(Chest. 2002;121:1847-1852.)
© 2002
American College of Chest Physicians
Analysis of Exhaled Nitric Oxide by the Helium Bolus Method*
Masaharu Shinkai, MD;
Shunsuke Suzuki, MD;
Akira Miyashita, MD, PhD;
Hideo Kobayashi, MD, FCCP;
Takao Okubo, MD, PhD, FCCP and
Yoshiaki Ishigatsubo, MD
* First Department of Internal Medicine (Drs. Shinkai, Suzuki, Miyashita, Okubo, and Ishigatsubo), Yokohama City University School of Medicine, Yokohama; and Third Department of Internal Medicine (Dr. Kobayashi), National Defense Medical College, Saitama, Japan.
Correspondence to: Shunsuke Suzuki, MD, First Department of Internal Medicine, Yokohama City University School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama 236-0004, Japan; e-mail: ssuzuki{at}med.yokohama-cu.ac.jp
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Abstract
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Study objectives: The precise anatomic sites contributing to exhaled nitric oxide (eNO) are still unknown. The present study was designed to analyze profiles of eNO by referring to the He exhalation curve and examining the effects of breath-holding and expiratory flow rates on eNO.
Participants: Healthy volunteers and patients with stable asthma.
Measurement and results: We used the He bolus method of the closing volume, and simultaneously analyzed the concentrations of exhaled He and nitric oxide (NO). By referring to the He exhalation curve, the expired gas was divided into three parts: airway dead space (phase 1), a mixture of airway and alveolar gas (phase 2), and alveolar gas (phase 3 and phase 4). The eNO profiles showed a peak in phase 2 (peak eNO) and decreased gradually to a plateau in the latter half of phase 3 (plateau eNO). The levels of peak eNO were higher than those of plateau eNO in both normal subjects and asthmatic patients. Breath-holding increased levels of peak eNO 2.5-fold in both normal subjects and asthmatic patients, but it did not affect the levels of plateau eNO. The levels of peak eNO increased as the expiratory flow rate decreased, and the levels of plateau eNO showed a similar flow dependency.
Conclusion: A peak value of eNO concentration profiles may directly express the production of NO in the airway.
Key Words: airway • asthma • breath-holding • exhaled nitric oxide • expiratory flow rate • helium bolus • nitric oxide • peak nitric oxide • plateau nitric oxide
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Introduction
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High levels of exhaled nitric oxide (eNO) have been observed in patients with various pulmonary diseases, such as bronchial asthma, viral respiratory infections, and bronchiectasis.1
2
3
However, the precise anatomic sites contributing to eNO are still unknown. Theoretically, all tissues adjacent to the respiratory tract could excrete nitric oxide (NO) into the exhaled gas. Earlier studies4
5
have indicated that a large portion of eNO arises from the nose. By analysis of NO with CO2, eNO has been reported to be produced in the airways, but not at the alveolar level.6
Some investigators7
8
have reported that eNO is mainly derived from the upper airway, while studies9
10
using bronchoscopy or intubation have suggested that the lower airway is the source of the increased eNO in patients with bronchial asthma.
A particular concern is that NO measured in exhaled air may be contaminated by NO derived from the nose.11
12
The heterogeneity of the sources of eNO complicates the interpretation of eNO profiles, and profiles of the NO exhalation curve have not been fully characterized. The profiles of eNO have been described as an initial peak (peak eNO) followed by a plateau, and the plateau level of the eNO curve (plateau eNO) has been used for analysis.11
13
14
After breath-holding, the levels of peak eNO increased to greater than plateau levels.15
However, this increase in peak eNO has been regarded to be the result of nasal contamination.4
10
The measurement of eNO has been improved by using a low flow rate and a positive airway pressure without the wearing of a nose clip.11
16
It has been also demonstrated that breath-holding does not allow any contamination of eNO by nasal NO.16
17
It is therefore possible that peak eNO represents the production of NO in the airway wall and is useful in evaluating airway inflammation. In the present study, to further characterize the peak eNO, we used the He bolus method of closing volume,18
in which the He exhalation profile divides the expired gas into a mixture of dead space and airway (phase 1), a mixture of airway and alveolar gas (phase 2), and alveolar gas (phase 3 and phase 4), and analyzed profiles of eNO by referring to the He exhalation curve. We also studied the effects of breath-holding and expiratory flow rates on eNO in normal subjects and asthmatic patients.
