Detection and analysis of endogenous polar volatile organic compounds (PVOCs) in urine for human exposome research.

Background: The human exposome, defined as '…everything that is not the genome', comprises all chemicals in the body interacting with life processes. The exposome drives genes x environment (GxE) interactions that can cause long-term latency and chronic diseases. The exposome constantly changes in response to external exposures and internal metabolism. Different types of compounds are found in different biological media. Objective: Measure polar volatile organic compounds (PVOCs) excreted in urine to document endogenous metabolites and exogenous compounds from environmental exposures. Methods: Use headspace collection and sorbent tube thermal desorption coupled with bench-top gas chromatography-mass spectrometry (GC-MS) for targeted and non-targeted approaches. Identify and categorize PVOCs that may distinguish among healthy and affected individuals. Results: Method is successfully demonstrated to tabulate a series of 28 PVOCs detected in human urine across 120 samples from 28 human subjects. Median concentrations range from below detect to 165 ng/mL. Certain PVOCs have potential health implications. Conclusions: Headspace collection with sorbent tubes is an effective method for documenting PVOCs in urine that are otherwise difficult to measure. This methodology can provide probative information regarding biochemical processes and adverse outcome pathways (AOPs) for toxicity testing.

Human biomarker interpretation: the importance of intra-class correlation coefficients (ICC) and their calculations based on mixed models, ANOVA, and variance estimates.

Human biomonitoring is the foundation of environmental toxicology, community public health evaluation, preclinical health effects assessments, pharmacological drug development and testing, and medical diagnostics. Within this framework, the intra-class correlation coefficient (ICC) serves as an important tool for gaining insight into human variability and responses and for developing risk-based assessments in the face of sparse or highly complex measurement data. The analytical procedures that provide data for clinical and public health efforts are continually evolving to expand our knowledge base of the many thousands of environmental and biomarker chemicals that define human systems biology. These chemicals range from the smallest molecules from energy metabolism (i.e., the metabolome), through larger molecules including enzymes, proteins, RNA, DNA, and adducts. In additiona, the human body contains exogenous environmental chemicals and contributions from the microbiome from gastrointestinal, pulmonary, urogenital, naso-pharyngeal, and skin sources. This complex mixture of biomarker chemicals from environmental, human, and microbiotic sources comprise the human exposome and generally accessed through sampling of blood, breath, and urine. One of the most difficult problems in biomarker assessment is assigning probative value to any given set of measurements as there are generally insufficient data to distinguish among sources of chemicals such as environmental, microbiotic, or human metabolism and also deciding which measurements are remarkable from those that are within normal human variability. The implementation of longitudinal (repeat) measurement strategies has provided new statistical approaches for interpreting such complexities, and use of descriptive statistics based upon intra-class correlation coefficients (ICC) has become a powerful tool in these efforts. This review has two parts; the first focuses on the history of repeat measures of human biomarkers starting with occupational toxicology of the early 1950s through modern applications in interpretation of the human exposome and metabolic adverse outcome pathways (AOPs). The second part reviews different methods for calculating the ICC and explores the strategies and applications in light of different data structures.

Linking physiological parameters to perturbations in the human exposome: Environmental exposures modify blood pressure and lung function via inflammatory cytokine pathway.

Human biomonitoring is an indispensable tool for evaluating the systemic effects derived from external stressors including environmental pollutants, chemicals from consumer products, and pharmaceuticals. The aim of this study was to explore consequences of environmental exposures to diesel exhaust (DE) and ozone (O3) and ultimately to interpret these parameters from the perspective of in vitro to in vivo extrapolation. In particular, the objective was to use cytokine expression at the cellular level as a biomarker for physiological systemic responses such as blood pressure and lung function at the systemic level. The values obtained could ultimately link in vivo behavior to simpler in vitro experiments where cytokines are a measured parameter. Human exposures to combinations of DE and O3 and the response correlations between forced exhaled volume in 1 second (FEV1), forced vital capacity (FVC), systolic and diastolic blood pressure (SBP and DBP, respectively), and 10 inflammatory cytokines in blood (interleukins 1ß, 2, 4, 5, 8, 10, 12p70 and 13, IFN-γ, and TNF-α) were determined in 15 healthy human volunteers. Results across all exposures revealed that certain individuals displayed greater inflammatory responses compared to the group and, generally, there was more between-person variation in the responses. Evidence indicates that individuals are more stable within themselves and are more likely to exhibit responses independent of one another. Data suggest that in vitro findings may ultimately be implemented to elucidate underlying adverse outcome pathways (AOP) for linking high-throughput toxicity tests to physiological in vivo responses. Further, this investigation supports assessing subjects based upon individual responses as a complement to standard longitudinal (pre vs. post) intervention grouping strategies. Ultimately, it may become possible to predict a physiological (systemic) response based upon cellular-level (in vitro) observations.

