Subtle changes in exhaled particle size and phospholipid composition of the respiratory tract lining fluid following moderate-intensity exercise in sub-zero conditions

Subtle changes in exhaled particle size and phospholipid composition of the respiratory tract lining fluid following moderate-intensity exercise in sub-zero conditions | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Subtle changes in exhaled particle size and phospholipid composition of the respiratory tract lining fluid following moderate-intensity exercise in sub-zero conditions Helen Hanstock, Angelos Gavrielatos, Iluta Ratkevica, Per Larsson, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8084613/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Exercise-induced bronchoconstriction (EIB) commonly develops following prolonged exercise in cold, dry environments, but the acute mechanisms underlying airway responses to such environmental stressors remain poorly understood. Exhaled particle analysis (PExA) offers a novel, non-invasive approach to assess respiratory tract lining fluid (RTLF) composition and may provide mechanistic insights into early airway responses before clinically detectable changes occur. This study investigated exhaled particle characteristics and lipid composition in response to moderate-intensity exercise in sub-zero conditions among healthy atopic and non-atopic individuals. Methods Eighteen recreationally active participants (14 male) aged 29 ± 6 years, performed two moderate-intensity exercise trials (30 and 90 min duration) in a climate chamber set to -15°C. Participants provided exhaled particle (PEx) samples using the PExA® method, before and 30 min after each exercise trial. The PExA® device analysed particle mass and count across eight size bins (0.4–5 µm) and the collected PEx samples subsequently underwent lipidomic analysis to assess differences in the RTLF composition from before to after exercise. Data were analysed using univariate and multivariate statistical methods. Results Exercise induced a significant increase in smaller particles in PEx samples (0.4-0.7µm; p < 0.01). Three lipid species were significantly altered after the 30-min exercise trial, and eleven after 90-min exercise. Phosphatidylethanolamine PE(16:1_18:0) was the only lipid that consistently increased across both exercise durations (30 min: p = 0.023; 90 min: p = 0.044; g = 0.97). No significant differences in overall particle characteristics were observed between atopic and non-atopic participants, though three specific lipid species showed differential exercise responses between groups, including an oxidised phosphatidylcholine species PC(16:0_9:0;O) (p = 0.006, q = 0.59, g = 0.86). Conclusions Moderate-intensity exercise in sub-zero conditions induced a consistent shift toward smaller exhaled particles and subtle alterations in respiratory tract lining fluid lipid composition, including increased phosphatidylethanolamine PE(16:1_18:0) and decreased lysophosphatidylcholine species. Despite these changes, the overall stability of exhaled particle composition suggests that airway surfactant systems are relatively robust to acute environmental stress in healthy individuals. Trial registration ISRCTN13977758, Retrospectively registered 01/02/2022. https//doi.org/10.1186/ISRCTN13977758 Airway health cold environment exercise-induced bronchoconstriction exhaled particles lipidomics winter sports Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background The combination of exercise and exposure to airway irritants is recognised as an occupational health risk to athletes ( 1 – 3 ) and the general population exposed through work or leisure activities ( 4 , 5 ). Athletes in high-performance endurance sports, such as running, swimming and cross-country skiing, which demand sustained high ventilation rates, have a high prevalence of asthma ( 6 – 9 ). However, the occurrence of respiratory symptoms, acute reductions in lung function, and airway inflammatory responses is particularly high in settings where athletes are exposed to swimming pool chemicals ( 10 ), environmental air pollution ( 11 ), pollen ( 12 ), and cold, dry air ( 12 , 13 ) during training. These irritants are identified as triggers for exercise-induced bronchoconstriction (EIB) and are likely involved in the initial development of lower airway dysfunction among athletes ( 12 , 14 – 16 ). While lower airway dysfunction, an operational definition encompassing asthma, EIB and airway hyperresponsiveness ( 8 ), has been well-recognised in winter endurance athletes, such as cross-country skiers, for over 30 years ( 17 – 19 ), its prevalence appears relatively unchanged over time ( 8 ), and we still lack complete understanding of the airway pathophysiology induced by exercise in sub-zero conditions. A handful of cross-sectional and longitudinal studies have used samples from bronchial biopsies using bronchoscopy and induced sputum to characterise the immune cell and inflammatory profile of the airways following chronic exposure to cold environments ( 20 – 23 ). However, bronchoscopy is invasive and induced sputum is highly dependent on subject cooperation and technique, risk of contamination from upper airways, and variable reproducibility. In this context, several studies have used the blood and urine biomarker club cell Clara protein 16 (CC16) to ascertain whether exercise and/or environmental exposures affect airway epithelial integrity ( 24 – 27 ). Indeed, exercise in cold dry air has been shown to acutely increase blood CC16 concentrations in healthy individuals and is attenuated when using a heat- and moisture-exchanger ( 24 ). However, CC16 has also been shown not to differ between athletes with and without bronchoconstriction in response to bronchial challenge testing ( 28 ), suggesting it is a physiological marker of acute exposure as opposed to a marker of pathology or functional impairment. Thus, the ability to identify, validate and assess a larger panel of biomarkers of airway integrity directly from the respiratory tract before and after acute exercise bouts, using minimally invasive methods, is desirable. The respiratory tract lining fluid (RTLF) covering the airway epithelium constitutes a central line of defence against environmental triggers, such as cold, dry air. Analysis of the composition of the RTLF can be evaluated using a novel, non-invasive method termed PExA®, to collect exhaled particles (PEx)( 29 ). PEx from the RTLF generated by opening and closing of the airways have been shown to reflect peripheral and small airway inflammation ( 30 , 31 ) and be affected by environmental triggers, such as smoking ( 32 ), allergens ( 30 ), and occupational exposures ( 33 ). To date, the method has not yet been employed to assess airway responses to acute exercise in cold environments, a condition known to induce hyperpnea leading to airway cooling and drying, that contributes to epithelial injury ( 34 ). Moreover, single biomarker and omics-based analysis of the obtained PEx samples has potential to explore of how airway surfactant phospholipid composition may be altered following acute, prolonged exercise in a cold environment. Pulmonary surfactant, composed primarily of phospholipids, helps to maintain alveolar stability and optimal gas exchange ( 35 , 36 ) as well as serving as a protective barrier for the airway epithelium against mechanical stress and environmental insults ( 35 , 37 ). Notably, alterations in airway surfactant composition have been associated with reduced lung function in asthma ( 38 ), raising the possibility that similar changes, potentially triggered by exercise hyperpnoea in the cold, could increase airway vulnerability to injury. Existing data suggest that exercise performed at high-intensity elicits greater bronchoconstriction and airway epithelial damage than low-intensity training ( 19 ). However, the majority of the training that skiers perform is in the low-intensity domain ( 39 – 41 ), with prolonged sessions > 1.5 h making up a substantial part of skiers’ total training ( 42 ). Single bouts of prolonged, low-intensity exercise have been associated with systemic immune disturbances ( 43 , 44 ), which could possibly impair the ability of immune cells to promptly engage in epithelial repair post-exercise. While the repeated damage-repair cycle likely elicited by frequent endurance training in sub-zero environments may offer an explanation as to why cross-country skiing has been highlighted as a risk factor for development of asthma ( 2 ), to our knowledge no studies have investigated effects of prolonged endurance exercise in sub-zero conditions on the composition of the RTLF. The purpose of this exploratory study was to investigate whether particles in exhaled air can be used to measure airway effects of exercise in cold air. Specifically, the aims were to investigate whether moderate-intensity exercise in sub-zero temperatures affects PEx count, mass and the lipidomic profile of PEx samples, and furthermore whether the profile was affected by exercise duration and participants’ atopic status. Methods Participants Eighteen participants (♂ = 14, ♀= 4, age 29 ± 6 years; mean ± SD) provided written, informed consent to participate in the study. Participants were currently engaged in regular endurance training but had never competed in winter endurance sports at elite level (Tier 1–3 according to McKay et al., ( 45 )). Atopic status was determined using the Allergy Questionnaire for Athletes (AQUA©) ( 46 ). Atopy was defined as a questionnaire score ≥ 5. A detailed description of participant’s anthropometric characteristics and training background has been previously reported ( 47 ). Study design The study employed a 2x2 randomised crossover design whereby participants performed a preliminary test to determine V̇O 2 max at room temperature, followed by two treadmill running exercise trials, of 30- and 90-min duration, separated by approximately one week, in an environmental chamber at -15°C and circa 67% relative humidity. PEx were collected at two time points, before and 30 min after exercise. An individualised, prescribed treadmill speed for the main exercise trials was determined by interpolation of data from the submaximal stages of a preliminary submaximal and maximal exercise test to determine a speed that would elicit approximately 60% V̇O 2 max during the subsequent visits. A detailed methodology of the pre-test and chamber exercise trials, as well as physiological responses to exercise, lung function results and blood biomarkers measured during the study has been previously published elsewhere (47). Exhaled particle collection The composition of the RTLF can be assessed using a novel and non-invasive method to collect and analyse particles in exhaled air using a patented instrument (PExA®, patent P17584SE00). The instrument can collect and discriminate larger (0.4–5 µm) particles from the RTLF generated by opening and closing of the small airways ( 33 , 48 ), herein referred to as PEx. The PExA® instrument can be used to obtain a particle mass and count, both in total and across different particle size domains, while collection and storage of the collected particles enables subsequent assessment of airway surfactant phospholipid composition. During PEx collection, participants performed multiple repetitions of a specific breathing manoeuvre, whereby the participant exhaled to achieve residual volume (RV) then held their breath for 5 seconds. This was then followed by a steady inhalation to vital capacity followed by a slow exhalation (sVC) into the device. Inhalation of particle-free filtered room air was achieved through a two-way non-rebreathing valve (1420, Hans