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Oral Presentation Room Europa 2 - 08:30-10:30
M ONDAY, O CTOBER 6 TH 2008
189. New frontiers in noninvasive
assessment of the lungs
1563
Metabonomic analysis of EBC in adults by nuclear magnetic resonance
spectroscopy
Guglielmo de Laurentiis 1,2 , Debora Paris 3 , Dominique Melck 3 ,
Mauro Maniscalco 1 , Serafino Marsico 2 , Gaetano Corso 4 , Andrea Motta 3 ,
Matteo Sofia 1 . 1 Respiratory Diseases, University “Federico II”, Naples, Italy;
2
Respiratory Diseases, Second University of Naples, Naples, Italy; 3 Institute of
Biomolecular Chemistry, Consiglio Nazionale delle Ricerche, Pozzuoli, Naples,
Italy; 4 Biomedical Sciences, University of Foggia, Foggia, Italy
Exhaled breath condensate (EBC) is a non-invasive method to study airway lining
fluid. NuclearMagneticResonance (NMR) based metabonomic analysis provides
biochemical fingerprint in biological fluids.
Aim: to evaluate EBC in healthy adults (HS) and in COPD patients using NMR
spectroscopy examining the role of preanalytical variables such as saliva or
disinfectant.
Material and Methods: EBC and saliva samples from 11 HS and 11 COPD were
analyzed by means of 1 H-NMR spectroscopy (Bruker DRX-600MHz).The effect
on EBC of disinfectant utilized to sterilize reusable parts was assessed.
Results: COPD and HS NMR saliva spectra were strikingly different from those
of corresponding EBC.
The disinfectant perturbation on EBC spectra was almost completely removed
using ethanol.COPD spectra differed from HS by five signals, which appear to be
able to clusterize the data in two different groups (94%).
Conclusions: EBC metabonomics in adults discriminate the potential perturbation
by preanalytical variables such saliva or disinfectants.The NMR-metabonomic ap-
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M ONDAY, O CTOBER 6 TH 2008
the mechanisms responsible for this change and its precise role requires further
studies.
The study was supported by OTKA (68808).
1566
Alpha-1-antitrypsin is detectable and can be quantified in exhaled breath
condensate
Sarah Noeske, Robert Bals, Christian Herr, Claus Vogelmeier, Andreas
Rembert Koczulla. Department of Internal Medicine, Division for Pulmonary
Diseases, Philipps-University Marburg, Marburg/Hesse, Germany
Rationale: Alpha-1-antitrypsin deficiency (AATD) is a genetic disorder that pre-
disposes to chronic obstructive pulmonary disease (COPD). The main function
of alpha-1-antitrypsin (AAT) is to protect the lungs against neutrophil elastase.
proach could disclose the metabolic fingerprint of EBC giving a clearcut separation Until now a non invasive method to detect AAT reliably in the epithelial lining
between HS and COPD patients. fluid (ELF) is missing. Measurement of exhaled breath condensate (EBC) is a non
invasive method to investigate parameters of the ELF of the lung.
Aim: It was the aim of the study to show that AAT is measurable with the non
1564 invasive method of EBC. A Western Blot and ELISA should be validated and
Breath analysis in renal failure: effect of hemodialysis standardized to detect very small amounts of AAT in EBC.
Enrico Heffler 1 , Nicoletta Ferrero 1 , Luisa Bommarito 1 , Massimiliano Bugiani 2 , Methods: 1ml of EBC from each patient was lyophilized. Afterwards Western Blot
Roberta Bergia 1 , Giuseppe Guida 1 , Stefano Pizzimenti 1 , Michele Bruno 3 , and ELISA were established. Design: Measurement was performed in 10 healthy
Michele Petrarulo 3 , Giovanni Rolla 1 . 1 Department of Allergy and Clinical controls (HC), 10 patients with AATD and 10 smokers without lung disease. EBC
Immunology, University of Torino–Azienda Sanitaria Ospedale Mauriziano from all individuals was collected with the RTube™ device.
