SYSTEMATIC REVIEW AND META-ANALYSIS
|Year : 2022 | Volume
| Issue : 1 | Page : 100-104
|Volatile Organic Compounds in Human Breath
Monika Karunagaran, Pratibha Ramani, S Gheena, R Abilasha, R Hannah
Department of Oral Pathology and Microbiology, Saveetha Dental College and Hospital, Saveetha Institute of Medical and Technical Science, Saveetha University, Chennai, Tamil Nadu, India
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|Date of Submission||18-May-2020|
|Date of Acceptance||06-Jun-2020|
|Date of Web Publication||09-Aug-2022|
| Abstract|| |
A comprehensive analysis of volatile organic compounds (VOCs) from the exhaled breath sample is termed as breathomics. Breath samples are a complex mixture composed of a multitude of VOCs and other molecules. The analysis of total VOCs in exhaled breath provides a promising tool for the diagnosis of many diseases because it enables the observation of biochemical processes in the body in a non-invasive way. VOCs are produced in various physiological and pathophysiological conditions thus making it a potential biomarker for several diseases.
Keywords: Breath analysis, GC-MS, VOCs
|How to cite this article:|
Karunagaran M, Ramani P, Gheena S, Abilasha R, Hannah R. Volatile Organic Compounds in Human Breath. Indian J Dent Res 2022;33:100-4
|How to cite this URL:|
Karunagaran M, Ramani P, Gheena S, Abilasha R, Hannah R. Volatile Organic Compounds in Human Breath. Indian J Dent Res [serial online] 2022 [cited 2022 Oct 4];33:100-4. Available from: https://www.ijdr.in/text.asp?2022/33/1/100/353539
| Introduction|| |
The exhaled air contains volatile compounds at a concentration related to the blood concentrations. Nearly 200 compounds can be detected in human breath and it is correlated to various diseases. The actual breath contains mixtures of oxygen, carbon dioxide, water vapour, nitrogen, inert gases, and may also contain various elements and more than 1000 trace volatile compounds. Concentration ranges from parts per million to parts per trillion. The VOCs are usually the metabolic products of the body and most commonly found in normal breath are acetone, ethane and isoprene.
The volatile fraction of breath comprises less than 1% of the total volume, but contains hundreds (potentially thousands) of low molecular weight compounds that can be detected, identified and quantified with appropriate instrumentation. These VOCs arise from multiple sources, which are broadly categorised as:
- Exogenous: Environmental VOCs are ubiquitous. Common sources include combustion of fuel, cigarettes, fragrances, and cooking. Many VOCs can arise from both biological and non-biological processes and their source is not usually possible to find. Further, VOCs may be used as a substrate for metabolic processes and so finding reduced levels in exhaled air may be as scientifically relevant as finding raised levels.
- Endogenous: These are the compounds that arise from host metabolism. Healthy metabolic processes will result in the production, consumption, and alteration of VOCs. Any changes in these processes such as up- or down-regulation due to any disease would cause an effect on the exhaled VOC profile. Targeting specific portions of the breath should facilitate sampling of VOCs from specific compartments, that is, the upper airway “deadspace” (including the mouth, and so potentially of interest to oral hygiene researchers); the conducting airways (of interest in airways diseases such as asthma); and the alveolar air. Volatiles in this latter zone will be in equilibrium with the lung interstitium as well as capillary blood.
- Biological/non-host: The airways are not sterile even in health. The metabolism of the lung (and oral/nasal) micro-biome will also affect the exhaled VOC profile. This micro-biome will be altered in airway/lung infectious disease and in the presence of antimicrobial therapy, it would lead to changes in exhaled VOCs.
