Breath biomarkers in diagnosis of pulmonary diseases

Clinica Chimica Acta 413 (2012) 1770–1780

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Invited critical review

Breath biomarkers in diagnosis of pulmonary diseases Meigui Zhou, Yong Liu, Yixiang Duan ⁎ Research Center of Analytical Instrumentation, Analytical and Testing Center, College of Chemistry, Sichuan University, Chengdu 610064, China

a r t i c l e

i n f o

Article history: Received 11 April 2012 Received in revised form 20 May 2012 Accepted 5 July 2012 Available online 14 July 2012 Keywords: Breath analysis Breath biomarker Disease diagnosis Pulmonary disease

a b s t r a c t Breath analysis provides a convenient and simple alternative to traditional specimen testing in clinical laboratory diagnosis. As such, substantial research has been devoted to the analysis and identification of breath biomarkers. Development of new analytes enhances the desirability of breath analysis especially for patients who monitor daily biochemical parameters. Elucidating the physiologic significance of volatile substances in breath is essential for clinical use. This review describes the use of breath biomarkers in diagnosis of asthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), lung cancer, as well as other pulmonary diseases. A number of breath biomarkers in lung pathophysiology will be described including nitric oxide (NO), carbon monoxide (CO), hydrogen peroxide (H2O2) and other hydrocarbons. © 2012 Elsevier B.V. All rights reserved.

Contents 1. 2.

Introduction . . . . . . . . . . . . . . . . Common breath biomarkers . . . . . . . . 2.1. Nitric oxide . . . . . . . . . . . . . 2.2. Carbon monoxide . . . . . . . . . . 2.3. Hydrocarbons . . . . . . . . . . . . 2.4. Hydrogen peroxide . . . . . . . . . 2.5. Ancillary breath markers . . . . . . . 3. Application in lung disease diagnosis . . . . 3.1. Asthma . . . . . . . . . . . . . . . 3.2. Chronic obstructive pulmonary disease 3.3. Cystic fibrosis . . . . . . . . . . . . 3.4. Lung cancer . . . . . . . . . . . . . 3.5. Primary ciliary dyskinesia . . . . . . 3.6. Pulmonary arterial hypertension . . . 3.7. Interstitial lung disease . . . . . . . 3.8. Lung transplant rejection . . . . . . 4. Conclusions . . . . . . . . . . . . . . . . Acknowledgment . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . .

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Abbreviations: FENO, the fragment of exhaled nitric oxide; VOCs, volatile organic compounds; NOS, NO synthase; NOS1, neuronal NOS; NOS3 or eNOS, endothelial NOS; NOS2 or iNOS, inducible NOS; cNOS, constitutive NOS; LPS, lipopolysaccharide; HO, heme oxygenase; ROS, reactive oxygen species; MDA, malondialdehyde; OFR, oxygen free radical; TBARs, thiobarbituric acid reactive substances; ARDS, acute respiratory distress syndrome; EBC, exhaled breath condensate; ICS, inhaled corticosteroids; BAL, bronchoalveolar lavage; SRA, severe refractory asthma; hs-CRP, high-sensitivity C-reactive protein; ET-1, endothelin-1; cysLTs, cysteinyl leukotrienes; COPD, chronic obstructive pulmonary disease; CalvNO, alveolar NO; TNF-α, tumor necrosis factor alpha; CF, cystic fibrosis; PGE2, prostaglandin E2; IL-6, Interleukin-6; TNM, tumor, node, metastasis; PCD, primary ciliary dyskinesia; CT, computed tomography; PAH, pulmonary arterial hypertension; PPH, primary pulmonary hypertension; PH, pulmonary hypertension; BOS, bronchiolitis obliterans syndrome. ⁎ Corresponding author at: Research Center of Analytical Instrumentation, Sichuan University, 29 Wangjiang Road, Chengdu 610064, China. Tel./fax: +86 028 85418180. E-mail address: [email protected] (Y. Duan). 0009-8981/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2012.07.006

M. Zhou et al. / Clinica Chimica Acta 413 (2012) 1770–1780

1. Introduction Because of their potential application in pulmonary diagnostics, the analysis of biomarkers in exhaled breath has captured increased interest in clinical practice [1]. As can be expected, the most common and well-known use of breath analysis is the forensic assessment of alcohol consumption. Other potential applications do exist including measurement of the fragment of exhaled nitric oxide (FENO) in breath for asthma. This technique, standardized by the American Thoracic and European Respiratory Societies [2], is noninvasive, simple to perform and highly reproducible. Breath analysis is well suited for testing of physiologic or pathophysiologic conditions especially in patients who monitor daily parameters such as glucose and urea [3,4]. Exhaled breath consists of two components. The first 150 mL is “dead-space” air from the upper airway in which there is no gaseous exchange between blood and air occurs. The remaining 350 mL, known as “alveolar” breath, comes from the lungs, where gaseous exchange between blood and air occurs [5]. Breath is primarily a mixture of nitrogen, oxygen, carbon dioxide, water, and inert gases. The remaining fraction of breath contains more than one hundred volatile organic compounds (VOC) in the parts-per-million (ppm) to parts-per-trillion (ppt) concentration range [3,6–8]. Matrix elements in breath vary qualitatively and quantitatively between individuals especially for VOC [3,6–8]. Among VOC detected in breath, only a small number are common including isoprene, acetone, ethane, and methanol [8,9]. Because these molecules represent products of core metabolism, their measurement can potentially provide important diagnostic information. Other endogenously produced inorganic gases are commonly found in breath including nitric oxide (NO), carbon monoxide (CO) and carbon dioxide (CO2). A series of VOC such as ethane, pentane, acetone, isoprene, and other normally nonvolatile substances such as peroxynitrite, isoprostanes and cytokines can also be detected in breath condensate [3]. Alternatively, volatile substances may be exogenously introduced into the breath via inhalation. Within the breath, the presence of exogenous molecules, especially halogenated organic compounds, can indicate recent exposure to drugs or environmental pollutants [10]. For more than a decade, the main issue confronting this approach was the difficulty in the separation and identification of discrete breath components. CO2 is relatively easy to detect due to its relative amount (~ 5%) in total breath, whereas most other volatile compounds occur at substantially lower concentration (ppm or less) [5]. In 1971, Linus Pauling used gas chromatography (GC) to identify hundreds of breath VOC (ranging in size from C2 to greater than C20) present at picomolar concentration [11]. Issues related to separation and identification of breath components gradually diminished as analytical methods progressed in subsequent decades. The physiologic significance of these volatile substances and correlation of clinical state remains an important topic of interest and worthy of further elucidation [3]. Despite these advances, only a few markers, to date, have proven reliable in disease diagnosis. In pulmonary disease, inflammation appears to increase with disease progression and exacerbation. Although the target of large multicenter studies, it is likely that not all breath biomarkers will not be equal with respect to analytical precision, ease of use, clinical correlation and ability to assess therapeutic efficacy [12]. In addition, alternative biomarkers may be identified by genomic and proteomic analysis in the future. In this article, we review a selection of exhaled-breath markers that could potentially be used for diagnosis and monitoring various pulmonary diseases. 2. Common breath biomarkers 2.1. Nitric oxide Nitric oxide (NO) is a gaseous molecule produced by resident cells of the large and peripheral airways as well as the alveoli [13]. These