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Materials and Methods
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Subjects
Patients with bronchial asthma (age range, 22 to 75 years) were enrolled at our outpatient clinic, and healthy volunteers (age range, 23 to 46 years) were also recruited from hospital employees. Patients with asthma were all in stable condition, and its severity was mild to moderate (Table 1
). All patients and subjects were nonsmokers. Written informed consent was obtained from all participants.
Measurement of eNO
eNO was measured according to the method of Silkoff et al.16
Levels of eNO were measured by a rapid-response chemiluminescence analyzer (NOA 270B; Sievers; Boulder, CO), and a calibration was performed between 520 parts per billion (ppb) and 0 ppb. The linearity of the analyzer response was verified by repeated calibrations. The apparatus of eNO measurement consists of a one-way valve (model 1400; Hans-Rudolph; Kansas City, MO), of which the inspiratory port is connected to a Douglas bag containing NO-free air, and of which the expiratory port is connected to a pneumotachograph and a spring-loaded valve (Threshold PEP; Healthscan Products; Cedar Grove, NJ) to give a constant positive pressure (14 cm H2O) in relation to the expiratory line. eNO was continuously sampled via the side port close to the mouth at a sample flow of 200 mL/min. The airway pressure was continuously monitored with a pressure transducer (Validyne MP-45; Validyne Engineering; Northridge, CA) connected to a tap of the mouthpiece. In addition, the concentrations of CO2 were measured from a side port close to the mouthpiece with a mass spectrometer (WSMR-1400; Westron; Chiba, Japan). To keep the flow rate constant, the flow rate was displayed in the front of the subject.
He Bolus Method of Closing Volume
Fifty milliliters of He was injected to the mouthpiece at the beginning of inhalation from residual volume (RV) to total lung capacity (TLC), and subjects were then asked to exhale to RV through the mouthpiece at a constant flow rate by observing the flow rate on the oscilloscope. He concentrations were measured via a side port close to the mouthpiece with the mass spectrometer. The expired volume was obtained from the integration of the pneumotachograph signal and was displayed, along with the He concentrations, on an XY recorder.
Flow rate, mouth pressure, and concentrations of NO, CO2, and He were simultaneously recorded at a sampling rate of 100 Hz by the MacLab System (AD Instruments; Castle Hill, Australia). Expiratory flow rate studied in the present study was 60 mL/s, which was slightly higher than the recommended flow rate by the American Thoracic Society,12
because the present study was performed before the publication of this recommendation.
Study Protocol
Test of Nasal NO Contamination:
We studied whether the nasal cavity is in communication with the airway during a slow exhalation against positive airway pressure or during breath-holding. While the nasal cavity was gently flushed with He, the subjects were asked to exhale from TLC without breath-holding at a flow rate of 60 mL/s against positive pressure. He concentrations were monitored simultaneously with both NO and CO2 throughout the exhalation. In addition, during a slow exhalation from TLC after breath-holding for 10 s, nasal contamination was examined in the same way as described above.
Simultaneous Measurement of eNO and the He Exhalation Profile:
Before the eNO measurement, the subjects breathed NO-free air for 1 min and then performed two vital capacity (VC) maneuvers. At RV, the subjects inhaled an He bolus of 50 mL and NO-free air to TLC and then, with or without breath-holding for 10 s, exhaled at a flow rate of 60 mL/s against positive airway pressure. By referring to the He exhalation curve, we divided the eNO curve into phases 1, 2, 3, and 4 (Fig 1
). The typical eNO trace showed a peak in phase 2 and then a slight decrease in phase 3, forming a plateau. We sampled the peak and plateau eNO levels, the latter of which was measured for at least 5 s during the latter part of phase 3.
