**6. Biomarkers of pulmonary diseases present in EBC**

The term biomarker herein refers to a measurable biomolecular factor applicable for the measurement of a disease progression or treatment-related biomolecular changes in the human organism. On a molecular scale, biomarker refers to "a subset of markers that might be discovered using metabolomics, proteomics, genomics and other -omics technologies or imaging technologies." Biomarkers play a major role in medicinal biology. Biomarkers may be foreseen as a promising tool in the near future due to their unique potential for early diagnoses, which obviously permit disease prevention, a drug target identification, a drug response monitoring, etc. The collection and analyses of substances present in EBC provide a simple, noninvasive, real-time, point-of-care clinical and research tool for the evaluation of lung pathophysiology.

LTs are derivatives of arachidonic acid that are synthetized *via* the 5-lipoxygenase pathway (**Figure 5**). The major problem in the determination of LTs in body matrices is their low stability due to their sensitivity toward oxidation. This explains challenging analytical determina-

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Lipoxins (LXs) function in our organism as "natural antiasthmatics" as they are the antiinflammatory mediators. Binding of LXs to their receptors also support the reconstructive

LXs and LTs are derivatives of arachidonic acid and they are generated in three different metabolic pathways [34] . The first one is enabled by acetylsalicylic acid (ASA, in aspirin induced

tion of the used assays and to a relatively high variability of the published data.

process that is initiated in lungs immediately after the asthma attack.

*6.1.2. Lipoxins*

**Figure 5.** Biosynthesis of leukotrienes.

**Figure 4.** Biomarkers generated from arachidonic acid.

Very significant role is played by some biomarkers that are produced from the arachidonic acid (some of them were already mentioned above). Arachidonic acid ((5Z,8Z,11Z,14Z)Eicosa-5,8,11,14-tetraenoic acid) is a polyunsaturated omega-6 fatty acid present in phospholipid cell membranes [11, 12]. The products of the metabolism of arachidonic acid are called eicosanoids. These molecules are characterized by the 20C chain. The production of eicosanoids is enabled by different enzymes (**Figure 4**), the only exception are isoprostanes which emerge through oxidation of arachidonic acid (non-enzymatic pathway).

#### **6.1. Arachidonic acid metabolites**

Arachidonic acid is a polyunsaturated fatty acid present in phospholipid bilayer. In human organism, arachidonic acid acts as a vasodilator or regulates inflammation as a key intermediate. There are several pathways which allow transformation of the arachidonic acid in a number of different metabolites (**Figure 4**). Among the most significant products of its metabolism can be classified leukotrienes, lipoxins, isoprostanes, and prostanoids [6, 33].

#### *6.1.1. Leukotrienes*

Leukotrienes (LTs) [6, 33] represent a group of biologically active molecules. LTs are produced by various tissue cells (e.g., leukocytes, macrophages, mastocytoma cells) as a response to both immunological and non-immunological stimuli. LTs are potent pro-inflammatory [33] mediators and their release is usually triggered by the organism coming in contact with an allergen. The interaction between LTs and their receptors can lead to a wide range of biological effects: leukocytes activation, bronchial smooth muscles contraction, vascular permeability stimulation and increased mucus production, etc. All of the described symptoms are typically connected not only to pathophysiology of bronchial obstruction, especially to asthma, but also to other lung inflammatory disorders.

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**Figure 4.** Biomarkers generated from arachidonic acid.

LTs are derivatives of arachidonic acid that are synthetized *via* the 5-lipoxygenase pathway (**Figure 5**). The major problem in the determination of LTs in body matrices is their low stability due to their sensitivity toward oxidation. This explains challenging analytical determination of the used assays and to a relatively high variability of the published data.

#### *6.1.2. Lipoxins*

Especially, these common characteristics cause that asthma and COPD are sometimes misdiagnosed [30–32]. This can cause an incorrect pharmacotherapy administration, followed by

