INTRODUCTION

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Clinical acute lung injury and the acute respiratory distress syndrome (ALI/ARDS) represent a common response of the lung to a variety of insults, including sepsis, endotoxemia, trauma, aspiration of gastric contents, and pneumonia. Pulmonary edema, a major component of ALI, results primarily from increased permeability of the alveolar-capillary barrier to plasma proteins.

Studies in a wide variety of experimental models have implicated increased production of reactive oxygen species and nitric oxide (·NO) in the development and progression of ALI (1). Most of the injurious effects of ·NO have been attributed to the formation of peroxynitrite, which is formed from the reaction of ·NO with superoxide (O₂^·), at a near diffusion-limited rate (k = 6.7 × 10⁹ M ¹ s ¹). Peroxynitrite is a potent oxidizing and nitrating agent that damages a wide spectrum of biological molecules such as DNA, lipids, and proteins (2). Recent evidence suggests that in addition to peroxynitrite, myeloperoxidase can catalyze the oxidation of nitrite to form a reactive intermediate that is capable of nitrating proteins (5). Nitrated proteins have been detected in the lung tissue and bronchoalveolar lavage of both adult and pediatric patients with ALI (6). Protein nitration and oxidation by reactive oxygen nitrogen species in vitro have been associated with diminished function of a variety of crucial proteins present in the alveolar space, including ₁-proteinase inhibitor and surfactant protein A (SP-A) (4, 9). More recently, Gole and coworkers (10) reported the presence of nitrated ceruloplasmin, transferrin, ₁-protease inhibitor, ₁-antichymotrypsin, and -chain fibrinogen in the plasma of patients with ALI/ARDS. However, nitration of specific proteins in the alveolar space of patients with ALI/ARDS has not been documented. Furthermore, there is no evidence linking nitrate (NO₃) and nitrite (NO₂) levels to the severity of ALI.

To address these issues we measured NO₂ and NO₃ levels as well as protein nitrotyrosine in both the pulmonary edema fluid and plasma from patients with ALI/ARDS and for comparison, in patients with hydrostatic pulmonary edema. The levels of NO₂ and NO₃ in pulmonary edema fluid and plasma were correlated with indices of systemic injury, as well as the ability of the alveolar epithelium to remove pulmonary edema fluid from the alveolar space. In addition, we immunoprecipitated pulmonary surfactant protein A (SP-A), the most abundant surfactant protein, from pulmonary edema fluid samples and assessed the extent of its nitration by Western blotting analysis. Our data provide evidence for significant up-regulation of ·NO production by the lung cells in patients with ALI/ARDS and hydrostatic pulmonary edema and, for the first time, demonstrate in vivo nitration of a key surfactant protein in the alveolar lining fluid. Furthermore, they show that increased levels of NO₂ + NO₃ in edema fluid samples were associated with slower rates of alveolar fluid clearance.

	METHODS

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Reagents

Nitrate reductase (from Aspergillus species) was from Boehringer-Mannheim (Indianapolis, IN). ECL Western blotting detection reagents were from Amersham-Pharmacia Biotech Inc. (Piscataway, NJ). Triton X-100 and ethylenediaminetetraacetic acid (EDTA) were from EM Science, a Division of EM Industries, Inc. (Cherry Hill, NJ). Protein A/G-agarose was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Sodium dodecyl sulfate (SDS), Tween-20, dry milk blocker, 2-mercaptoethanol, and PVDF membranes were from Bio-Rad Laboratories (Hercules, CA). Trifluoroacetic acid was from Pierce Chemical Co. (Rockford, IL). Centrifugal filter device (MW cut-off = 5 kD) was from Millipore Corp. (Bedford, MA). Sulfanilamide N-(1-naphthyl)ethylenediamine dihydrochloride, sodium nitrite, sodium bicarbonate, tris(hydroxymethyl)aminomethane (Tris), sodium deoxycholate, sodium dithionite, 3-nitro-L-tyrosine, and NADPH were from Sigma Chemical Co. (St. Louis, MO). Horseradish peroxidase-conjugated donkey anti-rabbit and mouse immunoglobulin G (IgG) were from Accurate Chemical and Scientific Corp. (Westbury, NY). Polyclonal rabbit anti-human SP-A and mouse monoclonal anti-nitrotyrosine antibody were kind gifts from Dr. David S. Phelps (Pennsylvania State University, Hershey, PA) and Dr. Joseph S. Beckman (University of Alabama at Birmingham, Birmingham, AL), respectively.

