A state-of-the-science review and guide for measuring environmental exposure biomarkers in dried blood spots.

BACKGROUND: Dried blood spot (DBS) sampling is a simple, cost-effective, and minimally invasive alternative to venipuncture for measuring exposure biomarkers in public health and epidemiological research. DBS sampling provides advantages in field-based studies conducted in low-resource settings and in studies involving infants and children. In addition, DBS samples are routinely collected from newborns after birth (i.e., newborn dried blood spots, NDBS), with many states in the United States permitting access to archived NDBS samples for research purposes. OBJECTIVES: We review the state of the science for analyzing exposure biomarkers in DBS samples, both archived and newly collected, and provide guidance on sample collection, storage, and blood volume requirements associated with individual DBS assays. We discuss recent progress regarding analytical methods, analytical sensitivity, and specificity, sample volume requirements, contamination considerations, estimating extracted blood volumes, assessing stability and analyte recovery, and hematocrit effects. METHODS: A systematic search of PubMed (MEDLINE), Embase (Elsevier), and CINAHL (EBSCO) was conducted in March 2022. DBS method development and application studies were divided into three main chemical classes: environmental tobacco smoke, trace elements (including lead, mercury, cadmium, and arsenic), and industrial chemicals (including endocrine-disrupting chemicals and persistent organic pollutants). DBS method development and validation studies were scored on key quality-control and performance parameters by two members of the review team. RESULTS: Our search identified 47 published reports related to measuring environmental exposure biomarkers in human DBS samples. A total of 28 reports (37 total studies) were on methods development and validation and 19 reports were primarily the application of previously developed DBS assays. High-performing DBS methods have been developed, validated, and applied for detecting environmental exposures to tobacco smoke, trace elements, and several important endocrine-disrupting chemicals and persistent organic pollutants. Additional work is needed for measuring cadmium, arsenic, inorganic mercury, and bisphenol A in DBS and NDBS samples. SIGNIFICANCE: We present an inventory and critical review of available assays for measuring environmental exposure biomarkers in DBS and NDBS samples to help facilitate this sampling medium as an emerging tool for public health (e.g., screening programs, temporal biomonitoring) and environmental epidemiology (e.g., field-based studies).

Exhaled breath condensate biomarkers in critically ill, mechanically ventilated patients.

Pneumonia is a significant risk for critically ill, mechanically ventilated (CIMV) patients. Diagnosis of pneumonia generally requires a combination of clinician-guided diagnoses and clinical scoring systems. Exhaled breath condensate (EBC) can be safely collected non-invasively from CIMV patients. Hundreds of biomarkers in EBC are associated with acute disease states, including pneumonia. We evaluated cytokines in EBC from CIMV patients and hypothesized that these biomarkers would correlate with disease severity in pneumonia, sepsis, and death. EBC IL-2 levels were associated with chest radiograph severity scores (odds ratio = 1.68; 95% confidence interval = 1.09-2.60; P = 0.02). EBC TNF-α levels were also associated with pneumonia (odds ratio = 3.20; 95% confidence interval = 1.19-8.65; P = 0.02). The techniques and results from this study may be useful for all mechanically ventilated patients.

Dataset of polycyclic aromatic hydrocarbon recoveries from a selection of sorbent tubes for thermal desorption-gas chromatography/mass spectrometry analysis.

This dataset contains raw area counts and percent recoveries of polycyclic aromatic hydrocarbon (PAH) standards desorbed from selected sorbent tubes and analyzed using thermal desorption-gas chromatography/mass spectrometry (TD-GC/MS). The results of this study were published in the article "Recovery and reactivity of polycyclic aromatic hydrocarbons collected on selected sorbent tubes and analyzed by thermal desorption-gas chromatography/mass spectrometry" in Journal of Chromatography A [1]. The sorbent tubes studied include stainless steel Carbograph 2TD/1TD, glass quartz wool-Carbograph 2TD, inert-coated stainless steel Carbograph 2TD, glass and stainless steel Tenax TA, PAH (chemical weapons), and glass and stainless steel XRO-440 sorbent tubes. Tables listing the experimental conditions, TD methods, and types of sorbent tubes are included in the manuscript. Data for experiments, including the investigation of incomplete desorption of PAHs from Carbograph 2TD/1TD and XRO-440 sorbent tubes, the comparison of PAH recoveries from three different TD methods, the analysis of PAH breakthrough from sorbent tubes, the investigation of the effect of heat on PAH percent recovery from sorbent tubes, and the formation of reaction products during PAH loading and desorption are included in Appendix A. These data can be used to guide sorbent tube selection for PAH analyses in future studies.