Rudolph, Shawnee, KS, USA), followed by immediate exhalation into the device. The breathing manoeuvre was repeated at self-regulated intervals. Due to the limited time window for particle collection and the need to collect particles within a similar time window for all participants, particle collection was terminated as soon as the participant reached one of the following end points: 120 ng particle mass had been collected (sufficient particles obtained), after 15–16 exhalations (to prevent fatigue that could affect later tests), or after approximately 10 minutes (due to time constraints and to match the post-exercise time point within- and between participants; i.e. 30–40 min post-exercise). The concentration of particles was measured in eight size bins between approximately 0.4–5 µm in diameter by optical particle counter in the PExA instrument (PExA AB, Gothenburg), as described by Almstrand et al ( 48 ). The PExA computer software calculates the sampled particle mass online and displays collected mass to the participant. Particles are collected by the inertia principle using a cascade impactor. Particles are impacted on a thin hydrophilic polytetrafluoroethylene membrane mounted on the impactor collection plate. When sampling had finished, the sampling membrane was taken out of the impactor, divided in two using a clean scalpel, and stored in a microcentrifuge tube at -80°C until later analysis. Lipidomics analysis of PEx samples A detailed procedure for analysing surfactant lipids from PEx samples of small airways has been described previously ( 49 ). The lipids included in the method were selected during a method development phase preceding this study. Lipid panel selection was based on screening pooled PEx samples using Waters LipidQuan Quanpedia lipid MRM database (Milford, MA, USA). In addition to the targeted screening, untargeted screening was conducted using lipid class specific fragmentation scanning experiments, such as neutral loss scanning and precursor ion scanning. The most abundant lipids in each class were selected for confirmation and further validation (selectivity, precision, linearity) before inclusion into the lipid panel used in the present study. In brief, samples were placed in the insert of centrifugal filter tubes (Millipore UFC30LG25). To the sampling membrane 10 µl of isotope labeled lipid standard was added and allowed to dry with the sample for 10 min (Avanti EquiSPLASH SKU:330731 and Avanti GlcCer(d18:1(d5)/18:0) SKU:860638). Lipids were extracted in a thermomixer using 225 µl of isopropanol. The eluate was recovered from the insert to the microtube bottom by centrifugation. The extraction was then immediately repeated with methanol for improved recovery of both the polar and the neutral lipids. The methanol/isopropanol eluate in the microtube was transferred into LC vials (Waters Total Recovery vial SKU: 186002805) and stored at -20°C before analysis. Before analysis, the solvent in the LC-vial was evaporated under a stream of nitrogen and the remaining lipid film was solubilised in 100 µl of solvent consisting of acetonitrile and isopropanol in a ratio of 2:1. The extracted lipids were analysed with a targeted LC-MSMS method using the Waters ACQUITY UPLC I-Class chromatography system coupled to an electrospray ionization source on a Waters Xevo TQ-XS triple quadrupole mass spectrometer detector (Milford, MA, USA). Reverse phase chromatography was used for separation of PC lipids based on fatty acyl groups. All other lipids were analysed using HILIC chromatography that separates the lipid classes. Quantification was based on the peak area of the analyte divided by the peak area of the internal standard, the response factor. The response factor was multiplied with the mol of internal standard spiked to the sample. One internal standard per lipid class was used. The results of the lipid analyses were calculated as relative amount ( 49 ): $$\:(mol\%=100\:\times\:\frac{\text{s}\text{p}\text{e}\text{c}\text{i}\text{e}\text{s}\_\text{m}\text{o}\text{l}}{\text{l}\text{i}\text{p}\text{i}\text{d}\_\text{s}\text{u}\text{m}\_\text{m}\text{o}\text{l}})$$ . Type II isotope correction factors were calculated with the online correction tool LICAR for the lipid classes PG,PE and PI, as described by Gao et al. ( 50 ). When isomers were found as two distinct peaks then the first peak was annotated with an “_A” and the second with a “_B”. For example: LPC(16:1)_A and LPC(16:1)_B corresponds to the SN1 and SN2 position of the 16:1 fatty acyl group on the glycerol backbone. Data analysis The PExA instrument provided data on total particle count and mass, as well as the distribution of particle count and mass across eight particle size bins. These data were normalised relative to the total volume of exhaled air during the collection. Three samples encountered technical errors during collection and were missing from analysis, one further sample was excluded from analysis due to suspected contamination. Data were then analysed in GraphPad Prism (version 10.6, GraphPad Software, Boston, MA, USA), using the linear mixed model implementation. This mixed model uses a compound symmetry covariance matrix, and is fitted using the Restricted Maximum Likelihood (REML). First, total particle count and mass were analysed with fixed effects for trial, timepoint and their interaction. Subsequently, the proportion of particles in within each particle size domain was analysed using two models: first, with fixed effects for trial, timepoint and particle size, including their interactions, and subsequently, with fixed effects for timepoint and particle size only. Fisher’s LSD (for 2x2 analyses) or Bonferroni post-hoc tests were used to follow up significant interactions. Lipidomics data were reported as mol% of total sample lipid content. Lipids with < 20% missing values were retained; remaining missing values were imputed using the lowest detected mol% divided by √2. Samples with a total collected mass of < 10% of the target (< 6 ng) were excluded (n = 2). Thus, analysis of lipid composition was ultimately performed on data from 66 samples. For baseline analyses, the mean lipid mol% from each participant’s baseline samples were calculated. For each trial, the change in mean mol% for each lipid from pre to post exercise was quantified using the log 2 logit difference, calculated as: $$\:{{log}_{2}\left(\frac{{\frac{mol\%}{100}}_{post}}{{1-\:\frac{mol\%}{100}}_{post}}\right)-\:log}_{2}\left(\frac{{\frac{mol\%}{100}}_{pre}}{{1-\frac{mol\%}{100}}_{pre}}\right)$$ . Lipid responses to each individual exercise trial were analysed using paired t-tests; a prerequisite was thus that samples for both pre- and post- time points within a trial were available (30 min: n = 14; 90 min: n = 17). To meet the assumptions of parametric testing, lipid mol% data were arcsine transformed prior to statistical analysis, as this transformation stabilises variance and improves normality for proportion data bounded between 0 and 1. Results were visualised against the log 2 logit difference using volcano plots in Prism. The log₂ logit difference was chosen to quantify the magnitude and direction of change from pre- to post-exercise, offering an interpretable magnitude of change for visualisation, as it linearises proportion changes near 0 and 1 and facilitates comparison across trials. Differences in lipid mol% between atopic and non-atopic individuals at baseline, and log 2 logit responses to exercise, were analysed using independent t-tests. We used a significance threshold of p < 0.05 as a threshold for reporting biomarker responses of interest. Raw p values and FDR-corrected q values (Benjamini-Hochberg, Q = 10%) are presented in parallel, but given no q values reached the threshold to be retained as true discoveries, p values were used to best describe changes in the data, but should be interpreted with caution. Hedge’s g effect sizes are reported for pairwise comparisons to aid interpretation. Principal component analysis (PCA) was performed in Prism to investigate potential clustering of lipids measured pre and post 30- and 90- min exercise, and to assess differences in the lipid response to exercise based on atopic status. When visual separation was observed in unsupervised PCA analyses, the analysis plan was to follow up with a supervised orthogonal partial least squares discriminant analysis (orthoPLS-DA); however, insufficient separation on PCA was observed in all cases and thus orthoPLS-DA analyses were not performed to avoid overfitting ( 51 ). Results Effects of exercise on exhaled particle mass and count The mean (± SD) number of exhalations during sample collection was 10 ± 2, although the obtained particle mass from each collection varied substantially between samples (80 ± 56 ng). Due to technical errors, particle data were not obtained at three collection time points. Particle mass and count, normalised to the total exhaled air volume during sample collection, are shown in Fig. 1 . The change in particle mass and count from pre- to post-exercise differed significantly between trials (PEx count: F( 1 , 13 ) = 6.90, p = 0.02; PEx mass: F( 1 , 13 ) = 5.53, p = 0.035). Post hoc analyses revealed a decrease in particle mass and count post-exercise in the 90 min trial, attributable to higher particle counts and mass at baseline in the 90 min trial compared to both post-exercise and before the 30-min exercise trial (Fig. 1 ). Analysis of the response to exercise for particles across different size domains revealed a significant interaction between timepoint and particle size (PEx count: F(7, 136) = 6.692, p < 0.0001; PEx mass: F (7, 136) = 12.22, p < 0.0001). Follow-up analysis of these effects indicated a greater proportional count and mass of smaller particles (0.41-0.70µm) pre-exercise, and a lower proportional mass (but not count) of larger (1.44-4.55µm) particles post-exercise (Fig. 2 ). Effects of exercise on exhaled particles assessed using lipidomics In total, 93 lipid species were detected and retained for analysis. Phospholipid composition in all samples was dominated by PC(16:0/16:0) which made up more than 50% of the total lipids at all four time points (Supplementary Fig. 1). In general, the relative proportions of the major lipids showed minimal change across the four sample time points. Analysis of lipid responses to individual trials revealed three lipids that significantly differed (p < 0.05) from pre- to post-exercise in the 30-min trial (Fig. 3 ), and eleven in the 90-min trial (Fig. 4 ). The only lipid consistently altered across both trials was PE(16:1_18:0), which significantly increased following exercise (30 min: p = 0.023, q = 0.865; 90 min: p = 0.044, q = 0.38; combined time effect, g = 0.97). Effect of atopic status on the PEx and lipid profile before and after exercise in a sub-zero environment Based on the pre-defined AQUA score cut-off, 10 participants were classified as atopic (56%, 7 men, 3 women) and eight as non-atopic. There were no significant differences between the mean baseline PEx count or mass between the atopic and non-atopic groups (Supplementary Fig. 2). Similarly, there were no significant differences in exhaled lipid composition at baseline between the two groups. Analysis of the mean PEx response to exercise also showed no significant differences between the atopic and non-atopic groups (Supplementary Fig. 2C and D). In addition, no differences in the distribution of PEx mass or count over the particle size domains were found between atopic and non-atopic participants (Supplementary Fig. 3). Three lipids were identified through volcano plots as potentially different in their response to exercise between the atopic and non-atopic groups (Fig. 5 