Abstract
Human airways produce nitric oxide (NO), and exhaled NO increases as expiratory flow rates fall. We show that mixing during exhalation between the NO produced by the lower, alveolar airways (V˙l _{NO}) and the upper conducting airways (V˙u _{NO}) explains this phenomenon and permits measurement ofV˙l _{NO},V˙u _{NO}, and the NO diffusing capacity of the conducting airways (Du _{NO}). After breath holding for 10–15 s the partial pressure of alveolar NO (Pa) becomes constant, and during a subsequent exhalation at a constant expiratory flow rate the alveoli will deliver a stable amount of NO to the conducting airways. The conducting airways secrete NO into the lumen (V˙u _{NO}), which mixes with Pa during exhalation, resulting in the observed expiratory concentration of NO (Pe). At fast exhalations, Pa makes a large contribution to Pe, and, at slow exhalations, NO from the conducting airways predominates. Simple equations describing this mixing, combined with measurements of Pe at several different expiratory flow rates, permit calculation of Pa,V˙u _{NO}, and Du _{NO}.V˙l _{NO}is the product of Pa and the alveolar airway diffusion capacity for NO. In seven normal subjects, Pa = 1.6 ± 0.7 × 10^{−6} (SD) Torr,V˙l _{NO}= 0.19 ± 0.07 μl/min,V˙u _{NO}= 0.08 ± 0.05 μl/min, and Du _{NO} = 0.4 ± 0.4 ml ⋅ min^{−1} ⋅ Torr^{−1}. These quantitative measurements ofV˙l _{NO}andV˙u _{NO}are suitable for exploring alterations in NO production at these sites by diseases and physiological stresses.
 nitric oxide diffusing capacity of airways
 nitric oxide production by airways
 lung nitric oxide
 breath holding
increased exhaled nitric oxide (NO) concentrations have attracted interest as a means for detecting inflammation of the airways in asthma (17). NO production by the lungs may be abnormal in diseases such as sepsis, cirrhosis, primary pulmonary hypertension, and interstitial lung diseases (18, 21, 26, 27). The exhaled concentration of NO (Pe) increases as expiratory flow rates (Q˙e) fall (24), so Q˙e must be kept constant to obtain reproducible measurements of Pe (Fig.1). The reason for this flow dependence has recently been elucidated by Tsoukias and coworkers (28, 29). They show that during exhalation the mixing between NO from the lower alveolar airways perfused by the pulmonary circulation (V˙l _{NO}) with NO produced in the upper conducting airways (V˙u _{NO}) perfused by the bronchial circulation explains this phenomenon. Simple equations can describe this mixing. When combined with multiple measurements of Pe at differentQ˙e, these equations permit calculation ofV˙u _{NO}and the partial pressure of NO in the lower alveolar airways (Pa). In this report, we describe an analysis of expired NO at differentQ˙e that also permits calculation of the diffusing capacity of the upper airways (Du _{NO}) andV˙l _{NO}.V˙l _{NO}is determined from the product of Pa and measurements of the pulmonary diffusing capacity of the lower airways (Dl _{NO}) (12). Because diseases and physiological stress may cause changes in NO production and diffusing capacity by the alveoli different from those by the conducting airways, measurement ofV˙l _{NO},V˙u _{NO}, Dl _{NO}, and Du _{NO} may provide new information about factors that alter NO production by the lungs.
