Exhaled breath analysis: The new interface between medicine and engineering

Abstract

Exhaled breath testing is becoming an increasingly important non-invasive diagnostic method that can be used in the evaluation of health and disease states in the lung and beyond. Potential advantages of breath tests over other conventional medical tests include their non-invasive nature, low cost, and safety. To advance in this area further, however, there has to be a close collaboration between technical experts and engineers who have devices looking for clinical application(s), the medical experts who have the clinical problems looking for a test/biomarker that can be helpful in diagnosis or monitoring, and industry/commercial experts who can build and commercialize the final product.

As we breathe out we expel thousands of molecules into the air. When correctly captured and analyzed these molecules make a “breath-print” that can tell a lot about the state of our health [1]. The synergies between medicine and engineering in this area have the potential to revolutionize the way we monitor health and disease and allow us to provide personalized care for each individual based on his or her own “breath-print” [1].

The current conditions in the breath analysis field could not be better: the science is mature, the technology is exploding, industry is interested, and the medical community is embracing this noninvasive method of testing. Thanks to major engineering and breakthroughs in the 20th century including new technologies (infrared, electrochemical, chemiluminescence, and others) and the development of very sensitive modern mass spectrometry (MS), gas chromatography (GC) and gas chromatography mass spectrometry (GC–MS) instruments, we can now identify thousands of unique substances in exhaled breath. These substances include elemental gases like nitric oxide and carbon monoxide and a multitude of volatile organic compounds (Table 1). Furthermore, exhaled breath also carries aerosolized droplets collected as “exhaled breath condensate” that have non-volatile compounds that can be captured by a variety of methods and analyzed for a wide range of biomarkers from metabolic end products to proteins to a variety of cytokines and chemokines and the possibilities continue to expand [2,3]. With various technologies available to test for any and all of these exhaled breath components (Table 2), several methods are now in clinical use or about ready to enter that arena. Breath analysis is now used to diagnose and monitor asthma, to check for transplant organ rejection, and to detect lung cancer, to mention a few applications. Thus, the 21st century promises to deliver a revolution in our understanding of the constituents of exhaled breath and the advancement of the field of breath analysis and testing.

Table 1.

Markers identified in exhaled breath with their chemical structure, the associated medical conditions, and the techniques used to measure them.

