A breath test is a type of test performed on air generated from the act of exhalation.Types include:Breathalyzer - By far the most common usage of this term relates to the legal breath test to determine if a person is driving under the influence of alcohol.Hydrogen breath test - it is becoming more and more common for people to undertake a medical test for clinical diagnosis of dietary disabilities such as fructose intolerance, fructose malabsorption, lactose intolerance and lactulose intolerance.The presence of Helicobacter pylori (in peptic ulcer disease) can be tested for with the urea breath test.Exhaled nitric oxide is a breath test that might signal airwayinflammation such as in asthma.
Breath tests are among the least invasive methods available for clinical diagnosis, disease state monitoring, and environmental exposure assessment. In recent years, interest in breath analysis for clinical purposes has increased. This review is intended to describe the potential applications of breath tests, including clinical diagnosis of diseases and monitoring of environmental pollutant exposure, with emphasis on oxidative stress, lung diseases, metabolic disorder, gastroenteric diseases, and some other applications. The application of breath tests in assessment of exposure to volatile organic compounds is also addressed. Finally, both the advantages and limitations of breath analysis are summarized and discussed.
The bulk matrix of breath is a mixture of nitrogen,oxygen, CO2, H2O, and inert gases. The remaining smallfraction consists of more than 1000 trace volatile organiccompounds (VOCs) with concentrations in the range ofparts per million (ppm) to parts per trillion (ppt) byvolume (5, 8–10).
In terms of their origin, these volatilesubstances may be generated in the body (endogenous) or may be absorbed as contaminants from the environment (exogenous). The composition of VOCs in breath varies widely from person to person, both qualitatively and quantitatively. These common VOCs, which include isoprene, acetone, ethane, and methanol, are products of core metabolic processes and are very informativefor clinical diagnostics (10 ). The bulk matrix and trace VOCs in breath exchange between the blood and alveolar air at the blood–gas interface in the lung. One exception is NO, which is released into the airway in the case of airway inflammation.
Because breath tests are among the least invasive methods for monitoring a person’s disease state or exposureto a drug or an environmental pollutant, interest in breath analysis for clinical diagnosis has increased in recent years.
Depending on the origin of the substances found in human breath, i.e., endogenous or exogenous, the typicalapplications of breath tests fall into two main categories: diagnosis of disease and assessment of exposure to environmental pollutants.
The detection of VOCs in breath for the purpose of diagnosis has a long history. Ancient Greek physicians already knew that the aroma of human breath could provide clues to diagnosis. The astute clinician was alert for the sweet, fruity odor of acetone in patients with uncontrolled diabetes; the musty, fishy reek of advanced liver disease; the urine-like smell that accompanies failing kidneys; and the putrid stench of a lung abscess (12 Modern breath analysis started in the 1970s when researchers, using gas chromatography (GC), identified more than 200 components in human breath [reviewed by Miekisch et al. (5 )]. For more than a decade the main problems were the effective separation and identification of exhaled substances, but because of technical advances in analytical methods in the 1980s and 1990s, this is no longer the case. Instead, issues concerning the physiologic meaning of breath substances and correlations of breath markers with patients’ clinical symptoms have become increasingly important (5, 13). Since the 1990s, researchers have worked hard to understand the relationship between various breath substances and physical condition.
As a result of extensive studies, a few breath markers have been discovered and successfully used in diagnosis of disease, and some of these markers are very promising. In this section, we reviews the links between substances measured by breath testing and disease. The breath markers for different diseases and applications are summarized in Table 1.