Glossary
 Dl_{NO}
 Diffusing capacity of the lower, alveolar airways recorded as milliliters of NO stpd moving from the air spaces into the tissues and blood per minute per Torr of NO in the air spaces
 Du_{NO}
 Diffusing capacity of the upper, conducting airways recorded as milliliters of NO stpd moving from the air spaces into the tissues and blood per minute per Torr of NO in the air spaces
 f
 Small fraction of Du_{NO}, Pu, orV˙u_{NO}
 FVC
 Forced vital capacity
 NO
 Nitric oxide
 Pa
 Partial pressure of NO in the alveoli
 Pb
 Barometric pressure
 Pe
 Partial pressure of NO in exhaled gas
 Pu
 Partial pressure on NO in all or a segment of the upper conducting airway
 Q˙e
 Expiratory flow rate
 RV
 Residual volume of gases in the lungs
 TLC
 Total capacity of gases in the lungs
 V˙l_{NO}
 Rate of production of NO by the lower alveolar airways that enters the airways
 V˙u_{NO}
 Rate of production of NO by the conducting airways that enters the airways
METHODS
NO Exchange in the Alveolar Airways
The alveolar airways are defined as those tissues and air spaces well perfused by the pulmonary circulation, such as the alveoli, alveolar ducts, and respiratory bronchioles. In this zone, some of the NO produced by these lower airways diffuses into the air spaces. The fraction of the total NO produced in this alveolar compartment that enters the air spaces is calledV˙l
_{NO}. The NO in the alveoli can react with the surrounding tissues (16) or diffuse rapidly through the alveolar capillary membrane into the perfusing blood. After elimination of ventilation by breath holding for 10–15 s, a steady state will develop, and the amount of NO entering the alveoli (V˙l
_{NO}) equals the amount of NO diffusing into the perfusing blood and surrounding tissues (12) or
NO Exchange in the Conducting Airways
The conducting airways are defined as those airways extending from the alveolar airways to the mouth. Strategies such as continuous positive pressure in the mouth (13, 24) or constant suction of gases from one nostril (9, 28, 29) can be used to avoid contamination of expired NO from the conducting airways by the much higher concentration in the nasopharynx (14). NO gas exchange in the conducting airways can be analyzed in the same manner as in the alveolar airways. Namely, a fraction of NO production by the conducting airways (V˙u
_{NO}) enters the lumen. Some of this NO can diffuse back into the tissues of the conducting airways and enter the bronchial circulation in proportion to the partial pressure of NO in the lumen of the conducting airways (Pu). If the bronchial blood flow maintains the partial pressure of NO in the blood perfusing the tissues of the conducting airways at a negligible level, the amount of NO in the lumen that diffuses back into these tissues will equal Pu ⋅ Du
_{NO}. With exhalation at a constant flow rate, Pu will reach a constant value, and during this steady stateV˙u
_{NO}will equal the amount of NO diffusing back into the tissues or
Model 1.
Model 1 assumes a uniform concentration of NO throughout the conducting airways (Fig.2), so Pu = Pe. After breath holding for 10–15 s, a constant Pa is achieved (12), and subsequent exhalation at a steady flow rate (Q˙e) delivers a constant amount of NO to the conducting airways equal toQ˙e[Pa/ (Pb − 47)], where Pb is the barometric pressure, 47 is the partial pressure of water at body temperature in Torr,Q˙e is expressed in milliliters per minutestpd, and Pa is expressed in Torr. This NO from the alveolar airways instantaneously mixes with NO in the conducting airways, resulting in a uniform partial pressure of NO in the conducting airways and the expired breath (Pe). The amount of NO exhaled at any instant (stpd) equalsQ˙e[Pe/ (Pb − 47)]. This equals the contribution from the alveolar airways {Q˙e[Pa/ (Pb − 47)]} plusV˙u
_{NO}less the NO diffusing from the lumen of the conducting airways back into the tissues and bronchial circulation of the conducting airways (Pe ⋅ Du
_{NO}) or
To determine whether a reliable measurement ofV˙u _{NO}was possible from just the faster values ofQ˙e used to calculate Pa,V˙u _{NO}was also calculated from these data with Eq.5 and compared withV˙u _{NO}determined with all values ofQ˙e by use ofEq. 4.
This method for measuring Pa,V˙u _{NO}, and Du _{NO}with model 1 assumes rapid arrival at a new steady state when the NO coming from the alveolar airways mixes with the NO in the conducting airways during exhalation.appendix describes an equation for calculating the changes in Peduring mixing and shows the amount of gas needed to be exhaled to reach a steady state. The equation shows that once ∼30% of the expiratory vital capacity has been exhaled after the initial breathholding period, Pe is within 99% of the constant equilibrated value, so Eqs. 4 and 5 are valid for measuring Pa,V˙u _{NO}, and Du _{NO}.
Model 2.
