Disease Markers in Exhaled Breath: Basic Mechanisms and Clinical Applications (Nato: Life and Behavioural Sciences, 346)

DISEASE MARKERS IN EXHALED BREATH

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Series I: Life and Behavioural Sciences - Vol. 346

ISSN: 1566-7693

Disease Markers in Exhaled Breath Basic Mechanisms and Clinical Applications

Edited by

Nandor Marczin National Heart and Lung Institute, Imperial College, London, United Kingdom

and

Magdi H. Yacoub National Heart and Lung Institute, Imperial College, London, United Kingdom

/OS

Press Ohmsha

Amsterdam • Berlin • Oxford • Tokyo • Washington, DC Published in cooperation with NATO Scientific Affairs Division

Proceedings of the NATO Advanced Study Institute on Disease Markers in Exhaled Breath 22 June- 1 July 2001 Limin Hersonissou, Crete, Greece © 2002, IOS Press All rights reserved. No part of this book may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, without the prior written permission from the publisher. ISBN 1 58603 273 9 (IOS Press) ISBN 4 274 90532 2 C3045 (Ohmsha) Library of Congress Control Number: 2002109991

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Preface This monograph contains the contributions of invited speakers and participants at the NATO Advanced Study Institute on Disease Markers in Exhaled Breath: Basic Mechanisms and Clinical Applications, held at the Knossos Royal Village in Crete, Greece, June 22-July 1, 2001. This ASI was designed to summarise and disseminate expert knowledge regarding this rapidly evolving field of lung biology. Breath testing dates from the earliest history of medicine and puzzled brilliant scientific minds including Linus Pauling. It has provided us with powerful techniques of immense medical and commercial applicability such as capnography and ethanol breath testing. Despite this, a wider concept of breath analysis only recently moved from scientific curiosity to clinical reality. The major events responsible for promoting the current knowledge of exhaled breath testing relate to evolution of the idea that molecules such as nitric oxide (NO) and carbon monoxide (CO), previously viewed only as environmental toxicants, are naturally occurring compounds and play a principal role in the normal and pathological regulation of lung function. Furthermore, we have made tremendous progress in our understanding of the biochemistry and molecular biology of disease processes and bioproducts, which are either released from or consumed by the tissues resulting in changes in exhaled breath. Finally, advances in biomedical engineering now allow us to reliably and sensitively identify and quantify these molecules in the parts per billion concentration range and more than 3000 volatile organic compounds (VOCs) at picomolar concentrations. These developments hold enormous promise that analysis of exhaled breath could open a valuable new window onto human metabolism and illuminate its functions in health and disease. Accordingly, analysis of NO, CO and VOCs in exhaled breath has become a primary focus of respiratory research and essential aspect of investigations into many systemic diseases both in the laboratory and in the clinics. These activities are reflected in the rapid expansion of presentations at international meetings, scientific and clinical publications, editorials and recommendations by major respiratory societies. With the rapid expansion of this field, we felt it was timely to organise this institute and to purposefully bring together technical and basic mechanistic aspects of breath analysis through interaction of industry representatives and scientific investigators in order to explore further its potential from basic science to clinical practice. Accordingly, plenary lectures, short oral communications, small group tutorials, poster sessions, lively pro and contra debates and technical discussions were devoted to discuss basic mechanisms underlying production and release to the gas phase of the proposed major disease markers such as NO, CO and VOCs. The remaining daily lectures examined the mechanisms of altered regulation of these molecules and pathways and evidence for the recommendation to use these changes as biomarkers of the broad spectrum of clinical problems. These included acute and chronic lung inflammation with major emphasis on asthma and chronic obstructive pulmonary disease, acute lung injury such as occurring during thoracic organ transplantation and related end stage lung disease, mechanisms of acute and chronic rejection, ischaemia-reperfusion injury and systemic inflammation. It is concluded that NO, CO and VOCs are important molecular players in many of these conditions and analysis of these molecules in the exhaled breath might provide

important insights into pathogenesis, progression and therapeutic responses of patients suffering from these conditions. The tremendous success of the meeting reflects the tireless work of many people. We are especially thankful for the prominent Organising Committee, which consisted of internationally renowned leaders covering expertise on NO, CO and VOCs. The members of the Organising Committee, which included Peter J. Barnes (London), Augustine M.K. Choi (Pittsburgh), Serpil Erzurum (Cleveland), Sergei A. Kharitonov (London) and Charis Roussos (Athens), assisted by the selected Scientific Advisory Panel have been essential in setting the main objectives of the ASI and putting together a stellar cast of lecturers and eminent scientific program. We would like to take this opportunity to thank the members of the Local Organising Committe: Christina Gratsiou, Antonia Koutsoukou, Stylianos Orfanos and Andreas Papapetropoulos for all their hard work. We are also grateful for the help of our close colleagues, Tamas Kovesi and Marianna Imre throughout the meeting and for their editorial assistance. A very special thanks to Ruth Bundy-Marczin for her efficient and meticulous help and passionate support not only during but much before and after the conference. We are especially indebted to Lydia Argyropoulou for working day and night for making sure that every aspect of the organisation was executed smoothly. Finally the organisers would like to salute all speakers and participants for making this event so special and a landmark in the history of dialogues on exhaled breath markers. Nandor Marczin Magdi H. Yacoub

We wish to extend our sincere thanks and gratitude to the following companies for their generous support of this NATO ASI.

SPONSORS

THE SCIENTIFIC AFFAIRS DIVISION OF NATO, Brussels, Belgium ABBOTT LABORATORIES, Greece AIR LIQUIDS HELLAS, Greece AEROCRINE AB, Smidesvagen, Sweden ASTRAZENECA, Prague, Czech Republic BOEHRINGER INGELHEIM, Greece ERICH JAEGER GMBH, Hochberg, Germany GLAXO WELCOME (Allen Pharmaceuticals), Greece LOGAN RESEARCH LTD., Rochester, UK NOVARTIS (HELLAS) A.E.B.E., Athens, Greece PAPAPOSTOLOU, Greece RESPIRATORY RESEARCH INC, Charlottesville, VA, USA START PROMOTION, Milan, Italy SERVIER POLSKA, Warsaw, Poland THE THORAX FOUNDATION, Athens, Greece

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Participants Participantsofofthe theNATO NATOASI ASIononDisease DiseaseMarkers MarkersininExhaled ExhaledBreath, Breath,Greece, Greece,2001 2001

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List of Participants Ian Adcock Thoracic Medicine National Heart and Lung Institute, Imperial College. Dovehouse St London SW3 6LY UNITED KINGDOM [email protected] Christofer L. Adding, MD, PhD Division of Physiology Departement of Physiology and Pharmacology Von Eulers vag 6 171 77 Karolinska institute! Solna Stockholm SWEDEN T: 08 728 7226 F: 08 33 20 47 [email protected] Veronica Agrenius, MD PhD, Acting chief, Thoracic Medicine, Karolinska Hospital, 17176 Stockholm SWEDEN Ernst Altman Senior Researcher St Petersburg State University St Petersburg RUSSIAN FEDERATION altniaii@ea4603. spb.edu Adam Antczak MD Med. Univ. Lodz, POLAND T: +48 42 6787505 F:+48 42 6782129 [email protected] Zoe Athanasa, MD Resident and Clinical Fellow Asthma and Allergy Center, Pulmonary and Critical Care Department, Medical School, Athens University, Evgenidio Hospital, 20 Papadiarnantopoulou,! 1528, Athens, GREECE T: (301)7236743 F: (301)7242785

Fritz H. Bach, MD Lewis Thomas Professor Harvard Medical School 99 Brookline Ave. Ste. 370D Boston MA. 02215 USA T: (617) 632-1199 F:(617)632-1198 Fritzhbach @ aol .com Beatrix Balint MD Dept. of Thoracic Medicine University of Szeged Deszk, Alkotmany u 36. HUNGARY T:+36-62-271-411 F:+36-62-271-344 [email protected] George Baltopoulos Professor of Critical Care & Pulmonary Diseases Athens University School of Nursing Director, ICU at "KAT" Hospital of Kifissia 2 Nikis Street Kifissia 14561 Athens GREECE T: 00301-6218381 F: 00301-6218381 [email protected] Adam Barczyk Department of Pneumology Silesian Medical Academy ul. Medykow 14 40-752 Katowice POLAND F:+48-32-2523831 [email protected] Peter J Barnes Professor of Thoracic Medicine National Heart and Lung Institute, Imperial College, Dovehouse St London SW3 6LY UK T:0207 351 8174 F: 0207 351 5675 [email protected]

Gunther Becher FILT Lung and Chest Diagnostics Ltd. Robert-Rossle-Str. 10 Haus 79 (Erwin-Negelein-Haus) 13125 Berlin GERMANY T:+49-30-94892114 [email protected] Ion Belenis Head of Dept of Thoracic and Vascular Surgery, Evangelismos Hospital, Athens, GREECE T:+30-17224449 F:+30-17201506 [email protected] Michael J. Berry Ph.D Chief Scientist, Quadrivium LLC, P.O.Box 1421, Pebble Beach, CA 93953. USA T: (831)625-1177 F: (831) 647-1777. MBcrry717 @cs.com Jorge Boczkowski Unite INSERM 408 Faculte X. Bichat BP416 75870 Paris CEDEX 18 FRANCE T:33 1 44856251 F: 33 1 42263330 [email protected] Nigel Bough ton-Smith AstraZeneca R&D Charnwood Discovery BioSceince, Bakewell Road, Loughborough, Leics. L E i l 5RH, UK T:+44 (0)1509 644332 F:+44 (0)1509 645574 [email protected] Ruth Bundy Heart Science Centre Imperial College Harefield Hospital UB9 6JH, Middlesex UNITED KINGDOM T: 44-(0)l 895-823-737 ext 5044 F:44-(0) 1895-828-900 ruth, bundv@ harcfield.nthames.nhs.uk

John D. Catravas, Ph.D. Regents Professor and Director Vascular Biology Center Medical College of Georgia Augusta, GA 30912-2500 USA T: 706-721-6338

F: 706-721-9799 [email protected] Vladimir Cerny, MD, PhD, FCCM Charles University, Faculty of Medicine University Hospital Dept. of Anesthesiology and Intensive Care 500 05 Hradec Kralove CZECH REPUBLIC T: 42 49 583 ext. 2147 or 3218 F: 42 49 583 2022 [email protected] Augustine M.K. Choi, M.D. Professor of Medicine, Chief, Pulmonary, Allery and Critical Care Medicine University of Pittsburgh School of Medicine MUH 628 NW, 3459 Fifth Ave Pittsburgh, PA 15213 USA T: (412) 692-2117 F: (412) 692-2260 pager (412) 958-6054 choiam @ msx. upmc.cdu Alexander G. Chuchalin Academician, Director Institute of Pulmonology, 32/61, 11 -th Parkovaya Street Moscow 105077, RUSSIAN FEDERATION T: +7 095 465 5208 F: +7 095 465 5364 aisanov @ telemed.ru Eric Demoncheaux, PhD Division of Clinical Sciences (CSUH) Section of Medicine and Pharmacology Respiratory Medicine Unit University of Sheffield Medical School Floor F, Beech Hill Road Sheffield, S10 2RX, UK T: 44(0)114 271 2451 F: 44(0)114 271 1711 [email protected] Christiana Dimitropoulou, Ph.D. Department of Pharmacology and Toxicology Medical College of Georgia Augusta, GA 30912-2300 USA T: 706-721-7361 F: 706-721-2347 cd i [email protected]

Raed A. Dweik, M.D. Staff Physician Pulmonary and Critical Care Medicine Cleveland Clinic Foundation Cleveland, OH, 44195 USA T: 216-445-5763 P: 216-445-8160 [email protected] Serpil C. Erzurum, MD, FCCP Director, Lung Biology Program, Pulmonary and Critical Care Medicine, Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Avenue A90, Cleveland, Ohio 44195 USA T: (216)445-5764, F: (216)445-6624

Dr.Cristian Falup-Pecurariu Department of Neurology "Transilvania" University 2200 Brasov ROMANIA cristp@di'urocon.sult.n) Dr. Oana Falup-Pecurariu Assistant, Depart, of Pediatrics "Transylvania" University, Brasov, ROMANIA. [email protected] Gerasimos S. Filippatos MD Attending Cardiologist Evangelismos Hospital, 28 Doukissis Plakentias 1 1523 Athens GREECE Tel: 30-1-6745222 Fax: 30-1-6745222 geros@compulink,gr Bryan Flaherty, Ph.D. Vice President, Research and Development Natus Medical Inc. USA T: 650-801-7262 [email protected] Ben Gaston, MD. Division oi'Pediatric Respiratory Medicine Box 800386 University of Virginia Charlottesville, VA 22908 USA [email protected]

Nakos George MD, Director, ICU of University Hospital of loannina loannina GREECE T: + 30651 99353, F:+30 651 99279 [email protected] Gianfranco Giubileo ENEA, Laboratory of Molecular Spectroscopy Via E.Fermi 45, 00044-Frascati(RM), ITALY T: +39-06-94005768 F: +39-06-94005400 [email protected] Liza Goderdzishvili Department of Intensive Care Central Military Hospital Navtlugi St. No 6, 380013, Tbilisi, GEORGIA T: 995 32 962356 F:995 32958016 eli/.aveua [email protected] Christina Gratziou Head of Asthma and Allergy Center, Pulmonary and Critical Care Department, Medical School, Athens University Evgenidio Hospital, 20 Papadiamantopoulou,! 1528, Athens, GREECE T: (301)7236743 F: (301)7242785 [email protected] Lars E Gustafsson Professor Department of Physiology and Pharmacology Karolinska Institutet 171-77 Stockholm SWEDEN T: 46 8 728 7226 F: 46-8 332 047 [email protected] Nihal El Habashi. MD. Assistant Lecturer, Department of Physiology, Faculty of Medicine, Alexandria University EGYPT [email protected]

Tryggve Hemmingsson, Project leader Aerocrine AB Smidesvagen 12 S-171 41 SOLNA SWEDEN T: +46 8 629 07 80

F: +46 8 629 07 81 [email protected]

Timothy W Higenbottam, MD MA DSc FRCP Professor Global Clinical Expert, AstraZeneca R&D Charnwood, Clinical Sciences, Bakewell Road, Loughborough, Leics LEI 1 5RH, UK T:+44 (0)1509 644846 F:+44 (0)1509 645563 tim.higenbottam @ astrazeneca.com Markus Hofer MD Fellow, Lung Transplant Program CHOER 19 University Hospital CH-809! Zurich SWITZERLAND T:+41-1-255-1 1 1 1 , markus.holcM @DlM.us/..ch) Marieann Hogman, PhD, Dept of Medical Sciences, Uppsala University, SWEDEN maricann.hognian(°'mcdsci.uu.se

Jiri Homolka,M.D.,Ph.D. Assoc. Professor First Lung Department Foundation Katerinska 19 120 00 Prague 2 CZECH REPUBLIC T/F:+4 20-2-24941500 [email protected] Dr Adriana Hristea National Institute of Infectious Diseases "Prof dr. Matei Bals" 1 Dr. Grozovici Sect 2, 72204 Bucharest ROMANIA [email protected] Dr Razvan Hristea Institute of Aeronautical Medicine Str M.Vulcanescu nr 88 Bucharest. ROMANIA [email protected]

John Hunt, MD Division of Pediatric Respiratory Medicine Box 800386 University of Virginia Charlottesville. VA 22908 USA JFH2M@hscmail. nice, virginia.edu Marianna Imre, MD Assistant Professor Dept. Radiology Medical University of Pecs 7643 Pecs, Ifjusag u. 13 HUNGARY [email protected] Tamas Jilting, MD Assistant Professor Department of Pediatrics The Evanston Hospital Northwestern University Medical School 2650 Ridge Ave. Evanston, IL 60201 USA T: 847-570-1643 F: 847-570-0231 [email protected] Lena Kajland Wilen Marketing Director Aerocrine AB Smidesvagen 12 S-171 41 SOLNA SWEDEN T: +46 8 629 07 80 F:+46 8 629 07 81 [email protected] Miklos KeUermayer Professor, Director Dept.Clinical Chemistry Medical University of Pecs 7643 Pecs, Ifjusag u. 13 HUNGARY T: 36-72-536-122 F: 36-72-536-121 [email protected] Irina A. Kharitonova MD Department of Sleep Disorders, Centre of Medical Rehabilitation of the President of Russian Federation, Poselok Getsena, 143088 Moscow RUSSIAN FEDERATION.

Sergei A. Kharitonov, MD PhD Lecturer, Department of Thoracic Medicine, National Heart and Lung Institute, Imperial College of Science, Technology and Medicine, Royal Brompton & Harefield NHS Trust, Dovehouse Street, London SW3 6LY, UK T: +44 020 7352 8 1 2 1 pager 0025 F: +440207351 8126 [email protected] Anastasia Kotanidou Critical Care Department, Evangelismos General Hospital, Medical School, University of Athens, GREECE. akotanicKg'mcd.noa.gr Nick Koulouris MD. PhD. Senior Lecturer in Respiratory Medicine Respiratory Medicine Dept National University of Athens Medical School "Sotiria" Hospital for Diseases of the Chest 152 Mesogion Ave. Athens GR-11527

GREECE"

T: 0030 1 777 8827 F: 0030 1 777 0423 koulniCfl-'.vtre mc.gr Antonia Koutsoukou Critical Care Department, Evangelismos General Hospital, Medical School, University of Athens, GREECE. T: +30-1-7201913 F: +30-1-7244941 [email protected]

Andrew B. Lindstrom, Ph.D. Exposure Methods and Monitoring Branch (MD44) National Exposure Research Laboratory U.S. Environmental Protection Agency Research Triangle Park, NC 2771 1 USA T: 919-541-0551 F: 919-541-3527 [email protected] Ron Logan-Sinclair Director Logan Research Ltd., Unit B2, Spectrum Business Centre, Anthony's Way, Rochester Kent, ME2 4NP. UK. T:+44(0) 1634294900 F: 444(0) 1634294906 [email protected] Stelios Loukidis MD Chest Physician Head of Pneumonology Dept Athens Army General Hospital GREECE T:003018954603 F: 003017494095 ssat@hol.«r Katerina Marathias, M.D. Associate Director Intensive Care Unit Onassis Cardiac Surgery Center 356 Sygrou Ave, Athens GREECE T: (01) 9493000 Beeper #142 F: (01)9493331

v [email protected] Tamas Kovesi, MD Head of Anaesthetics Dept.Paediatrics Medical University of Pecs 7643 Pecs, Jo/.sel'A. u7 HUNGARY Tamas [email protected] Dan Laskovvski Cleveland Clinic Foundation, 9500 Euclid Avenue A90 Cleveland, Ohio 44195 USA T: (216)445-5764, F: (216)445-6624 [email protected] Marilcna Lekka Dep. Of Biochemistry, University of loannina, loannina, GREECE

Sadis Matalon, Ph.D. Associate Dean for Post Doctoral Education Alice McNeal Professor of Anesthesiology Professor of Physiology and Biophysics, Pediatrics, Comparative Medicine and Environmental Health, University of Alabama at Birmingham, 940 THT, 619 19th St. S, Birmingham, AL 35249-6810 USA T:(205)-934-4231 F: (205)-934-7437 [email protected]

Karen McRae, MDCM, FRCP Head of Thoracic Anaesthesia, Department of Anaesthesia, The Toronto General Hospital 585 University Ave Toronto, Ontario, M5G 2C4 CANADA T: I 4163405164 F: 1 4163403698 Karen. McraeCq:uhn.on.ca Wolfram Miekisch, PhD Dept. of" Anaesthesia and Intensive Care Med. University Hospital of Rostock, Schillingallee 35 18057 Rostock GERMANY T:+49-381-494 6114, F:+49-381-4946434 [email protected] Manfred Muertz, Ph.D. Institute of Laser Medicine Diisseldorf University, Medical Department GERMANY F:+49-211-811-1374 rnuerlz @ uni-duesscldorl.de

Andreas Papapetropoulos, PhD GP LIVANOS Research Laboratory Ploutarchou 3 10 675 Athens, GREECE T: (301) 721 7467 F: (301)721 94 17 [email protected] Lara Pizurki, PhD GP LIVANOS Research Laboratory Ploutarchou 3 10 675 Athens, GREECE T: (301) 721 7467 F: (301)721 94 17 lara [email protected] Mieczysiaw Pokorski Professor Dept. of Neurophysiology Medical Research Center ' Polish Academy of Sciences 5, Pawinskiego St. 02-106 Warsaw POLAND mpokorski@medres,cmdik. pan.pl

Pavlos Myrianthefs, MD Attending Physician Athens University School of Nursing, ICU at "KAT" Hospital, Nikis 2, Kifissia, 14561, Athens GREECE T: 00301/6280685 F: 00301/6280702 pavlos myrianlhcfs(fthotmail.com

Victoria PolyakovaPhD, Senior Researcher Institute of Human Ecological Pathology Vasylkivska Street, 45 Kiev, 03022 UKRAINE T: +380 44 266 98 03, 266 98 48 F: +380 44 227 66 13 victoria [email protected]

Lothar Neumann Produktmanagment Pneumologie ERICH JAEGER GmbH Leibnizstr. 7 D-97204 Hochberg GERMANY T:+49 931/4972-189 [email protected]

Cristina Popescu, MD, Specialist National Institute of Infectious Diseases "Prof dr. Matei Bals" 1 Dr.Grozovici Sect 2, 72204 Bucharest ROMANIA [email protected]

Stylianos Orfanos, MD., PhD Dept. of Critical Care University of Athens Medical School, Athens, GREECE T: 30-1-7201919 [email protected]

Gabriel A. Popescu, MD, PhD Senior Physician National Institute of Infectious Diseases "Prof dr. Matei Bals" I Dr.Grozovici Sect 2, 72204 Bucharest ROMANIA [email protected]

Alfred Priftanji Professor UHC "Mother Teresa"; 372, Dibra Sir. Tirana. ALBANIA. T/F: +355 4 224590 pri ftan ji («\san.corn.al Jacques Rami PhD Service d'exploration fonctionnelle respiratoire CHU de Rangueil-Larrey 1 Avenue Jean Poulhes, 31062 Toulouse Cedex, FRANCE T: 33.05.61.32.28.42 (Hospital) F: 33.05.62.88.90.75 (University) [email protected] Fabio Ricciardolo, MD, PhD Department of Respiratory Disease Ospedali Riuniti di Bergamo Largo Barozzi, 1 24100 Bergamo ITALY T: +39-035-269989 F: +39-035-266825 Trice iardoloCq'ospcdaliriuniti.bergarnu.it Terence H. Risby, Ph.D. Professor of Toxicological Sciences Department of Environmental Health Sciences Johns Hopkins University School of Hygiene and Public Health, 615 North Wolfe Street Baltimore MD 21205 USA T: (410)955-0024 F: (410) 955-0027. Pager: (410) 283-7078 [email protected] Giovanni Rolla, MD Clinical Investigator Dipartimento di Scienze Biomedtche e Oncologia Uinana, Universita di Torino Divisione di Immunologia Clinica e Allergologia Ospedale Mauriziano Umberto I largo Turati 62, 10128 Torino ITALY F: +39011 5682588 i>rolht@mauri/iano.ii