Patient Population

Patients with acute pulmonary edema from either a hydrostatic mechanism or ALI/ARDS were identified prospectively from patients admitted to the intensive care units of University of California at San Francisco (UCSF) or San Francisco General Hospital between 1985 and 1998. Additional inclusion criteria included acute respiratory failure requiring mechanical ventilation and aspirable pulmonary edema fluid from at least two time points within the first 4 h after endotracheal intubation. As in our previous studies (11), criteria for hydrostatic edema included: central venous pressure  14 mm Hg or a pulmonary artery wedge pressure  18 mm Hg, a left ventricular ejection fraction  45%, no risk factor for ALI/ARDS, and initial edema fluid/ plasma protein ratio  0.65. Evidence for ALI/ARDS included an appropriate risk factor for acute lung injury, Pa_O2/FI_O2 ratio < 300, bilateral infiltrates on chest radiograph, no clinical evidence of increased left atrial pressure, and an initial edema fluid/plasma protein ratio > 0.65. The medical records of all patients were reviewed, and pertinent data were recorded including hospital survival, days of mechanical ventilation, hemodynamic, ventilatory, and laboratory data, and medications. The Simplified Acute Physiology Score II (SAPSII) was calculated according to published methods (12). Patients with shock were defined as those with a systolic blood pressure less than 90 mm Hg or the need for vasopressor medications during the first 24 h after intubation and initiation of mechanical ventilation. Patients with missing clinical data had to be excluded from some analyses. The number of patients that could be included in each analysis is noted. This study was approved by the Committee for Human Research at UCSF.

Collection of Pulmonary Edema Fluid

Pulmonary edema fluid was collected from each patient within 30 min after endotracheal intubation by passing a standard 14-Fr tracheal suction catheter through the endotracheal tube into a wedged position in a distal airway. Gentle suction was then applied to aspirate a few milliliters of edema fluid into a suction trap. A simultaneous plasma sample was obtained. A second sample was obtained within 4 h of the first sample in 85% (46 of 54) of the patients for calculation of the rate of alveolar fluid clearance. The samples were centrifuged at 3,000 × g, and the supernatants were collected and stored at 70° C. Total protein concentration was determined in plasma and edema fluid samples by the Biuret method as described previously (11).

Measurements of NO₂ and NO₃ in Edema Fluid and Plasma

Samples were filtered through a centrifugal filter device (MW cut-off = 5 kD). NO₂ alone and total NO₂ + NO₃, after the reduction of NO₃ to NO₂ with nitrate reductase, were measured with Griess reagents. Briefly, 250 µl of each sample was incubated with an equal volume of 0.2 mM NADPH in 50 mM phosphate buffer, pH 7.5, at room temperature for 2 h in the presence of 0.2 U/ml of nitrate reductase, followed by the addition of 500 µl of Griess reagent containing 0.5% sulfanilamide and 0.05% N-(1-naphthyl)ethylenediamine dihydrochloride for 10 min. Absorbence was measured at 550 nm, and NO₂ concentration was determined using NaNO₂ and NaNO₃ standards. For comparison, levels of NO₂ + NO₃ were also measured in plasma samples obtained from six healthy adult volunteers from the University of California at San Francisco and four healthy adult volunteers from the University of Alabama at Birmingham. One of the samples was discarded because its mean NO₂ + NO₃ value was more than two standard deviations higher than the group mean. The four samples from the University of Alabama at Birmingham have also been included in a previous study (13).