How do cancer-sniffing dogs sort biological samples? Exploring case-control samples with non-targeted LC-Orbitrap, GC-MS, and immunochemistry methods.

Early identification of disease onset is regarded as an important factor for successful medical intervention. However, cancer and other long-term latency diseases are rare and may take years to manifest clinically. As such, there are no gold standards with which to immediately validate proposed preclinical screening methodologies. There is evidence that dogs can sort samples reproducibly into yes/no categories based on case-control training, but the basis of their decisions is unknown. Because dogs are sniffing air, the distinguishing chemicals must be either in the gas-phase or attached to aerosols and/or airborne particles. Recent biomonitoring research has shown how to extract and analyze semi- and non-volatile compounds from human breath in exhaled condensates and aerosols. Further research has shown that exhaled aerosols can be directly collected on standard hospital-style olefin polypropylene masks and that these masks can be used as a simple sampling scheme for canine screening. In this article, detailed liquid chromatography-high resolution mass spectrometry (LC-HR-MS) with Orbitrap instrumentation and gas chromatography-mass spectrometry (GC-MS) analyses were performed on two sets of masks sorted by consensus of a four-dog cohort as either cancer or control. Specifically, after sorting by the dogs, sample masks were cut into multiple sections and extracted for LC-MS and GC-MS non-targeted analyses. Extracts were also analyzed for human cytokines, confirming the presence of human aerosol content above levels in blank masks. In preliminary evaluations, 345 and 44 high quality chemical features were detected by LC-MS and GC-MS analyses, respectively. These features were used to develop provisional orthogonal projection to latent structures-discriminant analysis (OPLS-DA) models to determine if the samples classified as cancer (case) or non-cancer (control) by the dogs could be separated into the same groups using analytical instrumentation. While the OPLS-DA model for the LC-HR-MS data was able to separate the two groups with statistical significance, although weak explanatory power, the GC-MS model was not found to be significant. These results suggest that the dogs may rely on the less volatile compounds from breath aerosol that were analyzed by LC-HR-MS than the more volatile compounds observed by GC-MS to sort mask samples into groups. These results provide justification for more expansive studies in the future that aim to characterize specific chemical features, and the role(s) of these features in maintaining homeostatic biological processes.

Firefighters' and instructors' absorption of PAHs and benzene during training exercises.

INTRODUCTION: Training fires may constitute a major portion of some firefighters' occupational exposures to smoke. However, the magnitude and composition of those exposures are not well understood and may vary by the type of training scenario and fuels. OBJECTIVES: To understand how structure fire training contributes to firefighters' and instructors' select chemical exposures, we conducted biological monitoring during exercises involving combustion of pallet and straw and oriented strand board (OSB) or the use of simulated smoke. METHODS: Urine was analyzed for metabolites of polycyclic aromatic hydrocarbons (PAHs) and breath was analyzed for volatile organic compounds (VOCs) including benzene. RESULTS: Median concentrations of nearly all PAH metabolites in urine increased from pre-to 3-hr post-training for each scenario and were highest for OSB, followed by pallet and straw, and then simulated smoke. For instructors who supervised three trainings per day, median concentrations increased at each collection. A single day of OSB exercises led to a 30-fold increase in 1-hydroxypyrene for instructors, culminating in a median end-of-shift concentration 3.5-fold greater than median levels measured from firefighters in a previous controlled-residential fire study. Breath concentrations of benzene increased 2 to 7-fold immediately after the training exercises (with the exception of simulated smoke training). Exposures were highest for the OSB scenario and instructors accumulated PAHs with repeated daily exercises. CONCLUSIONS: Dermal absorption likely contributed to the biological levels as the respiratory route was well protected. Training academies should consider exposure risks as well as instructional objectives when selecting training exercises.

Recovery and reactivity of polycyclic aromatic hydrocarbons collected on selected sorbent tubes and analyzed by thermal desorption-gas chromatography/mass spectrometry.

