INTRODUCTION

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Keywords: exhaled nitric oxide; asthma; liver cirrhosis; healthy subject

Nitric oxide (NO) is a molecular gas that can be detected in the exhaled gas of animals and humans, but its origin and physiological functions are incompletely understood. In the respiratory system, NO synthases are found in several cell types, including airway epithelial cells, vascular endothelial cells, macrophages, neutrophils, fibroblasts, and neuronal synapses (1). Virtually all these cells may contribute to the significant concentration of NO in exhaled air (FE_NO); consequently, lower respiratory tract NO can be of bronchial or alveolar origins. Besides its well known bronchial origin, which is responsible for high values in asthma (2), it has been postulated that despite its great affinity for hemoglobin, the NO produced by the vascular endothelial cells may diffuse into the alveolar space. Therefore, FE_NO has also been used as a marker of endothelial function in various settings (5). By contrast, several studies have claimed that most of the FE_NO is not of alveolar origin and thus FE_NO does not provide a marker of vascular endothelial function, at least in healthy humans (11, 12).

Recent guidelines have standardized the technique of FE_NO measurement to exclude nasal NO and to interpret the NO plateau during single flow exhalation (13, 14). Indeed, a specificity of the FE_NO is its marked flow dependency, FE_NO being inversely related to expiration flow rate (15). Recent theoretical studies have provided convincing proof that this flow dependency can be attributed to a double origin of NO from the alveoli and from the bronchial tree (3, 16). In these models, NO concentration, which is stable in alveolar space (FA_NO), is increased by bronchial excretion during the passage of exhalate through airways. This passive bronchial excretion depends on the gradient between NO concentration in the airway wall (CW) and bronchial lumen and on the diffusing capacity of NO transfer from the airways to the expired air (D_NO). The use of these models requires multiple flow rate measurements of FE_NO, allowing calculation of NO output from alveolar and bronchial origins. One approach takes advantage of the fact that the relationship between NO output and expiratory flow appears to be linear above a threshold > 50 ml/s (16, 17). In this range of flows, lumenal NO concentration can be considered negligible compared with CW, so that the bronchial NO output is maximal and constant, whatever the flow rate. Tsoukias and coworkers have shown that the slope of this linear relationship is representative of the constant alveolar NO concentration (FA_NO) and the intercept at zero flow of the maximal bronchial NO output (br,max_NO) (16, 17).

The aim of our study was to assess whether multiple flow analysis of NO output allows the origin of the increased FE_NO observed usually both in patients with cirrhosis (6, 7, 9) and patients with asthma (1, 3) to be differentiated.

	METHODS

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Subjects

A total of 110 subjects (three groups: healthy condition, asthma, and liver cirrhosis) were enrolled in this study; all of them provided written informed consent. This human study was approved by our Institutional Review Board.

Healthy nonsmokers (never or ex-smokers) and subjects who currently smokeAll the 56 subjects (25 males, 31 females) were normotensive, were taking no medication, and had no evidence of cardiopulmonary or allergic disease at the time of the study. The subjects had a normal spirometry.

Nonsmoking subjects with asthmaTwenty-eight subjects with mild atopic asthma were studied. All subjects had a history compatible with asthma, an FEV₁ > 80% predicted, and a positive skin test to common allergens (20). Subjects were not using any asthma medications, with the exception of inhaled -agonists.

Patients with liver cirrhosisTwenty-six patients with liver cirrhosis were referred for pulmonary function tests while they were awaiting orthotopic liver transplantation. All had biopsy-proven liver cirrhosis and none had primary lung disease or portopulmonary hypertension. Physical examination findings and blood data were analyzed to classify hepatocellular function in liver cirrhosis according to the Child criteria (21). Smoking history was recorded for all patients. The patients were included in the study if they had normal pulmonary function tests. Sixteen of 26 patients were considered to have hepatopulmonary syndrome defined as advanced liver disease, increased alveolar-arterial oxygen gradient (> 20 mm Hg) while breathing room air, and absence of other known lung disease (22).

FE_NO Measurement Procedure

The breathing circuit consisted of a mouthpiece with a bacterial filter connected to a one-way valve, through which subjects exhaled via an expiratory resistance while targeting a fixed-mouth pressure of 20 cm H₂O displayed on a water column. The fixed-mouth pressure and resistance create a constant expiratory flow rate. Subjects exhaled via six separate resistances in turn while maintaining the same expiratory pressure, giving six flows of 50, 100, 150, 200, 250, and 300 ml/s, as measured by a downstream pneumotachograph (Fleisch #1, Lausanne, Switzerland; connected to a Validyne ± 2 cm H₂O, Northridge, CA) and corrected to take into account the analyzer sampling flow. These flow rates were chosen because they were in a range acceptable to most subjects.

