Early alteration of single-breath nitrogen washout (ΔN₂/L) in smokers with preserved spirometry: Association with smoking burden

Abstract

Background

Tobacco-related small-airway dysfunction (SAD) may precede spirometric obstruction. The single-breath nitrogen washout (SBW N₂) captures ventilation heterogeneity and may detect early peripheral airway involvement through the phase III slope (ΔN₂/L) and closing volume (CV).

Aim

To describe SBW N₂ indices in smokers with preserved spirometry, examine their relationship with cumulative smoking exposure, and contextualise results against published reference (healthy predicted) values.

Methods

Cross-sectional study of current smokers (n=90) with preserved spirometry (pre-bronchodilator FEV₁/FVC ≥0.70). Spirometry followed SEPAR criteria and SBW N2 followed ERS/ATS recommendations. Associations with pack-years were assessed by Pearson correlations and linear regression. In the absence of a concurrently recruited healthy control group, SBW N2 outcomes were also expressed as percent-predicted using published reference equations only in the subsets for which those equations were applicable. To explore sex-related differences in ventilation heterogeneity, we compared key characteristics and size-independent functional indices between men and women (spirometry as % predicted and ratios) and fitted a multivariable OLS model for ΔN₂/L adjusting for pack-years, age and BMI.

Results

Mean age was 42 (11) years and mean smoking exposure 29.01 (16.95) pack-years. Mean ΔN₂/L was 2.674 (1.424) %N₂·L⁻¹ and correlated significantly with pack-years (r=0.452, p<0.001), as did CV/VC (r=0.389, p<0.001). In external-reference comparisons, ΔN₂/L in males aged 30–70 years (n=42) was markedly elevated (327.1 (165.7)% predicted; p<0.001), with 83.3% exceeding the proposed ULN (180% predicted). In multivariable regression adjusting for pack-years, age and BMI, male sex remained independently associated with higher ΔN₂/L (β=+0.946 %N₂·L⁻¹; 95% CI 0.400 to 1.492; p<0.001).

Conclusions

SBW N₂ indices-particularly ΔN₂/L-show a dose–response relationship with smoking burden and are markedly abnormal relative to healthy predicted values, even in smokers with preserved spirometry. These findings support SBW N₂ as a sensitive tool for detecting early peripheral SAD before spirometric obstruction becomes apparent. Sex-related differences in ΔN₂/L persisted after adjustment for smoking exposure and anthropometrics, supporting a sex-aware interpretation of this index.

Introduction

Chronic exposure to tobacco smoke induces inflammation and remodelling of the small airways (≈<2 mm), with loss of bronchiolar–alveolar attachments and luminal obstruction, which may precede for years the obstruction detected by spirometry. Classic demonstrations that peripheral resistance increases substantially in chronic obstructive disease supported the concept of ‘small airways disease’ as an early pathophysiological target [1]. Structure–function studies showed that histological changes in bronchioles correlate with subtle functional abnormalities, reinforcing that conventional spirometry may underestimate heterogeneous and patchy damage in early phases [2].

The single breath nitrogen washout test (SBW N2) quantifies ventilation heterogeneity. The phase III slope (SIII or ΔN₂/L) is expressed as the percentage increase in nitrogen per litre of expired volume (%N2•L⁻¹) and is calculated on the middle segment of expired volume to minimise phase IV influence [3]. Buist and Ross introduced quantitative analysis of the ‘alveolar plateau’ to detect early obstruction [4]. Longitudinal studies evaluated its ability to identify smokers susceptible to progress to chronic airflow limitation [5] and to predict FEV₁ decline in the general population [6].

Interest in small-airway involvement has been strengthened by contemporary histopathological evidence placing bronchiolar inflammatory thickening and obstruction as key determinants of COPD progression [7]. In parallel, multiple-breath washout techniques allow separation of conductive and acinar components of ventilation inhomogeneity [8].

Other standardised techniques, such as oscillometry (IOS/FOT), assess respiratory system impedance during tidal breathing [9]. In smokers with preserved spirometry, comparative work has shown a high prevalence of abnormalities in washout and in IOS/FOT, but with weak correlations between both, suggesting they capture different components of peripheral dysfunction [10].

