Wolfram Miekisch

Breath gas aldehydes as biomarkers of lung cancer

There is experimental evidence that volatile substances in human breath can reflect presence of neoplasma. Volatile aldehydes were determined in exhaled breath of 12 lung cancer patients, 12 smokers and 12 healthy volunteers. Alveolar breath samples were collected under control of expired CO2. Reactive aldehydes were transformed into stable oximes by means of on‐fiber‐derivatization (SPME‐OFD). Aldehyde concentrations in the ppt and ppb level were determined by means of gas chromatography‐mass spectrometry (GC‐MS). Exhaled concentrations were corrected for inspired values. Exhaled C1–C10 aldehydes could be detected in all healthy volunteers, smokers and lung cancer patients. Concentrations ranged from 7 pmol/l (161 pptV) for butanal to 71 nmol/l (1,582 ppbV) for formaldehyde. Highest inspired concentrations were found for formaldehyde and acetaldehyde (0–55 nmol/l and 0–13 nmol/l, respectively). Acetaldehyde, propanal, butanal, heptanal and decanal concentrations showed no significant differences for cancer patients, smokers and healthy volunteers. Exhaled pentanal, hexanal, octanal and nonanal concentrations were significantly higher in lung cancer patients than in smokers and healthy controls (ppentanal = 0.001; phexanal = 0.006; poctanal = 0.014; pnonanal = 0.025). Sensitivity and specificity of this method were comparable to the diagnostic certitude of conventional serum markers and CT imaging. Lung cancer patients could be identified by means of exhaled pentanal, hexanal, octanal and nonanal concentrations. Exhaled aldehydes reflect aspects of oxidative stress and tumor‐specific tissue composition and metabolism. Noninvasive recognition of lung malignancies may be realized if analytical skills, biochemical knowledge and medical expertise are combined into a joint effort.

Determination of volatile organic compounds in exhaled breath of patients with lung cancer using solid phase microextraction and gas chromatography mass spectrometry

Abstract Background: Analysis of exhaled breath is a promising diagnostic method. Sampling of exhaled breath is non-invasive and can be performed as often as considered desirable. There are indications that the concentration and presence of certain of volatile compounds in exhaled breath of lung cancer patients is different from concentrations in healthy volunteers. This might lead to a future diagnostic test for lung cancer. Methods: Exhaled breath samples from 65 patients with different stages of lung cancer and undergoing different treatment regimes were analysed. Mixed expiratory and indoor air samples were collected. Solid phase microextraction (SPME) with carboxen/polydimethylsiloxane (CAR/PDMS) sorbent was applied. Compounds were analysed by means of gas chromatography (GC) and mass spectrometry (MS). Results: The method we used allowed identification with the spectral library of 103 compounds showing at least 15% higher concentration in exhaled breath than in inhaled air. Among those 103 compounds, 84 were confirmed by determination of the retention time using standards based on the respective pure compound. Approximately, one third of the compounds detected were hydrocarbons. We found aromatic hydrocarbons, alcohols, aldehydes, ketones, esters, ethers, sulfur compounds, nitrogen-containing compounds and halogenated compounds. Acetonitrile and benzene were two of 10 compounds which correlated with smoking behaviour. A comparison of results from cancer patients with those of 31 healthy volunteers revealed differences in the concentration and presence of certain compounds. The sensitivity for detection of lung cancer patients based on eight different compounds not seen in exhaled breath of healthy volunteers was 51% and the specificity was 100%. These eight potential markers for detection of lung cancer are 1-propanol, 2-butanone, 3-butyn-2-ol, benzaldehyde, 2-methyl-pentane, 3-methyl-pentane, n-pentane and n-hexane. Conclusions: SPME is a relatively insensitive method and compounds not observed in exhaled breath may be present at a concentration lower than LOD. The main achievement of the present work is the validated identification of compounds observed in exhaled breath of lung cancer patients. This identification is indispensible for future work on the biochemical sources of these compounds and their metabolic pathways. Clin Chem Lab Med 2009;47:550–60.

