Evaluation of needle trap micro-extraction and solid-phase micro-extraction: Obtaining comprehensive information on volatile emissions from in vitro cultures.

Volatile organic compounds (VOCs) emitted from in vitro cultures may reveal information on species and metabolism. Owing to low nmol L-1 concentration ranges, pre-concentration techniques are required for gas chromatography-mass spectrometry (GC-MS) based analyses. This study was intended to compare the efficiency of established micro-extraction techniques - solid-phase micro-extraction (SPME) and needle-trap micro-extraction (NTME) - for the analysis of complex VOC patterns. For SPME, a 75 µm Carboxen®/polydimethylsiloxane fiber was used. The NTME needle was packed with divinylbenzene, Carbopack X and Carboxen 1000. The headspace was sampled bi-directionally. Seventy-two VOCs were calibrated by reference standard mixtures in the range of 0.041-62.24 nmol L-1 by means of GC-MS. Both pre-concentration methods were applied to profile VOCs from cultures of Mycobacterium avium ssp. paratuberculosis. Limits of detection ranged from 0.004 to 3.93 nmol L-1 (median = 0.030 nmol L-1 ) for NTME and from 0.001 to 5.684 nmol L-1 (median = 0.043 nmol L-1 ) for SPME. NTME showed advantages in assessing polar compounds such as alcohols. SPME showed advantages in reproducibility but disadvantages in sensitivity for N-containing compounds. Micro-extraction techniques such as SPME and NTME are well suited for trace VOC profiling over cultures if the limitations of each technique is taken into account.

Continuous real-time breath analysis in ruminants: effect of eructation on exhaled VOC profiles.

BACKGROUND: The analysis of volatile organic compounds (VOCs) in breath allows non-invasive investigations of diseases. Animal studies are conducted as a model to perform research of VOCs and their relation to diseases. In large animal models ruminants were often used as experimental targets. The effect of their physiological eructation on VOC exhalation has not been examined yet and is the objective of this study. METHODS: Continuous breath profiles of two young cattle, four adult goats and four adult sheep were measured through a mask, covering mouth and nose, in real-time (200 ms) by means of proton transfer reaction time of flight mass spectrometry. Each animal was analysed twelve times for 3 consecutive minutes. RESULTS: Real-time monitoring yielded a distinction of different episodes in the breath profiles of ruminants. An algorithm to separate eructation episodes and alveolar breath was established. In the first exhalation after eructation at least 19 VOC concentrations increased (up to 36-fold) and went back to initial levels in subsequent exhalations in all investigated ruminants. Decay of concentrations was substance specific. In goats, less VOCs were affected by the eructation compared to cattle and sheep. Breath profiles without exclusion of eructation episodes showed higher variations and median values than profiles where eructation episodes were excluded. CONCLUSION: Real-time breath analysis of ruminants enables the discrimination and characterisation of alveolar breath and eructation episodes. This leads to a better understanding of variation in breath data and possible origins of VOCs: breath or digestion related. To avoid impairment of breath gas results and to gain further information on bacterial products from the rumen, eructation and alveolar breath data should be analysed separately.

Comparative analysis of volatile organic compounds for the classification and identification of mycobacterial species.

