Cancer metabolism: the volatile signature of glycolysis-in vitro model in lung cancer cells.

Discovering the volatile signature of cancer cells is an emerging approach in cancer research, as it may contribute to a fast and simple diagnosis of tumors in vivo and in vitro. One of the main contributors to such a volatile signature is hyperglycolysis, which characterizes the cancerous cell. The metabolic perturbation in cancer cells is known as the Warburg effect; glycolysis is preferred over oxidative phosphorylation (OXPHOS), even in the presence of oxygen. The precise mitochondrial alterations that underlie the increased dependence of cancer cells on aerobic glycolysis for energy generation have remained a mystery. We aimed to profile the volatile signature of the glycolysis activity in lung cancer cells. For that an in vitro model, using lung cancer cell line cultures (A549, H2030, H358, H322), was developed. The volatile signature was measured by proton transfer reaction mass spectrometry under normal conditions and glycolysis inhibition. Glycolysis inhibition and mitochondrial activity were also assessed by mitochondrial respiration capacity measurements. Cells were divided into two groups upon their glycolytic profile (PET positive and PET negative). Glycolysis blockade had a unique characteristic that was shared by all cells. Furthermore, each group had a characteristic volatile signature that enabled us to discriminate between those sub-groups of cells. In conclusion, lung cancer cells may have different subpopulations of cells upon low and high mitochondrial capacity. In both groups, glycolysis blockade induced a unique volatile signature.

Cancerous glucose metabolism in lung cancer-evidence from exhaled breath analysis.

Cancer cells prefer hyperglycolysis versus oxidative phosphorylation, even in the presence of oxygen. This phenomenon is used through the FDG-PET scans, and may affect the exhaled volatile signature. This study investigates the volatile signature in lung cancer (LC) before and after an oral glucose tolerance test (OGTT) to determine if tumor cells' hyperglycolysis would affect the volatile signature. Blood glucose levels and exhaled breath samples were analyzed before the OGTT, and 90 min after, in both LC patients and controls. The volatile signature was measured by proton transfer reaction mass spectrometry (PTR-MS). Twenty-two LC patients (age 66.6 ± 12.7) with adenocarcinoma (n = 14), squamous (n = 6), small cell carcinoma (n = 2), and twenty-one controls (age 54.4 ± 13.7; 10 non-smokers and 11 smokers) were included. All LC patients showed a hyperglycolytic state in their FDG-PET scans. Both baseline and post OGTT volatile signatures discriminate between the groups. The OGTT has a minimal effect in LC (a decrease in m/z 54 by 39%, p v = 0.0499); whereas in the control group, five masses (m/z 64, 87,88, 142 and 161) changed by -13%, -49%, -40% and -29% and 46% respectively. To conclude, OGTT has a minimal effect on the VOC signature in LC patients, where a hyperglycolytic state already exists. In contrast, in the control group the OGTT has a profound effect in which induced hyperglycolysis significantly changed the VOC pattern. We hypothesized that a ceiling effect in cancerous patients is responsible for this discrepancy.

Monoterpene separation by coupling proton transfer reaction time-of-flight mass spectrometry with fastGC.

Proton transfer reaction mass spectrometry (PTR-MS) is a well-established technique for real-time analysis of volatile organic compounds (VOCs). Although it is extremely sensitive (with sensitivities of up to 4500 cps/ppbv, limits of detection <1 pptv and the response times of approximately 100 ms), the selectivity of PTR-MS is still somewhat limited, as isomers cannot be separated. Recently, selectivity-enhancing measures, such as manipulation of drift tube parameters (reduced electric field strength) and using primary ions other than H3O(+), such as NO(+) and O2 (+), have been introduced. However, monoterpenes, which belong to the most important plant VOCs, still cannot be distinguished so more traditional technologies, such as gas chromatography mass spectrometry (GC-MS), have to be utilised. GC-MS is very time consuming (up to 1 h) and cannot be used for real-time analysis. Here, we introduce a sensitive, near-to-real-time method for plant monoterpene research-PTR-MS coupled with fastGC. We successfully separated and identified six of the most abundant monoterpenes in plant studies (α- and ß-pinenes, limonene, 3-carene, camphene and myrcene) in less than 80 s, using both standards and conifer branch enclosures (Norway spruce, Scots pine and black pine). Five monoterpenes usually present in Norway spruce samples with a high abundance were separated even when the compound concentrations were diluted to 20 ppbv. Thus, fastGC-PTR-ToF-MS was shown to be an adequate one-instrument solution for plant monoterpene research.

