Andreas Sponring

TD-GC-MS Analysis of Volatile Metabolites of Human Lung Cancer and Normal Cells In vitro

The aim of this study was to confirm the existence of volatile organic compounds (VOC) specifically released or consumed by the lung cancer cell line A549, which could be used in future screens as biomarkers for the early detection of lung cancer. For comparison, primary human bronchial epithelial cells (HBEpC) and human fibroblasts (hFB) were included. VOCs were detected in the headspace of cell cultures or medium controls following adsorption on solid sorbents, thermodesorption, and analysis by gas chromatography mass spectrometry. Using this approach, we identified VOCs that behaved similarly in normal and transformed cells. Thus, concentrations of 2-pentanone and 2,4-dimethyl-1-heptene were found to increase in the headspace of A549, HBEpC, and hFB cell cultures. In addition, the ethers methyl tert-butyl ether and ethyl tert-butyl ether could be detected at elevated levels in the case of A549 cells and one of the untransformed cell lines. However, especially branched hydrocarbons and alcohols were seen increased more frequently in untransformed than A549 cells. A big variety of predominantly aldehydes and the ester n-butyl acetate were found at decreased concentrations in the headspace of all cell lines tested compared with medium controls. Again, more different aldehydes were found to be decreased in hFB and HBEpC cells compared with A549 cells and 2-butenal was metabolized exclusively by both control cell lines. These data suggest that certain groups of VOCs may be preferentially associated with the transformed phenotype. Cancer Epidemiol Biomarkers Prev; 19(1); 182–95

Release of volatile organic compounds (VOCs) from the lung cancer cell line CALU-1 in vitro

BackgroundThe aim of this work was to confirm the existence of volatile organic compounds (VOCs) specifically released or consumed by lung cancer cells.Methods50 million cells of the human non-small cell lung cancer (NSCLC) cell line CALU-1 were incubated in a sealed fermenter for 4 h or over night (18 hours). Then air samples from the headspace of the culture vessel were collected and preconcentrated by adsorption on solid sorbents with subsequent thermodesorption and analysis by means of gas chromatography mass spectrometry (GC-MS). Identification of altogether 60 compounds in GCMS measurement was done not only by spectral library match, but also by determination of retention times established with calibration mixtures of the respective pure compounds.ResultsThe results showed a significant increase in the concentrations of 2,3,3-trimethylpentane, 2,3,5-trimethylhexane, 2,4-dimethylheptane and 4-methyloctane in the headspace of CALU-1 cell culture as compared to medium controls after 18 h. Decreased concentrations after 18 h of incubation were found for acetaldehyde, 3-methylbutanal, butyl acetate, acetonitrile, acrolein, methacrolein, 2-methylpropanal, 2-butanone, 2-methoxy-2-methylpropane, 2-ethoxy-2-methylpropane, and hexanal.ConclusionOur findings demonstrate that certain volatile compounds can be cancer-cell derived and thus indicative of the presence of a tumor, whereas other compounds are not released but seem to be consumed by CALU-1 cells.

Molecular analysis of volatile metabolites released specifically by staphylococcus aureus and pseudomonas aeruginosa