Calculation of Rate of Alveolar Fluid Clearance

The rate of alveolar fluid clearance (AFC) was calculated as the percentage of alveolar fluid volume reabsorbed per hour, as described previously (11). Briefly, based on the observation that the rate of protein removal from the alveoli is very slow relative to the rate of removal of edema fluid, the percentage of alveolar edema fluid volume reabsorbed may be estimated by the equation AFC (%) = (1  C_i/C_f) × 100 where C_i and C_f are initial and final protein concentrations, respectively. For comparison of rates of alveolar fluid clearance to levels of edema fluid NO₂ + NO₃, alveolar fluid clearance was categorized as impaired (< 3%/h), submaximal ( 3%/h, < 14%/h), or maximal ( 14%/h) as we have done previously (14). To correlate these categories of alveolar fluid clearance with edema fluid NO₃ levels, a mean NO₂ + NO₃ level in the pulmonary edema fluid was calculated for each interval over which alveolar fluid clearance was measured. This mean NO₂ + NO₃ level was calculated from the arithmetic mean of NO₂ + NO₃ levels in the initial and final samples in which protein concentration was measured. This approach was necessary because the edema fluid NO₃ levels fluctuated over time (data not shown), likely due to ongoing production and clearance of NO₃ and NO₂ in the alveolar space.

Quantitative Enzyme-Linked Immunosorbent Assay (ELISA)

Protein-associated nitrotyrosine levels in edema fluid and plasma samples were measured by a quantitative ELISA as described previously (3) using a rabbit polyclonal antibody against nitrotyrosine and horseradish peroxidase-conjugated goat anti-rabbit IgG as primary and secondary antibodies, respectively. H₂O₂ and o-phenylenediamine were used as substrates for the peroxidase reaction, and the absorbance was measured at 490 nm (9). Nitrotyrosine content was expressed as picomoles nitrotyrosine per milligram protein. For comparison purposes, nitrotyrosine levels were also measured in plasma samples from five volunteers.

High-performance Liquid Chromatography (HPLC) with Electrochemical Detection

The extraction, digestion, HPLC separation, and electrochemical detection were carried out as previously described (15). In brief, pulmonary edema or plasma samples were precipitated by 10 volumes of ethanol at 4° C for 10 min. The protein precipitates were then pelleted by centrifugation at 3,000 × g for 5 min. The precipitation was repeated one more time and redissolved in a volume of water equivalent to the initial sample volume. Samples containing 100 µg of redissolved proteins were transferred to 6 × 35-mm hydrolysis tubes and dried by vacuum centrifugation. Dried protein samples were hydrolyzed using 0.2 ml high grade 6 M hydrochloric acid (HCl) at 110° C for 12 h. Hydrolyzed samples were dried and resuspended in 50 mM sodium acetate buffer, pH 3.5 (HPLC buffer). The solutions were centrifuged at 10,000 × g for 2 min to pellet any undissolved particulates, and the supernatants were injected into a C₁₈ column (4.6 × 250 mm, Tosohaas 80TM). An isocratic elution was carried out using 0.75 ml/min of 95% HPLC buffer and 5% HPLC-grade methanol. Water used in the whole experiment was purified by a Millipore reverse osmosis/filter system, polished with sep-pack C₁₈ cartridges, and filtered through 0.2-µm filters. The eluted nitrotyrosine was detected by a 12-channel electrochemical detector (ESA, Inc.) and quantified by comparing the combined areas of the three dominant peaks with authentic nitrotyrosine standard.

Immunoprecipitation of SP-A and Albumin

Pulmonary edema fluid or plasma samples were diluted to a protein concentration of 2 mg/ml in buffer A (50 mM Tris-HCl, pH 7.5, containing 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0.1% SDS, and 0.5% sodium deoxycholate). SP-A in the supernatant was precipitated with a polyclonal rabbit anti-human SP-A at 4° C for 2 h while rocking and followed by incubation with 50 µl/ml protein A/G-agarose for an additional hour. The protein A/G-agarose beads were then washed with buffer A three times and once more with buffer B (10 mM Tris-HCl, pH 7.5, containing 0.1% Triton X-100). Albumin in the supernatant was precipitated with a polyclonal rabbit anti-human albumin at 4° C for 2 h while rocking and followed by incubation with 50 µl/ml protein A/G-agarose for an additional hour. The protein A/G-agarose beads were then washed with buffer A for three times and one more time with buffer B (10 mM Tris-HCl, pH 7.5, containing 0.1% Triton X-100).

