Breath analysis science at PittCon 2012,Orlando, Florida

Abstract

BackgroundBreath analysis science was featured in three organized sessions at this year's PittsburghConference and Exposition, or 'PittCon 2012' (http://www.pittcon.org/). As described inprevious meeting reports, PittCon is one of the largest international conferences foranalytical chemistry and instrumentation, typically attracting about 20 000 attendees and1000 commercial exhibitors (Pleil 2010, 2011). This year the conference was held inOrlando, Florida, USA at the Orange County Conference Center.In the past few years, breath analysis science has taken on an ever-increasing role atPittCon through the success of the International Association of Breath Research (IABR) andthe Journal of Breath Research (JBR). We have also had a presence at a recent conference ofthe Submarine Air Monitoring and Air Purification (SAMAP) organization in Taranto, Italy,wherein breath analysis and other biomarker measurements are taking on an increasinglyimportant role in assessing exposures and health in artificial atmospheres includingsubmarines, aircraft and spacecraft (Pleil and Hansel 2012).In 2012, members of IABR organized or participated in three distinct PittCon events: acontributed technical session, a conferee networking session, and an invited symposiumseries. The common thread among these three different conferencing settings was theconcept of 'non-invasive' environmental, medical, and diagnostic biomarker assessment. Ofparticular interest was how breath is being considered more and more mainstream as abiological medium, and is often discussed favorably as complementary or alternative toblood and urine measurements.Technical session: non-invasive biomedical analysisThis was our JBR/IABR signature session organized and co-chaired by Dr Joachim Pleilfrom the US Environmental Protection Agency and Dr Wolfram Miekisch1 from the RostockUniversity Hospital in Rostock, Germany. Our speakers were carefully selected to cover abroad range on breath diagnostic techniques with some topics including urinary biomarkers,the 'other' non-invasive (or at least minimally invasive) biomarker approach.The first presentation, 'Non-invasive biomedical analysis—dawning of a new area ofdiagnostic information' was given by Professor Jochen K Schubert2 from the University ofRostock, Germany. Professor Schubert discussed the important role that diagnostic tests playin life-saving therapy and in early stage disease detection. He particularly stressed theincreasing need for non-invasive techniques (especially for critical care medicine) and forrapid assessments preferably performed at the bedside. He discussed new technologies forinstrument miniaturization for breath analysis and presented results showing the potential ofreal-time mass spectrometers based on proton transfer reactions (PTR). He also gave anintriguing example for a minimally invasive blood test based on a gas-phase (breath)technology wherein specially coated fibers are inserted directly into the blood stream toabsorb certain compounds of interest for subsequent desorption analysis using standard solidphase micro-extraction (SPME) methods.Dr Jens Herbig3 from Ionimed Analytik in Innsbruck, Austria gave the secondpresentation, entitled 'Applications of PTR-MS in medicine and biotechnology'. Dr Herbigbegan his talk with an introduction to proton-transfer-reaction mass spectrometers(PTR-MS), highlighting benefits of these analytical tools for real-time analysis of volatileorganic compounds (VOCs) in biological media, and articulating differences betweenavailable quadrupole and time-of-flight (ToF) based detections systems. He thendemonstrated how, via real-time monitoring of exhaled breath, PTR-MS instruments can beused to evaluate chemical uptake, distribution and metabolism in the human body. Finally, heshowed applications of the PTR-MS systems for real-time monitoring of in vitro systems. Anexample was given of the analysis of fermentation off-gas produced by micro-organisms.Together these descriptions and examples showed unique advantages of real-time monitoringequipment over classical offline tools, drawing considerable interest from session attendees.Dr Jon Sobus from the US Environmental Protection Agency presented 'Statisticalconsiderations for interpreting urinary biomarker concentrations'. He discussed how urine,like breath, is a preferred sample medium for biomedical analysis since it is available inabundant supply and collected using non-invasive techniques. He highlighted that urinarybiomarker measurements are generally reported in units of concentration, and are thereforesubject to variations from changing urine output, as well as changing exposure or healthstate. Dr Sobus stressed that, given this dependence, careful evaluation of urinary biomarkermeasurements is critical for decision making in medical and public health investigations. Hefurther proposed a mathematical approach to correct for changing urine output; correctionfactors, based in part on urinary creatinine measurements, used empirical observations ofhealthy adults made during a recent EPA study. In closing, Dr Sobus highlighted implicationsof using and not using correction factors for biomedical urine analysis.Dr Sobus was followed by Professor Cristina E Davis4 from the University of California,Davis with a presentation entitled 'Mammalian cell culture headspace volatile organiccompounds hold vital clues as putative biomarkers of cellular changes'. Professor Davisdescribed the analysis of VOCs from cell culture using