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Increased keratin content detected by proteomic analysis of exhaled breath condensate from healthy persons who smoke

2004; Elsevier BV; Volume: 117; Issue: 1 Linguagem: Inglês

10.1016/j.amjmed.2004.01.022

1555-7162

Elisabetta Gianazza, L. Allegra, Enrica Bucchioni, Ivano Eberini, L. Puglisi, Francesco Blasi, Claudio Terzano, Robin Wait, Cesare R. Sirtori,

Microbial Inactivation Methods

Conventional methods for detecting pulmonary changes in persons who smoke, using, for example, radiographs, computed tomography, and functional measures, can only detect gross changes. Measuring the composition of bronchoalveolar lavage fluid or exhaled breath condensate may be a more sensitive technique (1Kharitonov S.A. Barnes P.J. Exhaled markers of pulmonary disease.Am J Respir Crit Care Med. 2001; 163: 1693-1722Crossref PubMed Scopus (753) Google Scholar). Cellular and biochemical alterations of the epithelial lining in various lung airway disorders are reflected in bronchoalveolar and nasal lavage fluid (2Lindahl M. Ståhlbom B. Svartz J. et al.Protein pattern of human nasal and bronchoalveolar lavage fluids analyzed by two-dimensional gel electrophoresis.Electrophoresis. 1998; 19: 3222-3229Crossref PubMed Scopus (63) Google Scholar, 3Wattiez R. Hermans C. 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Identification of a new airway irritation marker, palate lung nasal epithelial clone protein, in human nasal lavage fluid with two-dimensional electrophoresis and matrix-assisted laser desorption/ionization-time of flight.Electrophoresis. 2001; 22: 1795-1800Crossref PubMed Scopus (81) Google Scholar, 7Ghafouri B. Ståhlbom B. Tagesson C. et al.Newly identified proteins in human nasal lavage fluid from nonsmokers and smokers using two-dimensional gel electrophoresis and peptide mass fingerprinting.Proteomics. 2002; 2: 112-120Crossref PubMed Scopus (105) Google Scholar). Compared with persons who do not smoke, bronchoalveolar lavage fluid from persons who do smoke contains decreased amounts of immunoglobulin A (2Lindahl M. Ståhlbom B. Svartz J. et al.Protein pattern of human nasal and bronchoalveolar lavage fluids analyzed by two-dimensional gel electrophoresis.Electrophoresis. 1998; 19: 3222-3229Crossref PubMed Scopus (63) Google Scholar, 8Wattiez R. Hermans C. Cruyt C. et al.Human bronchoalveolar lavage fluid protein two-dimensional database: study of interstitial lung disease.Electrophoresis. 2000; 21: 2703-2712Crossref PubMed Scopus (101) Google Scholar, 9Lenz A.-G. Meyer B. Costabel U. et al.Bronchoalveolar lavage fluid proteins in human lung disease analysis by two-dimensional electrophoresis.Electrophoresis. 1993; 14: 242-244Crossref PubMed Scopus (56) Google Scholar), albumin (10Lindahl M. Ekstrom T. Sorensen J. et al.Two dimensional protein patterns of bronchoalveolar lavage fluid from nonsmokers, smokers, and subjects exposed to asbestos.Thorax. 1996; 51: 1028-1035Crossref PubMed Scopus (44) Google Scholar), ceruloplasmin, pro–apolipoprotein AI (4Sabounchi-Schütt F. Åström J. Eklund A. et al.Detection and identification of human bronchoalveolar lavage proteins using narrow-range immobilized pH gradient DryStrip and the paper bridge sample application method.Electrophoresis. 2001; 22: 1851-1860Crossref PubMed Scopus (41) Google Scholar), and cystatin S (11Lindahl M. Ståhlbom B. Tagesson C. Newly identified proteins in human nasal and bronchoalveolar lavage fluids Potential biomedical and clinical applications.Electrophoresis. 1999; 20: 3670-3676Crossref PubMed Scopus (80) Google Scholar), but increased amounts of Clara cell phospholipid-binding protein (12Lindahl M. Svartz J. Tagesson C. Demonstration of different forms of the anti-inflammatory proteins lipocortin-1 and Clara cell protein-16 in human nasal and bronchoalveolar lavage fluids.Electrophoresis. 