INTRODUCTION

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Several studies have demonstrated elevated levels of nitric oxide (NO) in the exhaled breath of patients with a variety of inflammatory lung diseases, including asthma (1) and bronchiectasis (4). Further, anti-inflammatory therapy is associated with a reduction in exhaled NO concentration (5), suggesting that this may prove to be a useful index of disease activity (6). A substantial body of laboratory (7) and clinical (8, 9) evidence supports the hypothesis that inflammatory insults are associated with an upregulation of the inducible isoform of nitric oxide synthase (iNOS), the enzyme that generates NO from L-arginine. Specific to the lung, inflammatory insults produce a rapid increase in the expression of iNOS in pulmonary tissues (10). The acute respiratory distress syndrome (ARDS) is characterized by a well-described inflammatory process, which is associated with the sequestration of neutrophils into pulmonary extravascular tissues and increased alveolar-capillary permeability. We therefore hypothesized that NO would be present in the airways of ventilated patients with ARDS and that this concentration would be greater than that in ventilated subjects without inflammatory lung disease.

	METHODS

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Patients

Adult patients fulfilling the American-European Consensus definition (11) of ARDS and requiring mechanical ventilation were studied. A group of patients anesthetized and ventilated prior to cardiac surgery acted as control subjects. The latter patients were nonsmokers and, at the time of preoperative assessment, had no symptoms or signs suggestive of acute or chronic respiratory disease or cardiac failure. The protocol and procedures were approved by the Ethics Committee of the Royal Brompton Hospital.

Measurement of Exhaled NO Concentration

This was performed by chemiluminescence using a LR2000 fast- responding analyzer (Logan Research, Rochester, UK). The device contains an integral capnograph, and it produces a continuous output from the reaction vessel, which is digitalized and downloaded to a computer at 25 Hertz. Extensive validation experiments were carried out on the instrument prior to clinical study. Specifically, two characteristics describe the speed of response of a gas analyzer. The first is "lag time," defined as the interval between an analyzer being exposed to a change in concentration of gas and the start of a positive deflection or change in signal output, and which reflects length and bore of sampling tube and sampling flow rate. The second is "response time," which reflects the speed of the analyzer to peak deflection or to provide an estimation of a new gas concentration. The most widely accepted definition of response time is that for the instrument to register a change from 10% of peak to 90% of peak new gas concentration (Figure 1), which avoids difficulties in accurately determining the absolute start of deflection. The figures quoted by manufacturers are generally obtained from bench experiments using gas samples directly injected into the reaction vessel, which tends to produce an optimistic view of device performance. In the clinical setting, devices are required to sample along substantial lengths of tubing via bacterial filters. Further, the change in concentration of gas sampled is not reflected by a uniform gas "front" traveling up the sampling tube, in that a certain amount of mixing and dilution takes place. These factors lengthen both lag and response times.

View larger version (16K): [in this window] [in a new window]   Figure 1.   (Upper panel ) Data recorded during a validation experiment to examine analyzer response time. The relative differences in response times to achieve a display of the new concentration can be seen, and they are reflected in the slope of the upward deflection. (Lower panel ) This is marked to demonstrate 10 and 90% of the new peak concentration of NO being measured.

The response time was assessed by sampling from a reservoir bag filled with test gas via a tube that was clamped. The analyzer probe was placed against the clamp, which was released during sampling. The response time was defined as above. The mean (± SEM) response time for CO₂ was 300 ± 31.6 ms, and for NO it was 1.14 ± 0.02 s, sampling at 500 ml/min. The lag time delays the appearance of the data, but with short lengths of fine bore tubing it does not materially affect its accuracy. The lag time for the equipment used in these experiments was approximately 1.3 to 1.6 s. A more accurate assessment was not possible because the LR2000 lacks an event marker.