Breath marker	Condition(s)	Technique(s)	References
hydrocarbons
Methane CH4	Oxidative stress, irritated bowel syndrome, breast cancer, heart transplant rejection, bronchial asthma, rheumatoid arthritis, schizophrenia, acute myocardial infraction	GC–MS, laser spectroscopy, mid infrared technology, methane breath test, GC–FID	[23,63-65]
Ethane C2H6	Oxidative stress, breast cancer, diabetes COPD, heart transplant rejection, OSA acute myocardial infraction ARDS, schizophrenia, rheumatoid arthritis bronchial asthma, scleroderma/systemic sclerosis lipid peroxidation of unsaturated fatty acids	GC–MS, GC–FID MIR, laser absorption spectroscopy cavity ring down spectroscopy cavity leak-out spectroscopy (CALOS)	[23-28]
Propane C3H8	Oxidative stress, lipid peroxidation of unsaturated fatty acids	GC–MS, laser spectroscopy	[24,23]
Butane C4H10	Lipid peroxidation of unsaturated fatty acids	GC–MS, automated thermal desorption	[25,26]
Pentane C5H12	Oxidative stress, cystic fibrosis, bronchial asthma, breast cancer, heart transplant rejection, rheumatoid arthritis acute myocardial infraction, SIRS, cigarette smoking, lipid per oxidation of unsaturated fatty acids	GC–MS, laser spectroscopy, SIFT–MS, CALOS analyzer automated thermal desorption GC–FID GC–PID	[23,24,26-32]
Hexane C6H14	Lung cancer, lipid peroxidation of unsaturated fatty acids	GC–MS, automated thermal desorption	[23,26,33]
Heptane C7H16	Lung cancer, lipid peroxidation of unsaturated fatty acids	GC–MS, automated thermal desorption	[23,26,33,34]
Octane C8H18	Lung cancer, lipid peroxidation of unsaturated fatty acids	GC–MS, automated thermal desorption	[23,26,33]
Nonane C9H20	Lung cancer, lipid peroxidation of unsaturated fatty acids	GC–MS, GC–time of flight, automated thermal desorption	[23,26,33]
Decane C10H22	Lung cancer, lipid peroxidation of unsaturated fatty acids	GC–MS, automated thermal desorption	[23,26,33]
Undecane C11H24	Lung cancer, lipid peroxidation of unsaturated fatty acids	GC–MS, automated thermal desorption	[23,26,33]
1-Methyl-2-pentylcyclopropane C10H12	Lung cancer, breast cancer	GC–MS, automated thermal desorption	[26,33,23]
Isoprene 2-methyl-1,3-butadiene C5H8	Lung cancer, breast cancer, hypercholesterolemia, oxidative stress	GC–MS, automated thermal desorption, laser spectroscopy, SIFT–MS	[24,35,28,26,36,23]
2-Methylpropane CH3–CH(CH3)CH3	Breast cancer	GC–MS, automated thermal desorption	[33,23,26]
2-Methylpentane CH3CH(CH3)CH2CH2CH3	Lung cancer, breast cancer pulmonary TB	GC–MS, automated thermal desorption	[26,36,33,23,44]
Methylcyclopentane C5H9(CH3)	Lung cancer, breast cancer	GC–MS, automated thermal desorption	[34,23,26,33]
1,4-Dimethylcyclohexane	Pulmonary TB	GC–MS	[44]
2,5-Dimethylhexane C6H12(CH3)2	Active/passive smoking	GC–MS, GC–time of flight automated thermal desorption	[26,33]
Cyclohexane C6H12	Lung cancer, breast cancer	GC–MS, automated thermal desorption	[33,23]
2-Methylheptane C7H15(CH3)	Lung cancer, breast cancer	GC–MS, automated thermal desorption	[26,33,23]
3-Methylheptane	Pulmonary TB	GC–MS	[44]
2,2,4,6,6-Pentamethyl heptane CH3–C(CH3)2–CH2–CH(CH3)–CH2–C(CH3)2–CH3	Pulmonary TB, lung cancer, breast cancer	GC–MS, automated thermal desorption	[36,23,44]
2-Methyloctane CH3–CH(CH3)–CH2–CH2–CH2–CH2–CH2–CH3	Lung cancer, breast cancer	GC–MS, automated thermal desorption	[26,23]
3-Methyloctane CH3–CH2–CH(CH3)–CH2–CH2–CH2–CH2–CH3	Lung cancer, breast cancer	GC–MS, automated thermal desorption	[33,23]
3-Methylnonane CH3–CH2–CH(CH3)–CH2–CH2–CH2–CH2–CH2–CH3	Lung cancer, breast cancer	GC–MS, automated thermal desorption	[26,23]
