measuring exhaled nitric oxide (NO) at different flow rates has been proposed as an elegant method to estimate airway and alveolar NO contributions to exhaled NO. Different methods are already in use to actually derive airway NO production and alveolar NO concentration to reflect inflammation in both airway and alveolated lung compartments (5). Nevertheless, no definitive lung model has yet been proposed that can consistently simulate the exhaled NO values obtained under the various experimental conditions. One model assumption under debate concerns the distribution of NO production along the bronchial tree (20, 25). Another relates to the reduction in NO production available to the airway lumen in response to airway constriction (26). The lung models currently gaining importance over the simple tube-balloon type models are single-trumpet models reflecting the increasing cross-sectional airway area between the conductive and acinar airways of the lung. A limitation of a single-trumpet model is that portions of the upper (apical) lung of a normal subject sitting upright that may be underperfused and/or underventilated cannot be differentiated from the remainder of the lung. This would require at least two trumpets in parallel for accurate simulations. Recently, one such model has been proposed to study the effect of different ventilation and the effect of different NO production in different parts of the lung (21).

In the present experimental study, we aimed to assess the effect on exhaled NO of varying the relative volumes of underperfused and perfused lung zones while keeping ventilation distribution similar. We have previously shown that the prone suspended and supine posture lead to similar ventilation distribution (18) but that transfer factor for CO (TL_CO) increases by 9% from prone to supine when adequately corrected for lung volume (13). Irrespective of whether this effect stems from an increased membrane diffusing capacity or increased pulmonary capillary perfusion, the postural differences in NO concentration are expected to take effect in the alveolar compartment, which acts as a NO sink due the high reaction rate of NO with hemoglobin (1). We therefore tested the hypothesis that, if the method to differentiate between alveolar and bronchial components of exhaled NO holds, only alveolar NO concentration (FANO) should be affected by a postural change from the prone suspended to the supine posture. Also, if we do find that maximal transfer of NO across the blood-gas barrier (i.e., the lowest FANO) can be obtained in the supine posture, any increase of FANO with respect to this supine FANO value can then serve as a basis for estimating the proportion of lung that does not participate in the alveolar NO transfer in a normal subject, for instance in the sitting posture.

MATERIALS AND METHODS

Main protocol: exhaled NO measurements in different body postures.

The main protocol was approved by the local ethics committee (no. B14320071296), and informed consent was obtained from the 10 participating normal, never-smoking subjects. No dietary requirements were imposed since its potential effect on, for instance, the oral contribution to exhaled NO (11) was not expected to vary with body posture. An identical set of exhaled NO and lung volume measurements was performed in three body postures after having the subject remain in any given body posture for at least 15min. All subjects started in the sitting posture, followed by supine and prone in random order. The prone posture was in fact a prone suspended posture with the subject supported by arms and knees to avoid ventral compression and to mimic the prone posture previously employed in a study where TL_CO was seen to be lower with respect to supine (13). One set of measurements in each posture included exhaled NO measurements at six expiratory flows between 50 and 350 ml/s (two tests per flow) and one N₂ washout measurement of lung volumes.

Lung volumes were measured with a N₂ washout technique using standard equipment (Vmax Encore, Sensor Medics), extracting residual volume (RV) and total lung capacity (TLC). Each exhaled NO measurement started with a vital capacity inhalation of NO-free air (filter type A1B2E2K1HgCONO-P3, Draeger, Luebeck, Germany) followed by exhalation via six different restrictors with the subject maintaining a mouth pressure of 15 cmH₂O, so as to obtain exhaled NO for target flows between 50 and 350 ml/s (3). Exhaled NO concentration was continuously recorded (CLD, EcoPhysics) such that a NO plateau value could be readily identified. Alveolar NO concentration (FANO) and NO flux in the airway compartment [Jaw(NO)] were estimated using the two most common methods, described by Pietropaoli et al. (15) and Tsoukias et al. (22). In Pietropaoli's method, FANO and Jaw(NO) correspond to intercept and slope, respectively, of the exhaled NO vs. flow⁻¹ plot, whereas in Tsoukias' method FANO and Jaw(NO) correspond to slope and intercept, respectively, of the exhaled NO times flow vs. flow plot. We applied both techniques by alternatively using data corresponding to all flow rates and by following the recommendation (5) that only data points corresponding to flow of >100 ml/s (flow⁻¹ < 0.01 s/ml) should be used.

Follow up protocol: capillary volume measurements in different body postures.