Towards standardization in the analysis of breath gas volatiles.

Despite growing interest and considerable progress in breath research over the last decade, standardized practices for the sampling and analysis of breath gas volatiles remain elusive. The primary reasons for this are (a) the rich chemical diversity of exhaled breath that covers an extensive range of volatile organic compounds at highly varied concentrations, (b) the vast disparity in the analytical tools employed, (c) diverse study goals and (d) the presence of (unidentified) confounders. These aspects place stringent but divergent demands on sampling and analysis: each analytical tool, target compound and concentration range requires its own specific protocol and in many cases the latter two are not even known a priori. The ongoing rapid developments and constant discoveries in the field of breath research and the lack of established best practices in breath gas sampling and analysis currently preclude an acceptable overall standardization of these methods. This paper addresses these manifold issues and suggests a framework that separately considers individual stages of sampling and analysis with a view to establishing standardization in the analysis of breath gas volatiles to suit different target compounds and analytical technologies.

Mass spectrometry for real-time quantitative breath analysis.

Breath analysis research is being successfully pursued using a variety of analytical methods, prominent amongst which are gas chromatography with mass spectrometry, GC-MS, ion mobility spectrometry, IMS, and the fast flow and flow-drift tube techniques called selected ion flow tube mass spectrometry, SIFT-MS, and proton transfer reaction mass spectrometry, PTR-MS. In this paper the case is made for real-time breath analysis by obviating sample collection into bags or onto traps that can suffer from partial degradation of breath metabolites or the introduction of impurities. Real-time analysis of a broad range of volatile chemical compounds can be best achieved using SIFT-MS and PTR-MS, which are sufficiently sensitive and rapid to allow the simultaneous analyses of several trace gas metabolites in single breath exhalations. The basic principles and the ion chemistry that underpin these two analytical techniques are briefly described and the differences between them, including their respective strengths and weaknesses, are revealed, especially with reference to the analysis of the complex matrix that is exhaled breath. A recent innovation is described that combines time-of-flight mass spectrometry with the proton transfer flow-drift tube reactor, PTR-TOFMS, which provides greater resolution in the analytical mass spectrometer and allows separation of protonated isobaric molecules. Examples are presented of some recent data that well illustrate the quality and real-time feature of SIFT-MS and PTR-MS for the analysis of exhaled breath for physiological/biochemical/pharmacokinetics studies and for the identification and quantification of biomarkers relating to specific disease states.

Multi-capillary-column proton-transfer-reaction time-of-flight mass spectrometry.

Proton-transfer-reaction time-of-flight mass-spectrometry (PTR-TOFMS) exhibits high selectivity with a resolution of around 5000 m/Δm. While isobars can be separated with this resolution, discrimination of isomeric compounds is usually not possible. The coupling of a multi-capillary column (MCC) with a PTR-TOFMS overcomes these problems as demonstrated in this paper for the ketone isomers 3-heptanone and 2-methyl-3-hexanone and for different aldehydes. Moreover, fragmentation of compounds can be studied in detail which might even improve the identification. LODs for compounds tested are in the range of low ppbv and peak positions of the respective separated substances show good repeatability (RSD of the peak positions <3.2%). Due to its special characteristics, such as isothermal operation, compact size, the MCC setup is suitable to be installed inside the instrument and the overall retention time for a complete spectrum is only a few minutes: this allows near real-time measurements in the optional MCC mode. In contrast to other methods that yield additional separation, such as the use of pre-cursor ions other than H3O(+), this method yields additional information without increasing complexity.

Real-time metabolic monitoring with proton transfer reaction mass spectrometry.