BACKGROUND: Species of Mycobacteriaceae cause serious zoonotic diseases in mammals, for example tuberculosis in humans, dogs, parrots, and elephants (caused by Mycobacterium tuberculosis) and in ruminants and humans (caused by M. bovis and M. caprae). Pulmonary diseases, lymphadenitis, skin diseases, and disseminated diseases can be caused by non-tuberculous mycobacteria (NTM). Diagnosis and differentiation among Mycobacterium species are currently done by culture isolation. The established diagnostic protocols comprise several steps that allow species identification. Detecting volatile organic compounds (VOCs) above bacterial cultures is a promising approach towards accelerating species identification via culture isolation. The aims of this project were to analyse VOCs in the headspace above 13 different species of mycobacteria, to define VOC profiles that are unique for each species, and to compile a set of substances that indicate the presence of growing mycobacteria in general. MATERIALS & METHODS: VOCs were measured in the headspace above 17 different mycobacterial strains, all cultivated on Herrold's Egg Yolk Medium and above pure media slants that served as controls. For pre-concentration of VOCs, needle-trap micro-extraction was employed. Samples were subsequently analysed using gas chromatography-mass spectrometry. All volatiles were identified and calibrated by analysing pure reference substances. RESULTS: More than 130 VOCs were detected in headspace above mycobacteria-inoculated and control slants. Results confirmed significant VOC emissions above all mycobacterial species that had grown well. Concentration changes were measurable in vials with visually assessed bacterial growth and vials without apparent growth. VOCs above mycobacterial cultures could be grouped into substances that were either higher or equally concentrated, lower or equally concentrated, or both as those above control slants. Hence, we were able to identify 17 substances as potential biomarkers of the presence of growing mycobacteria in general. CONCLUSIONS: This study revealed species-specific VOC profiles for eleven species of mycobacteria that showed visually apparent bacterial growth at the time point of analysis.

Smell of cells: Volatile profiling of stem- and non-stem cell proliferation.

Bacterial and cell cultures are known to emit a large number of volatile organic compounds (VOCs). Conventional biochemical methods are often destructive, time-consuming and expensive. In contrast, VOC analysis of headspace over cultures may offer a non-destructive alternative for the monitoring of cell proliferation and metabolism. VOC profiles from cultures of murine pluripotent stem cells and fibroblasts were assessed every 24 h for 3 days. Pure cell media were measured as parallel controls. VOC analysis was highly standardized with respect to time of measurement and phases of cell growth. Cultures were grown in custom-made inert boxes. In order to determine the effects of fresh media supply on VOC emissions, both cell types were cultured with and without daily media exchange. VOCs from headspace were preconcentrated by means of needle trap micro-extraction and analysed by gas chromatography-mass spectrometry (GC-MS). Murine pluripotent stem cells emitted increasing concentrations of thiirane and methyl-methoxy-hydroxy-methyl-amine (MMHA). Substance concentration correlated with cell numbers. Murine fibroblasts did not emit thiirane or MMHA. Concentrations of aldehydes, especially benzaldehyde, were lower in both cell cultures than in pure media samples. Daily media exchange resulted in higher cell numbers, but had no major effects on VOC concentrations emitted from the cells. Investigation and monitoring of volatile substances such as thiirane and MMHA may enhance the understanding of stem cell properties and lead to a destruction-free characterization of pluripotent stem cells.

Retinal cell death dependent reactive proliferative gliosis in the mouse retina.

Neurodegeneration is a common starting point of reactive gliosis, which may have beneficial and detrimental consequences. It remains incompletely understood how distinctive pathologies and cell death processes differentially regulate glial responses. Müller glia (MG) in the retina are a prime model: Neurons are regenerated in some species, but in mammals there may be proliferative disorders and scarring. Here, we investigated the relationship between retinal damage and MG proliferation, which are both induced in a reproducible and temporal order in organotypic culture of EGF-treated mouse retina: Hypothermia pretreatment during eye dissection reduced neuronal cell death and MG proliferation; stab wounds increased both. Combined (but not separate) application of defined cell death signaling pathway inhibitors diminished neuronal cell death and maintained MG mitotically quiescent. The level of neuronal cell death determined MG activity, indicated by extracellular signal-regulated kinase (ERK) phosphorylation, and proliferation, both of which were abolished by EGFR inhibition. Our data suggest that retinal cell death, possibly either by programmed apoptosis or necrosis, primes MG to be able to transduce the EGFR-ERK activity required for cell proliferation. These results imply that cell death signaling pathways are potential targets for future therapies to prevent the proliferative gliosis frequently associated with certain neurodegenerative conditions.