Model 2 assumes stratification of the NO concentration in the conducting airways so the concentration of NO can gradually increase as the expired gas moves through the conducting airway (Fig. 5). In contrast to model 1, the conducting airway is considered to be a cylinder with a total volume K and an infinite number of uniform segments. Each segment has an equal fraction (f) of K, V˙u _{NO}, and Du _{NO}, so that the dimensions of any segment are fV, fV˙u _{NO}, and fDu _{NO}. At the start of exhalation at a constantQ˙e, Pa enters the first segment, where fV˙u _{NO}adds NO and fDu _{NO}removes NO at a rate proportional to the partial pressure of NO in the segment. The bronchial blood flow in the wall of the upper airway is assumed to keep its partial pressure of NO at a negligible level. The resultant partial pressure of NO in the lumen of the segment equals Pu _{1}. Pu _{1} then enters the next segment, and its fraction ofV˙u _{NO}and Du _{NO}results in Pu _{2}, and so forth. At the proximal end of the conducting airway, Pu = Pe.
For any segment of the conducting airways, the amount of NO in the segment of volume fK equals fK[Pu / (Pb − 47)]. It is changed by the NO production (fV˙u
_{NO}) entering the segment less the amount diffusing out (Pu ⋅ fDu
_{NO}) or
Measurement of NO
Details of methods for measuring NO have been recently published (9). Briefly, a rapidly responding chemiluminescence NO analyzer (Sievers NOA, model 270B, Sievers, Boulder, CO) operating at a sample rate of 250 ml/min measured exhaled levels of NO at the mouthpiece with a 150cmlong, 1.6mmID, 3.2mmOD Tygon inlet tube. Response time of the analyzer was <200 ms for a signal 90% of full scale. The analyzer was adjusted to provide 40 measurements of the NO concentration per second that could be averaged over any time interval. The NO analyzer was calibrated daily by serial dilutions of a gas containing 229 parts per billion (ppb) of NO. To obtain gas samples free of NO, air from a gas cylinder containing <2 ppb of NO (Scott Specialty Gases, Plumsteadville, PA) was passed through a filter constructed from a 5.8cmID, 19cmlong cylinder (Gas Drying Unit, VWR Scientific, Rochester, NY) packed with potassium permanganate (Purafil, Thermoenvironmental Instruments, Franklin, MA) (4).
Because the air signal free of NO could drift as much as 2 ppb in 10 min, measurements of NOfree air were performed within 1 min before and after each NO measurement from expired gas samples, and these values were averaged to obtain the zero NO signal. The lag time between the volume signal obtained from a potentiometer attached to the spirometer and the change in the NO signal was determined daily and equaled 0.8 ± 0.1 (SD) s. Multiple repetitive measurements of gas mixtures of 2.8 and 8.2 × 10^{−6}Torr of NO showed a standard deviation of 0.09 × 10^{−6} Torr. We assumed that the detection limit of our analyzer was two times the standard deviation of these multiple measurements or 0.2 × 10^{−6} Torr. During gas sampling the operator exhaled warm humidified gas from the mouth by the inlet of the NO analyzer approximately every 5–10 min, so the walls of the unheated inlet tubing were kept moist. This resulted in all gases being considered measured atatps. Measurements of NO in parts per billion atps were converted to partial pressure of NO in Torr btps as follows: NO in Torr = (NO in ppbatps)(Pb)(Pb− 47) / (Pb −
Maneuvers Used to Measure Pe andQ˙e
Subjects exhaled to residual volume (RV) through the mouthpiece of the apparatus into the room and then rapidly inhaled room air from a baginbox device to total lung capacity (TLC) (Fig.7). The subject then held this breath for 10–20 s, so that Pa reached a constant concentration irrespective of the inhaled ambient NO concentration (12). At the end of the breath hold, the mouthpiece valve was turned 90° into the spirometry circuit, and the subject exhaled, maintaining mouth pressure at +5 cmH_{2}O by watching a water manometer. Corks with varioussized holes bored through their centers were placed in the expiratory tubing and resulted in different expiratory resistances andQ˙e. Each subject performed measurements at seven different flow rates that were as low as 6 ml/s and as high as 1,355 ml/s. Exhalations at each flow rate were performed in triplicate, and the values for Pe andQ˙e were averaged. Pe was measured as described above, andQ˙e was obtained from the spirometer’s volume signal after the initial and final 10% of the expired volume were discarded (Fig. 6). The entire experiment for each subject was completed within 4 h on the same day.
