Biomarkers of some pulmonary diseases in exhaled breath

Analysis of various biomarkers in exhaled breath allows completely non-invasive monitoring of inflammation and oxidative stress in the respiratory tract in inflammatory lung diseases, including asthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), bronchiectasis and interstitial lung diseases. The technique is simple to perform, may be repeated frequently, and can be applied to children, including neonates, and patients with severe disease in whom more invasive procedures are not possible. Several volatile chemicals can be measured in the breath (nitric oxide, carbon monoxide, ammonia), and many non-volatile molecules (mediators, oxidation and nitration products, proteins) may be measured in exhaled breath condensate. Exhaled breath analysis may be used to quantify inflammation and oxidative stress in the respiratory tract, in differential diagnosis of airway disease and in the monitoring of therapy. Most progress has been made with exhaled nitric oxide (NO), which is increased in atopic asthma, is correlated with other inflammatory indices and is reduced by treatment with corticosteroids and antileukotrienes, but not (β₂-agonists. In contrast, exhaled NO is normal in COPD, reduced in CF and diagnostically low in primary ciliary dyskinesia. Exhaled carbon monoxide (CO) is increased in asthma, COPD and CF. Increased concentrations of 8-isoprostane, hydrogen peroxide, nitrite and 3-nitrotyrosine are found in exhaled breath condensate in inflammatory lung diseases. Furthermore, increased levels of lipid mediators are found in these diseases, with a differential pattern depending on the nature of the disease process. In the future it is likely that smaller and more sensitive analysers will extend the discriminatory value of exhaled breath analysis and that these techniques may be available to diagnose and monitor respiratory diseases in the general practice and home setting.     

Exhaled Breath Analysis Using Electronic Nose in Cystic Fibrosis and Primary Ciliary Dyskinesia Patients with Chronic Pulmonary Infections

The current diagnostic work-up and monitoring of pulmonary infections may be perceived as invasive, is time consuming and expensive. In this explorative study, we investigated whether or not a non-invasive exhaled breath analysis using an electronic nose would discriminate between cystic fibrosis (CF) and primary ciliary dyskinesia (PCD) with or without various well characterized chronic pulmonary infections. We recruited 64 patients with CF and 21 with PCD based on known chronic infection status. 21 healthy volunteers served as controls. An electronic nose was employed to analyze exhaled breath samples. Principal component reduction and discriminant analysis were used to construct internally cross-validated receiver operator characteristic (ROC) curves. Breath profiles of CF and PCD patients differed significantly from healthy controls p = 0.001 and p = 0.005, respectively. Profiles of CF patients having a chronic P. aeruginosa infection differed significantly from to non-chronically infected CF patients p = 0.044. We confirmed the previously established discriminative power of exhaled breath analysis in separation between healthy subjects and patients with CF or PCD. Furthermore, this method significantly discriminates CF patients suffering from a chronic pulmonary P. aeruginosa (PA) infection from CF patients without a chronic pulmonary infection. Further studies are needed for verification and to investigate the role of electronic nose technology in the very early diagnostic workup of pulmonary infections before the establishment of a chronic infection.     

Determination of proteomic and metabolic composition of exhaled breath condensate of newborns

Here, the possibility of proteomic and metabolomic analysis of the composition of exhaled breath condensate of neonates with respiratory support. The developed method allows non-invasive collecting sufficient amount of the material for identification of disease-specific biomarkers. Samples were collected by using a condensing device that was incorporated into the ventilation system. The collected condensate was analyzed by liquid chromatography coupled with high resolution mass spectrometry and tandem mass spectrometry. The isolated substances were identified with a use of databases for proteins and metabolites. As a result, a number of compounds that compose the exhaled breath condensate was determined and can be considered as possible biomarkers of newborn diseases or stage of development.     