Charis Roussos MD, PhD Professor Critical Care Department. Evagelismos General Hospital 45-47 Ipsilandou Str. Athens 10675 GREECE T: 30-1-72-43-320 F: 30-1-72-16-503 croussos <$ cc.uoa.gr Nikoletta Rovina, M.D. Asthma and Allergy Center, Pulmonary and Critical Care Department, Evgenidio Hospital, University of Athens, Greece, 20, Papadiamantopoulou str. 115 28 Ilisia, Athens, GREECE T: (01)7236743, 7293407-8 F: (01)7242785 [email protected] David Royston Director of Research Consultant Anaesthetist Harefield Hospital UB9 6JH Middlesex UNITED KINGDOM T:44-(0)l895-823-737 [email protected] Mark H. Sanders, M.D. Professor of Medicine and Anesthesiology Chief, Pulmonary Sleep Disorders Program University of Pittsburgh School of Medicine 3459 Fifth Avenue, Suite S-643, Pittsburgh, PA 15213, USA T: 412-692-2880 F: 412-692-2888 Pager 412-393-9437 [email protected] Jochen Schubert Dept. of Anaesthesia and Intensive Care Med. University Hospital of Rostock, Schiliingallee 35 18057 Rostock GERMANY T:+49-381-494 6114, F: +49-3 81-4946434 jochen. schubcrl @mcdi/in.uni-rostock.de Jigme M. Sethi, M.D. Instructor in Medicine, Division of Pulmonary, Allergy and Critical Care Medicine University of Pittsburgh Medical Center 628.2 NW MUH, 3459 Fifth Avenue Pittsburgh, PA 15213 USA T: (412) 692-2210 F: (412)692-2260 [email protected]

Nicolaos M. Siafakas University General Hospital Dept. of Thoracic Medicine POBox 1352 GR-7lllOHeraklion GREECE T: 30-81-392-433 F: 30-81-542-650 sial'akfe 5 mcd.uoc.gr

Miklos Tekeres, MD, PhD Professor, Director Department of Anesthesiology and Intensive Therapy Medical University of Pecs Pecs, HUNGARY F: 36-1-319-1922 [email protected]

Nickolaj F. Starodub Professor, Head of Department of Sensoric and Regulatoric System of Institute of Biochemistry Ukrainian National Academy of Sciences 9 Leontovicha Str.,01030 Kyiv, UKRAINE T: 380-44-229 47 43 F: 380-44-229 63 65

John Timbrell Professor of Biochemical Toxicology (Editor, Biomarkers) Pharmacy Dept. King's College London, Franklin Wilkins Building, Stamford Street London SE1 8WA UK T/F: 020 7848 4789/4881 John.tiinbreIKnfkcl.ac.uk

nstarod lib (g> holinail .com

Werner Steinhauser Marketing Manager Produktmanagment Pneumologie ERICH JAEGER GmbH Leibnizstr. 7 D-97204 Hochberg GERMANY T: +49 93174972-319 DS @jaeger-toennies.com Gregory Stratakos Dept. of Critical Care, University of Athens Medical School, Athens, GREECE [email protected] Steven Sunshine, Ph.D. CEO Cyrano Sciences, Inc. Pasadena, CA USA T: (626) 744-1700x226 http. //www.cvranosciences.com Camille Taille INSERM U408 Faculte de Medecine Xavier Bichat 16, rue Henri Huchard 75018-Pans FRANCE T: (33) 1 44.85.62.48 F: (33) I 42.26.33.30 [email protected]

Miroslava Tilkian Department of Anaesthesia and Critical Care Plovdiv 4000 "Leonardo da Vinchi" str.66 BULGARIA F:+35932238107 miratil(°) vahoo.co.uk Tatiana Tkacheva, PhD Senior Researcher, Toxicology department Institute of Occupational Health of Russian Academy of Medical Sciences Prosp.Budennogo, 31 Moscow 105275, RUSSIAN FEDERATION T: +7 095 365 1000 F: +7 095 366 0583 latkachcva<3'mtu-nct.ru John Vaughan Division of Pediatric Respiratory Medicine Box 800386 University of Virginia Charlottesville, VA 22908 USA j w v4c (£ Virginia.EDU Gavin Wright Consultant Anaesthetist Harefield Hospital UB9 6JH Middlesex UNITED KINGDOM T:44-(0) 1895-823-737

Magdi Yacoub, FRS British Heart Foundation Professor of Cardiothoracic Surgery National Heart and Lung Institute, Imperial College, Royal Brornpton and Hare field NHS Trust, Harefield Hospital. Harefield, UK UNITED KINGDOM T: 44-1895-823-737 F: 44-1X95-828-901 Carnal Abdel Naser Yamamah Assistant Professor of Pediatrics Department of Clinical Medical Sciences National Research Center, El Tahrir St. Gi/a, EGYPT [email protected]

Spyros Zakynthinos Dep of Critical Care, University of Athens Medical School, Athens, GREECE

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Contents Preface Sponsors List of Participants

v vii xi

Part I. Introduction to Disease Markers in Exhaled Breath Section 1. Nitric Oxide Regulation of Nitric Oxide Synthases in the Lung, Serpil C. Erzurum Nitric Oxide Production in the Lung and its Regulation by Oxygen, RaedA. Dweik S-Nitrosothiols in Respiratory Biology, Benjamin Gaston Inhibitors of Nitric Oxide Synthase: Experimental Findings and Clinical Implications, Nigel K. Boughton-Smith Inhaled NO as a Replacement Therapy, George J. Baltopoulos and Pavlos M. Myrianthefs Physiology of Exhaled Nitric Oxide, L. Christofer Adding and Lars E. Gustafsson The "Vascular" Origins of Exhaled Nitric Oxide, Tim Higenbottam and Eric Demoncheaux Exhaled NO originates in Airway Epithelium, L. Christofer Adding and Lars Gustafsson NO is Generated via NOS Enzymes, Sergei A. Kharitonov Technical Aspects of Exhaled NO: Investigator Point of View, Sergei A. Kharitonov Technical Aspects of Exhaled Nitric Oxide: Aerocrine Point of View, Lena Kajland-Wilen Section 2. Carbon Monoxide Cytoprotection by Heme Oxygenase / CO in the Lung, Judit K. Sarady, Leo E. Otterbein and Augustine M.K. Choi Role of Heme Oxygenase in Airway Smooth Muscle Contractility, Camille Taille, Michel Aubier and Jorge Boczkowski Exhaled Carbon Monoxide is Produced in the Lungs, Jigme M. Sethi Exhaled Carbon Monoxide is Delivered from Systemic Sources, Nandor Marczin Kinetics of Carbon Monoxide Accumulation in Exhaled Breath, RaedA. Dweik ETCOc: An Indicator of Hemolysis in Neonatal Hyperbilirubinemia, Molly McCarthy and Judith Hall Section 3. Volatile Organic Compounds (VOCs) Volatile Organic Compounds as Exposure Markers, Andrew B. Lindstrom and Joachim D. Pleil Volatile Organic Compounds as Markers in Normal and Diseased States, Terence H. Risbv

3 11 18 24 30 35 49 52 51 62 64

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Section 4. The "Living State" The Living State: Intimate Insights through Personal Discoveries, Miklos Kellermayer

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Part II. Asthma Biology of Asthma, Peter J. Barnes Transcriptional Regulation of Airway Inflammation, Serpil C. Erzurum Molecular Mechanisms of Steroid Actions, Ian M. Adcock and Kazuhiro Ito Nitric Oxide Reactions in the Asthmatic Airway, RaedA. Dweik Regulation of pH in the Human Airway: Mechanisms and Monitoring, John F. Hunt Markers in Exhaled Air and Condensate to Monitor Treatment in Asthma, Sergei A. Kharitonov Extended NO Analysis Applied to Patients with Known Altered Values of Exhaled NO, Marieann Hogman Exhaled Nitric Oxide and Atopy, Christina Gratziou Bradykinin and Exhaled Nitric Oxide in Asthma, Fabio L.M. Ricciardolo and Peter J. Sterk Exhaled NO is an Optimal Marker of Severity and Responsiveness to Therapy in Asthma, Serpil C. Erzurum Superoxide-NO Interactions in Paranasal Sinus Inflammatory Diseases, Jacques Rami

\ 33 143 151 159 167 177 \ 87 191 196 199 202

Part III. Chronic Lung Diseases Biology, Diagnosis and Management of COPD, N.M. Siafakas, G. Chrysofakis and N. Tzanakis ' Disease Markers in COPD: Exhaled Breath vs. Exhaled Condensate, Sergei A. Kharitonov The Biology of Cystic Fibrosis, Tamas Jilling Exhaled Markers in Cystic Fibrosis, Beatrix Balint, Sergei A. Kharitonov, lldiko Horvdth and Peter J. Barnes Lung Cancer Screening by Breath Analysis, Michael J. Berry Nitric Oxide and Rheumatic Diseases, Giovanni Rolla Nitric Oxide in Hepatopulmonary Syndrome, Giovanni Rolla Pathological Changes in the Airways Epithelium of Liquidators of the Chernobyl Catastrophe, Victoria Polyakova

209 218 223 234 242 246 257 261

Part IV. Transplantation Heme Oxygenase-1 and/or Carbon Monoxide can Promote Organ Graft Survival, Miguel P. Soares, Lukas Guenther, Pascal Berberat and Fritz H. Bach Mechanisms of and Clinical Efforts to Minimise Perioperative Lung Injury, /. Gavin Wright and Nandor Marczin Condensate Inflammatory Markers in Lung Transplantation, Karen McRae. Exhaled Nitric Oxide (NO) in Human Lung Ischemia-Reperfusion, Nandor Marczin In Situ Lung Autotransplantation Model in Pigs, E.N. Koletsis, A. Chatzimichalis, K. Kokinis, V. Fotopoulos, I. Bellenis and D. Dougenis

267 274 284 291 300

Pathogenetic Mechanisms Leading to Obliterative Bronchiolitis, Erkki A. Kallio, Jussi M. Tikkanen, Petri K. Koskinen, Karl B. Lemstrom and Magdi Yacoub

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Part V. ARDS and Oxidative Stress Clinical Pathology of ARD$,Stylianos E. Orfanos, Antonia Koutsoukou, Irene Mavrommati, Ekaterini Psevdi, loanna Korovesi, Anastasia Kotanidou and Charts Roussos Markers of Oxidative Stress in Exhaled Breath Condensate, Adam Antczak Volatile Organic Compounds as Prognostic Markers in ARDS, J.K. Schubert, W. Miekisch, G.F.E. Noldge-Schomburg Do Reactive Oxygen-Nitrogen Intermediates Contribute to the Pathogenesis of ARDS? Judy M. Hickman-Davis, Ian C. Davis, Phillip O'Reilly, Philip McArdle and Sadis Matalon Modulation of Active Alveolar Na+ Transport by Reactive Oxygen-Nitrogen Intermediates, Karin M. Hardiman, Ahmed Lazrak, Vance Nielsen and Sadis Matalon Luteolin Reduces LPS-Induced TNF-a Production and Protects Mice against LPS Toxicity, Angeliki Xagorari, Anastasia Kotanidou, Charis Roussos and Andreas Papapetropoulos Acute Lung Injury in the Setting of Cardiopulmonary Bypass, David Royston Oxidative Stress During Cardiac Surgery in Diabetic Patients, Tamds Kovesi, Ruth Bundy, Ginette Hoare, David Royston and Nandor Marczin

319 333 338

344

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Part VI. New Technological Developments Exhaled Breath Condensate, Gunther Becher Smell as a Diagnostic Tool in the 21st Century: The Portable Electronic Nose, Timothy E. Burch and Steven A. Sunshine Biosensors and Express Biochemical Diagnostics of Some Diseases, Nickolaj F. Starodub, Andrew V. Rebriev and Valentyna M. Starodub Infrared Laser Spectrometer for Real-Time Analysis of Ethane in Exhaled Human Breath, Manfred Miirtz, Hannes Dahnke, Sandra Stry and Peter Hering Methods of Tuneable Diode Laser Absorption Spectroscopy Applied to the Analysis of Exhaled Breath, Gianfranco Giubileo

383 387 391 395 400

Part VII. Industrial Forum Industrial Forum - Aerocrine AB, Tryggve Hemmingsson Biochemical Diagnostics of the Lung with ECoScreen™, L, Neumann and W. Steinhaeusser Portability and Flexibility in Exhaled Breath Condensate Collection, John W. Vaughan

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Author Index

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Part I. Introduction to Disease Markers in Exhaled Breath Section 1. Nitric Oxide

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Disease Markers in Exhaled Breath N. Marciin and M.H. Yacoub (Eds.) /OS Press, 2002

Regulation of Nitric Oxide Synthases in the Lung Serpil C. ERZURUM Pulmonary and Critical Care Medicine Cancer Biology Lerner Research Institute Cleveland Clinic Foundation Cleveland Ohio 44195 U.S.A. Abstract. Exhaled nitric oxide (NO) is derived from endogenous NO synthesis in the lung by nitric oxide synthases (NOS) which convert L-arginine to Lcitrulline and NO. Three NOS isoforms (1 - 3 ) have been identified in the human lung. NOS1 and 3 are dependent on increases in intracellular calcium for enzyme activation while NOS2 is calcium independent. Ail NOS isoforms require oxygen, NADPH, FAD, FMN, tetrahydrobiopterin, and calmodulin for activity. Airway NOS2 expression is responsible for increased exhaled NO in asthma. NOS2 is upregulated by inflammatory cytokines, and produces high levels of NO. Localization of NOS1-3 and regulation of NOS2 is reviewed.

1. Nitric Oxide Synthases Since the finding that endothelium derived relaxing factor (EDRF) is nitric oxide (NO) [1, 2], NO has been shown to be a multifunctional molecule that mediates a number of physiologic processes in almost all vertebrate organ systems, including diverse functions such as smooth muscle relaxation, platelet inhibition, central and autonomic neurotransmission, tumor cell lysis, bacterial killing, and stimulation of hormonal release [3-6]. NO is a diffusible gas that is produced by a group of enzymes known collectively as nitric oxide synthases [NOS, EC 1.14.13.39]. These enzymes convert the amino acid L-arginine to NO and L-citrulline in the presence of oxygen and NADPH as cosubstrates, a reaction that requires several cofactors including FAD, FMN, tetrahydrobiopterin and calmodulin [7]. NO is relatively unstable and rapidly oxidized in solution to the stable metabolic end-products nitrite (NOz") and nitrate (NOs"), which can be used as indirect markers to monitor NO formation [7]. Three isoforms of NOS, which are the products of individual genes [7, 8], have been identified, including 2 constitutive forms [neuronal (nNOS or NOS 1), and endothelial (eNOS or NOS 3) and an inducible form (iNOS or NOS 2) [3, 5, 7]. In general, NOS 1 and 3 are continuously expressed in endothelial and neuronal cells respectively, and are dependent on increases in intracellular calcium to bind calmodulin which results in enzyme activation leading to picomolar levels of NO production [7]. NOS 1 is also found at relatively abundant levels in skeletal muscle [9|.

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NOS 2 is regulated at the level of transcription and mRNA stability and is expressed after exposure of cells to specific cytokines and endotoxin [4, 5, 7, 10]. NOS 2 is calcium independent, avidly binding calmodulin even at low calcium concentrations intracellularly, and produces nanomolar levels of NO [7]. NOS 2 has been described in epithelial, endothelial, smooth muscle cells, macrophages, fibroblasts, and neutrophils. Regulation of expression of NOS 2 varies in different cell types, but typically is increased by cytokines such as TNFa, interferon y (IFNy), and interleukin-1 p. NOS 2 expression is decreased by glucocorticoids, transforming growth factor beta (TGFp), platelet derived growth factor, epidermal growth factor, insulin-like growth factor 1, and thrombin [5. 1113]. Regulation of NOS 3 at the level of transcription is less well studied, but the gene is induced by oxygen tension, shear stress, and IFNy [11, 12, 14, 15]. Both NOS 1 and 3 mRNA are increased by estrogens. NOS 3, unlike 1 and 2, is membrane associated due to myristoylation of its amino-terminus end [8]. 2. NO in the lung NO is produced in the human lung, evidenced by NO detectable in the exhaled air of humans (6-8 ppb) and NO metabolites detectable in the airway aspirate and bronchoalveolar lavage fluid from human lungs [3, 16]. NO is recognized to play key roles in virtually all aspects of lung biology and has been implicated in the pathophysiology of lung diseases [17]. NO in the lung is involved in pulmonary neurotransmission, host defense and bacteriostasis, airway and vascular smooth muscle relaxation, pulmonary capillary leak, inflammation, mucociliary clearance, airway mucus secretion and cytotoxicity [3-5]. Cellular sources of NO in the lung include epithelial cells, endothelial cells of pulmonary arteries and veins, inhibitory nonadrenergic noncholinergic neurons, smooth muscle cells, mast cells, mesothelial cells, fibroblasts, neutrophils, lymphocytes, and macrophages [18]. All three NOS isoforms are present in the human lung [3, 18, 19]. Specifically, NOS 1 is located in inhibitory nonadrenergic noncholinergic neurons in the lung, while NOS 3 is found in endothelial cells and the brush border of ciliated epithelial cells [18]. NOS 2 is found in the epithelial cells of the airway [6, 10, 18, 19]. Although NOS 2 may be induced in several types of cells in response to cytokines, endotoxin, or reactive oxygen species, NOS 2 is continuously expressed in normal human airway epithelium at basal airway conditions [19]. Once produced, NO is freely diffusible and enters target cells activating soluble guanylate cyclase to produce guanosine 3', 5'-cyclic monophosphate (cGMP) which mediates the majority of NO effects [7]. NO also diffuses into the airway and can be measured in the gas phase [16]. Potential anatomic sources of NO in exhaled breath include the pulmonary circulation, the lower airways, and the upper airways and paranasal sinuses. NO is formed in high concentrations in the upper respiratory tract (nasopharynx and paranasal sinuses), but several studies have conclusively demonstrated that NO is also produced in the lower respiratory tract [16, 19].

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3. Alterations of exhaled NO in lung diseases Alterations in NO levels have been found in pulmonary diseases such as asthma, pulmonary hypertension, bronchiectasis, cystic fibrosis and interstitial lung disease [3]. Exhaled NO is increased in inflammatory airway diseases, such as asthma and upper respiratory tract infections/bronchiectasis, and reflects an increase in NOS 2 activity in airway epithelial cells [3, 17]. Exhaled NO levels are also higher in women with lymphangioleiomyomatosis (LAM) than healthy women, and is related to increased NOS 3 expresion in lesional smooth muscle in the lung [20]. Exhaled NO levels are lower than normal in smoking individuals and the reduction is proportionate to the number of cigarettes currently smoked. Cigarette smoke contains high levels of NO [17], which may dysregulate NOS activity and contribute to the lung injury caused by smoking [21]. There is also evidence that patients with primary pulmonary hypertension (PPH) have lower levels of expression of NOS 3 in pulmonary vascular endothelial cells, and lower levels of exhaled NO [22, 23]. 4. Posttranslational mechanisms regulating NO synthesis Studies identify transcriptional and posttranslational mechanisms regulating NO synthesis in the human airway. NO biosynthesis is regulated at multiple levels in cells, i.e. NOS gene transcription, mRNA processing, protein expression and dimerization, and enzyme reaction kinetics [7]. NO synthesis is dependent upon post-translational modifications to generate active NOS. Specifically, NOSs are synthesized as monomers and must dimerize to generate NO [7]. Recently deletion of regions critical for NOS dimerization due to alternative splicing of the NOS 2 mRNA have been identified [24]. In tissue culture cells, NOS 2 induction by cytokines and endotoxin results in an increase in both constitutively and alternatively spliced mRNA transcripts [24, 25]. Furthermore, enzyme catalyzed NO synthesis involves hydroxylation of arginine to generate N-hydroxyarginine, an enzyme-bound intermediate, which is then converted to citrulline. Intracellular concentration of arginine (several hundred DM) [26 - 29] has been reported to far exceed the Km of the NO synthases (5 - 10 DM) [29]. In this context, it would seem unlikely that arginine is ever rate limiting to the enzyme. However, arginine administration drives NO synthesis in vivo and in cell culture systems [26 - 29]. Independent of substrate effects, arginine may regulate enzyme reaction kinetics through effects on enzyme dimerization or influences on the reduction potential of the enzyme [7]. The kinetics of NO production by NOS 2 in activated macrophages over a range of arginine concentrations reveal a Km for arginine in intact cells of 73 to 150 DM [30, 31]. Intracellular arginine can be increased by de novo synthesis through regeneration from citrulline, or transport from extracellular sources [27 - 29]. Arginine synthetic pathways and transporter systems are induced coordinately with NOS 2 induction in cell cultures. Argininosuccinate synthetase, the rate limiting enzyme in the synthesis of arginine, is induced by endotoxin and IFNy, suppressed by corticosteroids. and generally mirrors NOS induction in vitro [32]. Arginine is present in healthy control airway epithelial cells, but are increased over 3-fold in asthmatic epithelial cells

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suggesting coordinate induction of the arginine synthetic pathways and/or cationic ami no acid transporters to support a high rate of NO synthesis in asthma [33]. 5. Transcriptional regulation of NOS 2 Although translational and post-translational mechanisms are important in the regulation of NO synthesis, NOS 2 is subject to predominantly transcriptional regulation [7, 10, 33]. Healthy human airway epithelium in vivo expresses the NOS 2 gene continuously at abundant mRNA levels [10, 19]. The human NOS2 gene is actively transcribed in airway epithelial cells in vivo using run-on transcription analyses [33]. Transcription of the NOS2 gene is at 15% of the transcription rate of yactin, an abundantly expressed mRNA in the airway epithelium [33]. NOS 2 mRNA expression in asthmatic airway epithelium is higher than controls in vivo, but not increased in asthmatics receiving inhaled corticosteroid. Several studies have shown that inhaled or intravenous corticosteroids reduce exhaled NO. In situ analysis of the asthmatic airway suggested that NOS 2 expression is reduced by corticosteroids [34]. In general, mechanisms by which corticosteroids regulate NOS 2 gene expression in vivo are not known. In vitro. glucocorticoids inhibit NOS 2 expression at multiple levels including inhibition of gene transcription, reduction of mRNA translation and increased degradation of NOS 2 protein [35 - 37]. Increased NOS 2 mRNA in asthma, which is downregulated by corticosteroid, supports an association between NOS 2 expression and airway inflammation. Furthermore, although NOS 2 is continuously expressed in airway cells in vivo. expression in control and asthmatic airway epithelial cells placed ex vivo is lost, which substantiates a critical link between airway conditions and/or factors in vivo and NOS 2 expression [10]. Induction of the NOS 2 gene in vitro The molecular basis for induction of the human NOS 2 gene is only partially understood [35, 38, 39]. In contrast, regions in the murine macrophage NOS 2 promotor essential for conferring inducibility of NOS 2 to LPS and IFNyhave been well defined [40, 41]. A nuclear factor kappa B (NF-DB) element at positions -76 to -85 bp relative to the transcription start point binds members of the NF-DB/Rel family of proteins in response to LPS [42], and further upstream an IFN-stimulated response element site binds interferon regulatory factor 1 (IRF-1) upon stimulation of murine macrophage cell line (RAW 264.7 cells) with IFNy [43]. Originally identified as a transcriptional activator of IFN-p as well as IFN inducible genes [44], IRF-1 is essential for NOS2 activation in murine macrophages [43, 45]. Studies suggest that IRF-1 is also important in human NOS 2 gene expression [46, 47]. NOS 2 expression parallels IRF-1 expression, with IRF-1 expression preceding NOS 2 mRNA accumulation. Induction of NOS 2 expression varies in different human cell types, but typically is increased by cytokines [5, 10, 38, 39]. In general, cytokines induce NOS 2 in human cell lines in vitro through transcriptional up-regulation of the gene [35, 38, 39]. The majority of studies on the regulation of NOS 2 expression in lung cells are based on the human lung cancer cell line, A549. For example, the combination of IL-1, TNF and IFNy

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1

leads to NOS2 induction in the A549 [10]. In particular, regulation of NOS2 gene expression in A549 requires activation of activator protein-1 (AP-1), which is comprised of c-fos and c-jun proteins [38]. Other studies suggest that NFkB activation and binding to kB DNA elements in the 5' flanking region of the NOS 2 gene plays a role in the cytokine induction of NOS 2 in A549 in vitro [39]. Induction of NOS2 expression in cell lines derived from transformed normal human bronchial epithelial cells, BEAS-2B and BET-1A require multiple cytokines and mediators to activate multiple intracellular signal transduction pathways, including AP-1, and signal transducer and activator of transcription 1 (STAT-1) and/or IRF-1 [46]. Thus, coordinate activation of multiple signaling pathways is necessary for lung cell lines to express NOS2. In contrast, IFNy alone is essential and sufficient for induction of NOS 2 gene expression in primary human airway epithelial cells in vitro [10, 47]. IFNy signaling IFNy signaling to gene expression begins with a specific receptor interaction and oligomerization of receptor chains. This causes a tyrosine phosphorylation cascade, which involves activation of Janus kinases (Jak) 1 and 2, which in turn activate STAT-1. STAT-1 phosphorylation, dimerization and translocation to the nucleus, is followed by binding to regulatory DNA elements to activate transcription of interferon-stimulatedgenes [48]. IFNy leads to STAT1 activation in primary human airway epithelial cells in culture [10, 33, 46, 47], and tyrosine kinase inhibitor abolishes induction of NOS 2 in airway epithelial cells [33]. Activation of signal transducers in vivo important for NOS 2 gene expression Recently, STAT1 activation has been demonstrated in the asthmatic airway by nuclear localization of STAT1 in airway epithelial cells, and demonstration of phosphorylation of STAT1 by western analyses of epithelial cell lysates [49]. The STAT1 activation correlated with induction of IFNy/STATl-stimulated-genes, including IRF-1 which is essential for NOS 2 activation in murine macrophages. Similarly, STAT1 activation quantitated by electrophoretic mobility shift assays is present in controls but increased in asthmatic airway epithelial cell lysates [33]. These data provide strong support for STAT1 activation mediating NOS 2 gene expression in human airway epithelial cells in vivo. 6, Conclusions Multiple mechanisms function coordinately to support NO synthesis in healthy airways, and high level NO synthesis in the asthmatic airway. Human airway epithelium has abundant expression of NOS 2 due to continuous transcriptional activation of the gene in vivo. Increased NOS 2 gene expression in asthmatic airways is associated with increased STAT1 activation, perhaps related to increased cytokines e.g., IFNy. High levels of intracellular arginine may enhance enzyme reaction kinetics and drive NO synthesis.