Strategies for the identification of disease-related patterns of volatile organic compounds: prediction of paratuberculosis in an animal model using random forests.

Modern statistical methods which were developed for pattern recognition are increasingly being used for data analysis in studies on emissions of volatile organic compounds (VOCs). With the detection of disease-related VOC profiles, novel non-invasive diagnostic tools could be developed for clinical applications. However, it is important to bear in mind that not all statistical methods are equally suitable for the investigation of VOC profiles. In particular, univariate methods are not able to discover VOC patterns as they consider each compound separately. The present study demonstrates this fact in practice. Using VOC samples from a controlled animal study on paratuberculosis, the random forest classification method was applied for pattern recognition and disease prediction. This strategy was compared with a prediction approach based on single compounds. Both methods were framed within a cross-validation procedure. A comparison of both strategies based on these VOC data reveals that random forests achieves higher sensitivities and specificities than predictions based on single compounds. Therefore, it will most likely be more fruitful to further investigate VOC patterns instead of single biomarkers for paratuberculosis. All methods used are thoroughly explained to aid the transfer to other data analyses.

Oral or nasal breathing? Real-time effects of switching sampling route onto exhaled VOC concentrations.

There is a need for standardisation in sampling and analysis of breath volatile organic compounds (VOCs) in order to minimise ubiquitous confounding effects. Physiological factors may mask concentration changes induced by pathophysiological effects. In humans, unconscious switching of oral and nasal breathing can occur during breath sampling, which may affect VOC patterns. Here, we investigated exhaled VOC concentrations in real-time while switching breathing routes. Breath from 15 healthy volunteers was analysed continuously by proton transfer reaction time-of-flight mass spectrometry during paced breathing (12 breaths min-1). Every two minutes breathing routes were switched (Setup-1: Oral â†’ Nasal â†’ Oral â†’ Nasal; Setup-2: OralinNasalout â†’ NasalinOralout â†’ OralinNasalout â†’ NasalinOralout). VOCs in inspiratory and alveolar air and respiratory and hemodynamic parameters were monitored quantitatively in parallel. Changing of the breathing routes and patterns immediately affected exhaled VOC concentrations. These changes were reproducible in both setups. In setup-1 cardiac output and acetone concentrations remained constant, while partial pressure of end-tidal CO2 (pET-CO2), isoprene and furan concentrations inversely mirrored tidal-volume and minute-ventilation. H2S (hydrogen-sulphide), C4H8S (allyl-methyl-sulphide), C3H8O (isopropanol) and C3H6O2 increased during oral exhalation. C4H10S increased during nasal exhalations. CH2O2 steadily decreased during the whole measurement. In setup-2 pET-CO2, C2H6S (dimethyl-sulphide), isopropanol, limonene and benzene concentrations decreased whereas, minute-ventilation, H2S and acetonitrile increased. Isoprene and furan remained unchanged. Breathing route and patterns induced VOC concentration changes depended on respiratory parameters, oral and nasal cavity exposure and physico-chemical characters of the compounds. Before using breath VOC concentrations as biomarkers it is essential that the breathing modality is defined and strictly monitored during sampling.

Effects of biological and methodological factors on volatile organic compound patterns during cultural growth of Mycobacterium avium ssp. paratuberculosis.