1999; 20: 881-890Crossref PubMed Scopus (58) Google Scholar), lipocalin 1, and immunoglobulin-binding factor (11Lindahl M. Ståhlbom B. Tagesson C. Newly identified proteins in human nasal and bronchoalveolar lavage fluids Potential biomedical and clinical applications.Electrophoresis. 1999; 20: 3670-3676Crossref PubMed Scopus (80) Google Scholar). In nasal lavage fluid, increases in Clara cell phospolipid-binding protein, and decreases in lipocortin, α1-antitrypsin, cystatin S, and palate lung nasal epithelial clone protein have been reported (7Ghafouri B. Ståhlbom B. Tagesson C. et al.Newly identified proteins in human nasal lavage fluid from nonsmokers and smokers using two-dimensional gel electrophoresis and peptide mass fingerprinting.Proteomics. 2002; 2: 112-120Crossref PubMed Scopus (105) Google Scholar). However, none of these changes appear to be well correlated with smoking, and collection of bronchoalveolar lavage fluid is difficult. Analysis of exhaled breath condensate may be useful as a noninvasive way of monitoring pulmonary conditions. Some of the airspace surface liquid is aerosolized during turbulent airflow, so that the condensate reflects the composition of the fluid lining the lung. Although simple and well accepted by patients, this technique has mainly been used for analysis of nitric oxide metabolites, 8-isoprostane, hydrogen peroxide, and various proinflammatory cytokines (13Montuschi P. Corradi M. Ciabattoni G. Increased 8-isoprostane, a biomarker of oxidative stress, in exhaled condensate of asthma patients.Am J Crit Care Med. 1999; 160: 216-220Crossref Scopus (488) Google Scholar). Proteomic investigation (14Wilkins M.R. Williams K.L. Appel R.D. et al.Proteome Research: New Frontiers in Functional Genomics. Springer-Verlag, Berlin, Germany1997Crossref Google Scholar) has enabled characterization of biological fluids, including serum, urine, cerebrospinal fluid, and bronchoalveolar lavage fluid (www.expasy.org). Although proteomic technology has been applied to exhaled breath condensate (15Scheideler L. Manke H.G. Schwulera U. et al.Detection of nonvolatile macromolecules in breath. A possible diagnostic tool?.Am Rev Respir Dis. 1993; 148: 778-784Crossref PubMed Scopus (161) Google Scholar, 16Mutlu G.M. Garey K.W. Robbins R.A. et al.Collection and analysis of exhaled breath condensate in humans.Am J Respir Crit Care Med. 2001; 164: 731-737Crossref PubMed Scopus (272) Google Scholar), prior studies have not identified proteins by mass spectrometry or other methods (17Griese M. Noss J. von Bredow C. Protein pattern of exhaled breath condensate and saliva.Proteomics. 2002; 2: 690-696Crossref PubMed Scopus (64) Google Scholar). We enrolled 21 nonsmokers (9 men and 12 women; mean [± SD] age, 51 ± 6 years) and 25 smokers (11 men and 14 women; mean age, 55 ± 8 years). All subjects were in good general health, as assessed by clinical evaluation, chest radiographs, and biochemical and hematological tests. Smokers had at least a 40 pack-year history of cigarette smoking. All subjects had normal spirometric test results (mean forced expiratory volume in 1 second [FEV1] = 93% ± 6% of predicted values for smokers, and 95% ± 4% for nonsmokers) (18Quarner P.H. Tammeling G.J. Cotes J.E. et al.Official statement of the European Respiratory Society lung volume and forced expiratory flow.Eur Resp J. 1993; 6: 5-40PubMed Google Scholar). Exhaled breath condensate was also collected from two male former smokers (ages 55 and 75 years) who had undergone tracheostomy for laryngeal cancer. Informed consent was obtained from all subjects. Exhaled breath condensate samples were collected using an Ecoscreen condensing chamber (Jaeger, Hoechberg, Germany). The exhaled air entered and left the chamber through one-way inlet and outlet valves, thus keeping the chamber closed. The