Measurement of Dl_{NO}
Dl _{NO} for each subject was calculated from the expired NO concentration measured after inspiring 10 parts/million of NO in air placed in the bag in Fig.7 from RV to TLC, breath holding for 5 s, and then exhaling to RV at a constant flow rate of 500 ml/s with a modification of the singlebreath exhalation method for continuously measuring Dl _{CO} during exhalation described by Newth and coworkers (19) and Perillo and coworkers (20). Lung volume at any instant during exhalation used in the calculation of the multiple values of Dl _{NO} was obtained by adding the amount of exhaled gas remaining above RV recorded by the spirometer (Fig. 7) to the subject’s RV. RV was obtained from the subject’s functional residual capacity (FRC) measured with body plethysmography (5) by subtracting the expiratory reserve volume obtained from a spirometer (P. K. Morgan, Haverhill, MA) from FRC. The multiple measurements of Dl _{NO} during the exhalation were averaged and performed in triplicate, and the mean value was recorded.
Subjects
Pa,V˙u _{NO}, and Du _{NO}were measured in seven healthy, nonsmoking, 31 to 72yrold (mean 46 ± 18 yr) subjects. Five were men and two were women. All subjects were free of cardiopulmonary disease. Spirometry showed values >90% of predicted for the forced expiratory volume in 1 s, with a mean value of 104 ± 16 (SD)% (2). This study was approved by the University of Rochester’s committee for investigations involving human subjects.
Statistical Methods
Values are means ± SD. In experiments where subjects served as their own control, results were compared using a twotailed pairedttest. Groups of subjects were compared with an unpaired ttest.P < 0.05 was required for statistical significance. Regression lines and curves were fitted to the experimental data by the line of least mean squares referenced to Pe.
RESULTS
Pa,V˙l_{NO}, and Dl_{NO}
Figure 8 shows the values for Pe and the reciprocal ofQ˙e(1/Q˙e) used to determine Pa from the faster exhalations in the seven subjects. The linear regression of these points extrapolated to infinite flow, where 1/Q˙e = 0, equals Pa. The regression line fitted the data closely, withr ^{2} = 0.965–0.999. Pa was 1.6 ± 0.7 × 10^{−6} (SD) Torr. Dl _{NO}was 123 ± 19 ml ⋅ min^{−1} ⋅ Torr^{−1}.V˙l _{NO}(i.e., Pa ⋅ Dl _{NO}) was 0.19 ± 0.07 μl/min.
V˙u_{NO}and Du_{NO}
Figure 9 shows the paired values for Pe and 1/Q˙e for all exhalations by the seven subjects used to determineV˙u _{NO}and Du _{NO}.Q˙e ranged from 6 to 1,355 ml/s. For model 1,V˙u _{NO}was 0.077 ± 0.053 μl/min and Du _{NO} was 0.4 ± 0.4 ml ⋅ min^{−1} ⋅ Torr^{−1}; for model 2 the values were similar: 0.074 ± 0.052 μl/min and 0.5 ± 0.4 ml ⋅ min^{−1} ⋅ Torr^{−1}, respectively. The regression lines for both models fit the data closely, withr ^{2} > 0.998 in all subjects. The value ofr ^{2} for the two models did not differ significantly: 0.9996 ± 0.0003 formodel 1 and 0.9994 ± 0.0006 for model 2(P = 0.30).V˙u _{NO}calculated with just the faster values ofQ˙e shown in Fig. 8 with use of Eq. 4 was 0.070 ± 0.048 μl/min. Although this value is slightly lower than 0.077 ± 0.053 μl/min with model 1 and 0.074 ± 0.052 μl/min with model 2, the difference was not significant (P = 0.2).
Comparison ofV˙l_{NO}andV˙u_{NO}
Figure 10 shows thatV˙l _{NO}of 0.19 ± 0.07 μl/min was consistently greater thanV˙u _{NO}of 0.077 ± 0.053 μl/min with use of model 1 (P < 0.01). Calculating with model 2 gave similar results.V˙l _{NO}was 0.19 ± 0.07 μl/min compared withV˙u _{NO}of 0.074 ± 0.052 μl/min (P< 0.01).
Comparison of Dl_{NO} and Du_{NO}
Table 1 shows that Dl _{NO} is >100fold greater than Du _{NO}calculated with model 1 ormodel 2.
