Human Breath Odors and Their Use in Diagnosis

CHRIS L. WHITTLE,a STEVEN FAKHARZADEH,b JASON EADES,a AND GEORGE PRETIa,b

a Monell

Chemical Senses Center, 3500 Market Street, Philadelphia, Pennsylvania 19104, USA of Dermatology, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA
b Department

ABSTRACT: Humans emit a complex array of volatile and nonvolatile molecules that are influenced by an individuals genetics, health, diet, and stress. Olfaction is the most ancient of our distal senses and may be used to evaluate food and environmental toxins as well as recognize kin and potential predators. Many body odors evolved to be olfactory messengers, which convey information between individuals. Consequently, those practicing the healing arts have used olfaction to aid in their diagnosis of disease since the dawn of medical practice. Studies using modern instrumental analyses have focused upon analysis of breath volatiles for biomarkers of internal diseases. In these studies, a subjects oral health status appears to seldom be considered. However, saliva and properly collected alveolar air samples must pass over or come in contact with the posterior dorsal surface of the tongue, a site of bacterial plaque development and source of halitosis-related volatiles. Because of our basic research into the nature of human body odors, our lab has received referrals of people with idiopathic malodor production, from either the oral cavity or body. We developed a protocol to help differentiate individuals with chronic halitosis from those with the genetic, odor-producing metabolic disorder trimethylaminuria (TMAU). In our referred population, TMAU is the largest cause of undiagnosed body odor. Many TMAU-positive individuals present with oral symptoms of dysguesia and halitosis as well as body odor. We present data regarding the presentation of our referred subjects as well as the analytical results from a small number of these subjects regarding their oral levels of halitosis-related malodorants and trimethylamine. KEYWORDS: human breath; biomarkers; breath condensate; disease; pathology; trimethylamine; trimethylaminuria; choline; flavincontaining monooxygenase 3 (FMO3); SPME; GC/MS

Address for correspondence: George Preti, Ph.D., Monell Chemical Senses Center, 3500 Market Street, Philadelphia, PA 19104. Voice: 215-898-4713; fax: 215-898-2084. preti@monell.org Ann. N.Y. Acad. Sci. 1098: 252266 (2007). doi: 10.1196/annals.1384.011
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INTRODUCTION
Humans emit a complex array of nonvolatile and volatile molecules. The metabolic processes of an individual, and hence the compounds emitted into the environment, may be influenced by genetics, diet, stress, and the immune status of the individual. Human olfaction is the most ancient of our distal senses and does provide information from distant sources in real time. Olfactory information may be used to detect and evaluate food sources and environmental toxins as well as to recognize kin and potential predators. In addition, many body odors evolved to be olfactory cues that convey information between individuals.1 Large numbers of volatile compounds may be emitted from several areas of the body that are prone to odor production; these include the scalp, axillae, feet, groin, and oral cavity.2 Consequently, it is not surprising that physicians have used their olfactory and gustatory senses to aid in the differential diagnosis of diseases since the beginning of medical practice.35 Hippocrates is reported to have used exhaled breath and smelled the breath of patients as part of his assessment.6 Breath testing as a scientific tool was pioneered in the 18th century by Lavoisier and Laplace.7 These pioneers established the presence of CO 2 in exhaled breath air. In the second half of the 20th century, the development of more sophisticated analytical techniques, such as gas chromatography, has allowed the separation and identification of volatile compounds from complex biological matrices, such as exhaled breath. In the 1960s, the separating power of gas chromatography was combined with mass spectrometry to create combined gas chromatography/mass spectrometry (GC/MS); see Watson for a review.8 This development allowed the separation of complex mixtures as well as structural identification of separated, volatile components. Numerous investigators have captured and concentrated breath and salivary volatiles. Studies by these researchers have focused upon establishing the normal breath constituents and searching for biomarkers from the oral cavity to aid in the diagnosis of illness or severity of disease.913 Analysis of exhaled breath provides a unique opportunity to examine the organic constituents of blood because alveolar breath reflects the concentration of metabolites that have passively diffused across the pulmonary alveolar membrane. Breath primarily consists of nitrogen, oxygen, carbon dioxide, inert gases, water vapor, and a trace amount of volatile organic compounds (VOCs) (e.g., acetone, isoprene, and pentane) that are present in nano- to picomolar concentrations.14 Because the organic constituents of exhaled breath are representative of the blood-borne concentrations of metabolites, breath analysis provides a noninvasive means to examine blood-borne constituents relative to using blood and/or urine samples. Some of the advantages of using exhaled breath include the fact that lung air volatiles reflect the arterial concentrations of biological substances; also the VOCs are removed from complex fluid matrices, such as blood and urine; consequently, the complete

