3-nitrotyrosine and Shock--Septic

A new proteomics technique for analyzing 3-nitrotyrosine-containing peptides is presented here. This technique is based on the combined fractional diagonal chromatography peptide isolation procedures by which specific classes of peptides are isolated following a series of identical reverse-phase HPLC separation steps. Here dithionite is used to reduce 3-nitrotyrosine to 3-aminotyrosine peptides, which thereby become more hydrophilic. Our combined fractional diagonal chromatography technique was first applied to characterize tyrosine nitration in tetranitromethane-modified BSA and further led to a high quality list of 335 tyrosine nitration sites in 267 proteins in a peroxynitrite-treated lysate of human Jurkat cells. We then analyzed a serum sample of a C57BL6/J mouse in which septic shock was induced by intravenous Salmonella infection and identified six in vivo nitration events in four serum proteins, thereby illustrating that our technique is sufficiently sensitive to identify rare in vivo tyrosine nitration sites in a very complex background.

Topics: Amino Acid Sequence; Animals; Blood Proteins; Cattle; Cell Extracts; Chromatography, High Pressure Liquid; Chromatography, Reverse-Phase; Disease Models, Animal; Humans; Jurkat Cells; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Oxidation-Reduction; Peptides; Proteome; Proteomics; Salmonella; Serum Albumin, Bovine; Shock, Septic; Tetranitromethane; Thiosulfates; Tyrosine

To investigate ceftazidime in acute lung injury (ALI) and sepsis.. Prospective, randomized, controlled animal study in an investigational ICU at a university hospital.. Eighteen female Merino sheep were prepared for chronic study and subjected to smoke inhalation and septic challenge according to an established protocol.. Whereas global hemodynamics and oxygenation remained stable in sham animals (no injury, no treatment), the injury contributed to a hypotensive-hyperdynamic circulation in the control group (smoke inhalation and sepsis, no treatment), as indicated by a significant increase in cardiac index) and heart rate and a drop in mean arterial pressure. Treatment with ceftazidime (smoke inhalation and sepsis, treatment group) stabilized cardiac index and heart rate and attenuated the decrease in mean arterial pressure. The deterioration in PaO2/FiO2 ratio and pulmonary shunt fraction (Qs/Qt) was significantly delayed and blunted by ceftazidime. At 24 h after injury a significant increase in airway obstruction scores of bronchi and bronchioles in both injured groups was observed. Ceftazidime significantly reduced airway obstruction vs. control animals. Whereas plasma nitrate/nitrite levels increased similarly in the two injured groups, lung 3-nitrotyrosine content remained at the baseline level in the ceftazidime group.. In ovine lung injury ceftazidime improves global hemodynamics and oxygenation not only by bacterial clearance but also via reduction in toxic nitrogen species such as 3-nitrotyrosine. Therefore ceftazidime appears as a clinically relevant adjunct in the common setting of sepsis-associated lung injury.

Topics: Animals; Anti-Bacterial Agents; Blood Pressure; Ceftazidime; Disease Models, Animal; Female; Heart Rate; Hemodynamics; Lung; Nitrates; Nitrites; Oxygen Consumption; Random Allocation; Sheep; Shock, Septic; Smoke Inhalation Injury; Tyrosine