The technique for NO measurement was as recommended by ATS guidelines (14) (a nose clip was not used; after inhalation to total lung capacity, the subjects exhaled immediately without breathholding). The exhaled NO single-breath profile showed a washout phase followed by a steady plateau; the latter gave the exhaled NO value. The criteria used for FE_NO interpretation were those of recent ATS guidelines (14) (the duration of expiratory time was at least 6 s with a plateau duration of at least 3 s). It has to be noted that with the expiratory flows used, the duration of NO plateau was usually at least 5 to 15 s. In addition, the value of the plateau was selected after 30% of vital capacity was exhaled, allowing a constant concentration (90% mixing) of NO to be reached that is maintained during the remainder of exhalation (18). NO was detected with a chemiluminescent analyzer (EVA4000, Seres, France) with a lower limit of detection of 1 ppb and NO sampling rate of 30 L/h. Calibration was performed with a zero gas and standard 100 and 800 ppb NO gas (AGA, Sweden) in accordance with ERS task force guidelines (13). As subjects inhaled ambient air, ambient NO concentration was measured at the time of each test. If ambient NO concentration was high (> 50 ppb), the subjects then inhaled air free of NO as recommended (13). NO concentration, expiratory flow, and expired volume were displayed on a computer (Biopac Systems Inc., Santa Barbara, CA).

Modeling the NO Output-Flow Rate Relationship

Simultaneous measurements of FE_NO and expiratory flow () were used to calculate NO output (_NO) using the following formula:

where 0.06 is a correction factor, _NO is expressed in nl/min, FE_NO in ppb, and in ml/s.

The calculated _NO values were represented as a function of the flow rates. Least-square linear regression over the _NO versus data was performed for flow rates  50 ml/s. According to the analysis of Tsoukias and coworkers (16, 17), the slope of this regression line can be considered as the alveolar NO concentration (FA_NO) and the intercept to zero flow as the maximal bronchial NO output (br,max_NO). FE_NO50 and FE_NO200 were then computed as

for equal to 50 (FE_NO50) ml/s and 200 ml/s (FE_NO200), respectively.

These calculated values were compared with the measured values of FE_NO50 and FE_NO200.

Pulmonary Function

Spirometry, flow-volume curves, diffusing capacity for carbon monoxide (single-breath DL_CO measurement), and arterial blood gas analysis were performed using conventional methods. Pulmonary function testing was always performed after FE_NO measurement as repeated spirometry can modify exhaled NO (23).

Statistical Analysis

Data are expressed as mean ± SEMs. Multiple groups were analyzed through factorial analysis of variance (ANOVA), and Fisher's post hoc method was used for secondary analysis. Comparisons between smokers and nonsmokers were made by unpaired t tests. p Values of less than 0.05 were considered significant.

The sensitivity and specificity of possible cut-off points for br,max_NO, FA_NO, FE_NO50, and FE_NO200 in discriminating between subjects with different conditions were determined with receiver-operator characteristic (ROC) curves. The ROC curves plot all possible combinations between the true-positive ratio (sensitivity; y axis) and the false-positive ratio (1  specificity; x axis) as one varies the definition of positivity. Different points were therefore obtained by varying br,max_NO, FA_NO, FE_NO50, and FE_NO200 values taken as the criterion for positivity. The best compromise between sensitivity and specificity was defined as the point of the curve closest to the upper left-hand corner.

	RESULTS

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For all subjects, we found that the flow dependency of FE_NO over a wide range of flow rates ( 50 ml/s) resulted in a linear relationship (for all subjects R  0.97) between NO output and expiratory flow allowing br,max_NO and FA_NO to be computed. Due to the high correlation of this linear relationship, measured and calculated values of FE_NO (at 50 and 200 ml/s) were not significantly different (paired t test). For instance, in patients with asthma, calculated values of FE_NO50 and FE_NO200 were 50.7 ± 4.7 and 17.5 ± 1.4 ppb, respectively, these values being close to the measured FE_NO (see Table 1).