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Figure 1. Profiles of expired gases after breath-holding for 10 s (a patient with bronchial asthma). Concentrations of He, NO, and CO2 are displayed against the expired volume. The lines divide a dead space (phase 1), a mixture of airway and alveolar gas (phase 2), and the alveolar plateau (phase 3). In this patient, phase 4 did not appear.
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Effects of Expiratory Flow Rate on eNO:
To examine the effects of the expiratory flow rate on eNO, the subjects were asked to exhale at a flow rate of 60 mL/s, 120 mL/s, or 240 mL/s against positive airway pressure. The subjects controlled the expiratory flow rate by visual feedback. The order of flow rate was randomized. Before the NO measurement, the subjects breathed NO-free air for 1 min and then performed two VC maneuvers. Next, during exhalation from TLC to RV, eNO was measured.
Effects of Breath-Holding on eNO:
After breathing NO-free air for 1 min and carrying out two VC maneuvers, subjects were asked to hold their breath at TLC for 10 s and then to exhale to RV at a flow rate of 60 mL/s. The order of eNO measurements with or without breath-holding was randomized.
All measurements were performed with subjects in a seated position. The subjects did not wear a nose clip. After eNO measurement, spirometry was performed, and VC and FEV1 were obtained with a dry-seal spirometer (CHESTAC-25V; Chest; Tokyo, Japan).
Statistical Analysis
Results are expressed as the mean value ± SD. Where appropriate, data were analyzed using a two-way analysis of variance with repeated measures, followed by a post hoc comparison using the Newman-Keuls test. For the comparison between healthy subjects and asthmatic patients, a Mann-Whitney U test was used, and the paired data were examined with a paired t test. p < 0.05 was considered significant.
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Results
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Evidence of NO Contamination From Nasal NO
When the subjects exhaled from TLC without breath-holding while applying positive airway pressure without a nose clip, He injected into the nasal cavity was not detected in the expired gas. Figure 2 shows the trace concentrations of eNO, CO2, and He as well as the flow and mouth pressures after breath-holding for 10 s, and no He was detected in the expired gas. Thus, the present method can prevent any contamination from nasal NO in the eNO measurement with or without breath-holding for 10 s.
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Figure 2. Effects of breath-holding on nasal contamination of NO after breath-holding for 10 s. He was not detected during exhalation, indicating that no nasal contamination occurred during breath-holding. Paw = airway pressure.
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Effects of Sampling Site on eNO: Peak eNO vs Plateau eNO
After washout of the airway dead space, the He concentration rose sharply in phase 2 (Fig 1)
; phase 2 consists of gas from both the airways and alveoli.18
Profiles of eNO showed a peak in phase 2 and a drop to a plateau in phase 3. The mean value of peak eNO at 60 mL/s was 20.8 ± 5.3 ppb and that of the plateau eNO was 12.9 ± 3.8 ppb in normal subjects; this difference was statistically significant (p < 0.001; Fig 3
). In asthmatic patients, peak eNO was 91.1 ± 52.9 ppb and plateau eNO was 62.3 ± 41.6 ppb. This difference in eNO was also statistically significant (p < 0.001).
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Figure 3. Peak and plateau eNO levels. Peak eNO was sampled at phase 2, and plateau eNO was obtained from a plateau portion of the latter half of phase 3. The measurement of eNO was performed at a flow rate of 60 mL/s. Normal subjects (A) showed an increase in peak eNO compared to the plateau eNO (p < 0.001). Asthmatic patients (B) had a higher peak eNO compared to the plateau eNO (p < 0.001). Asthmatic patients also had higher peak and plateau eNO levels compared to normal subjects (p < 0.001). Levels of eNO are expressed on a logarithmic scale. Bars and boxes indicate mean and SD, respectively.