In an attempt to further investigate a possible reason for the obtained FANO results, we designed a follow-up protocol to measure pulmonary capillary volume (V_c) in the same body postures. This could only be obtained on a different laboratory site, with 10 different never-smoker test subjects as part of another study and for whom informed consent was also obtained via the local ethics committee (no. B40620072173). One subject who had participated in the main protocol (n = 10) was also able to perform the follow-up protocol (n = 11) at the other laboratory site. The follow-up protocol basically consisted of having the test subjects perform single-breath tests for V_c determination in the three above-mentioned postures via simultaneous transfer factor for NO (TL_NO) and TL_CO measurement as described in Aguilaniu et al. (1). As in the main protocol, subjects remained in any given body posture for at least 15 min before carrying out the test, starting with the sitting posture, followed by supine and prone in random order.

An automated apparatus was used (Medisoft, Dinant, Belgium). The inhaled gas was a mixture of a test gas containing 0.28% CO, 14% He, 21% O₂ balanced with N₂, and a test gas containing 450 parts/million of NO in N₂ (Air Liquide Santé, Paris, France). The final NO and O₂ concentration in the inspiratory bag were respectively 40 parts/million and 19.1%. The apparatus was calibrated for gas fractions using automated procedures. The subjects were requested to expire deeply, and at the onset of the following inspiration the subject inspired the mixture from the bag during a rapid deep inspiration, followed by a 4-s breathhold and a rapid expiration. Alveolar volume (Va) at the time of breathhold was calculated using the He-dilution technique. The first 0.8 liter of expired gas was discarded, and the following 0.6 liter was sampled in a bag, where it was analyzed for NO, CO, and He concentration; the delay in analysis of expired gas sample was constant at 35 s.

The effective breathhold time was calculated from the beginning of inspiration minus the first 30% of the inspiratory time to the middle of the expiratory sample time (8). The measurements of TL_NO and TL_CO were accepted if two successive measurements of TL_NO and TL_CO were within 10% of each other. Membrane diffusing capacity (D_m) and capillary volume (V_c) were calculated automatically as previously described (9). Essentially, these D_m and V_c computations are based on the equation of Roughton and Forster (19) and by assuming that 1) the reaction rate of NO with hemoglobin is infinitely high such that TL_NO = D_m,NO; 2) D_m,CO = D_m,NO/1.97, 3) the reaction rate of CO with hemoglobin could be set to 14.29 min⁻¹·kPa⁻¹ for females and 13.16 min⁻¹·kPa⁻¹ for males (by estimating mean capillary oxygen tension at 13.3 kPa).

To account for potential Va differences between different postures, two volume correction methods have been implemented. Method 1 is a conceptual method, which has convincingly been shown not to overcorrect as does the transfer coefficient (7, 13). It consists of dividing TL_CO by (2·)/(1 + ) and TL_NO by where TLC_pred is the predicted TLC for each subject sitting upright. This Va correction is applied to all body postures. Method 2 directly uses the average experimental TL_NO and TL_CO changes with Va obtained in upright normal subjects (6, 24) and assumes linearity of TL_NO and TL_CO over small Va changes to apply a TL_NO (TL_CO) change of 23.75 (2.88) ml·mmHg⁻¹·min⁻¹ per l Va change. With this method, the TL_NO (TL_CO) value obtained in the body posture with the greatest Va remains unchanged, whereas the TL_NO (TL_CO) values of the two other body postures are corrected according to their smaller Va.

Statistical analysis.

Using Statistica 5.1 (StatSoft, Tulsa, OK), one-way ANOVA was used to search for posture-dependent effects (sitting, prone, supine) on TLC and transfer factor measurements. A two-way ANOVA was carried out on log-transformed exhaled NO concentration (factors: posture; flow). In either case, post hoc comparisons were done using a Tukey correction. Between supine and prone postures, Wilcoxon paired tests were performed on slopes and intercepts obtained from plots of exhaled NO vs. reciprocal of flow. Significance was set at P = 0.05.

RESULTS

Experimental data: main protocol.

