**8. Case studies**

#### **8.1. Asthma phenotyping**

The first aim of the study was to determine levels of the pro-inflammatory cys-LTs and levels of the anti-inflammatory LXs in EBC of patients suffering from different asthma phenotypes (including also SRA), compare the obtained data between the groups of asthmatics, and make the comparisons with healthy control subjects.

As is presented in **Figure 8**, the study showed that both levels of cys-LTs and LXs were changing among different asthma phenotypes. According to the results, EBC of SRA patients contained the highest levels of the pro-inflammatory cys-LTs but at the same time the lowest levels of the anti-inflammatory LXs. The results of the analysis of EBC of healthy control subjects were inverse to these (i.e., EBC of health controls contained the highest levels of LXs but on the contrary the lowest levels of cys-LTs).

The remaining groups have spread in the interval from healthy control subjects to SRA. The order of these groups was based on the raising severity of the asthma phenotype (mild asthma → moderate persistent asthma → difficult asthma) (**Figure 8**).

According to the results (**Figure 15**), it is possible to use cys-LTs and LXs for the differential diagnostics of asthma and identify various asthma phenotypes. The diagnosis can be assessed on the phenomenon that the concentration levels of LXs and cys-LTs are complementary and connected by dynamic equilibrium (i.e., increasing levels of the inflammatory LTs lead to a corresponding decrease in the levels of the anti-inflammatory LXs). This occurs due to the fact that biochemical synthesis (both cys-LTs and LXs are generated from LTA4 ) enhancing the production of LXs simultaneously lower the generation of LTs. Combining cys-LTs with LXs offers an interesting alternative to the currently used methods of molecular diagnostics of bronchial asthma. **Figure 8** describes the principle of equilibrium between the pro-inflammatory LTs and the anti-inflammatory LXs. The developed method represents a potential tool for asthma phenotyping accuracy improvement, which was proved in a clinical study, which enabled the separation of patients into five groups:


each derivatization reagent GirT (c = 100 μl/ml), reagent EDC together with 10 μl of sulfo-Nhydroxysuccinimide, 10 μl of 1% hydrochloric acid and 270 μl of propan-2-ol. Derivatization proceeded for 30 minutes and in such way prepared sample was immediately analyzed by LC-ESI-MS/MS. Chromatographic column used was a Thermo Hypercarb (100 × 21 mm × 5 μm) with pre-column Hypercarb (Thermo Electron Corporation, USA). For separation of substances, was used the isocratic elution method with a mobile phase composed of methanol: water (40:60—v/v) (pH adjusted with ammonium hydroxide to 9). Flow rate of mobile phase was 150 μl/min. Chromatographic column temperature was 30°C and the sample volume was 10 μl. Mass spectrometer parameters were optimized to the following values: capillary voltage 3000 V, capillary inlet temperature 300°C; HESI evaporator temperature 300°C, sheath gas (nitrogen) pressure 45 psi and auxiliary gas (nitrogen) 10 ArbU. Measurement parameters were optimized for use in neutral loss mode in the interval 150–750 Da (Q1) → 91–691 Da (Q3)

The first aim of the study was to determine levels of the pro-inflammatory cys-LTs and levels of the anti-inflammatory LXs in EBC of patients suffering from different asthma phenotypes (including also SRA), compare the obtained data between the groups of asthmatics, and make

As is presented in **Figure 8**, the study showed that both levels of cys-LTs and LXs were changing among different asthma phenotypes. According to the results, EBC of SRA patients contained the highest levels of the pro-inflammatory cys-LTs but at the same time the lowest levels of the anti-inflammatory LXs. The results of the analysis of EBC of healthy control subjects were inverse to these (i.e., EBC of health controls contained the highest levels of LXs

The remaining groups have spread in the interval from healthy control subjects to SRA. The order of these groups was based on the raising severity of the asthma phenotype (mild asthma

According to the results (**Figure 15**), it is possible to use cys-LTs and LXs for the differential diagnostics of asthma and identify various asthma phenotypes. The diagnosis can be assessed on the phenomenon that the concentration levels of LXs and cys-LTs are complementary and connected by dynamic equilibrium (i.e., increasing levels of the inflammatory LTs lead to a corresponding decrease in the levels of the anti-inflammatory LXs). This occurs due to the

the production of LXs simultaneously lower the generation of LTs. Combining cys-LTs with LXs offers an interesting alternative to the currently used methods of molecular diagnostics of bronchial asthma. **Figure 8** describes the principle of equilibrium between the pro-inflammatory LTs and the anti-inflammatory LXs. The developed method represents a potential tool for asthma phenotyping accuracy improvement, which was proved in a clinical study, which

) enhancing

fact that biochemical synthesis (both cys-LTs and LXs are generated from LTA4

with CID energy − 16.5 eV in the positive electrospray ionization (ESI+).