The aim of this study was to describe early abnormalities in SBW N2 indices in smokers with preserved spirometry and to analyse their relationship with cumulative exposure (pack-years). Although the physiological basis of the SBW N2 test was established decades ago, its relevance has re-emerged within the current concept of small-airway dysfunction and the search for abnormalities that precede overt COPD and may complement oscillometry [9–11].

Materials and Methods

Study design and setting

This was an observational cross-sectional study conducted in a Smoking Cessation Unit over a 6-month period and reported according to STROBE recommendations [12].

Study population

Consecutive current smokers were evaluated. All participants underwent pre-bronchodilator spirometry and SBW N2 testing. Preserved spirometry was defined as a pre-bronchodilator FEV₁/FVC ≥0.70. We use the term preserved rather than normal spirometry because a fixed pre-bronchodilator ratio may misclassify airflow obstruction, particularly in younger adults. Demographic and exposure variables recorded included age, sex, body mass index (BMI), cigarettes/ day, and cumulative smoking exposure (pack-years).

Spirometry

Spirometry was performed pre-bronchodilator according to SEPAR recommendations [13]. FVC, FEV₁, the FEV₁/FVC ratio, and FEF^25–75 were recorded. Percent-predicted values were calculated using the Global Lung Function Initiative (GLI) 2012 reference equations [14].

Single-breath nitrogen washout (SBW N₂)

SBW N2 was performed following the ERS/ATS consensus recommendations [3] using a MasterScreen system ( Jaeger, Würzburg, Germany) equipped with a low-resistance pneumotachograph and a rapid nitrogen analyser. After stable tidal breathing, subjects exhaled to residual volume, inhaled 100% O₂ rapidly to total lung capacity, and then exhaled slowly and continuously to residual volume at a target flow of 0.3–0.5 L·s⁻¹ until completion. At least three acceptable manoeuvres were obtained; two reproducible curves (difference in closing volume <10%) were averaged. The main SBW N2 outcomes were ΔN₂/L (%N₂·L⁻¹) and airway-closure indices including CV (L) and CV/VC (%), using the quantitative approach described by Buist and Ross [4,5].

External-reference contextualisation

Because a concurrently recruited never-smoker control group was not available, SBW N2 outcomes were additionally contextualised against published reference equations. For ΔN₂/L, percent-predicted values were calculated only in males aged 30–70 years using the age-based equation reported by Sixt et al. [15]; an upper limit of normal (ULN) of 180% predicted was used as proposed in that work. No male reference equation was extrapolated to women. For CV/VC, percent-predicted values were calculated using published prediction equations for closing-volume indices [16].

Ethics

The study was approved by the Research Ethics Committee (CEI) of Hospital Universitario San Juan de Alicante. All participants provided written informed consent.

Statistical analysis

Statistical analyses were performed using IBM SPSS Statistics version 18.0. Continuous variables are presented as mean (SD) (and as median [IQR] when appropriate), and categorical variables as n (%). Normality was assessed using Shapiro–Wilk tests and visual inspection of distributions. Between-sex comparisons were performed using the Mann–Whitney U test for continuous variables and the chi-square (χ²) test for categorical variables (Fisher’s exact test when expected counts were <5). To minimise anthropometric bias when comparing sexes, spirometry was analysed primarily using percent-predicted indices (FVC, FEV₁ and FEF_25–75) and ratios (FEV₁/FVC and CV/VC), rather than absolute volumes. To evaluate whether sex-related differences in ventilation heterogeneity persisted after accounting for confounding, a multivariable linear regression model (ordinary least squares, OLS) was fitted with ΔN₂/L (%N₂·L⁻¹) as the dependent variable and sex (male vs female), pack-years, age and BMI as independent variables; coefficients (β) are reported with 95% confidence intervals (CI) and two-sided p values. Correlations between pack-years and functional indices were assessed using Pearson’s correlation coefficient (r) with 95% CI calculated using Fisher’s z transformation, and simple linear regression models were fitted to estimate β coefficients (change in outcome per pack-year). All tests were two-sided and p<0.05 was considered statistically significant; p values are reported to three decimals, with smaller values shown as p<0.001.

Results

A total of 90 subjects were analysed (45 men, 45 women). Mean age was 42 (11) years (range 26–78). Smoking exposure was 29.01 (16.95) pack-years and consumption was 23.9 (9.6) cigarettes/day. Exhaled CO was 20.4 (9.1) ppm and BMI was 26 (4) kg/m² (Table 1).

