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Exhaled breath condensate (EBC) describes any sample collected by cooling exhaled breath. Because the method of condensate collection is simple, non-invasive, repeatable and does not necessarily require patient cooperation, EBC is not only an interesting, but also challenging, biological sample. Despite a period of EBC research lasting for more than 15 years, there are still many open questions with respect to EBC collection and analysis, and many biomarkers are still awaiting careful validation. In veterinary research, EBC collection has been described in conscious animals including calves, pigs, horses, cats and dogs. Numerous studies performed in these domestic animals not only contributed substantially to the current knowledge about the potentials of EBC-based diagnoses but also demonstrated pitfalls in EBC collection, analysis and interpretation. This review summarizes information about the collection of EBC and the interpretation of EBC results, particularly with respect to proteins, leukotrienes, hydrogen peroxide, urea, ammonia and pH. Published data emphasize the need to standardize approaches to produce reproducible EBC data. Quantifying the concentration of the EBC component of interest exhaled in a defined volume of exhaled breath (instead of comparing concentrations of this component analysed in liquid EBC) is an important step of standardization that might help to overcome methodological limitations deriving from the EBC collection process. Although information is based on domestic animal studies, it contributes to the general understanding in EBC research—independent of any particular mammalian species—and opens new perspectives for further studies.

The metabolism of ingested xenobiotics is clinically significant to minimize risk and optimize therapeutic benefits. A majority of the drugs approved by the FDA are metabolized by phase I enzymes. Stable isotope-labeled xenobiotics can be used to provide rapid in vivo phenotype assessment of phase I enzymes. In this paper, we describe three simple, noninvasive phenotype breath tests using [¹³C]-dextromethorphan and [¹³C]-pantoprazole for assessing polymorphic CYP2D6 and CYP2C19 enzyme activity and [¹³C]-uracil to assess the enzyme activity of DPD, DPHD and β-ureidopropionase for identifying pyrimidine metabolic disorder. The results of the [¹³C]-dextromethorphan, [¹³C]-pantoprazole and [¹³C]-uracil breath test studies suggest that they have great potential for evaluating CYP2D6, CYP2C19 and DPD enzyme activities in a relatively short time with a single time point breath collection in a clinic or hospital setting. This would enable physicians to prescribe personalized therapy for each individual by selecting the ideal medication at the right dose for optimal efficacy of xenobiotics metabolized by these enzymes.

Halitosis can be subdivided into intra-oral and extra-oral halitosis, depending on the place where it originates. Most reports now agree that the most frequent sources of halitosis exist within the oral cavity and include bacterial reservoirs such as the dorsum of the tongue, saliva and periodontal pockets, where anaerobic bacteria degrade sulfur-containing amino acids to produce the foul smelling volatile sulfur compounds (VSCs), especially hydrogen sulfide (H₂S) and methyl mercaptan (CH₃SH). Tongue coating is considered to be the most important source of VSCs. Oral malodor can now be treated effectively. Special attention in this overview is given to extra-oral halitosis. Extra-oral halitosis can be subdivided into non-blood-borne halitosis, such as halitosis from the upper respiratory tract including the nose and from the lower respiratory tract, and blood-borne halitosis. The majority of patients with extra-oral halitosis have blood-borne halitosis. Blood-borne halitosis is also frequently caused by odorous VSCs, in particular dimethyl sulfide (CH₃SCH₃). Extra-oral halitosis, covering about 5–10% of all cases of halitosis, might be a manifestation of a serious disease for which treatment is much more complicated than for intra-oral halitosis. It is therefore of utmost importance to differentiate between intra-oral and extra-oral halitosis. Differences between intra-oral and extra-oral halitosis are discussed extensively. The importance of applying odor characterization of various odorants in halitosis research is also highlighted in this article. The use of the odor index, odor threshold values and simulation of bad breath samples is explained.

