Dependence of exhaled breath composition on exogenous factors, smoking habits and exposure to air pollutants^*

Breath samples

A cohort of 115 candidates (68 non-smokers, 47 active-smokers) was recruited for this study. Demographic data including age, sex, and additionally health and smoking status are summarized in table 1. All individuals gave informed consent of participation. The candidates completed a questionnaire describing their current smoking status (active smokers, non-smokers, ex-smokers) and the time elapsed since last smoking.

All volunteers consumed food not shorter than 1 h before breath sampling. No special dietary regimes were applied, which might be considered as a limitation of the study. The last tobacco smoking was not shorter than 2 h before sampling. The health status of the candidates is not considered in the data interpretation. In particular, a detailed comparison of breath samples derived from lung cancer patients and healthy controls is beyond the scope of this work. The proportion of healthy individuals in both groups compared (i.e. smokers and non-smokers) is the same, being at the level of approximately 40% (see table 1 for details). The samples were collected at different daytimes independent of the time of meals and were processed within 6 h after sampling. The study was approved by the ethics committee of Innsbruck Medical University.

Urine samples

The urine collection was approved by the Ethics Commission of Innsbruck Medical University. Volunteers’ morning urine (after overnight fasting) was collected into 10 ml plastic urine monovettes (Sarstedt, Germany) immediately after urinating. Prior to the use, the monovettes were thoroughly rinsed with high-purity air at 60 °C for 4 h to remove contaminants, which could distort the sample integrity. Additional effort was made to minimize the storage time of the urine samples in the monovettes to 3 h. After collection, samples were transferred into 10 ml glass vials (9 ml of urine per vial) and frozen at −80 °C.

Creatinine level in urine samples was measured to normalize the VOCs concentrations. For this purpose, 0.1 ml of urine was mixed with 1 ml of elution buffer containing 2 g l⁻¹ EDTA to dissolve urinary sediments. For determination of creatinine in urine samples, high-performance liquid chromatography (HPLC; ProStar Pumpe Model 210; Varian Palo Alto, CA and UV detector Jasco UV 975; Jasco Germany) on reversed phase (C18, LiChroCart, Merck) with Sörensen phosphate buffer (e.g., 0.015 M, pH = 6.4) as eluent (flow rate = 1.0 ml min⁻¹) was applied. Creatinine concentrations were measured by the detection of its UV-absorption at 235 nm wavelength. The creatinine concentration ranged from 0.74 to 44.86 mmol l⁻¹ (average 15.38 mmol l⁻¹).

Ketones

Acetone appears in everyone’s breath and urine samples, being the most abundant VOC among all detected. In addition, 2-butanone and 2-pentanone could be detected in breath with high frequencies of 94% and 88%, respectively (non+ex smokers group), and they were present in all the examined urine samples, but also often in inspired air. According to our results, significantly higher levels of both 2-butanone and 2-pentanone were observed for exhaled breath as compared to indoor air (p <10⁻³ for both compounds in non-smokers). The obtained results are in accordance to already published data suggesting the degradation of fatty acids as a potential endogenous source of both 2-butanone and 2-pentanone [25]. Intriguingly, the same authors report lower exhaled levels of 2-butanone in breath than its inhaled air in the case of few individuals involved in their study [25]. Another discrepancy is that 2-butanone was found by Buszewski et al [15] more often in the exhaled air of smokers than non-smokers pointing its putative relation to smoking habit. This cannot be unambiguously confirmed by us, since no statistical significance was found for the difference between 2-butanone levels for active smokers and non-smoking controls (p = 0.2, Kruskal–Wallis test), although slightly higher peak areas of this substance were indeed detected for smokers. Levels of 2-pentanone remain for both smoker and non-smoker groups similar as for most of other ketones detected.

A common constituent in urine is 4-heptanone that is supposed to originate from β-oxidation of 2-ethylhexanoic acid [26], and accordingly, it was found in 100% of urine but also in 30% of breath samples. Among all ketones, the significant difference between active smokers and non+ex-smokers was found only for 3-hexanone (found exclusively in smokers) and 3-pentene-2-one (detected mostly in smokers); however, their occurrence in the breath of smoking subjects was only 17% (for both analytes). Additionally, these two compounds could be detected in urine with a high occurrence for both smoker and non+ex-smoker groups with a higher mean value for smokers (table 3).