A): PG(18:0_20:4) (p = 0.048, q = 0.92, g = 1.01; Fig. 5 B), PC(18:1_18:2) (p = 0.026, q = 0.92, g = 0.99, Fig. 5 C) and PC(16:0_9;O), (p = 0.006, q = 0.59, g = 0.86, Fig. 5 D). However, the distribution of particle mass across particle size bins was not affected by atopy (i.e. no significant main effect of atopy, nor atopy x timepoint interaction; Supplementary Fig. 3). Multivariate analysis Analysis with PCA showed no clear separation between sample time points or trials (Supplementary Fig. 4a), nor the difference in the response to exercise between atopic and non-atopic individuals (Supplementary Fig. 4b). The lack of separation between groups and time-points in the PCA analysis precluded further analysis with orthoPLS-DA to avoid overfitting. Discussion The present study investigated exhaled particle responses to moderate-intensity exercise in sub-zero conditions among healthy non-atopic and atopic participants, representing the first application of the PExA method in an acute exercise setting. The main findings were that 1) a greater proportion of smaller particles were obtained after exercise 2) a higher number of lipids (eleven) were altered after 90 min exercise versus 30 min exercise (three), accompanied by a greater reduction in PEx mass and count post-exercise after 90 min exercise 3) there was no significant difference in the composition of the obtained PEx mass and count between atopic and non-atopic participants at baseline nor in response to exercise, but 4) three lipids showed differing exercise responses between non-atopic and atopic participants. The consistent shift toward smaller particles (0.5-1µm) following exercise across both trials represents a novel finding, suggesting that the physical properties of particle generation may have been altered by exercise in a subzero environment. The reduction in particle mass and count after 90-min exercise likely reflects subtle alterations in airway surfactant properties that, while not causing functional impairment in this acute setting, may provide mechanistic insights into the pathogenesis of exercise-induced bronchoconstriction (EIB) with repeated exposure to exercise in cold air. While no participants in the present study had clinically relevant changes in FEV1 post-exercise ( 47 ), previous observations have shown an association between decreased PEx mass and increased asthma severity ( 52 ), suggesting that PEx measurements may be sensitive to early perturbations in respiratory tract integrity even before clinically detectable lung function changes occur. The increased ventilation of exercise in combination with the cold, dry environment leads to cooling and drying of the airway epithelium, which could affect mucosal hydration and viscosity, surface tension and airway reopening dynamics ( 53 ). The volume of the RTLF in the lower airways has been modelled to be under 1ml, and thus susceptible to water loss and resultant osmotic stress ( 54 ), especially during high ventilation exercise in a cold, dry environment. It has been hypothesised that shear stress on the dehydrated airway epithelium can cause airway epithelial damage, leading to inflammation, release of mediators, increased epithelial permeability and plasma leakage into the airways, which over time can contribute to airway remodelling and the development of airway hyperresponsiveness characteristic of EIB ( 34 ). The impact of prolonged exercise in the cold on exhaled particle generation has not previously been explored, but we can speculate that drying may reduce the thickness of the RTLF during exercise, potentially leading to smaller particles being generated during airway reopening, whereas cooling and drying may also alter airway compliance and surface tension, that could also influence the mechanics of particle formation. The responses of the detected lipid species to exercise, while modest, may also offer some insight into RTLF response to exercise in cold air. The phosphatidylcholine PC16:0/16:0 (dipalmitoyl phosphatidylcholine or DPPC) was the most abundant lipid across all samples at all time points, representing just over 50% of the total lipids. Larsson et al ( 31 ) describe DPPC as the most abundant of the targeted lipids determined from PEx, accounting for just over 50% of total lipids. Only one lipid species, PE(16:1_18:0), showed a consistent increase after exercise across both trials. PE species are highly enriched in mitochondrial inner membranes ( 55 ), and the observed increase in PE(16:1_18:0) may indicate enhanced membrane biosynthesis or remodeling to support increased metabolic demands during and after exercise. In contrast, two lysophosphatidylcholine (LPC) species decreased post-exercise. LPCs are generated from phosphatidylcholines through phospholipase A2 (PLA2)-mediated hydrolysis and can impair surfactant function when elevated ( 56 ). Additionally, LPCs can be used to disrupt the epithelial barrier in experimental contexts ( 57 ). The observed decrease in LPC levels suggests reduced phospholipase activity and/or active reacylation of LPCs back to intact phospholipids during the recovery period, rather than ongoing epithelial disruption. The 30-minute post-exercise sampling timepoint may therefore have captured the resolution phase following any transient perturbations during exercise. Finally, atopic individuals showed a greater increase in an oxidised PC species, PC(16:0_9:0;O), potentially reflecting heightened oxidative stress, as previously reported in inflamed tissues ( 49 ). We previously reported that atopic individuals experienced greater intensity of respiratory symptoms after exercise ( 47 ), so it is possible that their airways had a greater propensity to epithelial disruption, inflammation or mucus function. As research into lipid regulation of airway integrity is still emerging, it remains uncertain whether the responses we observed may reflect a propensity to adapt to higher rates of ventilation, cold exposure, or cellular stress and the specific functional significance of the identified species in surfactant homeostasis requires further investigation. Despite these changes in specific species, the overall lipid profile remained relatively stable at all measurement timepoints, and there were no significant shifts in overall lipid classes post-exercise. The overall greater number of lipid species that were altered (p < 0.05) after 90-min exercise versus 30-min exercise may indicate a dose-response relationship between exposure of the airways to acute epithelial stress and acute changes in RTLF composition. Taken together, the reported changes in particle characteristics and lipid composition of PEx samples suggest that even a single bout of prolonged exercise in a subzero environment can perturb the RTLF. The overall stability of most lipid species and classes nevertheless suggests that the airway surfactant system is relatively robust and likely recovers quickly after environmental stress. To our knowledge, this study was the first time that the PExA method has been used to collect samples before and after exercise. Given this novelty, comparisons with studies that have used other breath sampling methodologies can provide important context for our findings. Previous studies have collected breath biomarkers using, for example, exhaled breath condensate (EBC). Although a detailed comparison of EBC versus PEx methodology is outside the scope of the current paper, a recent proof-of-concept study also reported no change in breath biomarkers (8-isoprostane and pentraxin-3) after moderate-intensity exercise in healthy participants ( 58 ). The authors noted that these findings were not unexpected and suggested that more extreme exercise intensities or co-introduction of environmental stressors would likely be needed to observe detectable biomarker changes. This interpretation is also supported by studies using other airway stressors. For example, acute exposure to wood smoke has been associated with pro-inflammatory and pro-oxidative responses in EBC in combination with exercise ( 59 ) but not at rest ( 60 ). Like cold weather exercise, wood smoke exposure disrupts lung function and the airway epithelium, being associated with increases in exhaled nitric oxide and CC16 ( 60 ). Thus, it is likely that the moderate-intensity exercise employed in the present study, even in sub-zero air, does not constitute a sufficiently potent stimulus to stress the airway mucosa and RTLF composition in healthy individuals. Our observations should also be interpreted in the context of previous findings from this project. In contrary to the original hypothesis, prolonged (i.e. 90 min) moderate-intensity exercise did not elicit decreased lung function (measured using dynamic spirometry) nor airway epithelial damage (assessed via plasma club cell Clara protein 16, CC16, concentrations), compared to 30 min exercise at -15°C ( 47 ). The effects of exercise in the cold on lung function and airway epithelial damage were also minimal, with no changes in spirometry measurements after exercise, and only a small increase in CC16, indicative of mild airway epithelial damage after both trials. These findings might suggest that the exercise challenge was not potent enough to elicit substantial acute stress on the airways of our healthy participants – short, high-intensity exercise, or possibly longer duration exercise, would likely have elicited greater decrements in lung function ( 24 , 61 , 62 ). This adds important context to the present findings of only subtle changes in PEx mass and composition; whether a more potent stimulus (i.e. higher intensity and/or longer duration) would cause a greater perturbation in PEx samples remains a question for future research. Several limitations should be acknowledged in interpreting the findings from the present study. The elevated baseline particle mass and count before the 90 min trial introduce possible complexities; apparent post-exercise alterations in PEx and lipid compositions may be attributable to this difference. The reason for the elevated baseline remains unclear, given all trials were conducted at the same time of day within individuals and the trial order was randomised, and previous studies have not reported diurnal variation in PEx measurements ( 63 ). However, within-subject coefficients of variation for PEx count and mass have been reported at 29% and 15%, respectively, suggesting that day-to-day variability may have contributed to this difference ( 63 ). Additionally, the shorter sampling window used in this study compared to previous work (10 min was the maximum allocated time in the study protocol for PEx collection to permit continuation to other time-sensitive measures) and the wide range in collected sample mass may have contributed to variation in sample composition. The study did not utilise a seated rest control trial, that may have highlighted any possible effects of time of day and/or other laboratory measurements (e.g. spirometry manoeuvres) on the PEx collection. Given the exploratory nature and multivariate dataset, as well as the proportion-shape of the mol% data, the relatively small sample size ( 64 ), and the lack of discoveries retained after FDR-correction, we should be aware that lipid species responses where p 0.05 require further investigation and replication to verify our observations; also given that only one lipid showed a sufficiently robust response to exercise to produce even a significant p value post-exercise in both trials. Finally, since this was the first study to incorporate PEx measurements after physical activity, we highlight that no studies have yet been performed to determine the optimal post-exercise timepoint to detect possible alterations in