Lipid peroxidation is a chain reaction that is initiated by removal of an allylic hydrogen atom through ROS. The radical generated inthis way is conjugated, peroxidized by oxygen, and undergoes further reactions. Eventually, saturated hydrocarbons such as ethane and pentane are generated in this way from -3 and -6 fatty acids, which are basic components of cell membranes (9, 24). Aldehydes such as malondialdehyde (MDA) are generated along the same pathway (9 ).H2O2 is formed by inflammatory cells in the upper and lower airways. In general, exhalation of H2O2 appears to increase during unstable disease and is related to the total number of eosinophils in the sputum in asthma and with the total number of polymorph onucleates in induced sputum in chronic obstructive pulmonary disease (COPD).Many lung diseases, including asthma, COPD, bronchiectasis, CF, interstitial lung disease, and ARDS, involve chronic inflammation and oxidative stress. Because peroxidation and other reactions of ROS are basic mechanisms of inflammatory processes, markers of organic lipid peroxidation should be increased under these conditions. In fact, increased concentrations of pentane and ethane have been measured in the breath of patients with asthma 32 ), COPD (33 ), obstructive sleep apnea (34 ), pneumonia (35 ), and ARDS (23 ).
Currently, breath acetone testing is carried out by GC followed by flame ionization detection (77, 81), ion mobility spectrometry (77 ), and MS detection ( Breath hydrogen measurements have been applied in clinical medicine for the detection of carbohydrate malabsorption(82 ). Theoretically, H2 in expired air occurs when dietary sugars escape absorption in the small intestine, thereby becoming available for bacterial fermentation. H2 produced by bacterial metabolism of carbohydrates is absorbed into the portal circulation and excreted in the breath. H2 breath tests can be used to diagnose clinical disorders of digestion and absorption, including lactase deficiency and other disorders of di- and monosaccharide malabsorption, starch malabsorption, and small-bowel bacterial overgrowth. Breath H2 testing has repeatedly been demonstrated to be the most accurate indirect indicator of lactase deficiency, and breath H2 measurementshave been widely applied in digestion studies on theentire spectrum of dietary carbohydrates (82–84).In addition to H2 breath tests, the UBT can be used for the diagnosis of gastroenteric diseases. UBT is one of the most successful breath tests used in clinical applications. The range of diseases that can be identified include H. pylori infection, lactose and fructose intolerance, bacterial overgrowth, bile salt wastage, pancreatic insufficiency, liver dysfunction, and abnormal small-bowel transit (4 ). Among these diseases, H. pylori has been studied extensively, and testing has been widely applied for clinical diagnosis. We will focus on H. pylori testing in thissubsection.
In addition to H2 breath tests, the UBT can be used for the diagnosis of gastroenteric diseases. UBT is one of the most successful breath tests used in clinical applications. The range of diseases that can be identified include H. pylori infection, lactose and fructose intolerance, bacterialovergrowth, bile salt wastage, pancreatic insufficiency, liver dysfunction, and abnormal small-bowel transit (4 ).Among these diseases, H. pylori has been studied extensively, and testing has been widely applied for clinical diagnosis. We will focus on H. pylori testing in this subsection.
The major disadvantage of the 13C UBT is the need for gas isotope ratio MS to analyze the breath samples and to calculate a ratio of 12C to 13C. These instruments are quite expensive (4 ). However, because the breath samples are nonradioactive and the isotope is stable, samples can becollected and sent safely to a central processing laboratory. Newer technologies, including nondispersive, isotope selective, infrared spectroscopy and laser-assisted ratio analysis, are less expensive alternatives for these applications.