SDS-PAGE and Western Blotting Detection of SP-A and Albumin Nitration

Immunoprecipitated SP-A or albumin was solubilized in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer, resolved on 12% SDS-PAGE gels and transferred to PVDF membranes. Membranes were probed with a polyclonal sheep anti-human albumin or with a polyclonal rabbit anti-human SP-A. Nitration of both albumin and SP-A were detected by a mouse monoclonal anti-nitrotyrosine antibody. As a control, a duplicate membrane was treated with 100 mM sodium dithionite in 50 mM Na₂CO₃-NaHCO₃ buffer, pH 10 (three times, 1 min each) to reduce nitrotyrosine to aminotyrosine. Immunoreactive protein complexes were detected using ECL Western blotting detection reagents with horseradish peroxidase-conjugated donkey IgG against appropriate IgGs.

Statistical Analysis

All values are means ± SEM unless otherwise noted. Statistical differences between two group means were determined using Student's t test, or the Mann-Whitney U test if values were not normally distributed. Significant differences between groups were defined as p < 0.05. Statistical differences among multiple groups were determined using the one-way analysis of variance (ANOVA). If the F value was statistically significant (p < 0.05), we used the Student-Newman-Keuls test for post hoc comparisons.

	RESULTS

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Patients

A total of 20 patients with hydrostatic pulmonary edema and 34 patients with ALI/ARDS were included in the study. Clinical characteristics of all 54 patients are summarized in Table 1. Interestingly, both groups of patients had a high severity of illness as evidenced by the high SAPSII scores and the very high incidence of systemic shock. The degree of acidemia was similar in the two groups.

NO₂ + NO₃ in Pulmonary Edema Fluid and Plasma

Values of total NO₂ + NO₃ levels in plasma samples obtained from ALI/ARDS patients (83 ± 14 µM), patients with hydrostatic edema (51 ± 3 µM), and normal volunteers (26 ± 2.7) are shown in Figure 1. Total NO₂ + NO₃ in plasma samples from patients with ALI/ARDS was significantly higher than the corresponding value in normal volunteers (p = 0.04). Pulmonary edema fluid samples from ALI/ARDS patients had significantly higher NO₂ + NO₃ levels than those from hydrostatic edema patients (108 ± 13 µM for ALI/ ARDS versus 66 ± 9 µM for hydrostatic edema, p = 0.04; Figure 1). The ratios of NO₂/ NO₃ in 11 edema and 9 plasma samples were 0.01 ± 0.005 versus 0.008 ± 0.004. These findings indicate that the majority (more than 95%) of stable ·NO metabolites were in the form of NO₃. In both groups of patients, the concentration of NO₂ + NO₃ in the edema fluid exceeded that of plasma indicating that at least some of the NO₂ + NO₃ was produced by epithelial and inflammatory cells present in the alveolar space. We have previously reported that NO₂ + NO₃ levels were nearly undetectable in the bronchoalveolar lavage fluid of normal volunteers (13).

View larger version (27K): [in this window] [in a new window]   Figure 1.   Nitrate and nitrite in pulmonary edema fluid and plasma samples from acute lung injury (ALI), hydrostatic edema patients (Hydr.), and normal volunteers. Numbers in parenthesis are sample numbers. Values are means ± SEM.

Correlation of Plasma and Edema Fluid NO₃ Levels with Alveolar Fluid Clearance, Shock, and Systemic Hypoperfusion

As shown in Figure 2, in both patients with hydrostatic pulmonary edema and those with ALI/ARDS, maximal alveolar fluid clearance ( 14%/h) was associated with significantly lower mean interval pulmonary edema fluid NO₂ + NO₃ concentrations than were measured in patients with submaximal (< 14%/h,  3%/h) or impaired (< 3%/h) alveolar fluid clearance (p < 0.05). Because studies in rats have demonstrated that systemic hypoperfusion due to hypovolemia can impair alveolar fluid clearance by an oxidant-mediated mechanism (16), we compared NO₂ + NO₃ levels in patients with and without systemic shock. In both patients with hydrostatic pulmonary edema and those with ALI/ARDS, plasma NO₂ + NO₃ levels were higher in patients with shock than those without shock. When all patients were considered together, this difference reached statistical significance (79 ± 11 µM versus 53 ± 12 µM, p < 0.05; Table 2). Systemic hypoperfusion, as evidenced by either acidemia or increased anion gap, was also associated with two-fold higher plasma NO₂ + NO₃ levels (Table 2).