We analysed the time evolution of several volatile organic compounds formed by the catabolism of ingested isotope-labelled ethanol using real-time breath gas analysis with proton-transfer-reaction mass spectrometry. Isotope labelling allowed distinguishing the emerging volatile metabolites from their naturally occurring, highly abundant counterparts in the human breath. Due to an extremely low detection limit of the employed technologies in the parts per trillion per volume range, it was possible to detect the emerging metabolic products in exhaled breath within ∼10 min after oral ingestion of isotope-labelled ethanol. We observed that ethanol was in part transformed into deuterated acetone and isoprene, reflecting the different fates of activated acetic acid (acetyl-coenzyme A), formed in ethanol metabolism. Using ethanol as a model clearly demonstrated the value of the here presented technique for the search for volatile markers for metabolic disorders in the exhaled breath and its potential usefulness in the diagnosis and monitoring of such diseases.

First observation of a potential non-invasive breath gas biomarker for kidney function.

We report on the search for low molecular weight molecules-possibly accumulated in the bloodstream and body-in the exhaled breath of uremic patients with kidney malfunction. We performed non-invasive analysis of the breath gas of 96 patients shortly before and several times after kidney transplantation using proton-transfer-reaction mass spectrometry (PTR-MS), a very sensitive technique for detecting trace amounts of volatile organic compounds. A total of 642 individual breath analyses which included at least 41 different chemical components were carried out. Correlation analysis revealed one particular breath component with a molecular mass of 114 u (unified atomic mass units) that clearly correlated with blood serum creatinine, which is the currently accepted marker for assessing the function of the kidney. In particular, daily urine production showed good correlation with the identified breath marker. An independent set of seven samples taken from three patients at the onset of dialysis and three controls with normal kidney function confirmed a significant difference in concentration between patients and controls for a compound with a molecular mass of 114.1035 u using high mass resolving proton-transfer-reaction time-of-flight mass spectrometry (PTR-TOF-MS). A chemical composition of C7H14O was derived for the respective component. Fragmentation experiments on the same samples using proton-transfer-reaction triple-quadrupole tandem mass spectrometry (PTR-QqQ-MS) suggested that this breath marker is a C7-ketone or a branched C7-aldehyde. Non-invasive real-time monitoring of the kidney function via this breath marker could be a possible future procedure in the clinical setting.

Data interpretation in breath biomarker research: pitfalls and directions.

Most--if not all--potential diagnostic applications in breath research involve different marker concentrations rather than unique breath markers which only occur in the diseased state. Hence, data interpretation is a crucial step in breath analysis. To avoid artificial significance in breath testing every effort should be made to implement method validation, data cross-testing and statistical validation along this process. The most common data analysis related problems can be classified into three groups: confounding variables (CVs), which have a real correlation with both the diseased state and a breath marker but lead to the erroneous conclusion that disease and breath are in a causal relationship; voodoo correlations (VCs), which can be understood as statistically true correlations that arise coincidentally in the vast number of measured variables; and statistical misconceptions in the study design (SMSD). CV: Typical confounding variables are environmental and medical history, host factors such as gender, age, weight, etc and parameters that could affect the quality of breath data such as subject breathing mode, effects of breath sampling and effects of the analytical technique itself. VC: The number of measured variables quickly overwhelms the number of samples that can feasibly be taken. As a consequence, the chances of finding coincidental 'voodoo' correlations grow proportionally. VCs can typically be expected in the following scenarios: insufficient number of patients, (too) many measurement variables, the use of advanced statistical data mining methods, and non-independent data for validation. SMSD: Non-prospective, non-blinded and non-randomized trials, a priori biased study populations or group selection with unrealistically high disease prevalence typically represent misconception of study design. In this paper important data interpretation issues are discussed, common pitfalls are addressed and directions for sound data processing and interpretation are proposed.

Implementation of proton transfer reaction-mass spectrometry (PTR-MS) for advanced bioprocess monitoring.