The purpose of this study was to examine whether selective iNOS inhibition can restore the hemodynamic changes and reduce the nitrotyrosine levels in the cerebral cortex of rats with streptozotocin-induced diabetes during endotoxin-induced shock. The study was designed to include three sets of experiments: (1) measurement of changes in systemic hemodynamics, (2) measurement of biochemical variables, including iNOS activity and nitrotyrosine formation in the brain, and (3) assessment of mortality rate. Rats were randomly divided into four groups: group 1, control; group 2, LPS: Escherichia coli endotoxin, 10.0 mg/kg (i.v.) bolus; group 3 (i.v.) LPS and L-N6-(1-iminoethyl)-lysine (L-NIL), 4mg/kg (i.p.); and group 4, LPS and NG-nitro-L-arginine methyl ester (L-NAME), 5 mg/kg (i.p.). In nondiabetic rats, administration of L-NIL prevented the hemodynamic and biochemical changes, and increases in plasma nitrite and cerebral nitrotyrosine levels induced by LPS. Administration of L-NAME partially prevented these LPS-induced changes. On the other hand, in diabetic rats, administration of L-NIL only partially prevented the hemodynamic and biochemical changes, and increases in plasma nitrite and cerebral nitrotyrosine levels associated with LPS. Administration of L-NAME, however, had no effects on these LPS-induced changes in diabetic rats. There was a significant difference in nitrotyrosine levels between nondiabetic and diabetic rats in groups 2, 3, and 4 at 2 and 3 h after the treatment (at 3 h; nondiabetic--control, 4.6 +/- 0.4; LPS (i.v.), 8.9 +/- 1.0, LPS (i.v.) + L-NIL, 4.7 +/- 0.5; LPS (i.v.) + L-NAME, 7.1 +/- 0.9; diabetic--control, 5.5 +/- 0.4; LPS (i.v.), 13.6 +/- 1.2; LPS (i.v.) + L-NIL, 9.0 +/- 0.9; LPS (i.v.) + L-NAME, 13.0 +/- 1.0; densitometric units). Insulin therapy resulted in a decrease in iNOS activity (at 3 h: 1.0 +/- 0.5 fmol mg min), nitrotyrosine formation (at 3 h; 5.0 +/- 0.5, densitometric units), and mortality rates (30% at 6 h, 50% at 12 h) in the LPS (i.v.) + L-NIL group of diabetic rats. Selective iNOS inhibition in diabetic rats could not improve hemodynamic instability, chemical changes, iNOS activity, and nitrotyrosine formation during septic shock compared with the improvements observed in nondiabetic rats. Tight glucose control along with administration of L-NIL can result in more effective restoration of the biochemical changes of septicemia in diabetic rats. Thus, hyperglycemia may be one of the mechanisms related to the aggrava

Topics: Animals; Brain Chemistry; Cerebral Cortex; Diabetes Mellitus, Experimental; Enzyme Inhibitors; Hemodynamics; Lipopolysaccharides; Lysine; Male; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase Type II; Random Allocation; Rats; Rats, Wistar; Shock, Septic; Tyrosine

The aim of the present study was to assess the relative contributions of peroxynitrite formation following induction of nitric-oxide synthase (iNOS) in the pathophysiology of endotoxin-induced shock in the rat. To this end, we used a selective inhibitor of iNOS, N-(3-(aminomethyl)benzyl)acetamidine (1400W), and a peroxynitrite decomposition catalyst, 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinato iron III chloride (FeTTPs). Intravenous (i.v.) administration of Escherichia coli lipopolysaccharide (LPS; 4 mg/kg) elicited a time-dependent fall in mean arterial pressure as well as liver, renal, and pancreatic tissue damage. 1400W (3-10 mg/kg i.v.) administered 30 min before LPS delayed the development of hypotension but did not improve survival. On the other hand, FeTTPs administered (10-100 mg/kg i.v.) inhibited in a dose-dependent manner LPS-induced hypotension, tissue injury, and improved mortality rate. In separate experiments, rats were treated with LPS (4 mg/kg) or saline for control, and their aortas were isolated and placed in organ baths 2 h later. Tissues from LPS-treated rats had significant inhibition of contractile activity to phenylephrine as well as a significantly impaired relaxation response to acetylcholine. FeTPPs, when administered (100 mg/kg i.v.) 1 h before LPS, prevented the LPS-induced aortic contractile and endothelial dysfunction. These results demonstrate that nitric oxide-derived peroxynitrite formation plays an important role in this model of endotoxemia. Our results also suggest that use of an iNOS inhibitor in this setting has little beneficial effect in part because, in the presence of a failing eNOS system, some NO is needed to maintain adequate organ function.