Aldehydes

Among aldehydes, acetaldehyde was found at the highest levels in almost every breath, urine and room-air sample, while the second in respect to abundance and occurrence was benzaldehyde. Concerning relation to smoking habit, diverse volatile aldehydes, such as formaldehyde, acetaldehyde, propanal, acrolein, butanal and crotonaldehyde, are the constituents of cigarette smoke [27] and as such can attain the body by smoking. Correspondingly, we found higher levels of acetaldehyde, propanal, acrolein and crotonaldehyde (2-butenal) in exhaled breath of smokers. Butanal and formaldehyde show even lower levels for smokers than for non-smokers which can be explained with the occasionally higher amounts of these compounds in the respective inspired air. Importantly, the breath level for vast majority of detected aldehydes was significantly lower than in respective indoor air and the correlation between these inhaled and exhaled air samples was found for few aldehydes.

Alcohols

Apart from 1-propanol and 2-propanol, which are commonly used solvents in lab environment and constituents of disinfection agents ant paints [28], methanol and ethanol have shown a great occurrence in breath samples. Different studies have been investigated for monitoring of methanol and ethanol concentration in exhaled air [29–31]. Surprisingly, we found the same inspiratory and expiratory levels for ethanol and a more than 3 times higher value of mean peak area of methanol in exhaled breath compared to indoor air. The difference in the concentrations of methanol and ethanol between smokers and non-smokers is not significant (p = 0.71 for methanol and p = 0.91 for ethanol). The mean peak area of 1-propanol was not significantly different for smokers and non-smokers (p = 0.28), but as a constituent of disinfectants this compound is present at a higher level in indoor air (especially in hospital environment) than in exhaled air.

Acids and esters

Substantially higher occurrences of acetic acid and propionic acid in breath samples (51% and 26%, respectively, for non-smokers) than in indoor-air samples (less than 3% for both) might indicate the endogenous origin of these compounds. On the other hand, both acids show significantly higher breath levels for active-smokers.

Although esters, such as methyl acetate and ethyl acetate, are the ingredients of cigarette, their measured level in exhaled breath of active smokers was not significantly higher than those of non-smokers (p = 0.21 and p = 0.96, respectively). Among these two analytes, methyl acetate was found in nearly all breath samples measured but in only 40% of indoor-air samples for non-smokers and 26% for active smokers; see table 2 for details). Consequently, its level in expired air was significantly higher than in inspired air for both groups of subjects. Incidentally, methyl acetate is the compound which increases in concentration during effort [32]. Its appearance in exhaled breath is in agreement with previously published data showing the release of methyl acetate from human bronchial epithelial cells [9].

Furans

It is very intriguing that some of breath-VOCs related to smoking habit (table 3) are also very often found in the headspace of urine samples. This predominantly concerns furans that were observed in almost every urine sample, while only 13 among 50 of urine donors were smokers. Generally, apart from 3-methylfuran and 2-ethyl-5-methylfuran, all compounds of this class were found at nearly the same level in exhaled breath and indoor-air samples for non-smokers (figure 1) with no indication of their endogenous origin. Importantly, practically all furans were found at over fivefold higher level in the breath of smokers compared to non-smokers. Among them only furfural was detected at lower level in breath of smokers, but the exhaled amount of this compound seems to be strongly dependent on inspired level, regardless of smoking status (see table 2 for details). For most compounds of this class, comprised of furan, 2-methylfuran, 3-methylfuran, 2,4-dimethylfuran, 2,5-dimethylfuran, 2-ethyl-5-methylfuran and 2,3,5-trimethylfuran, the significant differences between the smoker and the non-smoker groups were found for both sort of samples investigated, i.e. breath-gas and urine headspace, clearly demonstrating a relation to the smoking habit.

Aromatics

Similarly to furans, most of aromatic hydrocarbons were observed in this study at a significantly higher level in smokers compared to non-smokers (except 1-butenylbenzene found in only two samples of active-smokers). Importantly, apart from toluene, all measured aromatics show lower levels in exhaled breath than indoor air for non-smokers, confirming that the intake of these substances is mostly related to smoking habit. Supporting this assumption, benzene, toluene and styrene were detected at significant higher mean levels in the smokers’ urine samples compared to non+ex-smokers (table 3).

