Improved Pulmonary Gas Exchange at Altitude Is Due to Pulmonary Vascular Adaptation to Chronic Hypoxia in Urban Residents

Abstract

Willixhofer, Robin, Laura Gochicoa-Rangel, Anna Apostolo, Jeness Campodonico, Elisabetta Salvioni, Ada De-Los- Santos-Martínez, Alejandro Reyes-García, Luis Torre-Bouscoulet, Sergio Harari, Federico Tagariello, and Piergiuseppe Agostoni. Improved pulmonary gas exchange at altitudes is due to pulmonary vascular adaptation to chronic hypoxia in urban residents. High Alt Med Biol. 00:00–00, 2025.

Background:

Chronic exposure to high altitude induces physiological adaptations in the lung, but the specific mechanisms of alveolar-capillary gas exchange adaptation in urban populations remain incompletely understood.

Methods:

We assessed altitude-related alveolar capillary membrane gas diffusion adaptations in Milan (lowlanders, 120 m) and Mexico City (highlanders, 2,240 m). A 1:1 nearest-neighbor matching by age and sex was performed.

Results:

Comparison between healthy young adults (n = 246, age <40 years) showed higher diffusing capacity for carbon monoxide (D_LCO: 31.7 [27.4–39.0] vs. 28.2 [24.6–32.8] ml/min/mmHg, p < 0.0001) and pulmonary capillary blood volume (Vcap: 105 [87–113] vs. 84 [71–98] ml, p < 0.0001), but lower membrane diffusing capacity (Dm: 50 [45–60] vs. 61 [51.70] ml/min/mmHg, p < 0.0001) in highlanders. The matching procedure yielded 71 pairs (n = 142) with balanced age and sex distributions (standardized mean differences <0.1). These differences remained significant after matching: highlanders showed higher DLCO (31.7 [27.8–39.1] vs. 27.1 [23.7–32.4] ml/min/mmHg, p < 0.0001), higher Vcap (104 [85–124] vs. 78 [65–96] ml, p < 0.0001), and lower Dm (51 [46–60 vs. 57 [50–68] ml/min/mmHg, p = 0.045).

Conclusions:

These findings suggest vascular, rather than membrane, adaptation to chronic hypoxia in high-altitude urban residents.

Keywords

altitude air pollution capillaries carbon monoxide pulmonary diffusing capacity

Introduction

The alveolar-capillary diffusion capacity (D_LCO) is a critical parameter in assessing pulmonary gas exchange efficiency. It reflects the ability of the lungs to transfer gases from the alveoli into the bloodstream. D_LCO comprises two primary components: the diffusing capacity of the alveolar-capillary membrane (Dm), which reflects the conductance of the alveolar-capillary barrier itself, and the pulmonary capillary blood volume (Vcap), which represents the amount of blood available for gas exchange.(Tamhane et al., 2001) These components provide a more granular insight into the mechanisms of pulmonary gas transfer and can be differentially affected by various physiological and environmental factors, including exposure to high altitudes.(Richalet et al., 2024)

These adaptations can be reflected in altered D_LCO measures.(Laura et al., 2024) However, the extent to which these diffusion-based parameters vary between highlanders and lowlanders in real-world, urban settings remains inconclusive. Indeed, in lowlanders exposed to very high altitude, an initial reduction in D_LCO has been observed, followed by progressive normalization. After more than 2 weeks at altitude, D_LCO even exceeds sea-level values. (Agostoni et al., 2013)

While data on D_LCO and its components at high altitude exists, they remain limited, particularly in healthy subjects from large urban populations living at different altitudes.(de Bisschop et al., 2010) Therefore, we compared data of young healthy adults permanently living at 2,200 m above sea level (asl, Mexico City) with comparable subjects living in Milan (90 m asl). Milan and Mexico City are well-suited for comparison, as both are large, densely populated cities with high levels of air pollution, particularly PM2.5 and nitric oxide, which are known to affect pulmonary function. (Cromar et al., 2021; Tunesi et al., 2024) However, they differ significantly in altitude, providing a natural contrast in chronic hypoxic exposure. This allows for the valuation of altitude-related adaptations in alveolar-capillary diffusion, while minimizing confounding from urban environmental factors.