The term biomarker herein refers to a measurable biomolecular factor applicable for the measurement of a disease progression or treatment-related biomolecular changes in the human organism. On a molecular scale, biomarker refers to "a subset of markers that might be discovered using metabolomics, proteomics, genomics and other -omics technologies or imaging technologies." Biomarkers play a major role in medicinal biology. Biomarkers may be foreseen as a promising tool in the near future due to their unique potential for early diagnoses, which obviously permit disease prevention, a drug target identification, a drug response monitoring, etc. The collection and analyses of substances present in EBC provide a simple, noninvasive, real-time, point-of-care clinical and research tool for the evaluation of lung pathophysiology. Very significant role is played by some biomarkers that are produced from the arachidonic acid (some of them were already mentioned above). Arachidonic acid ((5Z,8Z,11Z,14Z)Eicosa-5,8,11,14-tetraenoic acid) is a polyunsaturated omega-6 fatty acid present in phospholipid cell membranes [11, 12]. The products of the metabolism of arachidonic acid are called eicosanoids. These molecules are characterized by the 20C chain. The production of eicosanoids is enabled by different enzymes (**Figure 4**), the only exception are isoprostanes which emerge

Arachidonic acid is a polyunsaturated fatty acid present in phospholipid bilayer. In human organism, arachidonic acid acts as a vasodilator or regulates inflammation as a key intermediate. There are several pathways which allow transformation of the arachidonic acid in a number of different metabolites (**Figure 4**). Among the most significant products of its metabolism

Leukotrienes (LTs) [6, 33] represent a group of biologically active molecules. LTs are produced by various tissue cells (e.g., leukocytes, macrophages, mastocytoma cells) as a response to both immunological and non-immunological stimuli. LTs are potent pro-inflammatory [33] mediators and their release is usually triggered by the organism coming in contact with an allergen. The interaction between LTs and their receptors can lead to a wide range of biological effects: leukocytes activation, bronchial smooth muscles contraction, vascular permeability stimulation and increased mucus production, etc. All of the described symptoms are typically connected not only to pathophysiology of bronchial obstruction, especially to asthma, but

However, several factors can be used to distinguish asthma from COPD (**Table 1**).

**6. Biomarkers of pulmonary diseases present in EBC**

through oxidation of arachidonic acid (non-enzymatic pathway).

can be classified leukotrienes, lipoxins, isoprostanes, and prostanoids [6, 33].

**6.1. Arachidonic acid metabolites**

also to other lung inflammatory disorders.

*6.1.1. Leukotrienes*

their health state not (or just slightly) improving.

148 Biomarker - Indicator of Abnormal Physiological Process

Lipoxins (LXs) function in our organism as "natural antiasthmatics" as they are the antiinflammatory mediators. Binding of LXs to their receptors also support the reconstructive process that is initiated in lungs immediately after the asthma attack.

LXs and LTs are derivatives of arachidonic acid and they are generated in three different metabolic pathways [34] . The first one is enabled by acetylsalicylic acid (ASA, in aspirin induced

**Figure 5.** Biosynthesis of leukotrienes.

asthma), the second one by the enzyme 15-lipoxygenase, and the last one by 5-lipoxygenase which transforms arachidonic acid into LTA4 and then into LXA4 eventually into LXB4 (**Figure 6**). On the other hand, the levels of LXs are usually lowered during inflammation.

**6.2. Resolvins and protectins**

**6.3. Oxidative stress biomarkers**

*6.3.1. Biomarkers of lipid peroxidation*

oxidative stress.

stress.

Although the resolution of inflammation may have been regarded as a passive process, it has been proved that it can be actually described also as an active process in which numerous chemical mediators are involved. An example of these molecules may be resolvins and protectins. Both of them are synthetized from ω-3-PUFA precursors. Based on the model systems, it has been proved that resolvins and protectins participate in the anti-inflammatory response. In connection, the disproportion in their molecular levels can lead to diseases that are characterized by prolonged inflammation [33, 35]. At the same time, resolvin receptors

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Isoprostanes are prostaglandin-like compounds formed *in vivo* from the free radical-catalyzed peroxidation of essential fatty acids (primarily arachidonic acid) without the direct action of cyclooxygenase (COX) enzymes [6, 8, 9, 33]. These non-classical eicosanoids possess potent biological activity as inflammatory mediators that augment the perception of pain. These compounds are accurate markers of lipid peroxidation in both animal and human models of

8-iso-prostaglandin F2α (also known as 8-epi-PGF2α or 8-isoprostane) is a biomarker that has been shown to be useful for the assessment of oxidative stress *in vivo*. It is produced in the phospholipid membranes from the non-cyclooxygenase peroxidation pathways derived from arachidonic acid. It is present in EBC in physiological concentration levels which grows in the course of lifetime as a consequence of aging. Pathological levels in EBC are reasonably increased as a result of several lung diseases and disorders that are induced by oxidative

