INTRODUCTION

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Nitric oxide (NO) concentration may be detected continuously in the air exhaled by human beings (1). Although the NO concentration detected at the mouth is the difference between what is produced in the respiratory system and what is transformed or eliminated continuously by other endogenous pathways, the NO concentration in exhaled air (ECNO) or the quantity of exhaled NO over time (EVNO) are generally assumed to reflect the NO produced by cells within the respiratory tract. In this tract, NO may be generated by three isoforms of enzyme NO synthases. Two isoforms are considered to be constitutive. One is found in pulmonary arterial and venous endothelial cells and in epithelial cells, and the other is found in nonadrenergic noncholinergic inhibitory neurons (4- 7). These two forms are activated by a rise in intracellular calcium, generally in response to physiologic stimuli such as shear forces exerted by the circulating blood (4). The third isoform is considered to be inductive. It exists in several inflammatory cells or in cells such as endothelial and smooth muscle cells, and it is generated in response to proinflammatory cytokines and endotoxin (4, 8). Thus, an increase in ECNO or in EVNO values has been found in patients with pulmonary diseases such as asthma (9), respiratory tract inflammation (17), or hepatopulmonary syndrome (18). These data suggest that the measurement of exhaled NO might be a useful marker for airway and pulmonary diseases (19, 20). From this point of view, it is very important to obtain an accurate measurement of exhaled NO.

The usual technique used to estimate exhaled NO, particularly in patients, is to measure ECNO either in a vital capacity volume exhaled directly into a NO analyzer (11, 17, 21, 22) or in the volume resulting from ventilation exhaled into a bag for a few minutes and measured subsequently (1, 9, 10, 14, 23). Under these conditions, the value of the expired volume is not usually taken into account, and the peak NO concentration obtained at the end of the vital capacity or the mean NO concentration measured in the bag are considered to represent the exhaled NO (9, 1, 17, 21, 22). On the other hand, although in some studies subjects inhaled a NO concentration as close as possible to NO-free air (3, 9, 15, 23), in other studies subjects inhaled the ambient air. The ambient air may be contaminated with concentrations of NO that vary between 6 and 200 ppb, depending on the weather and on the traffic conditions in the vicinity of the laboratory concerned. Thus, with the exception of some studies that took advantage of the presence of exceptionally pure ambient air (16, 23), in other studies the ambient NO concentrations were variable. Under these conditions, the estimation of ECNO varied according to the studies. In some studies, inhaled NO concentrations were subtracted from ECNO (2, 12, 27), and in other studies, inhaled NO concentration was not taken into account (11, 13, 14, 17, 21, 28, 29).

In fact, no systematic study has been carried out concerning the effect of low inhaled NO concentrations on ECNO and EVNO. Thus, the aim of the present study was to observe the effect that the contamination of air with the NO concentrations encountered in the normal environment has on ECNO and EVNO.

	METHODS

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Subjects

Ten healthy subjects, (four women and six men) gave their informed consent to participate in this study. Characteristics recorded were: age, mean 45 ± 6 yr; height, 169 ± 4 cm; weight, 67.8 ± 3.6 kg. None of the subjects was a smoker or had prior or present history of respiratory disease.

Experimental Procedure

All subjects were weighed and then sat comfortably in rocking chairs. Their noses were blocked with noseclips and they breathed into a regular mouthpiece attached to a unidirectional T-valve. The internal side of the T-valve was coated with Teflon.