Taxonomic applicability of inflammatory cytokines in adverse outcome pathway (AOP) development.

Cytokines, low-molecular-weight messenger proteins that act as intercellular immunomodulatory signals, have become a mainstream preclinical marker for assessing the systemic inflammatory response to external stressors. The challenge is to quantitate from healthy subjects cytokine levels that are below or at baseline and relate those dynamic and complex cytokine signatures of exposures with the inflammatory and repair pathways. Thus, highly sensitive, specific, and precise analytical and statistical methods are critically important. Investigators at the U.S. Environmental Protection Agency (EPA) have implemented advanced technologies and developed statistics for evaluating panels of inflammatory cytokines in human blood, exhaled breath condensate, urine samples, and murine biological media. Advanced multiplex, bead-based, and automated analytical platforms provided sufficient sensitivity, precision, and accuracy over the traditional enzyme-linked immunosorbent assay (ELISA). Thus, baseline cytokine levels can be quantified from healthy human subjects and animals and compared to an in vivo exposure response from an environmental chemical. Specifically, patterns of cytokine responses in humans exposed to environmental levels of ozone and diesel exhaust, and in rodents exposed to selected pesticides (such as fipronil and carbaryl), were used as case studies to generally assess the taxonomic applicability of cytokine responses. The findings in this study may aid in the application of measureable cytokine markers in future adverse outcome pathway (AOP)-based toxicity testing. Data from human and animal studies were coalesced and the possibility of using cytokines as key events (KE) to bridge species responses to external stressors in an AOP-based framework was explored.

Analysis of inflammatory cytokines in human blood, breath condensate, and urine using a multiplex immunoassay platform.

A change in the expression of cytokines in human biological media indicates an inflammatory response to external stressors and reflects an early step along the adverse outcome pathway (AOP) for various health endpoints. To characterize and interpret this inflammatory response, methodology was developed for measuring a suite of 10 different cytokines in human blood, exhaled breath condensate (EBC), and urine using an electrochemiluminescent multiplex Th1/Th2 cytokine immunoassay platform. Measurement distributions and correlations for eight interleukins (IL) (1ß, 2, 4, 5, 8, 10, 12p70 and 13), interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α) were evaluated using 90 blood plasma, 77 EBC, and 400 urine samples collected from nominally healthy adults subjects in North Carolina in 2008-2012. The in vivo results show that there is sufficient sensitivity for characterizing all 10 cytokines at levels of 0.05-0.10 ρg/ml with a dynamic range up to 100 ng/ml across all three of these biological media. The measured in vivo results also show that the duplicate analysis of blood, EBC and urine samples have average estimated fold ranges of 2.21, 3.49, and 2.50, respectively, which are similar to the mean estimated fold range (2.88) for the lowest concentration (0.610 ρg/ml) from a series of spiked control samples; the cytokine method can be used for all three biological media. Nine out of the 10 cytokines measured in EBC were highly correlated within one another with Spearman ρ coefficients ranging from 0.679 to 0.852, while the cytokines measured in blood had a mix of negative and positive correlations, ranging from -0.620 to 0.836. Almost all correlations between EBC and blood were positive. This work also represents the first successful within- and between-person evaluation of ultra trace-level inflammatory markers in blood, EBC, and urine.

Volatile Organic Compounds Off-gassing from Firefighters' Personal Protective Equipment Ensembles after Use.