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include airway epithelial cells, airway and circulatory endothelial cells, and trafficking inflammatory cells. NO is generated by three NO synthase (NOS) isoenzymes: neuronal NOS (NOS1), endothelial NOS (NOS3 or eNOS), and inducible NOS (NOS2 or iNOS). Each isoenzyme plays different pathologic roles due to differential regulation and expression in the airways [14]. All NOS isoenzymes convert L-arginine to L-citrulline with a net yield of NO. Constitutive NOS (cNOS) isoenzymes contain both NOS1 and NOS3 and produce small amounts of NO (pmol range) which plays a local regulatory role. Activation of NOS1 and NOS3 depends on calcium compounds linked to the pro-inflammatory effect. NOS2 is not considered a constitutively expressed NOS and is independent of calcium compounds. However, large amounts of NO (nmol range) are produced by NOS2 [13] pro-inflammatory cytokines and bacterial product lipopolysaccharide (LPS) induction [15]. NOS2 may also be expressed in other cell types such as alveolar macrophages, eosinophils, and inflammatory cells [16]. Although corticosteroids suppressed NOS2 in rodent cells, they did not directly inhibit NOS2 expression in human airway epithelial cells [17]. NOS3 activity is common in endothelial cells of the bronchial and pulmonary circulation thus regulating vascular flow [18]. It is expressed in alveolar endothelial cells and airway epithelial cells in the respiratory tract and following stimulation by cytokines, endotoxins and LPS in vitro. NOS3 in alveolar endothelial cells may contribute to the production of peripheral NO [19]. Moreover, NOS3 may also play a role in reducing plasma exudation in the airway [20] and in regulating ciliary beating, therefore, making it a relevant factor in mucociliary clearance [14]. NOS are not the only source of NO in exhaled gas. NO reacts with thiol-containing molecules such as cysteine and glutathione to form S-nitrosoproteins and S-nitrosothiols [21]. Approximately 70–90% of exhaled NO is released by S-nitrosothiols providing a major source of NO for tissues [22]. Exhaled NO may also be derived from nitrate protonation to form nitrous acid, which releases NO gas during acidification [23]. The paranasal sinuses also produce high levels of NO [24], as there is a dense innervation with NOS1 immunoreactive nerve fibers located around nasal blood vessels [25]. Additionally, exhaled NO could also be derived from the vasculature. Vasculature-derived NO, however, is a minor source of NO in nasal mucosa [26]. There appears to be constitutive expression of NOS2 and the transcription factor NF-κB in nasal mucosa [27]. Chemiluminescence and electrochemical analyzers measure NO breath concentration. Inexpensive and portable analyzers for exhaled NO assessment are also available and used with increased frequency. The American Thoracic Society and the European Respiratory Society have jointly published recommended procedures for the measurement of both exhaled and nasal NO to standardize analysis and allow for result comparison from various research centers and groups [28]. Standardization remains a challenge because a number of demographic, anthropometric and biological factors cause variations in FENO. For example, smoking reduces FENO in the short- and long-term periods [29,30]. Cessation of smoking increases FENO [31]. In the absence of smoking, both eNOS and iNOS have regulatory effects on FENO [32]. Increased FENO was associated with increased L-arginine, NO donor drugs such as nitrates and bronchodilators such as β-agonists [33–36]. Decreased levels are typically associated with inhalation or oral consumption of corticosteroids and/or bronchoconstriction [32,37–39]. 2.2. Carbon monoxide There are three major sources of CO in exhaled air: enzymatic degradation of heme, non‐heme-related release (such as lipid peroxidation, xenobiotics, and bacteria), and exogenous CO [40,41]. Approximately 85% of CO in the body comes from the degradation of hemoglobin via heme oxygenase (HO). The remaining 15% is a result of degradation of myoglobin, guanylyl cyclase and cytochromes [42]. Approximately

