Characterization of standardized breath sampling for off-line field use.

Due to several sources of potential variability associated with exhaled breath bag sampling procedures for off-line analysis, the Respiration Collector for in vitro Analysis (ReCIVA) sampler was developed. Although designed to improve upon several pitfalls of sampling with exhaled breath bags, the ReCIVA remains a minimally studied research tool. In this manuscript, several attributes of the ReCIVA sampler are investigated among three individual tests, such as background contamination, control software version, performance of different adsorbent tubes, duplicate sample production, and comparison to exhaled breath bags. The data shows greater than a 58% reduction in background siloxanes can be achieved with submersion of ReCIVA masks in ethyl alcohol or baking the masks at a high temperature (200 °C). The results illustrate the ReCIVA control software version plays a key role in the flow rates applied to thermal desorption (TD) tubes. Using exhaled isoprene as a representative analyte, the data suggest duplicate samples among ReCIVA pump banks can be achieved using two different thermal desorption tubes, Tenax TA and Tenax/Carbograph 5TD, when using an updated control software and manually calibrating the ReCIVA pumps to uniform flow rates (Tenax p = 0.3869, 5TD p = 0.3131). Additionally, using the updated control software and manual ReCIVA flow calibration, the data suggest the ReCIVA can produce statistically similar results among TD tube types (p = 0.3824) and compared to standard exhaled breath bags (p = 0.1534). Collectively, these results establish a method for manually calibrating the flow of the ReCIVA device to allow for the most consistent results. These data support further experimentation into the use of the ReCIVA sampler for exhaled breath research.

Exhaled isoprene for monitoring recovery from acute hypoxic stress.

Hypoxia-like incidents in-flight have increased over the past decade causing severe safety concerns across the aviation community. As a result, the need to monitor flight crews in real-time for the onset of hypoxic conditions is paramount for continued aeronautical safety. Here, hypoxic events were simulated in the laboratory via a reduced oxygen-breathing device to determine the effect of recovery gas oxygen concentration (21% and 100%) on exhaled breath volatile organic compound composition. Data from samples collected both serially (throughout the exposure), prior to, and following exposures yielded 326 statistically significant features, 203 of which were unique. Of those, 72 features were tentatively identified while 51 were verified with authentic standards. A comparison of samples collected serially between recovery and hypoxia time points shows a statistically significant reduction in exhaled breath isoprene (2-methyl-1,3-butadiene, log2 FC -0.399, p = 0.005, FDR = 0.034, q = 0.033), however no significant difference in isoprene abundance was observed when comparing recovery gases (21% or 100% O2, p = 0.152). Furthermore, examination of pre-/post-exposure 1 l bag breath samples illustrate an overall increase in exhaled isoprene abundance post-exposure (log2 FC 0.393, p = 0.005, FDR = 0.094, q = 0.033) but again no significant difference between recovery gas (21% and 100%, p = 0.798) was observed. A statistically significant difference in trend was observed between isoprene abundance and recovery gases O2 concentration when plotted against minimum oxygen saturation (p = 0.0419 100% O2, p = 0.7034 21% O2). Collectively, these results suggest exhaled isoprene is dynamic in the laboratory ROBD setup and additional experimentation will be required to fully understand the dynamics of isoprene in response to acute hypoxic stress.

Dimerization Products of Chloroprene are Background Contaminants Emitted from ALTEF (Polyvinylidene Difluoride) Gas Sampling Bags.

Gas sampling bags have been used for collecting air samples. Tedlar bags are most commonly used, but bleed background chemicals such as N,N-dimethylacetamide and phenol. It is often necessary to remove the contaminant by flushing the bags with pure nitrogen or air. In this study, we identified four chloroprene dimerization products as background contaminants emitted from ALTEF bags that are made of a proprietary polyvinylidene difluoride (PVDF). No monomer chloroprene was detected in the bags analyzed. All of the dimers gradually increased once bags were filled with nitrogen due to diffusion from the bag surface. Flushing the bags with nitrogen reduced their concentrations, but was not effective for removing the contaminants. When the bags that had been flushed with nitrogen 5 times were left for 24 h, they increased again, indicating that the dimers were constantly emitted from the ALTEF bag surface. To our knowledge, these compounds have never been demonstrated in ALTEF or other PVDF bags. Our finding indicates that ALTEF might be incorporated with Neoprene (chloroprene-based polymer) during its manufacturing process.