Firefighters' personal protective equipment (PPE) ensembles will become contaminated with various compounds during firefighting. Some of these compounds will off-gas following a response, which could result in inhalation exposure. This study was conducted to determine the magnitude and composition of volatile organic compounds (VOCs) generated during controlled structure burns that subsequently off-gassed from the firefighters' PPE, and were systemically absorbed and exhaled in firefighters' breath. Three crews of five firefighters performed entry, suppression, and overhaul during a controlled burn. We used evacuated canisters to sample air inside the burn structure during active fire and overhaul. After each burn, we placed PPE from two firefighters inside clean enclosures and sampled the air using evacuated canisters over 15 min. Firefighters' exhaled breath was collected ∼1 hr before and 4-14 min after each burn. Using gas chromatography/mass spectrometry, the evacuated canister samples were analyzed for 64 VOCs and the exhaled breath samples were analyzed for benzene, toluene, ethylbenzene, xylene, and styrene (BTEXS). Fourteen of the same VOCs were detected off-gassing from PPE in 50% or more of the samples. Compared to background levels, we measured >5 fold increases in mean off-gas concentrations of styrene, benzene, 1,4-dichlorobenzene, acetone, and cyclohexane. Several of the compounds detected off-gassing from PPE were also measured at concentrations above background during active fire and overhaul, including benzene, propene, and styrene. The overhaul and off-gas air concentrations were well below applicable short-term occupational exposure limits. Compared to pre-burn levels, we measured >2 fold increases in mean breath concentrations of benzene, toluene, and styrene after the burns. Air concentrations of BTEXS measured off-gassing from firefighters' used PPE and in firefighters' post-burn exhaled breath were significantly correlated. The firefighters may have absorbed BTEXS through both the dermal route (during firefighting) and inhalation route (from off-gassing PPE after firefighting). Firefighters should be made aware of the potential for inhalation exposure when doffing and traveling in confined vehicles with contaminated PPE and take measures to minimize this exposure pathway.

Estimating common parameters of lognormally distributed environmental and biomonitoring data: harmonizing disparate statistics from publications.

The progression of science is driven by the accumulation of knowledge and builds upon published work of others. Another important feature is to place current results into the context of previous observations. The published literature, however, often does not provide sufficient direct information for the reader to interpret the results beyond the scope of that particular article. Authors tend to provide only summary statistics in various forms, such as means and standard deviations, median and range, quartiles, 95% confidence intervals, and so on, rather than providing measurement data. Second, essentially all environmental and biomonitoring measurements have an underlying lognormal distribution, so certain published statistical characterizations may be inappropriate for comparisons. The aim of this study was to review and develop direct conversions of different descriptions of data into a standard format comprised of the geometric mean (GM) and the geometric standard deviation (GSD) and then demonstrate how, under the assumption of lognormal distribution, these parameters are used to answer questions of confidence intervals, exceedance levels, and statistical differences among distributions. A wide variety of real-world measurement data sets was reviewed, and it was demonstrated that these data sets are indeed of lognormal character, thus making them amenable to these methods. Potential errors incurred from making retrospective estimates from disparate summary statistics are described. In addition to providing tools to interpret "other people's data," this review should also be seen as a cautionary tale for publishing one's own data to make it as useful as possible for other researchers.

Systemic exposure to PAHs and benzene in firefighters suppressing controlled structure fires.

Turnout gear provides protection against dermal exposure to contaminants during firefighting; however, the level of protection is unknown. We explored the dermal contribution to the systemic dose of polycyclic aromatic hydrocarbons (PAHs) and other aromatic hydrocarbons in firefighters during suppression and overhaul of controlled structure burns. The study was organized into two rounds, three controlled burns per round, and five firefighters per burn. The firefighters wore new or laundered turnout gear tested before each burn to ensure lack of PAH contamination. To ensure that any increase in systemic PAH levels after the burn was the result of dermal rather than inhalation exposure, the firefighters did not remove their self-contained breathing apparatus until overhaul was completed and they were >30 m upwind from the burn structure. Specimens were collected before and at intervals after the burn for biomarker analysis. Urine was analyzed for phenanthrene equivalents using enzyme-linked immunosorbent assay and a benzene metabolite (s-phenylmercapturic acid) using liquid chromatography/tandem mass spectrometry; both were adjusted by creatinine. Exhaled breath collected on thermal desorption tubes was analyzed for PAHs and other aromatic hydrocarbons using gas chromatography/mass spectrometry. We collected personal air samples during the burn and skin wipe samples (corn oil medium) on several body sites before and after the burn. The air and wipe samples were analyzed for PAHs using a liquid chromatography with photodiode array detection. We explored possible changes in external exposures or biomarkers over time and the relationships between these variables using non-parametric sign tests and Spearman tests, respectively. We found significantly elevated (P < 0.05) post-exposure breath concentrations of benzene compared with pre-exposure concentrations for both rounds. We also found significantly elevated post-exposure levels of PAHs on the neck compared with pre-exposure levels for round 1. We found statistically significant positive correlations between external exposures (i.e. personal air concentrations of PAHs) and biomarkers (i.e. change in urinary PAH metabolite levels in round 1 and change in breath concentrations of benzene in round 2). The results suggest that firefighters wearing full protective ensembles absorbed combustion products into their bodies. The PAHs most likely entered firefighters' bodies through their skin, with the neck being the primary site of exposure and absorption due to the lower level of dermal protection afforded by hoods. Aromatic hydrocarbons could have been absorbed dermally during firefighting or inhaled during the doffing of gear that was off-gassing contaminants.