Topics: Amidines; Animals; Benzylamines; Endothelium, Vascular; Liver; Male; Metalloporphyrins; Multiple Organ Failure; Nitric Oxide; Nitric Oxide Synthase Type II; Peroxynitrous Acid; Rats; Rats, Sprague-Dawley; Shock, Septic; Tyrosine; Vasoconstriction

In this study, we evaluated the time course of changes in inducible nitric oxide synthase (iNOS) in the brain by using the rat model of sepsis induced by cecal ligation and puncture (CLP) and examined whether selective iNOS inhibition can prevent the hemodynamic and neurological changes induced by sepsis. Male Wistar rats were randomly divided into four groups: control, sham, CLP, and CLP + the selective iNOS inhibitor L-N6-(1-iminoethyl)-lysine (L-NIL). Septic shock was induced in the rats by CLP under pentobarbital anesthesia, and then we measured hemodynamic variables, neurological indicators, blood gases, plasma levels of nitrate/nitrite (an indicator of the biosynthesis of NO), and brain iNOS activity and nitrotyrosine levels after 1, 6, 12, and 24 h. Plasma nitrite was increased at 12 and 24 h in the CLP group. The activity of iNOS in the brain was increased at 12 and 24 h after CLP (at 12 h: control, 0.3 +/- 0.05; sham, 0.3 +/- 0.1; CLP, 1.3 +/- 0.08*; CLP + L-NIL, 0.33 +/- 0.1 fmol x mg(-1) x min(-1); at 24 h: control, 0.27 +/- 0.08; sham, 0.31 +/- 0.1; CLP, 1.0 +/- 0.3*; CLP + L-NIL, 0.34 +/- 0.1 fmol x mg(-1) x min(-1); mean +/- SD; *P < 0.05). Brain nitrotyrosine was increased at 24 h after CLP (at 24 h: control, 6.7 +/- 0.4; sham, 6.7 +/- 0.5; CLP, 11.2 +/- 2.8*; CLP + L-NIL, 7.52 +/- 0.5 densitometric units; means +/- SD; *P < 0.01). In contrast, in both the CLP and CLP + L-NIL groups, the consciousness reflex was significantly decreased at 24 h after CLP. Selective iNOS inhibition restored the hemodynamic changes induced by sepsis but could not improve neurological dysfunction.

Topics: Animals; Blood Pressure; Blotting, Western; Calcium; Cecum; Cerebral Cortex; Enzyme Inhibitors; Heart Rate; Hemodynamics; Interleukin-1; Lysine; Male; Nervous System Diseases; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Rats; Rats, Wistar; Shock, Septic; Tumor Necrosis Factor-alpha; Tyrosine

Septic shock, a severe form of sepsis, is characterized by cardiovascular collapse following microbial invasion of the body. The progressive hypotension, hyporeactivity to vasopressor agents and vascular leak leads to circulatory failure with multiple organ dysfunction and death. Many inflammatory mediators (e.g. TNF-alpha, IL-1 and IL-6) are involved in the pathogenesis of shock and, among them, nitric oxide (NO). The overproduction of NO during septic shock has been demonstrated to contribute to circulatory failure, myocardial dysfunction, organ injury and multiple organ failure. We have previously demonstrated with in vitro and in vivo studies that methylguanidine (MG), a guanidine compound deriving from protein catabolism, significantly inhibits iNOS activity, TNF-alpha release and carrageenan-induced acute inflammation in rats. The aim of the present study was to evaluate the possible anti-inflammatory activity of MG in a model of septic shock induced by lipopolysaccharide (LPS) in mice. MG was administered intraperitoneally (i.p.) at the dose of 30 mg/kg 1 h before and at 1 and 6 h after LPS-induced shock. LPS injection (10 mg/kg in 0.9% NaCl; 0.1 ml/mouse; i.p.) in mouse developed a shock syndrome with enhanced NO release and liver, kidney and pancreatic damage 18 h later. NOx levels, evaluated as nitrite/nitrate serum levels, was significantly reduced in MG-treated rats (78.6%, p < 0.0001; n = 10). Immunohistochemistry revealed, in the lung tissue of LPS-treated group, a positive staining for nitrotyrosine and poly(adenosine diphosphate [ADP] ribose) synthase, both of which were reduced in MG-treated mice. Furthermore, enzymatic evaluation revealed a significant reduction in liver, renal and pancreatic tissue damage and MG treatment also improved significantly the survival rate. This study provides evidence that MG attenuates the degree of inflammation and tissue damage associated with endotoxic shock in mice. The mechanisms of the anti-inflammatory effect of MG is, at least in part, dependent on the inhibition of NO formation.