View larger version (18K): [in this window] [in a new window]   Figure 2.   Box-plot summary of mean interval edema fluid nitrate and nitrite concentration versus two categories of alveolar fluid clearance. Maximal alveolar fluid clearance is  14%/h. Submaximal/impaired alveolar fluid clearance is < 14%/h. Horizontal line represents the median, the box encompasses the 25th to 75th percentile, and the error bars encompass the 10th to 90th percentile. *p < 0.05 by Mann-Whitney U test.

Nitration of Proteins in Both Edema and Plasma Samples from Edema Patients

Significant levels of protein-associated nitrotyrosine were found in pulmonary edema fluid and plasma samples from patients with ALI/ARDS and hydrostatic edema as measured by ELISA (Table 3). Nitrotyrosine values in pulmonary edema fluid and plasma were at least one order of magnitude higher than the corresponding values from bronchoalveolar lavage fluid of normal volunteers (28 ± 2 pmol/mg protein) (13) and from plasma of normal healthy subjects (30 ± 2 pmol/mg protein; Table 3). Considering the fact that the largest fraction of protein in the edema fluid is albumin (data not shown), and 1 mol of albumin contains 19 tyrosines, we calculated a nitrotyrosine to tyrosine ratio (NT/T) of 0.2% for the edema fluid values in patients with ALI/ARDS.

Simultaneous measurement of nitrotyrosine levels in 11 pulmonary edema fluid and plasma samples with both ELISA and HPLC showed similar nitrotyrosine values (446 ± 140 pmol/mg protein by HPLC versus 543 ± 62 pmol/mg protein by ELISA; means ± SEM), demonstrating the validity of our ELISA method. All nitrotyrosine detected was protein associated. Experience with hundreds of samples from many different human and animal tissues has revealed that hydrolysis of 10 µg of protein from a heterogeneous protein mixture in a homogenate yields about 1.4 nmol of tyrosine (J. P. Crow, personal communication). Based on this empirical but consistent relationship, we calculated a (NT/T) of 0.19 ± 0.06 mol% for the HPLC values, which was similar to the one we calculated for the ELISA measurements (see above).

SP-A in Pulmonary Edema Samples from ALI/ARDS Patients Was Nitrated

Human SP-A usually exhibits a characteristic monomer band around 35 kD and a dimer band around 62 kD on reducing SDS-PAGE. Immunoprecipitated SP-A from five pulmonary edema samples taken from five different ALI/ARDS patients is shown in Figure 3. In subsequent Western blotting studies utilizing an anti-nitrotyrosine antibody, we found nitration of both the 35- and 62-kD bands in four of the five patients with ALI/ARDS (Figure 3, lanes E₁-E₄). No nitration was seen when SP-A samples were treated with dithionite, which reduces nitrotyrosine to aminotyrosine, prior to Western blotting studies. In addition, SP-A isolated from the lungs of patients with alveolar proteinosis was not nitrated (Figure 3, lane C ). Western blotting studies did not detect normal or nitrated SP-A in three plasma samples from patients with ALI/ARDS (Figure 3, lanes P₁-P₃). These data demonstrate the in vivo nitration of human SP-A in pulmonary edema fluid, but not in plasma from patients with ALI/ARDS.

View larger version (47K): [in this window] [in a new window]   Figure 3.   Nitration of surfactant protein A (SP-A) in pulmonary edema fluid samples from ALI/acute respiratory distress syndrome (ARDS) patients. SP-A precipitation and Western blotting detection of SP-A (A) and nitrotyrosine (B) were performed as described in METHODS. SP-A was detected in the pulmonary edema fluid but not in the plasma of all patients. Lane E1-E5, pulmonary edema fluid samples from five different ALI/ARDS patients; lane P1-P3, plasma samples from three different ALI/ARDS patients; lane C, purified human SP-A from a patient with alveolar proteinosis. Notice the lack of nitration in the control sample.

Similar amounts of albumin were immunoprecipitated by a polyclonal anti-human albumin antibody from both edema and plasma samples from ALI/ARDS and hydrostatic edema patients. Western blotting studies with the above mentioned anti-nitrotyrosine antibody showed that in contrast to SP-A, immunoprecipitated albumin from both group of patients was barely nitrated (data not shown).