Flow Dependency of FE_NO in Healthy Nonsmoking Subjects and Subjects Who Smoke

Values for alveolar NO concentration, bronchial NO output, and calculated FE_NO at 50 and 200 ml/s for healthy nonsmoking subjects and subjects who currently smoke are given in Table 1. The average proportion of alveolar origin was 31 ± 2% at a flow rate of 50 ml/s and 61 ± 3% at a higher flow rate of 200 ml/s in healthy nonsmoking subjects. A significant decrease in bronchial NO output and a trend toward a decrease in alveolar NO concentration were evident in healthy smokers as compared with nonsmokers.

A significant positive relationship was evidenced in healthy nonsmokers between maximal bronchial NO output and height (Figure 1) and weight (r = 0.41; p = 0.02). No correlation was evident between bronchial NO output and body mass index (weight/height²) (p = 0.11). FE_NO50 also correlated with height and weight, although to a lesser extent (r = 0.35; p = 0.04, and r = 0.34; p = 0.04, respectively), and no correlation was observed between FE_NO200 and height or weight.

View larger version (17K): [in this window] [in a new window]   Figure 1.   Correlation between maximal bronchial NO output (nl/min) and height (cm) in healthy nonsmoking subjects, p = 0.02. Y = 77.979 + 0.666 X; R = 0.376.

Flow Dependency of FE_NO in Patients with Asthma

Increased FE_NO values were evident in subjects with asthma compared with healthy nonsmoking subjects. These increased values were mainly related to an increase in maximal bronchial output. An insignificant increase in alveolar NO concentration was also observed (Table 1).

In these patients with asthma, when the linear model took into account only values obtained with flow rates  100 ml/s, the correlation coefficient of the least-square linear regression showed a slight improvement. A small decrease in alveolar NO concentration (5.9 ± 0.8 ppb) and increase in the maximal bronchial NO output (142 ± 18 nl/min) were observed. This could indicate that the linearity of the relationship between NO output and expiratory flow could occur at higher flow rates than in healthy subjects (personal observations), which could artificially increase the value of alveolar NO concentration. However, this effect remains of small magnitude for our subjects.

Single flow measurements of FE_NO were as effective in differentiating patients with asthma from healthy subjects. Figure 2 shows ROC curves corresponding to the sensitivity and specificity of possible cut-off points for br,max_NO, FA_NO, FE_NO50, and FE_NO200 to discriminate between patients with asthma and healthy subjects. br,max_NO, FE_NO50, and FE_NO200 were similarly able to separate these two groups with excellent sensitivity and specificity. As expected, FA_NO was not able to separate these two groups.

View larger version (30K): [in this window] [in a new window]   Figure 2.   Comparison of receiver-operator curves (ROC) corresponding to the sensitivity and specificity of possible cut-off points for maximal bronchial NO output (br,max NO), alveolar NO concentration (FANO), and exhaled NO concentration at 50 ml/s (FENO50) and at 200 ml/s (FENO200) to discriminate between patients with asthma and healthy subjects. Each point indicates the sensitivity and specificity of a given p value derived from the logistic regression model and ranging from 0 to 1. The cut-off values producing the best sensitivity and specificity are given for br,max NO, FENO50, and FENO200. , br NO; , FENO200; , FENO50; , FANO.

Flow Dependency of FE_NO in Patients with Liver Cirrhosis

Pulmonary function tests of the 26 patients with liver cirrhosis demonstrated a normal total lung capacity (94 ± 2% of predicted value), a mean Pa_O2 of 83 ± 2 mm Hg, a decrease in DL_CO (68 ± 4% of predicted value) due to a decrease in K_CO (78 ± 4% of predicted value), with a normal alveolar volume (88 ± 3% of predicted value).

Nonsmokers and smokers were similarly distributed in healthy patients and patients with cirrhosis (² > 0.05). Increased FE_NO values were evident in patients with cirrhosis as compared with healthy subjects, whatever their smoking history. These increased values were related to an increase in alveolar NO concentration without any significant change in maximal bronchial output (Table 1).

Figure 3 shows ROC curves corresponding to the sensitivity and specificity of possible cut-off points for br,max_NO, FA_NO, FE_NO50, and FE_NO200 to discriminate between patients with cirrhosis with or without hypoxemia as defined by an alveolar-arterial oxygen difference larger than 20 mm Hg. The best results were obtained for FA_NO followed by FE_NO200. This latter result is consistent with the fact that alveolar NO concentration represented 70 ± 4% of FE_NO200 in these patients. As expected, br,max_NO and FE_NO50 were not able to separate these two groups of patients.