DISCUSSION
These data show that a model of the human airways where exhaled NO from the alveoli mixes with the NO produced by the conducting airways precisely predicts the Peobserved at differentQ˙e. Simple equations describing this mixing combined with values for Pe at different values ofQ˙e result in measurements ofV˙u _{NO}, Du _{NO}, and Pa. Pa multiplied by a separate measurement of Dl _{NO} gives a measurement ofV˙l _{NO}. Besides these separate quantitative measurements ofV˙l _{NO}andV˙u _{NO}, this model provides a reasonable physiological explanation for the rise in expired NO with slowerQ˙e and helps define the physiological basis for observed values of expired NO reported by many investigators (6, 13, 17, 24, 29).
Common practice is to measure expired NO at a single relatively slowQ˙e on the order of 100–250 ml/s (13). The resultant observed values of Pe are three to five times Pa and, therefore, predominantly representV˙u _{NO}. Although these measurements at single relatively slowQ˙e values provide a useful index ofV˙u _{NO}, they are at a disadvantage for detecting changes in Pa andV˙l _{NO}.
A number of studies suggest that the mechanisms alteringV˙u _{NO}andV˙l _{NO}may be different. The large increases in Pe seen in bronchial asthma likely come from upregulation of inducible NO synthase in the conducting airways (11, 30). Endothelialderived NO synthase is reported to be located in the alveolar capillary membrane (10) and is upregulated in a rat model of the hepatopulmonary syndrome (7). This upregulation could explain the high levels of exhaled NO observed in some patients with cirrhosis and the hepatopulmonary syndrome (18). Downregulation of endothelialderived NO synthase may account for the low levels of expired NO reported in primary pulmonary hypertension (3,21). The technique described in this report for measuringV˙u _{NO}andV˙l _{NO}should provide a quantitative method to localize alteration in NO production to the alveoli or the conducting airways. Such measurements may result in more precision in the use of exhaled NO to assess lung injury or alterations in regulation of NO production by the lungs than that obtained with observations at a singleQ˙e.
Choice of Lung Models to Explain the Change in Pe With Different Values ofQ˙e
The simpler model (model 1) of the airways, where the conducting airways are considered one single uniform compartment, precisely described the observed data obtained at different values ofQ˙e, withr ^{2} > 0.998 in all subjects. The multicompartment model of the conducting airways (model 2), with the more realistic assumption that NO concentration in the conducting airways gradually approaches Pe during exhalation, does not provide a better fit to the observed data. We also performed theoretical calculations to see if measurements of Pe atQ˙e in humans as low as the practical limit of ∼5 ml/s can be used to distinguish between the two models. These models generate different values for Pe shown in Fig.11 for the same assumed values of Pa,V˙u _{NO}, and Du _{NO}. Fitting the equation of one model to the data generated by the other model results in a very tight fit, withr ^{2} > 0.999 (Fig. 12). Therefore, observed values of Pe measured over a wide spectrum of Q˙e values cannot be expected to distinguish which model provides a more realistic prediction of the observed data.
Models 1 and2 have limitations in their assumed dimensions, because the conducting airways must contain multiple compartments where the ratio of the surface area of the conducting airways that secretes NO into the gas volume in the lumen decreases as exhaled gas moves from the alveoli through the trachea (6, 23, 31). This anatomy results in uneven distribution betweenV˙u _{NO}, Du _{NO}, and conducting airway gas volume. Because the simple onecompartment model of the conducting airways so accurately predicts Pe at different values ofQ˙e, use of more realistic models of the conducting airways is not likely to result in a better measurable prediction of the experimental data.
Lung Model Where NO Production Is Uniformly Distributed Throughout the Walls of the Conducting Airways
Tsoukias and George (28) reported what may be a more realistic model of the dynamics of pulmonary NO exchange in the conducting and alveolar airways. They define NO production as taking place uniformly throughout the walls of the lungs’ tissues. From the differential mass balance of NO in the tissue, they derive a secondorder partial differential equation (Eq. 1 in Ref. 28) that allows determination of the changes in Pe by interventions such as varying breathholding time before exhalation, accelerating or slowing flow rates during exhalation, and varying the inspired NO concentration. Their experimental data obtained by measuring expired NO concentrations in seven normal subjects at different constantQ˙e levels result in a fit close to their model, similar to that obtained usingmodels 1 and2 described above. Therefore, expired NO concentrations collected at differentQ˙e in normal subjects unfortunately do not provide a means to determine which of these various models most closely accounts for the observed profiles of expired NO concentration.
