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'-[(14)C]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.

Increased plasma gln and Leu Ra and inappropriately low muscle protein synthesis rate in AIDS wasting.

Muscle protein wasting occurs in human immunodeficiency virus (HIV)-infected individuals and is often the initial indication of acquired immunodeficiency syndrome (AIDS). Little is known about the alterations in muscle protein metabolism that occur with HIV infection. Nine subjects with AIDS wasting (CD4 < 200/mm3), chronic stable opportunistic infections (OI), and >/=10% weight loss, fourteen HIV-infected men and one woman (CD4 > 200/mm3) without wasting or OI (asymptomatic), and six HIV-seronegative lean men (control) received a constant intravenous infusion of [1-13C]leucine (Leu) and [2-15N]glutamine (Gln). Plasma Leu and Gln rate of appearance (Ra), whole body Leu turnover, disposal and oxidation rates, and [13C]Leu incorporation rate into mixed muscle protein were assessed. Total body muscle mass/fat-free mass was greater in controls (53%) than in AIDS wasting (43%; P = 0.04). Fasting whole body proteolysis and synthesis rates were increased above control in the HIV+ asymptomatic group and in the AIDS-wasting group (P = 0. 009). Whole body Leu oxidation rate was greater in the HIV+ asymptomatic group than in the control and AIDS-wasting groups (P < 0.05). Fasting mixed muscle protein synthesis rate was increased in the asymptomatic subjects (0.048%/h; P = 0.01) but was similar in AIDS-wasting and control subjects (0.035 vs. 0.037%/h). Plasma Gln Ra was increased in AIDS-wasting subjects but was similar in control and HIV+ asymptomatic subjects (P < 0.001). These findings suggest that AIDS wasting results from 1) a preferential reduction in muscle protein, 2) a failure to sustain an elevated rate of mixed muscle protein synthesis while whole body protein synthesis is increased, and 3) a significant increase in Gln release into the circulation, probably from muscle. Several interesting explanations for the increased Gln Ra in AIDS wasting exist.