The published results of breath isoprene studies, to date largely involving adults, are briefly reviewed with special attention given to the work done on this topic during the last 10 years using selected ion flow tube mass spectrometry, SIFT-MS. Then the new data recently obtained on isoprene levels in the exhaled breath of some 200 healthy children and young adults (pupils) with ages ranging from 7 to 18 years measured using SIFT-MS are presented in detail. A concentration distribution has been constructed from the data obtained and compared to that for healthy adults also obtained from SIFT-MS data. Although there is overlap between the two distributions, which are close to log normal in both cases, the median level for the young cohort is much lower at 37 parts-per-billion, pbb, geometric standard deviation, GSD, 2.5, compared to that for the adult cohort of 106 ppb with a GSD of 1.65. Further to this, there is a clear increase in the mean breath isoprene concentration with age for the young cohort with a doubling of the level about every 5–6 years until it reaches the age-invariant mean level of that for adult cohort. Should this trend be extrapolated downwards in age it would indicate a near-zero breath isoprene in the newborn that was indicated by a previous study. Indeed, in the present study isoprene was not detected on the breath of two young children. The results reveal mean breath isoprene levels (±SD) for pupils within the given age ranges as 7–10 years (28 ± 24 ppb), 10–13 years (40 ± 21 ppb), 13–16 years (60 ± 41 ppb) and 16–19 years (54 ± 31 ppb). The more rapid increase that occurs between the second and third age ranges is statistically highly significant (p = 0.001) and we attribute this phenomenon to the onset of puberty and the spurt in growth that occurs during this phase of development. There is no significant difference in mean breath isoprene between males and females for both the adult cohort and the younger cohort.

The present study was conducted to assess the efficacy of a new mouthrinse formulation in reducing oral malodour compared to that of commercially available products containing chlorhexidine (CHX) and a negative control. 174 healthy volunteers, each with an organoleptic score of at least 2 and an H₂S level as part of the volatile sulfur compounds (VSC) higher than 50 ppb, were divided into four groups. Participants were stratified according to their organoleptic ratings (OR). Group I: mouthrinse I (250 ppm F⁻ from amine fluoride/stannous fluoride (ASF), 0.2% zinc lactate, oral malodour counteractives); group II: mouthrinse II (0.05% CHX, 0.05% cetylpyridinium chloride, 0.14% zinc lactate); group III: mouthrinse III (0.12% CHX); group IV: tap water. All groups were instructed to perform standardized oral hygiene measures and to apply the respective test rinse twice daily after tooth brushing. Malodour was assessed by organoleptic measurement and by VSC levels at baseline, day 1, day 7, day 14 and day 21 into the study. To evaluate discolouration of the teeth, the colour was assessed at baseline and final visit. The ASF mouthrinse showed superior efficacy as compared to the negative control. A significant reduction in OR and VSC readings was achieved after single application as well as after 7 and 21 days of continuous use. Between test groups I–III no statistically significant differences were found at any time point. There was also a trend towards fewer side effects caused by the ASF product compared to the products containing CHX. The newly developed mouthrinse product significantly reduces oral malodour in patients with increased values both in OR and in VSC.

The loads and locations of bacterial types associated with oral malodour on the tongue surface and gingival crevice were investigated in man and the dog respectively. In the human study, samples were taken from 50 subjects with brushes at the dorsal anterior, dorsal middle, dorsal posterior, dorsal posterior to the circumvallate papillae (DPCP), lateral posterior and ventral posterior (VP) surfaces, and cultured appropriately. Malodour was assessed by trained judges. Mean volatile sulfur compound (VSC) producing bacterial counts (colony forming units/brush × 10⁵) were found to be highest (88.94) and lowest (0.33) at the DPCP and VP sites respectively. Anaerobic, gram-negative and VSC counts at DPCP surfaces increased with malodour intensity, whereas aerobic and S. salivarius counts decreased. The prevalence and populations of the VSC producing Porphyromonas and Prevotella species were determined in the dental plaque from 34 dogs. Porphyromonas gulae and Prevotella intermedia were present in 68% and 44% of dogs, and 47% and 23% of plaque samples respectively. P. gulae and Prev. intermedia counts increased with plaque quantity (P < 0.05) and gingivitis (P < 0.1). The close association observed between canine periodontal disease and measurements of oral malodour is supported.

The sensitive assay method was developed for a parallel, rapid and precise determination of the most prominent oxidative stress biomarkers: 8-iso-prostaglandin F_2α a lipid oxidation biomarker, o-tyrosine an amino acid oxidation biomarker and 8-hydroxy-2'-deoxy-guanosine a nucleic acid oxidation biomarker. The method consisted of a pre-treatment part, freeze drying (lyophilization), serving the purpose of biomarkers concentration from the exhaled breath condensate and detection method LC-ESI-MS/MS, where the selected reaction-monitoring mode was used for its extremely high degree of selectivity and the stable-isotope-dilution assay for its high precision of quantification. The developed method is characterized by the following parameters: the precision was higher than 84.3% and the mean accuracy (relative error) was determined lower than 11.6%. The method was tested on samples obtained from patients diagnosed with asbestosis and silicosis, occupational diseases induced by oxidative stress, and then compared with samples from healthy subjects. The difference in biomarkers' concentration levels found between the two groups was statistically significant.