Classification of volatile organic compound
- Saturated hydrocarbons (Ethane, pentane, and aldehydes) produced by lipid peroxidation of fatty acid components of cell membranes–triggered by reactive oxygen species
- Unsaturated hydrocarbons (Isoprene) produced by Mevalonic pathway of cholesterol synthesis
- Oxygen-containing (Acetone) produced by Decarboxylation of acetoacetate from lipolysis or lipid peroxidation
- Sulfur-containing (Ethyl mercaptane and dimethylsulfide) produced by incomplete metabolism of methionine
- Nitrogen-containing (Dimethylamine and ammonia) are elevated in liver impairment and uremia
Major VOCs in human exhaled breath
1. Saturated hydrocarbons
The major compounds are ethane and pentane and their fractions in exhaled breath are of systemic origin. These compounds are thought to originate from lipid peroxidation, a chain reaction that starts when an allylic hydrogen atom is eliminated by reactive oxygen species (ROS). The resulting radical is conjugated, peroxidised by oxygen, and undergoes further chemical reactions. The generation of ROS is described as the destructive aspect of oxidative stress. In oxidative stress, cells are damaged as a result of a chemical reaction with oxidative agents such as superoxide anion or hydroxyl radical. ROS bear free radicals and peroxides. These species may undergo further chemical changes and turn into more aggressive radical agents that can potentially cause extensive cellular damage. Saturated hydrocarbons are produced from × 3 (e.g., linolenic acid) and × 6 fatty acids (e.g., linoleic and arachidonic acid), respectively. These fatty acids have been described as the fundamental components of cell membranes.
Several diseases, for example, lung cancer, HIV, and inflammatory bowel disease are also influenced by oxidative stress induced by ROS generation. Such hydrocarbons are the end products of lipid peroxidation, which are stable and less soluble in blood and are released into the breath quickly after their formation in tissues. Thus, analysing the concentration of ethane and n-pentane in exhaled breath has the potential in monitoring the oxidative stress in the body. Elevated levels of ethane have been reported for patients with asthma, chronic obstructive pulmonary diseases (COPD), and cystic fibrosis. Pentane has also been observed at increased levels in asthma patients.
2. Unsaturated hydrocarbons
Isoprene (2-methyl-1, 3-butadiene) is the major unsaturated hydrocarbon found in human exhaled breath. It is thought to be formed in the mevalonic acid (MVA) pathway during cholesterol biosynthesis. Mevalonate is formed from acetic acid which is an important chemical reaction in the cholesterol biosynthesis. The compound 3-hydroxy-3-methyl-glutaryl coenzyme A (HMGCoA) catalyses the rate-limiting step of sterol synthesis. Then, mevalonate is converted in the cytosol to isopentenyl pyrophosphate, followed by isomerization to dimethylallyl pyrophosphate (DMPP). After passing through the formation of intermediate carbonium ion, DMPP is rapidly converted to isoprene via an acid-catalysed elimination reaction. Isoprene levels are decreased in patients with acute respiratory distress syndrome, cystic fibrosis, and asthma. The concentration of isoprene in breath appears to be age-dependent and is also increased during physical activity. Breath isoprene levels are also altered by physiological and pathophysiological states including haemodialysis, general anaesthesia, liver disease, and cancer.
3. Oxygen-containing compounds
Acetone is one of the major abundant VOCs in human breath. They originate from two major pathways. First is the decarboxylation of acetoacetate (major source in mammals) which arises from either lipolysis (lipid per oxidation) or amino acid degradation and the second pathway is the dehydrogenation of isopropanol or 2-propanol by liver alcohol dehydrogenase, ADH. Acetoacetate decarboxylation may occur both in an enzyme catalysed manner and non-enzymatically. Acetone which is formed is released mostly via urine and exhaled breath from the body. Acetone may also be further metabolized to acetate, formate, and carbon dioxide. Acetone, a natural metabolic intermediate of lipolysis, is used as a potential biomarker for monitoring the ketotic state of diabetic and fasting individuals, assessing fat loss, and measuring glucose levels. In the human body, the origins of ethanol and methanol can be exogenous or endogenous. The endogenous VOCs originate from microbial fermentation of the carbohydrates in the gastro-intestinal tract by the intestinal bacterial flora. Methanol derives from S-adenosyl methionine in the pituitary or from the breakdown of ethanol by intestinal flora. Sources of exogenous methanol in the healthy human body include fruits, vegetables, and alcoholic beverages.
