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To investigate alterations of the redox balance in osteoarthritic cartilage and synovial tissue, we prepared, purified, and quantified the redox buffers present in synovial fluid and cartilage tissue. We further compared the effects of redox buffers to other well-known inhibitors of articular cartilage degeneration. We developed a purification protocol that was based on the presence of copper ions within the oxidized redox buffer, as copper is known to be involved in redox signaling of the chondrocyte. Metal-free buffers contained mainly the reducing agent ascorbic acid, ascorbic acid reduced by glutathione, and the oxidized and reduced forms of glutathione. Hemoglobin was the only notable exception to the ascorbate-glutathione redox buffer system, possibly because it has a higher reducing power than ascorbate. The concentrations of ascorbic acid and ascorbate in synovial fluid were significantly higher than in plasma, but comparable with the concentrations in chondrocyte culture medium. Cartilage tissue extract showed a decreased concentration of reducing agents. In contrast, synovial tissue extract had significantly elevated concentrations of most of these reducing agents. The oxidized and reduced forms of glutathione together contributed over 95% of total glutathione in synovial fluid and cartilage extract. The amounts of oxidized and reduced glutathione in cartilage extracts decreased with increasing severity of osteoarthritis. These data demonstrate a higher concentration of reducing agents within synovial fluid and cartilage matrix as compared to plasma, and a decreasing concentration of reducing agents in chondro