Samples of exhaled breath are collected, and the isotopically labeled carbon in the exhaled CO2 is measured. If isotoImmediately after sampling the patient’s breath after an overnight fast the patient receives orally 75mg 13C-urea (99% 13C) dissolved in about 250 ml orange juice. 20 or 30 minutes after tracer intake asecond breath sample is taken. (If the rracer amount is enhanced to 100 mg 13C-urea, the time intervalbetween the first and the second breath sampling can be shortened to ten minutes without loss ofdiagnostic accuracy.) The difference between the δ-values of the two samples, the so-called delta overbaseline (DOB) value, is the diagnostic criterion, the cut-off value being 3 to 5 ‰. For children withtheir higher endogenous carbon dioxide production a cut-off value of 3.5‰ has to be assumed.Use of aqueous 13C-urea solutions, enlarging tracer dose to 100 mg 13C-urea, application of gelatinecapsuledsubstrate or rinsing oral cavity after tracer intake in order to prevent pre-gastric ureahydrolysis, shortening or prolonging the period of time between first and second breath sampling orsubstitution of orange juice by 0.1 N citric acid solution do not appreciably affect diagnostic results, nordoes the administration of the tracer together with special commercial test meals instead of orangejuice or citric acid solution for retarding gastric emptying. The posture of the patient during the test(supine, sitting and changed position by rolling) seems to influence the 5 and 10 minute δ13C-valuesrather than the 30 minute values.Application of film-coated tablets enablespically labeled CO2 is detected in the breath, it indicates that H. pylori is present in the stomach.
The Concept Behind 13C Breath TestingThe concept of 13C breath testing is based on measuring the ratios of two non-radioactive isotopes of the Carbon atom in the form of CO2:Carbon 12 (12C) and Carbon 13 (13C). Both isotopes naturally exist in normal breath, but their ratios are dissimilar: approximately 99% ofexhaled breath is 12C and only about 1% is isotope 13C. Breath testing is based on changing this ratio by giving patients a test meal or drinkthat is enriched with 13C. The 13CO2 components of the substrate are extracted during absorption, gastro-intestinal transit, or metabolismof the absorbed substrate. The exhalation of 13CO2 in patients’ breath over a period of time reflects the function of the studied organ. Thisrate may indicate a particular organ’s health and can be measured by devices such as the BreathID® to measure the 13C enrichment in theexhaled air.For the UBT, patients swallow a capsule containing urea labeled with 13C or 14C. If H. pylori is present in thestomach, the bacterium metabolizes the urea into nitrogen and carbon (as CO2). The CO2 is absorbed across the lining of the stomach and into the blood. It is then from the lungs in the breath. The study of pharmacokinetics is essential for understanding the fates of the carbon isotopes in the breath test. Irving and coworkers (87–89) have exhaustively investigated the bicarbonate kinetics and developed a pharmacokineticmodel. Their model consists of 3 pools of freely exchangeable bicarbonate: a central vascular pool, a “heart– brain– other” pool, and a muscle pool (87 ). CO2 production from the heart– brain– other pool and the muscle pool were127.5 and 25.5 mol kg1 min1, respectively, whichyielded a CO2 output rate of 153 mol kg1 min1.Metabolism of Substrate:Helicobacter pylori produces urease, an enzyme which cleaves urea to form carbon dioxide andammonia according to the equation:urease13CO(NH2)2 + H2O -> 13CO2 + 2 NH313C-urea water 13C-carbon dioxide ammoniaFast increase of 13C in exhaled breath indicates Helicobacter pylori infection.