the composition of the RTLF. Nevertheless, our findings may have some value to inform advice provided to athletes regarding the risks of training in cold environments. To achieve primary prevention of exercise-induced asthma, studies such as this can help to identify, or indeed discount possible training-related hazards to airway integrity. The results from the present study, in combination with our previous findings ( 47 ), suggest that a single bout of moderate-intensity exercise in sub-zero conditions triggers only subtle changes in the RTLF composition, which warrant further investigation of their biological importance in susceptible populations and with more intense exercise protocols and well-powered, robust study designs. Conclusion The present study represents the first, to our knowledge, application of the PExA method to exercise physiology research, and provides important preliminary data regarding the exhaled particle response to exercise under environmental stress. Our findings demonstrate that moderate-intensity exercise in sub-zero conditions induces subtle but measurable changes in exhaled particle composition, including a reduction in particle size and shifts in selected lipid species that may be used as a basis to inform future hypotheses and follow-up studies. The overall stability of the PEx composition nevertheless suggests the airway surfactant is relatively robust to, and recovers quickly, after environmental stress, in agreement with our earlier findings of minimal changes in lung function or airway epithelial damage from the study protocol. Thus, our data provide preliminary insight into airway responses to exercise in a cold climate and contribute to the growing understanding of how the PExA method can be applied as a non-invasive tool for respiratory health assessment in an acute setting. Declarations Ethics approval and consent to participate The study was approved by the Swedish Ethical Review Authority (2020–05205). All participants provided full written, informed consent to participate in the study and all procedures were conducted in accordance with the Declaration of Helsinki. Consent for publication Not applicable Competing Interests A-CO is a board member and shareholder of PExA AB, the company that produce and sell the PExA instrument used in the present study. PL is a shareholder of PExA AB. All other authors have no conflicts of interest to declare. Funding The study received funding from the Östersund municipality and Mid Sweden University financial agreement, Rolf and Gunilla Enström’s Foundation for Research and Development. Author Contribution HH: conceptualisation, design, data acquisition, data curation, data analysis, data interpretation, writing – original draft, writing – revising and editing.AG: conceptualisation, design, data acquisition, data curation, writing – revising and editing.IR: data acquisition, data curation.PL: data acquisition, data analysis, data interpretation, writing – revising and editingA-CO: conceptualisation, data interpretation.NS: conceptualisation, design, data interpretation, writing – revising and editing.All authors approved the submitted version of the manuscript and agree to be accountable for the work. Acknowledgement The authors thank the participants for their time, efforts, and patience in the lab. In addition, we acknowledge the support of Marianne Anderson for providing training in the PExA methodology. Data Availability The datasets generated and/or analysed during the current study are available on request from the corresponding author. 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08:12:35","extension":"png","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":14373,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8084613/v1/d1725de4df4f1a22e4f6d8db.png"},{"id":97124827,"identity":"74596c37-c7d8-4ed1-996e-0389d599f780","added_by":"auto","created_at":"2025-12-01 08:12:34","extension":"png","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":117264,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8084613/v1/a6148e5ba95af45e66791078.png"},{"id":97141897,"identity":"4668a611-e007-4756-b012-afc6c0d89642","added_by":"auto","created_at":"2025-12-01 10:07:09","extension":"png","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":75544,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8084613/v1/07b6beb6bc0a367e4df8546d.png"},{"id":97124830,"identity":"e1303219-461b-4188-9590-98ffb5275623","added_by":"auto","created_at":"2025-12-01 08:12:35","extension":"png","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":13366,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8084613/v1/2b5d5547319f7e63efef69c7.png"},{"id":97124822,"identity":"f370bdc7-d5df-49a4-a42e-41d41469a1fe","added_by":"auto","created_at":"2025-12-01 08:12:34","extension":"png","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":14036,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8084613/v1/3324b41ba9db4c86c5c81577.png"},{"id":97124825,"identity":"13148ffb-5559-4c3b-af7a-784292918e5a","added_by":"auto","created_at":"2025-12-01 08:12:34","extension":"xml","order_by":24,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":146566,"visible":true,"origin":"","legend":"","description":"","filename":"abac7b3c7c0341bd97dc01426415e29b1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8084613/v1/30db88ed5b5e93d71b1213e5.xml"},{"id":97142795,"identity":"f07b8c51-edaf-47b0-91b4-fbcdd63e9ec9","added_by":"auto","created_at":"2025-12-01 10:07:57","extension":"html","order_by":25,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":164221,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8084613/v1/a5fa9d9d86d87b4c08ae4547.html"},{"id":97124808,"identity":"4118376d-c702-4827-bae9-476c2e31154b","added_by":"auto","created_at":"2025-12-01 08:12:34","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":206728,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003ePEx count (A) and mass (B) pre and post- 30- and 90-min exercise in -15°C. Difference between time points, *, p \u0026lt; 0.05, **, p \u0026lt; 0.01.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8084613/v1/d1ad566973dbda829158ad6b.jpg"},{"id":97142859,"identity":"f8fb9e42-2771-485e-bc05-d36576fe247d","added_by":"auto","created_at":"2025-12-01 10:08:00","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":397047,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eParticle count and mass within specific particle size domains pre- and post 30- and 90-min exercise in -15°C (Mean + SD). A) Proportion of total particle count represented by particles in different size domains, across all four measurement time points. Significant post-hoc differences within size domains between timepoints are shown in panel C. B) Proportion of total particle mass represented by particles in different size domains, across all four timepoints. Significant post-hoc differences within size domains between timepoints are shown in panel D. *, p \u0026lt; 0.05; **, p \u0026lt; 0.01, ***, p \u0026lt; 0.001, ****, p \u0026lt; 0.0001.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8084613/v1/0f7d086739bb038e635dbcd2.jpg"},{"id":97124805,"identity":"fbe3a047-56c6-440b-bd17-b55413dfbb8d","added_by":"auto","created_at":"2025-12-01 08:12:34","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":185590,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eLipid responses to the 30 min trial. A: Volcano plot displaying lipid responses to exercise and t-test significance. B-D: show the three most significant lipid responses (Mean + SD).\u003c/em\u003e\u003c/p\u003e","description":"","filename":"fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8084613/v1/5feba5070d115d988d8729f3.jpg"},{"id":97141851,"identity":"2f253515-8651-482e-805b-4592938cc5b8","added_by":"auto","created_at":"2025-12-01 10:07:06","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":221521,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eLipid responses to the 90 min trial. A: Volcano plot displaying lipid responses to exercise and t-test significance, B-D: show the three most significant lipid responses (Mean + SD).\u003c/em\u003e\u003c/p\u003e","description":"","filename":"fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8084613/v1/6813717adc76b3184bd89ef5.jpg"},{"id":97124818,"identity":"fd7b6199-0867-4442-b8f1-d1bfc82868fa","added_by":"auto","created_at":"2025-12-01 08:12:34","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":245128,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eMean lipid responses to exercise in non-atopic and atopic participants. A: volcano plot showing significant lipids identified using unpaired t-tests (B-D). Visualisation of the mean (+ SD) exercise response for lipids that significantly differed between atopic and non-atopic participants.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"fig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8084613/v1/c4595b491b7a70d046c47191.jpg"},{"id":101512998,"identity":"56150183-edcf-4cb8-be2b-4e04abbb50de","added_by":"auto","created_at":"2026-01-30 15:26:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1939782,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8084613/v1/b7be8d69-be27-4784-83bf-df59c361aff8.pdf"},{"id":97124811,"identity":"fc735b76-3275-4d80-a1d2-a48e96fe10a2","added_by":"auto","created_at":"2025-12-01 08:12:34","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":365458,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-8084613/v1/b394f96b54d27034d63cf936.docx"}],"financialInterests":"Competing interest reported. A-CO is a board member and shareholder of PExA AB, the company that produce and sell the PExA instrument used in the present study. PL is a shareholder of PExA AB. All other authors have no conflicts of interest to declare.","formattedTitle":"Subtle changes in exhaled particle size and phospholipid composition of the respiratory tract lining fluid following moderate-intensity exercise in sub-zero conditions","fulltext":[{"header":"Background","content":"\u003cp\u003eThe combination of exercise and exposure to airway irritants is recognised as an occupational health risk to athletes (\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) and the general population exposed through work or leisure activities (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Athletes in high-performance endurance sports, such as running, swimming and cross-country skiing, which demand sustained high ventilation rates, have a high prevalence of asthma (\u003cspan additionalcitationids=\"CR7 CR8\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). However, the occurrence of respiratory symptoms, acute reductions in lung function, and airway inflammatory responses is particularly high in settings where athletes are exposed to swimming pool chemicals (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e), environmental air pollution (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e), pollen (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e), and cold, dry air (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e) during training. These irritants are identified as triggers for exercise-induced bronchoconstriction (EIB) and are likely involved in the initial development of lower airway dysfunction among athletes (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). While lower airway dysfunction, an operational definition encompassing asthma, EIB and airway hyperresponsiveness (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e), has been well-recognised in winter endurance athletes, such as cross-country skiers, for over 30 years (\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e), its prevalence appears relatively unchanged over time (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e), and we still lack complete understanding of the airway pathophysiology induced by exercise in sub-zero conditions.