Potential Errors inV˙l_{NO}Calculated With Eq. 2 With the Assumption That Dl_{NO} Is Constant
If the decrease with lung volume observed for Dl _{CO} is the same as that observed for Dl _{NO},V˙l _{NO}might be falsely high when values for Dl _{NO}obtained at high lung volumes are used and falsely low when measurements of Dl _{NO}measured at low lung volumes are used. In the calculation ofV˙l _{NO}with Eq. 2 , we used a mean value of Dl _{NO}obtained from Dl _{NO}continuously calculated from the expired NO concentration recorded during expiration. The calculation started at a maximum volume equal to the subject’s TLC less four times the subject’s estimated anatomic dead space and ended when the subject reached a volume equal to the RV plus 15% of the forced vital capacity (19, 20). Newth and coworkers (19) reported that Dl _{CO}measured with this method was unchanged as lung volume decreased. Preliminary measurements in nine subjects (20) showed that Dl _{NO}decreased 9% over this volume interval, but this change did not reach statistical significance (P = 0.3). Therefore, the change in Dl _{NO} with different lung volumes with use of the continuously calculated values during exhalation appears modest and would not be expected to result in large errors inV˙l _{NO}. However, use of singlebreath measurements of Dl _{NO}obtained at TLC could result in overestimation ofV˙l _{NO}.
Fraction of TotalV˙l_{NO}andV˙u_{NO}Measured From Analyses of Pe
This method of measuringV˙l _{NO}andV˙u _{NO}assumes that NO produced in the tissues enters the air spaces and then diffuses into the surrounding tissues and perfusing blood. Some of the NO produced in the alveoli and the conducting airways will react with the tissues and blood and never enter the air spaces (16). This NO will not be measured by analyses of NO in the airways; therefore,V˙l _{NO}andV˙u _{NO}are likely underestimates of the true amount of NO produced by the alveoli and conducting airways. We are unaware of methods that can measure the fraction of NO that does not communicate with airways, and its size may be increased by diseases that impair diffusion of NO from the tissues into the air spaces.
Comparison to Estimates ofV˙l_{NO}and Pa From Data of Others
Because determination of Parequires breath holding or rebreathing for 10–15 s to achieve a constant value as well as rapid exhalations, most published values of Pe do not permit calculations of Pa. However, Silkoff and coworkers (24) measured Pe in 10 subjects atQ˙e of 1,550 ml/s preceded by a 30s breath hold and obtained a Pe of 2.4 ± 1.0 × 10^{−6} Torr. With use of their mean data for Pe at slowerQ˙e, extrapolation of their data to an infinite value forQ˙e gives Pa of 1.9 ± 0.8 × 10^{−6} Torr, which is in close agreement with our value of 1.6 ± 0.7 × 10^{−6} Torr observed in our seven subjects.
Recently, Tsoukias and coworkers (28, 29) published a similar twocompartment model consisting of a nonexpansile compartment representing the conducting airways and an expansile compartment representing the alveolar region of the lungs. In their seven normal subjects, they determined Pafrom 8–12 measurements of Pe andQ˙e performed at constant values ofQ˙e that varied from 175 to 600 ml/s. With an equation equivalent toEq. 3 , they calculated Pa and the flux of NO from the tissues of the conducting airways to the lumen. Formodel 1, flux equalsV˙u _{NO}− (Pe ⋅ Du _{NO}). By plottingQ˙e[Pe/ (Pb − 47)] on the vertical axis vs.Q˙e on the horizontal axis, the intercept on the vertical axis equals flux and the slope equals Pa / (Pb − 47). Their values of Pa of 4.1 ± 2.3 × 10^{−6} Torr were significantly greater than 1.6 ± 0.7 × 10^{−6} Torr obtained in our seven normal subjects (P = 0.025). We have no explanation for the higher values of Pa obtained by Tsoukias and coworkers. However, their flow rates ranged from only 175 to 600 ml/s, whereas Q˙e for the subjects of Silkoff et al. (24) and our subjects varied from 4 ml/s to as high as 1,550 ml/s. This greater range inQ˙e may provide more precision in determining Pa.