人的粪便、血浆、唾液、呼出气体中短链脂肪酸的分析

目的建立一种顶空气相色谱-串联质谱法(HS-GC/MS)快速检测人的粪便、血浆、唾液、呼出气体中短链脂肪酸(SCFAs)的方法,初步探索人的粪便、血浆、唾液、呼出气体中短链脂肪酸的相关性。方法样品无需处理直接封存于顶空进样瓶中,顶空进样;采用DB-FFAP毛细管柱(30 m×0.25 mm×0.25μm)分离;全扫描模式检测。结果人的粪便、血浆、唾液、呼出气体中均含有短链脂肪酸。在人的粪便、唾液样本中均检测到8个短链脂肪酸(乙酸、丙酸、异丁酸、丁酸、异戊酸、戊酸、异己酸、己酸);血浆、呼出气体样本中均检测到7个短链脂肪酸(未检测到异己酸)。结论初步推测人的粪便、血浆、唾液、呼出气体中的短链脂肪酸具有一定的相关性。本方法简单、快速、灵敏,可用于人的生物样品中短链脂肪酸的快速检测。     

Heart rate variability in non-apneic snorers and controls before and after continuous positive airway pressure

Background

The effects of changes in cooling temperature on biomarker levels in exhaled breath condensate have been little investigated. The aim of the study was to test the effect of condensation temperature on the parameters of exhaled breath condensate and the levels of selected biomarkers.

Methods

Exhaled breath condensate was collected from 24 healthy subjects at temperatures of -10, -5, 0 and +5 C degrees. Selected parameters (condensed volume and conductivity) and biomarkers (hydrogen peroxide, malondialdehyde) were measured.

Results

There was a progressive increase in hydrogen peroxide and malondialdehyde concentrations, and condensate conductivity as the cooling temperature increased; total condensate volume increased as the cooling temperature decreased.

Conclusion

The cooling temperature of exhaled breath condensate collection influenced selected biomarkers and potential normalizing factors (particularly conductivity) in different ways ex vivo. The temperature of exhaled breath condensate collection should be controlled and reported.     

Influenza virus in human exhaled breath: an observational study

Background

Recent studies suggest that humans exhale fine particles during tidal breathing but little is known of their composition, particularly during infection.

Methodology/Principal Findings

We conducted a study of influenza infected patients to characterize influenza virus and particle concentrations in their exhaled breath. Patients presenting with influenza-like-illness, confirmed influenza A or B virus by rapid test, and onset within 3 days were recruited at three clinics in Hong Kong, China. We collected exhaled breath from each subject onto Teflon filters and measured exhaled particle concentrations using an optical particle counter. Filters were analyzed for influenza A and B viruses by quantitative polymerase chain reaction (qPCR). Twelve out of thirteen rapid test positive patients provided exhaled breath filter samples (7 subjects infected with influenza B virus and 5 subjects infected with influenza A virus). We detected influenza virus RNA in the exhaled breath of 4 (33%) subjects–three (60%) of the five patients infected with influenza A virus and one (14%) of the seven infected with influenza B virus. Exhaled influenza virus RNA generation rates ranged from <3.2 to 20 influenza virus RNA particles per minute. Over 87% of particles exhaled were under 1 µm in diameter.

Conclusions

These findings regarding influenza virus RNA suggest that influenza virus may be contained in fine particles generated during tidal breathing, and add to the body of literature suggesting that fine particle aerosols may play a role in influenza transmission.     

Korea red ginseng water extract increases nitric oxide concentrations in exhaled breath.

Panax ginseng is well known to enhance the release of nitric oxide (NO) from endothelial cells of the rat aorta and to reduce blood pressure in animals. In this study, we investigated the effects of water extract of Korea red ginseng (KRG) on NO concentration levels in the exhaled breath, blood pressure, and heart rate of human volunteers. We also are interested in whether NO levels in exhaled breath are increased by KRG extract, and correlated with blood pressure and heart rate. Twelve healthy, non-smoking male volunteers were recruited for this study. A single administration of KRG water extract (500 mg/50 kg) increased NO levels in exhaled breath, and concomitantly decreased mean blood pressure and heart rate. The correlation value between NO levels and heart rate (r = 0.94), and the correlation value between NO levels and heart rate (r = 0.84) are significant (P < 0.01). Linear regression analysis shows the clear conversed correlation between NO levels and blood pressure as well as heart rate. Therefore, present data suggest that KRG may be useful for the treatment of hypertension and pulmonary vascular obstruction.     

