NADPH Oxidase of Neutrophils Elevates o,o′-Dityrosine Cross-Links in Proteins and Urine during Inflammation

Reactive intermediates generated by phagocytic white blood cells are of central importance in destroying microorganisms, but they may also damage normal tissue at sites of inflammation. To investigate the potential role of such oxidants in tissue injury, we used gas chromatography/mass spectrometry to quantify levels of o,o′-dityrosine in mouse peritoneal neutrophils and urine. In wild-type animals, neutrophils markedly increased their content of protein-bound dityrosine when they were activated in vivo. This increase failed to occur in mice that were deficient in the phagocyte NADPH oxidase. Levels of o,o′-dityrosine in urine mirrored those in neutrophil proteins. When o,o′-[14C]dityrosine was injected intravenously into mice, the radiolabel was not metabolized or incorporated into tissue proteins: instead, it was recovered in urine with near-quantitative yield. Patients with sepsis markedly increased their output of o,o′-dityrosine into urine, suggesting that systemic inflammation also may be a potent source of oxidative stress in humans. These observations demonstrate that activated neutrophils produce o,o′-dityrosine cross-links in tissue proteins, which may subsequently be degraded into free amino acids and excreted into urine. Our results indicate that mouse phagocytes use oxidants produced by the NADPH oxidase to create o,o′-dityrosine cross-links in vivo and raise the possibility that reactive intermediates produced by this pathway promote inflammatory tissue damage in humans.