We report on the implementation of proton transfer reaction-mass spectrometry (PTR-MS) technology for on-line monitoring of volatile organic compounds (VOCs) in the off-gas of bioreactors. The main part of the work was focused on the development of an interface between the bioreactor and an analyzer suitable for continuous sampling of VOCs emanating from the bioprocess. The permanently heated sampling line with an inert surface avoids condensation and interaction of volatiles during transfer to the PTR-MS. The interface is equipped with a sterile sinter filter unit directly connected to the bioreactor headspace, a condensate trap, and a series of valves allowing for dilution of the headspace gas, in-process calibration, and multiport operation. To assess the aptitude of the entire system, a case study was conducted comprising three identical cultivations with a recombinant E. coli strain, and the volatiles produced in the course of the experiments were monitored with the PTR-MS. The high reproducibility of the measurements proved that the established sampling interface allows for reproducible transfer of volatiles from the headspace to the PTR-MS analyzer. The set of volatile compounds monitored comprises metabolites of different pathways with diverse functions in cell physiology but also volatiles from the process matrix. The trends of individual compounds showed diverse patterns. The recorded signal levels covered a dynamic range of more than five orders of magnitude. It was possible to assign specific volatile compounds to distinctive events in the bioprocess. The presented results clearly show that PTR-MS was successfully implemented as a powerful bioprocess-monitoring tool and that access to volatiles emitted by the cells opens promising perspectives in terms of advanced process control.

Proton transfer reaction-mass spectrometry applications in medical research.

Gathering information about a subject's physiological and pathophysiological condition from the `smell' of breath is an idea that dates back to antiquity. This intriguing concept of non-invasive diagnosis has been revitalized by `exhaled breath analysis' in recent decades. A main driving force was the development of sensitive and versatile gas-chromatographic and mass-spectrometric instruments for trace gas analysis. Ironically, only non-smelling constituents of breath, such as O(2), CO(2), H(2), and NO have so far been included in routine clinical breath analysis. The `smell' of human breath, on the other hand, arises through a combination of volatile organic compounds (VOCs) of which several hundred have been identified to date. Most of these volatiles are systemic and are released in the gas-exchange between blood and air in the alveoli. The concentration of these compounds in the alveolar breath is related to the respective concentrations in blood. Measuring VOCs in exhaled breath allows for screening of disease markers, studying the uptake and effect of medication (pharmacokinetics), or monitoring physiological processes. There is a range of requirements for instruments for the analysis of a complex matrix, such as human breath. Mass-spectrometric techniques are particularly well suited for this task since they offer the possibility of detecting a large variety of interesting compounds. A further requirement is the ability to measure accurately in the concentration range of breath VOCs, i.e. between parts-per-trillion (pptv) and parts-per-million (ppmv) range. In the mid 1990's proton transfer reaction-mass spectrometry (PTR-MS) was developed as a powerful and promising tool for the analysis of VOCs in gaseous media. Soon thereafter these instruments became commercially available to a still growing user community and have now become standard equipment in many fields including environmental research, food and flavour science, as well as life sciences. Their high sensitivity for VOCs with detection limits down to sub-pptv levels without pre-concentration and their highly linear signal response over seven orders of magnitude make PTR-MS instruments valuable tools for exhaled breath analysis. The `soft' chemical ionization process in PTR-MS largely avoids fragmentation, providing interpretable spectra without pre-separation. This is especially important for complex gas mixtures such as breath. Even more interesting, PTR-MS instruments analyse a gas sample in real-time and do not require any sample pre-treatment. This offers the possibility for online breath analysis with breath-to-breath resolution. This special issue on PTR-MS applications in medical research contains articles exploring different medical applications of PTR-MS. These applications include screening studies, where the breath composition of a large number of patients is investigated to, e.g., determine influences of demographic data on breath concentrations (Schwarz et al 2009 J. Breath Res. 3 027003). In online monitoring studies the breath of one subject is continuously measured, e.g., to study rapid changes in breath volatiles under physical exercise (King et al 2009 J. Breath Res. 3 027006). Other papers address more elementary breath research and discuss the interpretation of exhaled breath composition in the presence of fragmenting and overlapping compounds (Schwarz et al 2009 J. Breath Res. 3 027002), examine the different causes of variability in the measurement of breath samples (Thekedar et al 2009 J. Breath Res. 3 027007), and compare blood and breath concentrations directly (O'Hara et al 2009 J. Breath Res. 3 027005). Potential sources for breath markers are also explored, by analysing the head-space emissions from microbial culture samples (O'Hara and Mayhew 2009 J. Breath Res. 3 027001). Finally, a recent technological advancement in PTR-MS technology promises several advantages especially for breath gas analysis, which is demonstrated by on-line breath sampling with a PTR-time-of-flight (PTR-TOF) instrument (Herbig et al 2009 J. Breath Res. 3 027004).

On-line breath analysis with PTR-TOF.