呼吸中的疾病诊断标志物及检测技术

人类呼出气体中的各种化合物能提供各种疾病和健康状况的重要信息。近年来,由于红外、电化学、化学发光等新技术的重大突破和质谱仪的使用,使得在极低浓度下精确测量呼出的挥发性有机化合物(VOCs)和气溶胶颗粒成为可能,呼吸检测领域因而取得了重大进展:,呼吸检测因其可以作为一种实时、快速和无创的方法来评估和监测各种疾病与健康状况信息,在科学研究、临床运用中引起了广泛关注。本综述主要概述呼出气体成分分析方法及在疾病诊断中的研究与应用情况,旨在为将来疾病的实时、快速和无创诊断提供一种新的策略.     

Influenza Virus Aerosols in Human Exhaled Breath: Particle Size,Culturability, and Effect of Surgical Masks

The CDC recommends that healthcare settings provide influenza patients with facemasks as a means of reducing transmission to staff and other patients, and a recent report suggested that surgical masks can capture influenza virus in large droplet spray. However, there is minimal data on influenza virus aerosol shedding, the infectiousness of exhaled aerosols, and none on the impact of facemasks on viral aerosol shedding from patients with seasonal influenza.We collected samples of exhaled particles (one with and one without a facemask) in two size fractions (“coarse”>5 µm, “fine”≤5 µm) from 37 volunteers within 5 days of seasonal influenza onset, measured viral copy number using quantitative RT-PCR, and tested the fine-particle fraction for culturable virus.Fine particles contained 8.8 (95% CI 4.1 to 19) fold more viral copies than did coarse particles. Surgical masks reduced viral copy numbers in the fine fraction by 2.8 fold (95% CI 1.5 to 5.2) and in the coarse fraction by 25 fold (95% CI 3.5 to 180). Overall, masks produced a 3.4 fold (95% CI 1.8 to 6.3) reduction in viral aerosol shedding. Correlations between nasopharyngeal swab and the aerosol fraction copy numbers were weak (r = 0.17, coarse; r = 0.29, fine fraction). Copy numbers in exhaled breath declined rapidly with day after onset of illness. Two subjects with the highest copy numbers gave culture positive fine particle samples.Surgical masks worn by patients reduce aerosols shedding of virus. The abundance of viral copies in fine particle aerosols and evidence for their infectiousness suggests an important role in seasonal influenza transmission. Monitoring exhaled virus aerosols will be important for validation of experimental transmission studies in humans.     

Surfactant Protein A in Exhaled Endogenous Particles Is Decreased in Chronic Obstructive Pulmonary Disease (COPD) Patients: A Pilot Study

Background

Exhaled, endogenous particles are formed from the epithelial lining fluid in small airways, where surfactant protein A (SP-A) plays an important role in pulmonary host defense. Based on the knowledge that chronic obstructive pulmonary disease (COPD) starts in the small airway epithelium, we hypothesized that chronic inflammation modulates peripheral exhaled particle SP-A and albumin levels. The main objective of this explorative study was to compare the SP-A and albumin contents in exhaled particles from patients with COPD and healthy subjects and to determine exhaled particle number concentrations.

Methods

Patients with stable COPD ranging from moderate to very severe (n = 13), and healthy non-smoking subjects (n = 12) were studied. Subjects performed repeated breath maneuvers allowing for airway closure and re-opening, and exhaled particles were optically counted and collected on a membrane using the novel PExA^® instrument setup. Immunoassays were used to quantify SP-A and albumin.

Results

COPD patients exhibited significantly lower SP-A mass content of the exhaled particles (2.7 vs. 3.9 weight percent, p = 0.036) and lower particle number concentration (p<0.0001) than healthy subjects. Albumin mass contents were similar for both groups.

Conclusions

Decreased levels of SP-A may lead to impaired host defense functions of surfactant in the airways, contributing to increased susceptibility to COPD exacerbations. SP-A in exhaled particles from small airways may represent a promising non-invasive biomarker of disease in COPD patients.     