3-Methylundecane CH3–CH2–CH(CH3)C8H17	Breast cancer	GC–MS, automated thermal desorption	[33]
5-Methyltridecane	Breast cancer	GC–MS, automated thermal desorption	[26]
3-Methylnonadecane CH3–CH2–CH(CH3)C16H33	Breast cancer	GC–MS, automated thermal desorption	[26]
4-Methyldodecane CH3–CH2–CH2–CH(CH3)C16H33	Breast cancer	GC–MS, automated thermal desorption	[26]
Methylcyclododecane	Pulmonary TB	GC–MS	[44]
Ethylene	Anesthetics inhalation	GC–MS	[34]
Butadienes CH2=CH–CH=CH2	Periodontal disease	SIFT–MS	[23]
1-Hexene C6H13	Lung cancer, breast cancer	GC–MS	[23]
1-Heptene C7H15	Lung cancer, breast cancer	GC–MS	[23]
1-Octene	Lung cancer	GC–MS	[44]
Benzene C6H6	Lung cancer, breast cancer, smoke exposure	GC–MS, SIFT–MS	[24,37]
Trimethylbenzene C6H3(CH3)3	Lung cancer, breast cancer	GC–MS	[36,23]
1,2,3,4-Tetramethylbenzene	Pulmonary TB	GC–MS	[44]
Ethylbenzene C6H5–C2H5	Lung cancer, breast cancer	GC–MS	[23,44]
Propylbenzene C6H5C3H7	Lung cancer, breast cancer	GC–MS	[23,44]
Toluene C6H5CH3	Lung cancer	SIFT–MS, GC	[24,37,38]
o-Toluidine 2-amino-1-methylbenzene C7H9N	Lung cancer	GC–MS	[34]
m-Toluidine 3-amino-1-methylbenzene C7H9N	Lung cancer	GC–MS	[34]
Aniline C6H5NH2	Lung cancer	Gas chromatography	[34]
Styrene C6H5CH=CH2	Lung cancer	GC–MS	[36,34,23]
Xylenes C6H4(CH3)2	Lung cancer	GC–MS, SIFT–MS	[38,23]
Naphthalene C10H8	Occupational exposure pulmonary TB	GC–MS	[34,39]
Heteroarenes
Pyridine C5H5N	Periodontal disease	GC–MS	[40]
2-Methylfuran C5H6O	Smoking	GC–MS, GC–TOF, automated thermal desorption	[34,41]
2,5-Dimethylfuran C6H8O	Smoking	GC–MS, GC–time of flight automated thermal desorption	[41]
2,3-Dihydro-1-phenyl-4(1H)-quinazoline C8H6N2	Lung cancer	GC–MS	[23]
Indole C8H7N	Liver disorders cirrhosis	GC–MS, SIFT–MS	[38,34]
Skatole C9H9N	Aminuria	Gas chromatography	[34]
Alcohols
Methanol CH3OH	Overgrowth of intestinal bacterial flora	SIFT–MS, GC–MS	[24,23,35,42]
Ethanol C2H5OH	Overgrowth of intestinal bacterial flora, alcohol consumption	SIFT–MS GC–MS, SPME GC–MS, GC–PID	[24,23,42,43,44,35]
n-Propanol C3H7OH	Lung cancer	GC–MS	[42,45]
2-Propanol CH3CH(OH)CH3	Acetone reduction by bacterial flora, lung cancer, breast cancer, stomach cancer	GC–MS, SIFT–MS, SPME GC–MS	[24,44,23,53]
Dodecanol C12H25–OH	Ovulation	GC–MS	[34]
Tetradecanol C14H29–OH	Ovulation	GC–MS	[34]
Aldehydes
Formaldehyde HCHO	Lung cancer, prostrate cancer, bladder cancer	GC–MS, laser photoacoustics-quartz PTR-MS	[23]
Acetaldehyde CH3CHO	Lung cancer, alcohol consumption	GC–MS	[34,23,46]
Malondialdehyde CH2(CHO)2	Oxidative stress	GC–MS	[25]
Hexanal C6H12O	Lung cancer	GC–MS	[23]
Heptanal C7H14O	Lung cancer, breast cancer	GC–MS	[23,53]
Nonanal	Pulmonary T.B.	GC–MS	[44]
p-Tolualdehyde CH3–C6H4–CHO	Unknown	GC–MS	[34]
Ketones
Acetone 2-propanone OC(CH3)2	Diabetes mellitus, ketosis, halitosis	GC–MS, SIFT–MS laser spectroscopy, SPME GC–MS CALOS analyzer, CRDS	[24,23,35,47,45,44,34,42]
1-Phenyletanone C6H5COCH3	Lung cancer, breast cancer	GC–MS	[23,48]
2-Butanone CH3C(O)CH2CH3	Liver function disorder, lung cancer, halitosis	GC–MS	[47,45]
Ethyl, methyl ketone	Liver disorders	GC–MS	[34,47]
Penta-2-none C5H10O	Liver disorders, halitosis	GC–MS	[47,45]
1-Methyl,3-heptanone	Pulmonary TB	GC–MS	[39]
2,3-Dihydro-1-phenyl-4(1H)-quinazolinone	Breast cancer	GC–MS	[48]
Esters
Isopropyl myristate/ tetradecanoic acid C17H34O2	Breast cancer, lung cancer	GC–MS	[23,53]
Carboxylic acids
C2–C8 normal and branched org. acids	Upper respiratory/oropharyngeal carcinoma	Gas chromatography	[34]
Propanoic acid	Liver disorder	Gas chromatography	[34]