Methods

This prospective observational study was conducted across two cardiology centers, namely Centro Cardiologico Monzino in Milan, Italy (90 m asl), and the Department of Respiratory Physiology at the National Institute of Respiratory Diseases in Mexico City (2,240 m asl). Both cohorts consisted of healthy young volunteers. Participants in both cohorts were free of known cardiac or pulmonary disease. Patients or the public were not involved in the design, conduct, reporting, or dissemination plans of our research.

Pulmonary function testing

Pulmonary function testing was performed according to current guidelines. (Stanojevic et al., 2022) The assessments included D_LCO and the diffusion capacity for nitric oxide (D_LNO), conducted using a standardized single-breath technique. In both locations D_LCO and D_LNO were measured sequentially using a 4- to 10-second breath-hold, with D_LCO values corrected for hemoglobin. Gas mixtures were: 40 ppm NO balanced with N₂ for D_LNO, and 0.28% CO, 21% O₂, 9.0% helium balanced with N₂ for D_LCO. The two gas mixtures used with the MS-PFT system were administered simultaneously. The inspired NO concentration used was set, as a standard, at 40 ppm, a dose considered free of hemodynamic effects.

D_LNO is considered almost entirely limited by membrane diffusion and is therefore used to estimate the diffusing capacity of the alveolar-capillary membrane. Dm was calculated using the formula:

D m = \frac{D_{L} N O}{\propto}

Where α is the diffusivity ratio between nitric oxide and carbon monoxide, reflecting the higher diffusion and solubility of NO in the alveolar-capillary membrane compared to CO. The ratio allows the conversion of D_LNO to an equivalent membrane diffusing capacity for CO. An α ratio of 1.97 was used in both centers conforming with standard practice. (Zavorsky et al., 2017) Vcap was then derived from the Roughton-Forster relationship. (Hughes, 2022):

\frac{1}{D_{L} C O} = \frac{1}{D m} + \frac{1}{θ C O \times Vcap}

Where θCO is the conductance of carbon monoxide in blood, corrected for individual hemoglobin levels using this equation as described in the European Respiratory Society Task Force Report (equation 1). (Zavorsky et al., 2017)

In both locations the same devices (MS-PFT, Jaeger Master Screen, Vyaire Medical, Höchberg, Germany) were used. All procedures followed the same standards and were functionally comparable.

Ethical considerations

This study was conducted in accordance with the ethical principles outlined in the Declaration of Helsinki and complied with all relevant institutional and national research regulations. Written informed consent was obtained from all participants prior to their inclusion in the study. Ethical approval was granted by the institutional review boards at both study sites: in Milan, Italy, by the Comitato Etico Territoriale Lombardia 2 at the Istituto Europeo di Oncologia and Centro Cardiologico Monzino (Approval No. R853/18-CCM897), and in Mexico City, Mexico, by the local institutional ethics committee of the National Institute of Respiratory Disease (Approval No. C65-17).

Statistical analysis

Between-group comparisons were performed using t-tests or Mann–Whitney U tests for continuous variables (based on Shapiro–Wilk normality testing) and chi-squared tests for categorical data. To control for potential confounding by age and sex, a 1:1 nearest-neighbor matching was performed between cohorts (MatchIt R package). Matching was exact for sex and nearest on age within a caliper of 0.2 standard deviations. Results are reported as mean ± SD or median (IQR). D_LCO, Dm, and Vcap differences were visualized with boxplots; a contour overlay illustrated the Dm–Vcap relationship. To account for multiple comparisons, p-values were adjusted using the Benjamini–Hochberg procedure, which controls the false discovery rate (FDR). For the overall cohort, the Benjamini–Hochberg correction was applied across all variables to identify general cohort differences. For the matched cohort, FDR adjustment was applied only to physiological outcome variables, while matching variables (age and sex) were excluded from FDR adjustment because balance for these covariates was achieved by the matching procedure. An FDR level of 0.05 was accepted. Variables with Benjamini–Hochberg adjusted p-values <0.05 were considered statistically significant. Analyses were performed in R version 4.4.2 (“Pile of Leaves,” The R Foundation for Statistical Computing).