Method for determination of methadone in exhaled breath collected from subjects undergoing methadone maintenance treatment

At present drugs of abuse testing using exhaled breath as specimen is only possible for alcohol. However, we recently discovered that using modern liquid chromatography–mass spectrometry technique amphetamine and methamphetamine is detectable in exhaled breath following intake in drug addicts. We therefore undertook to develop a method for determination of methadone in exhaled breath from patients undergoing methadone maintenance treatment. Exhaled breath was collected from 13 patients after intake of the daily methadone dose. The compounds were trapped by filtering the air through a C18 modified silica surface. After elution of any trapped methadone the extract was analysed by a combined liquid chromatography–tandem mass spectrometry method. Recovery of trapped methadone from the filter surface was 96%, no significant matrix effect was observed, and the quantification using methadone-d3 as an internal standard was accurate (<10% bias) and precise (coefficient of variation 1.6–2.0%). Methadone was indisputably identified by means of the mass spectrometry technique in exhaled breath samples from all 13 patients. Identification was based on monitoring two product ions in selected reaction monitoring mode with correct relative ratio (±20%) and correct retention time. Excretion rates ranged from 0.39 to 78 ng/min. No methadone was detected in 10 control subjects. This finding confirms that breath testing is a new possibility for drugs of abuse testing. Collection of exhaled breath specimen is likely to be more convenient and safe as compared to other matrices presently in use.     

Exhaled Aerosol Pattern Discloses Lung Structural Abnormality: A Sensitivity Study Using Computational Modeling and Fractal Analysis

Background

Exhaled aerosol patterns, also called aerosol fingerprints, provide clues to the health of the lung and can be used to detect disease-modified airway structures. The key is how to decode the exhaled aerosol fingerprints and retrieve the lung structural information for a non-invasive identification of respiratory diseases.

Objective and Methods

In this study, a CFD-fractal analysis method was developed to quantify exhaled aerosol fingerprints and applied it to one benign and three malign conditions: a tracheal carina tumor, a bronchial tumor, and asthma. Respirations of tracer aerosols of 1 µm at a flow rate of 30 L/min were simulated, with exhaled distributions recorded at the mouth. Large eddy simulations and a Lagrangian tracking approach were used to simulate respiratory airflows and aerosol dynamics. Aerosol morphometric measures such as concentration disparity, spatial distributions, and fractal analysis were applied to distinguish various exhaled aerosol patterns.

Findings

Utilizing physiology-based modeling, we demonstrated substantial differences in exhaled aerosol distributions among normal and pathological airways, which were suggestive of the disease location and extent. With fractal analysis, we also demonstrated that exhaled aerosol patterns exhibited fractal behavior in both the entire image and selected regions of interest. Each exhaled aerosol fingerprint exhibited distinct pattern parameters such as spatial probability, fractal dimension, lacunarity, and multifractal spectrum. Furthermore, a correlation of the diseased location and exhaled aerosol spatial distribution was established for asthma.

Conclusion

Aerosol-fingerprint-based breath tests disclose clues about the site and severity of lung diseases and appear to be sensitive enough to be a practical tool for diagnosis and prognosis of respiratory diseases with structural abnormalities.     

Circadian Variation of the Human Metabolome Captured by Real-Time Breath Analysis

Circadian clocks play a significant role in the correct timing of physiological metabolism, and clock disruption might lead to pathological changes of metabolism. One interesting method to assess the current state of metabolism is metabolomics. Metabolomics tries to capture the entirety of small molecules, i.e. the building blocks of metabolism, in a given matrix, such as blood, saliva or urine. Using mass spectrometric approaches we and others have shown that a significant portion of the human metabolome in saliva and blood exhibits circadian modulation; independent of food intake or sleep/wake rhythms. Recent advances in mass spectrometry techniques have introduced completely non-invasive breathprinting; a method to instantaneously assess small metabolites in human breath. In this proof-of-principle study, we extend these findings about the impact of circadian clocks on metabolomics to exhaled breath. As previously established, our method allows for real-time analysis of a rich matrix during frequent non-invasive sampling. We sampled the breath of three healthy, non-smoking human volunteers in hourly intervals for 24 hours during total sleep deprivation, and found 111 features in the breath of all individuals, 36–49% of which showed significant circadian variation in at least one individual. Our data suggest that real-time mass spectrometric "breathprinting" has high potential to become a useful tool to understand circadian metabolism, and develop new biomarkers to easily and in real-time assess circadian clock phase and function in experimental and clinical settings.     

