Molecular diagnostics of lung diseases

Introduction

Nowadays there is an increasing interest in the development of noninvasive diagnostic tools for lung diseases. These tools are based on analysis of substances originating in lungs as an impact of pathological processes related to the disease. Diagnostic techniques that have been known so far (e.g. spirometry, bronchoprovocation tests, etc.) are considered invasive as they interfere with the human organism. The noninvasive alternative how to acquire samples is drawing of exhaled breath condensate (EBC), which is generally well tolerated even by children and older people. EBC is a bodily liquid with composition corresponding to the processes that take place directly in lungs and airways. Therefore it is a useful tool for gaining information about the progress of various pathological processes in lungs (oxidative stress, inflammatory processes), leading to our better understanding of the lung diseases.

Condensed water vapor and water-soluble substances form the majority of EBC (99 %). Only a small part of the EBC consists of drops containing substances insoluble in water. These substances are carried by the turbulent flow in alveoli in the form of aerosol and accumulate in the condensation tool (Fig. 1).

 



Fig. 1: Origin of the exhaled breath condensate. Volatile substances are evaporated from the surface of airways and the substances insoluble in water form binary system on the surface of the airways. The aerosol particles are carried away by the turbulent flow (on the left). Aerosol particles are collected by the condenser of exhaled breath, which also condenses water vapor and other volatile substances (on the right).

 

The condenser of exhaled breath (Fig. 2) is a commercially manufactured, easy-to-use portable device usable in various situations.

 



Fig. 2: Condenser of exhaled breath (left) and clinical taking of EBC sample (right).

 

During ten minutes of adults’ breathing into the device it is possible to draw 1–3 mL of EBC. This volume is enough to perform basic qualitative and quantitative analysis.

The aim of this project is to develop analytical methods for qualitative and quantitative analysis of substances (biomarkers) typical for damaged phospholipids, nucleic acids, and proteins as the consequence of oxidative stress and of substances that are present in lungs as a consequence of inflammatory and allergic reaction. Therefore, multimarker screening of EBC giving the clinical picture of the airways has to be developed.
Very important part of this project is testing the reliability of the developed diagnostic method performing clinical studies (in collaboration with the 1st Faculty of Medicine, Charles University in Prague), the aim of which is to compare concentration levels of various biomarkers in the group of patients diagnosed with lung diseases (diagnostics by the “classic” diagnostic tests – spirometry, results of bronchoprovocation tests, etc.) and in a control group of healthy people.

The developed method is based on the combination of a suitable derivatization and separation technique with a sensitive and selective detection method based on mass spectrometry, which represents reliable and highly selective qualitative and quantitative device for the detection of various biomarkers.

Projects

1. Analysis of biomarkers of oxidative stress (aldehydes, guanosines, isoprostanes, tyrosines)

Lungs are exposed to oxygen-rich environment, they have large surface and high blood supply. That is why they are predisposed to the damage by reactive oxygen species (ROS). Oxidation changes of proteins, nucleic acids, and phospholipids are connected with higher production of ROS. They can cause direct damage to lungs or induce other various cell responses through the production of secondary reactive metabolic substances. Detailed description of this problematic is to be found in the article Biomarkers of oxidative stress.

2. Analysis of biomarkers of enzymatic oxidation (leukotrienes)

Leukotrienes are substances released during inflammatory processes and allergic reactions. They are formed by enzymatic oxidation of arachidonic acid. Leukotriene A4 (LTA4) is the first substance produced. LTB4 is formed from LTA4 in higher concentrations during inflammatory processes. During the allergic reaction, the first cysteinyl leukotriene (LTC4) is formed and is further transformed to LTD4 which is subsequently transformed to LTE4. Cysteinyl leukotrienes interact with Cys-LT receptors which are located mainly in cells of smooth muscle tissues. The interaction of cysteinyl leukotrienes with Cys-LT1 receptors located in lungs and airways causes the contriction of bronchi, blood congestion, and higher secretion of viscous mucus, which causes the narrowing of airways and repeated conditions of expiratory shortness of breath.
Leukotrieny jsou látky uvolňované při zánětlivých procesech a alergických reakcích.

It is possible to determine higher concentration levels of leukotrienes e.g. in bronchial asthma.

3. Multimarker screening

The purpose of this project is to develop a single method able to determine high amount of substances in EBC. This method will enable to gain maximum information even from a minimal amount of sample.