Results

A total of 246 young (age <40 years) participants were included, with 106 lowlanders and 140 highlanders. Highlanders were younger [27 (22–31) vs. 31 (28–34) years, p < 0.0001] and had a higher body mass index [25.3 (23.5–27.1) vs. 23.0 (20.8–25.0) kg/m², p < 0.0001], though gender distribution and weight were comparable between groups. Albeit anthropometric differences, lung function parameters indicate in both groups a preserved and comparable function as indicated by percent-predicted forced vital capacity and forced expiratory flow (Table 1).

Table 1.

Comparison of Demographic Characteristics, Pulmonary Function, and Gas Exchange Parameters Between Lowlanders and Highlanders

Variable	Lowlanders (n = 106)	Highlanders (n = 140)	p-Value	FDR-adjusted p-Value^b
Demographics
Age (years)	31 (28–34)	27 (22–31)	<0.0001	<0.0001
Female Gender	56 (53%)	91 (65%)	0.0725	0.0915
Height (cm)	170 (164–178)	162 (156–170)	<0.0001	<0.0001
Weight (kg)	66 (56–77)	66 (61–76)	0.6577	0.7408
Body mass index (kg/m²)	23.0 (20.8–25.0)	25.3 (23.5–27.1)	<0.0001	<0.0001
Lung volumes and capacities
Total lung capacity (l)	6.04 (5.27–6.91)	5.11 (4.64–5.91)	<0.0001	<0.0001
Total lung capacity (% pred.)	100 (12)	99 (10)	0.5659	0.5659
Alveolar volume (l)	5.68 (4.85–6.62)	4.95 (4.48–5.67)	<0.0001	<0.0001
Alveolar volume (% pred)	99 (12)	102 (11)	0.0677	0.1354
Lung function measures
FEV₁ (l)	3.75 (3.22–4.53)	3.26 (2.90–3.96)	<0.0001	<0.0001
FVC (l)	4.45 (3.87–5.55)	3.82 (3.39–4.66)	<0.0001	<0.0001
FEV₁/FVCAP ratio	84.4 (80.7–88.2)	83.6 (81.1–88.1)	0.9702	0.9702
FEV₁ (% predicted)	105 (97–114)	104 (98–111)	0.9326	0.9702
FVC (% predicted)	106 (98–116)	107 (99–117)	0.6790	0.7408
Gas exchange and diffusing capacity
D_LCO (ml/min/mmHg)^a	28.15 (24.63–32.76)	31.71 (27.41–39.04)	<0.0001	<0.0001
D_LCO (% predicted)	92 (13)	102 (14)	<0.0001	<0.0001
D_LCO/VA (ml/min/mmHg/l)	5.02 (4.60–5.47)	6.50 (5.85–7.07)	<0.0001	<0.0001
D_LNO (ml/min/mmHg)	120 (100–139)	99 (89–119)	<0.0001	<0.0001
Vcap (ml)	84 (71–98)	105 (87–13)	<0.0001	<0.0001
Dm (ml/min/mmHg)	61 (51–70)	50 (45–60)	<0.0001	<0.0001
Other physiological measures
Oxygen saturation (%)	98 (98.−99)	94 (92–95)	<0.0001	<0.0001
Hemoglobin (g/dl)	14.1 (13.0–15.6)	14.9 (13.9–15.8)	0.0001	0.0002

Comparison of lowlanders and highlanders. Pulmonary function and gas exchange measures include spirometry, lung volumes, and diffusing capacity components (D_LCO, D_LNO, membrane diffusing capacity, and pulmonary capillary blood volume). D_LCO values were corrected for hemoglobin concentration.

Corrected for Hemoglobin.

Benjamini-Hochberg false discovery rate correction.

BMI, body mass index; D_LCO, diffusing capacity for carbon monoxide; D_LCO (% predicted), percent predicted diffusing capacity for carbon monoxide; D_LCO/VA, D_LCO per unit alveolar volume; D_LNO, diffusing capacity for nitric oxide; Dm, membrane diffusing capacity; FDR, false discovery rate; FEV₁, forced expiratory volume in 1 second; FEV₁ (% predicted), percent predicted FEV₁; FEV₁/FVC Ratio, ratio of FEV₁ to forced vital capacity; FVC, forced vital capacity; FVC (% predicted), percent predicted FVC; Height, body height; Hemoglobin, hemoglobin concentration; oxygen saturation, peripheral oxygen saturation; TLC, total lung capacity; Vcap, pulmonary capillary blood volume; Weight, body weight.