Preliminary clinical studies revealed that most subjects produced a peak in NO concentration that predominantly coincided with the end of the capnograph exhalation signal. However, this was not a universal finding, with some subjects producing an earlier peak. A time- averaged approached was therefore adopted. A controlled ventilation maneuver was performed with the subject's lungs ventilated at six breaths per minute with tidal volumes of 12 ml/kg. The inspiratory-to-expiratory time ratio was 1:2, and the level of positive end-expiratory pressure (PEEP) applied was 8 to 10 cm H₂O. The fractional inspired oxygen concentration (FI_O2) was 0.6 to 0.8. Air added to the ventilator was NO-free from cylinders (BOC, Beckenham, UK). The chemiluminescence device sampled (at 500 ml/min) from the tip of the endotracheal tube, via non-NO-absorbing Teflon tubing, for an 80-s epoch. The data were subsequently plotted, and the time period for five exhalation peaks were identified using the capnography trace. The mean of all NO concentration data points for this time period was calculated as an index of NO concentration (Figure 2). Each 80-s measuring maneuver was performed in triplicate. If the mean NO concentration was less than 1 ppb, a value of zero was recorded.

View larger version (25K): [in this window] [in a new window]   Figure 2.   Typical combined airway NO concentration and capnograph trace from a patient with normal lungs ventilated at a rate of six breaths per minute and a tidal volume of 12 ml/kg. The mean NO concentration is calculated for the period defined by the bar. In this example, the peak NO concentration occurs momentarily after the end of exhalation, defined by the capnogram; this may be a reflection in the different response rates of the analyzers.

Study Protocol

Patients were studied as soon as it was clinically practical and consistent with safety after the diagnosis of ARDS was made, or at the time the patient arrived in the intensive care unit after transfer from a referring hospital. Details of age, diagnosis, and number of days of mechanical ventilation were recorded. A full hemodynamic and blood gas tension profile, and lung injury severity score (LISS) (12) were performed, and a measurement of endogenous NO in the airways was carried out.

Control patients were studied after induction of anesthesia and tracheal intubation once cardiovascular stability had been confirmed. Measurement of exhaled NO was performed using a maneuver identical to that already described, with the exception that there was no PEEP applied, and the FI_O2 was 0.6.

Statistical Analysis

Data (presented as mean ± SEM) were assessed for parametricity, and parametric or nonparametric tests used as appropriate. Two-tailed tests were used when available.

	RESULTS

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Thirteen patients with ARDS (mean age, 38 ± 4 yr) and 18 control subjects (mean age, 66.6 ± 2 yr; 2 p < 0.001) were studied. Mean LISS in the ARDS group was 3.1 ± 0.2; other demographic and baseline clinical data are presented in Tables 1 and 2. Mean exhaled NO concentration was 1.13 ± 0.4 ppb in patients with ARDS and 5.5 ± 0.8 ppb in control subjects (2 p < 0.0001) (Figure 3). There was no correlation between exhaled NO concentration and LISS (r = 0.3, p = 0.3), Pa_O2/FI_O2 ratio (r = 0.33, p = 0.26) or pulmonary vascular resistance (PVR) (r = 0.12, p = 0.5). A combined capnogram and NO trace from a patient initially enrolled as a control subject is shown in Figure 4 (upper panel ); subsequently, she developed a severe (fatal) acute lung injury. The lower panel demonstrates a trace recorded after lung injury had become apparent, and it was typical of traces from such patients.

View larger version (10K): [in this window] [in a new window]   Figure 3.   Endogenous airway NO concentration for patients with ARDS and control subjects. Data are displayed as individual values and as box-and-whisker-plots (median and interquartile range) (2 p = 0.0001, Mann Whitney U test).

View larger version (26K): [in this window] [in a new window]   Figure 4.   The upper panel represents a combined NO concentration and capnograph trace from a patient anesthetized and ventilated prior to coronary artery bypass grafting. The small positive gradient exhibited by the CO2 plateau suggests that this patient may have a minor (subclinical) degree of obstructive pulmonary disease. The lower panel shows the same investigation 18 h later, after the patient had developed an overwhelming acute lung injury, from which she died. The very low concentration of NO seems typical of patients with ARDS.

	DISCUSSION

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In this study we were able to detect NO in the airways of a group of patients with ARDS at a concentration significantly lower than that found in control subjects (Figure 3). This difference was striking, almost no NO being detectable in some patients (Figure 4).

The measurement of NO concentration in exhaled gas from ventilated subjects is complicated by the very low concentrations involved and the effects of mechanical ventilation on the measurement technique. Alterations in tidal volume or respiratory frequency vary the dilution of NO in the ventilator gas, which renders the data produced uninterpretable. These and other difficulties undoubtedly contribute to the paucity of published data from ventilated subjects compared with those breathing spontaneously.