Exhaled breath and fecal volatile organic biomarkers of chronic kidney disease

BackgroundWhile much is known about the effect of chronic kidney disease (CKD) on composition of body fluids little is known regarding its impact on the gases found in exhaled breath or produced by intestinal microbiome. We have recently shown significant changes in the composition of intestinal microbiome in humans and animals with CKD. This study tested the hypothesis that uremia-induced changes in cellular metabolism and intestinal microbiome may modify the volatile organic metabolites found in the exhaled breath or generated by intestinal flora.MethodsSD rats were randomized to CKD (5/6 nephrectomy) or control (sham operation) groups. Exhaled breath was collected by enclosing each animal in a glass chamber flushed with clean air, then sealed for 45 min and the trapped air collected. Feces were collected, dissolved in pure water, incubated at 37 °C in glass reactors for 24 h and the trapped air collected. Collected gases were analyzed by gas chromatography.ResultsOver 50 gases were detected in the exhaled breath and 36 in cultured feces. Four gases in exhaled breath and 4 generated by cultured feces were significantly different in the two groups. The exhaled breath in CKD rats showed an early rise in isoprene and a late fall in linear aldehydes. The CKD animals' cultured feces released larger amounts of dimethyldisulfide, dimethyltrisulfide, and two thioesters.ConclusionsCKD significantly changes the composition of exhaled breath and gaseous products of intestinal flora.General significanceAnalysis of breath and bowel gases may provide useful biomarkers for detection and progression of CKD and its complications.     

Targeted eicosanoids lipidomics of exhaled breath condensate in healthy subjects

Background

Exhaled breath condensate collection is a non-invasive method of sampling the respiratory tract that can be repeated several times in a wide range of clinical settings. Quantitation of non-volatile compounds in the condensate requires highly sensitive analytical methods, e.g. mass spectrometry.

Objective

To validate cross-platform measurements of eicosanoids using high performance liquid chromatography or gas chromatography coupled with mass spectrometry in exhaled breath condensate sampled from 58 healthy individuals.

Methods

Twenty different eicosanoid compounds, representing major arachidonic acid lipoxygenation and cyclooxygenation pathways were measured using a stable isotope dilution method. We applied a free palmitic acid concentration as a surrogate marker for the condensate dilution factor.

Results

Eicosanoids concentrations in the condensates were consistent with their content in other biological fluids. Prostaglandin E₂ was the most abundant mediator, represented by its stable metabolite tetranor-PGEM. Prostaglandin D₂ products were at low concentration, while hydroxyacids derived from lipoxygenation were abundant. 5-HETE was elevated in current tobacco smokers. Leukotriene B₄ has the highest concentration of all 5-LO products. 15-LO analogues of cysteinyl leukotrienes–eoxins were detectable and metabolized to eoxin E₄. Two main vascular prostanoids: prostacyclin and thromboxane B₂ were present as metabolites. A marker for non-enzymatic lipid peroxidation, 8-iso-PGF_2α isoprostane was increased in smokers.

Conclusion

Presented targeted lipidomics analysis of exhaled breath condensate in healthy subjects justifies its application to investigation of inflammatory lung diseases. Measurements of non-volatile mediators of inflammation in the condensates might characterize disease-specific pathological mechanisms and responses to treatment.     

Real-time measurement of particulate matter deposition in the lung.

Air pollution and cigarette smoke are recognized health risks. A method was developed for the measurement of the deposition fraction (DF) of polydisperse particulate matter (PM) in human airways. Ten normal volunteers [three females, age range 18-67 years, mean age (SD) 43.9 (14)] made single breath exhalations after inhalation to total lung capacity. The exhaled breath was diverted to a multichannel laser diffraction chamber where the particulate profiler measured 0.3 - 1.0-microm particles. DF was inversely related to expiration flow-rate, 0.69 (0.02) at 4 l min-1 and 0.5 (0.01) at 13 l min-1, respectively (p<0.05), and was influenced by the inhalation flow-rate [0.70 (0.02) at 3 l min-1 and 0.59 (0.02) at 13 l min-1, respectively (p<0.05)], while no differences were found between nasal and oral inhalation (0.68 (0.05) versus 0.67 (0.06), p>0.05). Higher breath holding times were associated with elevated DF [0.74 (0.02) at 20 s, and 0.62 (0.05) without breath holding (p<0.01)]. When the expiratory flow was controlled and the breath hold time standardized, DF was reproducible (CV = 4.85%). PM can be measured in the exhaled breath and its DF can be quantified using a portable device. These methods may be useful in studies investigating the health effects of air pollution and tobacco smoke.     