subjects wore nose clips and breathed at tidal volumes (14 breaths per minute, guided by a metronome) through a mouthpiece connected to the condenser, for 10 minutes. Between 1 and 1.3 mL of condensate was collected per sample. One-mL aliquots of the sampled material were transferred to 15-mL tubes and stored at –70°C until lyophilization. Identical protein recoveries and electrophoretic patterns were obtained after concentration by trichloroacetic acid precipitation rather than by lyophilization. For one-dimensional electrophoresis, samples were reduced for 1 hour at 50°C with 0.5% 2-mercaptoethanol in pH 8.8 buffer, then carboxyamidomethylated by treatment with 100 mM iodoacetamide for 1 hour in the dark at room temperature before analysis on 4% to 20% polyacrylamide gradient gels. For two-dimensional electrophoresis, freeze-dried pellets were dissolved in 8 M urea and focused on a nonlinear 4-10 immobilized pH gradient in the presence of 8 M urea, then separated on polyacrylamide gels (19Gianazza E. Casting immobilized pH gradients.in: Walker J.M. The Protein Protocol Handbook. 2nd ed. Humana Press, Totowa, New Jersey2002: 169-180Crossref Google Scholar). Both anodic and cathodic sample applications were attempted; no spots were detected in the anodic samples, whereas results compatible with the banding pattern observed in one-dimensional sodium dodecyl sulfate-electrophoresis were obtained with the cathodic samples. For qualitative evaluation and mass spectrometry analysis, the gels were stained with silver nitrate (20Heukeshoven J. Dernick R. Neue ergebnisse zum mechanismus der silberfärbung.in: Radola B.J. Elektrophorese Forum '86. VCH, Weinheim, Germany1986: 22-27Google Scholar), with or without glutaraldehyde in the sensitization step and formaldehyde in the impregnation solution. Spot volumes were calculated using Image J 1.29x (W. Rasband, National Institutes of Health, Bethesda, Maryland) for one-dimensional gels, and with PDQUEST (Biorad, Hercules, California) for two-dimensional gels; in both cases, total spot volume was used for normalization. The correlation between clinical parameters and levels of exhaled keratin were evaluated using the Pearson test; P <0.05 was considered significant. In-gel trypsinolysis was performed using an Investigator Progest (Genomic Solutions, Huntingdon, United Kingdom) robotic digestion system (21Wait R. Gianazza E. Eberini I. et al.Proteins of rat serum, urine, and cerebrospinal fluid: VI. Further protein identifications and interstrain comparison.Electrophoresis. 2001; 22: 3043-3052Crossref PubMed Scopus (60) Google Scholar). The resulting mixtures of peptides were characterized by tandem electrospray high-performance liquid chromatographic mass spectrometry using a Q-TOF spectrometer interfaced with a Waters CapLC chromatograph (Waters, Manchester, United Kingdom). Uninterpreted tandem mass spectra were correlated to entries in SwissProt/TREMBL using ProteinLynx Global server (version 1.1, Waters) (22Wait R. Miller I. Eberini I. et al.Strategies for proteomics with incompletely characterized genomes the proteome of Bos taurus serum.Electrophoresis. 2002; 23: 3418-3427Crossref PubMed Scopus (90) Google Scholar). Candidate identifications were verified by manual interpretation of the spectra. Protein spots were detectable when breath condensates were analyzed by gel electrophoresis. To obtain enough protein for identification by mass spectrometry, condensates were pooled. This yielded 4-mL volumes from both the smoking and nonsmoking groups, which were analyzed by two-dimensional electrophoresis. The spots, labelled 1 through 5 (Figure A), from the smokers were excised, digested in gel with trypsin, and analyzed by tandem mass spectrometry. For spots number 2, 3, and 5, the best matches were to human keratins (Table). The pooled breath condensates from other groups of smokers and from the nonsmokers contained the same proteins, although the total spot intensity and relative abundance varied. Water controls processed identically in parallel with the breath condensates showed only trace amounts of stainable material. No spot corresponding to salivary amylase was observed in any of the analyzed pools, and zymography of selected condensate samples also failed to detect it.TableExhaled Breath Condensate Protein Identifications by Tandem Electrospray Mass Spectrometry in a Pooled Sample from 8 Healthy NonsmokersSpot No.