Evolution of environmental exposure science: using breath-borne biomarkers for "discovery" of the human exposome.

According to recent research, 70-90% of long-term latency and chronic human disease incidence is attributable to environmental (human exposome) factors through the gene-environment interaction. Environmental exposure science is now embarking on a new "discovery" path for decoding the human exposome using biomarkers in breath and other biological media.

Quality Control: Hand and Glove Sanitizing in Sterile Compounding, Part 2.

Selecting an appropriate sanitizer (i.e., "rub") for application to hands and gloves before and, if necessary, during sterile compounding is as important as is its consistent and judicious use. Alcohols and chlorhexidine gluconate, which have long been recognized as safe and powerful biocides, are often essential ingredients in such sanitizing products. In this second article in a 2-part series on alcohol-based hand and glove sanitizers, we review the selection of and need for those rubs in sterile compounding, present considerations for their safe storage, compare the features of several appropriate sanitizing agents, and answer compounders' frequently asked questions about their use. Glove sanitizing is discussed as part of the hand-sanitizing process. In part 1 of this series, we explained, among other topics, the mechanism of action and composition of alcohol-based sanitizers and presented a protocol for their application to hands and gloves.

Quality Control: Hand and Glove Sanitizing in Sterile Compounding, Part 1.

In pharmaceutical compounding, strict adherence to a protocol for hand hygiene and glove sanitizing is essential to ensure the purity, safety, and effectiveness of sterile preparations; reduce patient morbidity and mortality; and decrease the cost of health care. Alcohols and chlorhexidine gluconate are among the most effective bactericides, virucides, and fungicides, and acquired resistance to those agents has not been shown in clinical practice. This article, which is part 1 in a series of 2, pertains primarily to alcohol-based hand rubs that are appropriate for use in sterile compounding (glove sanitizing is discussed as part of the handsanitizing process). In a brief overview of those products, we define pertinent terminology, examine the necessity of and requirements for the use of sanitizers, review their mechanism of action and composition, consider factors pertinent to their selection, and present a protocol for their application. In part 2 of this series, the topics examined include a comparison of various alcohol-based sanitizers and answers to compounders' frequently asked questions about their use.

Predicting polycyclic aromatic hydrocarbons using a mass fraction approach in a geostatistical framework across North Carolina.

Currently in the United States there are no regulatory standards for ambient concentrations of polycyclic aromatic hydrocarbons (PAHs), a class of organic compounds with known carcinogenic species. As such, monitoring data are not routinely collected resulting in limited exposure mapping and epidemiologic studies. This work develops the log-mass fraction (LMF) Bayesian maximum entropy (BME) geostatistical prediction method used to predict the concentration of nine particle-bound PAHs across the US state of North Carolina. The LMF method develops a relationship between a relatively small number of collocated PAH and fine Particulate Matter (PM2.5) samples collected in 2005 and applies that relationship to a larger number of locations where PM2.5 is routinely monitored to more broadly estimate PAH concentrations across the state. Cross validation and mapping results indicate that by incorporating both PAH and PM2.5 data, the LMF BME method reduces mean squared error by 28.4% and produces more realistic spatial gradients compared to the traditional kriging approach based solely on observed PAH data. The LMF BME method efficiently creates PAH predictions in a PAH data sparse and PM2.5 data rich setting, opening the door for more expansive epidemiologic exposure assessments of ambient PAH.

Standardization of the collection of exhaled breath condensate and exhaled breath aerosol using a feedback regulated sampling device.

Exhaled breath condensate (EBC) and associated exhaled breath aerosols (EBA) are valuable non-invasive biological media used for the quantification of biomarkers. EBC contains exhaled water vapor, soluble gas-phase (polar) organic compounds, ionic species, plus other species including semi- and non-volatile organic compounds, proteins, cell fragments, DNA, dissolved inorganic compounds, ions, and microbiota (bacteria and viruses) dissolved in the co-collected EBA. EBC is collected from subjects who breathe 'normally' through a chilled tube assembly for approximately 10 min and is then harvested into small vials for analysis. Aerosol filters without the chilled tube assembly are also used to separately collect EBA. Unlike typical gas-phase breath samples used for environmental and clinical applications, the constituents of EBC and EBA are not easily characterized by total volume or carbon dioxide (CO2) concentration, because the gas-phase is vented. Furthermore, EBC and associated EBA are greatly affected by breathing protocol, more specifically, depth of inhalation and expelled breath velocity. We have tested a new instrument developed by Loccioni Gruppa Humancare (Ancona, Italy) for implementation of EBC collection from human subjects to assess EBC collection parameters. The instrument is the first EBC collection device that provides instantaneous visual feedback to the subjects to control breathing patterns. In this report we describe the operation of the instrument, and present an overview of performance and analytical applications.