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85% of the CO in the body is bound to hemoglobin in circulating erythrocytes and the remaining 15% is bound to other compounds (such as myoglobin) or present in tissues; less than 1% of all CO in the body is unbound and dissolved in body fluid [43]. Because exhaled CO is predominantly derived from heme degradation [44], the liver is considered the major organ for CO production, followed by the spleen, brain, and erythropoietic system [45]. Endogenous CO comes from the oxidative degradation of heme proteins by HO which acts as a defense mechanism against oxidative stress. HO activity is primarily induced by reactive oxygen species (ROS) [46]. HO exists in three isoforms: HO-1, HO-2, and HO-3. HO-2 is constitutively expressed in most tissues, while HO-1 is the only inducible form and HO-3 is detected in rats [47]. Increased endogenous CO production by means of HO-1 activity can be induced by oxidative stress, hypoxia, heavy metals, sodium arsenite, heme and heme derivatives, various cytokines, and even exogenous CO [48]. Research also suggest that upper respiratory tract viral infections may induce HO-1 activity, resulting in increased exhaled CO among both adults [49] and children [50]. CO produced in nose and paranasal sinus may contribute to exhaled CO concentrations [51]. Both superoxide anions and peroxynitrite can stimulate HO-1 activation, with the subsequent production of CO serving as an important negative-feedback regulatory mechanism that limits the release of these cytotoxic substances [52]. While HO-1 activation can be diminished by the presence of N-acetylcysteine, which is also a precursor of glutathione with antioxidant properties [53]. HO-1 and HO-2 are active in human airways and are found in most types of cells, with particularly strong immunofluorescence in airway epithelial cells [54]. Therefore, elevated exhaled CO levels might be an early indication of an acute infective episode, which may lead to exacerbation of asthma and COPD. A number of techniques can detect CO. Most employ electrochemical CO sensors that are both sensitive and inexpensive while providing reproducible results. However, these instruments are susceptible to interference from a large number of other substances. Hence, adjustable laser spectrophotometers [55] and/or near-infrared CO analyzers [56] have been used to detect exhaled CO at ppb level. Near-infrared instruments, commonly used to monitor atmospheric CO, have proven to be both sensitive and stable. Nonetheless, the effectiveness of a near-IR analyzer is limited due to its relatively large size, sensitivity to water and CO2, and need for large sample volume [57]. A method for measuring CO using nasally sampled exhaled air in non-cooperative neonates has been developed. By placing a small and relatively noninvasive catheter into the posterior of the nasopharynx, exhaled air samples can be collected, either manually or automatically [44]. 2.3. Hydrocarbons Breath methylated hydrocarbons have been described as lipid peroxidation markers [58,59]. Lipid peroxidation produces ethane and pentane as a result of a ROS chain reaction. The process is initiated by ROS removing an allylic hydrogen atom from unsaturated hydrocarbons, such as ω−3 and ω−6 fatty acids, i.e., the basic components of cell membranes [7]. Radicals are conjugated, peroxidized via oxygen and underwent subsequent reaction. Along this reaction pathway, aldehydes such as malondialdehyde (MDA) are generated [3]. Aghdassi et al. [60] have demonstrated that exhaled pentane and ethane correlated well with lipid peroxidation markers such as MDA, thiobarbituric acid reactive substances (TBARs) and glutathione. Other studies have shown that pentane is also a product of oxygen free radical (OFR)-mediated lipid peroxidation of ω−6 polyunsaturated fatty acids and is subsequently degraded by cytochrome P450 enzymes [61]. Increased ethane and pentane have been measured in the breath of patients with asthma, COPD, acute respiratory distress syndrome (ARDS), obstructive sleep apnea, and pneumonia [62–66]. Treatment with vitamins E and C as well as a low-fat, high-vegetable diet significantly decreased exhaled ethane and pentane. Smoking, on the other hand, is associated with the opposite effect:

increased exhaled ethane and pentane. This finding may result from a high concentration of hydrocarbons in cigarette smoke and oxidative damage caused by smoking [67]. Hydrocarbons produced from lipid peroxidation are stable and have low solubility in blood. However, they can easily mix with exhaled breath air within a few minutes of their formation. Therefore, exhaled ethane and pentane can be used to monitor oxidative stress in the body. Mendis et al. [68] have demonstrated that exhaled isoprene may be related to oxidative damage in the fluid lining of the lungs. Clinical studies have suggested that isoprene might serve as a marker of cellular damage and repair. Isoprene is significantly lower in ARDS [66]. Because abundant isoprene is produced following the mevalonic pathway of cholesterol biosynthesis, decreased concentrations in ARDS patients may be due to impairment of the membrane repair mechanism of alveolar cells. A similar explanation could account for decreased isoprene in chronic heart failure [69] and cystic fibrosis [70]. Contrary to what is expected, Senthilmohan et al. [71] reported that breath isoprene increased after smoking. 2.4. Hydrogen peroxide Hydrogen peroxide (H2O2) is a molecule detected in exhaled breath condensate (EBC). Various studies have reported that increased breath H2O2 is related to a variety of pulmonary diseases, making it a potentially non-invasive marker of airway inflammation and oxidative stress. H2O2 is generated via non-enzymatic and enzymatic dismutation of superoxides in the upper and lower airways. Enzymes including xanthine oxidase, cytochrome P450 (reduces/ converts O2 into H2O2), and flavoenzymes act as oxidases by using reduced NADPH or other reduced cofactors [72]. In healthy individuals, H2O2 production is an ongoing oxygen reduction process that occurs due to electron transport in mitochondrial respiration. Mitochondrial electron transfer is either spontaneous or catalyzed by cytosolic superoxide dismutase in the rapid reaction of superoxide anion with water-derived protons (2·O2− + 2 H+ → H2O2 + O2). Furthermore, induced H2O2 production is a result of increased activity of phagocytic NADPH oxidase in polymorphonuclear leukocytes, monocytes, macrophages, B lymphocyte and endothelial cells [73]. Conversely, degradation of H2O2 occurs at several cellular locations due to catalases and peroxidases. Catalases assist in conversion of H2O2 to water via the reaction: H2O2 + H2O2 → 2H2O + O2. Peroxidases, on the other hand, are capable of rapidly binding H2O2 to heme enzymes present in bacteria. In turn, a reduced form of H2O2 can be used to catalyze a number of oxidative reactions. Because of the lability of superoxides, direct detection is often difficult [74]. H2O2, however, is volatile, more stable, and enters the gas phase at physiological temperatures. Therefore, it can be measured by fluorometric assays, which utilize the oxidation of a substrate to a fluorescing compound by H2O2. Using this method, Antczak et al. [75] found increased EBC H2O2 in asthma, COPD, ARDS, CF, and lung cancer and in the EBC of healthy smokers. EBC H2O2 concentration was increased further when asthma and COPD were exacerbated. In COPD, H2O2 decreased after steroid treatment and long-term treatment with N-acetylcysteine. This finding may indicate that major sources of exhaled H2O2 are due to inflammatory cells (neutrophils and macrophages) in peripheral blood and airways [76]. Levels of inflammatory cytokines, activation of phagocyte, and endothelial NADPH oxidase are directly associated with exhaled H2O2 concentration. These species, in combination with the influence of age and gender, may contribute to the large inter-individual variability of exhaled H2O2 [77]. 2.5. Ancillary breath markers A volatile marker not found in the exhaled breath of healthy individuals is carbon disulfide (CS2). CS2 appears to be generated during