*6.3.2.1. 8-hydroxy-2′-deoxyguanosine, 8-hydroxyguanosine, and 5-hydroxymethyl uracil*

The steady-state levels of nucleic acids damage biomarkers represent the balance between formation and repair. As reviewed by Valavanidis et al. [36], increased levels of 8-hydroxy-2′-deoxyguanosine (8-OHdG), the principal product of DNA oxidation, represent a valuable

8-Hydroxyguanosine (8-OHG) is a nucleoside that is an oxidative derivative of guanosine. Measurement of the levels of 8-OHG is used as a biomarker of RNA damage by oxidative

In a rat model, 8-OHdG was found to have anti-inflammatory effect. Rats treated with lipopolysaccharide (LPS) exhibited inflammatory lung injury dependent on neutrophils with an increase in pro-inflammatory cytokines such as interleukins 6 and 18 (IL-6, IL-18) and

may represent interesting targets for the future pharmacotherapies.

*6.3.1.1. Isoprostanes; 8-iso-prostaglandin F2α (8-iso-PGF2α or 8-isoprostane)*

stress (asbestosis, silicosis, lung cancer, COPD, etc.).

biomarker of DNA damage by oxidative stress.

*6.3.2. Biomarkers of nucleic acids damage*

#### *6.1.3. Prostanoids*

Prostanoids represent another group of biomarkers that are generated from the arachidonic acid. The synthesis is enabled by the enzyme cyclooxygensases (COX1 , COX2 ) [6, 33]. Three major groups of biomarkers belong to the prostanoid family: prostacyclins, prostaglandins (PGD2 , PGE2 , and PGF2 ), and thromboxanes (TXA2 , TXB2 ). All of them represent significant participants in the inflammatory response. Thromboxanes are mainly responsible for vasoconstriction, while prostaglandins play an important role in the inflammatory and anaphylactic reactions. Another important function of thromboxanes and prostaglandins is their ability to adapt the inflammatory response and affect symptoms, such as fever, pain, or swelling.

The effect of prostanoids can be both pro- and anti-inflammatory with regard to the type of the inflammatory stimulus. Increased levels of some prostanoids with brocnhoconstrictive effects (PGE<sup>2</sup> , PGD2 , PGF2 , and TBX2 ) have been detected in EBC; however, the significance of their presence has not been sufficiently explained yet.

**Figure 6.** Biosynthesis of lipoxins.

#### **6.2. Resolvins and protectins**

Although the resolution of inflammation may have been regarded as a passive process, it has been proved that it can be actually described also as an active process in which numerous chemical mediators are involved. An example of these molecules may be resolvins and protectins. Both of them are synthetized from ω-3-PUFA precursors. Based on the model systems, it has been proved that resolvins and protectins participate in the anti-inflammatory response. In connection, the disproportion in their molecular levels can lead to diseases that are characterized by prolonged inflammation [33, 35]. At the same time, resolvin receptors may represent interesting targets for the future pharmacotherapies.

#### **6.3. Oxidative stress biomarkers**

#### *6.3.1. Biomarkers of lipid peroxidation*

#### *6.3.1.1. Isoprostanes; 8-iso-prostaglandin F2α (8-iso-PGF2α or 8-isoprostane)*

Isoprostanes are prostaglandin-like compounds formed *in vivo* from the free radical-catalyzed peroxidation of essential fatty acids (primarily arachidonic acid) without the direct action of cyclooxygenase (COX) enzymes [6, 8, 9, 33]. These non-classical eicosanoids possess potent biological activity as inflammatory mediators that augment the perception of pain. These compounds are accurate markers of lipid peroxidation in both animal and human models of oxidative stress.

8-iso-prostaglandin F2α (also known as 8-epi-PGF2α or 8-isoprostane) is a biomarker that has been shown to be useful for the assessment of oxidative stress *in vivo*. It is produced in the phospholipid membranes from the non-cyclooxygenase peroxidation pathways derived from arachidonic acid. It is present in EBC in physiological concentration levels which grows in the course of lifetime as a consequence of aging. Pathological levels in EBC are reasonably increased as a result of several lung diseases and disorders that are induced by oxidative stress (asbestosis, silicosis, lung cancer, COPD, etc.).