Saturated hydrocarbons

Propane, n-butane and n-pentane were found in the breath with high occurrence of 87%, 99% and 90%, respectively (table 2). The first two are considered as the metabolic products of bacteria and from protein oxidation [33]; however, observed in this work, the expired level of n-butane was found to be smaller than inspired (in the groups of non-smokers). Among remaining straight-chained saturated hydrocarbons (ordered in diminishing occurrence), n-hexane was found in 100%, n-decane in 99%, n-undecane in 90%, n-octane in 85%, n-nonane in 81% and n-heptane in 65% of non-smokers. The later one should be considered as exogenous contaminant, since its occurrence in breath was limited to the cases when it was also found in corresponding inspired air. n-decane was found at nearly identical levels in expired and inspired air with no statistical difference (p = 0.40 and p = 0.66 for non-smokers and active smokers, respectively). Although n-decane and n-octane have been reported as smoking-related compounds by Buszewski et al [15], no straight-chained saturated hydrocarbons were found to be significantly related to smoking habit in our study (table 3). In turn, for several methylated (and one cyclic) saturated hydrocarbons, the significant difference between smoking and non-smoking individuals was observed.

The importance of careful consideration of smoking habit for application of breath analysis in the medical diagnosis can be exemplified with 2-methylpentane. This compound was indicated as a smoking-related compound in [15] with four times higher concentrations reported for smokers’ breath. Interestingly, the same authors suggest this compound, and its isomer 3-methylpentane, to be cancer related, detecting them both at higher levels in expired air of lung cancer patients compared to healthy controls [6]. Considering their other findings and the fact that lung cancer patients have richer history of smoking than healthy subjects, the link of 2-methylpentane to lung cancer might result from a hidden correlation. Nevertheless, no significant difference between breath level of smokers and non-smokers was found for 2-methylpentane in our experiments, whereby its distributions in both groups compared were almost equal (87% and 83%, respectively, p = 0.11).

Unsaturated hydrocarbons

The unsaturated hydrocarbons detected in the cohort of tested subjects were generally classified in three groups (except aromatics), i.e. alkenes, dienes and alkynes, while the most abundant unsaturated hydrocarbon detected in practically everyone’s exhaled air (100% occurrence for both smoker and non-smoker) is isoprene.

In the group of alkenes, propene and 3-octene were often detected in breath samples; however, their abundance was found to be similar to that in indoor air. A big number of other branched but also linear alkenes could be detected at a higher expired level for smokers than non-smokers, indicating an exogenous, clearly smoking-related nature of these compounds (figure 3). The strong dependence on smoking habits is particularly explicable for the group of dienes, which, apart from few isomers of pentadiene and hexadiene, were almost never found in the breath of non-smokers (table 3). Hence, an impressive separation of the smokers from non- and ex-smokers was achieved according to the measured expired alkenes and especially dienes (figure 4). Additionally, three alkynes were found to be significantly related to smoking behavior: propyne, 2-butyne, 1-buten-3-yne (p <10⁻⁴, p < 10⁻⁶, p < 0.001, respectively) while only propyne was detected in non-smokers (3% of tested population).

Sulfur compounds

Among volatile sulfur compounds, dimethylsulfide (DMS), methanthiol, methyl propyl sulfide and allyl methyl sulfide could be detected most often in human breath. The first two are common constituents also in urine.

The mean peak area of methanethiol for expiratory air was found at nearly the same level as for inspiratory air, while dimethylsulfide, methyl propyl sulfide, allyl methyl sulfide, 3-methylthiophene and 1-(methylthio)-1-propene show elevated mean exhaled breath values. Furthermore, allyl methyl sulfide and both isomers of 1-(methylthio)-1-propene could be detected solely in breath and not in indoor air, excluding the exposure to air pollutants from their potential sources in human’s breath. Apparently, these compounds might be metabolized in the body in consequence of consumption of specific vegetables, such as onion and garlic [34]. The only volatile sulfur compound for which a significantly higher breath level was found for active smokers is ethyl methyl sulfide (p <0.001) but the occurrence of this analytes was low even in the groups of smokers (17% of population).

