Phillip Trefz

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.

Needle trap micro-extraction for VOC analysis: effects of packing materials and desorption parameters.

Combining advantages of SPE and SPME needle trap devices (NTD) represent promising new tools for a robust and reproducible sample preparation. This study was intended to investigate the effect of different packing materials on efficacy and reproducibility of VOC analysis by means of needle trap micro extraction (NTME). NTDs with a side hole design and containing different combinations of PDMS, DVB and Carbopack X and Carboxen 1000 and NTDs containing a single layer organic polymer of methacrylic acid and ethylene glycol dimethacrylate were investigated with respect to reproducibility, LODs and LOQs, carry over and storage. NTDs were loaded with VOC standard gas mixtures containing saturated and unsaturated hydrocarbons, oxygenated and aromatic compounds. Volatile substances were thermally desorbed from the NTDs using fast expansive flow technique and separated, identified and quantified by means of GC-MS. Optimal desorption temperatures between 200 and 290°C could be identified for the different types of NTDs with respect to desorption efficiency and variation. Carry over was below 6% for polymer packed needles and up to 67% in PDMS/Carboxen 1000 NTDs. Intra and inter needle variation was best for polymer NTDs and consistently below 9% for this type of NTD. LODs and LOQs were in the range of some ng/L. Sensitivity of the method could be improved by increasing sample volume. NTDs packed with a copolymer of methacrylic acid and ethylene glycol dimethacrylate were universally applicable for sample preparation in VOC analysis. If aromatic compounds were to be determined DVB/Carboxen 1000 and DVB/Carbopack X/Carboxen 1000 devices could be considered as an alternative. PDMS/Carbopack X/Carboxen 1000 NTDs may represent a good alternative for the analysis of hydrocarbons and aldehydes. NTME represents a powerful tool for different application areas, from environmental monitoring to breath analysis.

Volatile emissions from Mycobacterium avium subsp. paratuberculosis mirror bacterial growth and enable distinction of different strains.

Control of paratuberculosis in livestock is hampered by the low sensitivity of established direct and indirect diagnostic methods. Like other bacteria, Mycobacterium avium subsp. paratuberculosis (MAP) emits volatile organic compounds (VOCs). Differences of VOC patterns in breath and feces of infected and not infected animals were described in first pilot experiments but detailed information on potential marker substances is missing. This study was intended to look for characteristic volatile substances in the headspace of cultures of different MAP strains and to find out how the emission of VOCs was affected by density of bacterial growth. One laboratory adapted and four field strains, three of MAP C-type and one MAP S-type were cultivated on Herrold’s egg yolk medium in dilutions of 10-0, 10-2, 10-4 and 10-6. Volatile substances were pre-concentrated from the headspace over the MAP cultures by means of Solid Phase Micro Extraction (SPME), thermally desorbed from the SPME fibers and separated and identified by means of GC-MS. Out of the large number of compounds found in the headspace over MAP cultures, 34 volatile marker substances could be identified as potential biomarkers for growth and metabolic activity. All five MAP strains could clearly be distinguished from blank culture media by means of emission patterns based on these 34 substances. In addition, patterns of volatiles emitted by the reference strain were significantly different from the field strains. Headspace concentrations of 2-ethylfuran, 2-methylfuran, 3-methylfuran, 2-pentylfuran, ethyl acetate, 1-methyl-1-H-pyrrole and dimethyldisulfide varied with density of bacterial growth. Analysis of VOCs emitted from mycobacterial cultures can be used to identify bacterial growth and, in addition, to differentiate between different bacterial strains. VOC emission patterns may be used to approximate bacterial growth density. In a perspective volatile marker substances could be used to diagnose MAP infections in animals and to identify different bacterial strains and origins.

Physiological variability in volatile organic compounds (VOCs) in exhaled breath and released from faeces due to nutrition and somatic growth in a standardized caprine animal model.

Physiological effects may change volatile organic compound (VOC) concentrations and may therefore act as confounding factors in the definition of VOCs as disease biomarkers. To evaluate the extent of physiological background variability, this study assessed the effects of feed composition and somatic growth on VOC patterns in a standardized large animal model. Fifteen clinically healthy goats were followed during their first year of life. VOCs present in the headspace over faeces, exhaled breath and ambient air inside the stable were repeatedly assessed in parallel with the concentrations of glucose, protein, and albumin in venous blood. VOCs were collected and analysed using solid-phase or needle-trap microextraction and gas chromatograpy together with mass spectroscopy. The concentrations of VOCs in exhaled breath and above faeces varied significantly with increasing age of the animals. The largest variations in volatiles detected in the headspace over faeces occurred with the change from milk feeding to plant-based diet. VOCs above faeces and in exhaled breath correlated significantly with blood components. Among VOCs exhaled, the strongest correlations were found between exhaled nonanal concentrations and blood concentrations of glucose and albumin. Results stress the importance of a profound knowledge of the physiological backgrounds of VOC composition before defining reliable and accurate marker sets for diagnostic purposes.