Topics: Animals; Cell Line; Cell Survival; Disease Models, Animal; Lipopolysaccharides; Lung; Macrophages; Male; Methylguanidine; Mice; NF-kappa B; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Shock, Septic; Survival Rate; Tyrosine

Interrelation of plasma nitrotyrosine (NT) concentrations in patients of septic shock and their prognosis was examined. Blood samples were obtained from 12 patients during the first episode of septic shock at hospitalization, and their plasma NT concentrations were measured. Five patients died within five days after hospitalization, but seven patients recovered. Plasma NT concentrations (means +/- SE) of the non-survivors and survivors were 0.68 +/- 0.13 nmol/mL (n = 7), and 0.21 +/- 0.05 nmol/mL (n = 5), respectively, the former being significantly higher than the latter. The present results suggest that plasma concentration of NT relates to prognosis in human septic shock, although further studies with a larger patient population are necessary for confirmation of the suggestion.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers; Critical Illness; Female; Humans; Intensive Care Units; Male; Middle Aged; Nitric Oxide; Probability; Prognosis; Sampling Studies; Sensitivity and Specificity; Severity of Illness Index; Shock, Septic; Survival Analysis; Tyrosine

To assess the contribution of poly (adenosine 5'-diphosphate ribose) synthetase (PARS) to the development of bacterial lipopolysaccharide (LPS)-induced acute lung injury and vascular failure in pigs.. Four groups of anesthetized, paralyzed, and mechanically ventilated domestic white pigs. Group 1 served as control, whereas Escherichia coli LPS (20 microg/kg/h) was continuously infused in group 2. Group 3 received 20 mg/kg injection of 3-aminobenzamide (a selective inhibitor of PARS activity) 15 minutes before LPS infusion. Only 3-aminobenzamide and not LPS was injected in group 4. All animals were examined for 180 minutes. Systemic and pulmonary hemodynamics and lung mechanics were measured during the experimental period. Lung wet/dry ratio, bronchoalveolar lavage (BAL) protein levels and cell counts and lung nitrotyrosine (footprint of peroxynitrite) immunostaining were also measured in a few animals.. LPS infusion evoked a progressive decline in systemic arterial pressure, a small increase in cardiac output, and biphasic elevation of pulmonary arterial pressure. Lung compliance declined progressively, whereas lung and total respiratory resistance rose significantly after LPS infusion. Prominent nitrotyrosine immunostaining was detected around small airways and pulmonary endothelium of LPS-infused animals. No significant changes in lung wet/dry ratio and BAL protein levels and cell counts were produced by LPS infusion. Pretreatment with 3-aminobenzamide did not alter the systemic and pulmonary hemodynamic responses to LPS infusion but eliminated the rise in pulmonary and total respiratory resistance.. We concluded that PARS activation plays an important role in the changes of lung mechanics associated with LPS-induced acute lung injury but had no role in vascular failure.