4. Sulfur-containing compounds
Sulfur-containing compounds such as dimethyl sulfide, dimethyl disulfide, and ethyl mercaptan originate from the incomplete metabolism of methionine via the transamination pathway. These compounds give a distinctive odour in the breath of cirrhotic patients., So sulfur compounds can be suggested as major markers of liver failure. The breath of patients with hepatocellular failure has a sweet, musty, or slightly faecal aroma, termed fetor hepaticus and is mostly attributed to sulfur compounds. Impairment of liver function increases the level of these compounds, which have a characteristic smell, such as that of rotten cabbage. Bacteria and fungi related to lung infections, such as Haemophilus influenza and Streptococcus pneumoniae) also generate dimethyl sulfide. Normally, human blood and breath shows very low (a few parts per billion) concentrations of sulfur-containing compounds.
5. Nitrogen-containing compounds
Ammonia is the major nitrogen-containing volatile biomarker. It originates in the body as a breakdown product of proteins. A substantial amount of these proteins comes from the bacterial degradation of proteins in the intestine. In the liver, ammonia is converted to urea and released mostly via urine and partly through exhaled breath and the skin. Increased levels of ammonia have been observed in the blood when the liver fails to convert ammonia to urea as seen in subjects with cirrhosis or severe hepatitis.,, Patients with chronic renal failure have been reported to exhibit characteristic uremic breath odour, which is termed 'ammoniacal', 'fishy', or 'fetid' because of the presence of nitrogen-containing compounds (e.g., dimethylamine and trimethylamine) at elevated concentration levels. Cigarette smoking also may influence the ammonia concentration in the exhaled breath. It has been reported in a study that relatively high concentration range [245–2935 ppb] of breath ammonia is produced in normal healthy humans. Such elevated levels may also be related to bacterial production in the oral cavity.
Some exhaled breath VOCs and their concentration ranges in normal healthy subjects are given in [Table 1].
|Table 1: Various breath VOCs and their concentrations in healthy normal subjects|
Click here to view
| Breath Analysis|| |
The analysis of exhaled breath and associated VOCs enables the non-invasive observation of the biochemical processes of the human body. The first initiatives of breath analysis for determining the physiological state of humans originated during the time of Hippocrates (460-370 BC), when the ancient Greek physicians realized that some diseases could be diagnosed from the characteristic odour of patients' breath and found that the human breath might provide sound information on health conditions. In the period 1782–1783, Lavoisier for the first time analysed the breath CO2 of Guinea pigs and showed that the gas is a product of combustion in the body. Practically, it is not difficult for a skilled technician to recognize the characteristic 'fruity smell' of acetone, 'musty and fishy smell', 'urine-like smell', and 'putrid smell' in the breath of patients with diabetes, advanced liver disease, kidney failure, and lung abscess, respectively.
Numerous studies have reported the significance of analysing VOCs in human breath. In 1938, the 'drunkometer' was introduced as a roadside test of breath alcohol concentration. In the 1970s, Pauling et al. used gas chromatography (GC) to detect more than 200 VOCs in human breath. In 1997, Phillips estimated 1259 VOCs from 20 normal healthy subjects using gas chromatography–mass spectrometry (GC/MS). In 1999, using GC/MS, Phillips et al. described 3481 VOCs in the breath gas of healthy controls, with an average number of about 200 VOCs detectable in an individual's breath gas. Recently, 1765 VOCs of healthy humans have been published.
Breath VOCs can be analysed online or offline, with specific methods for capture and analysis for each method. Online measurement allows for rapid, near-patient sample analysis and negates the need for sample storage. For offline measurement, the sample must be captured, stored and transported to the analytical instrument. Examples of analytical methods suitable for online analysis include forms of ion-mobility spectrometry (IMS), such as Selected Flow Tube – Mass Spectrometry (SIFT-MS) and proton transfer reaction – MS (PTR-MS), which do not require sample pre-concentration and separation.