Nitrogen-containing compounds

Among the nitrogen-containing compounds acetonitrile, N,N-dimethylformamide and N,N-diethylformamide were often in the breath samples. Acetonitrile could be detected also in approximately one-third of the urine samples. Numerous studies support the smoking-related origin of acetonitrile. It was detected both in the smoke of a lit cigarette [35] and in the breath of smokers [6, 15] as well as in the headspace of urine of smoking persons [36, 37]. Correspondingly, we also detected a significantly higher value of mean peak area in breath and in urine headspace for a smoker than that for a non-smoker (p <10⁻³ for breath and urine). Furthermore, a significantly higher level in smokers’ breath was also observed for 2-cyano-1-propene and N-methylpyrrole, while lower for (dimethylamino)-acetonitrile and 4-propanenitrile (figure 5). Nevertheless, the mean breath level of 4-propanenitrile was not significantly different than corresponding indoor air and simultaneously both were correlated. Hence, although p < 0.05 was found (Kruskal–Wallis test) this analyte should not be considered as a smoking-related VOC. In turn, N,N-dimethylformamide and N,N-diethylformamide are common organic solvents and known artifacts released from Tedlar bags [7, 38]. Thus, the existence of mentioned amides in exhaled breath is rather doubtful and detected levels are most probably related to background contaminants.

Endogenous compounds

Acetone is the most abundant substance in the exhaled air and is produced via spontaneous decarboxylation of acetoacetate deriving from either lipolysis or amino acid degradation [39]. Acetone is a well-studied substance [2] and was suggested as a marker for uncontrolled diabetes mellitus exhibiting an enhanced concentration in the case of the disease [40]. Besides, it has been linked to other diseases, such as heart failure or liver illnesses [41–43]. The second most thoroughly investigated breath-VOC is isoprene which is also found in everyone’s breath at high concentrations, estimated by Gelmont et al at 2–4 mg/day, corresponding to 30–70% of all hydrocarbons [44]. Although considerable environmental sources of isoprene are known, mainly petrochemical industry (manufacturing of synthetic rubbers) and natural production by plants, it is commonly considered that the main source of isoprene is an endogenous synthesis probably from mevalonic acid, a precursor in cholesterol biosynthesis [45]. Investigations on adult populations show age and gender dependence but no relation to the cholesterol level was found [46]. Further studies demonstrated that the breath isoprene level rapidly increases during moderate workload [32, 47–49] suggesting that muscles may release substantial amounts of this analyte [50], particularly during the physical activity. Moreover, a study on patients with chronic liver disease has shown elevated isoprene concentrations in their exhaled breath compared to the breath of healthy volunteers [43]. A significantly lower level of isoprene was found in exhaled air of patients with heart failure [51] and lung cancer [7, 18]. Recent research with patients suffering from muscle dystrophy demonstrates that muscle tissues can be expected to act as extrahepatic sites responsible for substantial production of isoprene [50]. These findings exemplify the complexity of breath analysis and show that the role of isoprene in the human body is not yet understood [52]. It should also be mentioned that the partition constant of isoprene between blood and alveolar air is not exactly identical in different volunteers [53,54]. Nevertheless, results of the mentioned studies suggest the importance of this compound also for future diagnostic purposes.

As mentioned, 2-pentanone is expected to be produced via degradation of fatty acids [25], which was confirmed with breath analysis of fasting individuals [55] for which positive alveolar gradients of 2-pentanone and 2-butanone were reported. Moreover, 2-pentanone was found to be released from human bronchial epithelial cells and human fibroblasts (both are non-transformed lung cells) as well as from A549 lung cancer cells [9]. On the other hand, 2-butanone was significantly taken up by CALU-1 lung cancer cells [10], while its release was not observed from any of the investigated lung cell lines. Apparently, 2-butanone was either catabolized by CALU-1 cells or, as an important intermediate product, used in further metabolic cycles. The detail biochemical pathways for both ketones in lung cells remain, however, largely unknown.

Another endogenous compound is acetaldehyde, which was detected in almost every breath and urine sample. The major endogenous source of this substance is the oxidation of ethanol via the enzyme alcohol dehydrogenase [56]. Other aldehydes, such as acrolein and methacrolein are considered as the end products of lipid peroxidation [57]. Similarly, breath alcohols, such as methanol and ethanol, are the well-known metabolic products arising partly through fermentation in the gut [58]. Endogenous methanol is also generated through the methyltransferase enzymatic system [59]. Acetic acid, and also 2,3-butanedione, 1-butanol, 2-propanol, acetaldehyde and ethanol, can be formed through the glycolytic and non-glycolytic fermentations of carbohydrates (oxidation of acetaldehyde by the enzyme aldehyde dehydrogenase), lactate converting fermentations but also the fermentations of amino acids (e.g. alanine, glycine and especially glutamate) [56, 60]. The production of propionic acid occurs in the gut and is influenced by diet and nature of bacteria populations residing there [61]. Esters can also be generated endogenously in processes related to enzymatic reactions of hydroxyl compounds (e.g. alcohols) and short- and intermediate-chain free fatty acids [62], which are created in high concentrations via lipolysis of triglycerides [63].