#### *6.3.2. Biomarkers of nucleic acids damage*

**Figure 6.** Biosynthesis of lipoxins.

asthma), the second one by the enzyme 15-lipoxygenase, and the last one by 5-lipoxygenase

Prostanoids represent another group of biomarkers that are generated from the arachidonic

major groups of biomarkers belong to the prostanoid family: prostacyclins, prostaglan-

nificant participants in the inflammatory response. Thromboxanes are mainly responsible for vasoconstriction, while prostaglandins play an important role in the inflammatory and anaphylactic reactions. Another important function of thromboxanes and prostaglandins is their ability to adapt the inflammatory response and affect symptoms, such as fever, pain,

The effect of prostanoids can be both pro- and anti-inflammatory with regard to the type of the inflammatory stimulus. Increased levels of some prostanoids with brocnhoconstrictive

), and thromboxanes (TXA2

On the other hand, the levels of LXs are usually lowered during inflammation.

acid. The synthesis is enabled by the enzyme cyclooxygensases (COX1

and then into LXA4

, TXB2

) have been detected in EBC; however, the significance of

eventually into LXB4

, COX2

). All of them represent sig-

(**Figure 6**).

) [6, 33]. Three

which transforms arachidonic acid into LTA4

150 Biomarker - Indicator of Abnormal Physiological Process

, and PGF2

*6.1.3. Prostanoids*

dins (PGD2

or swelling.

effects (PGE<sup>2</sup>

, PGE2

, PGD2

, PGF2

, and TBX2

their presence has not been sufficiently explained yet.

#### *6.3.2.1. 8-hydroxy-2′-deoxyguanosine, 8-hydroxyguanosine, and 5-hydroxymethyl uracil*

The steady-state levels of nucleic acids damage biomarkers represent the balance between formation and repair. As reviewed by Valavanidis et al. [36], increased levels of 8-hydroxy-2′-deoxyguanosine (8-OHdG), the principal product of DNA oxidation, represent a valuable biomarker of DNA damage by oxidative stress.

8-Hydroxyguanosine (8-OHG) is a nucleoside that is an oxidative derivative of guanosine. Measurement of the levels of 8-OHG is used as a biomarker of RNA damage by oxidative stress.

In a rat model, 8-OHdG was found to have anti-inflammatory effect. Rats treated with lipopolysaccharide (LPS) exhibited inflammatory lung injury dependent on neutrophils with an increase in pro-inflammatory cytokines such as interleukins 6 and 18 (IL-6, IL-18) and tumor necrosis factor α (TNF-α). Rats pre-treated with 8-OHdG prior to LPS treatment showed inhibited LPS-induced inflammatory responses. 8-OHdG anti-inflammatory action was found to be higher than for aspirin and other nucleosides (8-OHG, deoxyguanosine, guanosine, adenosine). 8-OHG and adenosine also exhibited anti-inflammatory activity, but it was much lower than for 8-OHdG. Deoxyguanosine was found to be almost ineffective. Compared to aspirin, which acts through cyclooxygenase (COX) inhibition, 8-OHdG seems to be more versatile and, therefore, more effective as it was found that 8-OHdG suppresses ROS formation in human neutrophils. However, in human organism, 8-OHdG is excreted in much lower concentrations than in rats and, therefore, only exogenously administered 8-OHdG could have a therapeutic potential as an anti-inflammatory agent. 8-OHdG is also considered to be a potential biomarker of cancers related to smoking (e.g., lung cancer).

5-Hydroxymethyl uracil (5-OHMeU) is an example of oxidized-pyrimidines. Low levels of these molecules have been detected as a consequence of DNA oxidation initialized by oxidative stress. Oxidized-pyrimidines are more likely to be repaired than other relative molecules, which may represent an explanation of their low detected pathological concentration levels. As the excision rate from DNA is different for various bases, participation of specific excisionrepair enzymes might occur.