| Principle|| |
External chemical enters the body by three routes namely ingestion, inhalation, and topical contact. The chemicals absorbed into systemic circulation will distribute in the body or is directly excreted into the faeces. These absorbed chemicals are of two types volatile and non-volatile. The volatile compounds get exchanged with alveolar air and are excreted in the breath, known as VOCs. Therefore, breath analysis detects VOCs. The non-volatile compound analysis is done by blood and urine. The breath analysis can be explained by pharmacokinetic models related to the VOCs. Wallace et al. discover a linear pharmacokinetic model of exhaled breath. Here were assume that someone not previously exposed to particular VOCs is suddenly given a high concentration at a constant rate (cair), now the alveolar breath concentration (calv) is given in the following equation:
calv = ƒ cair Σ ai × 1 − exp (−t/τi) + (1)
where f is the fraction of parent compound exhaled at equilibrium; τi is residence time in the ith compartment; ai is the fraction of breath concentration contributed by the ith compartment at equilibrium (t = ∞); t is the time of exposure (t = 0 at start of exposure); and Σ ai = 1
This model was used in the quantitative estimation of VOC in the body or conversely to estimate previous exposure of exhaled breath. The advantage of this model is that it could measure the long-term inhalation at low or moderate concentration compared to instantaneous intake or intermittent exposure at high concentrations. Wallace et al. presented equations that can apply to the n-compartmental model, and at the same time, it gives a linear equation with increasing exposures at low or high concentrations.
The principle of the breath analysis explains physiological basis of the exchange of gases between air and blood. Gas exchange occurs at the surface of numerous tiny chambers known as alveoli, present at the tip of the bronchial air passage. Alveoli are lined with very thin membranes that are loaded with capillaries, which mean there are tiny distances between red blood cells moving through the capillaries and the air inside the alveolus, the large surface area and tiny distance associated with alveoli afford a ready opportunity for volatile organic compounds to diffuse from the air into the blood. The key challenge in the analysis of the breath is separating the alveolar breath from the breath contained in the upper airways namely mouth and pharynx that is uninvolved in gas exchange. Another challenge is separating and identifying the volatile breath components which tend to be present at just picomolar level concentrations.
| Diagnostic Uses|| |
- Breath carbon monoxide test can detect neonatal jaundice
- Breath hydrogen test can detect disaccharides deficiency, gastrointestinal transit time, bacterial overgrowth and intestinal statis
- Breath nitric oxide test can be used for asthma therapy
- Breath test can detect heart transplant rejection
- Urea breath test can be used for helicobacter pyloric infection
- Breath analysis monitoring blood sugar level in patients with diabetes
- Breath analysis with colorimetric array can be used in the diagnosis of lung cancer
The gold standard method for VOC identification, especially for biomarker discovery, is gas chromatography–mass spectrometry (GC–MS). While the equipment is bulky and expensive and requires considerable expertise, it allows confident identification of VOCs present at very low concentrations in a sample. However, it requires the sample to be collected for “off-line” analysis. Typically, breath is sampled onto a small, portable sorbent tube that traps the VOCs for this purpose. Alternative methods for VOC-capture are solid phase micro extraction (SPME) fibres, or needle-trap devices.
| Future Scope|| |
A large number of VOCs have been found in human breath. Although many of them have been reported to be biomarkers of several diseases, their exact physiological interpretation, including their biochemical status both in diseased and healthy conditions of the subjects, needs further study. Studies need to be conducted with a greater sample size to give an insight into the VOCs produced in the body and its role in the disease causation and progression. Thus breath analysis of a patient at an early stage of any disease may help in improving the survival period of the patient and treating the patients at an early stage.
| Conclusion|| |
Breath tests are currently not routinely used in day-to-day clinical practice. Further research and development in breath analysis and the analytical instrumentation in breathomics will establish breath analysis as a reliable approach for the detection and monitoring of various diseases.
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Conflicts of interest
There are no conflicts of interest.
| References|| |
Shaji J, Jadhav D. Breath biomarker for clinical diagnosis and different analysis technique. Res J Pharm Biol Chem Sci 2010;1:639.
Davis MD, Fowler SJ, Montpetit AJ. Exhaled breath testing – A tool for the clinician and researcher. Paediatr Respir Rev 2019;29:37-41.
Wang XR, Cassells J, Berna AZ. Stability control for breath analysis using GC-MS. J Chromatogr B Anal Technol Biomed Life Sci 2018;1097-8:27-34.
Lärstad MAE, Torén K, Bake B, Olin AC. Determination of ethane, pentane and isoprene in exhaled air-Effects of breath-holding, flow rate and purified air. Acta Physiol 2007;189:87-98.