Recent studies have shown an elevated amount of n-pentane in exhaled air of persons with breast cancer [16], but also a substantially higher level of n-hexane for lung cancer patients [6]. One of the endogenous origin of pentane is peroxidation of fatty acids [64, 65] and, therefore, it serves as a good marker of the so-called oxidative stress [33]. Some saturated hydrocarbons have been linked to lung cancer, e.g., the elevated level of n-decane in exhaled breath of cancer patients (compared to healthy control) was reported by Poli et al [66]. n-decane was also detected in cancer cell cultures and as such was considered to be the characteristic marker for apoptosis and necrosis stages [67]. Methylated hydrocarbons are concerned also as products of lipid peroxidation and supposed to be markers of lung cancer and oxidative stress [68]; however, the origin of their formation have not yet been completely revealed. Particularly interesting is the case of 2-methylpentane, which was found to be released by NCIH2087 lung cancer cells (causing non-small cell lung cancer: Adenocarcinoma) [12] and not by healthy (non-transformed) lung cells: human bronchial epithelial primary cells and human fibroblasts [9]. This important observation explicates findings of other researchers reporting a significantly higher level of 2-methylpentane in the breath of patients suffering from non-small cell lung cancer [66].

Recently performed in vitro experiments demonstrate that diverse volatile sulfur-containing compounds (VSCs), inter alia methanethiol, dimethylsulfide, dimethyldisulfide or dimethyltrisulfide, are produced by pathogenic bacteria colonizing upper and lower airways and causing lung infection, such as pneumonia [8]. VSCs can be produced endogenously through the metabolism of the sulfur-containing amino acids (methionine and cysteine) in the transamination pathway [69]. According to Tangermann, methanethiol is produced via demethiolation of methionine catalyzed by the enzyme l-methionine γ-lyase and from methylation of hydrogen sulfide (H₂S) (product of cysteine metabolism) by the enzyme S-methyltransferase [70]. However, the importance of hydrogen disulfide methylation was questioned by Levitt’s group [71, 72]. There is an evidence that transamination is the principal pathway of methionine catabolism in bacteria, yielding the intermediate product 4-methylthio-2-ketobutyrate which may be directly reduced to methanethiol or may undergo decarboxylation to methional prior final reduction [73]. Recently, Troccaz et al suggest the generation of methanethiol from l-cystathionine via recombinant cystathionine b-lyase in Staphyloccoci heamolyticus [74]. Thiol S-methyltransferase also forms dimethylsulfide via the methylation of methyl mercaptane [70]. These processes can be considered as a detoxification mechanism, removing toxic sulfur species (H₂S and methanethiol) from tissues. Dimethylsulfide was also found to be the main cause of extra-oral halitosis [70].

Environmental pollutants

Aromatics play a key role as environmental contaminants in both indoor air and outdoor air. Over the last 20 years, numerous studies were focused on the analysis of benzene, toluene, ethylbenzene and xylene (BTEX)—exposure by measurement of blood, urine and exhaled air [75–77]. These compounds could be detected in almost all breath and urine samples. Benzene is still counted to be a component of petrochemical industry products including gasoline, so inhalation is the most common exposure route. However, it also rapidly penetrates the skin and can contaminate water and food causing additional dermal and ingestion exposure [78,79]. Toluene is used as a solvent in the paint industry what considerably increase the risk of exposure on this substance in the certain group of employees [28]. o-xylene and p-xylene are also environmental pollutants that could be measured in the exhaled air and in urine. However, our results in urine are either doubtful, because these compounds are released also by the SPME-fiber.