The subjects in the control group described in the current study were significantly older than the patients with ARDS, but there was only a weak and nonsignificant positive correlation between age and NO concentration in the control subjects (r = 0.1, p = 0.07; individual data not shown). PEEP is unlikely to account for the differences observed, as it should increase the FRC and therefore the volume of gas diluting exhaled NO produced in the distal airways. However, the FRC in patients with ARDS is extremely low (13), and even with PEEP applied, it is almost certainly lower than that in patients with nondiseased lungs just after induction of anesthesia. The reduction in FRC (of around 15 to 20%) after induction of anesthesia is recognized, but of limited extent (reviewed in 14). If this effect was important, the sum of FRC plus tidal volume in patients with ARDS would still be smaller than that in the control subjects (assuming similar proximal anatomic dead space). Therefore, this smaller volume (FRC plus tidal volume) would dilute the NO evolved to a lesser extent, leading to higher measured values. It is possible that differences in dead space or pulmonary elastic recoil could have contributed to the clear difference observed, but we feel this is unlikely to have been a major factor. The use of patients about to undergo cardiac surgery as control subjects is a clear limitation. Although patients with respiratory disease were excluded, a subtle interaction between cardiac disease and endogenous NO in the airways, however unlikely, cannot be excluded. No control patients were receiving nitroprusside or nitroglycerine infusions. However, a number were receiving oral nitrate therapy (12 of 18). There was no difference between nitrate and non-nitrate-treated patients (5.4 ± 1 versus 5.8 ± 1.2 ppb; 2 p = 0.6).

Levels of NO detectable in the lower airways described in the current study are difficult to compare with other published data because these have generally not been obtained from patients undergoing mechanical ventilation. The data reported here are of the same order as those reported previously in spontaneously breathing subjects, with the upper respiratory tract excluded (3, 15). It is clear that the upper airway and the nasal sinuses contribute substantially to NO levels measurable in exhaled breath of conscious subjects (18). A previous study examined a variety of subjects, some of whom were ventilated for respiratory failure (17) and reported a mean NO concentration of 0.8 ppb in the respiratory failure group, compared with 4.6 ppb in a group of patients with chronic tracheostomies for the therapy of nocturnal hypoventilation. Previously published data from spontaneously breathing subjects, using an identical analyzer, have demonstrated an expiratory NO concentration plateau (19). These experiments involved subjects performing a controlled expiration against resistance (to isolate the nasopharynx). We were able to reproduce their findings in spontaneously breathing subjects using our own instruments. We did not observe such a plateau in our ventilated subjects (Figure 2), and we ascribe the difference to the ventilator-subject-analyzer interaction. We do not feel that the continuous rise in measured NO concentration during exhalation was due to differences in response time between the two analyzers, although this may have had a limited effect.

It is possible that less NO is produced in the respiratory systems of a patients with ARDS. However, given the inflammatory status of the lung in such patients, and the association of inflammation and iNOS, this seems relatively unlikely. An alternative explanation is that the NO produced reacts rapidly with neighboring molecular species, and it is thus not evolving in measurable form into proximal airways. The recruitment of neutrophils to the lung has been identified as a key pathologic process in acute lung injury. Activated neutrophils release molecular superoxide, derivatives of which have been identified in breath condensates of patients with ARDS (20). Moreover, NO and superoxide react rapidly together to produce the highly toxic peroxynitrite ion (21). Peroxynitrite cannot easily be measured directly, but the reaction products of peroxynitrite and amino acids such as nitrotyrosine have been identified in lung tissue from patients with ARDS (22).

A change in diffusing capacity for NO (DL_NO) has recently been invoked as a possible explanation for the altered exhaled NO measurements reported in some disease states (23). Given the perialveolar hemorrhage commonly seen in histologic specimens from patients with ARDS, NO-hemoglobin binding may produce an apparent increase in DL_NO without any other component altering. This would certainly have contributed to the low levels of NO seen in the lung injury group.

Data from fast response chemiluminescence analyzers have now been reported from a variety of settings (24), and the response times and bench-test accuracy are impressive. However, the interaction between the device and a breathing or ventilated subject render the data extremely difficult to interpret. There is an obvious requirement for further studies in this area before definitive conclusions may be drawn.

NO concentration in the lower airways of patients with ARDS was significantly lower than in those of the control subjects. This supports the hypothesis that exhaled NO is rapidly bound and therefore may prove to be of little value as a marker of underlying pulmonary inflammation in this setting.