Butyric acid CH3–CH2–CH2–COOH	Liver disorders	Gas chromatography	[34]
Isobutyric acid	Liver disorders	Gas chromatography	[34]
Isovaleric acid 3-methylbutanoic acid C5H10O2	Liver disorders	Gas chromatography	[34]
Pentanoic acid	Liver disorders	GC–MS	[34]
Ammonia
NH3	Uremia, peptic ulcer, kidney disease	Gas chromatography laser-cavity ring-down spectroscopy	[49,23,34,50,51]
Amines (Ammonia derivatives) R–NH2
Putrescine/1,4-diaminebutane	Aminuria	Gas chromatography	[34,23]
Cadaverine/1,5-diaminepentane	Aminuria	Gas chromatography CRDS	[34]
Dimethyl amine (CH3)2NH	Aminuria kidney failure	Gas chromatography CRDS	[23,34]
Trimethyl amine (CH3)3N	Aminuria kidney failure	Gas chromatography CRDS	[23,34]
13C Urea CO(NH2)2	Helicobacter pylori	Ammonia/urea breath test	[52]
Sulfides
Methylsulfide CH3S	Intestinal bacterial flora, liver disorder	GC–MS, SIFT–MS	[23]
Dimethylsulfide CH3–S–CH3	Intestinal bacterial flora, liver disorders, oral infection	SIFT–MS	[34]
Dimethyldisulfide S(CH3)2	Liver disorders, oral infection	GC–MS, SIFT–MS	[24,23]
Methanethiol CH4S	Liver disorders, ovulation and menstrual cycle	GC–MS, SIFT–MS	[34]
Ethanethiol C2H5S	Liver disorders	GC–MS	[23,34]
Hydrogen sulfide HS	Periodontal disease, ovulation and menstrual cycle	Gas chromatography	[35,34]
Carbonyl sulfide O=C=S	Acute lung transplant rejection	Mid infra red absorption spectroscopy, laser spectroscopy, SIFT–MS	[52]
Carbon disulfide CS2	Liver allograft rejection, schizophrenia, aminuria	SIFT–MS solid phase microextraction and gas chromatography-mass spectrometry (SPME GC–MS)	[49,45,44]
Hydrogen
H2	Disorder in digestion and absorption, small bowel bacterial overgrowth	H-breath test	[53,54-56,63-65]
Oxides of carbon
Carbon monoxide CO	Differentiation between current/passive smokers, hyperbillurubinemia, hemolysis, lung diffusion capacity, diabetes, asthma, inflammation, oxidative stress	Mid infra red spectroscopy (MIR) IR laser absorption spectroscopy quantum cascade laser technique CRDS QCL based sensor CALOS analyzer	[57,35,58,51]
Carbon dioxide CO2	Intestinal bacterial flora, H. pylori (13CO2)	Mid infra red absorption spectroscopy CALOS analyzer	[58,51,59,34,54,63-65]
Oxides of nitrogen
Nitric oxide (NO)	Asthma, pulmonary hypertension, inflammation, oxidative stress, cystic fibrosis, rhinitis, bronchitis, asbestos related lung disorders, end stage renal disease	MIR, chemiluminescence analyzer, laser-multi pass cell, laser cavity enhanced technique, LMRS, ring down spectroscopy (14NO and 15NO) CALOS, Faraday modulation spectroscopy. NO analyzer	[50,38,60,23]
Nitrogen dioxide (NO2)	Airway inflammation	Laser spectroscopy
Nitrate	Asthma, cystic fibrosis	SIFT–MS	[52]
Nitrite	Asthma, cystic fibrosis	SIFT–MS	[52]
Nitrous oxide (N2O)	Airway inflammation	Laser photoacoustics quartz Spectroscopy	[61]
Alkyl halides
Vinyl chloride H2C=CHCl	Exposure to VOCs from contaminated water	GC–MS	[62]
Cis-1,2-dichloroethane C2H4(Cl)2	Exposure to VOCs from contaminated water	GC–MS	[62]
1,1,1-Trichloroethylene	Exposure to VOCs from contaminated water	GC–MS	[37]
1,1,1-Trichloroethane	Exposure to VOCs from contaminated water	IR-spectrometry	[34]
Tetrachloroethylene	Exposure to VOCs from contaminated water	GC–MS	[37]
Bromodichloroethane Br–CH2–CH(Cl)2	Exposure to VOCs from contaminated water	GC–MS	[62]
Bromotrichloroethane Br–CH2–C(Cl)3	Exposure to VOCs from contaminated water	GC–MS	[62]
Dichloroethane	Exposure to VOCs from contaminated water	GC–MS	[34]
Chloroform CHCl3	Exposure to VOCs from contaminated water	GC–MS	[34,62]
Dimethyl selenide	Liver Disorders	GC–MS	[47]