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sample of all compounds are present in the collected sample with no work-up required prior to analysis of the sample. However, breath analysis also has limitations. One issue that hinders routine breath tests in clinical practice is a lack of standardized collection and analysis methods and the need for complex and unfamiliar (for clinicians) instrumentation. In addition, simple and automated chemical tests that are routinely used for blood and urine analyses are relatively inexpensive when compared to the most commonly used instrumentation for breath analyses, GC/MS. The latter requires an expensive initial investment (> $70,000) and maintenance (>$10,000/year) as well as skilled operators who must be able to interpret data from large complex data sets.9,15 A large number of studies using modern instrumental analyses have focused upon analysis of breath volatiles in search of biomarkers for disease states. The vast majority of these studies have examined respiratory system diseases such as asthma. Nitric oxide is the most extensively exhaled marker investigated, and has been linked to a host of respiratory ailments including chronic obstructive pulmonary disease,16,17 rhinitis,18,19 rhinorrhea,20 chronic cough (primary),21,22 asthma,23,24 cystic fibrosis,25 bronchiectasis,26,27 and lung cancer.28,29 Numerous exhaled markers of disease have been studied, such as several leukotrienes and nitrogenous compounds.3033 Many recent studies focused upon branched or methylated hydrocarbons or VOCs with broad diagnostic potential. These have been postulated to increase in breath effluvia as a function of oxidative stress or damage (see Miekisch et al. for a review).11 Although hydrocarbons are commonly found in the air of urban areas, where many of the above-cited studies have taken place, investigators appear certain that these products are products of human biological activity and not exogenous, environmental contaminants. These include the straightchained hydrocarbons, ethane and pentane, which are markers of oxidative damage and have also been linked to diseases, such as asthma,34,35 lung and breast cancer,33,36 interstitial lung disease,37 chronic obstructive pulmonary disease,35 and heart rejection.38 Numerous other volatile compounds have been found in exhaled breath and have been linked to different diseases. Although not exhaustive, TABLE 1 illustrates the range of VOCs that may serve as biomarkers for disease states. In these studies, however, a subjects oral health status appears to seldom be considered. Halitosis is one of the most frequent complaints expressed by dental patients. Approximately 90% of oral malodor is thought to originate from the oral cavity, with the remaining 10% originating from distal points in the digestive and respiratory systems.3941 Persistent halitosis may be indicative of underlying medical conditions, such as diabetes, leukemia, gastrointestinal ulcers, lung cancer, trimethylaminuria (TMAU), and several other idiopathic conditions.39,42 Even correctly collected alveolar air and saliva samples must pass over or come in contact with the posterior dorsal surface of the tongue. This is a site of bacterial plaque development that is a principal

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TABLE 1. Oral/breath volatiles identified in patients with systemic disease

Pathologic condition Diabetes mellitus

Compound(s) Acetone, other ketones Breath methylated alkane contour (BMAC) Interleukin IL-6, 8-isoprostane Nitrate, cyanide Carbon dioxide Carbon monoxide Dimethylsulfide

Reference Booth and Ostenson, 196663 Walsh, 200464 Phillips et al., 200438 Carpagnano et al., 200265 Lechner et al., 200566 Pathak et al., 199467 Sylvester et al., 200568 Chamberlin et al., 199646 Montuschi and Barnes, 2002;30 Hanazawa et al., 2000;69 Cap et al., 200470 Phillips et al., 200633

Sleep apnea H. pylori infection

Sickle cell disease Methionine adenosyltransferase deficiency Asthma Leukotrienes

Breast cancer

Lung carcinoma

Chronic obstructive pulmonary disease

2-propanol, 2,3-dihydro-1phenyl-4 (1H)-quinazolinone, 1-phenyl-ethanone, heptanal Acetone, methylethylketone, n-propanol Aniline, o-toluidine Alkanes, mono-methylated breath alkanes, alkenes Hydrogen peroxide Nitrosothiols Nitrosothiols nitric oxide 8-isoprostane Leukotriene B(4), interleukin8 Hydrogen disulfide, limonene Hydrogen disulfide

Gordon et al., 198571 Preti et al., 198872 Phillips et al., 200328 Dekhuijzen et al., 199773 Corradi et al., 200131 Liu and Thomas, 200574 Montuschi et al., 200075 Bodini et al., 200576 Friedman et al., 199447 Friedman et al., 199447 Chen et al., 1970a48 ; Kaji et al., 197877 Tangerman et al., 199478 Simenhoff et al., 197779 Leopold et al., 199056 Preti et al., 199512

Cystic fibrosis

Liver disease Noncholestatic Primary biliary cirrhosis Decompensated cirrhosis of the liver (foetor hepaticus) Uremia/kidney failure Trimethylaminuria