Table 1.

Baseline characteristics and smoking exposure.

Variable	Mean (SD)	Median [IQR]	Range
Sex (men/women)	45/45	-	-
Age (years)	42 (11)	41 [34.00–47.75]	26–78
Pack-years	29.01 (16.95)	25 [18.25–37.50]	3–90
Cigarettes/day	23.86 (9.61)	20 [20.00–30.00]	10–60
Exhaled CO (ppm)	20.41 (9.14)	20 [15.25–24.75]	1–57
BMI (kg/m 2 )	26 (4)	25 [23.00–29.00]	18–40

Spirometry showed preserved values: FVC 3.76 (0.79) L and FEV₁ 2.96 (0.63) L, with FEV₁/FVC 79 (5)% and FEF_25–75 87.39 (24.75)% predicted (Table 2).

Table 2.

Baseline demographic and functional indices stratified by sex.

Variable	Men (n=45)	Women (n=45)	p value
Age, years	44 (11)	40 (10)	0.095
Smoking duration, years (estimated)*	26.5 (12.3)	22.6 (8.2)	0.136
Smoking exposure, pack-years	34.2 (18.6)	23.8 (13.5)	0.003
Cigarettes/day	26.2 (9.8)	21.5 (8.9)	0.012
Exhaled CO, ppm	22.1 (9.2)	18.7 (8.9)	0.160
BMI, kg/m 2	27 (4)	25 (4)	0.011
FVC, % predicted	88 (8)	91 (10)	0.113
FEV 1 , % predicted	89 (9)	92 (10)	0.066
FEV 1 /FVC, %	78 (5)	80 (5)	0.036
FEF 25–75 , % predicted	83.49 (24.93)	91.29 (24.22)	0.111
CV/VC, %	17.07 (6.74)	15.42 (4.99)	0.389
Phase III slope (ΔN2/L), %N2•L−1	3.22 (1.64)	2.13 (0.90)	<0.001

Data are mean (SD). Between-sex comparisons: Mann–Whitney U test.

^*Smoking duration estimated as (pack-years × 20) / cigarettes per day.

Note: Spirometry is reported as % predicted and ratios (FEV₁/FVC, CV/VC) to minimise anthropometric bias between sexes. Absolute lung volumes in the text are reported in litres.

In SBW N2, CV was 0.58 (0.25) L and CV/VC 16.24 (5.96)%. Phase III slope (ΔN₂/L) was 2.674 (1.424) %N₂·L⁻¹ (range 0.670–8.400) (Table 3).

Table 3.

Single-breath nitrogen washout (SBW N₂) indices.

Variable	Mean (SD)	Median [IQR]	Range
Closing volume (CV) (L)	0.58 (0.25)	0.55 [0.42–0.67]	0.11–1.54
CV/VC, %	16.24 (5.96)	16.00 [12.00–19.00]	4.00–32.00
Phase III slope (ΔN2/L) (%N2•L−1)	2.67 (1.42)	2.42 [1.69–3.33]	0.67–8.40

ΔN₂/L correlated positively with pack-years (n=90; r=0.452; p<0.001), with β=0.038 %N₂·L⁻¹ per pack-year (Figure 1). CV/VC also showed a positive association with pack-years (n=90; r=0.389; p<0.001), with β=0.137 percentage points per pack-year (Figure 2). Correlations for additional indices are summarised in Table 4 (Figures 3, 4).

Figure 1.

Correlation between pack-years and the phase III slope (ΔN₂/L, %N₂·L⁻¹). Scatter plot with linear regression line and 95% confidence interval for the mean.

Figure 2.

Correlation between pack-years and CV/VC (%). Scatter plot with linear regression line and 95% confidence interval for the mean.

Table 4.

Summary of Pearson correlations with pack-years.

Variable	r	95% CI (lower)	95% CI (upper)	p
Phase III slope (ΔN2/L) (%N2•L−1)	0.452	0.270	0.603	<0.001
CV/VC, %	0.389	0.197	0.551	<0.001
Closing volume (CV) (L)	0.297	0.095	0.476	0.005
FEF 25–75 , % predicted	−0.376	−0.541	−0.183	<0.001
FEV 1 /FVC, %	−0.538	−0.671	−0.373	<0.001

Figure 3.

Correlation between pack-years and FEV₁/FVC (%). Scatter plot with linear regression line and 95% confidence interval for the mean.