Table 2.

Techniques and devices used in breath analysis.

Optical techniques

Laser absorption spectroscopy

Mid infra red absorption spectroscopy

Multi pass cell-laser absorption spectroscopy

Tunable diode-laser spectroscopy

Cavity ring-down spectroscopy

Cavity leak-out spectroscopy

Cavity enhanced comb spectrometer

Integrated cavity output spectroscopy

Laser magnetic resonance spectroscopy (LMRS)

Laser photoacoustics-quartz spectroscopy

Faraday-LMRS

Quantum cascade laser MIR spectroscopy

Proton transfer reaction mass spectroscopy

Faraday modulation spectroscopy

Non-optical techniques

Gas chromatography (GC)

Mass spectroscopy (MS)

GC–MS

GC–flame ionization

GC–photoionization

GC–solid phase micro extraction chromatography

GC–automated thermal desorption chromatography

GC–time of flight

Selected ion flow tube mass spectroscopy (SIFT)

Ion mobility spectrometry (IMS)

While the concept of using breath analysis as a medical test may be a surprise to engineering professionals, it is an easier to accept fact for medical professionals. The history of medicine is replete with discoveries that led to our current day understanding of the diagnostic potential of exhaled breath. Hippocrates described fetor oris and fetor hepaticus in his treatise on breath aroma and disease, Lavoisier and Laplace in 1784 showed that respiration consumes oxygen and eliminates carbon dioxide [4], Nebelthau in mid 1800s showed that diabetics emit breath acetone [5], and Anstie in 1874 isolated ethanol from breath (which is the basis of breath alcohol testing today) [6]. A major breakthrough in the scientific study of breath started in the 1970s when Linus Pauling demonstrated that there is more to exhaled breath than the classic gases of nitrogen, oxygen, carbon dioxide and water vapor. Using gas–liquid partition chromatography analysis, Pauling demonstrated the presence of 250 substances in exhaled breath [7].