The exhaled NO measurements obtained on all 10 subjects (5 women/5 men; 36 ± 17 yr; mean ± SD) are summarized in Fig. 1A; the raw data can be found on the online repository. Analysis of variance showed significant flow (P < 0.001) and posture (P = 0.006) effects on the log-transformed NO concentrations. Post hoc comparisons did not show a significant difference between sitting and prone (P > 0.1 at all flow levels) and a highly significant difference between supine and prone (P < 0.001 at all flow levels). Figure 1B illustrates the respective lung volumes at RV and TLC in the different postures. ANOVA on RV did not show a posture-dependent effect (P = 0.3), whereas TLC showed a posture-dependent effect (P = 0.02) with a post hoc significant difference between sitting and supine (P = 0.04) and between prone and supine (P = 0.03). The TLC decrease from prone to supine averaged 7%.

Figure 2 shows the FANO and Jaw(NO) values corresponding to Fig. 1A obtained by using the methods of Pietropaoli et al. (15) and Tsoukias et al. (22), alternatively including all data or only data points corresponding to flow of >100 ml/s (flow⁻¹ < 0.01 s/ml) for regression. In the case of Pietropaoli's method, using all data or only data corresponding to flow⁻¹ < 0.01 s/ml led to R values that averaged 0.99 and 0.98, respectively, across the 10 subjects under study. In the case of Tsoukias' method, using all data or only data corresponding to flow > 100 ml/s led to average R values of 0.98 and 0.97, respectively. The relative FANO and Jaw(NO) changes between prone and supine were very similar across both analysis techniques, with an absence of significant change in Jaw(NO) between both postures and a significant FANO decrease from prone to supine ranging from 23 to 33%.

Experimental data: follow-up protocol.

Table 1 summarizes the V_c measurements obtained on 10 new subjects and the 1 subject who had also performed the exhaled NO tests (n = 11; 5 women/6 men; 43 ± 13yr). There was no V_c difference between sitting and prone postures (P = 0.7). Average V_c increased from 102 ± 2 ml prone to 115 ± 20 ml supine (P = 0.002). The subject participating in both exhaled NO and V_c protocols showed a V_c increase from 109 to 133 ml (i.e., an individual 24-ml V_c change compared with the average 13-ml V_c change) and a FANO change decreasing from 6.1 to 4.3 parts/billion (i.e., 1.8 parts/billion change compared with the average 0.8 parts/billion change) when using Pietropaoli's method including all flows (leftmost bars in Fig. 2B).

The TL_NO and TL_CO values that were at the basis of the V_c results are also shown in Table 1, together with the corresponding Va to correct TL_NO and TL_CO for the volume at which they were measured, according to two Va correction methods. Significant changes were found between TL_NO^corr prone and supine (increasing by 4–7%; P ≤ 0.01 for both Va correction methods) and between TL_CO^corr prone and supine (increasing by 9–11%; P = 0.002 for both Va correction methods). The ratio TL_NO^corr/TL_CO^corr decreased by 3.6–4.8% (P ≤ 0.04 for both Va correction methods).

DISCUSSION

The present study shows a clear-cut decrease in alveolar NO concentration from prone to supine, as determined from the exhaled NO dependence on flow in either posture. In particular, the significant decrease in intercept (FANO) from prone to supine and the absence of change in the slope [Jaw(NO)], identifies the peripheral alveolar compartment as the sole location of change. If the slope had also shown a posture-dependent change, this would have signaled a modification in either NO production or in the constriction pattern of nonalveolated airways, which could have affected the NO back diffusion (10, 20, 25, 26). This was not the case, and it was thus confirmed experimentally that a postural change in exhaled NO from prone to supine, which was expected to be only operational in the alveolar compartment, duly resulted in a FANO change only. The degree of FANO decrease from prone to supine ranged from 23 to 33% (Fig. 2B), depending on the regression analysis used and on the data points that were included. Good quality of the regression analyses necessitated a sufficient number of data points on a sufficiently linear portion of NO concentration vs. flow⁻¹ [Pietropaoli method (15)] or flow [Tsoukias method (22)]. The high correlation coefficients obtained here indicate that both these criteria were met across all analyses.

A purely volumetric effect of the 7% TLC decrease from prone to supine postures could have affected FANO measurement in a number of ways, some of which are interrelated. First, the concentrating effect of a smaller volume supine would have increased rather than decreased FANO. Alternatively, a smaller lung volume could have diminished the NO production available to the airway lumen, potentially decreasing FANO. Using the rule of thumb that FANO roughly corresponds to alveolar NO production divided by TL_NO, the assumption that NO production is proportional to airway surface (10) or airway volume (20) would have resulted in a 4 or 7% FANO decrease, respectively. Alternatively, experimental studies (with subjects in the sitting posture) have consistently shown an increasing TL_NO with alveolar volume (6, 23, 24). Assuming this volumetric effect to be independent of posture, these studies predict that a 7% TLC decrease would result in a 11% FANO increase. Taken together, the above arguments suggest that the alveolar lung volume decrease observed experimentally between prone and supine cannot account for the corresponding FANO decrease.