overnight fast. The substrate is prepared from milk of cows fed with silo maize for at least two weeks.The δ13C-value is then –13.293 ± 0.002. The tracer is dissolved in 250 ml of water. In children thedose is 2 g/kg body mass in 50 ml of water. Breath samples are collected ten to fifteen and fiveminutes before tracer intake and then every 30 minutes for four hours, the subjects remaining quietlyseated during the test
Oral lactose (2g/kg)Breath hydrogen sampled at baseline and at 30 min intervals for three hoursBreath hydrogen value of 10ppm – normal, 10-20ppm – indeterminate unless symptomatic, >20ppm – diagnosticFalse positive – recent smoking, false negative – recent use of antibiotics, lung disorders, 1% non-hydrogen producersUnder the age of 5 years – abnormal test reflects an abnormal intestinal mucosa or bacterial overgrowth, both of which require further evaluation by appropriate diagnostic testsperiod.How does hydrogen breath testing work?The bacteria in the colon, including the anaerobic bacteria, are able to digest and use sugars and carbohydrates as food. When the anaerobic bacteria digest sugars and carbohydrates, they convert some of the sugars and carbohydrates into gases, most commonly hydrogen. They also may produce and release into the colon other substances, for example, chemicals that cause the colon to secrete water and cause diarrhea. As previously discussed, some of the hydrogen gas is absorbed by the colon into the blood and is eliminated in the breath where it can be measured. As long as little sugar or carbohydrate reaches the colon, the small amounts of gas and other substances that are produced do not cause a problem. When larger amounts of sugar or carbohydrate reach the colon because they are not digested and absorbed in the small intestine, larger amounts of gas and substances are formed in the colon.For example, if an individual digests and absorbs the sugar in milk (lactose) normally, then none of the lactose that is given for the lactose hydrogen breath test reaches the colon, and no increase in the concentration of hydrogen in the breath is seen during the breath test. On the other hand, if the individual does not digest and absorb the lactose completely, that is, he or she is lactose intolerant, the lactose travels through the small intestine and enters the colon where the bacteria digest it and produce hydrogen. An increase in hydrogen in the breath then is seen. Other sugars for which poor digestion can be diagnosed by breath testing include sucrose and fructose (found in corn syrup), and sorbitol (a sugar that is used as a low-calorie sweetener).There are ways other than abnormal digestion of dietary sugars by which the bacteria can cause problems. Unlike in the colon, the number of hydrogen-producing, anaerobic bacteria in the small intestine is small. If, however, large numbers of hydrogen-producing bacteria move into the small intestine from the colon, a condition called bacterial overgrowth of the small bowel, the bacteria may digest the sugars and carbohydrates before the small bowel has had a chance to digest and absorb them and produce large amounts of hydrogen.Finally, if individuals have abnormally rapid passage of food through the small intestine, there may not be enough time for the small intestine to digest and absorb sugars and carbohydrates. This results in the entry of larger amounts of sugar and carbohydrate into the colon where the bacteria can digest and convert them to gas.To diagnose bacterial overgrowth and rapid transit through the small intestine, a sugar that is not digested and absorbed by man, such as lactulose, usually is used for the test. In the case of rapid passage through the small intestine, the sugar passes quickly through the small intestine and into the colon so that hydrogen is found in the breath very soon after ingestion of the sugar. In the case of bacterial overgrowth, production of hydrogen occurs twice during the test. Once as the sugar passes the bacteria in the small intestine and again when the sugar enters the colon.
Procedure:After a nocturnal fasting period 100 or 75 mg of sodium[1-13C]acetate (90% 13C) are given togetherwith a 370 kcal semiliquid test meal consisting of 100 ml of coffee, in which the tracer is dissolved,20 ml of milk, 100 ml of orange juice, 45 g of mixed-grain bread, 20 g of butter and two scrambledeggs (370 kcal). This test meal should be taken within 15 minutes. Breath samples are taken at thebeginning of the test and then in 10 minute intervals over two hours.
Indication / Relevance to Medical Research and Diagnosis:The N, N-dimethyl-13C-aminopyrine breath test is used for studying hepatic microsomalbiotransformation, especially its demethylating and oxidative capacity, and for diagnosing andassessing therapy of liver diseases in which the activity of microsomal monoxygenases is diminished.It can be used to monitor inactivation of P450 enzymes in the liver. The test also detects altered livermetabolism caused by low-dose oral contraceptives. When the excretion of 13CO2 in breath of patientswith cirrhosis or chronic hepatitis diminishes in the course of time, the test predicts evolution to hepaticcoma and death. Moreover the test is helpful for determining optimum liver transplantation time and forthe preventive diagnosis of rejection reaction after liver transplantation.Suitability for clinical diagnosis: excellent.
[U-13C]galactose breath test can be used for the diagnosis of liver diseases, particularly for thediagnosis of liver fibrosis in chronic hepatitis C, for recognising presence or degree of severity ofalcoholic cirrhosis and for studying galactose oxidation in patients with galactose-1-phosphateuridyltransferase deficiency. In experiments with rats the test was used for investigating effects ofethanol and diabetes on galactose oxidation in rats.