Arterial lactate concentrations, taken as indicators of physical fitness, in athletes as well as in patients with cardio-respiratory or metabolic diseases, are measured invasively from arterialized ear lobe blood. Currently developed micro enzyme detectors permit a non-invasive measurement of hypoxia-related metabolites such as lactate in exhaled breath condensate (EBC). The aim of our study is to prove whether this technology will replace the traditional measurement of lactate in arterialized blood. Therefore, we determined the functional relation between lactate release in EBC and lactate concentration in blood in young and healthy subjects at rest and after exhausting bicycle exercise. During resting conditions as well as after exhausting bicycle exercise, 100 L of exhaled air along with blood samples from the ear lobe was collected after stationary load conditions in 16 healthy subjects. EBC was obtained by cooling the expired air volume with an ECoScreen I® (FILT GmbH, Berlin) condenser. The analysis was performed within 90 min using an ECoCheck® ampere meter (FILT GmbH, Berlin). Lactate measurements were performed using a bi-enzyme sensor after lactate oxidase-induced oxidation of lactate to pyruvate and H₂O₂. The rates of lactate release via the exhaled air were calculated from the lactate concentration, the volume and the collection time of the EBC. The functional relation of lactate release in exhaled air and lactate concentration of arterial blood was computed. At rest, the mean lactate concentration in arterialized blood was 0.93 ± 0.30 mmol L⁻¹. At a resting ventilation of 11.5 ± 3.4 L min⁻¹, the collection time for 100 L of exhaled air, Ts, was 8.4 ± 2.9 min, and 1.68 ± 0.40 mL EBC was obtained. In EBC, the lactate concentration was 21.4 ± 7.7 µmol L⁻¹, and the rate of lactate release rate in collected EBC was 4.5 ± 1.7 nmol min⁻¹. After maximal exercise load (220 ± 20 W), the blood lactate concentration increased to 10.9 ± 1.8 mmol L⁻¹ and the ventilation increased to 111.6 ± 21.4 L min⁻¹. The EBC collection time decreased to 3.9 ± 1.9 min, and 1.20 ± 0.44 mL EBC were obtained in the recovery period after termination of exercise. The lactate concentration in EBC increased to 40.3 ± 23.0 µmol L⁻¹, and the lactate release in EBC increased to 13.6 ± 8.6 nmol min⁻¹ (p < 0.01). Assuming a volume of 4.3 mL water in 100 L of exhaled air (saturated with water at 37 °C), we calculated a lactate release at rest of 11.5 ± 4.3 nmol min⁻¹ and 48.6 ± 30.7 nmol min⁻¹ (p < 0.01) after exhausting exercise. Detectable releases of lactate in exhaled breath condensate were found already under resting conditions. During exhausting external load on a bicycle spiroergometer, an increase in the lactate concentration was found in arterialized blood along with an increased lactate release in EBC. The correlation between expiratory lactate release via EBC and lactate concentration in arterialized blood is studied in pursuing investigations.

Volatile sulfur compounds (VSCs), specifically hydrogen sulfide, methyl mercaptan and dimethyl sulfide, are generally considered to be the primary volatiles responsible for 'morning' malodors in breath. To date, the 'gold standard' for detecting VSC concentrations in breath is the use of gas chromatography coupled with sulfur chemiluminescence detection. Breath gas is often collected in a polypropylene syringe and then aliquots are injected into a gas chromatograph for analysis. The objective of this work was to compare the Twister™ bar in-mouth extraction methodology for measurement of VSCs with the gas syringe breath-sampling collection technique. The Twister bar technology captures malodorous compounds in the mouth as opposed to breath gas. Using these techniques, comparable results were obtained in studies demonstrating the efficacy of a proprietary oral malodor counteraction system.