BackgroundThe routinely used microbiological diagnosis of ventilator associated pneumonia (VAP) is time consuming and often requires invasive methods for collection of human specimens (e.g. bronchoscopy). Therefore, it is of utmost interest to develop a non-invasive method for the early detection of bacterial infection in ventilated patients, preferably allowing the identification of the specific pathogens. The present work is an attempt to identify pathogen-derived volatile biomarkers in breath that can be used for early and non- invasive diagnosis of ventilator associated pneumonia (VAP). For this purpose, in vitro experiments with bacteria most frequently found in VAP patients, i.e. Staphylococcus aureus and Pseudomonas aeruginosa, were performed to investigate the release or consumption of volatile organic compounds (VOCs).ResultsHeadspace samples were collected and preconcentrated on multibed sorption tubes at different time points and subsequently analyzed with gas chromatography mass spectrometry (GC-MS). As many as 32 and 37 volatile metabolites were released by S. aureus and P. aeruginosa, respectively. Distinct differences in the bacteria-specific VOC profiles were found, especially with regard to aldehydes (e.g. acetaldehyde, 3-methylbutanal), which were taken up only by P. aeruginosa but released by S. aureus. Differences in concentration profiles were also found for acids (e.g. isovaleric acid), ketones (e.g. acetoin, 2-nonanone), hydrocarbons (e.g. 2-butene, 1,10-undecadiene), alcohols (e.g. 2-methyl-1-propanol, 2-butanol), esters (e.g. ethyl formate, methyl 2-methylbutyrate), volatile sulfur compounds (VSCs, e.g. dimethylsulfide) and volatile nitrogen compounds (VNCs, e.g. 3-methylpyrrole).Importantly, a significant VOC release was found already 1.5 hours after culture start, corresponding to cell numbers of ~8*106 [CFUs/ml].ConclusionsThe results obtained provide strong evidence that the detection and perhaps even identification of bacteria could be achieved by determination of characteristic volatile metabolites, supporting the clinical use of breath-gas analysis as non-invasive method for early detection of bacterial lung infections.

Analysis of volatile organic compounds (VOCs) in the headspace of NCI-H1666 lung cancer cells.

Analysis of volatile organic compounds (VOCs) provides an elegant approach for cancer screening and disease monitoring, whose use is currently limited by a lack of validated cancer-derived metabolites, which may serve as biomarkers. The aim of the experiments presented here was to investigate the release and consumption of VOCs from the non small cell lung cancer cell line NCI-H1666, which was originally derived from a bronchoalveolar carcinoma.Following detachment by trypsinization suspended cells were incubated in a sealed fermenter for 21 hours. 200 ml of headspace from the cell culture were sampled, diluted with dry, highly purified air and preconcentrated by adsorption on three different solid sorbents with increasing adsorption strength. VOC-analysis was performed by thermodesorption-gas chromatography mass spectrometry (TD-GC-MS). In contrast to our previous studies experiments with NCI-H1666 cells only confirmed the consumption of several aldehydes, n-butyl acetate and the ethers methyl tert-butyl ether and ethyl tert-butyl ether, but no unequivocal release of VOCs was observed. Together with our previously published work these data indicate that the consumption of certain VOCs is commonly observed while their release shows cell line-restricted patterns, whose underlying causes are unknown.

Characterization of volatile metabolites taken up by or released from Streptococcus pneumoniae and Haemophilus influenzae by using GC-MS.

Volatile organic compounds (VOCs) released from or taken up by Streptococcus pneumoniae and Haemophilus influenzae cultures were analysed by means of GC-MS after adsorption of headspace samples on multi-bed sorption tubes. Sampling was performed at different time points during cultivation of bacteria to follow the dynamics of VOC metabolism. VOCs were identified not only by spectral library match but also based on retention times of native standards. As many as 34 volatile metabolites were released from S. pneumoniae and 28 from H. influenzae, comprising alcohols, aldehydes, esters, hydrocarbons, ketones and sulfur-containing compounds. For both species, acetic acid, acetaldehyde, methyl methacrylate, 2,3-butanedione and methanethiol were found in strongly elevated concentrations and 1-butanol and butanal in moderately elevated concentrations. In addition, characteristic volatile biomarkers were detected for both bacterial species and exclusively for S. pneumoniae, also catabolism of aldehydes (3-methylbutanal and hexanal) was found. The results obtained provide important input into the knowledge about volatile bacterial biomarkers, which may become particularly important for detection of pathogens in upper airways by breath-gas analysis in the future.

Release and uptake of volatile organic compounds by human hepatocellular carcinoma cells (HepG2) in vitro.