C 2 C 5 aliphatic acids, methylmercaptan

Ethanethiol, dimethylsulfide Dimethylamine, trimethylamine Trimethylaminine

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source of halitosis-related volatiles.43 The bad breath mixture has been studied principally for its volatile sulfur compounds (VSCs): hydrogen sulfide, methylmercaptan, dimethyl sulfide, carbonyl sulfide. These compounds provide much of the impact odor of halitosis; however, the breath of individuals with halitosis does contain a variety of volatile organic odorants, not just VSCs.44,45 Consequently, VSCs as well as many other volatiles may be indicative of oral-related health issues rather than non-oral disorders and disease. Studies addressing markers from exhaled breath may want to address the subjects oral health status. Several authors examining exhaled breath of patients with liver diseases for VSCs have not addressed their subjects oral health.46,47 These authors found elevated levels of VSCs in these patients versus controls. However, the historical link of liver diseases with foul breath odor (foetor hepaticus) appears to exclude an oral cause for breath odor in these patients. In addition, Chen et al. used a methionine challenge to elicit VSC production in their subjects.48 Because of our basic research into the nature and origin of human body odors, our lab has become the focal point for a large number of referrals of people with idiopathic malodor production from either the body or oral cavity. Regardless of presenting symptoms and to help differentiate individuals with bad breath from other possible disorders, all individuals are examined for odor production from the oral cavity and upper body by the same protocol first described by Preti et al.12 A central part of this work-up is the choline challenge test for TMAU developed by Tjoa and Fennessey.49 Trimethylaminuria was first described by Humbert,50 and is a metabolic disorder characterized by the inability of individuals to oxidize and convert dietary derived trimethylamine (TMA) to trimethylamine N-oxide (TMAO) in the liver. This disorder results from an inherited autosomal recessive trait in the gene, which codes for the flavin-containing monooxygenase enzyme 3 (FMO3). The genetic changes range from gene mutations associated with the most severe cases to the more common single nucleotide polymorphic changes in the FMO3 gene that may be associated with the less severe cases.51,52 Malodorous TMA is formed in the gut by bacterial metabolism of dietary constituents, mainly choline. In normal individuals, TMA is converted/oxidized to TMAO at >95% efficiency by FMO3. Individuals that have FMO3 metabolic capacity <90% conversion of TMA to TMAO are considered positive for TMAU.53,54 TMAO is nonodorous, more polar and water-soluble than TMA, and readily excreted in the urine.55 Individuals suffering from TMAU have a reduced capacity to oxidize TMA to TMAO. TMA is a gas at body temperature and has a foul, rotten fish odor. At low concentrations it may be perceived as unpleasant or garbage-like. The inability to efficiently oxidize TMA results in the sporadic production of a body odor that is perceived as foul, unpleasant, and in its most extreme cases fish-like. This odor is caused by excess, unmetabolized TMA present in the circulatory system that is excreted in urine, sweat, breath, and saliva. Because there are

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many foods that are rich in choline (i.e., eggs, certain legumes, and organ meats), TMAU-affected individuals, family members, friends, and physicians are unlikely to associate the odor with food intake. Symptoms may include foul body odor, halitosis, and/or dysguesia that can produce social embarrassment and may only be temporarily relieved by normal hygienic procedures.56 The main difficulties experienced by TMAU-affected individuals are psychosocial ones that are caused by sporadic, undiagnosed odor production.57 To enable an easier diagnosis for TMAU and to examine whether or not elevated salivary levels of TMA might accompany oral symptoms in our referred subjects, we began collecting saliva from all subjects reporting to our lab with malodor production problems to examine this fluid for TMA.

METHODS
All procedures were approved by the Office of Regulatory Affairs at the University of Pennsylvania and informed consent was obtained from each subject before study participation. We adapted the method of Mills and Walker58 that employs the techniques of solid-phase microextraction (SPME) and gas chromatographymass spectrometry (GC/MS) to quantify salivary TMA levels.57 This adaption included the use of perdeuterated trimethylamine (D 9 -TMA) as an internal standard to aid in quantitation. We report herein on the data obtained from the saliva of several subjects using SPME-GC/MS. The linear range of the method was investigated by obtaining a calibration curve in the concentration ranges of interest. Trimethylamine concentrations (as trimethylamine hydrochloride) were made up from a stock solution of 2 mg/mL in acidified water (pH1). The following concentrations were used: 2 mL each of acidified, distilled water containing TMA concentrations of 0.006 mg/mL, 0.003 mg/mL, 0.0012 mg/mL, 0.0006 mg/mL, 0.0003 mg/mL, and 0.00012 mg/mL. TMA-HCl were combined with 2 mL of 0.002 mg/mL perdeuterated (D 9 )-TMA-DCl and 0.5 mL of 12 M NaOH. Each solution was equilibrated for 15 min at 50 C prior to exposing the SPME fiber to collect volatiles for 15 min. Each solution was analyzed a minimum of three times for both the calibration curve and patient samples. Gas Chromatography/Mass Spectrometry After collecting salivary volatiles containing the TMA, the SPME fiber was inserted into the hot injector of the GC/MS (held at 230 C) and exposed for 1 min to desorb volatiles collected on the fiber. A Thermoquest/Finnigan Voyager GC/MS with Xcalibur software (ThermoElectron Corp., San Jose, CA, USA) was used for all analyses. A polar, Stabilwax column, 30 M 0.32 mm