We report on on-line breath gas analysis with a new type of analytical instrument, which represents the next generation of proton-transfer-reaction mass spectrometers. This time-of-flight mass spectrometer in combination with the soft proton-transfer-reaction ionization (PTR-TOF) offers numerous advantages for the sensitive detection of volatile organic compounds and overcomes several limitations. First, a time-of-flight instrument allows for a measurement of a complete mass spectrum within a fraction of a second. Second, a high mass resolving power enables the separation of isobaric molecules and the identification of their chemical composition. We present the first on-line breath measurements with a PTR-TOF and demonstrate the advantages for on-line breath analysis. In combination with buffered end-tidal (BET) sampling, we obtain a complete mass spectrum up to 320 Th within one exhalation with a signal-to-noise ratio sufficient to measure down to pptv levels. We exploit the high mass resolving power to identify the main components in the breath composition of several healthy volunteers.

Buffered end-tidal (BET) sampling-a novel method for real-time breath-gas analysis.

We present a novel method for real-time breath-gas analysis using mass-spectrometric techniques: buffered end-tidal (BET) on-line sampling. BET has several advantages over conventional direct on-line sampling where the subject inhales and exhales through a sampling tube. In our approach, a single exhalation is administered through a tailored tube in which the end-tidal fraction of the breath-gas sample is buffered. This increases sampling time by an order of magnitude to several seconds, improving signal quality and reducing the total measurement time per test subject. Furthermore, only one exhalation per minute is required for sampling and the test subject can otherwise maintain a normal breathing pattern, thereby reducing the risk of hyperventilation. To validate our new BET sampling method we conducted comparative measurements with direct on-line sampling using proton-transfer-reaction mass spectrometry. We find excellent agreement in measured acetone and acetonitrile concentrations. High variability observed in breath-by-breath isoprene concentrations is attributed to differences in exhalation depth and influences of hyperventilation on end-tidal concentrations.

On the use of Tedlar® bags for breath-gas sampling and analysis.

The storage capability of Tedlar® bags for gaseous compounds was assessed using on-line proton-transfer-reaction mass spectrometry (PTR-MS). Sample bags were filled with a mixture of volatile organic compounds (VOCs) at known quantities in the ppbv range. The test gas included alcohol, nitrile, aldehyde, ketone, terpene and aromatic compounds. PTR-MS enabled frequent bag-direct measurements of compound abundances over a 70 h storage period. Concentrations of all compounds decreased with bag storage time, with compound-specific decay rates. The most rapid decline in concentration levels was seen for water vapour in the bag, i.e. sample humidity. Such a decrease is particularly relevant for breath-gas samples, where water vapour content is high. Compound losses were attributed to a combination of adsorption to and diffusion through the bag walls. Storage property observations suggest that sample analyses made within 10 h of sampling offer adequate sample authenticity replication. Based on observations, an appropriate bag-cleaning procedure was established and assessed. Results indicated that acceptable bag cleanliness for breath-gas sampling is achievable.

Three-body recombination at large scattering lengths in an ultracold atomic gas.

We study three-body recombination in an optically trapped ultracold gas of cesium atoms with precise magnetic control of the s-wave scattering length a. At large positive values of a, we measure the dependence of the rate coefficient on a and confirm the theoretically predicted scaling proportional to a(4). Evidence of recombination heating indicates the formation of very weakly bound molecules in the last bound energy level.

Preparation of a pure molecular quantum gas.

An ultracold molecular quantum gas is created by application of a magnetic field sweep across a Feshbach resonance to a Bose-Einstein condensate of cesium atoms. The ability to separate the molecules from the atoms permits direct imaging of the pure molecular sample. Magnetic levitation enables study of the dynamics of the ensemble on extended time scales. We measured ultralow expansion energies in the range of a few nanokelvin for a sample of 3000 molecules. Our observations are consistent with the presence of a macroscopic molecular matter wave.

Bose-Einstein condensation of cesium.

Bose-Einstein condensation of cesium atoms is achieved by evaporative cooling using optical trapping techniques. The ability to tune the interactions between the ultracold atoms by an external magnetic field is crucial to obtain the condensate and offers intriguing features for potential applications. We explore various regimes of condensate self-interaction (attractive, repulsive, and null interaction strength) and demonstrate properties of imploding, exploding, and non-interacting quantum matter.