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methionine metabolism and could act as a sensitive marker for organ rejection after a lung transplant [78]. Increased breath gases such as sulfur-containing compounds have been reported in liver transplant patients [78]. Ethanol has been of particular interest as a potential volatile breath marker. Increased ethanol is commonly found in the breath of subjects who recently ingested alcohol [3]. Because exhaled acetaldehyde results from oxidation of endogenous ethanol via intestinal flora [79], ethanol breath concentration is always higher than the corresponding acetaldehyde. EBC pH is currently considered a robust measure to determine breath acidification in patients with various inflammatory lung diseases. Abnormally decreased EBC pH has been reported in acute onset of asthma, COPD and CF, as well as in stable COPD, bronchiectasis and moderate asthma [80–82]. A number of EBC markers such as eicosanoids, products of lipid peroxidation, vasoactive amines, NO-related products, ammonia, electrolytes, hydrogen ions, proteins, and cytokines have been extensively researched [40]. 3. Application in lung disease diagnosis 3.1. Asthma Asthma is a heterogeneous chronic inflammatory disorder of the airways that produces airway hyperresponsiveness and reversible airway obstruction. Typical symptoms include wheezing, coughing and shortness of breath. The majority of patients with asthma suffer from mild to moderate forms of the disease and can well control respiratory issues with regular intake of low to medium doses of inhaled corticosteroids (ICS) [83]. Several mediators, including basic molecules such as purines, have been involved in the development and maintenance of airway inflammation due to asthma [84]. The FENO is an important and easily measured variable in exhaled breath. It is primarily related to eosinophilic airway inflammation as represented in blood, sputum, bronchoalveolar lavage (BAL) and mucosal esinophilia [85–88]. In patients with severe refractory asthma (SRA), FENO was found to be significantly higher vs patients with moderate asthma and healthy subjects [89]. Different FENO cutoff values could be used to identify individuals with predominant eosinophilia as well as neutrophilia [89]. FENO was found to decrease in patients with predominant neutrophilia, regardless of whether or not those patients had eosinophilia as well. Increased FENO was found in the exhaled breath of asthmatic individuals. It was further increased during acute attacks, but returned to normal following anti-inflammatory therapy. Although cNOS isoforms may influence exhaled NO, it appears directly related to severity due to increased NOS2 activity in the epithelial cells of the respiratory tract [13,16]. Exhaled NO may be used to identify subjects with atopic and non-atopic asthma. Both allergic and non-allergic asthma are associated with increased FENO, but only in subjects who never smoked [90]. Among adults, patients with atopic asthma have significantly increased FENO vs those with non-atopic asthma [91]. Similarly, FENO is higher in atopic children regardless of whether or not they have asthma [92]. Furthermore, FENO also increases in times of allergen exposure, such as during the late phase response to the allergen challenge [93], during grass pollen season [94], or during exposure to indoor allergens [95]. In asthmatic patients with both subclinical airway inflammation and increased FENO, early anti-inflammatory treatment may prevent subsequent development and further progression [63]. Likewise, exhaled NO can be used to monitor the effectiveness of anti-inflammatory treatment in asthma patients. Exhaled NO decreased significantly in response to corticosteroid inhalation (2–3 days), reaching maximum effectiveness 2–4 weeks of treatment [38]. Increased exhaled NO may be useful to distinguish asthma from other causes of chronic cough [96,97]. Dupont et al. [98] have

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shown that the measurement of FENO can differentiate healthy persons with or without respiratory symptoms and patients with asthma. This study obtained a 90% specificity and 95% positive predictive value when exhaled NO > 15 ppb was used as a cutoff value for asthma. In another study, exhaled NO at a cutoff level of 16 ppb had 90% specificity and >90% positive predictive value for asthma diagnosis. These findings suggest that exhaled NO can be used as a simple noninvasive tool for screening suspected asthma [99]. Increased exhaled CO has been reported in stable asthmatic patients treated with traditional ICS [100]. No difference in exhaled CO between normal and asthmatic subjects and the effect of inhaled steroids on exhaled CO in patients with mild asthma was notable [54]. Both HO-1 and HO-2 are extensively distributed in airways of normal and asthmatic subjects [54]. Thus, increased exhaled CO in stable asthma patients is likely due to preferential increase in HO-1 expression occurring in the alveolar macrophages of induced sputum [53]. The presence of increased bilirubin (a product of heme catabolism) in induced sputum suggested increased HO-1 activity. Inhalation of hemin, a substrate of HO, results in a significant increase in exhaled CO in normal and asthmatic subjects [53]. Episodes of acute asthma exacerbation increase exhaled CO as well, but can be reduced after treatment with oral corticosteroids [101]. Dramatically increased CO is found in patients with severe asthma, even those treated with 30 mg of oral steroid for 2 weeks [102]. Children with persistent asthma, despite steroid treatment (which reduces exhaled NO), have significantly increased exhaled CO vs children with infrequent episodic asthma [50]. As such, exhaled CO may be useful for noninvasive monitoring of asthma especially among children with chronic asthma. Studies have shown that ethane and pentane are the two hydrocarbons that have the greatest potential as asthma breath markers. Pentane has been reported to increase in patients during acute asthma attacks and declines to normal levels during recovery [103]. Exhaled ethane is increased in patients with mild steroid-naïve asthma vs steroid-treated patients and healthy subjects [62]. Increased H2O2 was detected in the EBC of adults and children with asthma [104–106]. In asthma, exhaled H2O2 was associated with the number of sputum eosinophils and airway hyperresponsiveness. Despite treatment with corticosteroids, which significantly reduced exhaled NO in patients with severely unstable asthma, H2O2 remained increased [104]. Steroid-naïve asthmatic children had over 5-times EBC H2O2 vs healthy control subjects [107]. In a study on adult asthmatics (clinically stable after a one month washout period using only short acting β-agonist on demand), H2O2 was more than 20-fold higher vs control subjects [105]. This may be related to the fact that neutrophils, which are prevalent in severe asthma [108], generate increased superoxide radicals and, in turn, H2O2 [109]. In one study from Robroeks et al., EBC H2O2 in asthmatic children was not increased vs healthy subjects [110]. This unexpected finding, however, may be due to differences in pharmacologic treatment. Asthmatic patients also exhale significantly higher TBAR which mimic increased H2O2 and indirectly reflects increased oxidative stress [105]. Recently, research has shown that CRP, H2O2 and nitrite/nitrate were significantly increased among asthmatic individuals vs healthy control groups [111]. In addition, H2O2 in EBC correlated significantly with nitrite/nitrate. EBC pH has been proposed as a promising and noninvasive tool in the assessment of patients with asthma due to its convenience, ease of use and assay performance [112]. pH has been found to be significantly increased in mildly persistent asthma. There is evidence that EBC pH correlates significantly with eosinophil number obtained from induced sputum of patients with moderate asthma [80]. Among individuals with severe and non-severe asthma, EBC pH was lower than that of control subjects [113]. Moreover, EBC pH was significantly lower in patients with SRA than in patients with moderate asthma and healthy subjects [89]. Previously published data on pH evaluation support decreased pH in patients with moderate asthma vs patients with mild asthma [80]. This result could be attributed to