Exhaled aerosol transmission of pandemic and seasonal H1N1 influenza viruses in the ferret

Person-to-person transmission of influenza viruses occurs by contact (direct and fomites) and non-contact (droplet and small particle aerosol) routes, but the quantitative dynamics and relative contributions of these routes are incompletely understood. The transmissibility of influenza strains estimated from secondary attack rates in closed human populations is confounded by large variations in population susceptibilities. An experimental method to phenotype strains for transmissibility in an animal model could provide relative efficiencies of transmission. We developed an experimental method to detect exhaled viral aerosol transmission between unanesthetized infected and susceptible ferrets, measured aerosol particle size and number, and quantified the viral genomic RNA in the exhaled aerosol. During brief 3-hour exposures to exhaled viral aerosols in airflow-controlled chambers, three strains of pandemic 2009 H1N1 strains were frequently transmitted to susceptible ferrets. In contrast one seasonal H1N1 strain was not transmitted in spite of higher levels of viral RNA in the exhaled aerosol. Among three pandemic strains, the two strains causing weight loss and illness in the intranasally infected 'donor' ferrets were transmitted less efficiently from the donor than the strain causing no detectable illness, suggesting that the mucosal inflammatory response may attenuate viable exhaled virus. Although exhaled viral RNA remained constant, transmission efficiency diminished from day 1 to day 5 after donor infection. Thus, aerosol transmission between ferrets may be dependent on at least four characteristics of virus-host relationships including the level of exhaled virus, infectious particle size, mucosal inflammation, and viral replication efficiency in susceptible mucosa.     

Comparison of aerosol and bioaerosol collection on air filters

Air filters efficiency is usually determined by non-biological test aerosols, such as potassium chloride particles, Arizona dust or di-ethyl-hexyl-sebacate (DEHS) oily liquid. This research was undertaken to asses, if application of non-biological aerosols reflects air filters capacity to collect particles of biological origin. The collection efficiency for non-biological aerosol was tested with the PALAS set and ISO Fine Test Dust. Flow rate during the filtration process was 720 l/h, and particles size ranged 0.246–17.165 μm. The upstream and downstream concentration of the aerosol was measured with a laser particle counter PCS-2010. Tested bioaerosol contained 4 bacterial strains of different shape and size: Micrococcus luteus, Micrococcus varians, Pseudomonas putida and Bacillus subtilis. Number of the biological particles was estimated with a culture-based method. Results obtained with bioaerosol did not confirmed 100% filters efficiency noted for the mineral test dust of the same aerodynamic diameter. Maximum efficiency tested with bacterial cells was 99.8%. Additionally, cells reemission from filters into air was also studied. Bioaerosol contained 3 bacterial strains: Micrococcus varians, Pseudomonas putida and Bacillus subtilis. It was proved that the highest intensity of the reemission process was during the first 5 min. and reached maximum 0.63% of total number of bacteria retained in filters. Spherical cells adhered stronger to the filter fibres than cylindrical ones. It was concluded that non-biological aerosol containing particles of the same shape and surface characteristics (like DEHS spherical particles) can not give representative results for all particles present in the filtered air.     

Testing and evaluation of off-gas filters for bioreactors by a new bacterial aerosol challenge test method (TBAC).

A TNO bacterial aerosol challenge (TBAC) filter test rig was developed for direct assessment of the effectiveness of bioreactor off-gas filters as an alternative to routinely applied indirect wet integrity testing (IT). This TBAC test rig is based on bacterial aerosol challenging with Pseudomonas diminuta and dual monitoring by laser particle counting (LPC) and Andersen microbial sampling (AMS) of viable cells. The TBAC filter test rig is able to reproduce the various conditions encountered in fermentation processes. In experiments with several filters from one class, it was demonstrated that some filters were actually penetrated by up to 3,000 viable cells per test, despite their approval by commercially available IT test equipment. Repetitive filter use, prolonged use, and autoclaving of filters resulted in an increase in pressure drop over the filter but improved the performance of leaking/deviant filters due to building up of a filter cake (this phenomenon was identified by electron microscopy). The integrity tests used were found inadequate for accurate assessment of filter quality. Certification of filter lots by random tests of commercially available off-gas filters using sensitive direct methods such as those presented here might be advisable, as not all filters purchased were of appropriate quality.