“Umberto I”, Torino, Italy; 2 Department of Respiratory Medicine, Results: AAT in EBC was detectable by Western Blot and ELISA in each in-
CPA–ASL-TO2, Torino, Italy; 3 Department of Nephrology, Azienda Sanitaria dividual. We could detect AAT in concentrations down to 0.2ng/ml. We found
Ospedale Mauriziano “Umberto I”, Torino, Italy differences regarding the AAT levels between HCs, patients with AATD and
smokers.
Background: Impaired nitric oxide (NO) signaling is thought to play a major Conclusion: We were able to detect and quantify AAT in EBC. The highly sen-
role in endothelial dysfunction of patients with renal failure (RF). A simple way sitive developed assay can be used for measuring levels of AAT from the lung of
to assess NO production is to measure its concentration in exhaled air (FENO). humans non-invasively. This could be of value for therapy monitoring.
Airway pH may influence FENO and in patients in hemodialysis (HD) it may have
significant shift, with possible influence on FENO. Ammonia (NH3) is involved
in airway pH homeostasis and it is present in great concentration in the breath of 1567
RF patients, and it is largely removed by HD An electronic nose distinguishes the exhaled breath condensates of AATD
Methods: Airway and arterial blood pH, metabolites of NO (NOx/NO2) and NH3 patients and healthy controls
in Exhaled Breath Condensate (EBC), and FENO were measured in 12 patients Rembert Koczulla, Akira Hattesohl, Sarah Noeske, Claus Vogelmeier,
with RF in mantainance HD 30 minutes before and after HD, and in 14 healthy Robert Bals. Department of Medicine, Division of Pulmonology,
controls (HC) Philipps-Universität Marburg, Marburg, Hessen, Germany
Results:
Background: Exhaled respiratory air contains countless volatile organic com-
RF before HD RF after HD HC p value† p value‡ pounds (VOCs). The composition of those VOCs varies dependent of different
pulmonary diseases. With the customizable electronic nose Cyranose 320 (C-320)
FENO, ppb (IC95%) 14.5 (12.2-21.0) 11.5 (7.2-19.0) 13.0 (10.7-21.0) >0.05 0.012 it is possible to detect VOCs and distinguish between different VOC patterns. It
EBC-pH (IC95%) 7.16 (6.8-7.4) 6.4 (5.6-6.8) 6.6 (6.4-6.8) 0.004 0.002
was the aim of this study to analyze and distinguish the VOCs in exhaled breath
Arterial blood pH
condensates (EBC) of patients with α1 -antitrypsin deficiency (AATD) and healthy
(IC95%) 7.35 (7.33-7.35) 7.46 (7.44-7.46) 7.41 (7.39-7.42) 0.03 0.002
controls (HC).
NOx, μM (IC95%) 12.3 (11.1-41.9) 9.9 (5.0-16.4) 9.4 (4.6-10.9) 0.007 0.01
NO2, μM (IC95%) 4.7 (1.2-8.2) 0.8 (0.6-1.2) 0.9 (0.7-1.2) 0.004 0.008
Material and Methods: EBC was collected from ten HCs and ten AATD patients
NH3, μM (IC95%) 2340 (1325-3922) 527 (318-895) 660 (406-872) <0.001 0.002 with the RTube by ten minutes of tidal breathing. 200 μl of EBC were heated
up 37° C and argon was passed through with a constant flow of 350 ml /min for
†
before RD vs HC; ‡ before RD vs after HD. two minutes. Subsequently, the samples were measured by holding the snout of
the C-320 above the surface of the samples and drawing gas for ten seconds. Ten
Conclusion: FENO is normal in RF, while EBC-NOx/NO2 are increased, suggest- samples of ambient lab air were taken as loading controls. A linear discriminant
ing oxidative stress of respiratory system. The huge amount of EBC-NH3 and its analysis (LDA) has been performed on the data.
dramatic decrease after HD may explain the higher than normal levels of EBC-pH Results: The analyses showed that AATD patients, HCs and lab air are clearly sep-
in RF before HD and its decrease after HD. arable. The predictive positive value (PPV) was calculated with a cross-validation
using ten percent of the training data as test data (see table).
1565 Mahalanobis distances (and PPVs) between the groups
Effect of exercise on exhaled breath condensate pH in young healthy subjects HC lab air
Andras Bikov 1 , Balazs Szili 1 , Balazs Antus 2 , Mark Kollai 1 , Ildiko Horvath 1 .
