INTRODUCTION

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Nitric oxide (NO) plays a pivotal role in the regulation of local blood flow and vasomotor tone in both the systemic and pulmonary circulation (1). NO is synthesized by different forms of nitric oxide synthase (NOS) by oxidative deamination of the amino acid L-arginine (4). A calcium/calmodulin-regulated isoform of NOS (ecNOS), expressed by endothelial cells, may contribute to the modulation of pulmonary vascular tone under normal conditions. However, induction of a calcium-independent isoform of NOS, referred to as inducible NOS (iNOS), by various cytokines (e.g., interleukin-1, interleukin-4, interferon-, tumor necrosis factor-) and bacterial endotoxin leads to NO overproduction in many inflammatory conditions. This, in turn, results in inappropriate systemic vasodilation, which is a clinical hallmark of bacterial sepsis. Recently using various NOS isoform-deficient mice, Steudel and coworkers (5) suggested that iNOS is the main contributor to the synthesis of exhaled NO. Activation of iNOS in lung tissues of patients with asthma is consistent with the higher production of NO observed in the exhaled air in these patients (6).

Measurement of exhaled NO is a simple, noninvasive means to assess asthmatic airway inflammation (6, 9). We have shown that the level of exhaled NO is a poor reflection of bronchial tone because it is not altered by pharmacologically induced bronchoconstriction (10). By contrast, exhaled NO strongly correlates with the expression of iNOS in alveolar macrophages and markers of eosinophilic airway inflammation present in induced sputum from subjects with asthma (11, 12). Measurement of exhaled NO can be applied to detect activation of iNOS of inflammatory cells from peripheral airways and alveolar tissue. Nasal contamination of NO in exhaled air often occurs. Therefore, maneuvers such as slow exhalation through the mouth against resistance is used to maintain uvular closure, thereby avoiding nasal contamination of expiratory gas (13). Still, this measurement maneuver does not allow for the measure of absolute NO in alveolar gas. Intubated and ventilated patients represent a population in which it is possible to definitively separate upper airways and alveolar space (14).

There is no "gold standard" for the diagnosis of pneumonia that is based on a combination of criteria, such as fever and leukocytosis, the results of tracheal-aspirate cultures, and the presence of infiltrate on chest roentgenograms. Each criterion is too nonspecific to be useful by itself, and when associated with each other they increase but do not definitively identify pneumonia in ventilated patients (15). New pulmonary infiltrates, leukocytosis, and fever are common in ventilated patients and are often related to other processes mimicking the diagnosis of pneumonia. Moreover purulent tracheal secretions with or without microorganisms isolated by analysis of endotracheal aspirates do not distinguish tracheobronchial colonization from pneumonia, especially in patients previously receiving antibiotics. The uncertainties related to this "clinical" approach have prompted new invasive methods in order to improve the identification of patients with true pneumonia and selection of appropriate antibiotics, but the value of such tests remains controversial (16).

Because inflammation is a primary process resulting from infection, and because NO is a primary mediator released during inflammation, we hypothesized that exhaled NO would be elevated in ventilator-dependent patients with pneumonia and could therefore represent a marker of pneumonia in this patient population. Accordingly, we measured the exhaled NO in tracheal gas in patients admitted and ventilated within 72 h in our intensive care unit (ICU). Patients were also diagnosed as having acute pneumonia or not. In a second group of similar patients, we determined the sensitivity and specificity of the NO threshold calculated from the first group of patients. We also investigated whether there is a correlation between exhaled NO levels, aspirated nasal air NO levels, and nitrite/ nitrate plasma concentrations. We reasoned that nonspecific inflammation could induce iNOS expression specifically in the airway epithelium, as manifest by an associated increase in NO levels in the nasal passages, or nonspecifically, as manifest by an increase in plasma nitrite levels.