Sarbach C, Stevens P, Whiting J, Puget P, Humbert M, Cohen-Kaminsky S, et al
. Evidence of endogenous volatile organic compounds as biomarkers of diseases in alveolar breath. Ann Pharm Fr 2013;71:203-15.
Ross BM, Maxwell R, Glen I. Increased breath ethane levels in medicated patients with schizophrenia and bipolar disorder are unrelated to erythrocyte omega-3 fatty acid abundance. Prog Neuro-Psychopharmacology Biol Psychiatry 2011;35:446-53.
Miekisch W, Schubert JK, Noeldge-Schomburg GFE. Diagnostic potential of breath analysis-Focus on volatile organic compounds. Clin Chim Acta 2004;347:25-39.
Deneris ES, Stein RA, Mead JF. In vitro
biosynthesis of isoprene from mevalonate utilizing a rat liver cytosolic fraction. Biochem Biophys Res Commun 1984;123:691-6.
Salerno-Kennedy R, Cashman KD. Potential applications of breath isoprene as a biomarker in modern medicine: A concise overview. Wien Klin Wochenschr 2005;117:180-6.
King J, Mochalski P, Unterkofler K, Teschl G, Klieber M, Stein M, et al
. Breath isoprene: Muscle dystrophy patients support the concept of a pool of isoprene in the periphery of the human body. Biochem Biophys Res Commun 2012;423:526-30.
Van den Velde S, Nevens F, Van hee P, van Steenberghe D, Quirynen M. GC-MS analysis of breath odor compounds in liver patients. J Chromatogr B Anal Technol Biomed Life Sci 2008;875:344-8.
Tangerman A, Meuwese-Arends MT, van Tongeren JHM. A new sensitive assay for measuring volatile sulphur compounds in human breath by Tenax trapping and gas chromatography and its application in liver cirrhosis. Clin Chim Acta 1983;130:103-10.
Mansoor JK, Schelegle ES, Davis CE, Walby WF, Zhao W, Aksenov AA, et al
. Analysis of volatile compounds in exhaled breath condensate in patients with severe pulmonary arterial hypertension. PLoS One 2014;9:3-10.
Wlodzimirow KA, Abu-Hanna A, Schultz MJ, Maas MAW, Bos LDJ, Sterk PJ, et al
. Exhaled breath analysis with electronic nose technology for detection of acute liver failure in rats. Biosens Bioelectron 2014;53:129-34.
Davies MP, Barash O, Jeries R, Peled N, Ilouze M, Hyde R, et al
. Unique volatolomic signatures of TP53 and KRAS in lung cells. Br J Cancer 2014;111:1213-21.
Dent AG, Sutedja TG, Zimmerman PV. Exhaled breath analysis for lung cancer. J Thorac Dis 2013;5(S5):S540-50.
Phillips M. Method for the collection and assay of volatile organic compounds in breath. Anal Biochem 1997;278:272-8.
Buszewski B, Ligor T, Jezierski T, Wenda-Piesik A, Walczak M, Rudnicka J. Identification of volatile lung cancer markers by gas chromatography-mass spectrometry: Comparison with discrimination by canines. Anal Bioanal Chem 2012;404:141-6.
Grabowska-Polanowska B, Faber J, Skowron M, Miarka P, Pietrzycka A, Śliwka I, et al
. Detection of potential chronic kidney disease markers in breath using gas chromatography with mass-spectral detection coupled with thermal desorption method. J Chromatogr A 2013;1301:179-89.
Song G, Qin T, Liu H, Xu GB, Pan YY, Xiong FX, et al
. Quantitative breath analysis of volatile organic compounds of lung cancer patients. Lung Cancer 2010;67:227-31.
Das S, Pal S, Mitra M. Significance of exhaled breath test in clinical diagnosis: A special focus on the detection of diabetes mellitus. J Med Biol Eng 2016;36:605-24.
Dr. Monika Karunagaran
Senior Lecturer, Department of Oral Pathology and Microbiology, Saveetha Dental College and Hospital, SIMATS University, Chennai - 600 077, Tamil Nadu
Source of Support: None, Conflict of Interest: None
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