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

Isoprene and acetone concentration profiles during exercise on an ergometer

A real-time recording setup combining exhaled breath volatile organic compound (VOC) measurements by proton transfer reaction-mass spectrometry (PTR-MS) with hemodynamic and respiratory data is presented. Continuous automatic sampling of exhaled breath is implemented on the basis of measured respiratory flow: a flow-controlled shutter mechanism guarantees that only end-tidal exhalation segments are drawn into the mass spectrometer for analysis. Exhaled breath concentration profiles of two prototypic compounds, isoprene and acetone, during several exercise regimes were acquired, reaffirming and complementing earlier experimental findings regarding the dynamic response of these compounds reported by Senthilmohan et al (2000 Redox Rep. 5 151-3) and Karl et al (2001 J. Appl. Physiol. 91 762-70). While isoprene tends to react very sensitively to changes in pulmonary ventilation and perfusion due to its lipophilic behavior and low Henry constant, hydrophilic acetone shows a rather stable behavior. Characteristic (median) values for breath isoprene concentration and molar flow, i.e., the amount of isoprene exhaled per minute are 100 ppb and 29 nmol min(-1), respectively, with some intra-individual day-to-day variation. At the onset of exercise breath isoprene concentration increases drastically, usually by a factor of ∼3-4 within about 1 min. Due to a simultaneous increase in ventilation, the associated rise in molar flow is even more pronounced, leading to a ratio between peak molar flow and molar flow at rest of ∼11. Our setup holds great potential in capturing continuous dynamics of non-polar, low-soluble VOCs over a wide measurement range with simultaneous appraisal of decisive physiological factors affecting exhalation kinetics. In particular, data appear to favor the hypothesis that short-term effects visible in breath isoprene levels are mainly caused by changes in pulmonary gas exchange patterns rather than fluctuations in endogenous synthesis.

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.

Breath biomarkers for lung cancer detection and assessment of smoking related effects--confounding variables, influence of normalization and statistical algorithms.

BACKGROUND Up to now, none of the breath biomarkers or marker sets proposed for cancer recognition has reached clinical relevance. Possible reasons are the lack of standardized methods of sampling, analysis and data processing and effects of environmental contaminants. METHODS Concentration profiles of endogenous and exogenous breath markers were determined in exhaled breath of 31 lung cancer patients, 31 smokers and 31 healthy controls by means of SPME-GC-MS. Different correcting and normalization algorithms and a principal component analysis were applied to the data. RESULTS Differences of exhalation profiles in cancer and non-cancer patients did not persist if physiology and confounding variables were taken into account. Smoking history, inspired substance concentrations, age and gender were recognized as the most important confounding variables. Normalization onto PCO2 or BSA or correction for inspired concentrations only partially solved the problem. In contrast, previous smoking behaviour could be recognized unequivocally. CONCLUSION Exhaled substance concentrations may depend on a variety of parameters other than the disease under investigation. Normalization and correcting parameters have to be chosen with care as compensating effects may be different from one substance to the other. Only well-founded biomarker identification, normalization and data processing will provide clinically relevant information from breath analysis.

Assessment of propofol concentrations in human breath and blood by means of HS-SPME–GC–MS

BACKGROUND Breath analysis could offer a non-invasive means of drug monitoring if adequate analytical methods and robust correlations between drug concentrations in breath and blood can be established. We therefore applied headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry (HS-SPME-GC-MS) to assess breath and blood concentrations of the intravenous drug propofol in patients under anesthesia or sedation. METHODS Arterial, central- and peripheral-venous blood and alveolar breath samples were drawn in parallel from 16 mechanically ventilated patients. In addition, six patients undergoing lung resection were investigated. Substances were preconcentrated by means of HS-SPME, separated by GC and identified by MS. RESULTS Propofol detection limits were 0.006 nmol/L in breath and 72.20 nmol/L in blood, the quantitation limits were 0.009 nmol/L and 75.89 nmol/L (end tidal breath/blood). Intraday precision was 8-11%, recovery 97-103%. Propofol concentrations were 0.04-0.5 nmol/L in breath and 2-120 micromol/L in blood. Only arterial propofol concentrations showed a correlation with concentrations in breath. Impaired ventilation/perfusion ratios in patients under lung resection resulted in changes of correlation coefficients. CONCLUSIONS Reliable and precise analytical methods such as HS-SPME-GC-MS represent basic requirements if breath analysis is to be set up for non-invasive monitoring of intravenous drugs and control of anesthesia.