Inflammatory Cytokines and White Blood Cell Counts Response to Environmental Levels of Diesel Exhaust and Ozone Inhalation Exposures.

Epidemiological observations of urban inhalation exposures to diesel exhaust (DE) and ozone (O3) have shown pre-clinical cardiopulmonary responses in humans. Identifying the key biological mechanisms that initiate these health bioindicators is difficult due to variability in environmental exposure in time and from person to person. Previously, environmentally controlled human exposure chambers have been used to study DE and O3 dose-response patterns separately, but investigation of co-exposures has not been performed under controlled conditions. Because a mixture is a more realistic exposure scenario for the general public, in this study we investigate the relationships of urban levels of urban-level DE exposure (300 µg/m3), O3 (0.3 ppm), DE + O3 co-exposure, and innate immune system responses. Fifteen healthy human volunteers were studied for changes in ten inflammatory cytokines (interleukins 1ß, 2, 4, 5, 8, 10, 12p70 and 13, IFN-γ, and TNF-α) and counts of three white blood cell types (lymphocytes, monocytes, and neutrophils) following controlled exposures to DE, O3, and DE+O3. The results show subtle cytokines responses to the diesel-only and ozone-only exposures, and that a more complex (possibly synergistic) relationship exists in the combination of these two exposures with suppression of IL-5, IL-12p70, IFN-γ, and TNF-α that persists up to 22-hours for IFN-γ and TNF-α. The white blood cell differential counts showed significant monocyte and lymphocyte decreases and neutrophil increases following the DE + O3 exposure; lymphocytes and neutrophils changes also persist for at least 22-hours. Because human studies must be conducted under strict safety protocols at environmental levels, these effects are subtle and are generally only seen with detailed statistical analysis. This study indicates that the observed associations between environmental exposures and cardiopulmonary effects are possibly mediated by inflammatory response mechanisms.

Exploratory breath analyses for assessing toxic dermal exposures of firefighters during suppression of structural burns.

Firefighters wear fireproof clothing and self-contained breathing apparatus (SCBA) during rescue and fire suppression activities to protect against acute effects from heat and toxic chemicals. Fire services are also concerned about long-term health outcomes from chemical exposures over a working lifetime, in particular about low-level exposures that might serve as initiating events for adverse outcome pathways (AOP) leading to cancer. As part of a larger US National Institute for Occupational Safety and Health (NIOSH) study of dermal exposure protection from safety gear used by the City of Chicago firefighters, we collected pre- and post-fire fighting breath samples and analyzed for single-ring and polycyclic aromatic hydrocarbons as bioindicators of occupational exposure to gas-phase toxicants. Under the assumption that SCBA protects completely against inhalation exposures, any changes in the exhaled profile of combustion products were attributed to dermal exposures from gas and particle penetration through the protective clothing. Two separate rounds of firefighting activity were performed each with 15 firefighters per round. Exhaled breath samples were collected onto adsorbent tubes and analyzed with gas-chromatography-mass spectrometry (GC-MS) with a targeted approach using selective ion monitoring. We found that single ring aromatics and some PAHs were statistically elevated in post-firefighting samples of some individuals, suggesting that fire protective gear may allow for dermal exposures to airborne contaminants. However, in comparison to a previous occupational study of Air Force maintenance personnel where similar compounds were measured, these exposures are much lower suggesting that firefighters' gear is very effective. This study suggests that exhaled breath sampling and analysis for specific targeted compounds is a suitable method for assessing systemic dermal exposure in a simple and non-invasive manner.

Clinical breath analysis: discriminating between human endogenous compounds and exogenous (environmental) chemical confounders.