\u003c/p\u003e\u003cp\u003eA handful of cross-sectional and longitudinal studies have used samples from bronchial biopsies using bronchoscopy and induced sputum to characterise the immune cell and inflammatory profile of the airways following chronic exposure to cold environments (\u003cspan additionalcitationids=\"CR21 CR22\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). However, bronchoscopy is invasive and induced sputum is highly dependent on subject cooperation and technique, risk of contamination from upper airways, and variable reproducibility. In this context, several studies have used the blood and urine biomarker club cell Clara protein 16 (CC16) to ascertain whether exercise and/or environmental exposures affect airway epithelial integrity (\u003cspan additionalcitationids=\"CR25 CR26\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Indeed, exercise in cold dry air has been shown to acutely increase blood CC16 concentrations in healthy individuals and is attenuated when using a heat- and moisture-exchanger (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). However, CC16 has also been shown not to differ between athletes with and without bronchoconstriction in response to bronchial challenge testing (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e), suggesting it is a physiological marker of acute exposure as opposed to a marker of pathology or functional impairment. Thus, the ability to identify, validate and assess a larger panel of biomarkers of airway integrity directly from the respiratory tract before and after acute exercise bouts, using minimally invasive methods, is desirable.\u003c/p\u003e\u003cp\u003eThe respiratory tract lining fluid (RTLF) covering the airway epithelium constitutes a central line of defence against environmental triggers, such as cold, dry air. Analysis of the composition of the RTLF can be evaluated using a novel, non-invasive method termed PExA\u0026reg;, to collect exhaled particles (PEx)(\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). PEx from the RTLF generated by opening and closing of the airways have been shown to reflect peripheral and small airway inflammation (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e) and be affected by environmental triggers, such as smoking (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e), allergens (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e), and occupational exposures (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). To date, the method has not yet been employed to assess airway responses to acute exercise in cold environments, a condition known to induce hyperpnea leading to airway cooling and drying, that contributes to epithelial injury (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). Moreover, single biomarker and omics-based analysis of the obtained PEx samples has potential to explore of how airway surfactant phospholipid composition may be altered following acute, prolonged exercise in a cold environment. Pulmonary surfactant, composed primarily of phospholipids, helps to maintain alveolar stability and optimal gas exchange (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e) as well as serving as a protective barrier for the airway epithelium against mechanical stress and environmental insults (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). Notably, alterations in airway surfactant composition have been associated with reduced lung function in asthma (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e), raising the possibility that similar changes, potentially triggered by exercise hyperpnoea in the cold, could increase airway vulnerability to injury.\u003c/p\u003e\u003cp\u003eExisting data suggest that exercise performed at high-intensity elicits greater bronchoconstriction and airway epithelial damage than low-intensity training (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). However, the majority of the training that skiers perform is in the low-intensity domain (\u003cspan additionalcitationids=\"CR40\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e), with prolonged sessions\u0026thinsp;\u0026gt;\u0026thinsp;1.5 h making up a substantial part of skiers\u0026rsquo; total training (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). Single bouts of prolonged, low-intensity exercise have been associated with systemic immune disturbances (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e), which could possibly impair the ability of immune cells to promptly engage in epithelial repair post-exercise. While the repeated damage-repair cycle likely elicited by frequent endurance training in sub-zero environments may offer an explanation as to why cross-country skiing has been highlighted as a risk factor for development of asthma (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e), to our knowledge no studies have investigated effects of prolonged endurance exercise in sub-zero conditions on the composition of the RTLF.\u003c/p\u003e\u003cp\u003eThe purpose of this exploratory study was to investigate whether particles in exhaled air can be used to measure airway effects of exercise in cold air. Specifically, the aims were to investigate whether moderate-intensity exercise in sub-zero temperatures affects PEx count, mass and the lipidomic profile of PEx samples, and furthermore whether the profile was affected by exercise duration and participants\u0026rsquo; atopic status.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eParticipants\u003c/h2\u003e\u003cp\u003eEighteen participants (♂ = 14, ♀= 4, age 29\u0026thinsp;\u0026plusmn;\u0026thinsp;6 years; mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD) provided written, informed consent to participate in the study. Participants were currently engaged in regular endurance training but had never competed in winter endurance sports at elite level (Tier 1\u0026ndash;3 according to McKay et al., (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e)). Atopic status was determined using the Allergy Questionnaire for Athletes (AQUA\u0026copy;) (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e). Atopy was defined as a questionnaire score\u0026thinsp;\u0026ge;\u0026thinsp;5. A detailed description of participant\u0026rsquo;s anthropometric characteristics and training background has been previously reported (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eStudy design\u003c/h3\u003e\n\u003cp\u003eThe study employed a 2x2 randomised crossover design whereby participants performed a preliminary test to determine V̇O\u003csub\u003e2\u003c/sub\u003emax at room temperature, followed by two treadmill running exercise trials, of 30- and 90-min duration, separated by approximately one week, in an environmental chamber at -15\u0026deg;C and circa 67% relative humidity. PEx were collected at two time points, before and 30 min after exercise. An individualised, prescribed treadmill speed for the main exercise trials was determined by interpolation of data from the submaximal stages of a preliminary submaximal and maximal exercise test to determine a speed that would elicit approximately 60% V̇O\u003csub\u003e2\u003c/sub\u003emax during the subsequent visits. A detailed methodology of the pre-test and chamber exercise trials, as well as physiological responses to exercise, lung function results and blood biomarkers measured during the study has been previously published elsewhere (47).\u003c/p\u003e\n\u003ch3\u003eExhaled particle collection\u003c/h3\u003e\n\u003cp\u003eThe composition of the RTLF can be assessed using a novel and non-invasive method to collect and analyse particles in exhaled air using a patented instrument (PExA\u0026reg;, patent P17584SE00). The instrument can collect and discriminate larger (0.4\u0026ndash;5 \u0026micro;m) particles from the RTLF generated by opening and closing of the small airways (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e), herein referred to as PEx. The PExA\u0026reg; instrument can be used to obtain a particle mass and count, both in total and across different particle size domains, while collection and storage of the collected particles enables subsequent assessment of airway surfactant phospholipid composition.\u003c/p\u003e\u003cp\u003e During PEx collection, participants performed multiple repetitions of a specific breathing manoeuvre, whereby the participant exhaled to achieve residual volume (RV) then held their breath for 5 seconds. This was then followed by a steady inhalation to vital capacity followed by a slow exhalation (sVC) into the device. Inhalation of particle-free filtered room air was achieved through a two-way non-rebreathing valve (1420, Hans Rudolph, Shawnee, KS, USA), followed by immediate exhalation into the device. The breathing manoeuvre was repeated at self-regulated intervals. Due to the limited time window for particle collection and the need to collect particles within a similar time window for all participants, particle collection was terminated as soon as the participant reached one of the following end points: 120 ng particle mass had been collected (sufficient particles obtained), after 15\u0026ndash;16 exhalations (to prevent fatigue that could affect later tests), or after approximately 10 minutes (due to time constraints and to match the post-exercise time point within- and between participants; i.e. 30\u0026ndash;40 min post-exercise).\u003c/p\u003e\u003cp\u003eThe concentration of particles was measured in eight size bins between approximately 0.4\u0026ndash;5 \u0026micro;m in diameter by optical particle counter in the PExA instrument (PExA AB, Gothenburg), as described by Almstrand et al (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e). The PExA computer software calculates the sampled particle mass online and displays collected mass to the participant. Particles are collected by the inertia principle using a cascade impactor. Particles are impacted on a thin hydrophilic polytetrafluoroethylene membrane mounted on the impactor collection plate. When sampling had finished, the sampling membrane was taken out of the impactor, divided in two using a clean scalpel, and stored in a microcentrifuge tube at -80\u0026deg;C until later analysis.\u003c/p\u003e\n\u003ch3\u003eLipidomics analysis of PEx samples\u003c/h3\u003e\n\u003cp\u003eA detailed procedure for analysing surfactant lipids from PEx samples of small airways has been described previously (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e). The lipids included in the method were selected during a method development phase preceding this study. Lipid panel selection was based on screening pooled PEx samples using Waters LipidQuan Quanpedia lipid MRM database (Milford, MA, USA). In addition to the targeted screening, untargeted screening was conducted using lipid class specific fragmentation scanning experiments, such as neutral loss scanning and precursor ion scanning. The most abundant lipids in each class were selected for confirmation and further validation (selectivity, precision, linearity) before inclusion into the lipid panel used in the present study.