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Effects of Flow Rate on eNO
In both normal subjects and asthmatic patients, as the expiratory flow rate increased, the levels of peak eNO decreased, with a similar decline in plateau eNO (Fig 4
). Peak eNO levels at 60 mL/s and 240 mL/s in the normal subjects were 20.8 ± 5.3 ppb and 13.0 ± 3.6 ppb, respectively, and this difference was statistically significant (p < 0.001). In asthmatic patients, peak eNO was also different between 60 mL/s and 240 mL/s (113.0 ± 100.3 ppb and 56.0 ± 39.9 ppb, respectively, p < 0.001). When eNO was normalized to the value of the lowest flow rate, both peak eNO and plateau eNO at the middle and highest flow rates decreased similarly in both normal subjects and asthmatic patients. At any flow rate (60 mL/s, 120 mL/s, or 240 mL/s), peak eNO was higher than plateau eNO in both normal subjects and asthmatic patients (both p < 0.05, analysis of variance). We therefore decided to adopt the eNO values at the lowest flow rate of 60 mL/s.
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Figure 4. Effects of the exhaled flow rate on eNO levels. Tested exhaled flow rates were 60 mL/s, 120 mL/s, and 240 mL/s. In both normal subjects (circles) and asthmatic patients (squares), exhaled peak eNO (open symbols) decreased as the expiratory flow rate increased. Also, exhaled plateau eNO (closed symbols) decreased with increases in the flow rate.
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Effects of Breath-Holding on eNO
The peak eNO concentrations after breathholding for 10 s in normal subjects was 54.3 ± 14.6 ppb, which was higher than the value without breath-holding (20.9 ± 5.0 ppb; p < 0.001). In asthmatic patients, peak eNO after breath-holding increased significantly from 90.1 ± 52.9 to 226 ± 162 ppb (p < 0.001). The increases in peak eNO in response to breath-holding were 2.6-fold in normal subjects and 2.5-fold in asthmatic patients, and were similar between these two groups (Fig 5
). Plateau eNO was not changed by breath-holding in either normal subjects or asthmatic patients.
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Figure 5. Effects of breath-holding (BH) on eNO. eNO with breath-holding is plotted against the eNO without breath-holding. Peak eNO with breath-holding was higher than that without breath-holding in both normal subjects and asthmatic patients (p < 0.001). The magnitude of increase was similar in both normal subjects and asthmatic patients. Plateau eNO (closed symbols) did not differ with respect to breath-holding in either normal subjects (circles) or asthmatic patients (triangles). The diagonal dashed line is a line of identity.
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Discussion
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We have demonstrated that by referring to the He exhalation curve, a peak value in the early part of the eNO curve comes from the airway. Breath-holding for 10 s causes an increase in peak eNO levels, but does not affect the plateau eNO levels. Therefore, the peak eNO levels after breath-holding are suggested to represent the actual concentrations of NO in the airway walls.
Several eNO measurement techniques have been suggested as standard methods.11
12
There are several key points in measuring eNO, including the expiratory flow rate and positive airway pressure. Silkoff et al16
demonstrated that eNO levels are dependent on the expiratory flow rate. However, the flow rate has varied among investigators, from 4 to 250 mL/s.11
16
Our lowest flow rate of 60 mL/s is close to the recommended flow rate (50 mL/s) from the American Thoracic Society.12
Positive airway pressure during expiration is critical to closing the soft palate, thus preventing nasal contamination.16
17
We confirmed that no nasal eNO contamination occurs during exhalation against a positive airway pressure of 14 cm H2O at any flow rate. In previous studies4
10
in which nasal contamination was suspected, the subjects were wearing nose clips. Rubinstein et al19
demonstrated that the use of a nose clip opens the nasopharyngeal velum. It has also been recommended,11
12
regarding NO measurement, that no nose clip be worn. Thus, in the measurement of eNO, a slow exhalation without the wearing of a nose clip is actually ideal.