In Vivo Volatile Organic Compound Signatures of Mycobacterium avium subsp. paratuberculosis

Mycobacterium avium ssp. paratuberculosis (MAP) is the causative agent of a chronic enteric disease of ruminants. Available diagnostic tests are complex and slow. In vitro, volatile organic compound (VOC) patterns emitted from MAP cultures mirrored bacterial growth and enabled distinction of different strains. This study was intended to determine VOCs in vivo in the controlled setting of an animal model. VOCs were pre-concentrated from breath and feces of 42 goats (16 controls and 26 MAP-inoculated animals) by means of needle trap microextraction (breath) and solid phase microextraction (feces) and analyzed by gas chromatography/ mass spectrometry. Analyses were performed 18, 29, 33, 41 and 48 weeks after inoculation. MAP-specific antibodies and MAP-specific interferon-γ-response were determined from blood. Identities of all marker-VOCs were confirmed through analysis of pure reference substances. Based on detection limits in the high pptV and linear ranges of two orders of magnitude more than 100 VOCs could be detected in breath and in headspace over feces. Twenty eight substances differed between inoculated and non-inoculated animals. Although patterns of most prominent substances such as furans, oxygenated substances and hydrocarbons changed in the course of infection, differences between inoculated and non-inoculated animals remained detectable at any time for 16 substances in feces and 3 VOCs in breath. Differences of VOC concentrations over feces reflected presence of MAP bacteria. Differences in VOC profiles from breath were linked to the host response in terms of interferon-γ-response. In a perspective in vivo analysis of VOCs may help to overcome limitations of established tests.

Monitoring of breath VOCs and electrical impedance tomography under pulmonary recruitment in mechanically ventilated patients

Analysis of exhaled VOCs can provide information on physiology, metabolic processes, oxidative stress and lung diseases. In critically ill patients, VOC analysis may be used to gain complimentary information beyond global clinical parameters. This seems especially attractive in mechanically ventilated patients frequently suffering from impairment of gas exchange. This study was intended to assess (a) the effects of recruitment maneuvers onto VOC profiles, (b) the correlations between electrical impedance tomography (EIT) data and VOC profiles and (c) the effects of recruitment onto distribution of ventilation. Eleven mechanically ventilated patients were investigated during lung recruitment after cardiac surgery. Continuous breath gas analysis by means of PTR-ToF-MS, EIT and blood gas analyses were performed simultaneously. More than 300 mass traces could be detected and monitored continuously by means of PTR-ToF-MS in every patient. Exhaled VOC concentrations varied with recruitment induced changes in minute ventilation and cardiac output. Ammonia exhalation depended on blood pH. The improvement in dorsal lung ventilation during recruitment ranged from 9% to 110%. Correlations between exhaled concentrations of acetone, isoprene, benzene sevoflurane and improvement in regional ventilation during recruitment were observed. Extent and quality of these correlations depended on physico-chemical properties of the VOCs. Combination of continuous real-time breath analysis and EIT revealed correlations between exhaled VOC concentrations and distribution of ventilation. This setup enabled immediate recognition of physiological and therapeutic effects in ICU patients. In a perspective, VOC analysis could be used for non-invasive control and optimization of ventilation strategies.

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.

Electrochemical sensor system for breath analysis of aldehydes, CO and NO.

Bulky and hyphenated laboratory-based analytical instrumentation such as gas chromatography/mass spectrometry is still required to trace breath biomarkers in the low ppbV level. Innovative sensor-based technologies could provide on-site and point-of-care (POC) detection of volatile biomarkers such as breath aldehydes related to oxidative stress and cancer. An electrochemical sensor system was developed for direct detection of the total abundance of aldehydes in exhaled breath in the ppbV level and for simultaneous determination of the airway inflammation markers carbon monoxide (CO) and nitric oxide (NO). The sensor system was tested in vitro with gaseous standard mixtures and in vivo in spontaneously breathing patients and under mechanical ventilation in an animal model. The sensor system provided in vitro and in vivo detection of trace levels of aldehydes, CO and NO. Inertness of the tubing system was important for reliable results. Sensitivity of the aldehyde sensor increased with humidity. Response time for analysis of breath samples was about 22 s and relative standard deviations of sensor amplitudes were <5%. Detection limits in the low ppbV range and a linear range of more than two orders of magnitude could be achieved for volatile aldehydes. Cross sensitivities were moderate for alcohols such as ethanol or isopropanol and negligible for other typical breath volatile organic compounds such as acetone, isoprene or propofol. In proof of concept analyses in patients suffering from lung cancer and diabetes, aldehyde and CO sensor signals differed between the groups. Elevated CO levels indicated previous smoking. In a mechanically ventilated pig, continuous monitoring of breath aldehyde concentrations in the low ppbV was realized. Cumulative aldehyde measurements may add interesting and complementary information to the conventional parameters used in clinical breath research. POC applicability, easy handling and low cost of sensors facilitate measurements in large patient cohorts.