\u003c/p\u003e\u003cp\u003eIn brief, samples were placed in the insert of centrifugal filter tubes (Millipore UFC30LG25). To the sampling membrane 10 \u0026micro;l of isotope labeled lipid standard was added and allowed to dry with the sample for 10 min (Avanti EquiSPLASH SKU:330731 and Avanti GlcCer(d18:1(d5)/18:0) SKU:860638). Lipids were extracted in a thermomixer using 225 \u0026micro;l of isopropanol. The eluate was recovered from the insert to the microtube bottom by centrifugation. The extraction was then immediately repeated with methanol for improved recovery of both the polar and the neutral lipids. The methanol/isopropanol eluate in the microtube was transferred into LC vials (Waters Total Recovery vial SKU: 186002805) and stored at -20\u0026deg;C before analysis. Before analysis, the solvent in the LC-vial was evaporated under a stream of nitrogen and the remaining lipid film was solubilised in 100 \u0026micro;l of solvent consisting of acetonitrile and isopropanol in a ratio of 2:1. The extracted lipids were analysed with a targeted LC-MSMS method using the Waters ACQUITY UPLC I-Class chromatography system coupled to an electrospray ionization source on a Waters Xevo TQ-XS triple quadrupole mass spectrometer detector (Milford, MA, USA). Reverse phase chromatography was used for separation of PC lipids based on fatty acyl groups. All other lipids were analysed using HILIC chromatography that separates the lipid classes.\u003c/p\u003e\u003cp\u003eQuantification was based on the peak area of the analyte divided by the peak area of the internal standard, the response factor. The response factor was multiplied with the mol of internal standard spiked to the sample. One internal standard per lipid class was used. The results of the lipid analyses were calculated as relative amount (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e):\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:(mol\\%=100\\:\\times\\:\\frac{\\text{s}\\text{p}\\text{e}\\text{c}\\text{i}\\text{e}\\text{s}\\_\\text{m}\\text{o}\\text{l}}{\\text{l}\\text{i}\\text{p}\\text{i}\\text{d}\\_\\text{s}\\text{u}\\text{m}\\_\\text{m}\\text{o}\\text{l}})$$\u003c/div\u003e\u003c/div\u003e.\u003c/p\u003e\u003cp\u003eType II isotope correction factors were calculated with the online correction tool LICAR for the lipid classes PG,PE and PI, as described by Gao et al. (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). When isomers were found as two distinct peaks then the first peak was annotated with an \u0026ldquo;_A\u0026rdquo; and the second with a \u0026ldquo;_B\u0026rdquo;. For example: LPC(16:1)_A and LPC(16:1)_B corresponds to the SN1 and SN2 position of the 16:1 fatty acyl group on the glycerol backbone.\u003c/p\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003eData analysis\u003c/h2\u003e\u003cp\u003eThe PExA instrument provided data on total particle count and mass, as well as the distribution of particle count and mass across eight particle size bins. These data were normalised relative to the total volume of exhaled air during the collection. Three samples encountered technical errors during collection and were missing from analysis, one further sample was excluded from analysis due to suspected contamination. Data were then analysed in GraphPad Prism (version 10.6, GraphPad Software, Boston, MA, USA), using the linear mixed model implementation. This mixed model uses a compound symmetry covariance matrix, and is fitted using the Restricted Maximum Likelihood (REML).\u003c/p\u003e\u003cp\u003eFirst, total particle count and mass were analysed with fixed effects for trial, timepoint and their interaction. Subsequently, the proportion of particles in within each particle size domain was analysed using two models: first, with fixed effects for trial, timepoint and particle size, including their interactions, and subsequently, with fixed effects for timepoint and particle size only. Fisher\u0026rsquo;s LSD (for 2x2 analyses) or Bonferroni post-hoc tests were used to follow up significant interactions.\u003c/p\u003e\u003cp\u003eLipidomics data were reported as mol% of total sample lipid content. Lipids with \u0026lt;\u0026thinsp;20% missing values were retained; remaining missing values were imputed using the lowest detected mol% divided by \u0026radic;2. Samples with a total collected mass of \u0026lt;\u0026thinsp;10% of the target (\u0026lt;\u0026thinsp;6 ng) were excluded (n\u0026thinsp;=\u0026thinsp;2). Thus, analysis of lipid composition was ultimately performed on data from 66 samples.\u003c/p\u003e\u003cp\u003eFor baseline analyses, the mean lipid mol% from each participant\u0026rsquo;s baseline samples were calculated. For each trial, the change in mean mol% for each lipid from pre to post exercise was quantified using the log\u003csub\u003e2\u003c/sub\u003e logit difference, calculated as:\u003cdiv id=\"Equb\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e\n$$\\:{{log}_{2}\\left(\\frac{{\\frac{mol\\%}{100}}_{post}}{{1-\\:\\frac{mol\\%}{100}}_{post}}\\right)-\\:log}_{2}\\left(\\frac{{\\frac{mol\\%}{100}}_{pre}}{{1-\\frac{mol\\%}{100}}_{pre}}\\right)$$\u003c/div\u003e\u003c/div\u003e.\u003c/p\u003e\u003cp\u003eLipid responses to each individual exercise trial were analysed using paired t-tests; a prerequisite was thus that samples for both pre- and post- time points within a trial were available (30 min: n\u0026thinsp;=\u0026thinsp;14; 90 min: n\u0026thinsp;=\u0026thinsp;17). To meet the assumptions of parametric testing, lipid mol% data were arcsine transformed prior to statistical analysis, as this transformation stabilises variance and improves normality for proportion data bounded between 0 and 1. Results were visualised against the log\u003csub\u003e2\u003c/sub\u003e logit difference using volcano plots in Prism. The log₂ logit difference was chosen to quantify the magnitude and direction of change from pre- to post-exercise, offering an interpretable magnitude of change for visualisation, as it linearises proportion changes near 0 and 1 and facilitates comparison across trials. Differences in lipid mol% between atopic and non-atopic individuals at baseline, and log\u003csub\u003e2\u003c/sub\u003elogit responses to exercise, were analysed using independent t-tests. We used a significance threshold of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 as a threshold for reporting biomarker responses of interest. Raw p values and FDR-corrected q values (Benjamini-Hochberg, Q\u0026thinsp;=\u0026thinsp;10%) are presented in parallel, but given no q values reached the threshold to be retained as true discoveries, p values were used to best describe changes in the data, but should be interpreted with caution. Hedge\u0026rsquo;s g effect sizes are reported for pairwise comparisons to aid interpretation.\u003c/p\u003e\u003cp\u003ePrincipal component analysis (PCA) was performed in Prism to investigate potential clustering of lipids measured pre and post 30- and 90- min exercise, and to assess differences in the lipid response to exercise based on atopic status. When visual separation was observed in unsupervised PCA analyses, the analysis plan was to follow up with a supervised orthogonal partial least squares discriminant analysis (orthoPLS-DA); however, insufficient separation on PCA was observed in all cases and thus orthoPLS-DA analyses were not performed to avoid overfitting (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003eEffects of exercise on exhaled particle mass and count\u003c/h2\u003e\u003cp\u003eThe mean (\u0026plusmn;\u0026thinsp;SD) number of exhalations during sample collection was 10\u0026thinsp;\u0026plusmn;\u0026thinsp;2, although the obtained particle mass from each collection varied substantially between samples (80\u0026thinsp;\u0026plusmn;\u0026thinsp;56 ng). Due to technical errors, particle data were not obtained at three collection time points.\u003c/p\u003e\u003cp\u003eParticle mass and count, normalised to the total exhaled air volume during sample collection, are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The change in particle mass and count from pre- to post-exercise differed significantly between trials (PEx count: F(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;6.90, p\u0026thinsp;=\u0026thinsp;0.02; PEx mass: F(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;5.53, p\u0026thinsp;=\u0026thinsp;0.035). Post hoc analyses revealed a decrease in particle mass and count post-exercise in the 90 min trial, attributable to higher particle counts and mass at baseline in the 90 min trial compared to both post-exercise and before the 30-min exercise trial (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAnalysis of the response to exercise for particles across different size domains revealed a significant interaction between timepoint and particle size (PEx count: F(7, 136)\u0026thinsp;=\u0026thinsp;6.692, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; PEx mass: F (7, 136)\u0026thinsp;=\u0026thinsp;12.22, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Follow-up analysis of these effects indicated a greater proportional count and mass of smaller particles (0.41-0.70\u0026micro;m) pre-exercise, and a lower proportional mass (but not count) of larger (1.44-4.55\u0026micro;m) particles post-exercise (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eEffects of exercise on exhaled particles assessed using lipidomics\u003c/h3\u003e\n\u003cp\u003eIn total, 93 lipid species were detected and retained for analysis. Phospholipid composition in all samples was dominated by PC(16:0/16:0) which made up more than 50% of the total lipids at all four time points (Supplementary Fig.\u0026nbsp;1). In general, the relative proportions of the major lipids showed minimal change across the four sample time points.\u003c/p\u003e\u003cp\u003eAnalysis of lipid responses to individual trials revealed three lipids that significantly differed (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) from pre- to post-exercise in the 30-min trial (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), and eleven in the 90-min trial (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The only lipid consistently altered across both trials was PE(16:1_18:0), which significantly increased following exercise (30 min: p\u0026thinsp;=\u0026thinsp;0.023, q\u0026thinsp;=\u0026thinsp;0.865; 90 min: p\u0026thinsp;=\u0026thinsp;0.044, q\u0026thinsp;=\u0026thinsp;0.38; combined time effect, g\u0026thinsp;=\u0026thinsp;0.97).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eEffect of atopic status on the PEx and lipid profile before and after exercise in a sub-zero environment\u003c/span\u003e\u003c/p\u003e\u003cp\u003eBased on the pre-defined AQUA score cut-off, 10 participants were classified as atopic (56%, 7 men, 3 women) and eight as non-atopic. There were no significant differences between the mean baseline PEx count or mass between the atopic and non-atopic groups (Supplementary Fig.\u0026nbsp;2). Similarly, there were no significant differences in exhaled lipid composition at baseline between the two groups.\u003c/p\u003e\u003cp\u003eAnalysis of the mean PEx response to exercise also showed no significant differences between the atopic and non-atopic groups (Supplementary Fig.\u0026nbsp;2C and D). In addition, no differences in the distribution of PEx mass or count over the particle size domains were found between atopic and non-atopic participants (Supplementary Fig.\u0026nbsp;3).\u003c/p\u003e\u003cp\u003eThree lipids were identified through volcano plots as potentially different in their response to exercise between the atopic and non-atopic groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA): PG(18:0_20:4) (p\u0026thinsp;=\u0026thinsp;0.048, q\u0026thinsp;=\u0026thinsp;0.92, g\u0026thinsp;=\u0026thinsp;1.01; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), PC(18:1_18:2) (p\u0026thinsp;=\u0026thinsp;0.026, q\u0026thinsp;=\u0026thinsp;0.92, g\u0026thinsp;=\u0026thinsp;0.99, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC) and PC(16:0_9;O), (p\u0026thinsp;=\u0026thinsp;0.006, q\u0026thinsp;=\u0026thinsp;0.59, g\u0026thinsp;=\u0026thinsp;0.86, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). However, the distribution of particle mass across particle size bins was not affected by atopy (i.e. no significant main effect of atopy, nor atopy x timepoint interaction; Supplementary Fig.