Impact of sampling procedures on the results of breath analysis.

The impact of different sampling techniques on the results of breath analysis was to be assessed in this study. Alveolar, mixed expiratory and time-controlled samples were collected from ten volunteers and from eight lung cancer patients. Breath sampling was visually controlled by means of capnometry. PCO(2) and 13 VOCs were determined. Mixed expiratory sampling yielded 25% lower concentrations of CO(2) and blood-borne VOCs. Time-controlled sampling generated high variation of results. Ratios C(alv)/C(mixed) were >1.5 for CO(2), acetone and isoprene, and <1 for isopropanol, 2-butanone and hexanal. Acetonitrile, butane, dimethylsulfide, pentane, butanal, benzene and hexane showed 1.5 > C(alv)/C(mixed) > 1. The ratio C(alv)/C(mixed) of CO(2), acetone and isoprene was different in healthy volunteers and lung cancer patients. Alveolar samples showed the highest concentrations of endogenous and lowest concentration of exogenous substances. Sampling can impact results in breath analysis. Valuable information can be obtained from ratios of alveolar and mixed expired concentrations.

Impact of inspired substance concentrations on the results of breath analysis in mechanically ventilated patients

Abstract A well-defined relationship has to exist between substance concentrations in blood and in breath if blood-borne volatile organic compounds (VOCs) are to be used as breath markers of disease or health. In this study, the impact of inspired substances on this relationship was investigated systematically. VOCs were determined in inspired and expired air and in arterial and mixed venous blood of 46 mechanically ventilated patients by means of SPME, GC/MS. Mean inspired concentrations were 25% of expired concentrations for pentane, 7.5% for acetone, 0.7% for isoprene and 0.4% for isoflurane. Only if inspired concentrations were <5% did substance disappearance rates from blood and exhalation rates correlate well. Exhaled substance concentrations depended on venous and inspired concentrations. Patients with sepsis had higher n-pentane and lower acetone concentrations in mixed venous blood than patients without sepsis (2.27 (0.37–8.70) versus 0.65 (0.33–1.48) nmol L−1 and 69 (22–99) versus 18 (6.7–56) µmol L−1). n-Pentane and acetone concentrations in breath showed no differences between the patient groups, regardless whether or not expired concentrations were corrected for inspired concentrations. In mechanically ventilated patients, concentration profiles of volatile substances in breath may considerably deviate from profiles in blood depending on the relative amount of inspired concentrations. A simple correction for inspired substance concentrations was not possible. Hence, substances having inspired concentrations >5% of expired concentrations should not be used as breath markers in these patients without knowledge of concentrations in blood and breath.

Multibed Needle Trap Devices for on Site Sampling and Preconcentration of Volatile Breath Biomarkers

To facilitate their use in trace gas analysis, the adsorption capacity of needle trap devices (NTDs) was increased by combining three adsorbent materials and increasing total adsorbent amount. The use of 22 gauge needles, application of internally expanding desorptive flow technique without cryofocusation and a new on site alveolar sampling method for NTDs provided sensitivity in the parts per trillion range of VOC concentrations without loosing precision or linearity. LODs were 0.4 ng/L for isoprene, 0.5 ng/L for dimethyl sulphide, 0.9 ng/L for 2-butenal, 1.0 ng/L for hexane, 1.2 ng/L for pentane, 2.3 ng/L for hexanal, 5.3 ng/L for pentanal, and 8.3 ng/L for acetone. R of calibration curves were consistently >0.98. Loss of volatile aldehydes during storage for 7 days was less than 10%. Needle trap devices packed with more than one adsorbent material represent a promising alternative to SPE and SPME for analysis of volatile organic compounds in the low parts per billion/parts per trillion range. Crucial problems of clinical breath analysis concerning sensitivity of analytical methods, limited stability, and decomposition of breath compounds during sampling and storage could be solved.