Volatile organic compounds (VOCs) in exhaled breath originate from current or previous environmental exposures (exogenous compounds) and internal metabolic (anabolic and catabolic) production (endogenous compounds). The origins of certain VOCs in breath presumed to be endogenous have been proposed to be useful as preclinical biomarkers of various undiagnosed diseases including lung cancer, breast cancer, and cardio-pulmonary disease. The usual approach is to develop difference algorithms comparing VOC profiles from nominally healthy controls to cohorts of patients presenting with a documented disease, and then to apply the resulting rules to breath profiles of subjects with unknown disease status. This approach to diagnosis has a progression of sophistication; at the most rudimentary level, all measurable VOCs are included in the model. The next level corrects exhaled VOC concentrations for current inspired air concentrations. At the highest level, VOCs exhibiting discriminatory value also require a plausible biochemical pathway for their production before inclusion. Although these approaches have all shown some level of success, there is concern that pattern recognition is prone to error from environmental contamination and between-subject variance. In this paper, we explore the underlying assumptions for the interpretation and assignment of endogenous compounds with probative value for assessing changes. Specifically, we investigate the influence of previous exposures, elimination mechanisms and partitioning of exogenous compounds as confounders of true endogenous compounds. We provide specific examples based on a simple classical pharmacokinetic approach to identify potential misinterpretations of breath data and propose some remedies.

Breath biomarkers in environmental health science: exploring patterns in the human exposome.

The human genome is the counterpart to the human exposome with respect to the gene × environment interaction that describes health state and outcome. The genome has already been sequenced and is in the process of being assessed for specific functionality; to similarly decode the exposome will require the measurement and interpretation of suites of biomarker compounds in biological media such as blood, breath and urine. Of these, exhaled breath provides some important advantages for community or population-based studies in that the supply is essentially unlimited, the sampling procedures are non-invasive and can be self-administered, and there are little, if any, infectious wastes generated. The main concerns are to document a variety of compounds in breath, to assess what compounds and concentrations are considered statistically 'normal' in the healthy or unremarkably exposed population, and what graphic and mathematical approaches can be applied to assess outlier measurements as perturbations to the healthy exposome. In this paper, we explore a data set of exhaled breath measurements of exogenous exposures to jet fuel and develop summary statistics and variable clustering methods to establish between-group and intrinsic within-sample patterns that could be used to assess the status of random subjects.

Heat map visualization of complex environmental and biomarker measurements.

Over the past decade, the assessment of human systems interactions with the environment has permeated all phases of environmental and public health research. We are invoking lessons learned from the broad discipline of Systems Biology research that focuses primarily on molecular and cellular networks and adapting these concepts to Systems Exposure Science which focuses on interpreting the linkage from environmental measurements and biomonitoring to the expression of biological parameters. A primary tool of systems biology is the visualization of complex genomic and proteomic data using "heat maps" which are rectangular color coded arrays indicating the intensity (or amount) of the dependent variable. Heat maps are flexible in that both the x-axis and y-axis can be arranged to explore a particular hypothesis and allow a fast overview of data with a third quantitative dimension captured as different colors. We are now adapting these tools for interpreting cumulative and aggregate environmental exposure measurements as well as the results from human biomonitoring of biological media including blood, breath and urine. This article uses existing EPA measurements of environmental and biomarker concentrations of polycyclic aromatic hydrocarbons (PAHs) to demonstrate the value of the heat map approach for hypothesis development and to link back to stochastic and mixed effects models that were originally used to assess study results.

Observing the human exposome as reflected in breath biomarkers: heat map data interpretation for environmental and intelligence research.

Over the past decade, the research of human system biology and the interactions with the external environment has permeated all phases of environmental, medical and public health research. Similar to the fields of genomics and proteomics research, the advent of new instrumentation for measuring breath biomarkers and their associated meta-data also provide very useful, albeit complex, data structures. The biomarker research community is beginning to invoke tools from system biology to assess the impact of environmental exposures, as well as from internal health states, on the expression of suites of chemicals in exhaled breath. This new approach introduces the concept of the exposome as a complement to the genome in exploring the environment-gene interaction. In addition to answering questions regarding health status for the medical community, breath biomarker patterns are useful for assessing public health risks from environmental exposures. Furthermore, breath biomarker patterns can inform security risks from suspects via covert interrogation of blood borne chemical levels that reflect previous activities. This paper discusses how different classes of exhaled breath biomarker measurements can be used to rapidly assess patterns in complex data. We present exhaled breath data sets to demonstrate the value of the graphical 'heat map' approach for hypothesis development and subsequent guidance for stochastic and mixed effect data interpretation. We also show how to graphically interpret exhaled breath measurements of exogenous jet fuel components, as well as exhaled breath condensate measurements of endogenous chemicals.