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Taken together, airway epithelial cells have highly efficient NO synthetic machinery which are amplified in airway inflammation. References [I] R.M.J. Palmer, A.G. Gerrige, and S. Moncada. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature 327( 1987)524-526. [2] L.J. Ignarro, G.M. Buga, K.S. Wood, R. Byrnes, and G. Chaudhuri. Endothelium derived relaxing factor produced and released from artery and vein is nitric oxide. Proc Nail Acad of Sci USA 84 (1987)9265. [3] B. Gaston, J.M. Drazen, J. Loscalzo, and J.S. Stamler. The biology of nitrogen oxides in the airways. Am J Respir Crit Care Med 149 (1994) 538. [4] H.H.H. Schmidt, and U. Walter. NO at work. Cell 78 (1994)919. [5] C. Nathan. Nitric oxide as a secretory product of mammalian cells. FASEB J 6 (1992) 3051. [6] F.H. Guo and S.C. Erzurum. Regulation of inducible nitric oxide synthase in human airway epithelium. Environmental Health Perspectives 106 (1998) 1119-1124. [7] D.J. Stuehr and O.W. Griffith. Mammalian nitric oxide synthases. Adv Enzymol Relat Areas Mol Biol 65(1992)287-346. [8] C. Nathan, and Q. Xie. Nitric oxide synthases: Roles, tolls, and controls. Cell 78 (1994) 915. [9] U. Frandsen, M. Lopez-Figueroa, and Y. Hellsten. Localization of nitric oxide synthase in human skeletal muscle. Biochem Biophys Res Commun 227 (1996) 88-93. [10] F. H. Guo, K. Uetani, S. J. Haque, B. R. G. Williams, R. A. Dweik, F. B. J. M. Thunnissen, W. Calhoun, and S. C. Erzurum. Interferon y and interleukin 4 stimulate prolonged expression of inducible nitric oxide synthase in human airway epithelium through synthesis of soluble mediators. J. Clin. Invest. 100 (1997) 829. [ I I ] B. Gaston, and J.S. Stamler. Nitrogen Oxides. In: Cyrstal RG, West JB eds. The Lung: Scientific Foundations 2d ed. Philadelphia:Lippincott-Raven, 1997; 239-253. [12] C. Nathan, and Q. Xie. Regulation of biosynthesis of nitric oxide. J Biol Chem. 269 (1994) 13725. [13] K. Asano, C.B.E. Chee, B. Gaston, C.M. Lilly, C. Gerard, J.M. Drazen, and J.S. Stamler. Constitutive and inducible nitric oxide synthase gene expression, regulation, and activity in human lung epithelial cells. Proc Natl Acad Sci USA 91 (1994) 10089. [14] N. Kim, Y. Vardi, H. Padma-Nathan, J. Daley, I. Goldstein, and I. Saenez de Tejeda. Oxygen tension regulates the nitric oxide pathway. J Clin Invest 91 (1993) 437. [15] P.W. Shaul, and L.B. Wells. Oxygen modulates nitric oxide production selectively in fetal pulmonary endothelial cells. Am J Respir Cell Mol Biol 11(1994) 432. [16] R.A. Dweik, D. Laskowski, H.M. Abu-Soud, F.T. Kaneko, R. Hutte, D.J. Stuehr, and S.C. Erzurum: Nitric oxide synthesis in the lung: regulation by oxygen through a kinetic mechanism. J Clin Invest 101(1998)660. [17] P. J. Barnes, and M.G. Belvisi. Nitric oxide and lung disease. Thorax 48 (1993)1034. [18] L. Kobzik, D.S. Bredt, C.J. Lowenstein, J. Drazen, B. Gaston, D. Sugarbaker, and J.S. Stamler. Nitric oxide synthase in human and rat lung: immunohistochemical and histochemical localization. Am J. Respir. Cell. Mol. Biol. 9 (1993)371. [19] F. H. Guo, H. R. De Raeve, T. W. Rice, D. J. Stuehr, F. B. J. M. Thunnissen, and S. C. Erzurum. Continuous nitic oxide synthesis by inducible nitric oxide synthase in normal human airway epithelium in vivo. Proc. Natl. Acad. Sci. USA. 92 (1995) 7809. [20] R.A. Dweik, D. Laskowski, M. Ozkan, C. Farver, and S.C. Erzurum. High Levels of Exhaled Nitric Oxide (NO) Associated with NO Synthase HI Expression in Lesional Smooth Muscle in Lymphangioleiomyomatosis. Am J Resp Cell Mol Biol 24 (2001)414-418. [21] H. Ischiropoulos, M. Ignacio, D. Fisher, A.B. Fisher, and S.R. Thorn. Role of neutrophils and nitric oxide in lung alveolar injury from smoke inhalation. Am J Respir Crit Care Med 150 (1994) 337. [22] A. Giaid, and D. Saleh. Reduced expression of endothelial nitric oxide synthase in patients with pulmonary hypertension. N Engl J Med 333 (1995) 214-221. [23] F.T. Kaneko, Arroliga, A.C., Dweik, R.A., Comhair, S.A., Laskowski, D., Oppedisano, R., Thomassen, M.J., and S.C. Erzurum. Correlation of nitric oxide reaction products to severity of pulmonary hypertension. Am J Respir Crit Care Med. 158 (1998) 917-923.

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[24] N. T. Eissa, J. W. Yuan, C. M. Haggerty, E. K. Choo, C. D. Palmer, et al. Cloning and characterization of human inducible nitric oxide synthase splice variants: A domain, encoded by exons 8 and 9, is critical for dimerization. Proc. Nad. Acad. Sci. USA. 95 (1998) 7625. [25] N. T. Eissa, A. J. Strauss, C. M. Haggerty, E. K. Choo, S. C, Chu, and J. Moss. Alternative splicing of human inducible nitric-oxide synthase mRNA. J. Bioi. Chem. 271 (1996) 27184. [26] S. Kurz, and D. G. Harrison. Insulin and the arginine paradox../ Clin. Invest. 99 (1997) 369. [27] M. Hecker, W. C. Sessa, H. J. Harris, E. E. Anggard, and J. R. Vane. The metabolism of L-arginine and its significance for the biosynthesis of endothelium-derived relaxing factor: Cultured endotheliai cells recycle L-citrulline to L-arginine. Proc. Natl. Acad. Sci. USA. 87 (1990) 8612. [28] R. Hammermann, J. Hirschmann, C. Hey, J. Mossner, G. Folkerts, et al. Cationic proteins inhibit Larginine uptake in rat alveolar macrophages and tracheal epithelial cells. Am. J. Respir. Cell Moi Biol. 21(1999) 155. [29] A. R. Baydoun, P. W. Emery, J. D. Pearson, and G. E. Mann. Substrate-dependent regulation of intracellular amino acid concentrations in cultured bovine aortic endotheliai cells. Biochem. Biophy. Res. Commun. 173(1990)940. [30] F. lyengar, D. J. Stuehr, and M. A. Marietta. Macrophage synthesis of nitrite, nitrate, and Nnitrosamines: Precursors and role of the respiratory burst. Proc. Natl. Acad. Sci. USA. 84 (1987) 6369. [31] D. L. Granger, J. B. Hibbs, J. R. Perfect, and D. T. Durack. Metabolic fate of L-Arginine in relation to microbiostatic capability of murine macrophages. J. Clin. Invest. 85 (1990) 264. [32] A. R. Baydoun, P. W. Emery, J. D. Pearson, and G. E. Mann. Substrate-dependent regulation of intracellular amino acid concentrations in cultured bovine aortic endotheliai cells. Biochem. Biophy. Res. Commun. 173(1990)940. [32] Y. Hattori, E. B. Campbell, and S. S. Gross. Argininosuccinate synthetase mRNA and activity are induced by immunostimulants in vascular smooth muscle. J. Biol. Chem. 269 (1994) 9405. [33] F.H. Guo, S.A.A. Comhair, S. Zheng, R.A. Dweik, N.T. Eissa, M.J. Thomassen, W. Calhoun, S.C. Erzurum. Molecular Mechanisms of Increased Nitric Oxide (NO) in Asthma: Evidence for Transcriptional and Post-translational Regulation of NO Synthesis. J Immunol 164 (2000) 5970. [34] D. Saleh, P. Ernst, S. Lim, P. J. Barnes, and A. Giaid. Increased formation of the potent oxidant peroxynitrite in the airways of asthmatic patients is associated with induction of nitric oxide synthase: effect of inhaled glucocorticoid. FASEBJ. 12 (1998) 929. [35] D. A. Geller, A. K. Nussler, M. D. Silvio, C. J. Lowenstein, R. A. Shapiro, et al. Cytokines, endotoxin, and glucocorticoids regulate the expression of inducible nitric oxide synthase in hepatocytes. Proc. Natl. Acad. Sci. USA. 90 (1993) 522. [36] M.W. Radomski, R. M. J. Palmer, S. Moncada. Glucocorticoids inhibit the expression of an inducible. but not the constitutive, nitric oxide synthase in vascular endotheliai cells. Proc. Natl. Acad Sci. USA. 87(1990) 10043. [37] D. Kunz, G. Walker, W. Eberhardt, J. Pfeiischifter.. Molecular mechanisms of dexamethasone inhibition of nitric oxide synthase expression in interleukin 1 p-stimulated mesangial cells: Evidence for the involvement of transcriptional and posttranscriptional regulation. Proc. Natl. Acad. Sci. USA. 93(1996)255. [38] J. Marks-Konczalik, S.C. Chu, and J. Moss. Cytokine-mediated transcriptional induction of the human inducible nitric oxide synthase gene requires both activator protein 1 and nuclear factor kappaBbinding sites. J. Biol. Chem. 273 (1998) 22201. [39] M. E. de Vera, R. A. Shapiro, A. K. Nussler, J. S. Mudgett, R. L. Simmons, S. M. Morris, Jr., T. R. Billiar, and D. A. Geller. Transcriptional regulation of human inducible nitric oxide synthase (NOS2) gene by cytokines: initial analysis of the human NOS2 promoter. Proc. Natl. Acad. Sci. USA. 93 (1996)1054. [40] C. J. Lowenstein, E. W. Alley, P. Raval, A. M. Snowman, S. H. Snyder, S. W. Russell, and W. J. Murphy. Macrophage nitric oxide synthase gene: two upstream regions mediate induction by interferon gamma and lipopolysaccharide. Proc. Natl. Acad. Sci. USA. 90 (1993) 9730. [41] Q. W. Xie, R. Whisnant, and C. Nathan.. Promoter of the mouse gene encoding calcium-independent nitric oxide synthase confers inducibility by interferon gamma and bacterial lipopolysaccharide. J. Exp. Med. 177(1993) 1779. [42] Q. W. Xie, Y. Kashiwabara, and C. Nathan. Role of transcription factor NF-kappa B/Rel in induction of nitric oxide synthase. J. Biol. Chem. 269 (1994) 4705.

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[43] E. Martin, C. Nathan, and Q. W. Xie. Role of interferon regulatory factor 1 in induction of nitric oxide synthase../. Exp. Med 180(1994)977. [44] H. Harada, T. Fujita, M. Miyamoto, Y. Kimura, M. Maruyama, A. Furia, T. Miyata, and T. Taniguchi. Structurally similar but functionally distinct factors, IRF-1 and IRF- 2, bind to the same regulatory elements of 1FN and IFN-inducible genes. Cell. 58 (1989) 729. [45] R. Kamijo, H. Harada, T. Matsuyama, M. Bosland, J. Gerecitano, D. Shapiro, J. Le, S. I. Koh, T. Kimura, S. J. Green, T. W. Mak, T. Taniguchi, and J. Vilcek.. Requirement for transcription factor IRF-1 in NO synthase induction in macrophages. Science. 263 (1994) 1612. [46] K. Uetani. M. Arroliga, and S.C. Erzurum. Double-stranded RNA Dependence of Nitric Oxide Synthase 2 Expression in Human Bronchial Epithelial Cell Lines BET-1A and BEAS-2B. Am J Resp Cell Mol Biol 24 (2001) 720. [47] K. Uetani, M.J. Thomassen, and S.C. Erzurum. Interferon-y Induction of Human Nitric Oxide Synthase 2 (NOS2) through an Autocrine Loop via Respiratory Epithelial Cell-derived Soluble Mediator. Am J Physiol 280 (2001) L1179. [48] S. J. Haque, and B. R. G. Williams. Signal transduction in the interferon system. Semin. Oncol. 25 1998) 14. [49] D. Sampath, M. Castro, D. C. Look, and M. J. Holtzman. Constitutive activation of an epithelial signal transducer and activation of transcription (STAT) pathway in asthma. J. Clin. Invest. 103 (1999) 1353.

Disease Markers in Exhaled Breath N. Marczin and M.H. Yacoub (Eds.) IOS Press, 2002

Nitric Oxide Production in the Lung and its Regulation by Oxygen Raed A. DWEIK Department of Pulmonary and Critical Care Medicine Cleveland Clinic Foundation 9500 Euclid Avenue Cleveland Ohio 44195 U.S.A.

Abstract. Nitric oxide (NO) in exhaled breath is primarily derived from the airway. NO is endogenously synthesized by nitric oxide synthases (NOS) which convert L-arginine to L-citrulline and NO. Three NOS isoforms (type I, II and III) have been identified in the human lung. Oxygen is a substrate for all NOS enzymes and it also regulates NOS expression and the enzyme activity in the iung. Rapid response of endogenous NO in direct proportion to inspired oxygen strongly supports a role for oxygen in regulating NO production in the lung through a kinetic mechanism.

1. Nitric Oxide (NO) in the Lung NO is produced in the human lung, evidenced by NO detectable in the exhaled air of humans (6-8 ppb) and NO metabolites detectable in the airway aspirate and bronchoalveolar lavage fluid from human lungs [1-8]. NO is recognized to play key roles virtually in all aspects of lung biology and has been implicated in the pathophysiology of lung diseases [2, 9-13]. NO in the lung is involved in pulmonary neurotransmission, host defense and bacteriostasis, airway and vascular smooth muscle relaxation, pulmonary capillary leak, inflammation, mucociliary clearance, airway mucus secretion and cytotoxicity [12, 13]. NO is endogenously synthesized by nitric oxide synthases (NOS) which convert L-arginine to L-citrulline and NO. Three NOS isoforms (type I, II and III) have been identified in the human lung [1-5]. NOSI and HI are dependent on increases in intracellular calcium for enzyme activation while NOSH is calcium independent [6]. All NOS isoforms require oxygen, NADPH, FAD, FMN, tetrahydrobiopterin, and calmodulin for activity [6, 7].

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R.A. Dweik/NO Production in the Lung and its Regulation by Oxigen

2. Source(s) of NO in Exhaled Breath Cellular sources of NO in the lung include epithelial cells, endothelial cells of pulmonary arteries and veins, inhibitory nonadrenergic noncholinergic neurons, smooth muscle cells, mast cells, mesothelial cells, fibroblasts, neutrophils, lymphocytes, and macrophages [2, 3, 12, 13]. All three NOS isoforms are present in the human lung [1,2, 7]. Specifically, NOSI is located in inhibitory nonadrenergic noncholinergic neurons in the lung, while NOSIII is found in endothelial cells and the brush border of ciliated epithelial cells [1, 2, 7]. NOSH is found in the epithelial cells of the airway. Although NOSH may be induced in several types of cells in response to cytokines, endotoxin, or reactive oxygen species, NOSH is continuously expressed in normal human airway epithelium at basal airway conditions [5]. Once produced, NO is freely diffusible and enters target cells activating soluble guanylate cyclase to produce guanosine 3', 5'-cyclic monophosphate (cGMP) which mediates the majority of NO effects [7]. NO also diffuses into the airway and can be measured in the gas phase [8]. Potential anatomic sources of NO in exhaled breath include the pulmonary circulation, the lower airways, and the upper airways and paranasal sinuses [7, 8, 14]. NO is formed in high concentrations in the upper respiratory tract (nasopharynx and paranasal sinuses) [14], but several studies have conclusively demonstrated that NO is also produced in the lower respiratory tract [7, 8]. Studies of gas phase NO in the airway have been helpful in determining the anatomic source(s) of NO in the lung. During a breathhold NO accumulates in bronchiolar gases in an exponential fashion with an initial linear rise, followed by a plateau [8]. The steady state NO levels in the bronchiolar gases of the lung are achieved within seconds. Attainment of steady-state levels indicates a constant rate of production balanced by a constant consumption or scavenging of NO [8. 15]. Since NO is freely diffusible, consumption of NO can occur at different sites within the cell, lung tissue, extracellular fluids, and intravascular compartments. Primary reactions that may consume NO intra- and extracellularly include its reaction with oxygen, superoxide, hemoglobin, another molecule of NO, enzymes containing ironsulfur centers, heme-containing proteins, and thiol proteins [16]. An especially important scavenger of NO in the lung is hemoglobin [8, 15-17]. NO produced in the lungs may diffuse into the lumen of blood vessels where most will be trapped by oxy- and deoxyhemoglobin in red blood cells [15, 17]. The addition of even very small amounts of hemoglobin results in substantial decrease in the steady-state distribution of NO in vitro [15]. Furthermore, bronchoscopic measurements of airway NO in humans reveal that bronchiolar but not alveolar gases accumulate NO during a breath-hold [8]. During an expiratory breath-hold, bronchiolar gases accumulate NO quickly to a plateau. At the end of expiratory breath-hold, complete exhalation to residual volume causes NO levels to decrease rapidly [8]. Near zero NO levels observed at the end of exhalation indicate alveolar gases do not accumulate NO [8]. Studies in rabbits also demonstrate that NO levels in exhaled gases remain after blood circulation was stopped by intravenous injection of air or helium [18] or when blood was replaced by a physiologic buffer [19]. Despite this, at least some of the endogenously produced NO diffuses into the airway and is eliminated via the gas phase.

R.A. Dweik /NO Production in the Lung and its Regulation by Oxigen

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Thus, while NO levels in the gas phase likely underestimate NO levels in the lung tissue and at pulmonary vascular sites, the gas phase levels reflect in an accurate and qualitative manner the dynamics of NO production and consumption in the lung. The capacity of the blood as a scavenger of NO can be estimated from experiments of isolated perfused lungs [19]. Exhaled NO output from blood-perfused lungs is less than half the amount from buffer-perfused lungs [19] attesting to the large scavenging capacity of the blood in the pulmonary circulation. Exhaled NO levels also increase in rabbits with experimental anemia [20]. A progressive fall in the hematocrit from 30% to 11% by serial isovolemic hemodilution in rabbits causes a significant and parallel increase in expired NO levels [20]. Considering the high diffusibility of NO, the extremely rapid rate of scavenging by hemoglobin, and the rich supply of blood vessels found in the lung, the pulmonary circulation is undoubtedly a significant biologic sink for NO, and not likely to contribute to NO in exhaled breath.

3. Regulation of NO Synthesis by Oxygfen All NOS isoforms require the presence of oxygen for activity [6]. Although it is recognized that oxygen is a substrate for NOS, its effects on regulation of NOS activity are more complex than simple enzyme-substrate interaction [8, 21-25]. One mechanism to explain oxygen's effect on NO levels in the lung is revealed by enzyme kinetic analyses. All NOS are comprised of an oxygenase domain that contains binding domains for iron protoporphyrin IX (heme), tetrahydrobiopterin, and L-arginine, and a reductase domain that contains binding domains for FMN, FAD, and calmodulin [6]. The NOS heme iron participates in catalysis by binding oxygen and catalyzing the oxidation of Larginine [21-25]. NOSI activity is dependent on oxygen concentration (KMO2 400 uM) [21, 22]. When NO is formed during catalysis, it binds to the heme iron in the catalytic site of NOSI and forms an inactive heme iron-NO complex [19, 20]. The rate of decay of the complex is subsequently dependent upon oxygen concentration to enter the active catalytic cycle [19, 20]. Studies of purified NOSH activity in vitro as determined by the rate of NADPH consumption demonstrate that NOSII activity is dependent on molecular oxygen concentrations in the physiologically relevant range (KMO2 135 jaM) [8]. Although the KMO2 of NOSII is lower than that of NOSI, NOSII has also been shown to form heme iron-NO complexes, however studies using scavengers of NO suggest that the mechanism of regulation by NO and oxygen is not similar between the two enzymes [8, 26]. Work utilizing rapid kinetic analysis of the purified NOSIII enzyme in vitro also confirms significant dependence on oxygen [24]. The formation of active NOSIII complex is first order with respect to oxygen, reversible, and follows a simple one-step mechanism. However, decay of the complex is independent of oxygen concentration and occurs via a one- or two-exponential process depending upon co factors or substrate availability [22, 24]. Based on double reciprocal plots of NOSIII activity and oxygen concentration in vitro, the apparent NOSIII KN^ is strikingly low (4 uM) and consistent

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R.A. Dweik/NO Production in the Lung and its Regulation by Oxigen

with very little heme-NO complex formation in this isoform [24]. oxygen is less likely relevant to NOSIII enzyme activity in vivo.

