oxide (NO) is an important mediator of joint inflammation and destruction in rheumatoid arthritis (RA) and osteoarthritis (OA). Peroxynitrite induced by NO can react with amino acids including tyrosine (Y) to form nitrotyrosine. Increased NO-related species have been found in the joint of patients with RA and OA, especially in synovial tissue.Objectives: The aim of this study was to develop a new immunoassay recognizing nitrosylated N-telopeptide fragment of type III collagen –a major constituent of synovial membrane- to monitor oxidative-related joint damage in arthritis.Methods: We produced a rabbit polyclonal antibody raised against a nitrosylated sequence specific of the N-telopeptide of human type III collagen (IIINys). We developed a competitive ELISA to measure IIINys levels in the synovial fluid, serum and urine of healthy controls and patients with RA or OA. Using IIINys antibody, immunohistochemistry was performed on synovial tissues from 12 patients with knee OA undergoing total joint replacement.Results: No recognition of IIINys antibody was observed with the non-nitrosylated sequence of type III collagen N-telopeptide, nitrosylated BSA and free nitrotyrosine, indicating a high specificity of the antibody for both nitrosylation and type III collagen sequence. The ELISA, developed for serum measurements, demonstrated intra and inter-assay variability below 15% and recovery of diluted serum samples ranged from 96.6 to 118.3% (mean: 99%). Detectable levels of IIINys were also measured in the synovial fluid, serum and urine of 12 patients with knee OA. Compared to 30 healthy postmenopausal women, serum IIINys levels were increased by an average of 195% (p<0.0001) in 30 postmenopausal women with early RA. Immunohistochemistry of synovial tissue from patients with knee OA, showed strong IIINys staining in the extracellular matrix, particularly around the synoviocytes and within macrophage-like cells.Conclusion: We have developed an immunoassay which detects specifically nytrosylated type III collagen N-telopeptide (IIINys). The strong immunoreativity of IIINys in the synovial tissue of patients with OA and the marked increased serum IIINys levels in patients with RA, suggest that this new biochemical marker should be useful for the investigation of oxidative-induced alterations of synovial tissue in patients with RA or OA.Citation: Ann Rheum Dis, volume 66, supplement II, year 2007, page 280Session: Cartilage and synovial-like fibroblasts