#### *6.3.3. Biomarkers of peptides damage*

#### *6.3.3.1. o-Tyrosine, 3-chlorotyrosine and 3-nitrotyrosine*

*o*-Tyrosine (*o*-Tyr), 3-chlorotyrosine (3-ClTyr), and 3-nitrotyrosine (3-NOTyr) are among the most prominent biomarkers of oxidative protein damage and are present in the body fluids of patients with diseases related to oxidative stress [6].

human organism, they can act as both inflammatory and anti-inflammatory molecules, however in the respiratory tract they are mainly considered to represent biomarkers of chronic

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A wide range of cytokines has been detected in EBC so far. An example of such cytokine can be tumor necrosis factor (TNF) or interferon (IFN). Low concentration levels [37–40] of both of these biomarkers have been detected in EBC. Specifically, TNF represents a biomarker of oncological diseases as its increased levels have been mainly described among lung cancer patients [41, 42]. Other cytokines that are detectable in EBC are various members of the inter-

Glutathione (GSH) is a tripeptide that functions in organism as an endogenous antioxidant. The main task of GSH is to prevent the cells to be damaged by free radicals and reactive oxygen species and thus protect the organism from oxidative stress. An important part of this process is the oxidation of GSH to glutathione disulfide (GSSG). This process occurs, for example, in the airway cells, where it is essential to protect the lungs and airways tissue which are exposed to the effect of external oxidants. Simultaneously, GSH is one of the regulators of the NO cycle. Decreased levels of GSH and proportionally increased levels of GSSG, which are mainly connected to the disproportion in the redox balance, represent a reliable biomarker of oxidative stress, usually coupled with inflammation [43]. Decreased levels of GSH in EBC have been mainly monitored

leukin family (IL-1, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10 and IL-13).

**Figure 7.** Formation of *o*-tyrosine, 3-chlorotyrosine, and 3-nitrotyrosine.

inflammation.

*6.4.2. Glutathione*

Free radicals cause alterations in cellular protein structure and function. Oxidized, nitrated, and chlorinated modifications of aromatic amino acids including phenylalanine and tyrosine are reliable biomarkers of oxidative stress and inflammation in clinical conditions. In human organism, tyrosine is formed from phenylalanine. Physiological *p*-tyrosine (*p*-Tyr) occurs by enzymatic oxidation of phenylalanine by phenylalanine hydroxylase. Important derivatives of tyrosine are catecholamines (dopamine, adrenaline, and noradrenaline) or thyroid hormones. *o*-Tyr and *m*-tyrosine (*m*-Tyr) are formed by the attack of ROS on phenylalanine. Unlike *p*-Tyr, *o*-Tyr and *m*-Tyr are not natural amino acids and are considered to be oxidative stress biomarkers. The biomarkers that are formed during protein oxidative damage are amino acids *o*-Tyr, 3-ClTyr, and 3-NOTyr (**Figure 7**).

#### **6.4. The other biomarkers**

#### *6.4.1. Cytokines*

Cytokines are proteins secreted by immune cells (e.g., B lymphocytes, T lymphocytes, macrophages, and mast cells) or fibroblasts and endothelial cells. Cytokines are fundamental regulators of the immune system and they play various roles in human organism (not only in immune system), as they influence: regeneration of the tissue, embryonal development, carcinogenesis, angiogenesis, etc. The function of numerous cytokines can be triggered by oxidative stress. In Molecular Diagnostics of Pulmonary Diseases Based on Analysis of Exhaled Breath Condensate http://dx.doi.org/10.5772/intechopen.74402 153

**Figure 7.** Formation of *o*-tyrosine, 3-chlorotyrosine, and 3-nitrotyrosine.

human organism, they can act as both inflammatory and anti-inflammatory molecules, however in the respiratory tract they are mainly considered to represent biomarkers of chronic inflammation.

A wide range of cytokines has been detected in EBC so far. An example of such cytokine can be tumor necrosis factor (TNF) or interferon (IFN). Low concentration levels [37–40] of both of these biomarkers have been detected in EBC. Specifically, TNF represents a biomarker of oncological diseases as its increased levels have been mainly described among lung cancer patients [41, 42]. Other cytokines that are detectable in EBC are various members of the interleukin family (IL-1, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10 and IL-13).