	METHODS

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Study Cohort

All patients hospitalized and mechanically ventilated within 72 h in one intensive care unit were included in the study. This study was approved by the ethics committee of Cochin Port-Royal hospital and informed consent was obtained from the patient's next of kin. Severity scores calculated for the first day in the ICU were the Simplified Acute Physiology Score (SAPS II) (19). Patients were stratified into several overlapping groups: pneumonia, acute respiratory distress syndrome (ARDS), sepsis, and smoking-related chronic obstructive lung disease (COPD). Post hoc diagnosis of pneumonia was made from a combination of clinical criteria including fever (> 38° C), new and persistent lung infiltrates on chest roentgenograms, macroscopically purulent tracheal secretions, and elevated white blood cell count (> 12,000/mm³) with or without microbiological evidence of bacterial infection. Independent observers blinded to the exhaled NO values interpreted these criteria for each subject. These criteria are similar to those used for ventilator-associated pneumonia described by Pugin and coworkers (15). Pneumonia was considered to be absent when an alternative cause for pulmonary infiltrates was established or no substantial growth occurred on a reliable airway culture in association with the resolution of fever, infiltrate, and leukocytosis.

The diagnosis of ARDS was made based on the American-European consensus conference recommendations (20), which included the following criteria: acute onset of illness, arterial hypoxemia with a Pa_O2/FI_O2 ratio lower than 200 (regardless of positive end-expiratory pressure [PEEP] level), bilateral infiltrates seen on chest roentgenogram, pulmonary artery occlusive pressure lower than 18 mm Hg or no clinical evidence of atrial hypertension, and compatible risk factors. Patients were classified as having the sepsis syndrome or septic shock if they met the criteria described by Bone and coworkers (21). Smoking habit within 1 mo before admission was quantified as following; 0, no smoking; 1, 1-10 cigarettes/d; 2, 10-20 cigarettes/d; 3, > 20 cigarettes/d. Finally organ dysfunctions were defined accordingly to the definition in the Logistic Organ Dysfunction system (22).

The extent of roentgenographic densities provides an assessment of the presence and severity of increased permeability pulmonary edema, the primary consequence of acute pulmonary injury, and of the subsequent resolution or organization of the process. We used the chest roentgenogram score established by Murray and coworkers (23) as follow: 0, no alveolar consolidation; 1 through 4, alveolar consolidation confined to 1, 2, 3, or 4 quadrants, respectively.

Measurements

Exhaled tracheal or nasal NO. Airway NO levels were measured by chemiluminescence using a fast-responding analyzer (NOA 280, Sievers Instruments Inc., CO). Quantification of NO by this method is based on the gas-phase interaction between NO and ozone (O₃) (Equation 1). Some of the NO₂ produced by this reaction is in the excited state (NO₂*) and emits light (h) as electrons return to ground state, as described by the following equation:

(1)

Light is measured by a cooled, photomultiplier tube. The analog signal is sampled 20 times/s, digitized (Sievers Instruments Inc., CO), permitting breath-by-breath detection of NO. The analyzer was calibrated with a certified 110 parts per billion (ppb) NO source (Air Liquide Santé, France) and oxygen (0 ppb) before each measurement.

We measured NO levels in both the airway via the endotracheal tube and the nasal cavity via a sampling catheter. The tracheal gas NO concentration was measured by sampling directly from the distal end of the tracheal tube for 1 min after overinflation of the tracheal tube cuff (40 cm H₂O). NO values at the end-inspiratory (minimal concentration) or end-expiratory period (maximal concentration) were recorded using simultaneous airway pressure detection. Mean exhaled NO concentration was computed from data collection from expired gas. To measure nasal NO levels, air was sampled through a nasal prong introduced ~ 2 cm from the aperture of each nostril sequentially. The nasal opening was then occluded in order to measure nasal NO production at a 200 ml/min sampling flow rate into the apparatus. We expressed NO concentration values averaged from the right and left nostrils. Preliminary studies showed good reproducibility for this method. We also measured NO levels in room air and medical air used for ventilating our patients.

NO production (VNOstpd) (L/min) was measured as

(2)

Where E is the minute ventilation, 0.826 is a conversion factor to adjust volumes to standard temperature and pressure (24), and [NO]_exh is the mean exhaled NO concentration.