	DISCUSSION

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Several experimental studies have provided evidence that reactive oxygen species play an important role in the pathogenesis of acute lung injury. For example, exposure of animals to normobaric hyperoxia, which leads to increased production of reactive oxygen and nitrogen species in the lung, has been shown to damage the alveolar epithelium and the pulmonary surfactant system (17), leading to the development of permeability-type alveolar edema, arterial hypoxemia, and death. Pulmonary surfactant abnormalities have also been demonstrated in patients with ALI/ARDS (18). Reduced and depleted antioxidants, which include decreased levels of reduced glutathione in the bronchoalveolar lavage fluid of patients with ALI/ARDS (19), decreased levels of ascorbate and ubiquinol-10 (20), the presence of oxidized glutathione in alveolar fluid (21), and elevated levels of hydrogen peroxide in the expired gas and the urine of ALI patients (22), indicate an increased oxidant stress and a compromised antioxidant system in patients with ALI/ARDS.

In addition to reactive oxygen species, reactive nitrogen species including ·NO have been implicated in the development and progression of several inflammatory diseases including acute lung injury. Initially, there was uncertainty and controversy as to whether human lung cells could generate ·NO (23). However, results of more recent studies have shown up-regulated production of reactive nitrogen intermediates by lung cells (7, 24). We have reported increased levels of nitrotyrosine staining in the lung sections of patients and animals with acute lung injury (6). In addition, alveolar macrophages isolated from the lungs of patients with ALI/ARDS, but not those of normal controls, immunostained positive for inducible nitric oxide synthase (8). In this study, patients with ALI/ ARDS also had significantly higher levels of NO₂ and NO₃ in their bronchoalveolar lavage fluid compared to normal healthy controls. However, definitive evidence that inflammatory human lung cells can generate sufficient levels of reactive oxygen-nitrogen intermediates to nitrate or oxidize proteins is still lacking.

In the current study, NO₂ and NO₃, stable byproducts of ·NO decomposition, were detected in the plasma and pulmonary edema fluid of both groups of patients, those with ALI/ ARDS and those with hydrostatic pulmonary edema. The finding of significant levels of NO₂ and NO₃ in hydrostatic edema patients was unexpected and will be discussed below. Overall, the levels of NO₂ and NO₃ were significantly higher in patients with ALI/ARDS than in patients with hydrostatic pulmonary edema, indicating that the cause of pulmonary edema is an important determinant of the degree of nitric oxide production. Furthermore, since edema fluid levels of NO₂ and NO₃ were consistently higher than simultaneous plasma levels, the higher levels seen in ALI/ARDS were not just a function of increased permeability of the alveolar capillary barrier compared to patients with hydrostatic edema. The source of enhanced nitric oxide production in the setting of ALI/ARDS cannot be determined from the present study, but previous work suggests that human alveolar macrophages may be an important source (8). The virtual absence of NO₂ from both edema fluid and plasma samples may be due to the oxidation of NO₂ to NO₃ by oxyhemoglobin (12), known to be present in the edema fluid of patients with ALI/ARDS and hydrostatic edema, or it may be due to the combination of ·NO with superoxide to form peroxynitrite, 95% of which decomposes to NO₃ (25).

Because pulmonary edema fluid was suctioned from a distal airway without the addition of saline, a comparison of simultaneous concentrations in the alveolar space with concentrations in the plasma was possible. In both ALI/ARDS and hydrostatic edema patients, NO₂ and NO₃ levels were higher in the pulmonary edema fluid than in the plasma, suggesting that there is local ·NO production in the lung. This is a novel and important finding, one which could not be documented previously in studies of bronchoalveolar lavage fluid where large dilutional factors precluded such comparisons. An additional benefit of sampling undiluted pulmonary edema fluid rather than diluted bronchoalveolar lavage fluid is the opportunity to measure alveolar fluid clearance by following serial changes in the protein concentration in the pulmonary edema fluid. This method has been well validated in prior studies of both hydrostatic pulmonary edema and ALI/ARDS (11, 14).