In early studies,15
20
21
the peak eNO value was reported as an eNO concentration. However, the peak eNO was regarded as a result of contamination from nasal NO of high concentrations.2
4
Thereafter, the peak eNO has been substituted for a plateau eNO. However, it has been revealed that the initial peak of eNO is derived from the airway wall, not from nasal NO.8
17
22
Theoretically, the initial rise of eNO in the first 200 mL is considered to be from the airway compartment and is due to diffusion from the airway wall to the airstream.23
We found that by referring to the He exhalation curve, eNO had a peak in phase 2 and then decreased to a plateau in the phase 3 and phase 4 in both normal subjects and asthmatic patients. It has been demonstrated that the source of eNO is primarily the airway wall when expiratory flow is slow.7
16
22
24
In the present study, the flow dependence of eNO levels was observed in both peak and plateau eNO values. As the expiratory flow decreases, there is more diffusion in the airway lumen from the airway wall due to a longer contact time. Further, the gas in the phase 2 stays in the airway for a longer time compared to the gas in the phase 3 or phase 4 in the VC maneuver of NO measurement. Thus, the peak eNO levels at a low flow rate express chiefly the NO level of the airway wall.
The maneuver of breath-holding had been reported to increase the levels of eNO.4
5
9
10
15
In a study insufflating an indicator gas to the nose,17
however, breath-holding itself did not cause any gas leak from the nose to the airway. Silkoff et al16
also confirmed that breath-holding does not allow nasal contamination. In the present study, we confirmed that during breath-holding for 10 s, no gas leak from the nose to the airway occurred. Persson et al15
reported that breath-holding increases the peak eNO in a duration-dependent manner. Massaro et al9
found a significant increase in eNO after breath-holding in patients with endotracheal intubation. Theoretically, a diffusion of NO occurs both during breathing and breath-holding in the airway.7
22
23
25
We studied the effects of breath-holding on eNO levels for 10 s alone. For a more quantitative study, we should have examined the breath-holding for longer periods. In the present study, breath-holding increases the peak eNO similarly in normal subjects and asthmatic patients, but does not change the plateau eNO. When the expiratory flow is fast, the plateau eNO level is determined primarily by NO levels of the alveolar gas (phase 3), irrespective of breath-holding time.7
When the expiratory flow rate is slow, phase 3 gas passes through the airway for a longer time, resulting in a substantial amount of airway NO possibly diffusing into the exhaling air, thus increasing eNO levels. In our normal subjects, the peak eNO level after breath-holding was 54 ppb, which is very close to the equilibrium concentration (56 ppb) in trachea during breath-holding reported by DuBois et al.7
The magnitude of the increase in peak eNO in response to breath-holding in our asthmatic patients was comparable to that of normal subjects, although no change in the plateau eNO was observed. During breath-holding, NO in the airway wall may diffuse into the exhaling gas, but after breath-holding, alveolar gas (phase 3) may pass through the airway for a given time, suggesting that the amount of NO diffusing from the airway may depend on the flow rate alone.
In conclusion, the present study has demonstrated that peak eNO, especially after breath-holding, more directly expresses NO levels in the airway wall.
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Acknowledgements
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The authors thank Drs. S. Komatsu, S. Inoue, A. Hashimoto, and M. Suzuki for their helpful suggestions.
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Footnotes
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Abbreviations: eNO = exhaled nitric oxide; NO = nitric oxide; ppb = parts per billion; RV = residual volume; TLC = total lung capacity; VC = vital capacity
Received for publication June 5, 2001.
Accepted for publication November 20, 2001.
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References
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Kharitonov, SA, Barnes, PJ (1997) Nasal contribution to exhaled nitric oxide during exhalation against resistance or during breath holding. Thorax 52,540-544[Abstract]
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Kharitonov, SA, Yates, D, Robbins, RA, et al (1994) Increased nitric oxide in exhaled air of asthmatic patients. Lancet 343,133-135[CrossRef][ISI][Medline]
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Tsoukias, NM, George, SC (1998) A two-compartment model of pulmonary nitric oxide exchange dynamics. J Appl Physiol 85,653-666[Abstract/Free Full Text]
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Pietropaoli, AP, Perillo, IB, Torres, A, et al (1999) Simultaneous measurement of nitric oxide production by conducting and alveolar airways of humans. J Appl Physiol 87,1532-1542[Abstract/Free Full Text]
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Abstract
Introduction