Taken together,

4. Effect of Oxygen on Exhaled NO As predicted from the above enzyme kinetic studies, changes in inspired oxygen concentration lead to significant changes in exhaled NO. NO in exhaled gases from lungs of anesthetized rabbits ventilated with hypoxic gas mixtures are only modestly reduced using 0.14 or 0.10 FiO2, but decrease markedly using 0.06 FiOi [18]. In addition, ventilation of an isolated neonatal pig lung with 0.075 FiO2 rapidly decreases NO in exhaled gases and NO2~/NO3~ in the recirculating perfusate as compared to ventilation with 0.21 FiO2 [27]. Similarly, exhaled NO levels correlate to oxygen levels in the hypoxic range in humans, decreasing as oxygen levels decrease below ambient air [«]• Interestingly, the effect of hypoxia on NO levels in the airway is primarily a result of airway and alveolar oxygen tension (FiO2) rather than vascular oxygen tension (pO2) [18, 19, 27, 29]. In models of isolated perfused lungs [18, 19], inspired hypoxic gases result in significant decline in NO output from the lung, while vascular hypoxia has no significant effect on NO lung output. The results are similar whether the lungs are perfused with blood or a physiologic buffer. On the other hand, NO metabolites continue to accumulate in the perfusate [29], suggesting a vascular source of NO that may not be affected by oxygen. Differential regulation of lung NO synthesis in response to hypoxia suggests a complex model for NO production in the lung that involves at least 2 different compartments. The airway compartment with predominant NOSH expression in the airway epithelium is capable of rapidly changing NO output depending on inspired oxygen, couples ventilation-perfusion, and thus mediates hypoxic pulmonary vasoconstriction. In contrast, the vascular compartment predominantly expresses NOSIII, which is less affected by changes in oxygen. NOSIII may be responsible for a continuous low level of NO which modulates the effect of high output NO production by NOSH on the pulmonary circulation. In support of this, genetically engineered NOSIII null mice have only a slight increase in pulmonary artery pressures [30], but an exaggerated vasoconstrictive response to hypoxia. Furthermore, NOSIII-null murine lungs have significantly higher NOSII expression than the wildtype mice [30]. Oxygen concentration in intact tissues ranges from 1 to 150 uM [2, 8, 31. 32], with the highest levels found in the lung. Airway epithelial cells are unique in their exposure to oxygen, since above a thin layer of epithelial lining fluid, the airway cells are exposed directly to air containing 21% oxygen. Based upon oxygen solubility and the low differential oxygen gradient between overlying fluid to intracellular endoplasmic reticulum (1-2 uM) [32], the levels of oxygen in airway epithelial cells may actually approach 260 uM. Thus, the KMO2 determined for NOS II, but not NOSIII or NOSI, is well within the physiologic range of oxygen concentrations in lung epithelial cells. Importantly, KMO2 for NO synthesis in the intact human lung (190 uM) is similar to NOSII KMO2 in vitro [8].

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15

Taken together, these studies support a primary role for NOSII in maintenance of low pulmonary vascular resistance, and in the pulmonary vasoconstrictor response to hypoxia.

5. Regulation of NOS Gene Expression by Oxygen The immediate effects of short-term changes in oxygen concentration on NOS enzymes activity are likely due to the effects of oxygen on NOS enzyme kinetics. However, prolonged hypoxia can have significant effects on the gene expression of the different NOS isoforms [33, 34]. These transcriptional effects may vary among species or among organ systems in the same species. While hypoxia produces a progressive decline in constitutive NOS mRNA levels in bovine pulmonary artery endothelial cells [33, 34, 35], chronic hypoxia upregulates constitutive NOS expression in rabbit hearts [36] and rat lung pulmonary arteries [37]. Chronic hypoxia also increases NOS expression and NOS activity in rat carotid bodies [38].

6. Conclusions NO in exhaled breath is primarily derived from the airway. The pulmonary circulation likely affects exhaled NO levels by acting as a biologic sink that consumes NO and creates a gradient towards the pulmonary circulation. Oxygen regulates NOS expression and the enzyme activity. Rapid response of endogenous NO in direct proportion to inspired oxygen strongly supports a role for NOSII as a biochemical oxygen sensor and critical mediator of ventilation-perfusion coupling in the lung.

References [1] Kobzik, L., D.S. Bredt, C.J. Lowenstein, J. Drazen, B. Gaston, D. Sugarbaker, and J.S. Stamler. Nitric oxide synthase in human and rat lung: immunohistochemical and histochemical localization. Am. J. Respir. Cell. Mol. Biol. 1993; 9:371-377. [2] Gaston, B., J.M. Drazen, J. Loscalzo, and J.S. Stamler. The biology of nitrogen oxides in the airways. Am. J. Respir. Crit. Care Med. 1994; 149:538-551. [3] Guo FH, Uetani K, Haque J, Williams BRG, Dweik RA, Thunnissen FBJM, Calhoun W, Erzurum SC. Interferon-g and interleukin-4 stimulate prolonged expression of inducible nitric oxide synthase in human airway epithelium through synthesis of soluble mediators. Journal of Clinical Investigation 1997; 100:829-38. [4] Dweik RA, Guo FH, Uetani K, Erzurum SC. Nitric oxide synthase in the human airway epithelium. Acta Pharmacologica Sinica 1997; 18: 550-2. [5] Guo, F.H., H.R. De Raeve, T.W. Rice, D.J. Stuehr, F.B.J.M. Thunnisssen, and S.C. Erzurum. Continuous nitric oxide synthesis by inducible nitric oxide synthase in normal human airway epithelium in vivo. Proc. Natl. Acad. Sci. U.S.A. 1995; 92:7809-7813. [6] Stuehr, D.J., and O.W. Griffith. Mammalian nitric oxide synthases. Adv. Enzymol. Relat. Areas Mol. Biol. 1992; 65:287-346.

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[7] Dweik, R.A., Erzurum, S.C. Effects of nitric oxide and cGMP on smooth muscle proliferation. In: LAM and other diseases characterized by smooth muscle proliferation. Moss J, editor. Marcel Dekker, Inc. New York. 1999; 131:333-349. [8] Dweik, R.A., Laskowski, D., Abu-Soud, H.M., Kaneko, F.T., Hutte, R., Stuehr, D.J., Erzurum, S.C. Nitric oxide synthesis in the lung: regulation by oxygen through a kinetic mechanism. Journal of Clinical Investigation 1998; 101: 660-666. [9] Kaneko, F.T., Arroliga, A.C., Dweik, R.A., Comhair, S.A., Laskowski, D., Oppedisano, R., Thomassen, M.J., Erzurum, S.C. Correlation of nitric oxide reaction products to severity of pulmonary hypertension. Am J Respir Crit Care Med. 1998; 158:917-923. [10]Raychaudhuri, B., Dweik, R., Connors, M.J., Buhrow, L.T., Malur, A., Drazba, J., Erzurum, S.C., Kavuru, M.S., Thomassen, M.J. Nitric Oxide blocks NFkB activation in alveolar macrophages in asthma and primary pulmonary hypertension. American Journal of Respiratory Cell and Molecular Biology 1999; 21: 311-316. [11] Thomassen, M.J., Raychaudhuri, B., Dweik, R.A., Farver, C., Buhrow, L.T., Malur, A., Hammel, J., Erzurum, S.C., Kavuru, M.S. Effect of segmental allergen challenge on airway nitric oxide, eosinophils, and cytokines in asthmatics. Journal of Allergy and Clinical Immunology 1999; 104:1174-1182. [12] Nathan, C. Nitric oxide as a secretory product of mammalian cells. FASEB. J. 1992; 6:3051-64. [13]Schmidt, H.H.H.W., and U. Walter. NO at work. Cell 1994; 78:919-925. [14]Lundberg, J.O.N., T. Farkas-Szallasi, E. Weitzberg, J. Rinder, J. Lidholm, A. Anggard, T. Hokfelt. J.M. Lundberg, and K. Alving. High nitric oxide production in human paranasal sinuses. Nat. Med. 1995; 1:370-373. [15] Lancaster, J.R. Jr. Simulation of the diffusion and reaction of endogenously produced nitric oxide. Proc. Natl. Acad. Sci. U.S.A. 1994; 91:8137-8141. [16]Wink, D.A., I. Hanbauer, M.B. Grisham, F. Laval, R.w. Nims, J. Laval, J. Cook, R. Pacelli, J. Liebmann, M. Krishna, P.C. Ford, and J.B. Mitchel. Chemical biology of nitric oxide: regulation and protective and toxic mechanisms. Current topics in cellular regulation. 1996; 34:159-187. [17] Goldstein, S., and G. Czapski. Kinetics of nitric oxide autoxidation in aqueous solutions in the absence and presence of various reductants. The nature of the oxidizing intermediates. J. Am. Chem. Soc. 1995; 117:12078-12084. [18]Gustafsson, L.E., A.M. Leone, M.G. Presson, N.P. Wilkund, and S. Moncada. Endogenous nitric oxide is present in the exhaled air of rabbits, guinea pigs and humans. Biochem. Biophys. Res. Commun. 1991; 181:852-857. [19]Ide, Hiroshi, Nakano, H., Ogasa, T., Osanai, S., Kikuchi, K., and Iwamoto, J. Regulation of pulmonary circulation by alveolar oxygen tension via airway nitric oxide. American Physiological Society. 1999; 8750-7587:1629-1636. [20]Deem, S., Hedges, R.G., McKinney, S., Polissar, N.L., Alberts, M.K., and Swenson, E.R. Mechanisms of improvement in pulmonary gas exchange during isovolemic hemodilution. J Appl Physiol 1999; 87: 132-141. [21] Abu-Soud, H.M., D.L. Rousseau, and D.J. Stuehr. Nitric oxide binding to the heme of neuronal nitricoxide synthase links its activity to changes in oxygen tension. J. Biol. Chem. 1996; 271:32515-32518. [22] Abu-Soud, H.M., J. Wang, D.L. Rousseau, J.M. Fukuto, L Ignarro, and D. J. Stuehr. Neuronal nitric oxide synthase self-inactivates by forming a ferrous-nitrosyl complex during aerobic catalysis. J. Biol. Chem. 1995; 270:22997-23006. [23] Adak, S., Ghosh, S., Wang, Q., and Stuehr, D.J. Molecular Basis for Hyperactivity in Tryptophan 409 Mutants of Neuronal NO Synthase. In Press. [24] Abu-Soud, Husam, M., Ichimori, K., Presta, A., and Stuehr, D.J. Electron Transfer, Oxygen Binding, and Nitric Oxide Feedback Inhibition in Endothelial Nitric Oxide Synthase. In press.. [25] Abu-Soud H.M., R. Gachhui, P.M. Raushel, and D.J. Stuehr. The ferrous-dioxy complex of neuronal nitric oxide synthase: divergent effects of L-arginine and tetrahydrobiopterin on its stability. J. Biol. Chem. 1997; 272:17349-17353. [26] Hurshman, A.R., and M.A. Marietta. Nitric oxide complexes of inducible nitric oxide synthase: spectral characterization and effect on catalytic activity. Biochemistry 1995; 34:5627-5634. [27]Nelin, L.D., C.J. Thomas, and C.A. Dawson. Effect of hypoxia on nitric oxide production in neonatal pig lung. Am. J. Physiol. 1996; 271 :H8-H14.

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[28]Tsujino, I., K. Miyamoto, M. Nishimura, H. Shinano, H. Makita, S. Saito, T. Nakano, and Y. Kawakami. Production of nitric oxide (NO) in intrathoracic airways of normal humans. Am. J. Respir. Crit. Care Med. 1996; 154:1370-1374. [29]Grimminger, F., Spriestersbach, R., Weissmann, N., Walmrath, D., and Seeger, W. Nitric oxide generation and hypoxic vasoconstriction in buffer-perfused rabbit lungs. The American Physiological Society. 1995; 1509-1515. [30]Pagan, K.A., Fouty, B.W., Tyler, R.C., Morris, K.G., Jr., Hepler, L.K., Sato, K., LeCras, T.D., Abman, S.H., Weinberger, H.D., Huang, P.L., McMurtry I.F., and Rodman, D.M. The pulmonary circulation of homozygous eNOS-null mice is hyperresponsive to mild hypoxia. The Journal of Clinical Investigation. 1999; 103:291-299. [31 ]Vanderkooi, J.M., M. Erecinska, and I.A. Silver. Oxygen in mammalian tissue: methods of measurement and affinities of various reactions. Am. J. Physiol. 1991; 260:C1131-1150. [32]Wakita, M., G. Nishimura, M. Tamura. Some characteristics of the fluorescence liftime of reduced pyridine nucleotides in isolated mitochondria, isolated hepatocytes, and perfused rat liver in situ. J. Biochem. 1995; 118:1151-1160. [33]Liao, J.K., J.L. Zulueta, F.S. Yu, H.B. Peng, C.G. Cote, and P.M. Hassoun. Regulation of bovine endothelial constitutive nitric oxide synthase by oxygen. J. Clin. Invest. 1995; 96:2661-2666. [34]Melillo, G., T. Musso, A. Sica, L.S. Taylor, G.W. Cox, and L Varesio. A hypoxia-responsive element mediates a novel pathway of activation of the inducible nitric oxide synthase promotor. J. Exp. Med. 1995; 182:1683-1693. [35]Phelan, M.W., and V. F. Fallen Hypoxia decreases constitutive nitric oxide synthase transcript and protein in cultured endothelial cells. J. Cell. Physiol. 1996; 167:469-476. [36]Baker, J.E., Holman, P., Kalyanaraman, B., Griffith, O.W., and Pritchard, K.A., Jr. Adaptation to chronic hypoxia confers tolerance to subsequent myocardial ischemia by increased nitric oxide production. Annals of the New York Academy of Sciences. 1999; 874:236-53. [37] Sato, K., Rodman, D.M., and McMurtry, I.F. Hypoxia inhibits increased ETB receptor-meadiated NO synthesis in hypertensive rat lungs. American Journal of Physiology. 1999; 276 (4 Pt 1):L571-80. [38] Di Giulio, C., Grilli, A., De Lutiis, M.A., Di Natale, F., Sabatino, G., and Felaco, M. Does chronic hypoxia increase rat carotid body nitric oxide? Comparative Biochemistry & Physiology. 1998: 120(2):243-7.

Disease Markers in Exhaled Breath N. Marczin and M.H. Yacoub(Eds.) 1OS Press. 2002

S-Nitrosothiols In Respiratory Biology Benjamin GASTON University of Virginia, Department of Pediatrics, Pediatric Respiratory Medicine, PO Box 800386, Charlottesville, Virginia 22908 Abstract. S-Nitrosothiol (SNOs) are nitric oxide (NO) -thiol adducts that mediate several cell and organ system signaling pathways relevant to respiration. SNOs are more selective and potent than NO in mediating many respiratory bioactivities, ranging from airway smooth muscle relaxation to regulation of central ventilatory drive [1-3]. Under physiological conditions, SNOs are also more stable and less cytotoxic than are NO and other bioactive nitrogen oxides [3, 4]. Their broad range of bioactivities arises, in part, from their potential to act stereoselectively [2], as nitrosonium (NO*) donors, as nitroxyl (NO') donors or as NO radical donors [5]. Additionally, specific cell systems regulate the bioactivity of SNOs by modifying their synthesis, breakdown and localization [2, 4, 6, 7].

1. Synthesis of S-nitrosothiols. SNOs may be formed by inorganic reactions, particularly in the oxidative environment of the airways. Specifically, NO produced by nitric oxide synthases (NOS) can react with relatively high concentrations of oxygen(O2) and superoxide (O2) - acting as electron acceptors - to S-nitrosylate reduced thiols - present in mM concentrations in the airways - forming extracellular SNOs such as S-nitrosoglutathione (GSNO) [2, 3]. Of note GSNO, was first identified and characterized as an endogenous, bioactive molecule in the airways. Concentrations are 0.1-0.5 uM at baseline and increase to the 1-10 uM range during acute lung inflammation [1]. Low mass, extracellular species such as GSNO are not the only SNOs formed in the lung: S-nitrosylated albumin is also present in the airway lining fluid, and airway epithelial cells, cartilage and inflammatory cells immunostain strongly for endogenously S-nitrosylated proteins under baseline conditions [8]. Synthesis of SNO bonds require an electron receptor. Under inorganic conditions, this acceptor generally represents nitrogen dioxide (NO2), superoxide (O2~) or iron sulfur precursors of iron nitrosyl species [9]. These relatively electropositive species accept an electron from NO radical and form a complex (such as dinitrogen trioxide or an ironnitrosyl species) from which a NO+ equivalent can serve to allow an electrophilic attack on a protein- or low mass- thiol, leaving a proton [4, 5, 9]. This kind of chemistry is also relevant to the organic synthesis of SNOs, where the electrophilic species can include a metal center (for example, in the case of ceruloplasmin [10]) or a redox-based amino acid motif in the primary or tertiary structure of a protein that favors NO transfer to thiol [11]. Among examples of the latter is the S-nitrosylation of the 6-93 cysteine of hemoglobin, which is favored by vicinal amino acids, stabilized during

B. Gaston / S-Nitrosothiols in Respiratory Biology

19

hemoglobin oxygenation and ("R" conformation) and destabilized by deoxygenation of the tetramer (switch to "T"-conformation) [12]. Of note, NO" can also react with more electropositive thiols to accomplish bioactivity [5, 13]. Finally, NOS itself, depending on the local redox environment—particularly including glutathione concentrations and superoxide dismutase activity—can produce S-nitrosomiols [14].

2. Breakdown of S-nitrosothiols. Several enzyme systems regulate the breakdown of GSNO and other Snitrosothiols. These include xanthine/ xanthine oxidase [15], the thioredoxin thyroredoxin reductase system [16], y-glutamyl transpeptidase (GOT), [2, 17] and glutathione peroxidase [18] as recently reviewed [4]. Additionally, Liu and coworkers have shown that glutathione-sensitive formaldehyde dehydrogenase (GS-FDH) tightly regulates intracellular SNO levels in a spectrum of organisms ranging from bacteria to mammals [7]. These enzymes are active in cells of relevance to the respiratory biology - ranging from airway epithelial cells, macrophages and lymphocytes [7, 19] to cells regulating control of breathing [2]. Of note, the catabolic products formed by these enzymes vary widely ( ranging from NO to ammonia) and have a broad spectrum of bioactivities, suggesting that expression of SNO catabolic enzymes may serve not only to regulate the cellular distribution and transnitrosation biochemistry of low-mass and protein SNOs, but also to localize the bioactivity of SNO catabolic products.

3. Bioactivities of S-nitrosothiols. Classically, SNOs exert their bioactivities through transnitrosation reactions, in which the NO group is transferred from one thiol residue to another [4, 5, 20]. During transfer, the target protein is modified through mechanisms ranging from inactivation of an active site thiol (6, 21) to modification of oxygen binding and delivery [22]. In the lung, SNOs have a broad range of functions, ranging from modulation of ventilation-perfusion matching [1,22-24] to alteration of inflammatory cell survival [19, 25]. The mechanisms by which these bioactivities are regulated involve precise control of the composition, compartmentalization and concentration of the SNO pool. Compositional regulation is exemplified by control of minute ventilation at the level of the nucleus tractus solitarius [2] where n-M GSNO levels [26] are only active to stimulate an increase in minute ventilation if activated by GGT to form S-nitrosocysteinyl glycine. An example of regulation of SNO bioactivity by compartmentalization is that of mitochondrial caspases 3 and 9, which are endogenously S-nitrosylated but are denitrosylated and activated with release into the cytosol [6, 19]. Additionally, release of S-nitrosothiols from neurons and epithelial cells into the extracellular space may be involved in nonadrenergicnoncholinergic mediated airway smooth muscle relaxation [27, 28]. The critical relevance of control of concentration is exemplified by increased DNA binding of the nuclear regulatory factor SP1 following exposure to physiological concentrations (nM to low (j.M) of GSNO, with paradoxical inhibition of binding by supraphysiological concentrations (>10 uM) (unpublished observations). Note that the bioactivities of SNOs may be cyclic GMP- dependent or cyclic GMPindependent, depending, in part, on whether the chemical basis for the bioactivity involves homolytic cleavage (to form NO) or transnitrosation reactions. An example is airway smooth muscle relaxation. In guinea pig trachealis, smooth muscle relaxation appears to be primarily cGMP- dependent, whereas in more peripheral airway smooth muscle in

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B. Gaston / S-Nitrosothiols in Respiratory Biology

humans and other organisms, relaxation is cGMP independent [3, 29-31]. Indeed, for peripheral airway smooth muscle relaxation, the extent to which SNO acts as a "NO donor" - releasing the NO radical - will be the extent to which it loses its bioactivity. In this regard, improved ventilation -perfusion matching associated with certain NO donors such as inhaled ethyl nitrite, inhaled GSNO - and perhaps even inhaled NO - may involve cGMP- independent mechanisms as much as cGMP- dependent mechanisms [8, 23, 32]. It is also important to appreciate that oxygen delivery to the periphery may be regulated by hemoglobin S-nitrosylation. That is, tissue hypoxemia will favor hemoglobin deoxygenation and transfer of the NO group from hemoglobin to anion exchange protein I on the red blood cell membrane, allowing ultimate transfer to - and relaxation - of peripheral vascular smooth muscle [12, 22, 33]. This transfer appears also to favor upregulation of hypoxia-inducible gene through stabilization of hypoxia-inducible factor 1a [34] and to increase minute ventilation through stereoselective neurostimulation [2]. Physiological concentrations of SNOs also affect a variety of other airway epithelial functions. For example, SNOs can modulate the activity of several airway epithelial ion channels [35-38] and increase airway epithelial ciliary beat frequency [39], primarily through cGMP-dependent mechanisms. The role of GSNO in cystic fibrosis (CF) epithelium may be particularly important. GSNO increases the expression and maturation of the CF transmembrane regulatory protein (CFTR) in cells homozygous for AF508, the most common mutation associated with CF [40]. Remarkably, maturation of this protein allows function of CFTR in AF508 homozygous epithelial cells [41]. Further, clinical restoration of normal levels of GSNO in the CF airway [42] is well tolerated and improves oxygenation [32]; there is hope that its long term administration could be of benefit to these patients. Cell signaling by SNOs is of relevance both to baseline respiratory cell signaling and to the pulmonary inflammatory response. Of note in this regard, physiological and supraphysiological concentrations of SNOs often have opposite effects, and levels can be increased or decreased in inflammation, depending on the location and metabolic flux. Examples of cell signaling events mediated by S-nitrosylation chemistry include 1) FAS ligand-signaled denitrosylation of caspases leading to lymphocyte apoptosis [6, 19]; 2) modulation of the expression of cell adhesion molecules, selectin, IL 1 and IL 8 by inhibition of transcription factor NFicB-specifically through S-nitrosylation of the P50 subunit [43-45]; 3) upregulation of HIF-1 binding by stabilization of the subunit HIFl-a [34] likely through S-nitrosylation of critical cysteines in the ubiquitin activation cascade; 4) inhibition of neutrophil NADPH oxidase by S-nitrosylation of the p47phoxsubunit [46]; and 5) inhibition of a variety of glutathione metabolic enzymes [4, 47, 28]. The size, localization and composition of the intracellular SNO pool, as described above, appears to be regulated in a manner loosely analogous to phosphorylation to coordinate the signaling processes. It is not surprising that the determinants of airway SNO concentrations during inflammation are complex. Therapy with NO donors and/or acute inflammation may be associated with high airway SNO levels [1,8,23]. On the other hand, while synthesis may be increased in the severely affected asthmatic airway, catabolism may be accelerated to a greater degree, resulting in high flux and low SNO levels [49, 50]. Low levels may also be found in the airways of patients with CF, where synthesis may be decreased and catabolism increased [42].