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with 1.0- coating, (Restek Corp., Bellefonte, PA, USA) was used for separation and analysis of the volatiles extracted from the samples. The separation of TMA from other components was done isothermally by holding the column at 50 C for 5 min. TMA elutes within the first 5 min; consequently, we rapidly increased the column temperature at 40 C/min to the final temperature of 220 C to bake-off undesired volatiles. The column was recycled back to the starting temperature of 50 C for the next analysis. The injection port was set at 230 C. Helium carrier gas was used at a constant column flow rate of 2.5 mL/min throughout the analysis. Data acquisition and operating parameters for the mass spectrometer were set as follows: scan rate 2/sec; scan range m/z 41 to m/z 440; ion source tempera. ture 200 C; ionizing energy 70eV Identification of structures/compounds was performed using both the NIST 02 library, as well as a manual interpretation of mass spectra compared with those reported in the literature. Gas Chromatography with Flame Photometric Detection to Measure Oral Volatile Sulfur Compounds We used gas chromatography with flame photometric detection (GC/FPD) to detect and quantify the amount of volatile sulfur compounds in the oral cavity of all subjects. The instrument used for these analyses was a Finnigan 9001 fitted with a flame photometric detector. We employed the method of Tonzetich,59 albeit with modifications previously reported by Kostelc et al. and Preti et al.60,61 Analysis of the subjects mouth air was performed in duplicate. Each analysis employed 10 mL of the subjects mouth air pulled into the chromatograph via an atmospheric sampling loop using a gas tight syringe. FMO3 Gene Sequence Analysis Genomic DNA was extracted from patient peripheral blood samples using the QIAamp DNA Mini Kit (Qiagen, Valencia, CA, USA). FMO3 coding exons 2-9 and flanking intron sequences were amplified by PCR using primers and conditions reported by Dolphin et al.62 PCR amplification products were purified from 1.5% agarose gels prior to sequencing. All samples were submitted to the DNA Sequencing Facility at the University of Pennsylvania School of Medicine for analysis. Sequencing reactions were performed in an ABI GeneAmp 9700 thermal cycler, resolved with an ABI 3730 DNA sequencer, and analyzed using ABI Sequencing Analysis software v 5.1 (ABI, Applied Biosystems, Incorporated, Foster City, CA, USA). RESULTS We have seen and tested more than 300 individuals in our laboratory using the protocol outlined in TABLE 2. One hundred two of these have been diagnosed with some form of TMAU using the choline challenge test.49 The presenting

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TABLE 2. Diagnostic protocol for referred individuals seen at the Monell Chemical Senses Center

1. First morning voided urine (base line for choline challenge test). 2. Individual presents in fasted condition (no cologne, cosmetics, fragrances, teeth and tongue plaque brushing); questionnaire and interview. 3. Organoleptic evaluation of individuals body odor and breath: 3 judges, scale of 110. 4. Collect axillary odors by placing a 4 4-inch cotton pad in individuals axillae. 5. Analysis of individuals mouth air by GC/FPD for volatile sulfur compounds (VSCs): H 2 S, CH 3 SH, (CH 3 ) 2 S. 6. Swab tongue for bacterial plaque and use for collection and analysis of volatiles. 7. Determination of resting whole mouth saliva flow rate using the methods described in Christensen and Navazesh, 1982.80 8. Determine and collect two, whole mouth-stimulated saliva samples using flavorless gum base. One of these is collected into a vial with 0.2 mL of 6N HCl for analysis of precholine challenge TMA. Other volatiles are examined by SPME-GC/FPD and SPME-GC/MS. 9. Lung air collection (10 L) for analysis of volatiles by GC/FPD. 10. Analyze tongue plaque volatiles by SPME-GC/MS and SPME-GC/FPD. 11. Administer 5 g of choline in 1216 oz of juice for choline challengea . 12. Remove axillary pads and perform organoleptic evaluation. 13. For the next 24 h individual must: (A) Collect urine in three 8-h aliquots (B) Collect two stimulated whole mouth saliva samples during each 8-h time period using vials with 0.2 mL of 6N HCl. Next day: Individual returns saliva and urine samples. Blood sample taken for genotyping of FMO3 gene.
a All urine samples collected during choline challenge testing were analyzed for trimethylamine and triemthylamine oxide in the laboratory of Dr. Paul Fennessey and Ms. Susan Tjoa,49 at the University of Colorado Health Sciences Center.

symptoms for the TMAU-positive individuals are illustrated in FIGURE 1. A majority of individuals had oral symptoms, complaining of either bad breath or bad taste, many in conjunction with body odors. In addition, as illustrated in FIGURE 2, the majority of our TMAU-positive individuals were females. However, regardless of gender, in our experience, TMAU was the largest cause for undiagnosed body odor. The large number of TMAU-positive individuals who listed oral complaints led us to hypothesize that salivary concentrations of TMA were responsible for these symptoms. During most individuals visits, saliva was collected in conjunction with urine as described in TABLE 2. This has resulted in an archive of more than 270 frozen (at 10 C) saliva samples. However, only a small number of the saliva samples collected have been examined thus far for TMA levels: six individuals each from TMAU-positive and -negative categories. We report these preliminary results here. TABLE 3 lists the mean salivary concentrations of TMA measured in each 8-h interval for six TMAU-positive and six TMAU-negative subjects. Clearly, the saliva of the positive subjects contains far more TMA than the saliva from

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FIGURE 1. Presenting symptoms and the numbers of subjects reporting each symptom, in the subjects own words. Body odor is the most common presenting symptom found in the literature pertaining to these patients. The symptoms were not always confirmed during subject evaluation using the protocol in TABLE 2.