Topics: Animals; Benzamides; Bronchoalveolar Lavage Fluid; Disease Models, Animal; Enzyme Inhibitors; Escherichia coli Infections; Female; Hemodynamics; Immunohistochemistry; Lipopolysaccharides; Male; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Proteins; Pulmonary Circulation; Respiratory Distress Syndrome; Respiratory Mechanics; Shock, Septic; Swine; Tyrosine

An enhanced formation of reactive oxygen species contributes to the multiple organ dysfunction syndrome (MODS) caused by endotoxin. We have recently discovered that two cell wall components, namely lipoteichoic acid (LTA) and peptidoglycan (PepG) of the gram-positive bacterium, Staphylococcus aureus, synergize to cause shock and MODS in the rat. Here, we investigate the effects of a membrane-permeable radical scavenger (tempol) on the circulatory failure and MODS (kidney, liver, lung) caused by coadministration of LTA (3 mg/kg i.v.) and PepG (10 mg/kg i.v.) in the anesthetized rat.. Prospective, randomized study.. University-based research laboratory.. Thirty-four anesthetized, male Wistar rats.. After surgical preparation, anesthetized rats were observed for 6 hrs. Control rats were given vehicle (control plus saline, 2 mL/kg bolus injection, followed by an infusion of 1.5 mL/kg i.v., n = 6) or tempol (control plus tempol, 100 mg/kg i.v. bolus injection, followed by an infusion of 30 mg/kg i.v., n = 6). Gram-positive septic shock was induced by coadministration of LTA (3 mg/kg i.v.) and PepG (10 mg/kg i.v.) (LTA/PepG plus saline, n = 12). Another group of rats was pretreated with tempol before shock was induced (LTA/PepG plus tempol, 100 mg/kg i.v. bolus injection, 15 mins before LTA/PepG administration, followed by an infusion of 30 mg/kg i.v., n = 10).. Within 6 hrs, administration of LTA/PepG resulted in hypotension, acute renal dysfunction, hepatocellular injury, pancreatic injury, and increased plasma concentrations of nitrite/nitrate. Pretreatment of rats with tempol augmented the hypotension but attenuated the renal dysfunction and the hepatocellular injury/dysfunction caused by LTA/PepG. Tempol did not affect the increase in nitrite/nitrate caused by LTA/PepG.. These results imply that an enhanced formation of reactive oxygen species (including superoxide anions) contributes to the kidney and liver injury and dysfunction caused by LTA/PepG in the anesthetized rat.

Topics: Animals; Blood Pressure; Cyclic N-Oxides; Free Radical Scavengers; Kidney; Lipopolysaccharides; Liver; Lung; Male; Multiple Organ Failure; Multiple Trauma; Nitrates; Nitrites; Peptidoglycan; Random Allocation; Rats; Rats, Wistar; Shock, Septic; Spin Labels; Staphylococcal Infections; Teichoic Acids; Tyrosine

To study the relation between nitrite, nitrate, nitrotyrosine, and nitrosothiols as NO indices in human septic shock.. A prospective clinical study.. Intensive care units in a university hospital and a central county hospital.. Sixteen patients admitted for septic shock. Nine healthy volunteers served as controls.. None.. Patients with septic shock had a hyperdynamic circulatory response and required infusion of at least two vasopressors to maintain systemic blood pressure. Four episodes of recurrent shock occurred in two patients. Heparinized plasma was collected once daily for analysis of NO indices. Peak plasma concentrations of nitrite + nitrate (NOx) were elevated in first episodes of septic shock; 144+/-39 microM vs. controls, 20+/-3 microM (p < .05). Peak plasma NOx concentrations in recurrent shocks were; 160+/-19 microM. Peak plasma concentrations of 3-nitrotyrosine (NT) were elevated in primary septic shock 102+/-19 pmol x mL(-1) vs. controls 14+/-6 pmol x mL(-1) (p < .05). Peak NT concentrations were 117+/-37 pmol x mL(-1) in recurrent septic shock. Peak plasma NT concentrations did not coincide with peak NOx concentrations in half of the episodes of septic shock. Plasma NT was elevated (59+/-15 pmol x mL(-1) vs. controls 14+/-6 pmol x mL(-1), p < .05) in patients with normal plasma NOx concentrations throughout septic shock. Plasma concentrations of nitrosothiols did not change during septic shock.. Plasma concentrations of NOx and NT are elevated in primary episodes of septic shock and may also be elevated in secondary septic shock, but too few episodes of recurrent septic shock occurred to allow firm conclusions. Plasma concentrations of NT are elevated in patients with septic shock with normal plasma NOx concentrations, indicating that plasma concentrations of NOx may not always accurately reflect NO production. Reactive nitrogen species may be formed in septic shock, and measuring both NOx and NT may give a better indication of NO production in septic shock than NOx alone. Plasma levels of nitrosothiols did not change during septic shock.