Comparison to Estimates ofV˙u_{NO}and Du_{NO} From Data of Others
Only a few investigators have measured Pe at a number of different constant Q˙ethat permit calculation ofV˙u _{NO}or Du _{NO}. Silkoff and coworkers (24) reported Pe at nine different values ofQ˙e between 4.2 and 1,550 ml/s in 10 subjects. Their data shown in Fig.13 permit calculation ofV˙u _{NO}and Du _{NO} by use of Eq. 4 or7. Note the similarity of their data to the findings in our subjects shown in Fig. 9. Model 1 closely fit the data of Silkoff and coworkers, with a mean r ^{2} of 0.996 for their 10 subjects.V˙u _{NO}from their data was 0.061 ± 0.056 μl/min compared with 0.076 ± 0.053 μl/min in our subjects and did not differ significantly (P = 0.22). Du _{NO} in their subjects was 0.4 ± 0.3 ml ⋅ min^{−1} ⋅ Torr^{−1}compared with 0.4 ± 0.4 ml ⋅ min^{−1} ⋅ Torr^{−1}in our subjects (P = 0.61).Model 2 gave similar results with a close fit to the data (r ^{2} = 0.995).V˙u _{NO}was 0.053 ± 0.039 μl/min compared with 0.074 ± 0.052 μl/min in our subjects (P = 0.20), and Du _{NO}was 0.5 ± 0.3 ml ⋅ min^{−1} ⋅ Torr^{−1}vs. 0.5 ± 0.4 ml ⋅ min^{−1} ⋅ Torr^{−1}in our subjects (P = 0.46). The data of Silkoff and coworkers and our data show a wide scatter for the values ofV˙u _{NO}and Du _{NO} in normal subjects, with coefficients of variation (CV) ranging from 60 to 90%. Pa andV˙l _{NO}show less scatter, with a CV on the order of 40%.
Tsoukias and coworkers (28, 29) calculated flux from the data in their seven subjects, as described above. With use of representative values of Pe in our subjects atQ˙e of 175–600 ml/s used by Tsoukias and coworkers, their values of flux would only be ∼1–3% smaller thanV˙u _{NO}. Flux in their subjects was 0.043 ± 0.015 μl/min and did not significantly differ from the values ofV˙u _{NO}of 0.070 ± 0.048 μl/min in our subjects with use of the fasterQ˙e shown in Fig. 8 (P = 0.20) or 0.077 ± 0.053 μl/min with model 1(P = 0.16) or 0.074 ± 0.52 μl/min with model 2(P = 0.18) with use of faster and slowerQ˙e.
Evaluation of a Simplified Method to Measure V˙u_{NO}by Use of Only FasterQ˙e
Measurement ofV˙u _{NO}with Q˙e > 80–100 ml/s would have the advantage of fewer measurements of Pe and elimination of the slow exhalations that are more difficult to perform because expiration must be continued for 25–150 s. The disadvantage is that Du _{NO} cannot be measured with any precision, because its accuracy requires the higher concentrations of NO in the conducting airways achieved with low values forQ˙e. In our subjects,V˙u _{NO}calculated with only the faster Q˙e shown in Fig. 8 with use of Eq. 4 was 0.070 ± 0.048 μl/min compared with 0.077 ± 0.053 μl/min formodel 1 and 0.074 ± 0.052 μl/min for model 2 by use of all the values of Pe andQ˙e shown in Fig. 9. The three values did not differ significantly (P = 0.2) and have similar CVs of ∼70%. MeasuringV˙u _{NO}with the useful expediency of using only fasterQ˙e provides acceptable values forV˙u _{NO}but at the expense of measurements of Du _{NO}.