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the reportedly close relationship between airway acidification and oxidative stress. Additionally, EBC pH variability in asthma is not influenced by changes in clinical status. Using a multiple linear regression model, it was determined that low EBC pH corresponded to high BAL neutrophil counts and allergy-related symptoms [113]. This finding suggests that pH is less dependent on steroid treatment and as such more reliable than exhaled NO [89]. Research from Zietkowski et al. [114] has shown that highsensitivity C-reactive protein (hs-CRP) in EBC and serum of asthmatic patients were significantly increased relative to healthy volunteers. In patients with unstable asthma, hs-CRP was significantly increased relative to steroid-naïve and ICS-treated patients with stable asthma. This observation was confirmed by Qian et al. [115], in which the highest serum CRP was found in patients with severe and persistent asthma. Moreover, there was a significant correlation between hs-CRP and exhaled NO in EBC and serum of patients with steroidnaïve, mild asthma and unstable, severe asthma [114]. It is worth noting that hs-CRP combined with exhaled NO, could better monitor the progression of asthma. Endothelins are a family of peptide mediators that have a number of biologic properties such as pro-inflammatory, pro-fibrotic, bronchoand vasoconstrictive influences in the human airway. Zietkowski et al. [116] demonstrated that endothelin-1 (ET-1) was significantly increased in patients with unstable asthma vs subjects with stable disease. Additionally, a significant correlation exists between ET-1 and FENO in asthmatic subjects as well as ET-1 and blood eosinophil count in patients with unstable asthma. As such, measurement of EBC ET-1 may provide another useful diagnostic tool for detecting and monitoring inflammation in asthma. Breath cysteinyl leukotrienes (cysLTs) are significantly increased in asthmatic children vs healthy subjects. The diagnostic accuracy of EBC cysLTs was 73.6% for asthma overall and improved to 78.2% for steroid-naïve asthmatic children [117]. As such, EBC cysLT can be used as an inflammatory marker to distinguish between asthmatic and healthy children. 3.2. Chronic obstructive pulmonary disease Chronic obstructive pulmonary disease (COPD) is an inflammatory disease of both the large and small airways as well as the alveoli characterized by progressive airflow obstruction and chronic inflammation [118]. Disease inflammation is predominantly mediated by cytokines and interleukins via neutrophilic pathways [119]. One study reported increased pro-inflammatory cytokines, including IL-1β, IL-6 and TNF-α, during COPD exacerbation. Unfortunately, these results was not been reproduced [120]. Measuring various biomarkers in the breath is a very attractive approach to monitoring COPD inflammation due to its noninvasive nature, simplicity and ease of use for repeat sampling [40,121]. Increased exhaled NO in COPD was correlated with increased eosinophil counts, increased bronchodilator response and increased steroid responsiveness. However, some studies reported an increase in these values among patients with stable COPD [122–125], while others reported unchanged or even reduced values [126]. These conflicting results can likely be explained by superoxide anions, produced at times of oxidative stress, combining with NO to form highly reactive peroxynitrite. The unstable anionic isomer can take the form of nitrate (NO3−), resulting in increasing EBC nitrate levels in COPD patients [127]. Moreover, nitrate production may also be responsible for increased tyrosine nitration in the peripheral lungs (confirmed by immunocytochemistry) [128]. This also explains why FENO is reduced in normal smokers compared to non-smokers [29]. However, FENO in patients with stable COPD is lower than FENO in both smoking and nonsmoking asthma subjects [29,129,130]. Moreover, patients with unstable COPD have higher NO concentrations than smokers or ex-smokers with stable COPD [131]. This could be a result of increased neutrophilic inflammation and oxidant/antioxidant imbalance [97].

While selective NOS2 inhibitors can reduce the concentration of exhaled NO in both asthmatic patients and normal subjects, they are less effective for COPD patients. This is because the formation of peroxynitrite removes NO from the gaseous phase, thus reducing its concentration in the airways at times of high oxidative stress [132,133]. Recent studies have looked at FENO measurements taken at different flow rates in order to discriminate between airway-derived NO, which is flow-independent, and peripheral NO, which stems from NOS1 and NOS2 activity in the alveoli and possibly the small airways. This means that even when airway NO levels are low or normal in COPD patients, there is an increase in peripheral NO that correlates with disease severity [124]. Brindicci et al. [124] found a significant correlation between alveolar NO (CalvNO) and both FEV1 levels and the Tiffeneau index (FEV1/FVC ratio), thus indicating that CalvNO in COPD patients may reflect peripheral inflammation and remodeling. Increased peripheral NO may be a result of increased NOS1 and NOS2 activity in COPD patients. Some studies have also revealed increased NOS2 expression presented in the peripheral lung and small airways of patients [19,128]. NOS3 activity, however, is reduced in the peripheral lung of patients with COPD, especially in severe forms of the disease since emphysema results in alveolar wall damage [19]. Decreased NOS3 activity also reflects an increase in inducible NO synthase in the lung periphery of COPD patients [134]. Although peripheral NO may prove to be a useful non-invasive biomarker of COPD inflammation, further studies testing on reproducibility and treatment effects are required. Only then a relationship between exhaled NO and disease severity can be established. Additionally, research from Olin et al. [135] have found that FENO value was significantly lower in current smokers and COPD patients. The results also indicate that the levels of exhaled NO reflect inflammatory changes in the peripheral airways for both non-smoking and smoking subjects. Increased levels of exhaled CO have been observed during acute exacerbation of COPD, with a decline after recovery [136]. Moreover, endogenous CO correlates well with COPD severity, and therefore, it is proposed as an indicator of oxidative stress processes in COPD patients [137,138]. However, there is a major limitation in using exhaled CO as a marker of COPD. While exhaled CO is elevated in patients with COPD, it is also elevated in normal smokers due to the high concentration of CO in cigarette smoke [63,139], mimicking the patterns of ongoing oxidative stress or inflammation. Moreover, cigarette smoke can also induce HO activity in the fibroblasts [49]. Due to a high variability in the sources of environmental CO and the effects of smoking on CO levels, further studies discerning endogenous and exogenous sources of CO are needed. Volatile hydrocarbons, such as ethane and pentane, have been detected in exhaled breath and are biomarkers of lipid peroxidation due to oxidative stress. Both pentane and ethane levels are increased in patients with COPD, while elevated ethane levels correlate with disease severity in patients with COPD [63]. However, measurements of ethane are generally made using an on-line GC–MS, limiting its potential application in clinical situations. Currently, smaller and more sensitive detectors for hydrocarbons are now under development. Some nonvolatile biomarkers such as hydrogen peroxide (H2O2) and 8-isoprostane can be detected in EBC. H2O2 is increased in EBC of COPD patients and is further increased during acute exacerbations of COPD [140], making it a reflective marker of COPD disease severity [141,142]. Exhaled H2O2 is reported reproducible over a period of 3 days [143]. In one study, COPD patients exhaled H2O2 10 times more than healthy subjects, but no difference was found when smoking status (current, former and non-smokers) was considered [144]. In one prospective trial performed to assess circadian and 21-day variability of EBC H2O2, mild stable COPD patients had H2O2 levels similar to that of control groups [145]. This finding supports a previous study claiming that increased H2O2 levels were found only in moderate and severe cases of COPD.