Mycobacterium avium ssp. paratuberculosis (MAP) causes chronic granulomatous enteritis in ruminants. Bacterial growth is still the diagnostic 'gold standard', but is very time consuming. MAP-specific volatile organic compounds (VOCs) above media could accelerate cultural diagnosis. The aim of this project was to assess the kinetics of a VOC profile linked to the growth of MAP in vitro. The following sources of variability were taken into account: five different culture media, three different MAP strains, inoculation with different bacterial counts, and different periods of incubation. Needle-trap microextraction was employed for pre-concentration of VOCs, and gas chromatography-mass spectrometry for subsequent analysis. All volatiles were identified and calibrated by analysing pure references at different concentration levels. More than 100 VOCs were measured in headspaces above MAP-inoculated and control slants. Results confirmed different VOC profiles above different culture media. Emissions could be assigned to either egg-containing media or synthetic ingredients. 43 VOCs were identified as potential biomarkers of MAP growth on Herrold's Egg Yolk Medium without significant differences between the tree MAP strains. Substances belonged to the classes of alcohols, aldehydes, esters, ketones, aliphatic and aromatic hydrocarbons. With increasing bacterial density the VOC concentrations above MAP expressed different patterns: the majority of substances increased (although a few decreased after reaching a peak), but nine VOCs clearly decreased. Data support the hypotheses that (i) bacteria emit different metabolites on different culture media; (ii) different MAP strains show uniform VOC patterns; and (iii) cultural diagnosis can be accelerated by taking specific VOC profiles into account.

FEV manoeuvre induced changes in breath VOC compositions: an unconventional view on lung function tests.

Breath volatile organic compound (VOC) analysis can open a non-invasive window onto pathological and metabolic processes in the body. Decades of clinical breath-gas analysis have revealed that changes in exhaled VOC concentrations are important rather than disease specific biomarkers. As physiological parameters, such as respiratory rate or cardiac output, have profound effects on exhaled VOCs, here we investigated VOC exhalation under respiratory manoeuvres. Breath VOCs were monitored by means of real-time mass-spectrometry during conventional FEV manoeuvres in 50 healthy humans. Simultaneously, we measured respiratory and hemodynamic parameters noninvasively. Tidal volume and minute ventilation increased by 292 and 171% during the manoeuvre. FEV manoeuvre induced substance specific changes in VOC concentrations. pET-CO2 and alveolar isoprene increased by 6 and 21% during maximum exhalation. Then they decreased by 18 and 37% at forced expiration mirroring cardiac output. Acetone concentrations rose by 4.5% despite increasing minute ventilation. Blood-borne furan and dimethyl-sulphide mimicked isoprene profile. Exogenous acetonitrile, sulphides, and most aliphatic and aromatic VOCs changed minimally. Reliable breath tests must avoid forced breathing. As isoprene exhalations mirrored FEV performances, endogenous VOCs might assure quality of lung function tests. Analysis of exhaled VOC concentrations can provide additional information on physiology of respiration and gas exchange.

Continuous real time breath gas monitoring in the clinical environment by proton-transfer-reaction-time-of-flight-mass spectrometry.

Analysis of volatile organic compounds (VOCs) in breath holds great promise for noninvasive diagnostic applications. However, concentrations of VOCs in breath may change quickly, and actual and previous uptakes of exogenous substances, especially in the clinical environment, represent crucial issues. We therefore adapted proton-transfer-reaction-time-of-flight-mass spectrometry for real time breath analysis in the clinical environment. For reasons of medical safety, a 6 m long heated silcosteel transfer line connected to a sterile mouth piece was used for breath sampling from spontaneously breathing volunteers and mechanically ventilated patients. A time resolution of 200 ms was applied. Breath from mechanically ventilated patients was analyzed immediately after cardiac surgery. Breath from 32 members of staff was analyzed in the post anesthetic care unit (PACU). In parallel, room air was measured continuously over 7 days. Detection limits for breath-resolved real time measurements were in the high pptV/low ppbV range. Assignment of signals to alveolar or inspiratory phases was done automatically by a matlab-based algorithm. Quickly and abruptly occurring changes of patients' clinical status could be monitored in terms of breath-to-breath variations of VOC (e.g. isoprene) concentrations. In the PACU, room air concentrations mirrored occupancy. Exhaled concentrations of sevoflurane strongly depended on background concentrations in all participants. In combination with an optimized inlet system, the high time and mass resolution of PTR-ToF-MS provides optimal conditions to trace quick changes of breath VOC profiles and to assess effects from the clinical environment.