Recent in vitro and in vivo studies have demonstrated that exposure of alveolar epithelial cells to reactive nitrogen species, such as ·NO or peroxynitrite, decreases their ability to actively transport Na⁺ ions (26, 27). The current study provides additional support for these findings. In those patients with the most rapid alveolar fluid clearance, edema fluid NO₂ and NO₃ levels were significantly lower than in the patients with slower alveolar fluid clearance. One possible explanation for this finding is that the generation of reactive oxygen and nitrogen species in the alveolar compartment leads to nitration and inactivation of proteins important in alveolar epithelial sodium transport, such as the epithelial sodium channel. Indeed, intratracheal instillation of DETANONOate, a ·NO donor, decreased amiloride-sensitive fluid clearance in rabbit lungs (28).

Because previous studies in rats have shown that shock due to hypovolemia is associated with impaired alveolar fluid clearance that is mediated by generation of oxidant species (29), we examined NO₂ and NO₃ levels in relation to the incidence of shock and systemic hypoperfusion in our patients. Interestingly, both shock and systemic hypoperfusion (as evidenced by acidemia or increased anion gap) were associated with significantly higher plasma NO₂ and NO₃ levels, regardless of the etiology of pulmonary edema. This finding suggests that hypoperfusion, regardless of the cause, may be associated with increased oxidant production, a finding that has previously been reported only in animal models (16). Importantly, it should be noted that the incidence of shock was similar in the two patient groups (Table 1), and thus, shock alone did not explain the differences in levels of NO₂ and NO₃ between patients with ALI/ARDS and hydrostatic edema.

Significant levels of protein-associated nitrotyrosine (~ 400- 500 pmol/mg protein) were detected by both quantitative ELISA and HPLC in samples from both ALI/ARDS and hydrostatic edema patients. These levels are at least one order of magnitude higher than those in proteins in normal human bronchoalveolar lavage fluid (28 pmol/mg protein) (13), normal rat lung tissue (~ 30 pmol/mg protein) (30), normal human serum albumin (~ 30 pmol/mg protein) (31), and normal human plasma low-density lipoprotein (~ 85 pmol/mg protein) (31). In a previous study, Lamb and coworkers (32) measured nitrotyrosine content in the bronchoalveolar lavage fluids of patients with severe ARDS and healthy volunteers using a high-performance liquid chromatography. Mean values in ALI/ ARDS and healthy volunteers were 2,650 and 170 pmol/mg proteins, respectively. These values are five-fold higher than ours. The reasons for these large differences are not clear as we also used an HPLC method to confirm our ELISA measurements. These authors also reported the presence of oxidized and chlorinated proteins in the bronchoalveolar lavage fluid of patients with ARDS. In a prior study, we found only minute quantitities of chlortyrosine in the lungs of patients who had undergone lung allotransplantation (13).

The finding that patients with hydrostatic pulmonary edema had substantial levels of NO₂ + NO₃ and nitrotyrosine in their edema fluid and plasma appears contradictory, as the primary etiology of noncardiogenic edema is elevated left atrial pressure and not lung injury. However, as noted above, the patients with hydrostatic edema did represent a severely ill group of patients with high SAPSII scores and a very high incidence of shock. Interestingly, there is some prior evidence for an inflammatory component to hydrostatic pulmonary edema. Cohen and coworkers (33) reported that pulmonary edema fluid from patients with congestive heart failure had significant numbers of neutrophils. Furthermore, in a recent study, Verghese and coworkers (14) reported that patients with severe hydrostatic edema, similar to the patients in our study, had a high severity of illness as measured by the Simplified Acute Physiology II score, a high mortality, and a high incidence of acidemia. In addition, 25% of patients with hydrostatic pulmonary edema had impaired alveolar fluid clearance, suggesting that some form of injury to the alveolar epithelium may have occurred. In light of these findings, it appears that severe hydrostatic pulmonary edema may involve an inflammatory component, although the degree of inflammation is far less than in ALI/ARDS.

Our data also indicate that SP-A, a pulmonary surfactant-associated protein with multiple biological functions, was nitrated. Although we previously demonstrated that SP-A is nitrated and oxidized in vitro when LPS-stimulated rat alveolar macrophages are used as the source of reactive species (34), this is the first evidence for nitration of a specific protein in the human alveolar space in vivo. Interestingly, our recent studies indicate that nitrated SP-A loses its ability to enhance the adherence of Pneumocystis carinii to rat alveolar macrophages (9) and inhibits killing of Mycoplasma pneumoniae by mouse alveolar macrophages (unpublished observations). Also, nitration of SP-A inhibited its lipid aggregation and mannose binding activities (3). Finally, SP-A isolated from the lungs of lambs exposed to high concentrations of inhaled ·NO, which is known to increase nitrotyrosine formation in patients with ARDS (7), had decreased ability to aggregate lipids (35).