View larger version (25K): [in this window] [in a new window]   Figure 3.   Comparison of receiver-operator curves (ROC) corresponding to the sensitivity and specificity of possible cut-off points for maximal bronchial NO output (br,max NO), alveolar NO concentration (FANO), and exhaled NO concentration at 50 ml/s (FENO50) and at 200 ml/s (FENO200) to discriminate between patients with cirrhosis with or without hypoxemia as defined by an alveolar-arterial oxygen difference larger than 20 mm Hg. Each point indicates the sensitivity and specificity of a given p value derived from the logistic regression model and ranging from 0 to 1. The cut-off values producing the best sensitivity and specificity are given for FANO and FENO200. , br NO; , FENO200; , FENO50; , FANO.

A significant positive relationship between the alveolar- arterial difference of partial pressure of oxygen and alveolar NO concentration was evident (Figure 4) (p = 0.01), and a decreased but significant negative relationship was evident between arterial partial oxygen pressure and alveolar NO concentration (p = 0.04). There was no correlation between FE_NO values at 50 ml/s or 200 ml/s with D(A-a)O₂ and Pa_O2, and no correlation was evident between alveolar NO concentration and DL_CO or K_CO.

View larger version (16K): [in this window] [in a new window]   Figure 4.   Correlation between alveolar NO concentration (ppb) and alveolar-arterial difference of partial pressure of oxygen (mm Hg) in patients with liver cirrhosis (n = 26), p = 0.007. Patients were classified according to their Child criteria: class A (open circles), class B (gray circles), and class C (black circles). Y = 14.184 + 1.182 X; R = 0.515.

	DISCUSSION

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The main finding of this study demonstrates that multiple flow rate analysis allows to differentiate the increase in exhaled NO from an alveolar source to be differentiated from the increase from a bronchial source. The increased maximal bronchial NO output observed during asthma has been previously shown (3, 4, 24); however, this is the first study demonstrating that increased NO output during liver cirrhosis is of alveolar origin and could participate in arterial hypoxemia.

Recent guidelines for measurement of exhaled NO have been provided by both the European Respiratory Society task force (13) and the American Thoracic Society (14). The two Societies have emphasized that a single breath method should be used, with a given flow, as measurement of FE_NO depends on the value of the expiratory flow. At the same time, this flow dependency of FE_NO has been attributed to a double origin of NO, namely, alveolar and bronchial, with the use of two-compartment models (19). In these models, NO concentration, which is stable in FA_NO, is increased by bronchial excretion during the passage of exhalate through airways. Thus, the determination of these model parameters, using multiple flow rates, should allow the increase in exhaled NO from an alveolar or a bronchial source to be differentiated. From a pathophysiological point of view, it seems evident that determining these parameters would be of value (19). Our aim was therefore to determine whether one of these models gives additional knowledge in a clinical sense.

Our data in normal subjects are in agreement with previous evaluation of alveolar and bronchial NO compartments. FA_NO values were found to be 5.1 ± 1.8, 5.6 ± 3.1, and 5.0 ± 1.0 ppb, in our study and in the studies by Tsoukias and coworkers (16) and Silkoff and coworkers (3), respectively. However, other investigators have reported lower values of alveolar NO concentration (2, 4); methodological issues, such as the use of higher flow rates, could explain these discrepancies. Maximal bronchial NO output was found to be 37 ± 16, 43 ± 15, and 42 ± 6 nl/min, in our study and in the studies by Tsoukias and coworkers (16) and Lehtimaki and coworkers (4), respectively. Thus, multiple flow analysis of NO output provides the opportunity to compare flow-independent NO exchange parameters between studies, a need that was emphasized by recent guidelines (13, 14). An interesting finding is the positive relationship evident between maximal bronchial NO output and height in healthy nonsmoking subjects. It can be hypothesized that the taller patients have a higher lumenal surface area and consequently higher D_NO. Along this line, one recent study has also demonstrated a positive relationship between FE_NO and height (25).