The pH of the tongue biofilm is likely to influence microbial composition and ecology with consequent effects on the metabolic activities and generation of volatile sulfur compounds (VSC) and other malodour gasses. The aim of this study was to identify the effects of pH on the development of biofilms and hydrogen sulfide production using an in vitro tongue-derived biofilm model. Community level physiological profiling (CLPP) was employed to examine the influence of pH on the collective metabolic fingerprint of each tongue-derived biofilm. A sorbarod perfusion system (n = 6 sorbarods) was inoculated from a single suspension of tongue scrape sample and mixed community tongue-derived biofilms were grown at pH 5.5, 6.0, 6.5, 7.0 7.5 and 8.0. Biofilms were perfused with medium for 120 h and gas phase samples (n = 4 per biofilm) removed and analysed with a portable sulfide gas chromatograph before being sacrificed into 10 ml sterile PBS-diluent and cells suspended by vortex mixing. Further ten-fold dilutions were made (down to 10⁻⁷) and dilutions plated out onto selective (fastidious anaerobic agar (FAA) + 0.0025% vancomycin) and non-selective (FAA) media for enumeration of strict and facultative anaerobes respectively. Biofilm suspensions were also mixed with Biolog inoculation fluid and distributed into 96 wells of Biolog AN plates for CLPP. Tongue biofilms developed at pH 7.5 produced significant (p < 0.05) concentrations of H₂S (≈52.2 ± SEM 5.6 µg H₂S per ml biofilm gas phase) followed by tongue biofilm developed at pH 7.0 and 8.0 (≈43.2 ± SEM 3.5 and ≈ 39.6 ± SEM 7.3 µg H₂S per ml biofilm gas phase respectively). Tongue biofilm developed at pH 6.0 and 6.5 produced approximately 21.5 ± SEM 2.3 and 37.1 ± SEM 1.7 µg H₂S per ml biofilm gas phase respectively and tongue biofilm developed at pH 5.5 produced approximately 0.19 ± SEM 0.09 µg H₂S per ml biofilm gas phase. Highest numbers of strict and facultative anaerobes were recovered from biofilms at pH 6.5 (1.10 × 10¹² and 2.07 × 10¹² cfu ml⁻¹ respectively), with a reduced number recovered from pH values above and below this range. CLPP and similarity index revealed biofilms at pH 6.5 and 7.0 most similar (S_j = 78%) and most diverse in terms of metabolic activity. The biofilm at pH 5.5 was the least related to all others and least diverse. The sorbarod perfusion system, in conjunction with H₂S analysis and CLPP, enables some of the physiological and ecological effects of pH at a local level within the biofilm on H₂S production to be identified.

Volatile sulfur compounds (VSCs) are produced by enzymes capable of transforming S-amino acids to corresponding sulfides. Protein degradation by periodontopathogens plays an important role in this process, and the proteolysis of glycoproteins depends on the initial removal of the carbohydrate side chains. In the present report, we tested the relationship between the β-galactosidase activity in saliva and parameters that influence oral malodor, including daily habits and oral conditions. The prevalence of periodontopathic bacteria was also examined. Forty-nine saliva samples were collected from halitosis patients. Patients were examined for breath odor and other associated parameters. Their breath odor was assessed using an organoleptic test, a portable sulfide monitor and gas chromatography. The presence of periodontopathic bacteria in the saliva was also examined. β-galactosidase activity was measured with the chromogenic substrates 5-bromo-4-chloro-3-indoyl-β-d-galactopyranoside and isopropyl-β-d-thiogalactopyranoside. β-galactosidase activity was positively correlated with malodor strength (organoleptic score, portable sulfide monitor score and VSC concentrations). Enzyme activity was also correlated with the degree of observable tongue coating. However, it showed no relationship with periodontal condition, saliva flow, tooth decay, unfitted restorations or the color of any tongue coating. While there was no relationship with Porphyromonas gingivalis and Treponema denticola, there was a negative correlation with Prevotella intermedia. These results indicate that β-galactosidase activity plays an important role in malodor production. Interestingly, the activity of this enzyme was not related to the presence of periodontopathic bacteria, which are the main malodor-producing organisms. The results obtained here may have been associated with physiologic halitosis, which is not necessarily associated with oral problems or with periodontopathic bacteria.