**8. Case studies**

**8.1. Asthma phenotyping**

156 Biomarker - Indicator of Abnormal Physiological Process

the comparisons with healthy control subjects.

but on the contrary the lowest levels of cys-LTs).

enabled the separation of patients into five groups:

→ moderate persistent asthma → difficult asthma) (**Figure 8**).


#### **8.2. Monitoring of efficacy of the used pharmacotherapy**

The developed method was used in a parallel study. The study was conducted to prove whether the method could be applied for monitoring of efficacy of the used pharmacotherapy. In this case, *per oral* and inhaled glucocorticoid treatments have been compared. Results of the study are present in **Figure 9**. In the clinical study of 35 patients with *per oral* glucocorticoid therapy, 35 patients with inhaled glucocorticoid therapy and 32 people from the healthy control group were involved.

From the results, it is obvious that the PCA analysis divided the subjects into three groups. The first group contained only healthy control subjects; however, the two remaining have

**Figure 8.** Statistically evaluated clinical results: levels of cys-LTs and LXs in different asthma phenotypes.

**Figure 9.** Monitoring of efficacy of the used pharmacotherapy.

been separated according to the type of glucocorticoid application. The results also show that on these terms more efficient was the *per oral* glucocorticoid therapy, as the cluster representing patients with *per oral* treatment is in the spectrum closer to the controls.

The study has also confirmed that the developed method can be used for such monitoring, which could in the future make the asthma pharmacotherapy more accurate. Furthermore, the method could also enable controlling of dosing and comparing of the efficacy of different anti-asthmatic drugs, which would globally improve asthma treatment.

According to the results (**Figure 10**), the PCA analysis has divided the patients into four groups based on their biomarker profiles. The results show that profiles of SRA and COPD patients were different, which allows an accurate separation of these two diseases. The figure also shows that the control groups were separated. Further, the results show that it is not possible with this method to separate (on the molecular level) the two phenotypes of COPD—

**Figure 10.** Results of clinical study: separation of COPD patients (light blue—bronchitis, dark blue—emphysema) and

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As a significant part of the study, a panel of biomarkers that can be used for differentiation of various pulmonary diseases was designed. The analyzed biomarkers are divided into two main groups. The first group contained biomarkers of eosinophil inflammation—

anti-inflammatory resolvins (RvD1). The second group contained biomarker of neutro-

to damage of phospholipid membrane, biomarkers of damage of proteins (Ʃ *o*-tyrosin, NO-tyrosin and Cl-tyrosin) and biomarkers of damage of nucleic acids (Ʃ 5-OHMeU,

The first two graphs show results of the analysis of EBC of patients suffering from SRA and moderate persistent asthma. The results are compared with the analysis of EBC of healthy

), the anti-inflammatory eicosanoids—LXs (Ʃ LXA4

, 8-isoprostane which is biomarker of oxidative stress connected

, LXB4

) and

**8.4. Biomarker panel for monitoring of pathogenesis of pulmonary diseases**

SRA patients (orange); control groups: green—COPD controls; yellow—SRA controls.

chronical bronchitis and emphysema.

, LTD4

, LTE4

cys-LTs (ƩLTC4

phil inflammation—LTB<sup>4</sup>

8-OHG and 8-OHdG).

control subjects.

#### **8.3. Asthma and COPD separation**

Apart from cys-LTs and LXs, EBC contains a wide range of other different biomarkers. The research has shown that biomarkers of oxidative stress play a significant role in the development of some pulmonary diseases. Examples of such biomarkers can be 8-isoprostan, MDA, HHE, HNE and other aldehydes and biomarkers connected to damage of proteins (*o*-Tyr, 3-ClTyr and 3-NOTyr) or nucleic acids (8-OHdG, 8-OHG and 5-OHMeU).