Storage stability of exhaled breath on Tenax TA.

Exhaled breath is coming to the forefront of non-invasive biomarker discovery efforts. Concentration of exhaled breath volatile organic compounds (VOCs) on thermal desorption (TD) tubes with subsequent analysis by gas chromatography-mass spectrometry (GC-MS) has dominated this field. As discovery experimentation increases in frequency, the need to evaluate the long-term storage stability of exhaled breath VOCs on thermal desorption adsorbent material is critical. To address this gap, exhaled breath was loaded on Tenax TA thermal desorption tubes and stored at various temperature conditions. 74 VOCs, 56 of which have been previously uncharacterized, were monitored using GC-MS over a period of 31 d. The results suggest that storage of exhaled breath at cold temperatures (4 °C) provides the most consistent retention of exhaled breath VOCs temporally. Samples were determined to be stable up to 14 d across storage conditions prior to gaining or losing 1-2 standard deviations in abundance. Through gene set enrichment analysis (GSEA), certain chemical classes were found to be positively (acids) or negatively (sulfur-containing) enriched temporally. By means of field sample collections, the effect of storage and shipping was found to be similar to those studies preformed in the laboratory at 4 °C. Collectively this study not only provides recommendations for proper storage conditions and storage length, but also illustrates the use of GSEA to exhaled breath based GC-MS data.

The identification of hypoxia biomarkers from exhaled breath under normobaric conditions.

Pilots have reported experiencing in-flight hypoxic-like symptoms since the inception of high-altitude aviation. As a result, the need to monitor pilots, in-flight, for the onset of hypoxic conditions is of great interest to the aviation community. We propose that exhaled breath is an appropriate non-invasive medium for monitoring pilot hypoxic risk through volatile organic compound (VOC) analysis. To identify changes in the exhaled breath VOCs produced during periods of reduced O2 levels, volunteers were exposed to simulated flight profiles, i.e. sea level for 5 min, O2 levels found at elevated altitudes for 5 min or placebo and 5 min at 100% O2 recovery gas, using a modified flight mask interfaced with a reduced O2 breathing device. During the course of these test events, time series breath samples from the flight mask and pre/post bag samples were collected and analyzed by gas chromatography/mass spectrometry (GC/MS). Seven compounds (pentanal, 4-butyrolactone, 2-pentanone, 2-hexanone, 2-cyclopenten-1-one, 3-methylheptane and 2-heptanone) were found to significantly change in response to hypoxic conditions. Additionally, the isoprene, 2-methyl-1,3-butadiene, was found to increase following the overall exposure profile. This study establishes an experimental means for monitoring changes in VOCs in response to hypoxic conditions, a computational workflow for compound analysis via the Metabolite Differentiation and Discovery Lab and MatLab(©) software and identifies potential volatile organic compound biomarkers of hypoxia exposure.

Detection of volatile organic compounds indicative of human presence in the air.

Volatile organic compounds were collected and analyzed from a variety of indoor and outdoor air samples to test whether human-derived compounds can be readily detected in the air and if they can be associated with human occupancy or presence. Compounds were captured with thermal desorption tubes and then analyzed by gas chromatography with mass spectrometry. Isoprene, a major volatile organic compound in exhaled breath, was shown to be the best indicator of human presence. Acetone, another major breath-borne compound, was higher in unoccupied or minimally occupied areas than in human-occupied areas, indicating that its majority may be derived from exogenous sources. The association of endogenous skin-derived compounds with human occupancy was not significant. In contrast, numerous compounds that are found in foods and consumer products were detected at elevated levels in the occupied areas. Our results revealed that isoprene and many exogenous volatile organic compounds consumed by humans are emitted at levels sufficient for detection in the air, which may be indicative of human presence.

Evaluation of Bio-VOC Sampler for Analysis of Volatile Organic Compounds in Exhaled Breath.