Pulmonary oedema is a medical condition characterized by abnormal accumulation of fluid in the extravascular space in the alveoli. Effective oxygenation is impaired and this leads to significant short- and long-term morbidity and mortality. The detection and monitoring of pulmonary oedema by measuring lung water volume is therefore crucial in the initiation and guidance of therapeutic intervention. The current gold standard bedside measurement of extravascular lung water volume (EVLW) is the dilution method using various indicators, but despite the good correlation of the results with those obtained using the post-mortem gravimetric method, the invasiveness of the dilution technique limits its general application in the wider clinical setting. In the present preliminary experiments, the dispersal kinetics of deuterium (actually HDO) in exhaled breath of three healthy participants following the inhalation of deuterium oxide (D₂O) vapour are explored as monitored using flowing afterglow mass spectrometry (FA-MS). Here, we present the basic ideas of lung water estimation using this novel technique, and briefly discuss its limitations and required future work.

The conferences of the International Association of Breath Research (IABR) have a tradition going back to 2004 when the the first conference was organized in Dornbirn, Austria. Subsequent conferences have taken place in Innsbruck (2005), Prague (2006), Cleveland (2007) and then in Dortmund in 2009.

The conference in Dortmund was organized sucessfully by Jörg Ingo Baumbach. With around 300 delegates it was also the largest conference in the series of IABR meetings. It was innovative insofar as it was taking place under the auspices of IABR and the International Society for Breath Odor Research (ISBOR). From ongoing cooperation between the two societies, there are now emerging joint projects in overlapping fields of research such as those related to bacteria in the oral cavity.

The Dortmund conference demonstrated that analytical technology for detection of volatile compounds is developing quickly. An example is the use of the time- of-flight (TOF) technique in mass spectrometry, which allows a reduction in measurement time and improvement of the mass resolution. Such developments on the analytical side are of enormous importance as they will push the field forward and will allow the analysis of breath samples with a very high sensitivity and within a few seconds. In addition to higher sensitivity and reduced measurement time, the miniaturization of instruments for clinical application is also showing considerable progress. Tunable lasers or miniaturized ion mobility spectrometers are examples of techniques which will revolutionize the clinical applications of breath research. Whereas in the past, accurate analysis of breath samples could only be done by large research instruments which need analytical expertise, the next few years will bring small instruments which are easily transportable and easily operable. The use of such instruments in clinical practice is not only restricted to medical diagnosis, but will also incorporate therapeutic monitoring even at intensive care units or during surgery.

Another very promising field is the use of isotopically labelled precursors (see the review article by Anil Modak [1]). ¹³C-labelled compounds, for example, can be engineered to end up as ¹³CO₂, which is observed in exhaled breath. The measurement of the ¹³CO₂-content in breath after ingestion of such precursor compounds gives information on the metabolization kinetics of drugs or specific foods. An example would be the tolerance of 5- fluorouracil (5-FU), which is used as a chemotherapeutic. People unable to metabolize 5-FU will also be unable to metabolize ¹³C- labelled uracil, which is now used for the respective breath test. The relevant enzyme dihydropyrimidine dehydrogenase (DPD) is genetically based on transcription of 23 exons. The respective genetic testing would lead to much higher costs than a simple breath test. In addition, the breath test determines the phenotype which sometimes differs from the genotype (due to low expression of the respective gene). With ¹³C-labelled compounds becoming less expensive, such tests can be developed for many different medications and will therefore allow a 'personalized medicine' and personalized medication regimes to become available at reasonable cost.

Another interesting field of research related to breath odor is the investigation of tongue biofilms (see the paper by Benjamin Taylor and John Greenman [2]). Using this technique for simulation of different environments for bacteria (e.g., with different O₂-content) will allow a much better understanding of the bacterial background of malodorous compounds in breath. Also the investigation of saliva (see the paper by M Yoneda et al [3]) gives way to new non-invasive information which complements results achieved through exhaled breath or exhaled breath condensate.

The Journal of Breath Research (JBR) is dedicated to all of these different exciting new developments. JBR is now established as an essential cornerstone in the timely publication of new results in the field and will help in the development of clinically applicable breath tests in the years to come.

References [1] Modak A 2010 Single time point diagnostic breath tests: a review J. Breath Res.4 017002 [2] Taylor B and Greenman J 2010 Modelling the effects of pH on tongue biofilm using a sorbarod biofilm perfusion system J. Breath Res.4 017107 [3] Yoneda M, Masuo Y, Suzuki N, Iwamoto T and Hirofuji T 2010 Relationship between the β-galactosidase activity in saliva and parameters associated with oral malodor J. Breath Res.4 017108