Topics: Adolescent; Adult; Humans; Nitrates; Nitric Oxide; Nitrites; Prospective Studies; Severity of Illness Index; Shock, Septic; Sulfhydryl Compounds; Tyrosine

The role of nitric oxide (NO) in lipopolysaccharide (LPS)-induced hepatic injury was studied in D-galactosamine (D-GalN)-sensitized mice. The inducible isoform of NO synthase (iNOS) was immunohistochemically detected on hepatocytes around blood vessels in livers of mice injected with D-GalN and LPS not on hepatocytes in mice injected with D-GalN or LPS alone, although mRNA for iNOS was found in those mice. Nitrotyrosine (NT) was also found in livers of mice injected with D-GalN and LPS. The localization of NT was consistent with that of iNOS, and the time courses of NT and iNOS expression were almost the same. Expression of iNOS and NT was detected exclusively in the hepatic lesions of mice injected with D-GalN and LPS. Anti-tumor necrosis factor alpha neutralizing antibody inhibited iNOS and NT expression and hepatic injury. The results suggested that NO from iNOS may play a role in LPS-induced hepatic injury on D-GalN-sensitized mice as an experimental endotoxic shock model.

Topics: Animals; Disease Models, Animal; Female; Galactosamine; Heat-Shock Proteins; Lipopolysaccharides; Liver; Mice; Mice, Inbred BALB C; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Rabbits; RNA, Messenger; Shock, Septic; Tumor Necrosis Factor-alpha; Tyrosine

Enhanced intestinal nitric oxide production observed during sepsis is thought to play a central role in lipopolysaccharide-induced intestinal damage. In contrast intestinal polyamines, both from endogenous and exogenous origin, are essential for the maintenance of mucosal integrity. Polyamines have been shown to inhibit lipopolysaccharide-induced nitric oxide release in vitro and have been claimed to exert additional antiinflammatory actions. In this study, the effect of the polyamine spermine on the release of the proinflammatory mediators nitric oxide and tumor necrosis factor-alpha by a murine macrophage cell line was investigated. Furthermore, we investigated whether oral spermine administration inhibits lipopolysaccharide-induced intestinal inducible nitric oxide synthase and nitrotyrosine expression and modulates the release of inflammatory mediators. Our results show that although spermine inhibited lipopolysaccharide-induced nitric oxide release in a murine macrophage cell line, no effect on tumor necrosis factor-alpha release was observed. In addition, oral spermine administration inhibited intestinal inducible nitric oxide synthase and nitrotyrosine expression suggesting a protective effect of spermine on lipopolysaccharide-induced intestinal damage. In parallel a decrease in serum levels of the proinflammatory mediators nitrate, nitrite, and interferon-gamma and an increase in the antiinflammatory cytokine interleukin-10 was observed, although tumor necrosis factor-alpha levels were unaffected. These results indicate that spermine inhibits lipopolysaccharide-induced nitric oxide release in vitro as well as in vivo. Further, intraluminally derived polyamines modulate the systemic immune response. It is concluded that oral spermine administration might have therapeutic perspectives for several disorders characterized by systemic inflammation and intestinal damage.