Measurement of plasma nitrate (NO₃) and nitrite (NO₂). NO does not persist per se in the blood once formed but is rapidly inactivated by interaction with hemoglobin and oxidized to form several nitrogen oxides (NO_x), in particular, nitrate (NO₃) and nitrite (NO₂). Thus, to measure the NO load in blood we used the vanadium reduction reaction as follows. After deproteinization by alcohol, NO_x was measured in a small volume of plasma by converting it to NO using a strong reducing environment: vanadium (iii) in the presence of 1 N HCl at 90° C (Equation 3) (25). An antifoaming agent is also added (FG-10, Dow Corning, Midland MI):

(3)

Validation cohort. We defined the threshold of nasal and tracheal NO levels associated with pneumonia in the study cohort of 49 patients. Subsequently, sensitivity, specificity, and positive and negative predictive values of these thresholds for pneumonia were evaluated in a second cohort of 60 patients consecutively admitted in our department (validation cohort).

Statistical Analysis

Values are expressed as the mean ± standard error. To analyze the relationship between categorical and continuous variables, the continuous variables were first plotted against the categorical variable, and the LOWESS smoothing function, using locally weighted least squares, was performed to identify the presence of a threshold. Continuous variable analyses with a threshold were then categorized using the suggested cut points. The characteristics of patients within each diagnosis group (e.g., pneumonia, ARDS, sepsis) were then compared using chi-square tests for categorical variables and Mann-Whitney or Kruskal-Wallis tests for numerical data. The relationship between two continuous variables was analyzed using Spearman's rank correlation tests. Relative risks and positive and negative predictive values were calculated using standard formulas. A two-tailed p value of less that 0.05 was considered to indicate significance.

	RESULTS

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Patient Characteristics

Forty-nine consecutive patients admitted within 72 h to our ICU were included. There were 22 females and 27 males, 56.6 ± 2.4 yr of age (mean ± SD). The SAPS II score was 54.9 ± 2.7. Thirteen patients had clinical features diagnostic of ARDS, whereas six had septic shock, six had COPD, and none of the patients had asthma either by history or physical examination. Twenty-one patients overall were treated for pneumonia, with the most common diagnosis being pneumonitis secondary to aspiration of gastric contents (Figure 1). Baseline characteristics of these patients are summarized in Table 1.

View larger version (19K): [in this window] [in a new window]   Figure 1.   Actual trace of NO measurement in a patient presenting with pneumonia. This is a 35-yr-old patient admitted for coma after voluntary toxic exposure to benzodiazepines. This patient presented with aspiration of gastric content secondary to consciousness impairment. The upper graph represents airway pressure measured to the tracheal tube and the lower graph represents NO detection by using a fast response analyzer.

Tracheal and Nasal NO

We found little NO in ambient air (8 ± 1.9 ppb) and none in compressed air delivered (0 ppb [0.2 ± 0.05 ppb]) to our patients through the hospital compressed air system. The lack of NO contamination in our medical gas, as compared with other medical centers, was due to the use of specific filters in our institution to eliminate NO contamination. The end-expiratory and mean expiratory NO levels measured at the trachea were very low (4.3 ± 0.5 and 2.3 ± 0.3 ppb, respectively), corresponding to a very low NO production in the lung (0.91 ± 0.1 nmol/min). Although the exhaled NO concentrations were low in all patient groups, patients with pneumonia had significantly higher tracheal NO levels and NO output (VNO) than patients who did not have pneumonia (Figure 2A and 2B). This difference was even more pronounced when considering nasal NO levels (Figure 3). However, there was only a weak but significant correlation between VNO and nasal NO levels (r = 0.33, p = 0.02). The concentrations of exhaled NO were not affected by the extent of alveolar consolidation as assessed by the Murray score. We did not observe any significant influence of smoking habits on tracheal or nasal NO levels. Specifically, a positive history of smoking was not associated with a decreased production of NO in either lung or nostrils (Table 2). There was no significant difference in exhaled or nasal NO between patients with and without COPD (Table 2) and between patients with and without sepsis (data not shown).

View larger version (31K): [in this window] [in a new window]   View larger version (25K): [in this window] [in a new window]   Figure 2.   Tracheal NO levels measured are elevated in patients with pneumonia compared with patients without pneumonia (A) and production of NO (VNO) expired per minute (B). NO was measured at the end-inspiratory (minimal concentration) or end-expiratory period (maximal concentration) using pressure detection. Mean exhaled NO concentration was computed from data collection by the analyzer. *p < 0.05 versus control group.