BackgroundVolatile organic compounds (VOCs) emitted by human body offer a unique insight into biochemical processes ongoing in healthy and diseased human organisms. Unfortunately, in many cases their origin and metabolic fate have not been yet elucidated in sufficient depth, thus limiting their clinical application. The primary goal of this work was to identify and quantify volatile organic compounds being released or metabolized by HepG2 hepatocellular carcinoma cells.MethodsThe hepatocellular carcinoma cells were incubated in specially designed head-space 1-L glass bottles sealed for 24 hours prior to measurements. Identification and quantification of volatiles released and consumed by cells under study were performed by gas chromatography with mass spectrometric detection (GC-MS) coupled with head-space needle trap device extraction (HS-NTD) as the pre-concentration technique. Most of the compounds were identified both by spectral library match as well as retention time comparison based on standards.ResultsA total of nine compounds were found to be metabolised and further twelve released by the cells under study (Wilcoxon signed-rank test, p<0.05). The former group comprised 6 aldehydes (2-methyl 2-propenal, 2-methyl propanal, 2-ethylacrolein, 3-methyl butanal, n-hexanal and benzaldehyde), n-propyl propionate, n-butyl acetate, and isoprene. Amongst the released species there were five ketones (2-pentanone, 3-heptanone, 2-heptanone, 3-octanone, 2-nonanone), five volatile sulphur compounds (dimethyl sulfide, ethyl methyl sulfide, 3-methyl thiophene, 2-methyl-1-(methylthio)- propane and 2-methyl-5-(methylthio) furan), n-propyl acetate, and 2-heptene.ConclusionsThe emission and uptake of the aforementioned VOCs may reflect the activity of abundant liver enzymes and support the potential of VOC analysis for the assessment of enzymes function.

Breath analysis for in vivo detection of pathogens related to ventilator-associated pneumonia in intensive care patients: a prospective pilot study

Existing methods for the early detection of infections in mechanically ventilated (MV) patients at intensive care units (ICUs) are unsatisfactory. Here we present an exploratory study assessing the feasibility of breath VOC analyses for the non-invasive detection of pathogens in the lower respiratory tract of ventilated patients. An open uncontrolled clinical pilot study was performed by enrolling 28 mechanically ventilated (MV) patients with severe intracranial disease, being at risk for the development of or already with confirmed ventilation-associated pneumonia (VAP). The recently developed sampling technique enabled the collection of breath gas with a maximized contribution of alveolar air directly from the respiratory circuit under continuous capnography control, adsorptive preconcentration and final analysis by means of gas chromatography-mass spectrometry (GC-MS).VAP was confirmed in 22/28 preselected patients (78%). The most common microorganisms were Staphylococcus aureus (5/22 VAP patients), Escherichia coli (5/22 VAP patients) and Candida spp. (5/22 VAP patients). 12/32 metabolites released by S. aureus in our previous in vitro studies were also detected in the end-tidal air of VAP patients infected with this pathogen. A similar overlap was seen in Candida albicans infections (8/29 VOCs). Moreover, the concentration profile of selected compounds correlated with the course of the infection.This prospective pilot study provides proof of the concept that the appearance and the concentration profile of pathogen-derived metabolites (elucidated from in vitro experiments) in the breath of ventilated patients during clinically confirmed VAP correlates with the presence of a particular pathogen.

Analysis of Volatile Organic Compounds Liberated and Metabolised by Human Umbilical Vein Endothelial Cells (HUVEC) In Vitro

Gas chromatography with mass spectrometric detection combined with head-space needle trap extraction as the pre-concentration technique was applied to identify and quantify volatile organic compounds released or metabolised by human umbilical vein endothelial cells. Amongst the consumed species there were eight aldehydes (2-methyl 2-propenal, 2-methyl propanal, 2-methyl butanal, 3-methyl butanal, n-hexanal, benzaldehyde, n-octanal and n-nonanal) and n-butyl acetate. Further eight compounds (ethyl acetate, ethyl propanoate, ethyl butyrate, 3-heptanone, 2-octanone, 2-nonanone, 2-methyl-5-(methylthio)-furan and toluene) were found to be emitted by the cells under study. Possible metabolic pathways leading to the uptake and release of these compounds by HUVEC are proposed and discussed. The uptake of aldehydes by endothelial cells questions the reliability of species from this chemical class as breath or blood markers of disease processes in human organism. The analysis of volatiles released or emitted by cell lines is shown to have a potential for the identification and assessment of enzymes activities and expression.