Figure 4.

Correlation between pack-years and FEF_25–75 (% predicted). Scatter plot with linear regression line and 95% confidence interval for the mean.

Table 2 summarises sex-stratified characteristics and size-independent functional indices. In multivariable linear regression adjusting for pack-years, age and BMI, male sex remained independently associated with higher ΔN₂/L (β=+0.946 %N₂·L⁻¹; 95% CI 0.400 to 1.492; p<0.001; adjusted R²=0.295). Pack-years were also associated with ΔN₂/L (β=0.030, p=0.005), whereas age was not (p=0.962) and BMI showed an inverse association (β=−0.079, p=0.012).

External-reference comparisons are shown in Table 5. In males aged 30–70 years (n=42), ΔN₂/L was markedly elevated (327.1 (165.7)% predicted; p<0.001) and 83.3% exceeded the proposed ULN (180% predicted) [15]. In the whole cohort (n=90), CV/VC expressed as percent-predicted was lower than predicted (81.80 (28.14)% predicted; p<0.001) [16].

Table 5.

External-reference comparison of SBW N₂ indices with published predicted values

Outcome	Subset (n)	Observed mean (SD)	Predicted mean (SD)	% predicted (mean (SD))	% abnormal	p vs 100% predicted
ΔN2/L (%N2•L−1) [15]	Males 30–70 y (n=42)	3.257 (1.684)	0.996 (0.136)	327.1 (165.7)	83.3% >180% predicted	<0.001
CV/VC (%) [16]	Whole cohort (n=90)	16.24 (5.96)	19.90 (3.42)	81.80 (28.14)	—	<0.001

SBW N₂ outcomes were contextualised using published reference equations because no never-smoker control group was available; for ΔN₂/L, external-reference analysis was restricted to the subgroup for which the selected male equation was applicable.

^*Upper limit of normal (ULN) defined as 180% predicted as proposed by Sixt et al. [15].

No male equation was extrapolated to women.

Discussion

In this cohort of smokers with preserved spirometry, SBW N2 indices showed abnormalities consistent with early SAD. In particular, ΔN₂/L showed a clear dose–response association with pack-years, supporting ventilation heterogeneity as an early peripheral functional marker that may precede spirometric obstruction [4–6].

Because a never-smoker control group was not available, we contextualised SBW N2 outcomes against published reference data. Using the Sixt et al. reference equation for healthy never-smoking males aged 30–70 years, ΔN₂/L was markedly elevated in the applicable male subset (mean 327% predicted) and most exceeded the proposed ULN (180% predicted) [15]. We restricted this external-reference analysis to the subgroup for which the selected equation was applicable and did not extrapolate the male equation to women.

To explore sex-related differences, we compared sexes using percent-predicted spirometry and ratios to reduce anthropometric confounding, and then fitted an OLS model for ΔN₂/L adjusting for pack-years, age and BMI. Male sex remained independently associated with higher ΔN₂/L (β=+0.946 %N₂·L⁻¹; 95% CI 0.400 to 1.492; p<0.001; adjusted R²=0.295). This finding should not be interpreted as evidence that women are less susceptible to smoking-related injury. Rather, sex-related differences in airway-to-lung size matching (dysanapsis), expiratory mechanics and alveolar plateau geometry may influence the single-breath nitrogen slope, while hormonal modulation of airway tone, ventilation and inflammatory responses may modify the phenotype of smoking-related abnormalities [17,18]. Thus, susceptibility to smoking and the magnitude of ΔN₂/L are not necessarily interchangeable constructs.

Our findings are consistent with the concept of SAD as an early pathophysiological target: classic studies showed that chronic obstruction is related to disproportionate peripheral changes relative to the baseline contribution of small airways to total resistance [1], and that bronchiolar structural abnormalities correlate with sensitive functional tests [2]. In established COPD, bronchiolar obstruction and remodelling are central determinants of progression [7]. In this context, SBW N₂ provides a functional window into inhomogeneity: Buist and Ross formalised quantitative analysis of the alveolar plateau to detect early obstruction [4], and its prognostic utility was later explored in susceptible smokers [5] and in relation to FEV₁ decline [6]. Subsequent studies reinforced the clinical relevance of the single-breath nitrogen test as a marker of functional decline and COPD development [12]. In parallel, Milic-Emili and colleagues synthesised decades of physiological evidence on closing volume, highlighting its role in understanding early closure and peripheral involvement in obstructive diseases [19].