The upward trajectory of the breath analysis field and related product development and manufacturing was very clear after a three-day Breath Analysis Summit hosted at the Cleveland Clinic in November 2007. The Summit was perhaps a turning point in the field as it attracted participants from 22 countries and 18 states [1]. The Summit brought together industry executives and entrepreneurs with scientists, engineers, and clinicians to discuss key trends, future directions, and upcoming technologies in breath analysis and medicine. The major focus of the Summit was on new technologies and medical applications and to address the major hurdles that faced this field as it transitions from the laboratory to clinical testing. Topics included exhaled nitric oxide, exhaled breath condensate, electronic nose and sensor arrays, mass spectrometry and bench top instrumentation, and cutting edge sensor technologies. Medical applications that were covered included asthma, COPD, pulmonary hypertension, other respiratory diseases, gastrointestinal diseases, occupational diseases, critical illness, and cancer [1]. The major conclusions of the summit were clear. Multidisciplinary collaboration is the only way to keep moving the field forward and to allow breath analysis and testing to achieve their potential and hold their rightful place among other accepted medical tests. The members of this team need to include scientist and engineers who have the technical knowledge and can build the appropriate devices, medical professionals who understand the disease processes and know the areas of greatest medical need, as well as industry to build devices for testing and later distribute the successful ones to a national and worldwide healthcare market. This is where the synergy between medicine and engineering is at its best.

Most likely to succeed in these endeavors are integrated multidisciplinary teams that join colleagues in industry and academia for the focused purpose of rapid development and commercialization of urgently needed non-invasive devices that will guide the diagnosis and monitoring of different diseases by exhaled breath testing. While each of these team members can independently make major contributions to their respective fields, the potential for having them work together is enormous and cannot be overstated. The process could start with a clinical need as identified by a physician; the appropriate sensor(s) can then be developed by team members with biochemical and engineering expertise. It is not uncommon, however, for the process to start from the engineering end when a new technology or device is found to be particularly sensitive or specific to detect a certain molecule or group of molecules and the medical team is approached for a possible medical application of this new technology. Regardless of where the process starts, however, it is likely to need continuous work and refinement from all involved. Once a sensor is developed a prototype is built and validated by an industrial partner and sent to the medical team for human testing. Depending on the human test results, the device may be sent back for further refinement and retesting. This process/cycle is repeated until a sensor/device achieves its expected goals. Once a device is ready, it goes back to the industry partner(s) for production on larger scale for clinical trials that will be needed for regulatory approval.

One great example of how the collaboration between technical, medical, and commercial professionals has resulted in a clinically useful tool is the measurement of exhaled nitric oxide (NO) in exhaled breath for monitoring airway inflammation. The advent of chemiluminescence analyzers in the early 1990s allowed the detection of low (ppb) levels of NO in exhaled breath [8]. This was quickly followed by the observation that patients with asthma had higher than normal levels of NO in their exhaled breath that was later linked to eosinophilic airway inflammation [9,10]. Standardization of the gas collection methods and measurement techniques allowed the industry to build the next generation of analyzers suitable for use in the clinical setting [11-14]. In 2003 the FDA approved the first desktop NO analyzer for monitoring airway inflammation in asthma [15]. This new test has evolved into an important tool in the modern management of asthma and airway inflammation. The use of exhaled NO in monitoring asthma is useful for several reasons. It is non-invasive, it can be performed repeatedly, and it can be used in children and patients with severe airflow obstruction where other techniques are difficult or not possible to perform. Exhaled NO may also be more sensitive than currently available tests in detecting airway inflammation which may allow more optimum therapy [13,16-22].

As breath analysis offers a window on lung physiology and disease, exhaled breath testing is becoming an increasingly important non-invasive diagnostic method that can be used in the evaluation of health and disease states in the lung and beyond. Potential advantages of breath tests over other conventional medical tests include their non-invasive nature, low cost, and safety. This is an area where the modern day advances in technology and engineering meet the ever expanding need in medicine for more sensitive, specific and non-invasive tests which makes this area a major front in the interface between medicine and engineering.