B. Gaston /S-Nitrosothiols in Respirator}' Biology

21

4. Summary In conclusion, SNOs are endogenously formed and highly regulated signaling molecules of substantial relevance to respiratory biology. They are present in the lung in physiologically relevant concentrations under normal conditions. They are involved in ventilation-perfusion matching, regulation of peripheral systemic oxygen delivery and central regulation of minute ventilation. Additionally, their cellular localization and metabolism is relevant to a broad range of cell signaling processes, particularly those involved in host defense. Appreciation of this chemistry has led not only to a more comprehensive understanding of the implications of various exhaled nitrogen oxide biomarkers to respiratory disease, but also to the development of new therapies.

Acknowledgment: This work was supported by the NIH/NHLBI: HL 59337. References 1.

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10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

Gaston, B., et al., Endogenous nitrogen oxides and bronchodilator S-nitrosothiols in human airways. Proceedings of the National Academy of Sciences of the United States of America, 1993. 90(23): p. 10957-61. Lipton, A.J., et al., S-nitrosothiols signal the ventilator/ response to hypoxia. Nature, 2001. 413(6852): p. 171-4. Gaston, B., et al., Relaxation of human bronchial smooth muscle by S-nitrosothiols in vitro. Journal of Pharmacology & Experimental Therapeutics, 1994. 268(2): p. 978-84. Gaston B. Nitric oxide andthiol groups. Biochim Biophys Acta 1999; 1411:323-333. Arnelle, D.R. and J.S. Stamler, NO+, NO, and NO- donation by S-nitrosothiols: implications for regulation of physiological functions by S-nitrosylation and acceleration of disulfide formation. Archives of Biochemistry & Biophysics, 1995. 318(2): p. 279-85. Mannick, J.B., et al., S-Nitrosylation of mitochondria! caspases. J Cell Biol, 2001. 154(6): p. 1 1 1 1 6. Liu, L., et al., A metabolic enzyme for S-nitrosothiol conserved from bacteria to humans. Nature. 2001.410(6827): p. 490-4. Lorch, S.A., et al., Immunohistochemical Localization of Protein 3-Nitrotyrosine and Snitrosocysteine in a Murine Model of Inhaled Nitric Oxide Therapy. Pediatric Research June, 2000. 47(6): p. 798-805. Vanin, A.F., I.V. Malenkova, and V.A. Serezhenkov, Iron catalyzes both decomposition and synthesis of S-nitrosothiols: optical and electron paramagnetic resonance studies. Nitric Oxide, 1997. 1(3): p. 191-203. Inoue, K., et al., Nitrosothiol formation catalyzed by ceruloplasmin. Implication for cytoprotective mechanism in vivo. J Biol Chem, 1999. 274(38): p. 27069-75. Perez-Mato, I., et al., Methionine adenosyltransferase S-nitrosylation is regulated by the basic and acidic amino acids surrounding the target thiol. J Biol Chem, 1999. 274(24): p. 17075-9. Jia, L., et al., S-nitrosohaemoglobin: a dynamic activity of blood involved in vascular control. Nature, 1996. 380(6571): p. 221-6. Choi, Y.B., et al., Molecular basis of NMDA receptor-coupled ion channel modulation by Snitrosylation. Nat Neurosci, 2000. 3(1): p. 15-21. Schmidt, H.H., et al., No .NO from NO synthase. Proc Natl Acad Sci USA, 1996. 93(25): p. 14492-7. Trujillo, M., et al., Xanthine oxidase-mediated decomposition of S-nitrosothiols. J Biol Chem, 1998. 273(14): p. 7828-34. Nikitovic, D. and A. Holmgren, S-nitrosoglutathione is cleaved by the thioredoxin system with liberation of glutathione and redox regulating nitric oxide. J Biol Chem, 1996. 271(32): p. 19180-5. Hogg, N., et al., S-Nitrosoglutathione as a substrate for gamma-glutamyl transpeptidase. Biochem J, 1997. 323(Pt2):p. 477-81. Hou, Y., et al., Seleno compounds and glutathione peroxidase catalyzed decomposition of Snitrosothiols. Biochem Biophys Res Commun, 1996, 228(1): p. 88-93. Mannick, J.B., et al., Fas-induced caspase denitrosylation. Science, 1999. 284(5414): p. 651-4. Stamler, J.S., Redox signaling: nitrosylation and related target interactions of nitric oxide. Cell, 1994. 78(6): p. 931-6.

22

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Nikitovic, D., A. Holmgren, and G. Spyrou, Inhibition of AP-1 DNA binding by nitric oxide involving conserved cysteine residues in Jun and Fos. Biochemical & Biophysical Research Communications, 1998. 242(1): p. 109-12. Stamler, J.S., et al., Blood flow regulation by S-nitrosohemoglobin in the physiological oxygen gradient. Science, 1997. 276(5321): p. 2034-7. Moya, M.P., et al., S-nitrosothiol repletion by an inhaled gas regulates pulmonary function. PNAS, 2001.98(10): p. 5792-5797. Emery, C.J., Vasodilator action of the S-nitrosothiol, SNAP, in rat isolated perfused lung. Physiological Research, 1995. 44(1): p. 19-24. Hunt J, Fang K, Malik R, Snyder A, Malhotra N, Platts-Mills TAE, Gaston B. Endogenous airway acidification: implications for asthma pathophysiology. Am J Respir Crit Care Med 2000; 161:694699. Kluge, I., et al., S-nitrosoglutathione in rat cerebellum: identification and quantification by liquid chromatography-mass spectrometry. J Neurochem, 1997. 69(6): p. 2599-607. Lilly, C.M., et al., Modulation of vasoactive intestinal peptide pulmonary relaxation by NO in tracheally supervised guinea pig lungs. AmJPhysiol, 1993. 265(4 Pt 1): p. L410-5. Thompson, D.C. and R.J. Altiere, Differential susceptibility of tracheal contraction to nonadrenergic noncholinergic relaxation. J Pharmacol Exp Ther, 1998. 284(1): p. 19-24. Jansen, A., et al., The relaxant properties in guinea pig airways of S-nitrosothiols. Journal of Pharmacology & Experimental Therapeutics, 1992. 261(1): p. 154-60. Bannenberg, G., et al., Characterization of bronchodilator effects and fate of S-nitrosothiols in the isolated perfused and ventilated guinea pig lung. Journal of Pharmacology & Experimental Therapeutics, 1995. 272(3): p. 1238-45. Perkins, W.J., et al., cGMP-independent mechanism of airway smooth muscle relaxation induced by S-nitrosoglutathione. AmJPhysiol, 1998. 275(2 Pt 1): p. C468-74. Snyder A, McPherson ME, Hunt JF, Johnson M, Stamler JS, Gaston B. Acute effects of aerosolized S-nitrosoglutathione in cystic fibrosis. Am J Respir Crit Care Med 2002, in press. Pawloski J, Hess D, Stamler J. Export by red blood cells of nitric oxide bioactivity. Nature 2001;409:622-626.

34.

Palmer, L.A., B. Gaston, and R.A. Johns, Normoxic stabilization of hypoxia-inducible factor-1 expression and activity: redox-dependent effect of nitrogen oxides. Molecular Pharmacology, 2000. 58(6): p. 1197-203.

35.

Jain, L., et al., Nitric oxide inhibits lung sodium transport through a cGMP-mediated inhibition of epithelial cation channels. Am J Physiol Lung Cell Mol Physiol, 1998. 274(4): p. L475-484.

36.

Duszyk, M., Regulation of anion secretion by nitric oxide in human airway epithelial cells. Am J Physiol Lung Cell Mol Physiol, 2001. 281 (2): p. L450-457. Kamosinska, B., et al., Role of Inducible Nitric-Oxide Synthase in Regulation of Whole-Cell Current in Lung Epithelial Cells. J Pharmacol Exp Ther, 2000. 295(2): p. 500-505. Kamosinska, B., et al., Nitric oxide activates chloride currents in human lung epithelial cells. American Journal of Physiology, 1997. 272(6 Pt 1): p. LI 098-104. Li, D., et al., Regulation of Ciliary Beat Frequency by the Nitric Oxide-Cyclic Guanosine Monophosphate Signaling Pathway in Rat Airway Epithelial Cells. Am. J. Respir. Cell Mol. Biol., 2000. 23(2): p. 175-181. Zaman, K., et al., S-nitrosoglutathione increases cystic fibrosis transmembrane regulator maturation. Biochem Biophys Res Commun, 2001. 284(1): p. 65-70. Gaston B, A.C., Zaman K, Hunt J, Palmer L, Roomans G., S-Nitrosoglutathione increases expression and activity of deltaF 408 CFTR. Pediatric Pulmonology, 2001. 22 (Supp): p. 193. Grasemann, H.M.D., et al., Decreased levels of nitrosothiols in the lower airways of patients with cystic fibrosis and normal pulmonary function. Journal of Pediatrics December, 1999. 135(6): p. 770-772. Marshall, H.E. and J.S. Stamler, Inhibition of NF-kappa B by S-nitrosylation. Biochemistry. 2001. 40(6): p. 1688-93. Marshall, H.E., K. Merchant, and J.S. Stamler, Nitrosation and oxidation in the regulation of gene expression. FASEBJ, 2000. 14: p. 1-12. delaTorre, A., et al., Endotoxin-mediated S-nitrosylation of p50 alters NF-kappa B-dependent gene transcription in ANA-1 murine macrophages. Journal of Immunology, 1999. 162(7): p. 4101-4108. Park, J.W., Attenuation of p47phox and p67phox membrane translocation as the inhibitory mechanism of S-nitrosothiol on the respiratory burst oxidase in human neutrophils. Biochemical & Biophysical Research Communications, 1996. 220(1): p. 31-5.

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47.

48.

49. 50.

Butzer, U., et al., Increased oxidative stress in the RAW 264.7 macrophage cell line is partially mediated via the S-nitrosothiol-induced inhibition of glutathione reductase. FEES Letters, 1999. 445(2-3): p. 274-278. Moellering, D., et al., The induction of GSH synthesis by nanomolar concentrations of NO in endothelial cells: a role for [gammaj-glutamylcysteine synthetase and [gamma]-glutamyl transpeptidase. FEES Letters, 1999. 448(2-3): p. 292-296. Gaston, B., et al., Bronchodilator S-nitrosothiol deficiency in asthmatic respiratory failure The Lancet, 1998.351(9112): p. 1317-1319. Fang K, Johns R, Macdonald T, Kinter Michael, Gaston B. S-Nitrosoglutathione breakdown prevents airway smooth muscle relaxation in the guinea-pig. Am J Physiol Lung Cell Molec Biol 2000;279:L716-L721.

Disease Markers in Exhaled Breath N. Marczin andM.H. Yacoub (Eds.) IOS Press, 2002

Inhibitors of Nitric Oxide Synthase: Experimental Findings and Clinical Implications Nigel K BOUGHTON-SMITH Discovery BioScience, AstraZeneca R&D Charnwood, Loughborough, Leicestershire LEI 1 5RH, U.K. Abstract Inhibitors of nitric oxide synthase (NOS) have been a major tool in investigating the importance of excessive NO and reactive products in the pathology of chronic inflammatory disease and in identifying the potential of selective iNOS inhibitors as potential therapy for these diseases

1. Introduction Excessive NO production occurs in disease states and has been implicated as an important mediator of immune and inflammatory responses and in the pathology of a variety of chronic inflammatory diseases. The role of NO is largely determined by the level and extent of synthesis. Under physiological conditions the levels of intracellular calcium regulates the constitutive NO synthase isoforms (nNOS or NOS-1 and eNOS or NOS-3 ). In contrast, following induction by inflammatory cytokines the activity of inducible NOS (iNOS, NOS-2) is largely controlled by the level of available substrate, Larginine, and produces much greater amounts of NO for longer periods. Excessive NO production following iNOS induction can result in a variety of reactive products which can interact with a number of molecular targets resulting in altered cell function and in some situation in cell death [1].

2. Inhibitors of NOS NOS inhibitors have been a major tool in determining the role of NO in both physiology and pathology [2,3,4,5]. Initially studies used analogues of L-arginine, such as, monomethyl Larginine (L-NMMA) or L-nitroarginine (L-NA) and its methyl ester (L-NAME), which are potent and specific inhibitors of NOS activity. Another inhibitor, which occurs naturally in some disease conditions, is asymm dimethylarginine (L-ADMA). These L-arginine analogues inhibit all the isoforms of NOS with similar potency and are therefore non-selective.

N.K. Boughton-Smith / Inhibitors of Nitric Oxide Synthase

25

Nitric Oxide Synthase inhibitors related to L- arginine R

R'

H

H

L-Arginine

H

CHS

N-Methyl-L-arginine(L-NMMA)

H

N02

N-Nitro-L-arginine(L-NA)

NH

R

S\r i

R.

11

,.

T**-^ ^-^ H

,^C02H IT 1

NH2

CH3CH3

as/mmdimethylarginin^L-ADMA)

Figure 1. Non-Selective Inhibitors of NO synthase

a. Protective Role for NO from Constitutive NOS Removal of endogenous NO by administration of non-selective NOS inhibitors such as L-NMMA and L- NAME potentiates acute intestinal vascular damage produced by endotoxin [5]. Furthermore, inhibition of NOS by the non-selective inhibitors promotes basal neutrophil endothelial adhesion and vascular leakage in the intestinal vasculature [6,7]. In addition, NO donors, a source of exogenous NO, protect against acute intestinal damage produced by endotoxin. These studies suggest that endogenous NO from constitutive NOS has a protective role in the vasculature. The mechanism of protection by NO is probably due to the vasodilator and anti-aggregatory action of NO as well as inhibition of cellular adhesion.

b. Pathological Role for excessive NO from induced NOS in acute inflammation The role of the excessive NO production from iNOS in models of vascular leakage and inflammation has also been investigated using more selective inhibitors of iNOS.

Miscellaneous Nitric Oxide Synthase Inhibitors

CQjH N-(IminoethyI)-L-lysine (L-NIL)

1400W (Glaxo-Wellcome)

2-Amino-4-mcthylpyridine

Figure 2. NO Synthase Inhibitors with some Selectivity for iNOS

26

N. K. Boughton-Smith / Inhibitors of Nitric Oxide Synthase

Examples of inhibitors with some selectivity for iNOS are the weak inhibitor of NOS aminoguanidine (AG) and the more potent inhibitors, N-iminoethyl-L-lysine (L-NIL), 2-amino4-methylpyridine, and the GSK compound 1400W [2,3,4]. Although some of these inhibitors are very potent in-vitro, they have only marginal selectivity in-vivo and at higher doses can produce adverse side effects. These inhibitors have, however, been used extensively as experimental tools to investigate the potential role of iNOS in normal physiology and in inflammatory disease. In contrast to non-selective NOS inhibitors, such as L-NMMA and LNAME which potentiate vascular leakage and oedema formation produced by endotoxin. the selective iNOS inhibitor, 1400W attenuates these effects [8]. Anti-inflammatory activities of NOS inhibitors have been described in a variety of models of acute inflammation in which there is iNOS induction. In zymosan peritonitis the vascular leakage, which is related to iNOS activity and increases in NO, is attenuated by L-NAME. The selective NOS inhibitor L-NIL also reduces oedema produced by carrageenan in the rat paw and air pouch in which increases in iNOS activity have also been detected [9,10,11]. The anti-inflammatory activity of non-selective and selective NOS inhibitors suggests that iNOS induction and the subsequent formation of NO and other reactive breakdown products play an important role in vascular leakage and oedema formation, the hall marks of the acute inflammatory response.

3. Role of iNOS in chronic inflammatory disease a. Role of iNOS in Inflammatory Bowel Disease Increases in iNOS activity and the consequential excessive production of NO and reactive products have been observed in a variety of chronic inflammatory disease. In Ulcerative Colitis (UC) and Crohn's Disease (CD), there are marked increases in iNOS activity in the inflamed colon which are extensively located in the mucosal epithelium. Furthermore, incubation of colonic epithelial cells with appropriate cytokines results in iNOS expression. There are increases in iNOS activity in animal models of IBD including; the TNB model. PG/PS granulomatous colitis, HLA-B27 transgene colitis and a model of colitis in the guinea pig. Inhibitors of NOS are beneficial in these models. In PG/PS granulomatous colitis both LNAME and AG reduce macroscopic inflammation and neutrophil infiltration. Neutrophil infiltration in HLA-B27 transgene colitis is also inhibited by L-NAME or AG. However, whereas AG reduces colonic permeability in this model, the non-selective inhibitor L-NAME increased permeability [12,13]. These results support the concept for a protective role of constitutive NOS and a pathological role for iNOS. The induction of NOS in the colonic mucosa, with the excessive production of NO, a potent vasodilator, may have an important role to play in the mucosal hyperaemia, vascular permeability and colonic mucosal oedema observed in active ulcerative colitis. Excessive NO production may also affect epithelial function and viability in this disease.

b. Role of iNOS in Rheumatoid and Osteoarthritis Induction of iNOS in arthritis results in increased nitrite levels in the synovial fluid, sera and urine from RA and OA patients. The iNOS is expressed in chondrocytes, type A synoviocytes and fibroblasts. Immunoreactive nitrotyrosine in the inflamed synovium indicates that the highly reactive product peroxynitrite, produced by interaction of NO with the

N. K. Boughton-Smith / Inhibitors of Nitric Oxide Synthase

27

superoxide radical( O2-), has been formed. Increase in plasma nitrite and iNOS activity in a variety of RA models including, adjuvant arthritis, collagen induced arthritis, streptococcal cell wall arthritis, and the spontaneous arthritis and nephritis produced in MLR Ipr/lpw mice are related to disease activity. Furthermore, paw swelling, synovial inflammation and cartilage degradation are reduced by L-NAME and L-NMMA and also by L-NIL, a NOS inhibitor with some selectivity for the induced NOS isoform [2,3,4]. In the dog cruciate ligament model of OA there is iNOS induction in the cartilage and L-NIL reduces the cartilage degradation and inflammatory [14]. The excessive formation of NO following iNOS induction may play an important role in arthritis [15]. The local vasodilator actions of NO may contribute to oedema and NO or reactive products may increase vascular leakage. NO may either directly, or through activation of cyclooxygenase-2 (COX2), also contribute to the pain of arthritis. NO inhibits chondrocyte proliferation and induces chondrocyte apoptosis. Furthermore, NO can both reduce proteoglycan synthesis and activate the metalloprotease enzymes, collagenase and stromolysin and thereby enhance cartilage degradation. Excessive NO production may play a role in cartilage destruction in the arthritic joint. NO may play a role in bone resorption since Inflammatory Disease Joint Lung

Pathology

Bowe|

UC

CD

RA

Vasodilatation

+

_|-

4.

OA

ASTHMA COPD _j.

-j_

Vascular Permeability

_j_

.j.

_j_

_j_

_j_

Epithelial Cell Function

,

+

+

Mucus Secretion

+

+

+

r

Mucosal Permeability

+

4-

+

+

Pain

?

?

Impaired Motility

+

+

+

Cartilage Degradation

+

+

Bone Loss/ Remodelling

.

,

T Cell Response

+

+

+

+

enhancement of IL-1(5 induced calverial bone resorption by TNFa is inhibited by L-NMMA [16]

Table 1 Potential role of iNOS in the pathology of chronic inflammatory disease (UC = Ulcerative Colitis; CD = Crohn's Disease; RA = Rheumatoid Arthritis; OA = Osteoathritris, COPD = Chronic Obstructive Pulmonary Disease)

c. Role of iNOS in Asthma Several clinical studies have shown a marked increase in exhaled NO from patients with asthma, which is closely associated with reduced lung function and eosinophil infiltration. Improvements in lung function produced by corticosteriods are accompanied by a concomitant reduction in exhaled NO [17]. The increase in NO is derived from iNOS in the airways and is located predominantly in the bronchial epithelium with some activity in inflammatory cells.

28

N. K. Boughton-Smith / Inhibitors of Nitric Oxide Synthase

Stimulation of bronchial biopsies, primary cultures of epithelial cells or epithelial cell line results in the induction of iNOS mRNA, protein and iNOS enzyme activity [18]. The induction of iNOS is related to the activation of the transcription factor NFicB [19]. Increases in the local production of NO by the bronchial epithelium of asthmatic could increase the permeability or have cytotoxic effects and contribute to loss of function. Increased NO may contribute to hyperaemia, plasma exudation and mucus production and in the imbalance in the T cell response that underlies the immunological basis of asthma [20].

4. Conclusions In chronic inflammatory diseases increased NO following iNOS induction may produce vasodilatation, vascular permeability and oedema. In addition, NO from iNOS may contribute directly to inflammatory pain. Selective inhibitors of iNOS have considerable potential as anti-inflammatory therapy. Since increased NO and reactive products can fundamentally affect cells to produce a loss of function and cell death inhibitors of iNOS also have the potential to restore cell function in chronic inflammatory disease and be disease modifying. The description of the iNOS crystal structure has heralded a new era in the design of novel inhibitors, which have the greater potency, selectivity, specificity and robust pharmacokinetics required to exploit the clinical potential of iNOS inhibition in chronic inflammatory disease.

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

11.

12.

13.

Moncada S, Palmer RMJ, Higgs EA. Nitric Oxide: physiology, pathophysiology and pharmacology Pharmacol. Rev. 1991, 43, 109-141. Cochran, FR, Selph J, Sherman P. Insights into the role of nitric oxide in inflammatory arthritis Med. Res. Rev. 1996, 16, 547-563. Fretland DJ, Connor JR, Pitzele BS, Currie MG, Manning PT. Inhibition of nitric oxide synthase and prospects for therapy in inflammatory diseases. Cur Pharm Design. 1997, 3, 447-462. Boughton-Smith NK, Tinker AC. Inhibitors of nitric oxide synthase in inflammatory arthritis. I Drugs 1998, 1, 321-333. Hutcheson IR, Whittle BJR, Boughton - Smith NK. Role of nitric oxide in maintaining vascular integrity in endotoxin - induced intestinal damage in the rat. Br. J. Pharmacol. 1990, 101,815- 820. Kubes,P. Granger DN. Nitric oxide modulates microvascular permeability. Am. J. Physiol.1992,262. H611-H615. Kubes P, Suzuki M, Granger DN. Nitric oxide: an endogenous modulator of leukocyte adhesion. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 4651-465. Garvey EP, Oplinger JA, Furfme ES, Kiff RJ, Laszlo F, Whittle BJR, Knowles RG. 1400W is a slow, tight binding inhibitor of inducible nitric oxide synthase. J. Biol. Chem. 1997, 21,4959-4963. Boughton-Smith NK, Ghelani A. Role of induced nitric oxide synthase and increased NO levels in zymosan peritonitis in the rat. Inflamm. Res. 1995, Suppl 2, S149-S150. Salvamini D, Manning PT, Zweifel BS, Seibert K, Connor J, Currie MG, Needleman P. Dual inhibition of nitric oxide and prostaglandin production contributes to the anti-inflammatory properties if nitric oxide synthase inhibitors. J. Clin. Invest. 1995, 96, 301-308. Salvemini D, Wang Z-Q, Wyatt PS, Bourden DM, Marino MH, Manning PT, Currie MG, Nitric oxide: a key mediator in the early and late phase of carrageenan-induced rat paw inflammation. Br..J.Pharmacol. 1996, 118,829-838. Grishham MB, Specian RD, Zimmerman TE. Effects of nitric oxide synthase inhibition on the pathophysiology observed in a model of granulomatous colitis. J Pharmacol. Exp. Ther. 1994, 271, 1114-1221. Aiko S, Fuseler J, Grisham MB. Effcet';s of nitric oxide inhibition or sulphasalzine on spontaneous colitis observed in HLA-B27 transgenic rats. J. Pharmacol & Exp. Thera. 1998, 284, 722 -727.