These biomarkers allowed extension of the developed method, which was originally based on the detection of levels of cys-LTs and LXs. An example of such extensions can be separation of asthma and COPD on molecular level.

The metabolic fingerprinting of EBC of patients suffering from COPD showed a significant increase of biomarkers of neutrophil inflammation—LTB<sup>4</sup> and also biomarkers of oxidative stress (mainly *o*-Tyr and 8-isoprostane). The developed method was used in a clinical study that was aimed at detection and description of differences between COPD patients and SRA (SRA were chosen because their profile is quite similar to the profile of COPD patients and thus their diagnosis is often altered). The obtained results were compared to the analysis of EBC of healthy control subjects (two control groups were chosen—one for COPD patients and one for SRA).

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**Figure 10.** Results of clinical study: separation of COPD patients (light blue—bronchitis, dark blue—emphysema) and SRA patients (orange); control groups: green—COPD controls; yellow—SRA controls.

According to the results (**Figure 10**), the PCA analysis has divided the patients into four groups based on their biomarker profiles. The results show that profiles of SRA and COPD patients were different, which allows an accurate separation of these two diseases. The figure also shows that the control groups were separated. Further, the results show that it is not possible with this method to separate (on the molecular level) the two phenotypes of COPD chronical bronchitis and emphysema.

#### **8.4. Biomarker panel for monitoring of pathogenesis of pulmonary diseases**

**Figure 9.** Monitoring of efficacy of the used pharmacotherapy.

158 Biomarker - Indicator of Abnormal Physiological Process

**8.3. Asthma and COPD separation**

asthma and COPD on molecular level.

increase of biomarkers of neutrophil inflammation—LTB<sup>4</sup>

been separated according to the type of glucocorticoid application. The results also show that on these terms more efficient was the *per oral* glucocorticoid therapy, as the cluster represent-

The study has also confirmed that the developed method can be used for such monitoring, which could in the future make the asthma pharmacotherapy more accurate. Furthermore, the method could also enable controlling of dosing and comparing of the efficacy of different

Apart from cys-LTs and LXs, EBC contains a wide range of other different biomarkers. The research has shown that biomarkers of oxidative stress play a significant role in the development of some pulmonary diseases. Examples of such biomarkers can be 8-isoprostan, MDA, HHE, HNE and other aldehydes and biomarkers connected to damage of proteins (*o*-Tyr,

These biomarkers allowed extension of the developed method, which was originally based on the detection of levels of cys-LTs and LXs. An example of such extensions can be separation of

The metabolic fingerprinting of EBC of patients suffering from COPD showed a significant

stress (mainly *o*-Tyr and 8-isoprostane). The developed method was used in a clinical study that was aimed at detection and description of differences between COPD patients and SRA (SRA were chosen because their profile is quite similar to the profile of COPD patients and thus their diagnosis is often altered). The obtained results were compared to the analysis of EBC of healthy control subjects (two control groups were chosen—one for COPD patients and one for SRA).

and also biomarkers of oxidative

ing patients with *per oral* treatment is in the spectrum closer to the controls.

anti-asthmatic drugs, which would globally improve asthma treatment.

3-ClTyr and 3-NOTyr) or nucleic acids (8-OHdG, 8-OHG and 5-OHMeU).

As a significant part of the study, a panel of biomarkers that can be used for differentiation of various pulmonary diseases was designed. The analyzed biomarkers are divided into two main groups. The first group contained biomarkers of eosinophil inflammation cys-LTs (ƩLTC4 , LTD4 , LTE4 ), the anti-inflammatory eicosanoids—LXs (Ʃ LXA4 , LXB4 ) and anti-inflammatory resolvins (RvD1). The second group contained biomarker of neutrophil inflammation—LTB<sup>4</sup> , 8-isoprostane which is biomarker of oxidative stress connected to damage of phospholipid membrane, biomarkers of damage of proteins (Ʃ *o*-tyrosin, NO-tyrosin and Cl-tyrosin) and biomarkers of damage of nucleic acids (Ʃ 5-OHMeU, 8-OHG and 8-OHdG).

The first two graphs show results of the analysis of EBC of patients suffering from SRA and moderate persistent asthma. The results are compared with the analysis of EBC of healthy control subjects.