The inspiratory side of the T-valve was connected via a three-way tap to polyethylene bags. The stability of these materials was checked beforehand to ensure that there was no interference with NO measurements. The stability was found to last for more than 24 h. One 2,000-L-capacity polyethylene bag was emptied with NO-free air, which was delivered through a teflon tube from a pressurized gas cylinder containing 21% O₂ and 79% nitrogen (certified; Air Liquide, Lyon, France). Four other 500-L-capacity polyethylene bags were filled with air containing NO concentrations as close as possible to 20, 50, 100, and 150 ppb. These different concentrations were carried out mixing the NO-free air (< 3 ppb) with appropriate amounts of the ambient air when the NO concentration in the ambient air was high. Under this condition, NO-free air from the pressurized gas cylinder containing 21% O₂ and 79% nitrogen was added slowly into the bag while the bag was shaken, and the NO concentration in the bag was continuously recorded. When the concentration reached the expected NO concentration, the bag was closed, shaken again, and the final NO concentration was controlled. When the NO concentration in the ambient air was low, the different concentrations were carried out mixing appropriate amounts of NO delivered from a pressurized gas cylinder containing 4,000 ppm NO into the 500-L bag filled with NO-free air. The precise inhaled NO concentrations obtained were 20 ± 1, 49 ± 1, 98 ± 2, and 148 ± 2 ppb, respectively. Each subject was connected at random to each bag via the inspiratory T-valve for 10 min. The subjects were encouraged to breathe as quietly as possibly, and no attempt was made to standardize the expiratory flow rate. However, between each bag, because some subjects had difficulty in tolerating the mouthpiece or the noseclips, the subjects were given a choice of either being connected immediately to another bag or to stop for 1 or 2 min. The total duration of the test was approximately 1 h.

For NO analysis of exhaled air, samples were drawn continuously from the expiratory part of the T-valve, at a flow rate of 400 ml/min. Water vapor was removed upstream from the analyzer by warming the sample. The NO concentration was measured by a NO chemiluminescence analyzer (Model Topaze 2020; Cosma, Igny, France). The detection limit for NO concentration was 1 ppb. Calibration was carried out before each experiment using a gas mixture of 883 ppb (certified calibration; Air Liquide). Moreover, the linearity of the measurement system for low concentrations was checked weekly, using 50 and then 100 ml of the same calibration NO gas injected into a teflon bag filled with 3 L of nitrogen with a 3-L calibration syringe (Hans Rudolph, St. Louis, MO). Only Teflon-coated tubes were used for calibration. The NO signal, which became stable after 10 s, was recorded continuously throughout the test by using a Mac Lab data-acquisition system and was calculated by averaging the samples every 30 s. In addition, the expiratory part of the T-valve was connected to a standard open circuit using an automated computerized analysis system (Brainware, Toulon, France). During the test, tidal volume, respiratory rate, oxygen consumption (O₂), and carbon dioxide production (CO₂) were determined continuously, and their values were calculated by averaging the samples every 30 s. Expiratory flow was measured with a pneumotachograph (Hans Rudolph). Before each test, calibration of the pneumotachograph was carried out using a 3-L syringe (Hans Rudolph), and the zirconium oxide cell O₂ (Servomex) and infrared CO₂ analyzers (Servomex) were calibrated by using gases of known concentrations. All measurements were corrected for ambient temperature, barometric pressure, and water vapor and expressed in BTPS units for minute ventilation and STPD for O₂ and CO₂. The level of NO in the expired gas was expressed both as a concentration in parts per billion (ppb) (ECNO) and as an amount expired per time unit (EVNO). The molar rate of output of NO was calculated by multiplying the concentration of exhaled NO and the minute ventilation (E) after correcting for atmospheric pressure and temperature. Moreover, the molar rate of inspired NO per unit time (InspVNO) was calculated by multiplying the inspired minute ventilation, by the concentration of inhaled NO in the different bags, after correcting for atmospheric pressure and temperature. The inspired minute ventilation was calculated from the E, taking into account the O₂ and CO₂ when the respiratory exchange ratio was not equal to 1. To allow comparison with previously published values, the molar rate were expressed in nanomoles per minute.

Statistical Analysis

All data are expressed as mean ± SE. The variations in ECNO and EVNO over the range of the different inhaled NO concentrations underwent ANOVA analysis for repeated measurements. When F value of ANOVA was significant, Student's t test for paired observations was used to determine differences between values obtained for the different inhaled NO concentrations. The relationships between ECNO and EVNO and inhaled NO concentrations were analyzed by standard linear regression methods. The accepted level of significance for all statistical tests was set at 5%.