View larger version (23K): [in this window] [in a new window]   Figure 3.   Nasal NO concentrations were higher in ventilated patients with pneumonia. Nasal air was drawn into the NO analyzer through a nasal prong introduced ~ 2 cm in each nostril sequentially, which was then occluded in order to measure the nasal production at the 200 ml/ min sampling flow rate of the apparatus. *p < 0.05 versus control group.

An end-expiratory tracheal threshold value of 5 ppb, a mean tracheal threshold value of 2.5 ppb, a VNO threshold value of 1.0 nmol/min, and a nasal threshold value of 500 ppb were considered definitive for the diagnosis of pneumonia (as summarized in Table 3). Using an independent cohort of 60 consecutive patients (validation cohort), similar to the first cohort in all parameters studied (Table 4), we further determined the sensitivity and specificity of these threshold values. The sensitivity and specificity of the threshold values are summarized in Table 5. Maximum (end-expiratory) tracheal NO values appear to be the best markers of acute pneumonia with a sensitivity of 88% and a specificity of 76%.

Plasma NO₂/NO₃ Concentrations

Mean plasma concentration of NO₂/NO₃ (NO_x) was 38.1 ± 6.9 µM in our study group. There was no significant difference between patients with or without pneumonia, although the trend was for NO_x levels to be lower in patients with pneumonia (Table 3). Furthermore, we did not observe any correlation between exhaled NO levels and plasma NO_x concentrations in either the total population or within subgroups. However, there was a significant inverse relationship between VNO and plasma NO_x in patients with pneumonia. Those with the highest NO_x levels had the lowest VNO (r = 0.53, p = 0.01) (Figure 4).

View larger version (12K): [in this window] [in a new window]   Figure 4.   Correlation between VNO and plasma NOx in patients with pneumonia. We found a significant inverse relationship between the two variables (r = 0.53, p = 0.01), which means that those with the highest NOx levels had the lowest VNO.

We did not observe a significant difference between plasma NO_x levels obtained from patients with and without sepsis (50 ± 12 and 32 ± 5 µM, respectively, NS) probably because among the patients without sepsis there were many other causes of increased plasma NO_x, such as renal failure or shock from other origins that may have independently led to elevated plasma NO_x levels.

	DISCUSSION

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In this study, we show that ventilator-dependent patients with pneumonia have significantly higher exhaled and nasal NO levels without associated increased systemic NO production, as assessed by plasma nitrate levels, as compared with patients with ARDS or other forms of acute respiratory failure. Although the levels of exhaled NO in all ventilator-dependent acutely ill patients are very low, the level of exhaled NO in ventilated patients with pneumonia is elevated relative to the levels seen in ventilated patients without pneumonia. The identification of threshold values of exhaled NO may help to distinguish patients with or without pneumonia. This increase was associated with higher nasal NO production in the same group of patients, suggesting that this phenomenon reflects a generalized airway epithelial response rather than a specific distal airway response.

In support of this hypothesis, although there was no correlation between exhaled NO and plasma NO_x in the total population, we found an inverse correlation with plasma NO_x levels in patients with pneumonia. This may be specific for pneumonia because this inverse correlation was not seen in patients with sepsis. These data agree with our previous findings (26) in an animal model of acid aspiration acute lung injury in which plasma NO_x levels fell following induction of acute lung injury but remained constant in control animals. More recently, Delen and coworkers (27) showed that exhaled nitric oxide was increased in chronic bronchitis but not in patients with COPD, which goes along with the increase of exhaled air observed in human subjects with upper respiratory tract infections (28). This study supports the fact that inflammation secondary to infection disease may well increase exhaled NO.

The detection of endogenously produced NO in expired air by Gustafsson and coworkers (29) indicated a new and noninvasive means of analyzing endogenous NO production in the lungs (9). However, if it arises from the pulmonary circulation production during intravenous NO donor administration, then it may represent a marker of organic nitrate tolerance (30). Importantly, little or no NO is released from the pulmonary capillary blood into the alveolar space (31). Accordingly, expired NO reflects production within lung tissue itself in normal lungs.

