Exhaled breath is dynamic and its molecular profile changes during exhalation. The first part of exhaled breath is composed of the molecules that were present in the inhaled gas contained in the anatomic dead space. As the process of exhalation continues, the breath profile is defined by molecules derived from the oral cavity and the airways. Monitoring the concentration of carbon dioxide in the breath can be used to follow the progress of exhalation. When the concentration of carbon dioxide plateaus this portion of exhaled breath is known as end-tidal breath.
The molecular profile of end-tidal breath is defined by the concentrations and identities of the volatiles present in blood. The sources of the volatiles in blood are: molecules and/or their metabolites that have been inhaled (exposome), or have entered the bloodstream via the skin (exposome), have entered the bloodstream from ingested foods or beverages (metabolome); and molecules produced by foreign cells (viruses, bacteria, fungi, and yeasts) (microbiome) or by tissues in the body including the mouth, nose, sinuses, airway and the gastrointestinal tract (human metabolome). The majority of mixed-expired breath (99.995%) consists of nitrogen (78%), oxygen (13%), carbon dioxide (5%), water vapor (4%), and the inert gases, and the remainder (<50 ppmv) is a mixture of as many as 1000 different compounds. The rates of excretion of molecules in breath are directly related to rates of ventilation and cardiac output. The physical and chemical properties of molecules also affect their rates of excretion. If a molecule is lipid soluble, it could be stored in tissues not well perfused by blood, such as adipose tissue, and be released into breath more slowly than a different molecule with hydrophilic properties that is not lipid stored.
In general, the concentrations of endogenously produced molecules in breath are lower than the concentrations of molecules from exogenous sources. Unique molecules in breath can only originate from the ingestion, inhalation, or dermal absorption of exogenous substances or be metabolically produced by foreign cells (bacteria, viruses, or eukaryotic organisms). Normal cellular biochemistry can only be induced or suppressed by abnormal physiology and although disease states may appear to be producing unique molecules these results are only a reflection of the detection limit of the analytical method.
Collection of representative breath samples under controlled conditions is an a priori requirement for successful breath research. Ideally, breath should be sampled when the concentration of carbon dioxide reaches a plateau (end-tidal phase) and it is important that the person providing the sample (e.g., the patient) does not hyperventilate. Normally, the depth and frequency of breathing are under autonomic control. However, when the patient is asked to provide a breath sample this action invariably results in a change from autonomic breathing to conscious breathing. Standardized breath sampling methods or guidelines on best practices do not exist, but endeavors are currently underway to establish a common consensus, as will be disseminated via this website in due course.
Many of the modern analytical methods based upon spectroscopy, mass spectrometry, electrochemistry and chromatography have sufficient speed, sensitivity and selectivity to allow real-time breath analyses to be performed on a single breath. Standardized breathing protocols or guidelines on best practices do not exist, but endeavors are currently underway to establish a common consensus, as will be disseminated via this website in due course.
If comparisons of the results of breath analysis are to be used to study therapeutic intervention or to compare different study subjects, it is important to normalize concentrations of breath molecules to oxygen consumption or carbon dioxide production.
The use of breath for diagnosing disease will first require that the concentration profiles of breath molecules for normal healthy human subjects be established and these studies should include such variables as age, gender, ethnicity and body mass index. In order to recognize potential confounders, well-described cohorts of participants identified as “healthy” or “diseased” will need to provide breath samples that are evaluated for validity and reproducibility. Ideally, control subjects will be matched to the disease group for better comparability. A recent direction has been to use partners of patients, who are typically of similar age and, critically, generally share domestic exposure history with the patient, thereby reducing the likelihood of exogenous confounders being falsely attributed to disease.
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