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 cen tral importance in destroying microorganisms, but they may also damage norm al tissue at sites of inflammation. To investigate the potential role of su ch 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 protein s. When o,o '-[C-14]dityrosine was injected intravenously into mice, the ra diolabel was not metabolized or incorporated into tissue proteins: instead, it was recovered in urine with near-quantitative yield. Patients with seps is markedly increased their output of o,o ' -dityrosine into urine, suggest ing that systemic inflammation also may be a potent source of oxidative str ess in humans. These observations demonstrate that activated neutrophils pr oduce o,o ' -dityrosine cross-links in tissue proteins, which may subsequen tly be degraded into free amino acids and excreted into urine. Our results indicate that mouse phagocytes use oxidants produced by the NADPH oxidase t o create o,o ' -dityrosine cross-links in vivo and raise the possibility th at reactive intermediates produced by this pathway promote inflammatory tis sue damage in humans. (C) 2001 Academic Press.