This article describes the optimization of methodology for extending the measurement of volatile organic compounds (VOCs) to increasingly heavier polycyclic aromatic hydrocarbons (PAHs) with a detailed focus on recent sorbent tube technology. Although PAHs have lower volatility than compounds such as benzene, toluene, ethylbenzene and xylenes, these semi-volatile compounds can be detected in air and breath samples. For this work, PAHs were captured on sorbent tubes and subsequently analyzed using automated thermal desorption gas chromatography - mass spectrometry (ATD-GC/MS). While many different sorbent tubes are commercially available, optimization for airborne PAH sampling using sorbent tubes has not been previously considered. Herein, several commercially available sorbent tubes, including Carbograph 2 TD/1TD, Tenax TA, XRO-440, and inert-coated PAH tubes are compared to determine the relative recovery for eight PAHs commonly found in the environment. Certain types of sorbent materials were found to be better suited for PAH recovery during thermal desorption, and PAH reaction products were observed on several types of sorbent tubes, including graphitized carbon black sorbents with stainless steel tube materials. As such, selection of sorbent tube media should be carefully considered prior to embarking on a PAH study.

Targeted GC-MS analysis of firefighters' exhaled breath: Exploring biomarker response at the individual level.

Biomarker measurements can provide unambiguous evidence of environmental exposures as well as the resultant biological responses. Firefighters have a high rate of occupational cancer incidence, which has been proposed to be linked in part to their increased environmental exposure to byproducts of combustion and contaminants produced during fire responses. In this article, the uptake and elimination of targeted volatile organic compounds were investigated by collecting the exhaled breath of firefighters on sorbent tubes before and after controlled structure burns and analyzing samples using automated thermal desorption-gas chromatography (ATD-GC/MS). Volatile organic compounds exposure was assessed by grouping the data according to firefighting job positions as well as visualizing the data at the level of the individual firefighter to determine which individuals had expected exposure responses. When data were assessed at the group level, benzene concentrations were found to be elevated post-exposure in both fire attack, victim search, and outside ventilation firefighting positions. However, the results of the data analysis at the individual level indicate that certain firefighters may be more susceptible to post-exposure volatile organic compounds increases than others, and this should be considered when assessing the effectiveness of firefighting protective gear. Although this work focuses on firefighting activity, the results can be translated to potential human health and ecological effects from building and forest fires.

Evolution of clinical and environmental health applications of exhaled breath research: Review of methods and instrumentation for gas-phase, condensate, and aerosols.

Human breath, along with urine and blood, has long been one of the three major biological media for assessing human health and environmental exposure. In fact, the detection of odor on human breath, as described by Hippocrates in 400 BC, is considered the first analytical health assessment tool. Although less common in comparison to contemporary bio-fluids analyses, breath has become an attractive diagnostic medium as sampling is non-invasive, unlimited in timing and volume, and does not require clinical personnel. Exhaled breath, exhaled breath condensate (EBC), and exhaled breath aerosol (EBA) are different types of breath matrices used to assess human health and disease state. Over the past 20 years, breath research has made many advances in assessing health state, overcoming many of its initial challenges related to sampling and analysis. The wide variety of sampling techniques and collection devices that have been developed for these media are discussed herein. The different types of sensors and mass spectrometry instruments currently available for breath analysis are evaluated as well as emerging breath research topics, such as cytokines, security and airport surveillance, cellular respiration, and canine olfaction.

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.

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.

Estimating central tendency from a single spot measure: A closed-form solution for lognormally distributed biomarker data for risk assessment at the individual level.

Exposure-based risk assessment employs large cross-sectional data sets of environmental and biomarker measurements to predict population statistics for adverse health outcomes. The underlying assumption is that long-term (many years) latency health problems including cancer, autoimmune and cardiovascular disease, diabetes, and asthma are triggered by lifetime exposures to environmental stressors that interact with the genome. The aim of this study was to develop a specific predictive method that provides the statistical parameters for chronic exposure at the individual level based upon a single spot measurement and knowledge of global summary statistics as derived from large data sets. This is a profound shift in exposure and health statistics in that it begins to answer the question "How large is my personal risk?" rather than just providing an overall population-based estimate. This approach also holds value for interpreting exposure-based risks for small groups of individuals within a community in comparison to random individuals from the general population.

Immunochemistry for high-throughput screening of human exhaled breath condensate (EBC) media: implementation of automated Quanterix SIMOA instrumentation.