#### *6.4.2. Glutathione*

tumor necrosis factor α (TNF-α). Rats pre-treated with 8-OHdG prior to LPS treatment showed inhibited LPS-induced inflammatory responses. 8-OHdG anti-inflammatory action was found to be higher than for aspirin and other nucleosides (8-OHG, deoxyguanosine, guanosine, adenosine). 8-OHG and adenosine also exhibited anti-inflammatory activity, but it was much lower than for 8-OHdG. Deoxyguanosine was found to be almost ineffective. Compared to aspirin, which acts through cyclooxygenase (COX) inhibition, 8-OHdG seems to be more versatile and, therefore, more effective as it was found that 8-OHdG suppresses ROS formation in human neutrophils. However, in human organism, 8-OHdG is excreted in much lower concentrations than in rats and, therefore, only exogenously administered 8-OHdG could have a therapeutic potential as an anti-inflammatory agent. 8-OHdG is also considered

5-Hydroxymethyl uracil (5-OHMeU) is an example of oxidized-pyrimidines. Low levels of these molecules have been detected as a consequence of DNA oxidation initialized by oxidative stress. Oxidized-pyrimidines are more likely to be repaired than other relative molecules, which may represent an explanation of their low detected pathological concentration levels. As the excision rate from DNA is different for various bases, participation of specific excision-

*o*-Tyrosine (*o*-Tyr), 3-chlorotyrosine (3-ClTyr), and 3-nitrotyrosine (3-NOTyr) are among the most prominent biomarkers of oxidative protein damage and are present in the body fluids of

Free radicals cause alterations in cellular protein structure and function. Oxidized, nitrated, and chlorinated modifications of aromatic amino acids including phenylalanine and tyrosine are reliable biomarkers of oxidative stress and inflammation in clinical conditions. In human organism, tyrosine is formed from phenylalanine. Physiological *p*-tyrosine (*p*-Tyr) occurs by enzymatic oxidation of phenylalanine by phenylalanine hydroxylase. Important derivatives of tyrosine are catecholamines (dopamine, adrenaline, and noradrenaline) or thyroid hormones. *o*-Tyr and *m*-tyrosine (*m*-Tyr) are formed by the attack of ROS on phenylalanine. Unlike *p*-Tyr, *o*-Tyr and *m*-Tyr are not natural amino acids and are considered to be oxidative stress biomarkers. The biomarkers that are formed during protein oxidative damage are

Cytokines are proteins secreted by immune cells (e.g., B lymphocytes, T lymphocytes, macrophages, and mast cells) or fibroblasts and endothelial cells. Cytokines are fundamental regulators of the immune system and they play various roles in human organism (not only in immune system), as they influence: regeneration of the tissue, embryonal development, carcinogenesis, angiogenesis, etc. The function of numerous cytokines can be triggered by oxidative stress. In

to be a potential biomarker of cancers related to smoking (e.g., lung cancer).

repair enzymes might occur.

**6.4. The other biomarkers**

*6.4.1. Cytokines*

*6.3.3. Biomarkers of peptides damage*

152 Biomarker - Indicator of Abnormal Physiological Process

*6.3.3.1. o-Tyrosine, 3-chlorotyrosine and 3-nitrotyrosine*

patients with diseases related to oxidative stress [6].

amino acids *o*-Tyr, 3-ClTyr, and 3-NOTyr (**Figure 7**).

Glutathione (GSH) is a tripeptide that functions in organism as an endogenous antioxidant. The main task of GSH is to prevent the cells to be damaged by free radicals and reactive oxygen species and thus protect the organism from oxidative stress. An important part of this process is the oxidation of GSH to glutathione disulfide (GSSG). This process occurs, for example, in the airway cells, where it is essential to protect the lungs and airways tissue which are exposed to the effect of external oxidants. Simultaneously, GSH is one of the regulators of the NO cycle. Decreased levels of GSH and proportionally increased levels of GSSG, which are mainly connected to the disproportion in the redox balance, represent a reliable biomarker of oxidative stress, usually coupled with inflammation [43]. Decreased levels of GSH in EBC have been mainly monitored in case of patients suffering from bronchial asthma. The results of another conducted study showed that significantly increased levels of GSSG occur in EBC of alcoholics [44].

<2 μm) and by shortening the time of LC-MS analysis. When using the so-called "stableisotope-dilution assay," the accuracy and precision of the LC-MS method can be increased by suitable deuterated internal standards. However, the main disadvantage of the LC-MS analysis is the inclusion of the pre-treatment step (SPE, immunoaffinity extraction, etc.), when the EBC sample is recommended to exclude a contact to room temperature, ideally temperature above 0°C. This problem can be prevented by using the 2D technology for liquid chromatography. In the first dimension, an on-line SPE is carried out and the subsequent dimension uses the UHPLC. For detection of selected biomarkers, 2D UHPLC-MS method was developed and

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because of the sensitivity of biomarkers mentioned above, it is highly recommended.

