SUPEROXIDE AND BONE-RESORPTION

The reaction of superoxide with nitroblue tetrazolium produces an elec tron-dense diformazan precipitate which can be used to localize areas of superoxide production. Transmission electron microscopy was used to demonstrate that diformazan granules formed by the reaction of nitrob lue tetrazolium with excess superoxide are electron dense, whereas mon oformazan granules generated by hydrogen peroxide were not. On the bas is of these observations, superoxide formed along the osteoclast-bone interface was localized by demonstrating the electron-dense diformazan granules between the osteoclastic membrane and the bone surface. The formation of this reaction product was inhibited by a superoxide scave nger, the deferoxamine mesylate-manganese complex (the ''green'' compl ex), confirming the specificity of the reaction product. The scavenger also inhibited bone resorption. High concentrations of superoxide gen erated in vitro at a neutral pH degraded osteocalcin into numerous pep tide fragments, demonstrating the ability of superoxide to break pepti de bonds. These studies localize superoxide production to the ruffled border space and suggest that superoxide generated at the osteoclast-b one interface is involved in bone matrix degradation.