TMAU-negative individuals, but there is a great deal of variation in the data, particularly from the positive subjects. Two of the positive subjects account for much of this variation because they demonstrated more than a 100-fold increase in salivary TMA levels from their precholine challenge base line to their highest levels in the 2nd or 3rd 8-h interval: Positive male 3 went from a base line of 25 ng/mL to 4,812 ng/mL in the 3rd, 8-hour segment; and positive male 6 went from a base line of 46.7 ng/mL to 4, 511 ng/mL in the 2nd, 8-h segment. Each of these subjects had a very low conversion of TMA to TMAO, indicative of one or more genetic mutations present in the gene for FMO3: male 3 had 25% TMAO conversion; male 6 had only 11% TMAO conversion. We also measured the volatile sulfur compounds in the oral cavity associated with each of these subjects. Results are summarized in TABLE 4. In the subjects chosen for these analyses, we found that, on average, TMAU-negative subjects had greater concentrations of each VSC measured; these concentrations were also converted to parts per billion (ppb) levels and are presented in TABLE 4. The difference in VSC levels between the TMAU-negative and positive individuals is a result of the subjects chosen for these analyses: in our clinical experience, both TMAU-positive and -negative individuals may have halitosis. However, the two conditions, TMAU and chronic halitosis caused by bacterial tongue plaque, are independent of each other.

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FIGURE 2. The gender distribution of TMAU-positive subjects seen in our laboratory is shown in this figure. We do not know whether or not the larger number of females affected is due to hormonal differences in the regulation of the FMO3 gene. Females report symptoms before and after menopause.

DISCUSSION
Our findings regarding the presenting symptoms of TMAU-affected individuals are in contrast to results found in most of the medical literature. Articles discussing TMAU suggest to the reader that all sufferers have a fishy body odor presentation. In our population, all of whom have been seen in person, the fish odor presentation was present in only about 10% of individuals who are TMAU-positive. Further to this point, these individuals emitted a strong fish odor recognizable at social distances only after choline challenge. Consequently, the assumption that the individual with TMAU will always smell like fish is incorrect and is often the reason that many TMAU-affected individuals
TABLE 3. Mean salivary trimethylamine concentrations: precholine and for each 8-h period (ng/mL)

Precholine Negative (n = 6) SE Positive (n = 6) SE 37.41 15.27 31.2 12.74

1st 8 hour 48.37 19.74 595.61 243.10

2nd 8 hour 61.07 24.93 799.20 742.39

3rd 8 hour 68.40 27.92 1736.57 823.88

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TABLE 4. Volatile sulfur compounds (VSCs) in the mouth air of referred subjects: ng/10 mL of mouth air

COS 5a

H2S

CH 3 SH

(CH 3 ) 2 S 0.21 0.10

TMAU-negative subjects: N = Mean 1.08 2.17 1.97 SE 0.67 1.38 1.04 Mean parts per billion (ppb) of total VSC in the mouth air of TMAU-negative subjects = 453 COS H2S CH 3 SH

(CH 3 ) 2 S 0.0 0.0

TMAU-positive subjects: N = 5a Mean 0.416 0.83 0.0 SE 0.415 0.57 0.0 Mean ppb of total VSC in the mouth air of TMAU-positive subjects = 104
a

Instrument malfunction caused one subject in each group not to be sampled for VSC levels. ABBREVIATION: COS = carbonyl sulfide; H 2 S = hydrogen sulfide; CH 3 SH = methylmercaptan; (CH 3 ) 2 S = dimethylsulfide.

are sent from one clinical specialist to another: quite often they are sent to a psychiatrist since their reported symptoms are thought to be subjective. The choline challenge test for TMAU provides a recognized means for diagnosing this disorder. The diagnosis currently relies upon a 24-h urine collection, divided into three 8-h aliquots.49 In our initial attempt to extend this diagnosis to saliva, we collected whole mouth-stimulated saliva to determine salivary TMA levels. Our hypothesis that the oral symptoms of many TMAU-affected individuals appears to be supported by the preliminary results presented here, although the numbers of subjects analyzed is still small. The data in TABLE 3 show much larger variation in the salivary TMA concentrations of TMAUpositive versus TMAU-negative individuals. TMAU is known to be caused by a spectrum of genetic changes to the gene that codes for FMO3;51 consequently, this variation may be due, in part, to differences in the genotype of each of the TMAU-positive individuals. This is supported by our clinical observations regarding the odor of different individuals as well as genotyping data. Two of the six TMAU-positive individuals whose saliva was analyzed presented with overt fish odor from their upper body and oral cavity after (22 h) choline challenge. As noted above, each of these male subjects had a low conversion of TMA to TMAO (<25%) and documented mutations in their FMO3 gene (data not shown).51
ACKNOWLEDGMENTS

This research was supported, in part, by the NIH Institutional Training Grant 2T32DC00014 as well as unrestricted funds from the Monell Chemical Senses Center.