Topics: Administration, Oral; Animals; Disease Models, Animal; Escherichia coli; Female; Immunohistochemistry; Inflammation; Interferon-gamma; Interleukin-10; Intestinal Mucosa; Intestines; Lipopolysaccharides; Macrophages; Mice; Neutrophils; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitrites; Shock, Septic; Spermine; Tumor Necrosis Factor-alpha; Tyrosine

The effect of hyperoxia on nitric oxide (NO) production in intact animals is unknown. We described the effects of hyperoxia on inducible nitric oxide synthase (iNOS) expression and NO production in the lungs of rats exposed to high concentrations of oxygen. Animals were placed in sealed Plexiglas chambers and were exposed to either 85% oxygen (hyperoxic group) or 21% oxygen (negative control group). Animals were anesthetized after 24 and 72 h of exposure and were ventilated via a tracheotomy. We measured NO production in exhaled air (E(NO)) by chemiluminescence. The lungs were then harvested and processed for detection of iNOS by immunohistochemistry and Western blotting analysis. The same experiments were repeated in animals exposed to hyperoxia for 72 h after they were infused with L-arginine. We used rats that were injected intraperitoneally with Escherichia coli lipopolysaccharide to induce septic shock as a positive control group. Hyperoxia and septic shock induced expression of iNOS in the lung. However, E(NO) was elevated only in septic shock rats but was normal in the hyperoxic group. Exogenous infusion of L-arginine after hyperoxia did not increase E(NO). To exclude the possibility that in the hyperoxic group NO was scavenged by oxygen radicals to form peroxynitrite, lungs were studied by immunohistochemistry for the detection of nitrotyrosine. Nitrotyrosine was found in septic shock animals but not in the hyperoxic group, further suggesting that NO is not synthesized in rats exposed to hyperoxia. We conclude that hyperoxia induces iNOS expression in the lung without an increase in NO concentration in the exhaled air.

Topics: Animals; Blotting, Western; Hyperoxia; Immunohistochemistry; Lipopolysaccharides; Luminescent Measurements; Lung; Male; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Rats; Rats, Sprague-Dawley; Respiration; Shock, Septic; Time Factors; Tyrosine

The production of both nitric oxide (NO) and superoxide increases in septic shock. The cogeneration of these molecules is known to yield peroxynitrite, which preferentially nitrates tyrosine residues of protein and non-protein origins. We present evidence of peroxynitrite production in septic shock by measuring plasma nitrotyrosine. The nitrotyrosine was measured by an HPLC C-18 reverse-phase column and ultraviolet detector in chronic renal failure patients with or without septic shock, and in healthy volunteers. Plasma nitrite + nitrate (NOx) was also measured to evaluate NO production. Nitrotyrosine was selected as an index for production of peroxynitrite because the direct measurement of peroxynitrite in vivo is difficult. Patients with renal failure were selected in order to minimize nitrotyrosine excretion through the kidney. Plasma nitrotyrosine levels were not detectable in volunteers, 28.0 +/- 12.3 microM (1.6 +/- 1.1% of total tyrosine) in renal failure patients without septic shock, and 118.2 +/- 22.0 microM (5.5 +/- 1.2% of total tyrosine) in patients with septic shock. NOx levels were also higher in patients with septic shock than in patients without septic shock (173.9 +/- 104.7 vs. 75.6 +/- 19.1 microM). Although renal failure itself increases plasma concentrations of both molecules, the higher levels in patients with septic shock suggest that peroxynitrite is generated and the nitration of tyrosine residues is increased in this disease.

Topics: Adult; Aged; Aged, 80 and over; Case-Control Studies; Female; Free Radicals; Humans; Kidney Failure, Chronic; Male; Middle Aged; Nitrates; Nitric Oxide; Shock, Septic; Superoxides; Tyrosine