According to the graph (**Figure 11**), it is obvious that EBC of patients who suffer from asthma contained increased levels of cys-LTs (the highest levels—SRA, this confirms the study mentioned above). On the contrary, EBC of asthmatics contained lowered levels of the anti-inflammatory LXs and resolvins. Considering the asthma-phenotyping-study, it can be also said that the results of analysis of EBC of SRA and controls were inverse.

**Figure 12** shows the results of the monitoring of LTB4 , 8-isoprostane, biomarkers of damage of proteins and nucleic acids. Levels of LTB4 showed the same trend as cys-LTs, for example, the highest levels were detected in EBC of SRA and the lowest in EBC of healthy controls. At the same time, levels of 8-isoprostane were slightly elevated among the group of patients with moderate persistent asthma and even more among SRA. The differences in levels of biomarkers responsible for damage of proteins and nucleic acids were slightly higher in EBC of asthmatics, but the differences were not so significant, which means that these biomarkers are not so specific and influential in case of bronchial asthma.

**Figures 13** and **14** show same biomarkers as the previous **Figures 11** and **12**, but in EBC of patients who suffer from COPD, asbestosis and lung cancer.

From **Figure 13**, it is quite obvious that biomarkers cys-LTs, LXs and resolvins do not play a significant role in pathogenesis of these diseases, as their levels are comparable to those detected among healthy control subjects (the levels are just slightly elevated and only COPD patients show some more noticeable deviations).

**Figure 12.** Evaluated clinical results: levels of LTB4

patients.

EBC of SRA, moderate persistent asthma and healthy controls.

, 8-isoprostane, biomarkers of proteins and nucleic acids damage in

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**Figure 13.** Evaluated clinical results: levels of cys-LTs, LXs, and resolvins in EBC of COPD, asbestosis, and lung cancer

**Figure 14** shows that illnesses characterized by damage of the pulmonary tissue are usually connected to increased levels of biomarkers of oxidative stress. One of these significant

**Figure 11.** Evaluated clinical results: levels of cys-LTs, LXs and resolvins in EBC of SRA, moderate persistent asthma and healthy controls.

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According to the graph (**Figure 11**), it is obvious that EBC of patients who suffer from asthma contained increased levels of cys-LTs (the highest levels—SRA, this confirms the study mentioned above). On the contrary, EBC of asthmatics contained lowered levels of the anti-inflammatory LXs and resolvins. Considering the asthma-phenotyping-study, it can be also said

the highest levels were detected in EBC of SRA and the lowest in EBC of healthy controls. At the same time, levels of 8-isoprostane were slightly elevated among the group of patients with moderate persistent asthma and even more among SRA. The differences in levels of biomarkers responsible for damage of proteins and nucleic acids were slightly higher in EBC of asthmatics, but the differences were not so significant, which means that these biomarkers are

**Figures 13** and **14** show same biomarkers as the previous **Figures 11** and **12**, but in EBC of

From **Figure 13**, it is quite obvious that biomarkers cys-LTs, LXs and resolvins do not play a significant role in pathogenesis of these diseases, as their levels are comparable to those detected among healthy control subjects (the levels are just slightly elevated and only COPD

**Figure 14** shows that illnesses characterized by damage of the pulmonary tissue are usually connected to increased levels of biomarkers of oxidative stress. One of these significant

**Figure 11.** Evaluated clinical results: levels of cys-LTs, LXs and resolvins in EBC of SRA, moderate persistent asthma

, 8-isoprostane, biomarkers of damage

showed the same trend as cys-LTs, for example,

that the results of analysis of EBC of SRA and controls were inverse.

**Figure 12** shows the results of the monitoring of LTB4

not so specific and influential in case of bronchial asthma.

patients who suffer from COPD, asbestosis and lung cancer.

patients show some more noticeable deviations).

and healthy controls.

of proteins and nucleic acids. Levels of LTB4

160 Biomarker - Indicator of Abnormal Physiological Process

**Figure 12.** Evaluated clinical results: levels of LTB4 , 8-isoprostane, biomarkers of proteins and nucleic acids damage in EBC of SRA, moderate persistent asthma and healthy controls.

**Figure 13.** Evaluated clinical results: levels of cys-LTs, LXs, and resolvins in EBC of COPD, asbestosis, and lung cancer patients.