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REFERENCES
1. WYSOCKI, C.J. & G. PRETI. 2004. Facts, fallacies, fears, and frustrations with human pheromones. Anat. Rec. 281A: 12011211. 2. WYSOCKI, C.J. & G. PRETI. 2000. Human body odors and their perception. Jpn. J. Taste Smell Res. 7: 1942. 3. LIDDELL, K. 1976. Smell as a diagnostic marker. J. Postgrad. Med. 52: 136138. 4. PHILLIPS, M. 1992. Breath tests in medicine. Sci. Am. 267: 7479. 5. PENN, D. & W.K. POTTS. 1998. Chemical signals and parasite-mediated sexual selection. Trends Ecol. Evol. 13: 391396. 6. GEIST H.C.I. 1957. Halitosis in ancient literature. Dent. Abst. 2: 417418. 7. CASPARY, W.F. 1978. Breath tests. Clin. Gastroenterol. 7: 35174. 8. WATSON, J.T. 1998. A historical perspective and commentary on pioneering developments in gas chromatography/mass spectrometry at MIT. J. Mass Spect. 33: 103108. 9. CAO, W. & Y. DUAN. 2006. Breath analysis: potential for clinical diagnosis and exposure assessment. Clin. Chem. 52: 800811. 10. RAHMAN, I. & F. KELLY. 2003. Biomarkers in breath condensate: a promising new non-invasive technique in free radical research. Free Rad. Res. 37: 1253 1266. 11. MIEKISCH, W., J.K. SCHUBERT & G.F.E. NOELDGE-SCHOMBURG. 2004. Diagnostic potential of breath analysisfocus on volatile organic compounds. Clin. Chim. Acta 347: 2539. 12. PRETI, G., H.J. LAWLEY, C.A. HORMANN, et al. 1995. Non-oral and oral aspects of oral malodor. In Bad Breath Research Perspectives. M. Rosenberg, Ed.: 149174. Ramot Publishing. Tel Aviv, Israel. 13. KHARITONOV, S.A. & P.J. BARNES. 2002. Biomarkers of some pulmonary diseases in exhaled breath. Biomarkers 7: 132. 14. MUKHOPADHYAY, R. 2004. Dont waste your breath. Anal. Chem. 76: 273A276A. 15. WOLFRAM, M., J.K. SCHUBERT & G.F.E. NOELDGE-SCHOMBURG. 2004. Diagnostic potential of breath analysisfocus on volatile organic compounds. Clin. Chim. Acta 347: 2539. 16. KHARITONOV, S.A. & P.J. BARNES. 2001. Exhaled markers of pulmonary disease. Am. J. Resp. Crit. Care Med. 163: 16931722. 17. MARCZIN, N., S.A. KHARITONOV & S.M.H. YACOUB. 2003. Disease Markers in Exhaled Breath. Dekker. New York. 18. BARALDI, E., N.M. AZZOLIN, S. CARRA, et al. 1998. Effect of topical steroids on nasal nitric oxide production in children with perennial allergic rhinitis: a pilot study. Resp. Med. 92: 558561. 19. HENRIKSEN, A.H., M. SUE-CHU, T. LINGAAS HOLMEN, et al. 1999. Exhaled and nasal NO levels in allergic rhinitis: relation to sensitization, pollen season, and bronchial hyperresponsiveness. Eur. Resp. J. 13: 13011306. 20. FRANKLIN, P.J., S.W. TURNER, G.L. HALL, et al. 2005. Exhaled nitric oxide is reduced in infants with rhinorrhea. Pediat. Pulmonol. 39: 117119. 21. DUPONT, L.J., F. ROCHETTE, M.G. DEMEDTS & G.M. VERLEDEN. 1998. Exhaled nitric oxide correlates with airway hyperresponsiveness in steroid-naive patients with mild asthma. Am. J. Resp. Crit. Care Med. 157: 894898. 22. CHATKIN, J.M.A.K., P.E. SILKOFF, P. MCCLEAN, et al. 1999. Exhaled nitric oxide as a noninvasive assessment of chronic cough. Am. J. Resp. Crit. Care Med. 159: 18101813.