gas chromatography-massspectrometry (GC-MS) to develop an odor fingerprint. She illustrated how VOC headspaceanalysis can distinguish between human B cells differing by a single human leukocyteantigen (HLA) gene, demonstrating how VOC analysis can illustrate changes occurring on acellular level. Professor Davis then showed that different viral infections result in uniquefluctuations to VOC fingerprints indicating tremendous biomedical potential. She suggestedthe potential for such a VOC analysis as a non-invasive technique for diagnosing infectionsgiven the development of appropriate sensor devices.Next, Dr Tzipporah M Kormos from the US Environmental Protection Agency presented'Metabolomics evaluation: perturbations of organic metabolites in human breath and urine'.Dr Kormos provided an overview of the attempts underway in method development at the USEPA for applying non-targeted top-down approaches to link environmental exposures tohuman health effects. She described the goal of characterizing the human exposome in orderto effectively link exposure to health effects. However, she also denoted how the eclecticchemical composition of the exposome poses an analytical challenge requiring a range ofinstrumentation to effectively characterize its components (including liquidchromatography-mass spectrometry (LC-MS), nuclear magnetic resonance spectroscopy(NMR), immunochemistry, and GC-MS). Dr Kormos stressed the need for non-invasivesampling in such exposure studies as the only way to obtain a sufficient number of biologicalsamples from the general populace in field studies.The sixth presentation of the session, entitled 'Real-time measurements andmathematical modeling of breath biomarkers to address the impact of physiological effects',was presented by Dr Julian King5 of the Austrian Academy of Sciences, Dornbirn, Austria.Here, Dr King presented real-time measurements of VOCs in breath samples, collectedduring periods of rest, exercise and sleep, and determined using PTR-MS and solid phasemicro-extraction (SPME)/GC-MS. Dr King focused on acetone and isoprene as targetmolecules, and showed how measurements of these analytes, combined with mathematicalmodels, can be used to explore mechanistic relationships governing the general behaviors of VOCs in breath. Dr King suggested that these results provide a basis for experimental designand general guidance for interpreting empirical results. Specifically, he recommended breathgas analysis for evaluating metabolic processes of VOCs, namely storage, transport, andbiotransformation, in vivo.Matthew Stiegel from the University of North Carolina at Chapel Hill, NC, gave theseventh talk, entitled 'Correlations of inflammatory cytokines in blood, exhaled breathcondensate, and urine'. This talk focused on multi-media biological measurements ofinterleukins, interferon-γ, and tumor necrosis factor-a made as part of a controlled chamberexperiment using human volunteers. Here, the TH1/TH2 cytokines were evaluated as makersof inflammatory/endogenous response to various controlled exposures, including dieselexhaust, ozone, and diesel exhaust plus ozone. Stiegel showed that cytokine levels andpatterns changed across media and subjects, and suggested that these changes reflectsubject-specific responses to the controlled exposures. He concluded that cytokine levels inblood, exhaled breath condensate (EBC), and urine are probative indicators of exposureand/or endogenous response, and thus, are useful for exposure and health monitoring.Phillip Trefz6 from the University of Rostock, Germany gave the final talk 'Microextraction techniques as a link between clinical application and hyphenated analyticaltechniques'. Mr Trefz explained how a current hindrance to the biomedical application ofbreath analysis is sampling. As analytical instrumentation is not yet a bedside technique,reliable tools are required for breath collection, concentration, and storage in order to enablethe detection of trace VOCs. He explained the benefits of needle trap devices (NTDs) oversolid phase extraction (SPE) and SPME techniques in terms of sensitivity and sampling time.A comparison of different sorbents showed the possibility of storing samples over a weekwhile retaining high levels of recovery and reproducibility. He then described an interestingpilot study using NTDs to sample from ventilators of patients with various health conditions.Networking session: non-invasive biomedical analysis—the fast, the furious,and the brave—innovative analytical instrumentation for breath gas testingAs in past PittCon meetings, the networking session was organized and facilitated by IABR'sown Dr Wolfram 'Wolfie' Miekisch. The theme this year was the spectrum of novelinstrumentation spanning the range from biomarker discovery, high-throughput analysis, andeventual outpatient or homecare devices. The play on words in the title (taken from recentmovies) referred to the three featured underlying analytical regimes for breath-discoveryusing two dimensional gas chromatography and time of flight mass spectrometry(2D-GC-ToFMS), on-line assessment and screening using PTR-MS, and the use of specificchemical sensors for outpatient monitoring and continuing therapy assessment.After an opening statement and introduction by Dr Miekisch, Dr Jens Herbig began thenetworking session with a brief introduction of what it takes to have an 'ideal' breathanalysis instrument. He stated that the PTR-MS has all four main components of this idealinstrument: sensitivity, specificity, real-time practicality, and accuracy. The fact