**Figure 14.** Evaluated clinical results: levels of LTB4 , 8-isoprostane, biomarkers of proteins and nucleic acids damage in EBC of COPD, asbestosis, and lung cancer patients.

surprisingly, the levels were significantly elevated (in case of SRA patients) which is against all expectations (it was expected to detect lower levels of serotonin, which would provide a possible explanation of positive SRA's responsiveness to SSRI antidepressants therapy). Probably, even more interesting is the fact the levels of serotonin of other asthma phenotypes and health

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**Figure 15.** Evaluated clinical results: levels of 5-HT in EBC of SRA, moderate persistent asthma and healthy controls.

The interpretation of these results is quite complicated. One of the possible hypotheses is that SRA could be a different disease that would only demonstrate itself as asthma (i.e., patients have similar symptoms as asthmatics, but the cause of the disease could be different). However, this theory will require further research in the future. One of the possible extensions could be monitoring of levels of serotonin in cerebrospinal fluid, which would provide information about the process behind the blood-brain barrier. On the other hand, the study proved that there many significant physiological differences between SRA and other asthmatics, which could be used in the future for the development of a possible drug against SRA.

Measurements of biomarkers in EBC offer a novel way of monitoring lung inflammation, damage by oxidation stress with an insight into the pathophysiology of different diseases. The described diagnostic method was based on the detection and quantification of biomarkers in a matrix specific for the respiratory tract—EBC. As the collection of EBC is completely noninvasive, the method offers a broad spectrum of application. The method is applicable to children as well as to senior people and it is appropriate also in case of longitudinal studies that are trying to precisely understand the processes occurring on the molecular level in the respiratory tract. The method can be easily repeated which proves its suitability for regular monitoring of the pharmacotherapy efficiency or the impact of various allergens. The results obtained from the EBC analysis represent reliable characterization of the exhaled biomarkers

controls were the same, which indicates that the deviation appears only among SRA.

**9. Conclusions**

indicators of ongoing tissue necrosis processes is 8-isoprostane. The analysis of EBC showed that the levels of this biomarker are increased among COPD and asbestosis patients and even more among people suffering from lung cancer. Similar information is provided by the biomarkers of proteins damage (tyrosines) and nucleic acids damage (5-OHMeU, 8-OHG, and 8-OHdG). The levels of these molecules were elevated in EBC of patients with COPD and asbestosis and it can be said that the highest levels are specific for lung cancer (average concentration of tyrosines is approximately 75 pg/ml of EBC for healthy controls and 160 pg/ml of EBC for patients with lung cancer).

#### **8.5. Serotonin in EBC of SRA**

Based on the clinical experience, it is proved that SRA patients positively respond to SSRI (selective serotonin reuptake inhibitors) antidepressants therapy. SSRI antidepressants usually improve physical state of patients, which may seem as a quite logical coincidence. However, much more surprising is the fact that when SRA patients are prescribed SSRI antidepressants, their breath functions improve significantly. This phenomenon prompted to performed research aimed at the detection of serotonin in EBC of SRA. The obtained results were compared with serotonin levels in EBC of other asthma phenotypes and healthy control subjects.

According to the results (**Figure 15**), it is obvious that the levels of serotonin in EBC of SRA are different as compared to other asthma phenotypes and healthy control subjects. However,

**Figure 15.** Evaluated clinical results: levels of 5-HT in EBC of SRA, moderate persistent asthma and healthy controls.

surprisingly, the levels were significantly elevated (in case of SRA patients) which is against all expectations (it was expected to detect lower levels of serotonin, which would provide a possible explanation of positive SRA's responsiveness to SSRI antidepressants therapy). Probably, even more interesting is the fact the levels of serotonin of other asthma phenotypes and health controls were the same, which indicates that the deviation appears only among SRA.

The interpretation of these results is quite complicated. One of the possible hypotheses is that SRA could be a different disease that would only demonstrate itself as asthma (i.e., patients have similar symptoms as asthmatics, but the cause of the disease could be different). However, this theory will require further research in the future. One of the possible extensions could be monitoring of levels of serotonin in cerebrospinal fluid, which would provide information about the process behind the blood-brain barrier. On the other hand, the study proved that there many significant physiological differences between SRA and other asthmatics, which could be used in the future for the development of a possible drug against SRA.