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8-Isoprostane is a stable marker of oxidative stress and increased in EBC of COPD patients. Concentrations of 8-isoprostane are greater in COPD patients than normal smokers and are reflective of disease severity [146–148], showing the largest increase during acute exacerbation [149]. Studies from Kostikas et al. [141] examined the effect of varying concentrations of EBC 8-isoprostane and H2O2 on the indices of inflammatory process and disease severity among COPD patients. The results showed an increase in both 8-isoprostane and H2O2 concentrations in the EBC of patients with COPD. However, H2O2 measurements seemed more reproducible and more sensitive to the indices of inflammatory process and disease severity. Dowak et al. [144] evaluated the mean concentrations of TBARs and H2O2 in the EBC of COPD subjects and found a 10 to 12 fold increase of both TBARs and H2O2 when compared to healthy control subjects. Moreover, TBARs levels had tendency to be higher in the EBC of smoking COPD subjects than in the EBC of former smokers and COPD subjects who had never smoked. These findings suggest an overload of oxidants and an increased amount of lipid peroxidation in the airways of COPD subjects. MDA, one of the most important volatile TBARs [144], is an end-product of polyunsaturated fatty acid peroxidation. Compared with normal smokers, MDA levels are typically elevated among COPD patients [150]. Cigarette smokers have increased number of H2O2 releasing macrophages and neutrophils in the lower airways [151]. Released H2O2 can be converted to hydroxyl radicals, which, in turn, cause peroxidation of the polyunsaturated fatty acids in cell membranes [152]. Moreover, increased nitrosative stress in COPD is indicated by increased concentrations of nitrite and nitrosothiols in EBC [153]. In patients with COPD or bronchiectasis, a lower EBC pH value is observed when compared with healthy individuals or patients with other airway conditions such asthma [80]. The decreased EBC pH was also significantly greater in patients colonized by Pseudomonas aeruginosa. Moreover, EBC tumor necrosis factor alpha (TNF-α) levels in COPD patients decreased after treatment with steroid and antibiotic therapy for acute exacerbations of COPD. These findings suggest a potential role for EBC TNF-α in non-invasive monitoring of disease activity. 3.3. Cystic fibrosis Cystic fibrosis (CF) is characterized by abnormal ion transport across the respiratory epithelium and chronic bacterial colonization within the lungs. Progressive destruction of the airways results in respiratory failure, increased airway mucus viscosity, chronic infection, and inflammation. Airway inflammation, moreover, plays a central role in the initiation and end stage respiratory failure of CF [154]. In CF, in spite of the intense inflammation in the airways, exhaled NO levels have been found to be the same or lower than those of control subjects [155,156]. Although one study has demonstrated that EBC nitrite levels are higher in patients with cystic fibrosis than in normal subjects, this was not the case with exhaled NO [157]. In CF patients, excess amounts of secretions and mucus in the airways may inhibit diffusion of gaseous NO into the airway lumen and increase the chance of NO interaction with the aqueous epithelial fluid lining. This interaction converts NO to nitrite and nitrate before it can be exhaled, thus explaining the increase in EBC nitrite levels with a decrease in EBC NO. Unfortunately, monitoring EBC nitrate levels has not proven to be useful in diagnosing mild cases of CF, especially in children [158]. The dramatic reduction of iNOS expression in airway and nasal epithelium of CF patients may hinder an important primary defense mechanism and increase airway susceptibility to bacterial infections [2]. Reduction of NO formation in CF is also associated with severe CF transmembrane regulator gene mutations, airway obstruction, pancreatic insufficiency, and Pseudomonas infection [159]. The expression of NOS2 is reduced in CF airway epithelial cells, which results in an increased risk in lower airway infection caused by P. aeruginosa [160]. L-arginine is the