that it has allfour of these necessary elements makes it the instrument to use when doing real-timebiomarker discovery or monitoring. The discussion moved from an instrumentationperspective to a more philosophical one as audience members wondered why it wasimportant to have the ability, or even the desire, to use a discovery tool such as the PTR-MS.Opinions given ranged from the need for top-down discovery approaches that involve patternanalysis and perturbations of normal biomarker ranges, to the use of targetedsingle-compound, or 'low-hanging fruit', analysis that could be perceived as more effective.It was further suggested that PTR-MS is more of an operational instrument, in that it tradesoff speed of analysis for highly resolved specificity. Other discussions revolved around theissue of 'competition' with blood and urine analysis; the strength of breath analysis inreal-time is that it provides access to reactive/intermediary metabolites that otherwise canonly be inferred from stable compounds measurements.Joe Binkley7 from Leco Corporation in St. Joseph, MI, followed this enlighteningdiscussion with an introduction to the details of 2D-GC-ToFMS. The instrumentation waspresented as a great discovery tool that allowed for a comprehensive analysis of difficult matrices such as that of human breath. The increased chromatographic resolution ofGC×GC made possible by orthogonal modes of separation (i.e. boiling point and polarity)as well as increased sensitivity due to the cryo-focusing effects of thermal modulation havelead to increasing use of this technology amongst breath researchers. The practicalapplications of the instrumentation were immediately compared to those of the PTR-MS.The 2D-GC-ToFMS was shown to be excellent not only in discovery but also withquantification; however, it lacked the ability to do real-time analysis, potentially losing anyreactive compounds during the downtime from sampling to analysis. The PTR-MS shines inthe real-time arena; however, it lacks the quantification ability of the 2D-GC-ToFMS. Theconsensus was that neither style of instrumentation was intrinsically 'better' than the otherbut that they both served the research needs well within their niche applications. Commentswere made that 2D GC creates so much data that it takes quite some time to interpret it, butthat it is probably the best way to get an appreciation for the breath exposome.The last segment of the session was introduced briefly by Dr Andreas Hengstenberg8from Draeger, Lübeck, Germany. Draeger is well known for a variety of laboratory andindustrial safety products including handheld or portable instrumentation for making airmeasurements. Dr Hengstenberg commented that blood analysis is still considered to be thegold standard in medical practice and that this is a difficult hurdle to overcome. He presentedsome thoughts as to how chemical-specific biosensors could be implemented for breathbiomonitoring applications with handheld devices that could be used for outpatient orhomecare monitoring where blood analysis cannot effectively compete. Dr Hengstenberg ledthe discussion to the concept of the 'triangle' of breath analysis comprised of the largelaboratory discovery instruments (e.g. GC-MS or ToF, LC-MS-MS or ToF, 2D-GC-ToFMS),the real-time multi-components instruments (e.g. PTR-MS or ToF, SIFT-MS), and finally thesmall, targeted instruments (e.g. chemical sensors, sensor arrays, optical absorption,photoacoustic). The subsequent discussions revolved around the need for all three types ofmethodologies to be used in parallel. Although the eventual goal is the penetration ofnon-invasive breath measurement into the home at the personal level, this cannot occurwithout the continuous discovery and validation from the other two research arenasimplementing broad-based analysis. The ensuing audience discussion concluded that specificbiosensors could be used to support all three arenas but that the targeted sensors were limitedby their compound-specific characteristicsSymposium: breath analysis as a non-invasive alternativefor medical diagnosticsThe symposium session for breath analysis was organized by Professor Janusz Pawliszyn9from University of Waterloo, Canada. Professor Pawliszyn is the inventor of SPMEtechniques for measuring trace-level organic compounds in complex gas-phase, in theheadspace of liquid and solid phase matrices, and even immersed in complex liquid phasessuch as blood and urine. SPME is now a mainstream technique for all forms ofenvironmental and biomarker research including blood, breath and urine metabolites.Professor Pawliszyn gave a brief introduction about breath analysis and then proceeded toserve as the moderator for the symposium presentations.The first speaker was Professor Raed A Dweik10 from the Cleveland Clinic in Ohio. Hepresented an overview entitled 'The state of breath analysis: achievements and challenges'wherein he discussed some of the most pressing issues in furthering the acceptance of breathanalysis for clinical practice and diagnosis. He used the development of the standardizationfor nitric oxide (NO) analysis as an example that the rest of breath applications methodologyshould follow. In particular, he described the painstaking process undertaken by theAmerican Thoracic Society (ATS) and the European Respiratory Society (ERS) as a jointeffort to develop specific guidance for NO analysis. Without such efforts and assurances ofunambiguous quality, he suggests that breath analysis will not be readily accepted in theclinical and diagnostic communities. In