Highlanders exhibited significantly higher D_LCO, D_LCO per unit alveolar volume, and percent-predicted D_LCO values compared to lowlanders (Fig. 1, top panel), while no significant difference in percent-predicted total lung capacity and alveolar volume was evident. In contrast, D_LNO and Dm were lower in highlanders, while Vcap was markedly higher (Fig. 1, middle and lower panels).

FIG. 1.

Boxplots comparing key pulmonary diffusion parameters between lowlanders and highlanders. Figure 1: Hemoglobin-corrected D_LCO, Dm, and Vcap were compared between participants residing in Milan (120 m) and Mexico City (2,240 m). Highlanders exhibited significantly higher D_LCO and Vcap, but lower Dm values, compared to lowlanders (p < 0.0001 for all comparisons). Abbreviations: D_LCO, diffusing capacity for carbon monoxide; Dm, membrane diffusing capacity; Vcap, pulmonary capillary blood volume.

This inverse relationship between Dm and Vcap was further illustrated in the overlay plot (Fig. 2), where highlanders clustered toward higher capillary volume but lower membrane diffusing capacity.

FIG. 2.

Overlay of membrane diffusing capacity (Dm) versus pulmonary capillary blood volume (Vcap) in lowlanders and highlanders. This scatter and density contour plot illustrates the relationship between membrane diffusing capacity (y-axis) and pulmonary capillary blood volume (x-axis) across cohorts. Lowlanders (red) cluster toward higher Dm and lower Vcap, while highlanders (blue) exhibit a rightward shift, reflecting increased capillary volume and reduced membrane conductance. The divergent distributions suggest altitude-specific physiological adaptation, with highlanders demonstrating a predominantly vascular response to chronic hypoxia.

Oxygen saturation was lower in highlanders, while hemoglobin levels were modestly elevated, consistent with compensatory hematological adaptation to chronic hypoxia at altitudes (Table 1).

Matched cohort

To account for potential confounding by age and sex, a 1:1 nearest-neighbor matching was performed between lowlanders and highlanders, resulting in 71 matched pairs (n = 142) with balanced age and sex distributions (standardized mean differences <0.1). In the matched cohort, height remained significantly greater among lowlanders (169 [163–175] cm vs. 163 [159–170] cm, p = 0.0022), while body mass index was higher in highlanders (25.4 [23.5–28.0] kg/m² vs. 23.0 [20.8–25.0] kg/m², p < 0.0001).

Among lung volumes, total lung capacity was lower in highlanders (5.10 [4.70–5.91] L vs. 5.84 [5.19–6.71] L, p = 0.002), while percent-predicted TLC and alveolar volume were comparable between groups (p > 0.05).

Marked differences were observed in gas exchange and diffusing capacity (Table 2). Highlanders demonstrated higher D_LCO (31.69 [27.76–39.08] ml/min/mmHg vs. 27.05 [23.68–32.41], p < 0.0001), percent-predicted D_LCO (101 [15] vs. 90 [13], p < 0.0001), and D_LCO/VA (6.30 [5.68–6.92] ml/min/mmHg/L vs. 5.05 [4.59–5.47], p < 0.0001). Conversely, D_LNO (100 [91–118] vs. 112 [98–135], p = 0.045) and Dm (51 [46–60] vs. 57 [50–68], p = 0.045) were lower in highlanders, whereas Vcap was substantially higher (104 [85–124] ml vs. 78 [65–96] p < 0.0001).

Table 2.