To understand whether the FANO changes could possibly be explained by changes in the volume of capillary blood available for carrying NO into the blood stream, a follow-up experiment was conducted. In this experiment, we showed that V_c increased by 13% from prone to supine in a group of 11 normal subjects, one of whom also participated in the main protocol. In fact, the subject who had participated in both exhaled NO and V_c protocols showed an above-average V_c increase accompanied by an above-average FANO decrease. The average V_c change between supine and prone observed here is greater than that reported by Peces-Barba et al. (13), where the average 3.3% V_c increase did not reach significance. In that study, V_c and D_m,CO components were obtained by performing normoxic and hyperoxic TL_CO measurements, and, despite a significant TL_CO increase, neither of its components was significantly different between prone and supine.

The greater V_c supine than prone observed here could have resulted from a redistribution of blood between perfused and unperfused pulmonary capillaries (i.e., capillary recruitment) or from a redistribution between fully perfused (engorged) and normally perfused capillaries. However, the smaller FANO supine than prone (Fig. 1A) can only be explained by an increased V_c supine if this V_c increase was brought about by capillary recruitment. Indeed, the degree of NO uptake in the blood is determined not by the amount of blood in the capillaries (or capillary engorgement) but rather by the absence or presence of blood for NO transit across the blood-gas barrier, given the high reaction rate of NO with hemoglobin (1). Therefore, the experimental FANO decrease from prone to supine observed here (Fig. 1B) strongly indicates that the observed V_c increase (Fig. 2) is due to capillary recruitment when changing from the prone to the supine posture.

In the prone posture, a greater proportion of lung volume is physically available to harbor capillaries in an un(der)perfused condition due to lung shape and the space occupied by the heart (2, 13). It has also been shown that un(der)perfused capillaries may well be present throughout the lungs (4, 12, 14, 17), but it is unclear whether nongravitational un(der)perfused capillaries would be more present prone than supine. The relative changes of V_c and TL_NO/TL_CO can provide a clue as to whether capillary recruitment is occurring when changing from prone to supine. Indeed, an increased V_c with a relatively constant TL_NO/TL_CO is expected to signal capillary recruitment, whereas an increased V_c with a similarly decreasing TL_NO/TL_CO is indicative of capillary distention (6). The latter was illustrated by Glénet el al. (6) by means of a hemodilution experiment, where an 8% V_c increase (corrected for hemoglobin concentration) was accompanied by a TL_NO/TL_CO decrease to more or less the same extent (7%). In our study, the 13% V_c increase from prone to supine (P = 0.002) is accompanied by a comparatively small 3.6–4.8% TL_NO/TL_CO decrease, barely reaching the border of significance (P ≤ 0.04). This indicates that the V_c increase observed here from prone to supine is in large part due to capillary recruitment.

Because the observed V_c increase between sitting and supine postures, consistent with previous reports (13, 16), is accompanied by a mere 2.7–3.9% decrease in TL_NO/TL_CO between those postures (Table 1), it is fair to assume that capillary recruitment is also occurring from sitting to supine. The supine posture thus appears to be the posture providing maximal capacity for NO uptake, resulting from recruitment of capillaries that are unperfused in the sitting posture. Therefore, trumpet models for the simulation of exhaled NO in the sitting posture should incorporate at least two compartments, such that a portion of the lungs can be set to be unperfused. The extent of unperfused lung can then be inferred from V_c measurements relative to the supine posture and based on anatomical data regarding the pulmonary vasculature such that its effect on exhaled NO can be simulated.

In summary, we induced a change in NO uptake in the peripheral lung compartment by changing body posture between supine and prone and assessed its impact on exhaled NO measurement. This was shown to affect only the estimate of alveolar NO concentration and not of NO airway production, in support of the method to extract airway and alveolar components of exhaled NO. The greater extent of NO uptake in the blood observed in the supine posture could not be explained by a mere distention of capillaries supine that were already open prone. Supplementary measurements of lung capillary volume suggested a greater pulmonary capillary recruitment supine than prone, consistent with the exhaled NO experiments.

GRANTS

This study was funded by the Fund for Scientific Research, Flanders, and the NO Microgravity Application Project of the European Space Agency.