N.K. Boughton-Smith /Inhibitors of Nitric Oxide Synthase

14. Pelletier J-P, Joanovic D, Fernandas JC, Manning P, Connor JR, Currie MG, DiBattista JA, MartelPelletier J. Reduced progression of experimental osteoarthritis in-vivo by selective inhibition of induced nitric oxide synthase. Arth & Rheum. 1998, 41, 1275-1286. 15. Amin, AR, Abramson SB. The role of nitric oxide in articular cartilage breakdown in osteoarthritis. Curr. Opin. Rheumatol. 1998, 10, 263-272. 16. Ralston SH, Ho LP, Helfrich MH, Grabowski PS, Johnston PW, Benjamin. Nitric oxideia cytokineinduced regulator of bone resorption. J. Bone & Mineral Res. 1995, 10, 1040-1049. 17. Kharitonov SA, Barnes PJ. Exhaled Markers of Pulmonary Disease. Am. J. Crit. Care Med. 2001, 163, 1693-1722. 18. Dweik RA, Comhair SAA, Gaston B, Thunnissen FBJM, Farver C, Thomassen MJ, Kavuru M, Hammel J, Abu-Soud HM, Erzurum SC. NO chemical events in the human airway during the immediate and the late antigen-induced asthmatic response. Proc. Natl. Acad. Sci. USA, 2001, 98, 2622-2627. 19. Adcock IM, Brown CR, Kwon O, Barnes PJ. Oxidative stress induces NFkB DNA binding and inducible NOS mRNA in human epithelial cells. Biochem. Biophys. Res, Comm. 1994, 199, 15181523 20. Barnes J, Liew FY. Nitric oxide and asthmatic inflammation. Immunology Today, 1995, 16, 128-130.

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Disease Markers in Exhaled Breath N. Marczin and M.H. Yacoub (Eds.) IOS Press, 2002

Inhaled NO as a Replacement Therapy George J. BALTOPOULOS and Pavlos M. MYRIANTHEFS Athens University School of Nursing Intensive Care Unit at K.A.T General Hospital ofKifissia, 2 Nikis Street 14561, Athens Greece Abstract. Nitric oxide, normally produced by the body in small quantities having significant physiologic functions, may express altered production or metabolism dependent on disease process, O2 concentration, L-arginine, and NOS isoforms function. Inhaled NO may cause selective pulmonary vasodilatation reducing PVR, PAP, increasing CO and improving V/Q matching and oxygenation. Inhaled NO replacement therapy may have significant supporting role in several critical illnesses associated with pulmonary hypertension, right ventricular failure and severe hypoxemia. However, data are lacking concerning iNO replacement based on actual NO deficiency due to reduced production or increased consumption during disease process; substrates or NOS deficiency or dysfunction.

1. Introduction Nitric oxide (NO) was first identified in 1987 as the endothelial-derived relaxing factor -EDRF [1,2]. NO is a small, colourless, almost odourless, unstable, highly reactive, gaseous free radical, slightly soluble to water, that is freely diffusible across cell membranes. NO can be found normally in the atmosphere as well as in the environment of industrial areas, smog, heavy traffic areas or cigarette smoke produced from combustion processes [3]. Thus, until about 10 years ago, NO was considered as a highly reactive toxic molecule to the body and an environmental pollutant. Work-place limits for exposure to NO have been set to 25 ppm [4]. NO is also commercially produced for medical purposes in inhaled form. NO rapidly reacts with O2 and superoxide converting respectively to nitrites/nitrates (NO2/NO3~) and peroxynitrite (a well known oxidizing and nitrating species with toxic properties) having a half-life period of 3 to 50 seconds [5, 6]. NO is normally produced in small quantities by the body being an endogenous molecule that has important physiologic functions concerning the cardiovascular, immune and nervous system [7]. There are three isoforms of NO synthases (endothelial, inducible and neuronal) which catalyses the synthesis of NO from the terminal quanidine nitrogen of L-arginine producing also L-citruline. Both eNOS and nNOS are constitutive forms that produce small amounts of NO for short periods of time when stimulated maintaining physiologic functions such as vascular tone and neurotransmission. Contrary, iNOS when stimulated produces large amounts of NO for longer periods of time and is thought to be involved in inflammatory responses and immune defence mechanisms against invading pathogens including bacteria and parasites [8]. NO has been the focus of intense basic and clinical research due to its important role as a mediator in inflammation, as a neurotransmitter and a regulator of vasomotor tone.

G.J. Baltopoulos and P. M. Myrianthefs / Inhaled NO as a Replacement Therapy

31

2. Rational of inhaled nitric oxide replacement therapy In blood NO produced by the endothelium, causes vasodilatation by increasing cyclic guanosine 3', 5' monophosphate (cGMP) which subsequently decreases intracellular calcium in the vascular smooth muscles [3, 7, 8]. The understanding of endogenous NOcGMP pathway and its significant role in the modulation of vascular tone led to the idea of using exogenous inhaled NO as a therapeutic pulmonary vasodilator. This assumption was based on the knowledge that NO rapidly binds to hemoglobin and consequently the vasodilatation would be limited to the pulmonary circulation and not to systemic circulation [5]. There are early data suggesting that supplemental inhaled NO vasodilate the pulmonary circulation in a dose depended fashion, decreasing pulmonary artery pressure (PAP) and pulmonary vascular resistance (PVR) in patients with primary pulmonary hypertension [9], improving ventilation/perfusion (V/Q) matching and oxygenation [6, 10] and increasing right ventricular injection fraction (RVEF) [11]. NO replacement therapy could be beneficial to control pulmonary vascular tone, pressure and resistance and improve arterial oxygenation if NO levels proved to be inadequate or reduced in the lungs due to disease process which means that NO may be inadequately produced or extremely consumed or metabolized to nitrites/nitrates and peroxinitrate, Inhaled NO could be also beneficial in idiopathic or secondary pulmonary hypertension in the presence of pulmonary vasoconstriction and right ventricle dysfunction. Inhaled NO may also be beneficial in acute lung injury because of its properties in mediating diverse antioxitant and anti-inflammatory reactions protecting from hyperoxia induced apoptosis and vascular leakage, down regulating the expression of adhesion molecules and reducing extravasation and tissue accumulation of leukocytes. Potential beneficial uses of iNO in clinical practice are: 1) mild bronchodilatation, 2) improved ventilation/perfusion ratio (V/Q) 3) decreased intrapulmonary shunt, 4) decreased right ventricular pressure (RVP), 4) decreased intracardiac shunt, 5) increased right ventricular ejection fraction (RVEF), 6) decreased pulmonary artery pressure (PAP) 7) decreased pulmonary vascular resistance (PVR), 8) increased cardiac output (CO) and 9) increased arterial oxygen tension (PaOi). Inhaled NO received approval by FDA for clinical use in 1999 for term and near term (older than 34 weeks) neonates with hypoxemic respiratory failure associated with pulmonary hypertension. 3. Deficient formation and excessive consumption of endogenous NO in the lungs NO production requires Oi, NADPH, cofactors, calmodulin, L-arginine, and functional NO synmases isoforms [3, 5, 12, 13]. In the lungs NO is produced from the vasculature by the action of eNOS and from the airways by the action of all three NO synthases isoforms [14]. Oxygen is an essential substrate for NO synthesis by NOS and thus hypoxia may contribute to significant decrease in NO production in the hypoxic lung. Acute hypoxia in terms of low inspired fraction of oxygen (FiO2) in normal humans resulted in significant decrease in exhaled NO when inhaled hypoxic gas was 5 % of oxygen [15] but not when inhaled oxygen was 10 % [15, 16]. Also, chronic hypoxia limits NO production in the lungs [17, 18] despite increased expression of all three NOS isoforms [19, 20, 21]. Same findings in chronically hypoxic rat lungs observed by Sato et al [24] at FiOi of 3 % which were most markedly at FiO2 of 0%. L-arginine substrate deficiency or uptake is also associated with reduced NO production which is reversed by L-arginine supplementation [23] suggesting an additional mechanism of decreased NO production and deficiency in the human body.

32

G.J. Baltopoulos and P.M. Myrianthefs / Inhaled NO as a Replacement Therapy

NO production is also dependent on the presence and function of NOS isoforms and it has been shown that eNOS immunostaining of pulmonary arterial endothelial cells was consistently less in patients who died with ARDS compared to non ARDS patients. On the other hand iNOS expression was higher in alveolar macrophages and airway epithelial cells in patients who died with ARDS compared to non ARDS patients [24]. These data suggest an impaired NOS expression in ARDS contributing to changes in NO levels in human lungs. In patients with chronic pulmonary hypertension iNOS expression is also reduced resulting in reduced NO production [25]. Aikio et al [26] showed that macrophages iNOS expression in neonates with fulminant early-onset pneumonia differed showing an impaired production of NO and peroxynitrite due to immature iNOS production. Steudel et al [27] showed that congenital absence of eNOS results in pulmonary hypertension and in combination with long-term hypoxia to increased right ventricular remodelling. In COPD patients exhaled NO production is reduced and NO production from the airways is lower and inversely related to the development of cor pulmonale [28]. In transplant patients endogenous production of NO by cadaver lung allografts in the perioperative period is significantly diminished following one of three patterns which correlates to clinical behaviour [29]. Recently Sittipunt et al [8] showed that NO and its products accumulates in the lungs before and after onset of ARDS, that iNOS is expressed at high levels in alveolar macrophages during ARDS and that nitration of intracellular and extra cellular proteins occurs in the lungs of ARDS. These data indicate an altered production and metabolism of NO in the lungs of ARDS patients. 4. Possible indications for inhaled NO replacement therapy Possible indications of iNO in perioperative, intensive care and ambulatory patients are listed in table 1. Table 1. Indications, underlying pathophysiology and related literature. Indication Comments Pathophysiology Mitral valve surgery, Coronary artery bypass grafting (CABG) Heart transplantation

Pulmonary hypertension Pre-existing pulmonary hypertension

Left ventricular assist device Lung transplantation

Reperfusion injury

Congenital heart disease surgery

Pulmonary hypertension

ALI/ARDS

Hypoxemia, Pulmonary hypertension Hypoxemia, Pulmonary hypertension

Persistent primary pulmonary hypertension of the newborn, Hypoxic respiratory failure Massive pulmonary embolism COPD Sickle cell disease, acute syndrome Cystic fibrosis

chest

Pulmonary hypertension, RV failure Pulmonary hypertension Infarction, hypoxemia, pulmonary hypertension Reduced expression of iNOS, and exhaled NO

Decreased PVR, improved CO in RV failure [7, 30] protection from RV failure [29, 3 1 ] Prevention of RV failure [32] Prevention from pulmonary hypertension and RV failure [33] Decrease in PVR, increase in cardiac output [34] Improve oxygenation, decrease PVR, [35, 36, 37, 38] Improve oxygenation, decrease PVR [39,40,41,42] Decrease PAP, increase CO [43] Improve oxygenation, decrease PVR [44] Improve oxygenation [45] Reversion of airway obstruction [46]

GJ. Baltopoulos and P.M. Myrianthefs / Inhaled NO as a Replacement Therapy

33

5. Conclusions Inhaled NO therapy could be an assisting-supporting therapeutic measure in several clinical conditions of critical illness in adult and paediatric patients associated with pulmonary hypertension, RV failure and hypoxemia. Despite lack of benefit in terms of outcome, iNO has several certain indications as a rescue therapy in patients non responding to other therapeutic measures. More studies are needed to determine the need of iNO in terms of absolute or relative NO deficiency (decreased production, increased consumption) in these conditions. References [I] RM Palmer, AG Ferrige, S Moncada. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature (1987) 327:524-526. [2] LJ Ignarro, GM Buga, KS Wood, RE Byrns, G Chaudhuri. Endotheliurn-derived relaxing factor produced and released from artery and vein is nitric oxide. Proc Natl Sci USA (1987) 84:9265-9269. [3] W Steudel, WE Hurford, WM Zapol. Inhaled nitric oxide. Basic biology and clinical applications. Anaesthesiology (1999) 91:1090-1121. [4] NIOSH recommendations for occupational safety and health standards. MMWR Morb Mortal Wkly Rep (1998)37:1. [5] E Anggard. Nitric oxide: mediator, murderer, and medicine. Lancet (1994) 343:1199-1206. [6] N Jindal, RP Dellinger. Inhalation of nitric oxide in acute respiratory failure. J Lab Clin Med (2000) 136:21-28. [7] E Haddad, SM Lowson, RA Johns, GF Rich. Use of inhaled nitric oxide perioperatively and in intensive care patients. Anaesthesiology (2000) 92:1821-1825. [8] C Sittipunt, KP Steinberg, JT Ruzinski, et al. Nitric oxide and nitrotyrosine in the lung of patients with acute respiratory distress syndrome. Am J Respir Crit Care Med (2001) 163:503-510. [9] J Pepke-Zaba, TW Higenbottam, AT Dihn-Xuan, D Stone, J Wallwork. Inhaled nitric oxide as a cause of selective pulmonary vasodilatation in pulmonary hypertension. Lancet (1991) 338:1173-1174. [10] Rossaint R, Falke KJ, Lopez F, et al. Inhaled nitric oxide for the adult respiratory distress syndrome. N Engl J Med 1993;328:399-405. I I 1 ] L Fierobe, F Brunet, J Daihnaut, et al. Effects of inhaled nitric oxide on right ventricular function in adult respiratory distress syndrome. Am J Respir Crit Care Med (1995) 151:1414-1419. [12] CM Hart. Nitric oxide synthesis in the lung. Chest (1999) 115:1407-1414. [13] RA Dweik, D Laskowski, HM Abu-Soud, et al. Nitric oxide synthesis in the lung. J Clin Invest (1998) 101:660-666. [14] TD Le Grass, IF McMurtry. Nitric oxide production in the hypoxic lung. Am J Physiol Lung Cell Mol Physiol (2001) 280:L575-L582. [15] L Schmetterer, K Strenn, J Kastner, H Eichler, M Woltz. Exhaled NO during graded changes in inhaled oxygen in man. Thorax (1997) 52:736-738. [16] I Tsujino, K Miyamoto, M Nishimura, et al. production of nitric oxide in intrathoracic airways of normal humans. Am J Respi Crit Care Med (1996) 154:1370-1374. [17] TC Isaacson, V Hampl, EK Weir, DP Nelson, SL Archer. Increased endothelium-derived NO in hypertensive pulmonary circulation of chronically hypoxic rats. J Appl Physiol (1994) 76:933-940. [18] M Muramatsu, RC Tayler, DM Rodman, IF McMurtry. Trapsigargin stimulates increased NO activity in hypoxic hypertensive rat lungs and pulmonary arteries.. J Appl Physiol (1998) 80:1336-1344. [19] TD Le Grass, C Xue, A Rengasamy, RA Johns. Chronic hypoxia upregulates endothelial and inducibie nitric oxide synthase gene and protein expression in rat lung. Am J Physiol Lung Cell Mol Physiol (1996) 270:L164-L170. [20] TC Resta, RJ Gonzales, WG Dail, TC Sander, BR Walker. Selective upregulation of arterial endothelial nitric oxide synthase in pulmonary hypertension. Am J Physiol Heart Circ Physiol (1997) 272:H806-813. [21] PW Shaul, AJ North, TS Brannon, et al. Prolonged in vivo hypoxia enhances nitric oxide synthase type 1 and type III gene expression in adult rat lung. Am J Respir Cell Mol Physiol (1995) 13:167-174. [22] K Sato, K Nair, J Hiddinga, et al. Hypoxia inhibits ETB receptor mediated NO synthesis in hypertensive rat lungs. Am J Respir Cell Mol Physiol (1999) 276:L571-L581. [23] CD Fike, MR Kaplowitz, LA Rehorst-Paea, LD Nelin. L-arginine increases nitric oxide production in isolated lung of chronically hypoxic newborn pigs. J Appl Physiol (2000) 88:1797-1803. [24] KH Albertine, ZM Wang, JR Michael. Expression of endothelial nitric oxide syntahse, inducibie nitric oxide synthase, and endothelin-1 in lungs of subjects who died with ARDS. Chest (1999) 116:1018-1025.

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G.J. Baltopoulos and P.M. Myrianthefs / Inhaled NO as a Replacement Therapy

[25] A Giad, D Saleh. Reduced expression of endothelial nitric oxide synthase in the lungs of patients with pulmonary hypertension. N Engl J Med (1995) 333:214-221. [26] O Aikio, K Vuopala, M-L Pokela, M Hallman. Diminished inducible nitric oxide synthase expression in fulminant early-onset neonatal pneumonia. Pediatrics (2000) 105:1013-1019. [27] W Steudel, M Scherrer-Crosbie, KD Bloch, et al. sustained pulmonary hypertension and right ventricular hypertrophy after chronic hypoxia in mice with congenital deficiency of nitric oxide synthase 3. J Clin Invest (1998) 101:2468-2477. [28] E Clini, G Cremona, M Campana, et al. Production of endogenous nitric oxide in COPD and patients with cor pulmonale correlates with echo Doppler assessment. Am J Respir Crit Care Med (2000) 162:446-450. [29] N Marczin, B Riedel, J Gal, J Polak, M Yacoub. Exhaled nitric oxide during lung transplantation. Lancet (1997)350:1681-1682. [30] DA Fullerton, J Jaggers, MM Wollmering, F Piedaule, FL Groer, RC Mclntyre. Variable response to inhaled nitric oxide after cardiac surgery. Ann Thorac Surg (1997) 63:1251 -1256. [31] N Keiler-Jensen, S Lundin, SE Ricksten. Vasodilator therapy after heart transplantation: Effects of inhaled nitric oxide. J Heart Lung Transplant (1995) 14:436-443. [32] M Argenziano, A Choundhri, N Moazami, EA Rose, CR Smith, HR Levin. Randomised, double blind study trial of inhaled nitric oxide in LVAD recipients with pulmonary hypertension. Ann Thorac Surg (1998) 65:340-345. [33] H Date, AN Triantafillou, EP Trulock, MS Pohl, JD Copper, GA Patterson,. Inhaled nitric oxide reduces human allografts dysfunction. J Thorac Cardiovasc Surg (1996) 111:913-919 [34] IA Russel, MS Zwass, JR Fineman, M Balea, K Roine-Rapp, M Brook. The effects of inhaled nitric oxide on postoperative pulmonary hypertension in infants and children undergoing surgical repair of congenital heart disease. Anaesth Analg (1998) 87:46-51. [35] RP Dellinger, JL Zimmerman, RW Taylor et al Effects of inhaled nitric oxide in patients with acute respiratory distress syndrome: Results of a randomised phase II trial. Inhaled Nitric Oxide in ARDS Study Group. Crit Care Med (1998) 26:15-23. [36] S Lundin, H Mang, M Smithies, O Stenqvist, C Frostell. For the European Study Group of Inhaled Nitric Oxide. Inhalation of nitric oxide in acute lung injury: results of a European multicentre study. Intensive Care Med (1999) 25:911-919. [37] D Payen, B Vallet and "Group d' Etude du NO dans I' ARDS". Results of the French prospective multicentric randomised double blind placebo controlled trial on inhaded nitric oxide (NO ) in ARDS.Intensive Care Med (1999) SI66. [37] R Ullrich, C Lorber, G Roder et al. Controlled airway pressure therapy, nitric oxide, prone position and extracorporeal membrane oxygenation as components of an intergrated approach to ARDS. Anaesthesiology (1999) 91:1577-1586. [38] E Troncy, JP Collet, S Shapiro, et al. Inhaled nitric oxide in acute respiratory distress syndrome. A pilot randomised controlled study. Am J Respir Crit Care Med (1998) 157:1483-1488. [39] R Clark, T Kueser, M Walker et al for the Clinical Inhaled Nitric Oxide Research Group. Low dose nitric oxide therapy for persistent pulmonary hypertension of the newborn. N Engl J Med (2000) 342:469-474. [40] R Smyth. Inhaled nitric oxide treatment for pre-term infants with hypoxic respiratory failure. Thorax (2000)55:S51-S55. [41] The Neonatal Inhaled Nitric Oxide Study Group. Inhaled Nitric Oxide in Full-Term and Nearly Full-Term Infants with Hypoxic Respiratory Failure. N Engl J Med (1997) 336:597-604. [42] D Cornfield, R Maynard, R deRegnier et al. Randomized, controlled trial of low dose of inhaled nitric oxide in the treatment of term and near-term infants with respiratory failure and pulmonary hypertension. Pediatrics (1999) 104:1089-1094. [43] G Capellier, T Jacques, P Balvay, G Blasco, E Belle, F Barale. Inhaled nitric oxide in patients with pulmonary embolism. Intensive Care Med (1997) 23:1089-1092. [44] M Yoshida, O Taguchi, EC Gabazza, T Kobayashi, T Yamakami, H Kobayashi. Combined inhalation of nitric oxide and oxygen in COPD. Am J Respi Crit Care Med (1997) 155:526-529. [45] AM Atz, DL Wessel. Inhaled nitric oxide in sickle cell disease with acute chest syndrome. Anesthesiology (1997) 87(4):988-990. [46] MJ Mhanna, T Ferkol, RJ Martin, et al. Nitric oxide deficiency contributes to impairement of airway relaxation in cystic fibrosis mice. Am J Respir Cell Biol (2001) 24 (5):621-626.

Disease Markers in Exhaled Breath N. Marczin and M.H. Yacoub (Eds.) IOS Press, 2002

35

Physiology of Exhaled Nitric Oxide L. Christofer ADDING and Lars E. GUSTAFSSON Department of Physiology and Pharmacology, Karolinska Institute, Stockholm, Sweden.

Abstract. Nitric oxide in exhaled air denotes the ongoing enzymatic production of this "simple" but essential mediator molecule in the respiratory system where it exerts important physiological functions. To appreciate the value of exhaled nitric oxide measurements one must comprehend that nitric oxide in exhaled air does not represent a waste product from systemic sources passively excreted by the blood. Nitric oxide production in the respiratory system is a necessity at birth and continuous throughout life. In fact, post mortem during circulatory arrest in experimental animals airway levels of exhaled nitric oxide increases provided that ventilation of the lungs are maintained. Experimental studies of exhaled nitric oxide show that the enzymatic production of nitric oxide in the lung is a constant process that endure many investigational provocations. However, there are a few intriguing physiological and pathophysiological conditions, pharmacological treatments and other experimental manoeuvres (e.g. stretch, carbon dioxide, inflammation, catecholamines) that per se alter (increase or decrease) the levels of exhaled nitric oxide. These biological observations and clinical findings becomes even more intriguing considering the fact that for some of these alterations in respiratory NO production we do not know if they are beneficial or detrimental. The standard method for measurements of exhaled nitric oxide is based on the highly specific and sensitive chemiluminescence technique and was first described in 1991. However, the method is not without pitfalls and precaution must be taken when interpreting exhaled nitric oxide data. Particular attention should be paid to physiological factors that are known to confound exhaled nitric oxide measurements and consequently predispose for erroneous conclusions. Thus, in this chapter we summarize the fundamental findings of exhaled nitric oxide measurements and give the reader a basic survey of the physiological regulation of exhaled nitric oxide.