Comparison of Demographic Characteristics, Pulmonary Function, and Gas Exchange Parameters Between a Matched Cohort of Lowlanders and Highlanders

Variable	Lowlanders (n = 71)	Highlanders (n = 71)	p-Value	FDR-adjusted p-Value^b
Demographics
Age (years)	30 (4)	30 (4)	0.6515	—
Female Gender	44 (62)	49 (69)	0.4801	—
Height (cm)	169 (163–175)	163 (159–170)	0.0010	0.0022
Weight (kg)	65 (55–74)	66 (61–80)	0.1364	0.1579
Body mass index (kg/m²)	23.0 (20.8–25.0)	25.4 (23.5–28.0)	<0.0001	<0.0001
Lung volumes and capacities
Total lung capacity (l)	5.84 (5.19–6.71)	5.10 (4.70–5.91)	0.0009	0.0022
Total lung capacity (% pred.)	100 (12)	97 (9)	0.1100	0.1344
Alveolar volume (l)	5.35 (4.60–6.36)	4.95 (4.54–5.66)	0.0804	0.1040
Alveolar volume (% pred)	99 (12)	100 (10)	0.4011	0.4412
Lung function measures
FEV₁ (l)	3.56 (3.09–4.36)	3.27 (2.92–3.81)	0.0176	0.0352
FVC (l)	4.19 (3.62–5.25)	3.84 (3.44–4.63)	0.0529	0.0776
FEV₁/FVCAP ratio	84.9 (82.0–88.8)	83.0 (81.0–86.4)	0.0609	0.0837
FEV₁ (% predicted)	106 (12)	105 (11)	0.4854	0.5085
FVC (% predicted)	107 (12)	107 (11)	0.7632	0.7632
Gas exchange and diffusing capacity
D_LCO (ml/min/mmHg)^a	27.05 (23.68–32.41)	31.69 (27.76–39.08)	<0.0001	<0.0001
D_LCO (% predicted)	90 (13)	101 (15)	<0.0001	<0.0001
D_LCO/VA (ml/min/mmHg/l)	5.05 (4.59–5.47)	6.30 (5.68–6.92)	<0.0001	<0.0001
D_LNO (ml/min/mmHg)	112 (98–135)	100 (91–118)	0.0264	0.0448
Vcap (ml)	78 (65–96)	104 (85–124)	<0.0001	<0.0001
Dm (ml/min/mmHg)	57 (50–68)	51 (46–60)	0.0265	0.0448
Other physiological measures
Oxygen saturation (%)	98 (98–99)	94 (93–95)	<0.0001	<0.0001
Hemoglobin (g/dl)	13.9 (12.5–14.8)	15.0 (13.9–16.2)	<0.0001	<0.0001

Comparison of a 1:1 nearest-neighbor matched cohort (matched for age and sex) of lowlanders and highlanders. Pulmonary function and gas exchange measures include spirometry, lung volumes, and diffusing capacity components (D_LCO, D_LNO, membrane diffusing capacity, and pulmonary capillary blood volume). D_LCO values were corrected for hemoglobin concentration.

Corrected for Hemoglobin.

Benjamini-Hochberg false discovery rate correction.

Furthermore, highlanders exhibited lower oxygen saturation (94 [93–95]% vs. 98 [98–99]%, p < 0.0001) and higher hemoglobin concentration (15.0 [13.9–16.2] g/dl vs. 13.9 [12.5–14.8], p < 0.0001), consistent with chronic altitude adaptation.

Discussion

In this prospective observational study comparing healthy individuals living in two polluted cities at vastly different altitudes, we found that highlanders exhibited distinct pulmonary diffusion profiles compared to lowlanders. Indeed, highlanders demonstrated increased D_LCO and Vcap, while Dm and D_LNO were significantly reduced. These differences remained even after matching for age and sex.

To reduce the role of confounders, we limited our observation to young, healthy subjects living in Mexico City and Milan, i.e., major metropolitan areas with high levels of ambient air pollution (Miljkovic, 2021), yet they differ substantially in altitudes. This contrast provides a natural experiment to examine how chronic high-altitude exposuremodifies pulmonary diffusion capacity in the presence of shared environmental stressors. Indeed, pollution is known to impair lung function and D_LCO (Zheng et al., 2023), its shared presence across both cities reduces its likelihood as a confounding factor in this specific comparison. (Cromar et al., 2021; Tunesi et al., 2024)

The present study findings support the notion that chronic high-altitude exposure results in alveolar capillary membrane adaptations. The observed increase in D_LCO and Vcap in highlanders, with comparable percent predicted lung and alveolar volumes, likely reflects enhanced capillary recruitment and distension in response to chronic hypoxia. This aligns with prior evidence suggesting that capillary blood volume expands to compensate for reduced oxygen availability at altitude. (de Bisschop et al., 2010) At the same time, chronic hypoxia induces sustained hypoxic pulmonary vasoconstriction and elevated pulmonary arterial pressures, which can drive vascular remodeling and promote the proliferation and recruitment of pulmonary capillaries. This adaptive process may underlie the observed increase in Vcap in highlanders, representing a compensatory mechanism to maintain gas exchange efficiency despite reduced membrane diffusing capacity. (Richalet et al., 2024)