1
Human Physiology and Clinical Experimental Research, Semmelweis University, AATD 2.390 (63.0%) 2.393 (93.5%)
Budapest, Hungary; 2 Pathophysiology, National Korányi Institute for TB and HC 2.402 (79.0%)
Pulmonology, Budapest, Hungary
Conclusion: Using the C-320, we could distinguish between EBCs from AATD
Introduction: Asthma-like symptoms are frequent in healthy people performing patients and HCs. This approach also shows EBC contains enough VOCs to
regular exercise in cold temperature. However the mechanism responsible for this separate two different patient groups. An advantage of using the EBC is the lower
link is not known. Based on current knowledge changes may involve an increase in contamination risk by not measuring for example infectious patients directly with
oxidative stress and in association with it a decrease in pH that may be represented the C-320 and the easiness to sample EBC.
in exhaled biomarker concentrations.
Objective: We aimed to follow changes in exhaled breath condensate (EBC) pH
and 8-isoprostane, a biomarker of oxidative stress, together with lung function in 1568
young healthy subjects performing exercise challenge at cold temperature. Repeated eNose assessment in the monitoring of O2 -induced changes in
Methods: Ten healthy subjects (21.4±1.1 years) participated in the study. At rest, exhaled breath metabolites in COPD
immediately and 30 minutes after exercise challenge at 5±1 °C FEV1 , FVC was Marten de Vries 1 , Jan van der Maten 1 , Peter J. Sterk 2 , Anneke Ten Brinke 1 .
1
measured and EBC was collected to determinate the changes in airway pH and Department of Pulmonary Diseases, Medical Centre Leeuwarden, Leeuwarden,
8-isoprostane levels. EBC pH was measured as previously described (Kullmann et Netherlands; 2 Department of Pulmonary Diseases, Academic Medical Center,
al, Eur Resp J, 2007; 29:185-7) and 8-isoprostane was determined by commercially Amsterdam, Netherlands
available ELISA. Data are expressed as mean±SD.
Results: Exercise caused a small decrease in FEV1 , while FVC did not change. Background: COPD is a complex disease in which oxidative stress-induced in-
There was a significant increase in EBC pH (from 5.9±0.13 to 6.07±0.12, flammation may play a pathophysiological role. Oxygen (O2 ) administration has
p=0.002), but there was no significant change in 8-isoprostane concentration. been shown to increase markers of oxidative stress in exhaled breath condensate
Conclusion: The increased airway pH suggested by changes in EBC pH could (Carpagnano, Thorax 2004). Electronic nose (eNose) technology allows online
play a role in airway regulation during and after exercise. However to understand pattern recognition of volatile organic compounds (VOCs). We hypothesized that
257s
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Oral Presentation Room Europa 2 - 08:30-10:30
M ONDAY, O CTOBER 6 TH 2008
the O2 -induced changes in exhaled metabolites in COPD can be detected by observed in healthy smokers even without having airways obstruction detected
changes in VOC profiles (smellprints). with PFTs. Similar characteristics of dynamic lung images have been recorded in
Aim: We investigated in 10 ex-smoking COPD patients (GOLD stage II/III: 7/3; COPD patients but with higher intensity and proportion. Spirometric values and
smoking: 30 (10-45) pck yrs) whether smellprints before and repeatedly after short VRI characteristics are shown. VRI seems to be a valuable system applicable in
term O2 supply can be discriminated by eNose. airways obstructive diseases and might be an additional tool for screening smokers.
Methods: After baseline sampling (t0 ), O2 (3L/min, VOC-free) was supplied for 40
min through a nasal canula. Thereafter exhaled breath was collected at 0(t1 ), 10(t2 ),
30(t3 ), 50(t4 ), 70(t5 ) and 100(t6 ) min, by expiratory VC according to Dragonieri
(JACI 2007), and sampled by eNose (Cyranose 320). Smellprints were analysed
by linear discriminant analysis on principal component reduction. Cross-validation
values (CVV) and Mahalanobis distance (M-dist) were calculated.
Results: After 40 min of O2 supply, pO2 changed from 9.1±1.3 to 13.0±2.5 kPa
(p<0.001). Smellprints at all time points after O2 administration could be well
discriminated from baseline (M-dist 2.69-4.27; CVV ≥90%), except for t3 (M-dist
1.70; CVV 75%).