Interestingly, the external-reference comparison suggested a dissociation between ventilation inhomogeneity and airway-closure indices in this sample: while ΔN₂/L was clearly abnormal when expressed as percent-predicted in the male subset, CV/VC expressed as percent-predicted based on published prediction equations showed a mean of 81.8% predicted in the full cohort. This apparent lower-than-predicted airway closure index, despite its positive association with smoking burden within the cohort, may reflect that ΔN₂/L and CV/VC capture different physiological components of peripheral dysfunction, and that reference equations for CV/VC are method- and protocol-dependent, limiting comparability across laboratories [16]. Overall, these findings reinforce the value of treating SBW N₂ indices as continuous variables and highlight that ΔN₂/L may be particularly sensitive for detecting early peripheral abnormalities in smokers.

Oscillometry (IOS/FOT) measures respiratory impedance during tidal breathing and provides indices of peripheral airway mechanics (e.g., R5–R20, X5, AX, Fres), with well-established technical standards [9]. Available literature suggests complementarity rather than substitution: in smokers with preserved spirometry, washout and oscillometry abnormalities can coexist but show only modest correlations, indicating that they capture different domains of peripheral dysfunction [10]. Tanaka et al. highlighted that heterogeneous bronchiolar narrowing can be reflected by both ventilation inhomogeneity and oscillometry indices, supporting a combined approach for more complete phenotyping [20].

Although the present study focuses on smokers, the conceptual framework is transferable to other conditions such as asthma, where small-airway and acinar involvement may persist even with good clinical control. Hanon et al. reported persistent acinar abnormalities despite optimised inhaled therapy and stable asthma control, with limited concordance across techniques [21]. Similarly, small-airway dysfunction and hyperinflation have been reported in Long COVID despite preserved conventional spirometry, supporting the role of sensitive small-airway tests when spirometry is non-diagnostic [22].

Strengths of this study include a balanced sample with equal numbers of men and women (n=90, 45/45), enabling sex-stratified analyses using size-independent functional metrics (percent-predicted spirometry and ratios) to minimise anthropometric confounding. We incorporated a multivariable model adjusting for pack-years, age and BMI to test whether sex differences in ΔN₂/L persisted beyond smoking burden and basic anthropometrics, strengthening interpretability of the sex comparison.

This study has limitations. First, a concurrently recruited never-smoker control group was not available; therefore, external-reference contextualisation relied on published reference equations, and for ΔN₂/L these were applied only to the male subset for which the selected equation was appropriate [15]. Second, preserved spirometry was defined using a pre-bronchodilator fixed ratio (FEV₁/FVC ≥0.70). Because a fixed ratio may underestimate obstruction in younger adults compared with the lower limit of normal, our cohort should be interpreted as smokers with preserved spirometry by study definition rather than unequivocally normal airways [23]. Third, while sex comparisons were performed using percent-predicted spirometry and ratios to minimise anthropometric bias, ΔN₂/L differed by sex even after adjustment for pack-years, age and BMI, suggesting that sex-related physiological or methodological factors may influence ventilation heterogeneity. Finally, the cross-sectional design precludes causal inference or prognostic assessment, residual confounding cannot be fully excluded, and IOS/FOT were not measured in the same cohort; therefore, comparisons with oscillometry rely on published evidence.

In conclusion, in smokers with preserved spirometry by fixed ratio, SBW N2 indices—particularly ΔN₂/L—show a dose–response association with smoking burden and, when interpreted against external reference values, demonstrate a high prevalence of abnormal ventilation heterogeneity in a subset where reference application is valid. These results reinforce that tobacco exposure can provoke early peripheral functional alterations that may be missed by spirometry alone and that interpretation of ΔN₂/L should be sex-aware.

Abbreviations

SBW

single-breath washout

SIII

phase III slope

CV

closing volume

SAD

small-airway dysfunction

IOS/FOT

impulse oscillometry/forced oscillation technique

SEPAR

Spanish Society of Pneumology and Thoracic Surgery.

Availability of data and material

The de-identified datasets generated and/or analysed during the current study are available from the corresponding author upon reasonable request. Data are not publicly available due to ethical and data-protection restrictions, as they contain information that could compromise participant privacy.