Conversely, the reduction in Dm and D_LNO observed here contrasts with studies of acute or subacute high-altitude exposure, where transient increases in Dm and D_LCO have been reported due to short-term membrane adaptation, likely reflecting early membrane recruitment under hypoxic stress. (Agostoni et al., 2011; Taylor et al., 2016, 2017). The modest Dm increase observed during exercise after acclimatization indicates that most capillary units are already recruited at rest, leaving little diffusion reserve available for further adaptation. (Taylor et al., 2016)

Changes with acute altitude exposure likely reflect the complex interplay between hypoxic vasoconstriction, interstitial fluid balance, and capillary recruitment during early acclimatization. Taken together, these findings indicate that acute altitude exposure elicits dynamic but reversible diffusion changes, whereas chronic residence, as in our study population, results in long-term remodeling characterized by increased vascular capacity but limited membrane conductance. (Agostoni et al., 2013; Stewart et al., 2024)

Previous research already suggested chronic altitude exposure related to differences in diffusing capacity. Dempsey et al. reported that lifelong high-altitude residents exhibit higher D_LCO and Vcap compared to lowlanders, indicating structural and functional adaptations of the alveolar-capillary interface to chronic hypoxia. (Dempsey et al., 1971) Our present findings are consistent with this early evidence, extending it to contemporary populations exposed to comparable urban environmental stressors.

The overlay of Dm versus Vcap (Fig. 2) further highlights this dissociation: while lowlanders demonstrated higher membrane diffusing capacity, highlanders shifted toward a capillary-dominant diffusion pattern. This suggests that at high altitude, pulmonary gas exchange becomes more dependent on blood volume expansion than on membrane conductance, as previously suggested. (de Bisschop et al., 2010) The persistence of these findings after age- and sex-matching strengthens the interpretation that they reflect altitude-related physiological adaptions rather than demographic bias; however, differences in ethnicity have to be considered. Therefore, due to the observational nature of the present study, findings should be interpreted as hypothesis-generating.

Limitations

Although this study is hypothesis-generating and provides novel insights into pulmonary gas exchange adaptation at altitude, echocardiographic parameters such as mPAP, sPAP, and PVR were not available. Their inclusion would have allowed a more comprehensive assessment of pulmonary vascular physiology and should be considered a valuable addition in future protocols.

Conclusion

High-altitude residence in an urban environment is associated with increased Vcap and D_LCO but reduced Dm compared to lowlanders. These findings suggest a primarily vascular adaptation to chronic hypobaric hypoxia.

Authors’ Contributions

R.W.: Conceptualization, formal analysis, original draft and revision. A.A.: Data curation and investigation. J.C.: Review and editing. E.S.: Review and editing. L.G.R.: Investigation, data curation, review and editing. A.D.L.S.M.: Data curation review and editing. A.R.G.: Data curation review and editing. L.T.B.: Data curation review and editing. S.H.: Review and editing. F.T.: Review and editing. P.A.: Overall supervision, conceptualization, writing, review and editing.

Footnotes

Acknowledgments

The authors want to thank the participating centers for their effort.

Author Disclosure Statement

All authors have completed the ICMJE uniform disclosure form and declare no conflicts of interest.

Funding Information

Fondazione Istituto Europeo di Oncologia e Centro Cardiologico Monzino-Career development fellowship—Robin Willixhofer; Italian Ministry of Health (ricercar Corrente, CUP= B43C24000090001)—Piergiuseppe Agostoni.

Ethical Approval

Local Ethics Committee Centro Cardiologico Monzino (R853/18-CCM897), and the Science and Bioethics Committee of the Instituto Nacional de Enfermedades Respiratorias (C65-17)

Data Sharing Statement

Data will be shared upon reasonable request to the corresponding author. Involvement of the public or patients: Patients or the public were not involved in the design, conduct, or reporting, or dissemination plans of our research.

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