fact, he suggests that physicians want a simpleanswer for a diagnosis from breath analysis methods like a traffic light where green means healthy, yellow means maybe, and red means disease; he then added, ' . but without theyellow'.He was followed by Professor Anton Amann11 from the Austrian Academy of Sciences,Innsbruck Austria who continued with the overview theme with his presentation 'The state ofbreath analysis: achievements and challenges—analytical perspective'. He discussed recentwork in his laboratory that implemented new proton transfer reaction-time of flight massspectrometry (PTR-ToF-MS) instrumentation to monitor endogenous breath markers in realtime. As an example, he demonstrated how the metabolic pathway for isoprene in breathcould be traced back to muscle tissue acting as both source and reservoir. His experimentsalso included analysis of breath during sleep cycles wherein he showed that exhaled isopreneis directly affected by sleep stage, increasing during deep sleep, and decreasing during REMstages.Professor Terence H Risby12 from Johns Hopkins University, Baltimore, gave the nextpresentation 'Methodological aspects of VOCs collection in real-time breath analysis'.Initially, Professor Risby explained that exhaled organic biomarkers are not unique fordifferent diseases but all reflect specific human biochemical pathways; the probativedifferences between sick and healthy patients are seen in changes in patterns that depend onhow the pathways are affected by the disease. He also stressed the importance of regulatingand monitoring the breathing technique of patients during breath collection because humanstend to hyperventilate when they focus on their own breathing. Feedback for depth ofinhalation, breath pacing, and exhalation volume is necessary to assure that samples arecomparable within and between subjects and among different laboratories. This isaccomplished by monitoring exhaled carbon dioxide and differential pressure. He alsostressed that external analytical standards made up to simulate the breath matrix are necessaryto assure that cross-laboratory results are comparable. Only if diagnostic results can beshown as consistent and accurate will diagnostic breath methods ever be broadly accepted.Dr Joachim D Pleil presented the environmental aspects of discovery research inbiomonitoring; his presentation was entitled 'Breath biomarkers in environmental healthscience: decoding the human exposome'. He discussed new discovery research fordistinguishing between the human pulmonary microbiome and endogenous breathbiomarkers derived from human metabolism. His examples showed GC-MS results forheadspace analyses of cultured anaerobic bacteria known to infect humans, and contrastedthem to aerosols collected directly from humans and grown under anaerobic conditions. Healso followed up on Professor Risby's concepts regarding breath sampling by introducingnew instrumentation from Loccioni Group, San Rosario, Italy, that was adapted to collectEBC using feedback 'coaching' of the subjects. He showed preliminary data that variabilityin collected volume was reduced by a factor of 10.The final symposium speaker was Dr Heather L Lord13 from University of Waterloo,Canada who presented her research implementing various novel sampling techniques forbreath analysis. Her talk was entitled 'Micro-sampling/sample preparation devices for breathanalysis' which focused on micro-scale sampling of breath wherein the organic compoundsare partitioned away from the bulk breath matrix either through transmission or absorptionfor subsequent analysis. She explained that the novel membrane and sorbent-basedtechnologies provide a fast and efficient concentration step due to the high surface to volumeratio in contrast to other absorptive devices. This technology is expected to be well suited forminiaturization and application to handheld devices as the sampling technology is essentiallypassive and requires little, if any, power.Concluding remarksThis year's foray of IABR members into the world of PittCon and analytical instrumentationwas a successful venture. We had the opportunity to interface with the broader instrumentand laboratory equipment manufacturing community. Breath work was well received and wehope to continue to disseminate the message that non-invasive biomarker methodology,especially using breath as a biological medium, is a valuable tool. We intend to continue our relationship with PittCon and encourage the JBR readership to consider attending andcontributing to future meetings. We also encouraged our PittCon colleagues to participate inbreath-specific endeavors by looking at JBR, the IABR website, and possibly attending thenext IABR meeting.The United States Environmental Protection Agency through its Office of Research andDevelopment has subjected this article to Agency administrative review and approved it forpublication.ReferencesPleil J D 2010 Meeting Report: breath biomarkers networking sessions at PittCon 2010, OrlandoFlorida J. Breath Res. 4 029001Pleil J D 2011 Meeting Report: breath biomarkers networking session at PittCon 2011, Atlanta, GA J. Breath Res. 5 029001Pleil J D and Hansel A 2012 Submarines, spacecraft and exhaled breath J. Breath Res. 6 0190011 wolfram.miekisch@uni-rostock.de2 jochen.schubert@uni-rostock.de3 jens.herbig@ionimed.com4 cedavis@ucdavis.edu5 julian.king@assoc.oeaw.ac.at6 phillip.trefz@uni-rostock.de7 joe_binkley@leco.com8 andreas.hengstenberg@draeger.com9 janusz@uwaterloo.ca10 dweikr@ccf.org11 anton.amann@i-med.ac.at12 thrisby@jhmi.edu13 hlord@uwaterloo.ca