The lung is an important interphase between the environment and the human body, and it serves as a major getaway for different biomolecules. Complex biological processes in different body organs have their fingerprints on exhaled breath by releasing gas phase mediators and other biomolecules that are transported to the lungs and released into the exhaled breath through the alveoli. The lung parenchyma and the airways are major sources of mediators released to the airways and make a substantial contribution to the content of exhaled breath.

The potential of using exhaled breath to obtain information about different body functions was first recognized at the time of ancient Greek medicine when special odors were linked with different diseases such as liver cirrhosis and diabetes. It took centuries to identify and quantify the biomolecules responsible for the signals sensed by human olfaction. A landmark study was published by Pauling L et al. [1] in 1971 demonstrating the presence of hundreds of volatiles in exhaled breath samples using gas–liquid partition chromatography. With the advent of gas chromatography and mass spectrometry researchers have identified and quantified thousands of volatile organic compounds (VOCs) in the breath, most of them in picomolar (10–12 mol/L or particles per trillion) concentrations [2,3,4]. Different diseases have characteristic metabolic profiles that can be captured by using exhaled VOC profiles (“breathprints”). For the interpretation of huge datasets arising from a complex mixture of thousands of widely different volatile molecules to provide clinically relevant information for discrimination between health and disease and for the prediction of therapeutical responses, several statistical algorithms have been used resulting in variable levels of diagnostic accuracy [5,6]. The large size of mass spectrometers, and the substantial expense and heavy workload required for sample processing have represented a major bottleneck for the point-of-care (POC) clinical applicability of these measurements. Two small molecules, hydrogen (H₂) and methane (CH₄), represent good examples of this transition, as they have made their way to be measured by POC tests and are widely used in the differential diagnosis of gastrointestinal disorders [7,8]. Hydrogen and methane-based breath tests are used to diagnose and monitor small intestinal bacterial overgrowth and carbohydrate maldigestion and guide clinicians to prescribe appropriate medication [9,10]. These tests are based on the observation that H₂ and CH₄ are produced by the bacterial fermentation of unabsorbed carbohydrate in the small intestine during digestion and diffused to the blood that carries them to the alveoli from where they are exhaled. Since human cells do not produce them, their concentrations in breath are related to the interstitial bacterial flora.

As well as VOCs, the environmental-pollutant-free radical nitric oxide (NO), a known gaso-transmitter in the body, was also detected in exhaled breath with trace concentrations in healthy subjects and elevated levels in asthmatic patients [11,12]. Determination of fractional exhaled NO (FeNO) has generated great interest as a potential biomarker of asthma. This was mainly based on its correlation with eosinophils and its increase after allergen exposure, suggesting that it may be useful as a predictive marker of asthma attacks and the therapeutic response [13,14,15]. FeNO has served as a prototype of exhaled biomarkers for disease monitoring and medical decision making. Several machines have US Food and Drug Administration approval and/or a European Union CE-mark as medical device for its measurement [16]. The other two toxic environmental pollutants with known gaso-transmitter functions in the human body, carbon monoxide (CO) and hydrogen sulfide (H₂S), can also be detected in exhaled breath. Their levels are altered in different diseases, such as asthma, chronic obstructive pulmonary disease (COPD) and cystic fibrosis [17,18,19,20,21]. However, the use of exhaled CO as a biomarker of heme oxygenase activity is hampered by the strong and long-standing effect of smoking on the exhaled CO level, and the exhaled H₂S level is profoundly influenced by its oral and gastrointestinal bacterial production [20,22].

Several species have a lot more sensitive olfactory systems than humans including dogs, rats and different insects. The specific coupling of large numbers of receptors with the brain neural network enables these species to recognize minor changes in the volatome of different human samples including the breath. This led to the idea of involving trained animals in human medicine and diagnostics. Sniffer dogs have been trained successfully to distinguish biological samples obtained from healthy and diseased individuals. They have been shown to identify patients with Parkinson’s disease [23], lung cancer [24], prostate cancer [25], ovarian cancer [26] and different infectious diseases [27] from samples such as urine, blood, serum, cell lines and bacterial cultures with very high sensitivity. Dogs can also be trained to alert to hypoglycemic periods in type 1 diabetics [28]. Moreover, even untrained dogs have been shown to sniff out the prodromal phase of seizures and respond to the unusual odor changes with an increase in affiliative behaviour directed at their owners [29]. As well as dogs, other animal species have been tested in odor-pattern-based diagnostics. For instance, African giant pouched rats can detect Mycobacterium tuberculosis, the pathogens causing tuberculosis very sensitively [30,31], and are indeed used for first line diagnostics in Africa. Insects, such as mosquitos and honeybees also have a very sensitive olfaction with great discriminatory power to detect a tremendous amount of chemical signals [32,33]. Moreover, bees have already been successfully trained to detect specific odors [34].

Compared to the detection and quantification of individual molecules, using the biological olfactory systems of animals as a model to build artificial olfaction systems, so-called electronic noses, is a completely different approach. Electronic noses consist of arrays of chemical vapor sensors that respond to certain characteristics of odorant molecules including exhaled VOCs. Sensors are not specific to a given molecule, a sensor may react with several different molecules and a given molecule may also generate responses from several sensors. In this approach individual molecules are not identified and quantified as they are by mass spectrometry; only the pattern of sensor responses (“breathprint”) induced by a complex mixture of different volatiles is clustered. Despite the limitation of the black box approach due to the versatile nature of potential arrays of chemosensitive sensors, their small size and low cost, they have gained great attention as potential point-of-care clinical tools [35,36,37]. Their integration with artificial intelligence for data analysis has contributed importantly to the rapid development of this field [38].

2. Exhaled Gaso-Transmitters