Conclusion: eNose technology is able to discriminate smellprints before and after
short term O2 supply in COPD patients. This suggests that oxidative stress can be
readily assessed from VOC patterns in exhaled air, which provides a monitoring
tool in the clinical research of COPD.
1569
Breath analysis by ion mobility spectrometry in sarcoidosis – results of a
feasibility study
Michael Westhoff 1 , Patric Litterst 1 , Barbara Obertrifter 1 ,
Alexander Bunkowski 2 , Joerg Ingo Baumbach 2 . 1 Pneumology and Sleep
Medicine, Lung Clinic Hemer, Hemer, Germany; 2 Metabolomics Department,
ISAS–Institute for Analytical Sciences, Dortmund, Germany
Purpose: The development of non-invasive diagnostic tools for the differentiation
of pulmonary disorders is desirable. We examined if ion mobility spectrometry can
detect volatile organic compounds (VOC) in human exhaled air that characterize
patients with sarcoidosis.
Methods: An ion mobility spectrometer (IMS) coupled to a multi-capillary-column
(MCC) was used to identify and quantify volatile metabolites occurring in human
breath down to the ng/L- and pg/L-range of analytes within less than 500 s and
without any pre-concentration. The IMS investigations are based on different drift
times of swarms of ions of metabolites formed directly in air at ambient pressure.
The exhaled breath of 20 patients with mediastinal lymph node enlargement (11
patients with sarcoidosis and 9 with exclusion of sarcoidosis) was investigated.
Results: A procedure related to a single peak in the IMS-chromatogram delivered
a differentiation into two groups of mediastinal lymph node enlargement (con-
firmed and excluded sarcoidosis) with a sensitivity of 0.91 and a specifity of 0.89.
The potential biomarker for sarcoidosis is characterised by the parameters inverse
mobility (1/K0 ) 0.53±0.01 Vs/cm2 and retention time 22±5 s.
Conclusion: The data show that ion mobility spectrometry may provide a typical
chromatogram in patients with sarcoidosis. This might allow differentiation of
mediastinal lymph node enlargement related to sarcoidosis from other diseases.
These first data need further confirmation by prospective studies.
Clinical Implications: Ion mobility spectrometry might gain an important role in
the non-invasive diagnosis of sarcoidosis.
1570
Is vibration response imaging (VRI) valuable in early detection of airways
obstruction in smokers?
Angeliki Florou, Sofia Vakali, Dimitrios Michailidis, Charis Roussos,
Christina Gratziou. Asthma and Allergy Center, Pulmonary and Critical Care
Department, Medical School-University of Athens-Evgenidion Hospital, Athens,
Greece
Vibration Response Imaging (VRI) is a new system that provides dynamic image
of the lung by a detection of vibrations produced by airflow during the respiratory
cycle.This study was undertaken in order to validate if VRI can be valuable for
detection of airways obstruction in smokers and in COPD patients and to investi-
gate if correlates with PFTs.25 “healthy”smokers (mean age 45,6±9,6),23 COPD
patients (mean age 59,6±8,) and 10 healthy non-smokers were included in the
study.Healthy smokers were definned those with no spirometric evidence of airways
obstruction.64% of this group reported one or more respiratory symptoms.The VRI
recordings were blindly evaluated characterized as normal and abnormal accord-
ing to dynamic image and maximal energy frame.Abnormal images have been
Spirometric values and VRI characteristics
Non-smokers N=10 Smokers N=25 COPD N=23
FEV1%pred mean (SD) 96,8 (7,6) 98,8 (17,9) 60,6 (20,3)
FEV1/FVC mean (SD) 81,5 (8,8) 78,7 (4,7) 58,4 (10,5)
MMEF75/25%pred mean (SD) 72,7 (18) 71,3 (26,7) 26,3 (12,6)
VRI characteristics %cases %cases %cases
Dynamic disturbance 0 64 95,7
Uneven distribution of vibration energy 0 28 82,6
Low vibration energy 0 24 73,9
Abnormal progression of the image 0 20 43,5
Wheezing 0 8 21,7
Prologation of expiration 0 8 30,4
258s
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