	RESULTS

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The mean E obtained during inhalation of NO-free air was 8.58 ± 0.67 L/min with a mean rate of 16.2 ± 1.6 breaths/min. The mean O₂ and respiratory exchange ratio (CO₂/O₂) values obtained during inhalation of NO-free air were 261 ± 22 ml/min and 0.87 ± 0.02, respectively. These values were identical for inhalation of the different inhaled NO concentrations.

EVNO versus time for each inhaled NO concentration is illustrated in Figure 1. This EVNO value became stable after approximately 3 min. Thus, to avoid any possible interference with the previous inhaled NO concentration, only the values obtained for the last 5 min of breathing into the bag were taken into consideration when calculating the mean ECNO and EVNO values. For the breathing of NO-free air ECNO was 15.4 ± 2.7 ppb and EVNO was 4.18 ± 0.69 nmol/min. In each subject, these ECNO and EVNO values are considered as the share of the endogenous NO production rejected by the ventilatory pathway (endoECNO and endoEVNO).

View larger version (26K): [in this window] [in a new window]   Figure 1.   Mean values ± SE of quantity of exhaled NO over time (EVNO) versus time obtained for 10 subjects inhaling different NO concentrations (InspCNO).

Mean ECNO and EVNO values versus inhaled NO concentration are illustrated in Figure 2. Compared with endoECNO and endoEVNO, the mean values were significantly higher for an inhaled NO concentration of 20 ppb. Above 20 ppb, ECNO and EVNO increased with inhaled NO concentration. In each subject, a linear correlation was found between inhaled NO concentration, and ECNO and EVNO, respectively (Table 1).

View larger version (16K): [in this window] [in a new window]   Figure 2.   Mean values ± SE of exhaled NO concentration (ECNO) (A) and quantity of exhaled NO over time (EVNO) (B) versus inspired NO concentrations (InspCNO).

InspVNO, EVNO, and EVNO values obtained in each subject for NO-contaminated air after subtracting endoEVNO are illustrated in Figure 3. Indeed, the EVNO obtained for contaminated air may be considered as being composed of the endoEVNO and of an additional amount of NO because of air contamination. For 20 ± 1, 49 ± 1, 98 ± 2, and 148 ± 2 ppb inspired NO concentrations, this additional amount may be estimated as being 35, 35, 31, and 29% of InspVNO, respectively.

View larger version (17K): [in this window] [in a new window]   Figure 3.   Quantity of inspired NO over time (InspVNO), quantity of exhaled NO over time (EVNO), and the difference between EVNO obtained by inhalation of contaminated air and EVNO obtained by inhalation of NO-free air (endoEVNO) versus inspired NO concentrations (InspCNO).

	DISCUSSION

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The data of the present study were obtained by making continuous, simultaneous measurements of ECNO and E during normal breathing, over several minutes. Although the subjects wore noseclips, this method, as with other methods that leave the posterior nasopharynx open, does not preclude nasal contamination of the measurements. Thus, as much as 50% of the exhaled NO may come from the nose during mouth-breathing (20). In addition to other methods such as direct exhalation into a NO analyzer with the measurement of peak NO, or exhalation into a collection bag and the measurement of the mean ECNO concentration collected, this method seems to allow accurate estimation of the quantity of NO rejected by the ventilatory pathway. It has the advantage that it does not generate pressure increases in the NO analyzer, as occurs with direct exhalation into the apparatus. Moreover, when measuring vital capacity, holding the breath prior to expiration and the duration of exhalation have been found to affect the ECNO (16). In addition, in several studies, and particularly when single-breath measurement is involved, the exhaled volume is not taken into account, and exhaled NO is expressed only as a concentration (9, 11, 17, 21, 22). However, in order to estimate the rate of entry of NO into the airway, it seems more accurate to estimate the amount of exhaled NO over time.