1. Introduction Direct measurements of nitric oxide (NO) have been a problem in NO research, due to NO's rapid oxidation in biological tissues with a half-life for NO only of seconds. Several assays have been used that indirectly measure NO production e.g. cGMP, nitrite (NO2") or citrulline conversion, reflecting the effect of NO, NO metabolism or NO synthesis, respectively, see [1]. In 1987 Palmer et al. [2] introduced a chemiluminescence method, originally developed by environmental scientists to detect nitrogen oxides in the atmosphere [3, 4], and direct measurements of NO released from cells could be made. However, the method determined the sum of NO and NO2" in liquid samples from cells or tissues and recovery of NO was dependent on rapid sample acquisition to avoid oxidation. In 1991 Gustafsson and colleagues used this method to analyse lung perfusate during hypoxic

36

L C. Adding and L E. Gustafsson / Physiology of Exhaled NO

conditions to elucidate the role of NO during hypoxic pulmonary vasoconstriction (HPV). During these experiments it was postulated that NO, being a gas with a low solubility in aqueous solution, would escape into the airways and be detectable in exhaled gas. This hypothesis proved to be right and direct evidence for the production of NO in humans was thus presented [5-7]. The discovery of NO in exhaled gas has not only offered a new approach to directly study features of NO metabolism in vivo, but also exhaled NO measurements seem to be a valuable non-invasive diagnostic tool of airway inflammation [8, 9]. Almost 600 studies in the field of exhaled NO research have been published with sometimes opposing results leading to conflicting interpretation. This chapter gives a review of the most important physiological factors that will influence exhaled NO.

2. Chemilummescence measurement of exhaled NO 1. Identity of exhaled NO Measurements of NO in respiratory gas by the chemiluminescence reaction is based on the finding that ozone reacting with NO yields excited NO2 which subsequently emits a photon of preferentially infrared light. The number of emitted photons are thus directly proportional to the original NO abundance, and the photons can be counted by a photomultiplier tube. Many compounds may react with ozone under the emission of light, but when the method employs red filters with a cut-off such that only light with wavelength above 600 nm is measured the method becomes quite specific although not unique to NO. For another substance to elicit chemiluminescence such a substance must be volatile and have chemical properties, not likely to occur in biological systems, that will make it react with ozone [1]. In the initial study [5], the presence of NO in exhalates of humans and guinea pigs was demonstrated by three separate methods including reacting exhaled gas with thioproline and subsequently demonstrating the presence of nitrosothioproline by gas chromatography/mass spectrometry (GcMS). In a later study [7], direct GcMS on exhaled gas was performed using a cooled gas chromatography column to separate NO from other molecules having a mass of 30 and present in normal athmosphere in significant amounts, e.g. ISI5 N 2 , and the presence of NO in exhalates from humans was thus confirmed in the most direct manner. A limitation of this method was that it was not linearly quantitative. Thus, there was therefore a need to evaluate exhaled NO during quantitative chemiluminescence measurements and using physicochemical means to test for its presence. This was initially done in humans and rabbits using a cold trap at -80 C [5]. A later study was done in humans, rats, guinea pigs, and rabbits [10]. Here the methodology employed was to carry exhaled gas through a cold trap cooled to temperatures where other gases than NO would be expected to be frozen out of exhaled gas, finally taking the temperature down where also the NO signal disappeared. The cooling trap consisted of a copper or stainless steel loop, having a configuration allowing counter-current exchange of heat from afferent to efferent limb to ascertain attainment of desired temperatures. Mixtures of dry ice, acetone and liquid nitrogen allowed temperatures of the coil down to -196 C. During these experiments it was verified that the chemiluminescence signal disappeared only when coil temperature was brought below the freezing point for NO, giving a more facile verification that the chemiluminescence signal derives from exhaled NO. Another support for exhaled NO as the cause for chemiluminescence is testing for the possibility to make it disappear by pharmacological inhibition of its biochemical formation.

LC. Adding and L.E. Gustafsson / Physiology of Exhaled NO

37

The chemiluminescence signal of exhalate is inhibited or disappears during administration of NO-synmase (NOS) inhibitors of L-arginine-derivatives type, and the signal can be partially or fully restored by a surplus of the natural substrate for NO formation, L-arginine (see table 1 and 2 for refs.). 2. Interfering compounds during NO measurements The chemiluminescence signal in NO measurement is reduced by water vapor, carbon dioxide and nitrous oxide [11-13]. Therefore sampled exhaled gas should preferably be led into a dehumidifying tube before entering the NO analyzer and a correction of the NO signal for the quenching effect by CO2 is a necessity if large variations of end-tidal CO2 are encountered. Many chemiluminescence analyzers have a cooled PMT tube, and if not adequately insulated this leads to a cooling of the window/filter between the reaction chamber and the PMT tube, causing condensation or even frost and significant loss in signal when measuring in biological, humid, systems. For this and several other reasons, in clinical measurements only clinically approved equipment should be used in a procedure approved by appropriate regulatory authorities, and in experimentation the investigator should be keenly aware of this kind of problem source. Some studies show that atmospheric NO levels may bias exhaled NO measurements so that exhaled NO increases during days with high NO levels in ambient air [14, 15] whereas others have found no such influence of ambient NO [16]. Recent studies suggest that outdoor air pollution per se may increase endogenous exhaled NO in healthy subjects [17, 18]. To exclude these uncertainties in exhaled NO measurements NO-free inspired air should be used when exhaled NO measurements are performed. A proper charcoal filter, using a sufficiently large filter with the right proportions of length to diameter can be used to make NO free air out of ambient air. Although recent data suggest that a charcoal filter does not remove NO from hospital compressed air [19], our own experience shows that this can be achieved [20]. With these considerations, pollutant-induced changes in exhaled NO should, in our view, be taken as biological effect measures and not as interference with the determination method. It is obvious that ventilatory or exhalation flow rates [21-26] and the pulmonary diffusing capacity of NO into the blood [27, 28], the diffusion capacity being 4.5 times that of carbon monoxide [29], influence the concentrations of NO in exhaled gas. This might be of importance when exhaled NO concentration decreases and NO excretion increases during heavy exercise or hyperventilation [30-35]. This phenomenon has recently been reviewed [36]. It might be that this effect is merely due to the relative changes in ventilation which will decrease the NO concentration gradient between the NO producing cells and the capillary blood, thereby decreasing the net diffusion of NO to the blood [28, 33]. Whether these changes in exhaled NO reflect an increased NO synthesis per se, the postulated mechanisms involving stretch- and catecholamine-induced NO formation [37, 38], will be discussed below. 3. Bioactive NO concentrations Once NO formation is completed in any tissue, regardless of the site of formation, NO diffuses in all directions until it reacts with potential target molecules and is further metabolised. Nitric oxide is lipophilic, has a modest solubility in aqueous solution (similar with oxygen) and NO molecules readily diffuse across cell-membranes. However, net

38

L.C. Adding and LE. Gustafsson /Physiology of Exhaled NO

diffusion of NO molecules, from the site of formation, will preferentially occur toward areas where NO concentrations are lowest. Consequently, if NO production and elimination in the respiratory tissue is constant, in time equilibrium of NO concentration with the surroundings would occur. However, in the respiratory system due to ventilation, measurements of NO will not give the absolute tissue concentration of NO, but will only reflect part of it.

Table 1. Physiological factors regulating exhaled endogenous nitric oxide in experimental animals, t (increase) ^(decrease), <-> (unchanged) Condition Acute airway hypoxia

Chronic airway hypoxia Selective gas- phase vs. vascular hypoxia Alveolar/ airway hyperoxia Carbon dioxide inhalation

Gas emboli i.v. pH (acidosis)

Stretch activation (PEEP, NEEP, CNETP, HFOV) Stretch inhibition (CPETP, Gd i.v.) «radrenoceptors ^-adrenoceptors (i.v.) Endothelin A-rec. Germ-free animals Endotoxin (LPS)

Ethanol (i.v.) Adenosine (i.v.) Angiotensin II i.v. Allergen, Histamine, LTC4 inhal.

Species Rabbit

Exh. NO 1

Pig Cat Pig (newborn) Rabbit

1

Rat Rabbit Guinea pig Dog Rabbit/Dog Rabbit Dog Rat Rabbit Guinea pig Rabbit/ guinea pig Horse Rabbit Rat Rat Pig Pig Rat Dog Rabbit Rabbit Guinea pig Guinea pig

T 4

; <-> i i i t t t t t t i i t t <-> «-» t t t i t t t <2 min 4> 10 min

Reference (5, 44) (45, 46) (47-49) (50) (5!) (46)

(52, 53) (12,45,54) (55) (56) (5, 57) (54) (58) (59) (12,45,60) (55,61) (12,61,62)

(63) (38) (64) (10) (65) (65, 66) (64, 67-72) (73) (74) (75) (76) (77-80)

L.C. Adding and L.E. Gustafsson / Physiology of Exhaled NO

Table 2. Factors affecting exhaled nitric oxide in healthy humans, t (increase) ^(decrease), <-> (unchanged). Provocation Acute alveolar hypoxia Post-moderate altitude stay Circadian rythm Menstrual midcycle Pregnancy Physical exercise

Oral NO

; 4 «t t*> <->

Nasal NO <->

<->

Texcretion iconcentr. texcretion ^concentr.

1

Breathhold Cold air inhal./ hypothermia Spirometry Cigarette smokers vs. nonsmokers

T 1 1 4

t

Cessation of smoking Cigarette smoke inhal. Outdoor air pollution Indoor formaldehyde levels Acute ozone inhalation Chronic ozone inhalation Influenza virus (nasal/ vaccination) Asthma; allergen challenge L-Arg p.o./ i.v./ inhal. Nitrovasodilators (i.v.) Dobutamine (i.v.) Corticosteroids (p.o./ nasal) Ethanol (p.o.) Ibuprofen (p.o.) Enalapril (ACE inhibitor p.o.) /?^agonist (inhal.) Prostacyclin (inhal.) Prostaglandin £2 & ¥2°(inhal.) L-NMMA (inhal./ i.v.) L-NAME (inhal./ nasal) Aminoguanidine (inhal.) «r agonists (nasal) Capsaicin (nasal) Histamine (nasal) Papaverin (nasal)

t t t t

Hyperventilation

(30-32, 34)

4

(31,96-106) (107, 108) (109) (110-116)

^>4

(117, 118) (119, 120) (17,18) (121) (122) (123) (124, 125)

4-> t t t t t

t <-»

<-> <->

<->

<-» 4 t <-»

4

<-> 4 1

4 <->

References (81,82) (33) (83) (84, 85) (86-89) (90) (30-35,91-95)

1^

4 •f^ •e> t

(110, 126-128) (129-133) (134, 135) (34) (130, 136) (122, 137) (138, 139) (140) (200) (141-144) (145) (146) (37, 137, 147-151) (152-159) (153) (154, 158, 159) (154) (154) (158,159)

39

40

L C. Adding and LE. Gustafsson /Physiology of Exhaled NO

In general it is the total dose of NO delivered to the site of action which is likely to be responsible for any biological effect [39]. Murad and colleagues found that the half maximal concentration (apparent K.,,.,) of NO required to activate purified soluble guanylate cyclase was in the 1-10 nM range [40]. Therefore, the most important question is, can airway NO concentrations in the ppb range exert biological effects? Indeed, in rabbits half-maximal reversal of pulmonary vasoconstriction by NO synthase inhibition was obtained by inhalation of approximately 15 nM NO gas [41] and other NO inhalation studies show that that NO concentrations below 30 ppb can influence pulmonary circulation [41-43]. Actions of low concentrations of NO are probably favoured by its high affinity for heme (e.g. in guanylate cyclase) which is 10 000-fold that of oxygen.

3. Physiological factors regulating exhaled NO formation 7. Pulmonary blood flow and exhaled NO Nitric oxide is rapidly inactivated by haemoglobin and therefore a major determinant of NO elimination in the airways and alveolus would be the abundance of blood in the pulmonary circulation. However, in animals neither volume loading (160) nor moderate pulmonary artery obstruction (60) and in humans neither modifying pulmonary blood flow by increased gravity (2 G) nor head-out water immersion affected the exhaled NO concentrations (161). Thus, changes in pulmonary blood-flow within physiological range will not alter exhaled NO. Nevertheless, extreme reductions in pulmonary blood flow, due to severe hypovolemic haemorrhage, increase exhaled NO concentrations (Figure 1 and [162]). Also, in patients with atrial septal defect, closure of the defect decreased pulmonary blood flow which was associated with a decrease in exhaled NO [163].

2. Airway hypoxia and exhaled NO Molecular oxygen is a substrate for NOS and it has been shown that oxygen concentrations likely to occur in hypoxic tissue can fall below the Km value for oxygen of type III NOS [164]. Furthermore, a similar value of Km was found when plotting a Lineweaver-Burke diagram between alveolar oxygen tension and exhaled NO in rabbits ex vivo [46]. Thus oxygen availability is necessary for an optimal pulmonary NO production. In accordance, acute hypoxia has been shown to decrease exhaled NO in vivo [5, 33, 48, 82], in perfused lungs [44, 46, 47, 51, 165] and in pulmonary endothelial cells [166, 167]. Doseresponse studies of FIO2 on exhaled NO show that NO production markedly ceases below FIO2 < 5 % [44, 46, 201]. Along similar lines chronic hypoxia in newborn piglets reduced exhaled NO [51] but chronic hyperoxia has not been found to increase exhaled NO [52]. Nitric oxide and oxygen are two powerful vasodilators in the pulmonary circulation. In fact NO can reverse pulmonary vasoconstriction induced by O2-deficiency (HPV) [168, 169] and, likewise, oxygen can reverse pulmonary vasoconstriction induced by NO-deficiency [41, 62]. Therefore, the most efficacious vasoconstriction in the lung occurs when oxygen and NO are not present as is the case for the fetus (whole lung) and in any hypo-ventilated part of a lung. Thus, exhaled NO production is maintained as long as

L, C, Adding and L.E. Gustafsson / Physiology of Exhaled NO

41

90- Start of bleeding 6030-

0

o u H

5 0J

Figure 1. Recording of the effect of haemorrhage (arrow indicates start of bleeding), until circulatory arrest and post-mortem, on exhaled NO production and end tidal carbon dioxide (ETCO2) in an anaesthetized and mechanically ventilated rabbit {in vivo). Shown is also the effect of carbon dioxide inhalation (CO2, black bars) before and after circulatory collapse, Note the marked effect of CO2-inhalation even after circulation ceased.

airway oxygen tensions are high enough, FIO2 > 5 % , [46] to contribute to the oxygenation of blood. In a recent study it was found that the alveolar-to-arterial difference for oxygen was reciprocally correlated with exhaled NO thus favouring the idea of positive relation between exhaled NO and blood oxygenation efficiency [170], However, in dogs a NO synthase inhibitor given by nebulization unexpectedly improved ventilation perfusion ratio thereby disputing the role for endogenous NO promoting ventilation perfusion ratio regulation [56]. These differences could be due to species differences since it seems that in dogs vasodilator prostaglandin is more important than NO during basal conditions [171], In man a clear-cut increase in pulmonary vascular resistance has been demonstrated upon administration of NO synthase inhibitor [148, 172, 173]. 3. Stretch related effects and exhaled NO As mentioned above it has been speculated that an increased flow rate of respiratory gas will induce NO formation in airway epithelial cells, resembling the flow (shear stress) induced NO production in vascular endothelial cells [31]. Electron microscopy studies show that type III NOS is associated with microtubuli of the cilia in bronchial epithelial cells [174], suggesting that epithelial cilia may in some way regulate epithelial NO production. Indeed, there is evidence for increased NO production from airway epithelial cells during increased ciliary activity in response to ^-adrenoceptor stimulation [175].

42

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9
in vivo

buffer perfused lung

6030-

0J

O(N X

20-1

s 2 min

PEEP

PEEP

Figure 2. Recording of the effect of PEEP (black bars) on exhaled NO production and insufflation pressure (IP) in an anaesthetized and mechanichally ventilated rabbit (in vivo, left) and, later in the same animal, during buffer perfusion of the lungs (buffer perfused lungs, right). Note the higher basal levels of exhaled NO in the perfused lung and that the effect of PEEP on exhaled NO is still pronounced though the blood is no longer present.

A recent study in rabbits showed a significant increase in exhaled NO in response to increased time-weighted tidal volume during pressure-controlled inverse ventilation [176]. This study lends support to another even more speculative possibility of a stretch-dependent and breathing-cycle differentiated formation of airway NO, with a rapid activation of NO production and an increased NO release during inspiration, locally yielding very high NO concentrations in well-ventilated (stretched) distal airways and alveoli. This would mean that we profoundly underestimate lower airway NO concentrations when we measure it in the exhaled gas during an active expiration. A stretch-dependent cellular mechanism in the airways would be in accordance with the mechanically stimulated endothelial NO synthesis encountered under physiological conditions [177, 178, 179, 180], and similar stretchresponsive mechanisms influencing NO release have been established in both striated muscle cells and osteoblastic cells of bone [181, 182]. Our group observed that positive end-expiratory pressure (PEEP) in guinea pigs and rabbits increased lower airway NO formation [60]. The mechanism of these PEEP-induced dose-dependent increases of exhaled NO is still not known but the observation has been confirmed in the blood perfused rabbit lung [45]. The authors of the latter study pointed to the possibility that pulmonary capillaries may become compressed by PEEP and lose their blood, resulting in less scavenging of NO by haemoglobin. The interpretation is likely, especially in situations when high PEEP pressures that interfere with pulmonary blood flow are applied [60, 61]. Nevertheless, the effect of PEEP on exhaled NO is still present in saltbuffer perfused lungs in situ, with no haemoglobin present (Figure 2), and therefore reduced capillary blood filling cannot fully subserve as the mechanism involved. The involvement of the vagus nerves [60] and a correlation with changes in functional residual capacity (FRC) [12] led to the suggestion that mechanical stretching of lung tissue might be one underlying

L. C. Adding and L.E. Gustafsson / Physiology of Exhaled NO

43

mechanism modulating exhaled NO in response to PEEP see [6]. In addition we recently demonstrated that the process of high frequency oscillatory ventilation (HFOV), which is mechanically characterized by small amplitudes and a very high frequency of tidal variation, significantly increased intra-airway NO concentrations and total excretion of NO, in both rabbits in vivo and in buffer perfused lung, in comparison with conventional ventilation suggesting an increase of total pulmonary NO production rather than an increased elimination of NO from the respiratory system. The effect of HFOV on exhaled NO production significantly exceeds the PEEP-related FRC effect during conventional ventilation. Therefore the existence of preferentially high frequency stretch-responsive mechanisms which regulate pulmonary NO production seems likely since such a mechanism has been revealed in endothelial cells [183]. A similar mechanism in the lung, the cellular basis of which remains to be shown, could explain all observations made in relation to stretch responses on exhaled NO, provided moderate degrees of PEEP and the alike are considered. Moreover, it would be in agreement with the suggested role of NO during pulmonary vascular adaptation at birth [184] and the recent findings of pulmonary vascular smooth muscle potassium-channels which are sensitive to both stretch and NO [185-187]. Specific stretch-activated channels in the endothelial cell membrane convert the mechanical stimuli into intracellular calcium mobilization in the micromolar range [188, 189] sufficient to activate the NOS [190]. Stretch-activated channels have also been identified in several pulmonary cells, including alveolar, endothelial, smooth muscle and airway epithelial cells [191-195]. For that reason we performed experiments with gadolinium chloride (GdCl3), an inhibitor of stretch-activated channels, and found that GdCl3 significantly inhibited basal and PEEP-induced pulmonary NO production in guineapigs and rabbits [61, 62]. In the latter study GdCl3 induced a major increase in pulmonary vascular resistance and decreased arterial PO2. 4. Effect of Carbon Dioxide on exhaled NO production Inhaled carbon dioxide (CO2) has been shown to inhibit formation of NO in exhaled air of rabbits [12], guinea-pigs [55] and dogs [56]. Furthermore, a specific CO2-dependent regulatory mechanism on pulmonary NO production could be separated from non-specific actions elicited by CO2-inhalation in vivo, e.g. changes in blood pH, sympathetic activation or pulmonary blood flow, in a study with HEPES buffer-perfused lungs [54]. During control conditions we also observed higher concentrations of exhaled NO in HEPES-buffer solution perfused lungs (~ 80 ppb), where no CO2 was added to respiratory gas, than previously reported in bicarbonate-buffer solution perfused lungs (~ 50 ppb) where CO2 was added to the inspired gas to achieve end-tidal CO2 readings within the normal range [44, 45, 196]. These observations support the findings that CO2 exerts an inhibitory effect on pulmonary NO production. Another interesting observation is the rapid and large increase in exhaled NO when blood flow through the pulmonary circulation ceases at the start of buffer-solution perfusion. The common opinion is that the increase in exhaled NO is a consequence of decreased scavenging by haemoglobin due to the reduced pulmonary blood flow. However, inhaled CO, can reverse a major part of the increment in exhaled NO seen post-mortem (Figure 1).

44

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100

0

3

6

9

12

% CO2 in inhaled air Figure 3. Concentration dependence of the inhibitory effect of inhaled CO2 on basal levels (solid circles) and on PEEP-induced formation (open circles) of exhaled lower airway nitric oxide in guinea pigs in vivo. Values are means ± SEM; n=5. *, significant difference (P< 0.05) between the nitric oxide concentration in exhaled tracheal air during inhalation of CO2 and the pre-exposure value; paired t-test. (From (55), with permission).