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23. DUPONT, L.J.F., M.G. DEMEDTS & G.M. VERLEDEN. 1999. Prospective evaluation of the accuracy of exhaled nitric oxide for the diagnosis of asthma. Am. J. Resp. Crit. Care Med. 159: 861. 24. DUPONT, L.J., M.G. DEMEDTS & G.M. VERLEDEN. 2003. Prospective evaluation of the validity of exhaled nitric oxide for the diagnosis of asthma. Chest 123: 751756. 25. THOMAS, S.R., S.A. KHARITONOV, S.F. SCOTT, et al. 2000. Nasal and exhaled nitric oxide is reduced in adult patients with cystic fibrosis and does not correlate with cystic fibrosis genotype. Chest 117: 10851089. 26. TRACEY, W.R., C. XUE, V. KLINGHOFER, et al. 1994. Immunocytochemical detection of inducible NO synthase in human lung. Am. J. Physiol. 266: L722L727. 27. HO, L.P., J.A. INNES & A.P. GREENING. 1998. Exhaled nitric oxide is not elevated in the inflammatory airways diseases of cystic fibrosis and bronchiectasis. Eur. Resp. J. 12: 12901294. 28. PHILLIPS, M., R.N. CATANEO, B.A. DITKOFF, et al. 2003. Volatile markers of breast cancer in the breath. Breast J. 9: 184191. 29. PHILLIPS, M., K. GLEESON, J.M. HUGHES, et al. 1999. Volatile organic compounds in breath as markers of lung cancer: a cross-sectional study. Lancet 353: 1930 1933. 30. MONTUSCHI, P. & P.J. BARNES. 2002. Exhaled leukotrienes and prostaglandins in asthma. J. Allergy Clin. Immunol. 109: 615620. 31. CORRADI, M., P. MONTUSCHI, L.E. DONNELLY, et al. 2001. Increased nitrosothiols in exhaled breath condensate in inflammatory airway diseases. Am. J. Resp. Crit. Care Med. 163: 854858. 32. BALINT, B., S.A. KHARITONOV, T. HANAZAWA, et al. 2001. Increased nitrotyrosine in exhaled breath condensate in cystic fibrosis. Eur. Resp. J. 17: 12011207. 33. PHILLIPS, M., R.N. CATANEO, B.A. DITKOFF, et al. 2006. Prediction of breast cancer using volatile biomarkers in the breath. Breast Cancer Res. Treat. 99: 19 21. 34. OLOPADE, C.O., M. ZAKKAR, W.I. SWEDLER & I. RUBINSTEIN. 1997. Exhaled pentane levels in acute asthma. Chest 111: 862865. 35. PAREDI, P., S.A. KHARITONOV, D. LEAK, et al. 2000. Exhaled ethane, a marker of lipid peroxidation is elevated in chronic obstructive pulmonary disease. Am. J. Resp. Crit. Care Med. 162: 6973. 36. PHILLIPS, M., R.N. CATANEO, A.R.C. CUMMIN, et al. 2003. Detection of lung cancer with volatile markers in the breath. Chest 123: 21152123. 37. KANOH, S., H. KOBAYASHI & K. MOTOYOSHI. 2005. Exhaled ethane: an in vivo biomarker of lipid peroxidation in interstitial lung diseases. Chest 128: 2387 2392. 38. PHILLIPS, M., J.P. BOEHMER, R.N. CATANEO, et al. 2004. Heart allograft rejection: detection with breath alkanes in low levels (the HARDBALL study). J. Heart Lung Transplant. 23: 701708. 39. TONZETICH, J. 1995. Preface. In Bad Breath Research Perspectives. M. Rosenberg, Ed.: xixviii. Ramot Publishing. Tel Aviv, Israel. 40. SCULLY, C., M. EL-MAAYTAH, S.R. PORTER & J. GREENMAN. 1997. Breath odor: etiopathogenesis, assessment and management. Eur. J. Oral Sci. 105: 287 293. 41. FELLER, L. & E. BLIGNAUT. 2005. Halitosis: a review. SADJ. 60: 1719. 42. MESSADI, D.V. 1997. Oral and non-oral sources of halitosis. J. Calif. Dent. Assoc. 25: 127131.

WHITTLE et al.

265

43. LOESCHE, W. & C. KAZOR. 2002. Microbiology and treatment of halitosis. Periodontol. 2000 28: 256279. 44. KOSTELC, J.G., G. PRETI, P.R. ZELSON, et al. 1980. Salivary volatiles as indicators of periodontitis. J. Periodontal Res. 15: 185192. 45. PAYNE, R.K., J.N. LABOWS & X. LIU. 1999. Released oral malodors measured by solid phase microextraction/gas chromatography mass spectrometry (HS-SPMEGC-MS). ACS Symp. Series 76: 373386. 46. CHAMBERLIN, M.E., U. TSUNEYUKI, S.H. MUDD, et al. 1966. Demyelination of the brain is associated with methionine adenosylation I/II deficiency. J. Clin. Invest. 98: 10211027. 47. FRIEDMAN, M.I., G. PRETI, R.O. DEEMS, et al. 1994. Limonene in expired lung air of patients with liver disease. Digest. Dis. Sci. 39: 16721676. 48. CHEN, S., L. ZIEVE & V. MAHADEVAN. 1970a. Mercaptans and dimethyl sulfide in the breath of patients with cirrhosis of the liver. J. Lab. Clin. Med. 75: 628635. . 49. TJOA, S. & P.V FENNESSEY. 1991. The identification of trimethylamine excess in man: quantitative analysis and biochemical origins. Anal. Biochem. 197: 7782. 50. HUMBERT, J.R., K. B. HAMMOND, W.E. HATHAWAY, J.G. MARCOUX & D. OBRIEN. 1970. Trimethylaminuria: the fish-odour syndrome. Lancet 2: 770771. 51. CASHMAN, J.R., K. CAMP, S.S. FAKHARZADEH, et al. 2003. Biochemical and clinical aspects of the human flavin-containing monooxygenase form 3 (FMO3) related to trimethylaminuria. Curr. Drug Metab. 4: 151170. 52. LATTARD, V., J. ZHANG, Q. TRAN, et al. 2003. Two new polymorphisms of the FMO3 gene in Caucasian and African-American populations: comparative genetic and functional studies. Drug Metab. Disp. 31: 854860. 53. ZHANG, A.Q., S. MITCHELL & R. SMITH. 1995. Fish odour syndrome: verification of carrier detect test. J. Inherit. Metab. Dis. 18: 669674. 54. ZSCHOCKE, J., D. KOHLMUELLER, E. QUAK, et al. 1999. Mild trimethylaminuria caused by common variants in FMO3 gene. Lancet 354: 834835. 55. CASHMAN, J.R. & J. ZHANG. 2002. Interindividual differences of human flavincontaining monooxygenase 3: genetic polymorphisms and functional variation. Drug Metab. Disp. 30: 10431052. 56. LEOPOLD, D.A., G. PRETI, M.M. MOZELL, et al. 1990. Fish-odor syndrome presenting as dysosmia. Arch. Otolaryngol. Head Neck Surg. 116: 354355. 57. WALKER, V. 1993. The fish odour syndrome: the problems are psychosocial. Br. Med. J. 307: 539. 58. MILLS, G.A., V. WALKER & H. MUGHAL. 1999. Quantitative determination of trimethylamine in urine by solid-phase microextraction and gas chromatographymass spectrometry. J. Chromat. B. 723: 281285. 59. TONZETICH, J. 1971. Direct gas chromatographic analysis of sulphur compounds in mouth air in man. Arch. Oral Biol. 16: 587597. 60. KOSTELC, J.G., G. PRETI, P.R. ZELSON, et al. 1984. Oral odors in early experimental gingivitis. J. Periodontal Res. 19: 303312. 61. PRETI, G., L. CLARK, B.J. COWART, et al. 1992. Non-oral etiologies of oral malodor. J. Periodontol. 63: 790796. 62. DOLPHIN, C.T., J.H. RILEY, R.L. SMITH, et al. 1997. Structural organization of the human flavin-containing monooxygenase 3 gene (FMO3), the favored candidate for fish-odor syndrome, determined directly from genomic DNA. Genomics 46: 260267. 63. BOOTH, G. & S. OSTENSON. 1966. Acetone in alveolar air and the control diabetes. Lancet 2: 11021105.