\u0026nbsp;3).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eMultivariate analysis\u003c/h2\u003e\u003cp\u003eAnalysis with PCA showed no clear separation between sample time points or trials (Supplementary Fig.\u0026nbsp;4a), nor the difference in the response to exercise between atopic and non-atopic individuals (Supplementary Fig.\u0026nbsp;4b). The lack of separation between groups and time-points in the PCA analysis precluded further analysis with orthoPLS-DA to avoid overfitting.\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003e The present study investigated exhaled particle responses to moderate-intensity exercise in sub-zero conditions among healthy non-atopic and atopic participants, representing the first application of the PExA method in an acute exercise setting. The main findings were that 1) a greater proportion of smaller particles were obtained after exercise 2) a higher number of lipids (eleven) were altered after 90 min exercise versus 30 min exercise (three), accompanied by a greater reduction in PEx mass and count post-exercise after 90 min exercise 3) there was no significant difference in the composition of the obtained PEx mass and count between atopic and non-atopic participants at baseline nor in response to exercise, but 4) three lipids showed differing exercise responses between non-atopic and atopic participants.\u003c/p\u003e\u003cp\u003eThe consistent shift toward smaller particles (0.5-1\u0026micro;m) following exercise across both trials represents a novel finding, suggesting that the physical properties of particle generation may have been altered by exercise in a subzero environment. The reduction in particle mass and count after 90-min exercise likely reflects subtle alterations in airway surfactant properties that, while not causing functional impairment in this acute setting, may provide mechanistic insights into the pathogenesis of exercise-induced bronchoconstriction (EIB) with repeated exposure to exercise in cold air. While no participants in the present study had clinically relevant changes in FEV1 post-exercise (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e), previous observations have shown an association between decreased PEx mass and increased asthma severity (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e), suggesting that PEx measurements may be sensitive to early perturbations in respiratory tract integrity even before clinically detectable lung function changes occur. The increased ventilation of exercise in combination with the cold, dry environment leads to cooling and drying of the airway epithelium, which could affect mucosal hydration and viscosity, surface tension and airway reopening dynamics (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e). The volume of the RTLF in the lower airways has been modelled to be under 1ml, and thus susceptible to water loss and resultant osmotic stress (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e), especially during high ventilation exercise in a cold, dry environment. It has been hypothesised that shear stress on the dehydrated airway epithelium can cause airway epithelial damage, leading to inflammation, release of mediators, increased epithelial permeability and plasma leakage into the airways, which over time can contribute to airway remodelling and the development of airway hyperresponsiveness characteristic of EIB (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). The impact of prolonged exercise in the cold on exhaled particle generation has not previously been explored, but we can speculate that drying may reduce the thickness of the RTLF during exercise, potentially leading to smaller particles being generated during airway reopening, whereas cooling and drying may also alter airway compliance and surface tension, that could also influence the mechanics of particle formation.\u003c/p\u003e\u003cp\u003eThe responses of the detected lipid species to exercise, while modest, may also offer some insight into RTLF response to exercise in cold air. The phosphatidylcholine PC16:0/16:0 (dipalmitoyl phosphatidylcholine or DPPC) was the most abundant lipid across all samples at all time points, representing just over 50% of the total lipids. Larsson et al (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e) describe DPPC as the most abundant of the targeted lipids determined from PEx, accounting for just over 50% of total lipids. Only one lipid species, PE(16:1_18:0), showed a consistent increase after exercise across both trials. PE species are highly enriched in mitochondrial inner membranes (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e), and the observed increase in PE(16:1_18:0) may indicate enhanced membrane biosynthesis or remodeling to support increased metabolic demands during and after exercise. In contrast, two lysophosphatidylcholine (LPC) species decreased post-exercise. LPCs are generated from phosphatidylcholines through phospholipase A2 (PLA2)-mediated hydrolysis and can impair surfactant function when elevated (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e). Additionally, LPCs can be used to disrupt the epithelial barrier in experimental contexts (\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e). The observed decrease in LPC levels suggests reduced phospholipase activity and/or active reacylation of LPCs back to intact phospholipids during the recovery period, rather than ongoing epithelial disruption. The 30-minute post-exercise sampling timepoint may therefore have captured the resolution phase following any transient perturbations during exercise.\u003c/p\u003e\u003cp\u003eFinally, atopic individuals showed a greater increase in an oxidised PC species, PC(16:0_9:0;O), potentially reflecting heightened oxidative stress, as previously reported in inflamed tissues (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e). We previously reported that atopic individuals experienced greater intensity of respiratory symptoms after exercise (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e), so it is possible that their airways had a greater propensity to epithelial disruption, inflammation or mucus function. As research into lipid regulation of airway integrity is still emerging, it remains uncertain whether the responses we observed may reflect a propensity to adapt to higher rates of ventilation, cold exposure, or cellular stress and the specific functional significance of the identified species in surfactant homeostasis requires further investigation.\u003c/p\u003e\u003cp\u003e Despite these changes in specific species, the overall lipid profile remained relatively stable at all measurement timepoints, and there were no significant shifts in overall lipid classes post-exercise. The overall greater number of lipid species that were altered (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) after 90-min exercise versus 30-min exercise may indicate a dose-response relationship between exposure of the airways to acute epithelial stress and acute changes in RTLF composition. Taken together, the reported changes in particle characteristics and lipid composition of PEx samples suggest that even a single bout of prolonged exercise in a subzero environment can perturb the RTLF. The overall stability of most lipid species and classes nevertheless suggests that the airway surfactant system is relatively robust and likely recovers quickly after environmental stress.\u003c/p\u003e\u003cp\u003eTo our knowledge, this study was the first time that the PExA method has been used to collect samples before and after exercise. Given this novelty, comparisons with studies that have used other breath sampling methodologies can provide important context for our findings. Previous studies have collected breath biomarkers using, for example, exhaled breath condensate (EBC). Although a detailed comparison of EBC versus PEx methodology is outside the scope of the current paper, a recent proof-of-concept study also reported no change in breath biomarkers (8-isoprostane and pentraxin-3) after moderate-intensity exercise in healthy participants (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e). The authors noted that these findings were not unexpected and suggested that more extreme exercise intensities or co-introduction of environmental stressors would likely be needed to observe detectable biomarker changes. This interpretation is also supported by studies using other airway stressors. For example, acute exposure to wood smoke has been associated with pro-inflammatory and pro-oxidative responses in EBC in combination with exercise (\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e) but not at rest (\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e). Like cold weather exercise, wood smoke exposure disrupts lung function and the airway epithelium, being associated with increases in exhaled nitric oxide and CC16 (\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e). Thus, it is likely that the moderate-intensity exercise employed in the present study, even in sub-zero air, does not constitute a sufficiently potent stimulus to stress the airway mucosa and RTLF composition in healthy individuals.\u003c/p\u003e\u003cp\u003eOur observations should also be interpreted in the context of previous findings from this project. In contrary to the original hypothesis, prolonged (i.e. 90 min) moderate-intensity exercise did not elicit decreased lung function (measured using dynamic spirometry) nor airway epithelial damage (assessed via plasma club cell Clara protein 16, CC16, concentrations), compared to 30 min exercise at -15\u0026deg;C (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). The effects of exercise in the cold on lung function and airway epithelial damage were also minimal, with no changes in spirometry measurements after exercise, and only a small increase in CC16, indicative of mild airway epithelial damage after both trials. These findings might suggest that the exercise challenge was not potent enough to elicit substantial acute stress on the airways of our healthy participants \u0026ndash; short, high-intensity exercise, or possibly longer duration exercise, would likely have elicited greater decrements in lung function (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e). This adds important context to the present findings of only subtle changes in PEx mass and composition; whether a more potent stimulus (i.e. higher intensity and/or longer duration) would cause a greater perturbation in PEx samples remains a question for future research.