Immunochemistry is an important clinical tool for indicating biological pathways leading towards disease. Standard enzyme-linked immunosorbent assays (ELISA) are labor intensive and lack sensitivity at low-level concentrations. Here we report on emerging technology implementing fully-automated ELISA capable of molecular level detection and describe application to exhaled breath condensate (EBC) samples. The Quanterix SIMOA HD-1 analyzer was evaluated for analytical performance for inflammatory cytokines (IL-6, TNF-α, IL-1ß and IL-8). The system was challenged with human EBC representing the most dilute and analytically difficult of the biological media. Calibrations from synthetic samples and spiked EBC showed excellent linearity at trace levels (r(2) > 0.99). Sensitivities varied by analyte, but were robust from ~0.006 (IL-6) to ~0.01 (TNF-α) pg ml(-1). All analytes demonstrated response suppression when diluted with deionized water and so assay buffer diluent was found to be a better choice. Analytical runs required ~45 min setup time for loading samples, reagents, calibrants, etc., after which the instrument performs without further intervention for up to 288 separate samples. Currently, available kits are limited to single-plex analyses and so sample volumes require adjustments. Sample dilutions should be made with assay diluent to avoid response suppression. Automation performs seamlessly and data are automatically analyzed and reported in spreadsheet format. The internal 5-parameter logistic (pl) calibration model should be supplemented with a linear regression spline at the very lowest analyte levels, (<1.3 pg ml(-1)). The implementation of the automated Quanterix platform was successfully demonstrated using EBC, which poses the greatest challenge to ELISA due to limited sample volumes and low protein levels.

Commentary on the contributions and future role of occupational exposure science in a vision and strategy for the discipline of exposure science.

Exposure science is a holistic concept without prejudice to exposure source. Traditionally, measurements aimed at mitigating environmental exposures have not included exposures in the workplace, instead considering such exposures to be an internal affair between workers and their employers. Similarly, occupational (or industrial) hygiene has not typically accounted for environmental contributions to poor health at work. Many persons spend a significant amount of their lifetime in the workplace, where they maybe exposed to more numerous chemicals at higher levels than elsewhere in their environment. In addition, workplace chemical exposures and other exogenous stressors may increase epigenetic and germline modifications that are passed on to future generations. We provide a brief history of the development of exposure science from its roots in the assessment of workplace exposures, including an appendix where we detail current resources for education and training in exposure science offered through occupational hygiene organizations. We describe existing successful collaborations between occupational and environmental practitioners in the field of exposure science, which may serve as a model for future interactions. Finally, we provide an integrated vision for the field of exposure science, emphasizing interagency collaboration, the need for complete exposure information in epidemiological studies, and the importance of integrating occupational, environmental, and residential assessments. Our goal is to encourage communication and spur additional collaboration between the fields of occupational and environmental exposure assessment. Providing a more comprehensive approach to exposure science is critical to the study of the "exposome", which conceptualizes the totality of exposures throughout a person's life, not only chemical, but also from diet, stress, drugs, infection, and so on, and the individual response.

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.

Editorial: new analytical and statistical approaches for interpreting the relationships among environmental stressors and biomarkers.

The broad topic of biomarker research has an often-overlooked component: the documentation and interpretation of the surrounding chemical environment and other meta-data, especially from visualization, analytical and statistical perspectives. A second concern is how the environment interacts with human systems biology, what the variability is in "normal" subjects, and how such biological observations might be reconstructed to infer external stressors. In this article, we report on recent research presentations from a symposium at the 248th American Chemical Society meeting held in San Francisco, 10-14 August 2014, that focused on providing some insight into these important issues.

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.

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.

Estimating lifetime risk from spot biomarker data and intraclass correlation coefficients (ICC).

Human biomarker measurements in tissues including blood, breath, and urine can serve as efficient surrogates for environmental monitoring because a single biological sample integrates personal exposure across all environmental media and uptake pathways. However, biomarkers represent a "snapshot" in time, and risk assessment is generally based on long-term averages. In this study, a statistical approach is proposed for estimating long-term average exposures from distributions of spot biomarker measurements using intraclass correlations based upon measurement variance components from the literature. This methodology was developed and demonstrated using a log-normally distributed data set of urinary OH-pyrene taken from our own studies. The calculations are generalized for any biomarker data set of spot measures such as those from the National Health and Nutrition Evaluation Studies (NHANES) requiring only spreadsheet calculations. A three-tiered approach depending on the availability of metadata was developed for converting any collection of spot biomarkers into an estimated distribution of individual means that can then be compared to a biologically relevant risk level. Examples from a Microsoft Excel-based spreadsheet for calculating estimates of the proportion of the population exceeding a given biomonitoring equivalent level are provided as an appendix.

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.