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substrate of both NOS and arginase. In the sputum of CF patients, arginase activity increases, thus reducing the availability of L-arginine for NO synthesis in the airways. Although antibiotic treatment that improves pulmonary function in CF patients also increases FENO [161], individual responses to the treatment are highly varied. Moreover, the relationship between FENO and pulmonary function is not strong enough to validate FENO as a marker of CF. There is a tendency for both exhaled and nasal NO to be higher in patients who are not homozygous for the ΔF508 CF transmembrane regulator mutation [162], however, there is no strong association between exhaled NO and disease severity (for either CF [162] or infections due to Pseudomonas) [163]. In contrast to NO, exhaled CO levels were markedly elevated in patients with stable CF [164,165]. Moreover, CO levels also tend to reflect disease severity, further increasing during CF exacerbation and declining after antibacterial treatment [162]. Sergei et al. [164] found that patients who are homozygous for the CF transmembrane regulator ΔF508 mutation have higher exhaled CO levels than heterozygous patients. Gene therapy has gained great interest as a possible treatment for cystic fibrosis. However, further studies are needed to investigate the role of CO levels in effective therapeutic gene delivery or to diagnosis patients with borderline sweat test result (where an alternative genetic analysis is not available). Patients with CF have elevated levels of exhaled ethane, which correlates significantly with exhaled CO levels and airway obstruction [165], indicating that oxidative stress and lipid peroxidation increase in the airways of patients with CF. The primary reactive oxygen metabolites formed in vivo are superoxide and H2O2 [154]. EBC H2O2 is considered a marker of oxidative stress in lung diseases such as CF [75]. A study from Ho et al. shows that the concentration of H2O2 in breath condensate of CF patients was lower than that of normal subjects [154]. A phenomenon with several possible explanations: firstly, it is possible that H2O2 is still generated in regular amounts among CF patients but is not detected because of rapid reaction with other reactive species such as superoxide. Alternatively, large amounts of airway secretions may prevent H2O2 from diffusing into the airway lumen. Lastly, increased levels of antioxidants such as catalase, glutathione, or myeloperoxidase may be present in the airways of CF patients and neutralize H2O2. For example, myeloperoxidase generates hypohalous acids in the presence of H2O2 and halides, thus effectively preventing H2O2 exhalation. Compared with control subjects, 8-isoprostane levels were higher in the EBC of stable CF patients. Furthermore, unstable CF patients had higher exhaled 8-isoprostane levels than stable CF patients. Exhaled prostaglandin E2 (PGE2) was also elevated in the EBC of stable and, to an extent, unstable CF patients. In patients with CF, exhaled 8-isoprostane and PGE2 could be useful markers of disease severity. Lastly, 2-aminoacetophenone and hydrogen cyanide are both promising breath biomarkers for detecting P. aeruginosa lung infections in CF patients [166,167]. 3.4. Lung cancer Levels of nitrite in epithelial lining fluid and exhaled NO are significantly higher in patients with lung cancer than in control subjects, and correlate with the amount of NOS2 expression in alveolar macrophages [168]. While increased nitrite levels are directly associated with cancer, increased NO production is not specifically related to tumor activity and might be attributed to a tumor-associated nonspecific immunological and inflammatory mechanism. An empirical study of primary lung cancer from Phillips et al. identified a set of 22 VOCs in breath as lung cancer biomarkers. A mathematical model employing these VOCs (principally composed of alkanes, alkane derivatives and benzene derivatives) was sensitive and specific for primary lung cancer, and was, moreover, equally accurate in early and advanced stage disease detection [169]. An additional study utilized

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the same mathematical model but incorporated a set of oxidative stress breath markers rather than a defined set of 22 VOCs. These markers, nine alkanes and methylated alkanes, proved to be just as sensitive, specific, and accurate as the previously identified 22 VOCs for detection of lung cancer at all TNM (tumor, node, metastasis) stages [170]. A logic model using breath biomarkers, primarily alkane derivatives, could detect primary lung cancer with 84.6% sensitivity, 80.0% specificity, and 0.88 area under curve of the receiver operating characteristic curve [171]. Moreover, the abundance of most of these VOCs was decreased in patients with lung cancer, and the predictive accuracy of the breath test was similar in all TNM stages of disease. Results from both mathematical and logic models suggest that VOC products of oxidative stress may have undergone accelerated catabolism due to cytochrome P450 mixed oxidases during the pathogenesis of lung cancer [172,173]. Interleukin-6 (IL-6) levels are reportedly elevated in EBC of non-small-cell lung cancer patients compared with control subjects, and, moreover, correlate significantly with disease severity [174]. IL-2, TNF-α and leptin levels were also significantly elevated in the EBC of non-small-cell lung cancer patients compared to those of control groups [175]. Much like IL-6, the levels of these three markers also correlated with stages (Stages I–III) of lung cancer [177]. 3.5. Primary ciliary dyskinesia Primary ciliary dyskinesia (PCD), also known as Kartagener syndrome, is an autosomal recessive condition in which abnormal or absent beating of cilia results in poor mucociliary clearance. Studies have consistently revealed low levels of exhaled bronchial NO in PCD compared to healthy control subjects. Moreover, decreased EBC NO levels can differential PCD patients from individuals with forms of non-PCD bronchiectasis who have elevated exhaled bronchial NO levels. Low NO levels among individuals with PCD are primarily attributed to reduced bronchial iNOS activity rather than alveolar eNOS activity [176,177]. Because NO plays an important role in bactericidal activity and ciliary beating in the lungs, a lack of endogenous NO production might contribute to chronic chest infections in patients with PCD. Both exhaled and nasal NO are considerably reduced in subjects with PCD compared with healthy subjects as well as patients with other lung diseases (e.g. non-PCD bronchiectasis or cystic fibrosis) [176,178]. Because reduced NO levels may relate to mucociliary dysfunction [179], measurements of exhaled NO might be a useful method for detecting PCD among patients with recurring chest infections. 3.6. Pulmonary arterial hypertension Pulmonary arterial hypertension (PAH) is a hemodynamic state characterized by elevation of pulmonary arterial pressure and is associated with increased pulmonary vascular resistance, leading to deterioration in cardiopulmonary function and premature death [180]. PAH is commonly caused by, or associated with, an underlying pulmonary or systemic disease. Elevated pulmonary vascular resistance seems to result from an imbalance between locally produced vasodilators and vasoconstrictors, in addition to cellular proliferation and vascular remodeling [181]. NO produced by the airway epithelium or the vascular endothelium freely diffuses into pulmonary vascular smooth muscle cells to activate soluble guanylate cyclase. Guanylate cyclase yields guanosine 3,5-cyclic mono-phosphate, a molecule that causes vascular smooth muscle relaxation and vasodilation. Interestingly, patients with PAH have reduced airway wall concentrations of NO [182–184]. Moreover, compared with normal subjects, individuals with PAH also have lower NO reaction product concentrations in bronchoalveolar lavage (BAL) fluid, which is inversely related to the degree of pulmonary hypertension [185]. Nonetheless, successful therapy of PAH is associated with increased NO levels. The low FENO levels in patients with PAH and the improvement