Very important for correct interpretation of breath analysis is careful consideration of aldehydes emission from floor materials [80], wood furniture and plasticizers [81], which results in the high background level of indoor air. Consequently, diverse aldehydes are detectable in inspired air at substantially higher levels than in exhaled breath for non-smokers. Benzaldehyde found with the second highest abundance among the aldehydes might derive from exogenous intake as it is released to the environment in emissions from combustion processes, such as gasoline and diesel engines, incinerators and wood burning to the environment [82], which is revealed in the higher inspiratory than the expiratory level. Other aldehydes detected often in exhaled air, such as acrolein and methacrolein, can also be found in the environment as plasticizers and aggregate in the water supply of some industrial plants. Besides this, disposal in the environment occurs when organic matters like plants and fuels, such as gasoline and oil, are burned. Aldehydes can be generated in the oxidative degradation process of e.g. constituents of linoleum such as oleic acid and linoleic acids both saturated and unsaturated [83, 84]. Supporting this, all aldehydes detected in our study apart from 2-methyl-2-butenal and 3-methyl-2-butenal were at a higher level in indoor air than in exhaled breath pointing out the possibly exogenous origin. A very prominent example of dependence of breath-aldehydes level on the amount in ambient air is propanal, detected in almost 100% of all samples measured in this study (p <10⁻³). This observation is in agreement with other works, where propanal was found to be emitted from the combustion of gasoline, diesel fuel and wood [85]. Similarly, alcohols, such as isomers of propanol, are commonly occurring compounds in the indoor environment as, e.g., disinfectants or solvents [86]; hence, their inspired level was found to be higher from expired.

Finally, exhaled volatile sulfur compounds may be related to environmental exposure of both natural sources (soils, marshes) and pollutants. It is known that several industrial processes produce carbon disulfide as a by-product, including coal blast furnaces and oil refining.

Food and beverages

A large number of VOCs of diverse chemical classes are present as flavors in food and beverages substantially contributing to metabolism within humans. Alcohols, such as ethanol, are commonly known food ingredients. Also ketones are omnipresent in beverages (beer, wine, rum, whisky, coffee, tea) and in numerous types of food, particularly in fruit, vegetables, cheese, milk, meat and bread [87–89]. Aldehydes might be used directly as a flavoring agent, such as benzaldehyde particularly for artificial cherry or almond flavors but also several other foods, like sausages, wines [90, 91]. Numerous VSCs present in breath gas are the natural ingredients of vegetables such as methyl propyl sulfide and especially allyl methyl sulfide constituting leek and garlic oils. In turn, methanethiol, dimethylsulfide and dimethyldisulfide are present in cheese, fish, meat, baked goods and beverages, such as coffee, wine, beer, milk [87]. Besides, furan derivates have long been known to occur in heat-treated foods, like in biscuits, bread crust or roasted coffee beans, contributing to their sensory properties [92]. In this respect, high occurrence of furans in urine headspace samples may not necessary reflect the smoking habit but also the consumption of beverages and foodstuff.

The limitations resulting from diet or exposure to environmental pollutants are the common problems accompanying breath analysis in general and are inevitable in the clinical study, regardless of its purpose and analytical method applied. Thereby, the elevated levels of aldehydes might reflect exposure to indoor-air artifacts instead of lung cancer, while high concentrations of breath-VSCs might be related to specific diet rather than bacterial lung infection or liver malfunction.

Smoking-related compounds

According to here-presented results, diverse unsaturated hydrocarbons are related to smoking habit, comprising 26 alkenes, 3 alkynes and 29 dienes (table 3). The values of specificity determined for single compounds being above 90% (with exemption of six substances with values >79%) enable successful classification of smoking individuals. Importantly, a cohort of 30 compounds (comprising 19 dienes) were never found in the exhaled air of non-smokers (specificity = 1) excluding all non-smokers from the classified smoker group.

While many of the unsaturated hydrocarbons are constituents of cigarette smoke (created mostly during combustion of tobacco), other can originate from oxidation of phospholipids in membrane [93] caused by reactive chemicals in the smoke of a cigarette.

Apart from unsaturated hydrocarbons, aromatic compounds also exhibit relation to smoking habit. The most important aromatics occurring with the highest peak area values in smoker’s breath are BTEX. Because of the toxicity and carcinogenicity of these compounds, their analysis is still a topic [94, 95]. Since aforementioned aromatic hydrocarbons are also present in the smoke of a cigarette, passive smokers are also exposed to these compounds [15]. BTEX were also found in urine headspace of smokers, which can testify that substances released from the tar covering the lungs enter the bloodstream and are (in small part) further removed from the organism via urinary tract. Although saturated hydrocarbons such as n-decane or 2-methylpentane were reported to be smoking-related breath VOCs by Buszewski et al [15], we did not find the statistically significant difference between their breath levels in smokers and non-smokers.

Several furans and VNCs were found at elevated levels in exhaled breath and urine samples, what is in agreement with the works of other researchers supporting the smoking-related origin of these VOCs. In particular, acetonitrile was detected in the smoke of a lit cigarette [35] and in the breath of smokers [6, 15] as well as in the headspace of urine of smokers [36, 37].