Besides the scientific interest in determining the relative contribution of bronchial and alveolar sources of NO, the determination of alveolar NO concentration and bronchial NO output through a model analysis of multiple-flow determination of FE_NO could enhance the ability to detect small increases in FE_NO from alveolar or bronchial sources. We were unable to demonstrate the superiority of these calculated parameters in our clinical settings (healthy condition, asthma, and liver cirrhosis), provided an adequate flow rate for single-flow determination of FE_NO is chosen. Indeed, our data clearly show that in the case of measurements of FE_NO at a single expiratory flow as recommended by ERS and ATS guidelines (13, 14), the choice of the expiratory flow should be based on the type of disease studied, that is, a low flow of 50 to 100 ml/s for bronchial diseases where FE_NO is mainly dependent on the bronchial output and a higher flow of  200 ml/s for alveolar diseases where FE_NO is mainly dependent on alveolar NO concentration.

The importance of the choice of the value of the single flow rate to measure FE_NO is illustrated by the debate on the alveolar contribution to exhaled NO (11). For instance, Sartori and coworkers have evaluated whether exhaled NO is a marker of vascular endothelial function in healthy humans using epithelial or endothelial NO synthase inhibition (12). These authors concluded that NO is mostly of epithelial rather than endothelial origin as NO output decreased by 10% from 40 ± 5 to 36 ± 5 nl/min when endothelial NOS was inhibited. Although the value of expiratory flow was not given, it can be inferred from our data that the NO output value of 40 ± 5 nl/min was obtained with an expiratory flow < 50 ml/s. Similarly, Byrnes and coworkers concluded that NO is produced in airways and not at an alveolar level in healthy humans using expiratory flow of 440 ml/min (~ 7 ml/s) and 665 ml/min (11 ml/s) (11). The conclusion of these two studies, namely, a weak contribution of the alveolar origin of NO, seems therefore due to their experimental design, which allowed the assessment of NO output primarily from a bronchial origin, and it appears that using a higher expiratory flow could have led to different conclusions: our results clearly show that the "alveolar" NO contribution to exhaled NO increases from low to high flow rates, reaching 61 ± 3% of an expiratory flow of 200 ml/s in healthy subjects.

The two-compartment model of pulmonary NO exchange dynamics has been validated with the demonstration that patients with asthma are characterized by an increase in maximal bronchial NO output (3, 4, 24), and recently by Lehtimaki and coworkers who demonstrated that alveolar NO concentration was higher in alveolitis than in asthma or in a healthy condition (4).

The main finding of this study is that the enhanced NO output observed during liver cirrhosis that has been previously shown (6, 7, 26) is of alveolar origin. This suggests either increased production of NO locally in the alveolar region together with an impairment of diffusion across the alveolar capillary wall, or alternatively, increased excretion of NO from the pulmonary circulation into the alveolar region. A recent experimental study provides arguments for the latter hypothesis as increased exhaled NO in cirrhotic rats has been mainly related to increased production by pulmonary intravascular macrophages (inducible NO synthase), together with a moderate increase in pulmonary expression of endothelial NO synthase (27). The correlation between the impairment of oxygenation and alveolar NO concentration strongly suggests a causal relationship in this setting, as previously shown by Rolla and coworkers (26), which is also supported by the fact that the improvement in oxygenation observed after liver transplantation is associated with a decrease in exhaled NO (21). Moreover, a recent case report by these authors has suggested that smoking, by decreasing respiratory NO, apparently contributed to improved oxygenation in a 44-year-old man with alcohol-induced cirrhosis, complicated by hepatopulmonary syndrome, which further reinforces the hypothesis that NO is the most important vasodilating mediator in the hepatopulmonary syndrome (28). Along this line, a correlation between exhaled NO and cardiac index has been demonstrated in patients with liver cirrhosis (7, 26), and an enhanced endothelium-dependent vasodilatation of forearm resistance vessels in patients with cirrhosis has been shown, suggesting also an increased synthesis of NO in the vascular endothelium (29). It can be concluded that NO could be one of the main factors contributing to the hyperdynamic circulation seen in patients with cirrhosis by mediating the decreases in vascular tone and reactivity (22). All these data suggest that alveolar NO concentration might be used to assess the severity of the hepatopulmonary syndrome.

In summary, the linear model analysis we used was based on six flow NO measurements that were easily obtained in our patients, and it provided two flow-independent parameters characterizing NO excretion, alveolar concentration of NO, and maximal bronchial NO output. Such analysis allowed us to differentiate the increase in exhaled NO from alveolar or bronchial sources, allowed us to assess the severity of the hepatopulmonary syndrome, and could help in standardizing the evaluation of exhaled NO. Further studies are needed to evaluate the recently described single-breath technique with variable flow rate to characterize these flow-independent parameters (30).