following effective therapeutic intervention suggest that serial monitoring of NO may be useful. Among patients with various pulmonary diseases, the level of FENO in PAH patients was significantly lower than that of non-PAH individuals and control subjects. Conversely, the levels of ET-1 was higher in patients with PAH, suggesting that pulmonary hypertension was a consequence of an output imbalance between the pulmonary epithelium and endothelium [186]. In patients with primary pulmonary hypertension (PPH), biochemical reaction products of NO are inversely correlated with both pulmonary artery pressure and number of years since disease diagnosis [182]. This may explain the reportedly reduced level of NOS3 activity in patients with PPH [187,188]. 3.7. Interstitial lung disease Patients with cryptogenic fibrosing alveolitis and systemic sclerosis due to lung fibrosis were reported to have increased exhaled NO levels compared to normal, non-smoking subjects and patients on corticosteroids [189]. Moreover, the concentration of EBC NO also correlated with active BAL fluid levels [189]. Increased exhaled NO levels in systemic sclerosis patients is a result of reduced NO diffusion capacity in the alveoli, hence lowering the alveolar NO concentration. In this case, airway NO levels remain unaffected. The increase is also inversely related to the total lung capacity and directly related to the computerized tomogram (CT) fibrosis scan score. Alveolar concentration, therefore, can be used to non-invasively assess the extent of interstitial lung damage in patients with systemic sclerosis [190,191]. However, systemic sclerosis patients who have developed pulmonary hypertension (PH) exhibit reduced exhaled NO compared to both normal subjects and patients with non-PH interstitial lung disease [192,193]. This particular effect may be explained by reduced NOS3 activity in either the pulmonary vessels or the pulmonary vascular endothelial surface. There is an up-regulation of NOS2 activity, induced by cytokines such as TNF-α and interferon-γ, in the respiratory epithelium and granulomata of patients with sarcoidosis [194]. One study from Wilsher et al. have shown that levels of exhaled NO are not increased in patients with active pulmonary sarcoidosis and that no relationship exists between levels of exhaled NO and the extent of lung fibrosis (as measured by CT scan) [195]. These findings also support previous claims from smaller studies conducted by Odonnell et al. [196]. Studies from Ziora et al. showed an increase in exhaled NO levels among pulmonary sarcoidosis patients but no relationship to disease severity [197], while another small study showed an increase in exhaled NO levels that declined with sarcoidosis treatment [194]. Research from Paredi et al. reveals elevated levels of exhaled CO in patients with fibrosing alveolitis that correlate with interstitial lung disease activity (determined by BAL cell count) [189]. While exhaled NO levels may have limited uses in assessing interstitial lung diseases, exhaled CO could be a viable marker for monitoring disease progression and therapeutic response. 3.8. Lung transplant rejection In lung transplant patients, early detection of infection, bronchiolitis obliterans syndrome (BOS), and organ rejection is important for successful treatment and recovery. Fisher et al. [198] have shown that exhaled NO increases in lung transplant recipients with lymphocytic bronchiolitis, an early precursor of bronchiolitis obliterans. In addition, lung transplant recipients with pulmonary or upper airway infections had significantly elevated NO levels (compared to control subjects) that fell with treatment [199]. BOS is the greatest long-term threat to lung transplant patients. A variety of other factors such as early and late acute organ rejection, cytomegalovirus infection and airway ischemia affect the development of BOS. Early detection, however, can greatly reduce the risk of this long-term threat and improve survival rates.

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Intense airway inflammation and exhaled NO are higher than that in either control subjects or stable lung transplant recipients. In stable lung transplant recipients, exhaled NO concentrations are highly dependent on the severity of BAL neutrophilia and the intensity of NOS2 activity in the bronchial epithelium (but not in the subepithelial area) [200]. Research from Verleden et al. [201] have shown that exhaled NO measurements can accurately detect early BOS. Consistently measuring and monitoring exhaled NO levels has proven to be valuable in both BOS detection and deterioration [202,203]. Furthermore, exhaled NO has also been shown to correlate with the degree of acute lung allograft rejection, independent of other complications such as infection or BOS [204]. Because exhaled NO measurements may have a role in the early detection of both obliterative bronchiolitis and acute organ rejection, an additional marker is required to discern one condition from the other. In lung transplant recipients, Studer et al. [78] have found increased concentrations of exhaled carbonyl sulfide in patients with acute organ rejection compared to stable patients. Hence, carbonyl sulfide may also be a useful non-invasive marker for monitoring pulmonary allograft dysfunction. 4. Conclusions Breath analysis has gained considerable attention in both scientific and clinical studies. The breath test is noninvasive, easily repeatable, and is not associated with the discomfort or embarrassment that typically comes with blood and/or urine tests. Moreover, it is also an inexpensive way to rapidly screen for certain diseases. Exhaled breath content contains various biomarkers of respiratory function that are not present in serum or urine. Breath analysis can dynamically monitor the decay of volatile toxic substances in the body. Before breath tests can be applied in clinical practice as a diagnostic tool, various obstacles need to be overcome [205]. Firstly, a more in-depth understanding of the relationship between diseases and breath biomarkers is required. In addition, a normalized set of procedures for sampling, preconcentration, analysis, and background corrections are currently lacking. Lastly, standardization of exhaled breath content data remains a difficult but necessary challenge that must be addressed before breath markers can be used in the clinical diagnostics. Although a few problems remain unanswered, the use of breath test for diagnosis of pulmonary diseases shows great promise as new developments and improvements arise. In the future, pulmonary biomarkers may be useful in predicting disease progression, indicating disease in stability, and in predicting response to current therapies and novel therapies, many of which are now in development. Acknowledgment The authors are grateful to the financial support from National Major Scientific Instruments and Equipments Development Special Funds (No. 2011YQ030113), National Recruitment Program of Global Experts (NRPGE), the Hundred Talents Program of Sichuan Province (HTPSP), and the Startup Funding of Sichuan University for setting up the Research Center of Analytical Instrumentation. References [1] Cao W, Duan Y. Breath analysis: potential for clinical diagnosis and exposure assessment. Clin Chem 2006;52:800-11. [2] Abba AA. Exhaled nitric oxide in diagnosis and management of respiratory diseases. Ann Thorac Med 2009;4:173-81. [3] Miekisch W, Schubert JK, Noeldge-Schomburg GFE. Diagnostic potential of breath analysis — focus on volatile organic compounds. Clin Chim Acta 2004;347:25-39. [4] Guilbault GG, Palleschi G, Lubrano G. Non-invasive biosensors in clinical analysis. Biosens Bioelectron 1995;10:379-92. [5] Phillips M. Breath tests in medicine. Sci Am 1992;267:74-9.

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