Exhaled volatile substances mirror clinical conditions in pediatric chronic kidney disease

Monitoring metabolic adaptation to chronic kidney disease (CKD) early in the time course of the disease is challenging. As a non-invasive technique, analysis of exhaled breath profiles is especially attractive in children. Up to now, no reports on breath profiles in this patient cohort are available. 116 pediatric subjects suffering from mild-to-moderate CKD (n = 48) or having a functional renal transplant KTx (n = 8) and healthy controls (n = 60) matched for age and sex were investigated. Non-invasive quantitative analysis of exhaled breath profiles by means of a highly sensitive online mass spectrometric technique (PTR-ToF) was used. CKD stage, the underlying renal disease (HUS; glomerular diseases; abnormalities of kidney and urinary tract or polycystic kidney disease) and the presence of a functional renal transplant were considered as classifiers. Exhaled volatile organic compound (VOC) patterns differed between CKD/ KTx patients and healthy children. Amounts of ammonia, ethanol, isoprene, pentanal and heptanal were higher in patients compared to healthy controls (556, 146, 70.5, 9.3, and 5.4 ppbV vs. 284, 82.4, 49.6, 5.30, and 2.78 ppbV). Methylamine concentrations were lower in the patient group (6.5 vs 10.1 ppbV). These concentration differences were most pronounced in HUS and kidney transplanted patients. When patients were grouped with respect to degree of renal failure these differences could still be detected. Ammonia accumulated already in CKD stage 1, whereas alterations of isoprene (linked to cholesterol metabolism), pentanal and heptanal (linked to oxidative stress) concentrations were detectable in the breath of patients with CKD stage 2 to 4. Only weak associations between serum creatinine and exhaled VOCs were noted. Non-invasive breath testing may help to understand basic mechanisms and metabolic adaptation accompanying progression of CKD. Our results support the current notion that metabolic adaptation occurs early during the time course of CKD.

Applied upper-airway resistance instantly affects breath components: a unique insight into pulmonary medicine

Respiratory parameters such as flow or rate have complex effects on the exhalation of volatile substances and can hamper clinical interpretation of breath biomarkers. We have investigated the effects of progressively applied upper-airway resistances on the exhalation of volatile organic compounds (VOCs) in healthy humans. We performed real-time mass-spectrometric determination of breath volatiles in 50 subjects with parallel, non-invasive hemodynamic monitoring, breath-resolved spirometry and capnometry during controlled tidal breathing (12 breaths/min). Airway resistance was increased by changing the mouthpiece diameters from 2.5 cm to 1.0 cm and to 0.5 cm. At the smallest diameter, oxygen uptake increased (35%↑). Cardiac output decreased (6%↓) but end-tidal PCO2 (8%↑) and exhalation of blood-borne isoprene (19%↑) increased. Carbon dioxide production remained constant. Furan, hydrogen sulphide mirrored isoprene. Despite lowered minute ventilation (4%↓) acetone concentrations decreased (3%↓). Exogenous acetonitrile, propionic acid, isopropanol, limonene mimicked acetone. VOC concentration changes could be modelled through substance volatility. Airway resistance-induced changes in hemodynamics, and ventilation can affect VOC exhalation and thereby interfere with breath biomarker interpretation. The effects of collateral ventilation, intra-alveolar pressure gradients and respiratory mechanics had to be considered to explain the exhalation kinetics of CO2 and VOCs. Conventional breath sampling via smaller mouthpiece diameters (≤1.0 cm, e.g. via straw in Tedlar bags or canisters, etc) will immediately affect VOC exhalation and thereby mislead the analysis of the obtained results. Endogenous isoprene may probe respiratory muscle workload under obstructive conditions. Breath-gas analysis might enhance our understanding of diagnosis and management of obstructive lung diseases in the future.