The extent of SP-A nitration was not determined in these studies. However, based on the observations that 0.2% nitrotyrosine/tyrosine is present in the edema fluid, we can estimate that 0.2% of the SP-A molecules could be nitrated. This calculation was based on the consideration that each monomeric SP-A (MW ~ 35 kD) molecule contains eight tyrosine residues. In our previous in vitro studies, exposure of human SP-A to sufficient quantities of nitrating agents to nitrate 0.24% of its tyrosine residues resulted in significant inhibition of the ability of SP-A to bind mannose (3).

It is interesting to note that we were unable to detect significant levels of albumin nitration in the lung epithelial fluid of patients with ALI/ARDS, in spite of its much higher abundance compared to SP-A. These data confirm previous reports showing selective nitration of proteins in vivo. This is most likely due to accessibility of tyrosine residues in the protein to the nitrating agents (36). A recent study reported the nitration of a number of specific proteins in the plasma of patients with ARDS, including nitrated ceruloplasmin, transferrin, ₁-protease inhibitor, ₁-antichymotrypsin, and -chain fibrinogen (10). Furthermore, the ferroxidase activity of ceruloplasmin and the elastase-inhibiting activity of ₁-protease inhibitor of plasma sample from ARDS patients were reduced considerably compared with controls. These results agree with our observations of significant levels of NO₂ and NO₃ in the plasma of ARDS patients.

There are several limitations of our current study. First, as mentioned above, due to the very small amount of immunoprecipitated SP-A we could not obtain sufficient levels of SP-A for functional studies. Further studies are needed to determine the functional impact of nitration of SP-A in this patient population. Second, it is possible that reactive oxygen-nitrogen intermediates may either nitrate or oxidize a number of other key SP-A amino acids, including tryptophan and methionine (SP-A does not contain any free cysteines). Indeed, incubation of LPS-stimulated alveolar macrophages with SP-A resulted in nitration and oxidation (asssesed by carbonyl formation) of both of its monomer (35 kD) and dimer (58 kD) (34). Physiological concentrations of carbon dioxide (PCO₂ = 40 mg Hg) enhanced SP-A nitration by alveolar macrophages and decreased carbonyl formation. Furthermore, SP-A exposed to peroxynitrite in the presence of carbon dioxide (PCO₂ = 40 mg Hg) was significantly more nitrated and had significantly lower ability to aggregate lipids than SP-A exposed to similar concentrations of peroxynitrite, but in the absence of carbon dioxide (34). Taken as a whole, these in vitro observation indicate that the extent of SP-A nitration correlates with SP-A function dependent on an intact carbodydrate recognition domain. Third, because we are unable to directly measure activity of nitric oxide synthases in our patients, the presence of elevated NO₃ levels does not provide definitive evidence of increased ·NO production. There are several other possible explanations for the increase in NO₂ and NO₃ levels. For example, it is possible that clearance of NO₃ could be impaired in the setting of ALI/ARDS or that increased production of NO₃ resulted from the decomposition of peroxynitrite, formed by the interaction of nitric oxide and superoxide (25). A final limitation of our study is that the number of patients studied was small, which limited our ability to draw any conclusions about the association of levels of NO₂ and NO₃ with clinical outcomes such as mortality or duration of mechanical ventilation.

In summary, this study provides the first direct evidence that reactive nitrogen species may play a role in the pathogenesis of human lung ALI. Markedly elevated levels of NO₂ and NO₃ in plasma and pulmonary edema fluid were measured in patients with ALI/ARDS, and there was a significant correlation between the elevated plasma NO₂ + NO₃ levels and the presence of shock and systemic hypoperfusion. Also, patients with slower rates of alveolar fluid clearance had higher levels of NO₃ in their pulmonary edema fluid than patients with maximal alveolar fluid clearance. Furthermore, the presence of nitrated SP-A in the pulmonary edema fluid of patients with lung injury provides novel evidence that proteins are nitrated by reactive nitrogen species in human ALI.