The global baseline values of exhaled NO in our patients are markedly lower than that in some recent reports (27, 32- 35). There are at least two main factors that might explain variation in the results. First, the exhaled NO has been measured using different techniques producing widely different values. The main contributors to these differences are contamination either from nasopharynx production or from ambient NO levels (14, 36). In our population, NO contamination from the nasopharynx was prevented by endotracheal intubation and NO contamination from compressed gas used to drive a positive-pressure ventilator was abolished by using filters that effectively scrubbed all NO from the compressed gas. This allowed us to measure the actual NO produced in the lungs.

Our study shows that exhaled NO is higher in patients with pneumonia within the first 72 h of mechanical ventilation. The discovery that exhaled NO is raised in patients with asthma has led to the expectation that it may provide a tool for measurement of inflammation in the lower respiratory tract. This was further strengthened by the increase of exhaled NO before any decrement in lung function or increase in asthma symptoms, suggesting that monitoring of exhaled NO may be useful in assessing asthma control (9, 34). However, other forms of airways inflammation may not be reflected in increased exhaled NO. Exhaled NO is indeed lower in patients with cystic fibrosis or COPD even during exacerbation of airway inflammation or its treatment by antibiotics (37). Despite signs of active inflammation of the airways, patients with bronchiectasis do not exhibit higher exhaled NO concentrations (38). The lack of increase in exhaled NO in suppurative airway diseases may be explained by a decrease in diffusion of NO across secretions of increased viscosity, protein concentration, and volume. Such aqueous solutions promote the reaction of NO with other reactive species or simply with water, leading to the formation of NO₂/NO₃. Limitation of diffusion into the airway lumen may also favor NO uptake in the bronchial or pulmonary circulation. Alternatively, in a suppurative and inflamed environment, the presence of reactive oxygen species in the vicinity of NO production may result in a chemical interaction, with the effective removal of free NO by formation of peroxynitrite. Indeed, NO has been shown to be a potent scavenger of reactive oxidants in the lung (41). The increase of exhaled NO observed in patients with pneumonia strengthens this hypothesis as suppurative processes occur more rarely and are more localized than those observed in bronchiectasis or chronic obstructive pulmonary disease. Furthermore, the inverse correlation between tracheal NO levels and plasma NO_x observed in our group of patients with pneumonia, as described in patients with anorexigen-associated pulmonary hypertension (42), supports the hypothesis that the more plasma NO_x is produced, the less NO is exhaled. However, this point should be interpreted very cautiously because of the very complex metabolism of NO in a whole body and the wide array of pathologies observed in our patients.

We also found an increase in nasal production of NO in patients with pneumonia that may be explained either by a general inflammation of both upper and lower airway tracts or by systemic inflammation. The lack of correlation between tracheal NO levels and nitrates in the whole population suggests that stimuli of NO production are more likely to be localized to the airway tract rather than secondary to a systemic inflammation. This is further strengthened by the absence of an increase in exhaled NO in the group of patients with septic shock in which iNOS is known to be diffusely overexpressed.

In contrast to previous reports (8, 43), we did not find any significant influence of smoking habit on exhaled NO. This lack of correlation may be explained by the fact that patients usually stop smoking at the occurrence of the first respiratory symptoms for a few hours to a few days or because our sample size was too small to observe this small effect (Table 2). In support of the hypothesis that smoking may induce only minimal changes in airway NO release, Crater and coworkers (32) found no relationship between exhaled NO and self-reported smoking habits in their control group. Patients with COPD may also have altered airway NO metabolism. However, the data supporting this interaction are inconsistent at best. Some workers report decreased NO production (35, 44), whereas others do not (27, 45, 46). Furthermore, we did not find any significant difference between patients with COPD and others. This discrepancy may also be explained by too small a sample size and the variability of the underlying or acute illness bringing the patients to the ICU independent of any effect of airway NO production (Table 2).

In conclusion, we found that when administering contamination-free NO gas to ventilator-dependent patients and measuring isolated tracheal gas, the exhaled NO levels are very low. However, patients with pneumonia exhaled significantly more NO than those without pneumonia. This dichotomy might be useful clinically to rapidly discriminate patients with pneumonia from those without pneumonia.