*7.1.1. Determination of the amino compounds*

*7.1.2. Determination of aldehydes and carboxylic acids*

Tyr), 3-nitrotyrosine (3-NO2

Analysis of substances were realized on the LC-MS system consisting of quaternary pump and mass spectrometer operating on the principle of triple quadrupole equipped with electrospray ionization (ESI). To implement multimarker screening, it was necessary to carry out two types of analyses. The first one were determined substances containing amino group in its structure. The second one serves to determine substances with aldehyde and carboxylic groups. These two analyses were necessary because of the different conditions of derivatization reactions (acid vs. alkaline environment) and the resulting liquid chromatography at different conditions (different composition of the mobile phase used on different chromatographic columns).

For the derivatization of compounds containing an amino group in its structure (*o*-tyrosine (*o*-

8-hydroxy-2′-deoxyguanosine (8-OHdG) were used as derivatization reagent 3-aminopyridyl-N-hydroxysukcinimidyl carbamate (= APDS). To 500 μl of the EBC sample-containing deuterium labeled analyte analogues was added to 450 μl of borate buffer (pH 8.5) and 50 μl of APDS derivatization agent (concentration of 1 mg/ml of acetonitrile). Derivatization reactions were carried out for 10 min at 4°C. Thus prepared sample was subjected to LC-ESI-MS/MS analysis on chromatographic column XTerra MS (C18 50 × 1 mm × 3.5 μm) (Waters, Republic of Ireland). The substances were subjected to analysis where isocratic elution method with a mobile phase composed of acetonitrile: water (60:40—v/v) (water = 0.1% formic acid) was used. The column was tempered to 25°C. Mobile phase flow rate was 150 μl/min. The volume of the analyzed samples was 10 μl. Mass spectrometer parameters were optimized to the following values: capillary voltage −2500 V, the inlet capillary temperature 300°C, the temperature of the evaporator HESI 300°C, sheath gas (nitrogen) pressure 45 psi, auxiliary gas (nitrogen) 10 ArbU. Measurement parameters were optimized for use in neutral loss mode in the interval 250–500 Da (Q1) → 130–

380 Da (Q3) with CID energy 15 eV in the negative electrospray ionization (ESI-).

group in its structure 8-isoprostane (8-iso-PGF2α), cys-LTs, LTB<sup>4</sup>

Derivatization of aldehydes (n-aliphatic aldehydes (C6–C12), malondialdehyde (MDA), 4-hydroxynonenal (4-HNE), 4-hydroxyhexenal (4-HHE) and substances with a carboxyl

derivatization with Girard's reagent T (GirT) in the presence of N-(3-dimethylaminopropyl)- N′-ethylkarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide. To the sample containing 100 μl of EBC with deuterium-labeled internal standards were added 10 μl of


was carried out using

#### **6.5. Other molecules determined in EBC**

#### *6.5.1. Proteins and metabolites*

The majority of pulmonary diseases is also characterized by alternations in the protein profile of the patients. Many of these changes are measurable in EBC and can be used for monitoring of pathological process occurring (mainly) in the respiratory tract. The changes in the structure and concentration levels of various proteins have recently become a popular and reliable tool for monitoring of the process and molecular alterations in lungs and airways. Based on the proteomic analysis of EBC, 44 unique proteins [45, 46] have been detected so far. Many of these proteins might become steady biomarkers of inflammation or oxidative stress, when scanning of the differences between the proteome profiles of healthy control subjects and subjects with various pulmonary diseases may represent a significant shift toward detecting new prognostic and/or diagnostic biomarkers.

#### *6.5.2. Serotonin*

Serotonin (5-hydroxytryptamin (5-HT)) is a neurotransmitter that is predominantly located in central nervous system and gastrointestinal tract (GIT). In GIT, 5-HT regulates bowel movements. In CNS, it is responsible for the regulation of mood, sleep, muscle contraction, and some cognitive functions (involving memory and learning abilities). It is also present in thrombocytes, where it is involved in the regulation of homeostasis and coagulation [47].

5-HT plays a significant role in many pathological and neuropsychiatric diseases [47, 48]. The serotonergic substances are also important in pharmacology. The genes that code various components of 5-HT system are the subject of the study as they could be factors of depression, schizophrenia, obsessive–compulsive disorder, aggression, alcoholism, migraine, and autism [49].