Choice of Analytic Method to Determine Pa,V˙u_{NO}, and Du_{NO} From Measurements of Pe andQ˙e Performed at Different ConstantQ˙e
Tsoukias and coworkers (28, 29) measured Pa and flux by plotting the quantity of NO exhaled, which is the product ofQ˙e and Pe / (Pb − 47) vs.Q˙e, so that the slope of the graph equaled Pa / (Pb − 47) and the intercept equaled flux (Eq. 3 ). We rearranged Eq. 3 to the form inEqs. 4 and 5 and plotted Pe vs. 1/Q˙e so thatQ˙edid not appear on both axes, thus eliminating potential errors of mathematical coupling that can lead to erroneous conclusions (1, 22). However, in our normal subjects the two analytic techniques provide essentially the same values forV˙u _{NO}or flux and Pa. For example, the data using the higher values ofQ˙e shown in Fig. 8 with the analytic technique applied by Tsoukias and coworkers (28, 29) using Eq. 3 resulted in flux of 0.065 ± 0.045 μl/min compared withV˙u _{NO}of 0.070 ± 0.048 μl/min by use of Eq.5 (P = 0.21). Pa was 1.78 ± 0.77 × 10^{−6} Torr with the method of Tsoukias and coworkers compared with 1.60 ± 0.72 × 10^{−6} Torr with Eq. 6 (P = 0.14). Calculations with all seven sets of values ofQ˙e and Pe resulted in flux of 0.067 ± 0.046 μl/min with the method of Tsoukias and coworkers with use of Eq. 3 compared withV˙u _{NO}of 0.077 ± 0.053 μl/min with Eq.4 . Therefore, in normal subjects the two analytic methods result in similar data. Measurements in less welltrained subjects are prone to greater variations in Pe andQ˙e; therefore, it may be wise to analyze data with both methods to determine whether mathematical coupling is influencing the results.
Alternate Models to Explain Expired NO Levels at DifferentQ˙e
The models shown in Figs. 2 and 5 precisely predict expired NO concentrations in normal subjects. An alternate model of NO exchange in the upper conducting airways has been proposed that in preliminary reports shows a similar close fit to the experimental data (15, 25). These authors assume that the NO production in the conducting airways results from a constant partial pressure of NO in the tissue wall (Pti) that can diffuse into the lumen at a rate proportional to the concentration gradient. Then, for any small segment of the conducting airways of volume fV
In conclusion, these experiments show that NO production into the lungs’ airways can be measured and divided into contributions from the alveoli (V˙l _{NO}) and the conducting airways (V˙u _{NO}).V˙l _{NO}shows less scatter in measurements in normal subjects and is two to fourfold greater thanV˙u _{NO}. Dl _{NO} is >100fold greater than Du _{NO}. Because diffusion and control of NO production in the alveoli and conducting airways are likely governed by different mechanisms, this technique may provide new information about processes that control and alter NO production by the lungs.
Acknowledgments
The authors thank Dr. Phillip E. Silkoff for providing the values of expired NO at different expiratory flow rates previously reported in Ref. 24 and illustrated in Fig. 13. Ann Bauman contributed expert editorial assistance.
Footnotes

Address for reprint requests and other correspondence: A. P. Pietropaoli, Pulmonary and Critical Care Unit, University of Rochester Medical Center, 601 Elmwood Ave., Box 692, Rochester, NY 146428692 (Email: anthony_pietropaoli{at}urmc.rochester.edu).

This study was supported by National Institutes of Health Grants R01HL51701, R01ES02679, and T32HL07216.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.
 Copyright © 1999 the American Physiological Society
Appendix
Rate of Mixing of Alveolar Airway NO With Conducting Airway NO in a Twocompartment Model
Model 1 assumes that, at the initiation of expiration at a constant flow rate, NO in the conducting airways rapidly arrives at a constant value that is maintained throughout expiration. To determine the time required to reach this constant value, we calculated the rate of change of NO in the conducting airways (Pe) as NO enters from the alveolar airways. This instantaneous rate of change in the amount of NO in the conducting airways equals d/dt[(Pe ⋅ K)/(Pb− 47)], where K is the volume of gas in the conducting airways and Pe is the partial pressure of NO in the conducting airways. In this model, Pe is determined by four variables: 1) NO from the alveoli entering the conducting airways at a constant flow rate [Q˙e ⋅ Pa/ (Pb − 47)],2) NO produced in the conducting airway that enters its lumen (V˙u
_{NO}),3) NO diffusing out of the lumen of the conducting airway into the surrounding tissues (Pe ⋅ Du
_{NO}), and 4) NO leaving the conducting airway via exhalation [Q˙e ⋅ Pe/ (Pb − 47)]. Therefore
Appendix
Determination of Pe in a Twocompartment Model of the Airways with Stratification of the Concentration of NO Along the Lumen of the Conducting Airways
In any segment of the conducting airways illustrated in Fig. 5