*Spots 1, 4, 6, and 7 were unidentified; however, spot 1 appeared to migrate to the same position as human serum albumin.IdentityPeptides Sequenced by Tandem Mass Spectrometry2K1CI_HUMAN (keratin, type I cytoskeletal 9)TLLDIDNTRQGVDADINGLRFSSSSGYGGGSSRHGVQELEIELQSQLS KKNYSPYYNTIDDLKDQIVDLTVGNNK3K1CI_HUMAN (keratin, type I cytoskeletal 9)TLLDIDNTRFSSSSYGGGSSRHGVQELEIELQSQLSKHGVQELEIELQSQLSKK5K2CI_HUMAN, keratin, type II cytoskeletal 1SLVNLGGSKSLDLDSIIAEVKTNAENEFVTIKKSLNNQFASFIDKVRSLNNQFASFIDKVR(F)FSSCGGGGGSFGAGGGFGSRTHNLEPYFESFINNLRR* Spots 1, 4, 6, and 7 were unidentified; however, spot 1 appeared to migrate to the same position as human serum albumin. Open table in a new tab One-dimensional electrophoresis of exhaled breath condensate samples showed higher mean concentrations of proteins in smokers than in nonsmokers (Figure B). These are largely attributable to keratin isoforms. Mean (± SD) keratin spot volumes (in volume units) were 567 ± 673 in nonsmokers and 1981 ± 2241 in heavy smokers (P = 0.005). No correlation was found between keratin concentration and age (P = 0.99), pack-years of smoking (P = 0.93), or FEV1 (P = 0.37). The two samples obtained from the tracheostomized patients who had stopped smoking less than 3 years ago (lanes 37 and 38 in Figure B) contained large amounts of protein, with an identical banding pattern to the condensate collected from the smokers. We found that the protein composition of exhaled breath condensates from smokers contained more than three times as much keratin as did condensates from nonsmokers. In addition to tryptic peptide sequencing by mass spectrometry, the positions of three spots in the two-dimensional electrophoretic map were consistent with the isoelectric points and molecular masses of keratins. Using immobilized pH gradients, the standard deviation for the position parameters (i.e., the x and y coordinates in a two-dimensional electrophoresis map) was about 0.5 mm (23Gianazza E. Astrua-Testori S. Caccia P. et al.On the reproducibility of band position in electrophoretic separations.Electrophoresis. 1986; 7: 76-83Crossref Scopus (34) Google Scholar); in the absence of charged post-translational modifications, good agreement was obtained between isoelectric points computed from known protein sequences and amino acid compositions, and experimental values estimated from their migration behavior on two-dimensional gels (24Bjellqvist B. Hughes G.J. Pasquali C. et al.The focusing positions of polypeptides in immobilized pH gradients can be predicted from their amino acid sequences.Electrophoresis. 1993; 14: 1023-1031Crossref PubMed Scopus (830) Google Scholar). Our experiments were designed to exclude extraneous contamination. We used disposable parts and washed thoroughly between collection of samples. The possibility of subsequent contamination during the analytical procedures was checked by processing distilled water controls in parallel with each experiment. That keratin is the major component of the samples, rather than a background contaminant introduced during postelectrophoretic processing, is consistent with the correspondence between the positions of the spots on the two-dimensional gels and the calculated isoelectric points and molecular masses of cytokeratins. Moreover, extraneous