Our data show that when the ambient air is contaminated with various NO concentrations, ECNO and EVNO are higher than when the ambient air is NO-free. Thus ECNO and EVNO values increase linearly with inspired NO concentrations. This increase may be reasonably ascribed to the fact that the amount of inhaled NO over time is not absorbed totally by the lung and that a share is rejected into the expired air. This share may be estimated theoretically by subtracting the EVNO value obtained when the subject inhaled NO-free air from the EVNO value obtained when the subject inhaled NO-contaminated air. In fact, such an estimate is possible only if InspVno does not affect endogenous production or the metabolic fate of NO in the airway. No experimental data support the possibility that breathing contaminated air induces a further production of NO endogenously. However, the possibility that breathing contaminated air induces a decrease in the endogenous production of NO is suggested in smoking men who exhale less NO than nonsmoking men (21). It has been suggested that increased NO concentrations in the inspired air may decrease NO synthesis. However, if true, this downregulation in NO synthases probably takes time to occur. Consequently, given the fact that endogenous production of NO did not change within the range of the inspired NO concentration studied, the share of the InspVNO that is rejected by the ventilatory pathway may be estimated as being approximately one third of the InspVNO. As a consequence, an increased NO in the ambient air overestimates the EVNO values. These results show the importance of using NO-free air to make estimates of the lung output of NO. This condition was not always taken into account since in several studies the subjects inhaled the ambient air. Under these conditions, whereas some studies took advantage of exceptionally pure air (16, 26), other studies used air contaminated with various NO concentrations. In these cases, the estimates of EVNO varied according to the investigators. In some studies, endogenous ECNO and EVNO were estimated by subtracting inspired NO concentration from the ECNO measured in the apparatus (2, 12, 27). From our data, it can be asserted that this method underestimates the endogenous ECNO. Moreover, for inspired NO concentration values higher than 25 ppb, no endogenous ECNO could be obtained during normal tidal breathing. In other studies inspired NO concentration was not taken into account (11, 13, 14, 17, 21, 28, 29). This last procedure was based on a few experiments which suggested that low inspired NO concentration values do not affect ECNO. Thus, Iwamoto and colleagues (28) reported that when NO was inhaled at a concentration of 100 ppb for 30 min, ECNO and EVNO remained similar to the values obtained during breathing of NO-free air. Unfortunately, these results were those of preliminary studies and few details were given about the exact procedure and the number of subjects. Kharitonov and colleagues (13) reported that when two healthy subjects inhaled 800 ppb NO and exhaled into the analyzer, after holding their breath for 15 s, there was no change in exhaled peak NO when compared with the inhaled ambient air, and that ambient-air NO levels as great as 68 ppb had no effect on ECNO (21). In the above study, NO measurement was performed 5 min and 15 min after inhalation of NO. If we look at Figure 1, which shows the evolution of EVNO over time, it can be seen that less than 5 min seem to be sufficient to obtain a new stable rate for EVNO when inspired NO concentration has increased or decreased. Robbins and colleagues (22) reported that inhalation of NO at 113 ppb did not affect the peak exhaled NO value. Massaro and coworkers (14) and Trolin and colleagues (29) found no differences in ECNO, whether the subjects inspired NO free air or ambient air. It can be noticed that all these investigators measured the peak NO concentration obtained at the end of a single vital capacity while we measured ECNO during tidal breathing. The possibility exists that the inspired NO concentration remaining in the dead space affects more the EVNO measurements during tidal breathing than during a complete vital capacity. However, recently, in disagreement with the above previous observations, Dötsch and colleagues (10) found a correlation between ECNO obtained during maximal breath maneuvers and the inspired NO concentration encountered in ambient air, in asthmatic patients, in patients with cystic fibrosis, and in control subjects. Above all, the opinions concerning the lack of effect of inhaled NO concentration on exhaled NO concentrations seem to be based on the fact that reaction kinetics of NO with hemoglobin are extremely rapid, and that inspired NO disappears rapidly from the respiratory tract because of this very high affinity (30).

This high affinity probably explains why the major share of the InspVNO is taken up by the endogenous pathway. It does not explain why approximately one third of InspVNO was rejected by the ventilatory pathway in our study.

In summary, this study has clearly shown that when inspired NO is greater than 20 ppb, both exhaled NO concentration and exhaled NO over time are artifactually increased. Therefore, in order to make a precise estimate of both, the concentration and the amount of exhaled NO from the lung over time, it is absolutely essential that inspired NO concentration should be as close as possible to NO-free air.