266

ANNALS OF THE NEW YORK ACADEMY OF SCIENCES

64. WALSH, J. 2004. A breath of fresh air in diabetes detection. Spectroscopy 19: 50. 65. CARPAGNANO, G.E., S.A. KHARITONOV, O. RESTA, et al. 2002. Increased 8isoprostane and interleukin-6 in breath condensate of obstructive sleep apnea patients. Chest 122: 11621167. 66. LECHNER, M., A. KARLSEDER, D. NIEDERSEER, et al. 2005. H. pylori infection increases levels of exhaled nitrate. Helicobacter 10: 385390. 67. PATHAK, C.M., D.K. BHASIN, D. PANIGRAHI & R.C. GOEL. 1994. Evaluation of 14 C-urinary excretion and its comparison with 14 CO2 in breath after 14 C-urea administration in Helicobacter pylori infection. Am. J. Gastroenterol. 89: 734 738. 68. SYLVESTER, K.P. 2005. Exhaled carbon monoxide levels in children with sickle cell disease. Eur. J. Pediat. 164: 162165. 69. HANAZAWA, T., S.A. KHARITONOV, W. OLDFIELD, et al. 2000. Nitrotyrosine and cysteinyl leukotrienes in breath condensates are increased after withdrawal of steroid treatment in patients with asthma. Am. J. Resp. Crit. Care Med. 161: A919. 70. CAP, P., J. CHLADEK, F. PEHAL, et al. 2004. Gas chromatography/mass spectrometry analysis of exhaled leukotrienes in asthmatic patients. Thorax 59: 465470. 71. GORDON, S.M., J.P. SZIDON, B.K. KROTOSZYNSKI, et al. 1985. Volatile organic compounds in exhaled air from patients with lung cancer. Clin. Chem. 31: 1278 1282. 72. PRETI, G., J.N. LABOWS, J.G. KOSTELC, et al. 1988. Analysis of lung air from patients with bronchogenic carcinoma and controls using gas chromatographymass spectrometry. J. Chromat. B: Biomed. Appl. 432: 111. 73. DEKHUIJZEN, P.N., K.K. ABEN, I. DEKKER, et al. 1996. Increased exhalation of hydrogen peroxide in patients with stable and unstable chronic obstructive pulmonary disease. Am. J. Resp. Crit. Care Med. 154: 813816. 74. LIU, J. & P.J. THOMAS. 2005. Exhaled breath condensate as a method of sampling airway nitric oxide and other markers of inflammation. Med. Sci. Monitor 11: MT53MT62. 75. MONTUSCHI, P., S.A. KHARITONOV, G. CIABATTONI, et al. 2000. Exhaled 8isoprostane as a new non-invasive biomarker of oxidative stress in cystic fibrosis. Thorax 55: 205209. 76. BODINI, A., C. DORAZIO, D. PERONI, et al. 2005. Biomarkers of neutrophilic inflammation in exhaled air of cystic fibrosis children with bacterial airway infections. Pediat. Pulmonol. 40: 494499. 77. KAJI, H., M. HISAMURA, N. SATO & M. MURAO. 1978. Gas chromatographic determination of volatile sulfur compounds in the expired alveolar air in the hepatopathic subjects. J. Chromat. B: Biomed. Appl. 78. TANGERMAN, A., M.T. MEUWESE-ARENDS & J.B. JANSEN. 1994. Cause and composition of foetor hepaticus. Lancet 343: 730. 79. SIMENHOFF, M.L., J.F. BURKE, J.J. SAUKKONEN, et al. 1977. Biochemical profile of uremic breath. N. Engl. J. Med. 247: 132135. 80. CHRISTENSEN, C. & M.A. NAVAZESH. 1982. A comparison of whole mouth resting and stimulated salivary measurement procedures. J. Dent. Res. 61: 11581162.