keratin contamination is normally characterized by a complex mixture of isoforms from skin and hair, together with sequences of sheep origin derived from woollen clothing. The absence of oropharyngeal contaminants (i.e., amylase) was confirmed both by zymography and by the absence of spots corresponding to amylase spots on the gels (16Mutlu G.M. Garey K.W. Robbins R.A. et al.Collection and analysis of exhaled breath condensate in humans.Am J Respir Crit Care Med. 2001; 164: 731-737Crossref PubMed Scopus (272) Google Scholar, 25Ghafouri B. Tagesson C. Lindahl M. Mapping of proteins in human saliva using two-dimensional gel electrophoresis and peptide mass fingerprinting.Proteomics. 2003; 3: 1003-1015Crossref PubMed Scopus (120) Google Scholar). Possible contamination internal to the respiratory tract was tested by analyzing exhaled breath condensates collected via the trachea from tracheostomy patients. The high protein concentrations in these samples, together with the similarity of the protein pattern to that obtained by mouthpiece sampling, suggest that the upper respiratory tract makes little contribution to the protein content of the condensate. The keratin detected in exhaled breath condensate therefore appears to be of pulmonary origin. While the increased keratin content in the exhaled breath of smokers was striking, our study was not designed to determine whether these differences could provide a noninvasive method for identifying early changes in the pulmonary tract of smokers. Changes in specific cytokeratin expression by respiratory tract epithelial cells are correlated with progression from preneoplasia to neoplasia (26Fisseler-Eckhoff A. Erfkamp S. Muller K.M. Cytokeratin expression in preneoplastic lesions and early squamous cell carcinoma of the bronchi.Pathol Res Pract. 1996; 192: 552-559Crossref PubMed Scopus (18) Google Scholar); however, this could simply be a response to inflammation (27Nakamura H. Abe S. Shibata Y. et al.Elevated levels of cytokeratin 19 in the bronchoalveolar lavage fluid of patients with chronic airway inflammatory diseases a specific marker for bronchial epithelial injury.Am J Respir Crit Care Med. 1997; 155: 1217-1221Crossref PubMed Scopus (40) Google Scholar), attributable to smoke exposure (28Schlage W.K. Bulles H. Friedrichs D. et al.Cytokeratin expression patterns in the rat respiratory tract as markers of epithelial differentiation in inhalation toxicology. II. Changes in cytokeratin expression patterns following 8-day exposure to room-aged cigarette sidestream smoke.Toxicol Pathol. 1998; 26: 344-360Crossref PubMed Scopus (24) Google Scholar) among other causes. Assessing the quantitative meaning of our results is also difficult. The absolute amount of nonvolatile solutes in the condensates is determined by the number and size of aerosolized droplets of the fluid lining the lungs, while the concentration depends on the extent of dilution by gaseous water vapor (29Effros R.M. Hoagland K.W. Bosbous M. et al.Dilution of respiratory solutes in exhaled condensates.Am J Respir Crit Care Med. 2002; 165: 663-669Crossref PubMed Scopus (301) Google Scholar), which is largely a function of ventilation rate. The variation in the volume of exhaled breath condensate collected over a 10-minute period (1 to 1.3 mL) was far lower than the differences in the amount of keratin detected (at least threefold), suggesting that the controlled breathing rate during sampling resulted in exhalation of similar amounts of water vapor (and hence dilution) among subjects. However, it is possible that the increased mucus in the airways of smokers could cause greater turbulence, which could result in increased aerosolization of respiratory droplets, and thus higher yields of keratin in the condensates. Further investigation will be required to eliminate this possibility.