Monitoring volatile organic compounds (VOCs) from exhaled breath has been used to determine exposures of humans to chemicals. Prior to analysis of VOCs, breath samples are often collected with canisters or bags and concentrated. The Bio-VOC breath sampler, a commercial sampling device, has been recently introduced to the market with growing use. The main advantage for this sampler is to collect the last portion of exhaled breath, which is more likely to represent the air deep in the lungs. However, information about the Bio-VOC sampler is somewhat limited. Therefore, we have thoroughly evaluated the sampler here. We determined the volume of the breath air collected in the sampler was approximately 88 mL. When sampling was repeated multiple times, with the succeeding exhalations applied to a single sorbent tube, we observed linear relationships between the normalized peak intensity and the number of repeated collections with the sampler in many of the breath VOCs detected. No moisture effect was observed on the Tenax sorbent tubes used. However, due to the limitation in the collection volume, the use of the Bio-VOC sampler is recommended only for detection of VOCs present at high concentrations unless repeated collections of breath samples on the sampler are conducted.

Aptamer selection express: a novel method for rapid single-step selection and sensing of aptamers.

Here we describe a new DNA capture element (DCE) sensing system, based on the quenching and dequenching of a double-stranded aptamer. This system shows very good sensitivity and thermal stability. While quenching, dequenching, and separating the DCE systems made from different aptamers (all selected by SELEX), an alternative method to rapidly select aptamers was developed-the Aptamer Selection Express (ASExp). This process has been used to select aptamers against different types of targets (Bacillus anthracis spores, Bacillus thuringiensis spores, MS-2 bacteriophage, ovalbumin, and botulinum neurotoxin). The DCE systems made from botulinum neurotoxin aptamers selected by ASExp have been investigated. The results of this investigation indicate that ASExp can be used to rapidly select aptamers for the DCE sensing system.

Titanium and zirconium complexes of a phosphorus-containing p-tert-butylcalix[5]arene ligand: importance of metal and conformation on ligand/metal binding.

Binding of a calix[5]arene containing a single phosphorus ligand and three hydroxyl groups, calix[5]PNMe2(OH)3, 1, toward titanium and zirconium is investigated to yield insight into the factors that determine the strength of the phosphorus/metal interaction within the constraint of the calix[5]arene. Treatment of 1 with tetrakis(dimethylamino)titanium yields three complexes, 4a, 4b, and 4c, each of which shows the loss of 3 mol of dimethylamine in the reaction with the titanium bound to three oxygens. Treatment of 1 with tetrakis(diethylamino)zirconium proceeds similarly, although only two products, 5a and 5b, were isolated. X-ray structures of the products were obtained. Complexes 4a and 5a show similar geometries, with the calix[5]arene in an approximate cone conformation and the phosphorus lone pair directed toward the metal. The P...M distances are, however, markedly different: 3.69 A in 4a and 3.18 A in 5a, the former indicative of no interaction and the latter a weak one. Complexes 4b and 5b each are dimers, featuring a planar four-membered M-O-M-O ring; however, the titanium is five-coordinate in 4b with no phosphorus/metal bond, while the zirconium in 5b is six-coordinate with a P-Zr distance of 2.95 A. Complex 4c is monomeric with the calix[5]arene in an approximate 1,2-alternate conformation with a P...Ti distance of 2.90 A. The two most significant aspects controlling the phosphorus/metal contact are the metal, with the larger zirconium showing stronger interaction, and the calix[5]arene conformation, with the cone conformation showing the weaker interaction.

Phosphorus-based p-tert-butylcalix[5]arene ligands.

New calix[5]arene trivalent phosphorus derivatives have been synthesized which should be excellent ligands with which to study and control the interaction of a ligand atom with a metal. The larger cavity of the calix[5]arene (compared to calix[4]arene) provides a good balance between constraint and flexibility. Treatment of p-tert-butylcalix[5]arene with 2 equiv of either tris(dimethylamino)phosphine or dichlorophenylphosphine inserts two RP moieties into the calix[5]arene framework to give calix[5](PR)2(OH) (1, R = Me2N; 2, R = Ph). Further treatment of 1 with 4 equiv of HCl gives calix[5](PCl)2(OH) (3). Heating a solution of the monophosphorus compound calix[5](PNMe2)(OH)3 (4) releases dimethylamine to yield both monomeric calix[5](P)(OH)2 (6) and dimeric [calix[5](P)(OH)2](2) (7), the latter having a tubelike geometry. X-ray crystallographic studies confirm the structures and show that 1 and 2 have approximate cone conformations while 3 has an approximate 1,2-alternate conformation. The orientations of the phosphorus lone pairs and oxygen atoms in all derivatives provide a framework for both soft and hard ligand interactions within the calix[5]arene.