Breath test and their applications
Dr Vijaya Marakala MD
A breath test is a type of test
performed on air generated from
the act of exhalation.
The bulk matrix
of breath is a
CO2, H2O, and
• Depending on the origin of the substances
found in human breath, fall into two main
Diagnosis of disease
Assessment of exposure
• Ancient Greek physicians already knew that
the aroma of human breath could provide
clues to diagnosis.
Sweet, fruity odor
Musty, fishy reek
Modern breath analysis
started in the 1970s when
using gas chromatography
more than 200
components in human
As a result of
a few breath
Disease or application
Asthma, COPD, bronchiectasis, ARDS H2O2
Disease or application
NO, CO, H2O2, isoprostanes,
NO, H2O2, eicosanoids
NO, CO, H2O2,
Pulmonary allograft dysfunction
Disease or application
Disease or application
Disorders of digestion and
Gastritis, duodenal ulcer, gastric
ulcer, and gastric cancer
Isotopes of carbon (13C or 14C)
• VOCs enter the body mainly through
• Exposure to
• Exposure are
Para• Moth cakes/room
dichlorobenzene air deodorizers
Lactose and fructose intolerance
Bile salt wastage
Abnormal small-bowel transit
Almost all of the routine diagnostics and treatments for these
diseases have been replaced by studies focusing on the
epidemiology, isolation, and eradication of this single
Currently, two breath tests are available to
Intake of 13C labeled
urea together with
30 minutes later
13CO in breath
• Blow up the first breath
• Drink 13C- labeled test
• Wait for 30 minutes
• Blow up the second
The concept of 13C breath testing is based on
measuring the ratios of
two non-radioactive isotopes of the Carbon atom in
the form of CO2:
Carbon 12 (12C) and Carbon 13 (13C).
lactase deficiency in patients with gastrointestinal
symptoms and of measuring lactase activity of brush
• Lactase deficiency
• Lactose malabsorption
Measure fasting breath H2
Give lactose solution 50g in 180ml water
Rinse mouth with 20ml water and swallow
Measure breath H2 at15,30,60,90 & 120min
of liquid meals
100 mg of sodium
acetate[13C] with a
370 kcal semiliquid
Samples are taken
intervals over two
acid and protein
Metabolism of Substrate:
• Test meal and 100 mg of [13C]glycine
• Sample-every 15 min for 4 hours
• Useful for the diagnosis of
Procedure After a 12 h overnight fast- basal sample
Give 500 mg labeled cholesteryl octanoate[13C]
Sample-every 15 minutes thereafter for six hours
Measure 13CO2 recovery
• Studying hepatic microsomal biotransformation
Indication • Diagnosing and assessing therapy of liver diseases
After an overnight fast-fasting breath
Subjects ingest 2 mg per kg body mass
Breath sample at 30 minute intervals for
two to three hours
• Diagnosis of liver diseases
• Particularly for the diagnosis of liver
Overnight fast-Basal sample
Give aqueous solution of 10 g 13C-galactose per
m2 of body area
Sample-at 30 minute intervals over three
to four hours.
Breath testing is noninvasive, easily repeated
Breath samples closely reflect the arterial
Breath is a much less complicated mixture than
serum or urine
Breath analysis provides direct information on
Breath analysis can dynamically real-time monitor
the decay of volatile toxic substances in the body.
Problem in use of breath tests in clinical
High water content of breath
Instruments for breath
analysis are expensive.
Lack of established links between breath
substances and disease
Tests in Medical Research and
By Klaus Wetzel and Heinz Fischer
Breath Analysis: Potential for Clinical Diagnosis
and Exposure Assessment
By Wenqing Cao and Yixiang Duan
TIETZ Fundamental of CLINICAL CHEMISTRY