\u003c/p\u003e\u003cp\u003eSeveral limitations should be acknowledged in interpreting the findings from the present study. The elevated baseline particle mass and count before the 90 min trial introduce possible complexities; apparent post-exercise alterations in PEx and lipid compositions may be attributable to this difference. The reason for the elevated baseline remains unclear, given all trials were conducted at the same time of day within individuals and the trial order was randomised, and previous studies have not reported diurnal variation in PEx measurements (\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e). However, within-subject coefficients of variation for PEx count and mass have been reported at 29% and 15%, respectively, suggesting that day-to-day variability may have contributed to this difference (\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e). Additionally, the shorter sampling window used in this study compared to previous work (10 min was the maximum allocated time in the study protocol for PEx collection to permit continuation to other time-sensitive measures) and the wide range in collected sample mass may have contributed to variation in sample composition. The study did not utilise a seated rest control trial, that may have highlighted any possible effects of time of day and/or other laboratory measurements (e.g. spirometry manoeuvres) on the PEx collection. Given the exploratory nature and multivariate dataset, as well as the proportion-shape of the mol% data, the relatively small sample size (\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e), and the lack of discoveries retained after FDR-correction, we should be aware that lipid species responses where p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 but q\u0026thinsp;\u0026gt;\u0026thinsp;0.05 require further investigation and replication to verify our observations; also given that only one lipid showed a sufficiently robust response to exercise to produce even a significant p value post-exercise in both trials. Finally, since this was the first study to incorporate PEx measurements after physical activity, we highlight that no studies have yet been performed to determine the optimal post-exercise timepoint to detect possible alterations in the composition of the RTLF.\u003c/p\u003e\u003cp\u003eNevertheless, our findings may have some value to inform advice provided to athletes regarding the risks of training in cold environments. To achieve primary prevention of exercise-induced asthma, studies such as this can help to identify, or indeed discount possible training-related hazards to airway integrity. The results from the present study, in combination with our previous findings (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e), suggest that a single bout of moderate-intensity exercise in sub-zero conditions triggers only subtle changes in the RTLF composition, which warrant further investigation of their biological importance in susceptible populations and with more intense exercise protocols and well-powered, robust study designs.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe present study represents the first, to our knowledge, application of the PExA method to exercise physiology research, and provides important preliminary data regarding the exhaled particle response to exercise under environmental stress. Our findings demonstrate that moderate-intensity exercise in sub-zero conditions induces subtle but measurable changes in exhaled particle composition, including a reduction in particle size and shifts in selected lipid species that may be used as a basis to inform future hypotheses and follow-up studies. The overall stability of the PEx composition nevertheless suggests the airway surfactant is relatively robust to, and recovers quickly, after environmental stress, in agreement with our earlier findings of minimal changes in lung function or airway epithelial damage from the study protocol. Thus, our data provide preliminary insight into airway responses to exercise in a cold climate and contribute to the growing understanding of how the PExA method can be applied as a non-invasive tool for respiratory health assessment in an acute setting.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003cp\u003e The study was approved by the Swedish Ethical Review Authority (2020\u0026ndash;05205). All participants provided full written, informed consent to participate in the study and all procedures were conducted in accordance with the Declaration of Helsinki.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003cp\u003eNot applicable\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eCompeting Interests\u003c/h2\u003e\u003cp\u003eA-CO is a board member and shareholder of PExA AB, the company that produce and sell the PExA instrument used in the present study. PL is a shareholder of PExA AB. All other authors have no conflicts of interest to declare.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003e The study received funding from the \u0026Ouml;stersund municipality and Mid Sweden University financial agreement, Rolf and Gunilla Enstr\u0026ouml;m\u0026rsquo;s Foundation for Research and Development.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eHH: conceptualisation, design, data acquisition, data curation, data analysis, data interpretation, writing \u0026ndash; original draft, writing \u0026ndash; revising and editing.AG: conceptualisation, design, data acquisition, data curation, writing \u0026ndash; revising and editing.IR: data acquisition, data curation.PL: data acquisition, data analysis, data interpretation, writing \u0026ndash; revising and editingA-CO: conceptualisation, data interpretation.NS: conceptualisation, design, data interpretation, writing \u0026ndash; revising and editing.All authors approved the submitted version of the manuscript and agree to be accountable for the work.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors thank the participants for their time, efforts, and patience in the lab. In addition, we acknowledge the support of Marianne Anderson for providing training in the PExA methodology.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets generated and/or analysed during the current study are available on request from the corresponding author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBougault V, Adami PE, Sewry N, Fitch K, Carlsten C, Villiger B, et al. Environmental factors associated with non-infective acute respiratory illness in athletes: A systematic review by a subgroup of the IOC consensus group on acute respiratory illness in the athlete. J Sci Med Sport. 2022 June;25(6):466\u0026ndash;73.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEriksson LM, Irewall T, Lindberg A, Stenfors N. Prevalence, age at onset, and risk factors of self-reported asthma among Swedish adolescent elite cross-country skiers. 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PLoS ONE. 2025;20(8):e0325153.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGao L, Ji S, Burla B, Wenk MR, Torta F, Cazenave-Gassiot A. LICAR: An Application for Isotopic Correction of Targeted Lipidomic Data Acquired with Class-Based Chromatographic Separations Using Multiple Reaction Monitoring. Anal Chem. 2021;93(6):3163\u0026ndash;71.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWorley B, Powers R. PCA as a practical indicator of OPLS-DA model reliability. Curr Metabolomics. 2016;4(2):97\u0026ndash;103.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCarpaij OA, Muiser S, Bell AJ, Kerstjens HAM, Galban CJ, Fortuna AB, et al. Assessing small airways dysfunction in asthma, asthma remission and healthy controls using particles in exhaled air. ERJ Open Res. 2019;5(4):00202\u0026ndash;2019.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHaut B, Nonclercq A, Buess A, Rabineau J, Rigaut C, Sobac B. 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Scand J Med Sci Sports. 2015;25(4):e374\u0026ndash;80.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKokelj S, Kim JL, Andersson M, Runstr\u0026ouml;m Eden G, Bake B, Olin AC. Intra-individual variation of particles in exhaled air and of the contents of Surfactant protein A and albumin. PLoS ONE. 2020;15(1):e0227980.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBroadhurst DI, Kell DB. Statistical strategies for avoiding false discoveries in metabolomics and related experiments. Metabolomics. 2006;2(4):171\u0026ndash;96.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Airway health, cold environment, exercise-induced bronchoconstriction, exhaled particles, lipidomics, winter sports","lastPublishedDoi":"10.21203/rs.3.rs-8084613/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8084613/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eExercise-induced bronchoconstriction (EIB) commonly develops following prolonged exercise in cold, dry environments, but the acute mechanisms underlying airway responses to such environmental stressors remain poorly understood. Exhaled particle analysis (PExA) offers a novel, non-invasive approach to assess respiratory tract lining fluid (RTLF) composition and may provide mechanistic insights into early airway responses before clinically detectable changes occur. This study investigated exhaled particle characteristics and lipid composition in response to moderate-intensity exercise in sub-zero conditions among healthy atopic and non-atopic individuals.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eEighteen recreationally active participants (14 male) aged 29\u0026thinsp;\u0026plusmn;\u0026thinsp;6 years, performed two moderate-intensity exercise trials (30 and 90 min duration) in a climate chamber set to -15\u0026deg;C. Participants provided exhaled particle (PEx) samples using the PExA\u0026reg; method, before and 30 min after each exercise trial. The PExA\u0026reg; device analysed particle mass and count across eight size bins (0.4\u0026ndash;5 \u0026micro;m) and the collected PEx samples subsequently underwent lipidomic analysis to assess differences in the RTLF composition from before to after exercise. Data were analysed using univariate and multivariate statistical methods.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eExercise induced a significant increase in smaller particles in PEx samples (0.4-0.7\u0026micro;m; p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Three lipid species were significantly altered after the 30-min exercise trial, and eleven after 90-min exercise. Phosphatidylethanolamine PE(16:1_18:0) was the only lipid that consistently increased across both exercise durations (30 min: p\u0026thinsp;=\u0026thinsp;0.023; 90 min: p\u0026thinsp;=\u0026thinsp;0.044; g\u0026thinsp;=\u0026thinsp;0.97). No significant differences in overall particle characteristics were observed between atopic and non-atopic participants, though three specific lipid species showed differential exercise responses between groups, including an oxidised phosphatidylcholine species PC(16:0_9:0;O) (p\u0026thinsp;=\u0026thinsp;0.006, q\u0026thinsp;=\u0026thinsp;0.59, g\u0026thinsp;=\u0026thinsp;0.86).\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eModerate-intensity exercise in sub-zero conditions induced a consistent shift toward smaller exhaled particles and subtle alterations in respiratory tract lining fluid lipid composition, including increased phosphatidylethanolamine PE(16:1_18:0) and decreased lysophosphatidylcholine species. Despite these changes, the overall stability of exhaled particle composition suggests that airway surfactant systems are relatively robust to acute environmental stress in healthy individuals.\u003c/p\u003e\u003ch2\u003eTrial registration\u003c/h2\u003e\u003cp\u003eISRCTN13977758, Retrospectively registered 01/02/2022. https//doi.org/10.1186/ISRCTN13977758\u003c/p\u003e","manuscriptTitle":"Subtle changes in exhaled particle size and phospholipid composition of the respiratory tract lining fluid following moderate-intensity exercise in sub-zero conditions","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-01 08:12:29","doi":"10.21203/rs.3.rs-8084613/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1cf450b2-020c-4627-b5d5-8897cca54079","owner":[],"postedDate":"December 1st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-01-30T15:25:52+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-01 08:12:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8084613","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8084613","identity":"rs-8084613","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}