The inhibitory effect of CO2 on exhaled NO is dependent on the level of stretch (PEEP) applied to the lungs. At a moderately high PEEP, when pulmonary NO production is increased but pulmonary vasculature is yet not compressed by PEEP, CO2 is a more effective inhibitor of NO production (Figure 3) [55]. However, the mechanism whereby inhaled CO2 inhibits pulmonary NO formation is still unknown. It is noteworthy that the concentration response curve is steep in the biologically relevant interval. When high levels of PEEP are applied (10 cm H2O and above) in vivo it is our experience that effects of CO2 are minor, as well as are further effects of PEEP as discussed above. It might well be that under these conditions contribution to exhaled NO from an alveolar macrophage dependent non-calcium dependent component is quite significant, due i.a. to less scavenging from nearby capillary blood hemoglobin, and the responses therefore become blunted. In artificial ventilation systems there might be very significant inherent apparatus auto-PEEP. If this goes unnoticed, it is not surprising that it might be difficult to demonstrate PEEP or CO, effects, on exhaled NO. This might explain some controversies in the literature on PEEP and CO2 effects on exhaled NO. 5. Adrenoceptors and exhaled NO Stimulation of a-adrenoceptors by nasally nebulized methoxamine, an a,-adrenergic agonist, reduced nasally exhaled NO in horses [63]. Likewise, it has been shown that the

LC. Adding andLE. Gustafsson / Physiology of Exhaled NO

45

nasal decongestant, oxymetazolin (a,-adrenergic agonist), significantly lowers the concentrations of NO in the nasal cavities of humans [154, 158, 159]. It is not clear whether these changes in NO occur in response to a reduced local blood flow and/or reduced temperature in the nasal cavity [158] or if a more direct a-adrenergic regulatory effect on the NO synthase is involved. Inhalation of salbutamol (f32-adrenoceptor agonist) does not increase exhaled NO in healthy humans [141, 143] but may increase exhaled NO in asthmatics taking inhaled glucocorticosteroids [142]. A recent in vitro study of cultured tracheal epithelial cells concluded that isoproterenol, an unselective B-adrenoceptor agonist, could stimulate NO production [175] and that the mechanism involved cAMP dependent activation of a

pulmonary arteriole

terminal bronchiole

Poorly ventilated Hypoxia Hypercapnea Low stretch

Well ventilated Oxygen

Pulmonary veins Figure 4. Local regulation of nitric oxide production. Nitric oxide is acting in concert with oxygen to dilate pulmonary arteriole in a well ventilated region. In the poor ventilated region, carbon dioxide accumulates, airway/alveolar hypoxia occurs and stretch is minimal. Therefore nitric oxide production decrease and HPV can fully develop promoting V/Q-matching.

calcium-independent NO-synthase [197]. We have found that adrenaline via p,adrenoceptors stimulates the release of NO into the airways of anaesthetised rabbits [38]. In this study forskolin (an activator of adenylate cyclase), even in concentrations causing profound hypotension and tachycardia, did not stimulate NO production. The lack of effect of an adenylate cyclase stimulator suggests that elevation of cAMP is not a key event in rabbit exhaled NO production. However, administration of the calcium-channel blocker

46

L.C. Adding and LE. Gustafsson /Physiology of Exhaled NO

nimodipine reduced the stimulatory effect on NO production in response to pVadrenoceptor activation. In addition to a pure pharmacological effect of (5-adrenoceptor stimulated NO production we have recently demonstrated a role for endogenous adrenaline released by the adrenals in exhaled NO production and found a correlation between plasma concentrations of adrenaline and exhaled NO [198]. Taken together our data suggest that endogenous adrenaline activates pulmonary [3,-adrenoceptors that in turn stimulate calcium influx without the involvement of cAMP. Indeed, in other systems there is evidence for the existence of fJ,-adrenoceptors with direct G-protein coupling to calcium-channels [199].

4. Summary Together these observations suggest that in well ventilated areas of the lung, where stretch and oxygen tension are optimal, the largest amounts of NO will be synthesized. In poorly ventilated areas of the lung CO2 accumulates, oxygen concentrations are low and the small airways and alveoli collapse or exhibit only minor tidal excursion. These conditions would lead to a deficiency in local pulmonary NO production which may enhance HPV and thereby improve ventilation-perfusion matching (Figure 4). However further studies are needed to elucidate the role of CO2 on local NO production in insufficiently ventilated areas. In considering the role of endogenous NO one should consider methodological aspects such as interfering processes in chemiluminesce NO methodology itself, and also physiological alterations caused by the experimental situation, and in the basic physiology that is altered concomitant with experimental maneuvers. NO is a locally formed vasodilator, smooth muscle relaxant and immune modulator. Application of NO synthase inhibitors may thus not be regarded as identical with apllication of a vasoconstrictor, since the removal of NO will have largest effects in places where its formation is largest. Furthermore, NO synthase inhibition might have effects on relese of mediators or on activities in immune cells, effects some of which may not be instantly apparent.

Acknowledgement Supported by the Swedish MRC (proj no. 7919) and the Swedish Heart-Lung Foundation. References 1. 2. 3. 4. 5. 6. 7. 8. 9.

Archer S: Measurement of nitric oxide in biological models. Faseb J 1993;7:349-60. Palmer RM, Ferrige AG, Moncada S: Nitric oxide release accounts for the biological activity of endotheliumderived relaxing factor. Nature 1987;327:524-6. Fontijin A SA, Ronco RJ: Homogenous chemiluminescent measurement of nitric oxide with ozone: implications for continuous selective monitoring of gaseous air pollutants. Anal Chem 1970;42:575-579. Downes MJ, Edwards MW, Elsey TS, Walters CL: Determination of a non-volatile nitrosamine by using denitrosation and a chemiluminescence analyser. Analyst 1976; 101:742-8. Gustafsson LE, Leone AM, Persson MG, Wiklund NP, Moncada S: Endogenous nitric oxide is present in the exhaled air of rabbits, guinea pigs and humans. Biochem Biophys Res Commun 1991; 181:852-7. Gustafsson LE: Exhaled nitric oxide production by the lung, in Zapol W. Bloch K (eds): Nitric oxide and the lung. Basel, Marcel Dekker, 1997, pp 185-201. Leone AM, Gustafsson LE, Francis PL, Persson MG, Wiklund NP, Moncada S: Nitric oxide is present in exhaled breath in humans: direct GC-MS confirmation. Biochem Biophys Res Commun 1994;201:883-7. Barnes P), Kharitonov SA: Exhaled nitric oxide: a new lung function test [editorial]. Thorax 1996;51:233-7. Gustafsson LE: Exhaled nitric oxide as a marker in asthma. Eur Respir JSuppl 1998:26:495-525.

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Persson MG, Midtvedt T, Leone AM, Gustafsson LE: Ca(2+)-dependent and Ca(2+)-independent exhaled nitric oxide, presence in germ-free animals, and inhibition by arginine analogues. Eur JPharmacol 1994;264:13-20. van der Mark TW, Kort E, Meijer RJ, Postma DS, Koeter GH: Water vapour and carbon dioxide decrease nitric oxide readings. EurRespirJ 1997;10:2120-3. Stromberg S, Lonnqvist PA, Persson MG, Gustafsson LE: Lung distension and carbon dioxide affect pulmonary nitric oxide formation in the anaesthetized rabbit. Acta Physiol Scand 1997; 159:59-67. Binding N, Muller W, Czeschinski PA, Witting U: NO chemiluminescence in exhaled air: interference of compounds from endogenous or exogenous sources [In Process Citation]. Eur Respir J 2000; 16:499-503. Baraldi E, Azzolin NM, Dario C, Carra S, Ongaro R, Biban P, Zacchello F: Effect of atmospheric nitric oxide (NO) on measurements of exhaled NO in asthmatic children. Pediatr Pulmonol 1998;26:30-4. Corradi M, Pelizzoni A, Majori M, Cuomo A, de' Munari E, Pesci A: Influence of atmospheric nitric oxide concentration on the measurement of nitric oxide in exhaled air. Thorax 1998;53:673-6. Piacentini GL, Bodini A, Vino L, Zanolla L, Costella S, Vicentini L, Boner AL: Influence of environmental concentrations of NO on the exhaled NO test. Am J Respir Crit Care Med 1998; 158:1299-301. Steerenberg PA, Snelder JB, Fischer PH, Vos JG, van Loveren H, van Amsterdam JG: Increased exhaled nitric oxide on days with high outdoor air pollution is of endogenous origin. Eur Respir J 1999; 13:334-7. Van Amsterdam JG, Verlaan BP, Van Loveren H, Elzakker BG, Vos SG, Opperhuizen A, Steerenberg PA: Air pollution is associated with increased level of exhaled nitric oxide in nonsmoking healthy subjects. Arch Environ Health 1999;54:331-5. Thibeault DW, Rezaiekhaligh MH, Ekekezie I, Truog WE: Compressed air as a source of inhaled oxidants in intensive care units. Am J Perinatal 1999; 16:497-501. Schedin U, Norman M, Gustafsson LE, Jonsson B, Frostell C: Endogenous nitric oxide in the upper airways of premature and term infants [see comments]. Acta Paediatr 1997; 86:1229-3 5. Hdgman M, StrOmberg S, Schedin U, Frostell C, Hedenstierna G, Gustafsson E: Nitric oxide from the human respiratory tract efficiently quantified by standardized single breath measurements. Acta Physiot Scand 1997; 159:345-6. Tsoukias NM, George SC: A two-compartment model of pulmonary nitric oxide exchange dynamics. J Appl Physiol 1998;85:653-66. Silkoff PE, McClean PA, Slutsky AS, Furlott HG, Hoffstein E, Wakita S, Chapman KR, Szalai JP, Zamel N: Marked flow-dependence of exhaled nitric oxide using a new technique to exclude nasal nitric oxide. Am J Respir Crit Care Med I997;155:260-7. Byrnes CA, Dinarevic S, Busst CA, Shinebourne EA, Bush A: Effect of measurement conditions on measured levels of peak exhaled nitric oxide. Thorax 1997;52:697-701. Jorres RA: Modelling the production of nitric oxide within the human airways [In Process Citation]. Eur Respir J 2000; 16:555-60. Kissoon N, Duckworth LJ, Blake KV, Murphy SP, Taylor CL, Silkoff PE: FE(NO): relationship to exhalation rates and online versus bag collection in healthy adolescents. Am J Respir Crit Care Med 2000; 162:539-45. Rimar S, Gillis CN: Selective pulmonary vasodilation by inhaled nitric oxide is due to hemoglobin inactivation. Circulation 1993;88:2884-7. Hyde RW, Geigel EJ, Olszowka AJ, Krasney JA, Forster RE, 2nd, Utell MJ, Frampton MW: Determination of production of nitric oxide by lower airways of humans-theory. J Appl Physiol 1997;82:1290-6. Borland CD, Higenbottam TW: A simultaneous single breath measurement of pulmonary diffusing capacity with nitric oxide and carbon monoxide. Eur Respir J 1989;2:56-63. Iwamoto J, Pendergast DR, Suzuki H, Krasney JA: Effect of graded exercise on nitric oxide in expired air in humans. Respir Physiol 1994;97:333-45. Persson MG, Wiklund NP, Gustafsson LE: Endogenous nitric oxide in single exhalations and the change during exercise. Am Rev Respir Dis 1993; 148:1210-4. Bauer JA, Wald JA, Doran S, Soda D: Endogenous nitric oxide in expired air: effects of acute exercise in humans Lj/e5crl994;55:1903-9. St Croix CM, Wetter TJ, Pegelow DF, Meyer KC, Dempsey JA: Assessment of nitric oxide formation during exercise. Am J Respir Crit Care Med 1999; 159:1125-33. Phillips CR, Giraud GD, Holden WE: Exhaled nitric oxide during exercise: site of release and modulation by ventilation and blood flow. J Appl Physiol 1996;80:1865-71. Chirpaz-Oddou MF, Favre-Juvin A, Flore P, Eterradossi J, Delaire M, Grimbert F, Therminarias A: Nitric oxide response in exhaled air during an incremental exhaustive exercise. J Appl Physiol 1997;82:1311-8. Sheel AW, Road J, McKenzie DC: Exhaled nitric oxide during exercise. Sports Med 1999;28:83-90. Jilma B, Dirnberger E, Eichler HG, Matulla B, Schmetterer L, Kapiotis S, Speiser W, Wagner OF: Partial blockade of nitric oxide synthase blunts the exercise-induced increase of von Willebrand factor antigen and of factor VIII in man. Thromb Haemost 1997;78:1268-71. Adding LC, Agvald P, Artlich A, Persson MG, Gustafsson LE: Beta-adrenoceptor agonist stimulation of pulmonary nitric oxide production in the rabbit. BrJ Pharmacol 1999; 126:833-9. Hobbs AJ, Ignarro LJ: The nitric oxide-cyclic GMP signal transduction system, in Zapol WM, Bloch KD (eds): Nitric oxide and the lung. Basel, Marcel Dekker, 1997, pp 1-57.

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40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68.

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Murad F: The 1996 Albert Lasker Medical Research Awards. Signal transduction using nitric oxide and cyclic guanosine monophosphate. Jama 1996;276:1189-92 Persson MG, Kalz£n H, Gustafsson LE: Oxygen or low concentrations of nitric oxide reverse pulmonary vasoconstriction induced by nitric oxide synthesis inhibition in rabbits. Acta Physiol Scand 1994; 150:405-11. Gerlach H, Rossaint R, Pappert D, Falke KJ: Time-course and dose-response of nitric oxide inhalation for systemic oxygenation and pulmonary hypertension in patients with adult respiratory distress syndrome [see comments]. EurJClin Invest 1993;23:499-502. Lundberg JO, Settergren G, Gelinder S, Lundberg JM, Alving K. Weitzberg E: Inhalation of nasally derived nitric oxide modulates pulmonary function in humans. Acta Physiol Scand 19%; 158:343-7. Grimminger F, Spriestersbach R, Weissmann N, Walmrath D, Seeger W: Nitric oxide generation and hypoxic vasoconstriction in buffer-perfused rabbit lungs. JAppI Physiol 1995;78:1509-15. Carlin RE, Ferrario L, Boyd JT, Camporesi EM, McGraw DJ, Hakim TS: Determinants of nitric oxide in exhaled gas in the isolated rabbit lung. Am J Respir Crit Care Med \997; 155:922-7. Ide H, Nakano H, Ogasa T, Osanai S, Kikuchi K, Iwamoto J: Regulation of pulmonary circulation by alveolar oxygen tension via airway nitric oxide. JAppl Physiol 1999;87:1629-36. Cremona G, Higenbottam T, Takao M, Hall L, Bower EA: Exhaled nitric oxide in isolated pig lungs. J Appl Physiol 1995;78:59-63. Nelin LD, Thomas CJ, Dawson CA: Effect of hypoxia on nitric oxide production in neonatal pig lung. Am J Physiol 1996,27 \.m-U. Pearl JM, Nelson DP, Wellmann SA, Raake JL, Wagner CJ, McNamara JL, Duffy JY: Acute hypoxia and reoxygenation impairs exhaled nitric oxide release and pulmonary mechanics. J Thorac Cardiovasc Surg 2000; 119:931-8. Toga H, Watanabe T, Okazaki H, Ishigaki M, Noguchi T, Matsuda M, Huang J, Takahashi K, Ohya N, Fukunaga T, et al.: [Effect of endogenous and inhaled nitric oxide on pulmonary microcirculation]. Nihon Kyobu Shikkan GakkaiZasshi 1995;33 Suppl: 184-9. Fike CD, Kaplowitz MR, Thomas CJ, Nelin LD: Chronic hypoxia decreases nitric oxide production and endothelial nitric oxide synthase in newborn pig lungs. Am J Physiol 1998;274:L517-26. Cucchiaro G, Tatum AH, Brown MC, Camporesi EM, Daucher JW, Hakim TS: Inducible nitric oxide synthase in the lung and exhaled nitric oxide after hyperoxia. Am J Physiol 1999;277:L636-44. Bernareggi M, Radice S, Rossoni G, Oriani G, Chiesara E, Berti F: Hyperbaric oxygen increases plasma exudation in rat trachea: involvement of nitric oxide. Br J Pharmacol 1999; 126:794-800. Adding LC, Agvald P, Persson MG, Gustafsson LE: Regulation of pulmonary nitric oxide by carbon dioxide is intrinsic to the lung. Acta Physiol Scand 1999; 167:167-74. Bannenberg GL, Giammarresi C, Gustafsson LE: Inhaled carbon dioxide inhibits lower airway nitric oxide formation in the guinea pig. Acta Physiol Scand 1997; 160:401-5. Brogan TV, Hedges RG, McKinney S, Robertson HT, Hlastala MP, Swenson ER: Pulmonary NO synthase inhibition and inspired CO2: effects on VVQ1 and pulmonary blood (low distribution [In Process Citation). Eur Respir J 2000; 16:288-95. Deem S MS, Polissar NL, Hedges RG, Swenson ER: Hcmodilution during venous gas embolization improves gas exchange, with altering V(AVQ or pulmonary blood flow distributions. Anesthesiology 1999;91:I724-I732. Lee KH, Rico P, Billiar TR, Pinsky MR: Nitric oxide production after acute, unilateral hydrochloric acid-induced lung injury in a canine model. Crit Care Med 1998;26:2042-7. Pedoto A, Caruso JE, Nandi J, Oler A, Hoffmann SP, Tassiopoulos AK, McGraw DJ, Camporesi EM, Hakim TS: Acidosis stimulates nitric oxide production and lung damage in rats. Am J Respir Crit Care Med 1999:159:397402. Persson MG, Lonnqvist PA, Gustafsson LE: Positive end-expiratory pressure ventilation elicits increases in endogenously formed nitric oxide as detected in air exhaled by rabbits. Anesthesiology 1995;82:969-74. Bannenberg GL, Gustafsson LE: Stretch-induced stimulation of lower airway nitric oxide formation in the guinea-pig: inhibition by gadolinium chloride. Pharmacol Toxicol 1997;81:13-8. Adding LC, Bannenberg GL, Gustafsson LE: Gadolinium chloride inhibition of pulmonary nitric oxide production and effects on pulmonary circulation in the rabbit. Pharmacol Toxicol 1998,83:8-15 Mills PC, Martin DJ, Demoncheaux E, Scott C, Casas I, Smith NC, Higenbottam T: Nitric oxide and exercise in the horse. J Physiol (Lond) 1996;495:863-74. Fujii Y, Magder S, Cemacek P, Goldberg P, Guo Y, Hussain SN: Endothelin receptor blockade attenuates lipopolysaccharide-induced pulmonary nitric oxide production. Am J Respir Crit Care Med 2000; 161:982-9. Fujii Y, Goldberg P, Hussain SN: Intrathoracic and extrathoracic sources of exhaled nitric oxide in porcine endotoxemic shock. Chest 1998;! 14:569-76. Metha S JD, Datta P, Levy RD, Magder S: Porcine endotoxemic shock is associated with increased expired nitric oxide. Crit Care Med 1999;27:385-393. Stewart TE, Valenza F, Ribeiro SP, Wener AD, Volgyesi G, Mullen JB, Slutsky AS: Increased nitric oxide in exhaled gas as an early marker of lung inflammation in a model of sepsis. Am J Respir Crit Care Med 1995;! 51:713-8. Stitt JT, Dubois AB, Douglas JS, Shimada SG: Exhalation of gaseous nitric oxide by rats in response to endotoxm and its absorption by the lungs. JAppl Physiol 1997;82:305-16.

Disease Markers in Exhaled Breath N. Marczin and M.H. Yacoub (Eds.) 1OS Press, 2002

The "Vascular" Origins of Exhaled Nitric Oxide Tim HIGENBOTTAM and Eric DEMONCHEAUX AstraZeneca R&D Charnwood and University of Sheffield Respiratory Medicine Unit

Abstract. The origin of an exhaled gas is a classical physiological question. The biology and chemistry of nitric oxide (NO) is complex. In physiological conditions, NO acts while dissolved in aqueous biological solutions [1]. At high picomolar concentrations, it causes relaxation of vascular smooth muscle [2]. This is a result of activation of intra-cellular soluble guanylyl cyclase that increases the concentration of cyclic guanosine monophosphate (cGMP) which in turn initiates relaxation [1]. The evidence that NO normally regulates pulmonary vascular resistance depends on two observations. Firstly, changes in resistance are seen with inhibition of the endothelial enzyme nitric oxide synthase (eNOS). Secondly, pulmonary vascular disease is associated with changes in NO production.

1. Pulmonary endothelium derived nitric oxide regulates pulmonary vascular resistance and contributes to exhaled nitric oxide In many species, including man but excluding dog and rat, inhibition of endothelium derived NO results in a rise in pulmonary vascular resistance to blood flow [3]. The difference between species is important as it implies that NO is not unique in mammals in determining pulmonary vascular resistance. Other endothelial products, such as prostacyclin, may act to determine the basal vascular tone in the lungs in species such as the rat and possibly the cat. Increased blood flow, as occurs with exercise-induced rise in cardiac output, and increased release of NO, in species where endothelium-derived NO modulates pulmonary vascular resistance, may allow the pulmonary circulation to adapt without a rise in resistance [4]. Nitric oxide is relatively insoluble in aqueous solutions. At one atmosphere over water, only a small fraction of NO will dissolve and less than 5% remains in solution. In the lungs, with the massive surface area of the epithelial surface, NO is likely to be evolved from the aqueous surface liquid of the epithelial surface into the airspace where it can be measured in an exhaled breath [5]. Using an' isolated perfused and ventilated lung (porcine) it is possible to demonstrate that during inhibition of endothelium derived NO release by infusion of a nonspecific inhibitor, both the pulmonary vascular resistance rises and the exhaled NO concentration falls [6]. Furthermore, addition of blood to the lung perfusate reduces exhaled NO concentration, presumably due to reaction between NO with circulating haemoglobin within the red blood cells.

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2. Circulating nitric oxide "donors" can contribute to exhaled nitric oxide When NO gas is dissolved in aqueous solutions under conditions of continuous flow, the dissolved gas concentration rises to a plateau. The amount of NO dissolved is determined by the concentration of gas in the mixture overflowing the solution, the flow rate, the mass transfer characteristics and reaction kinetics. Micromolar concentrations of NO in the gas rapidly lead to nanomolar concentrations of dissolved NO, apparently at steady state, while nitrite quickly reaches micromolar concentrations in solution [2]. Interestingly, nitrite can generate free NO even at physiological pH [2]. This phenomenon is under great scrutiny as it may provide nitrite with an important physiological function as a NO carrier. This may well provide nitrite with an important role in ischaemic injury [7] or in chronic inflammatory disorders such as asthma [8]. Using the isolated perfused and ventilated lung (porcine) addition of nitrite to the perfusate lead to an increase in exhaled NO [2]. This shows that both circulating nitrite and endothelium derived NO may contribute to exhaled NO concentrations.

3. Exhaled nitric oxide and pulmonary vascular disease Measurement of the concentration of NO in collected exhaled air from patients with pulmonary arterial hypertension, a rare disease where there is a loss of up to 80% of the pre-capillary arteries, reveals significantly lower values compared to healthy subjects [9]. In the "explanted" lungs obtained from recipient patients at the time of lung transplant surgery, the release of endothelium derived NO is reduced in both isolated perfused lungs and isolated vascular rings of pulmonary arteries [10, 11]. Similarly in histological studies, immuno-staining for eNOS reveals a reduction in the amount of enzyme in the endothelium in lungs from patients with PPH [12]. The final piece of evidence comes from the measurement of the amount of NO formed from I5N labelled L-arginine which has been used to study NO production in patients with systemic hypertension [13]. This technique confirms a reduction in NO production in PPH patients when compared with age and sex matched healthy subjects [14]These findings link under-expression of NOS with 'under-activity of NO release" from pulmonary endothelium and lower exhaled NO concentrations.

4. Sources of exhaled nitric oxide other than the pulmonary circulation The exhaled air passes from the alveoli through the complex branching system of airways towards the mouth. Once the gas has left the alveolus no further pulmonary circulation derived NO will enter the exhaled air. The alveolar contribution cannot be changed. There is evidence that at high expiratory flow rate, low concentrations of NO are recorded [15]. A rise in NO concentrations at lower flow rates may indicate an important role for the airway epithelial surface as another source of NO [15]. Epithelial cells express the inducible form of NOS even under normal conditions [16]. In asthmatics, NOS expression is greatly increased and is associated with higher exhaled concentrations of NO [17]. Allergen challenge increases exhaled NO and treatment with inhaled steroids reduces the exhaled levels [17].

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51

These data provide quantitative information on the relative source of NO. In the asthmatic, the airway contributes through iNOS a hundred fold higher amount of exhaled NO than alveolar sources.

References 1. 2.

3. 4. 5.

6. 7. 8. 9. 10.

11.

12. 13. 14.

15. 16.

17.

Moncada, S., and A. Higgs. 1993. The L-arginine-nitric oxide pathway. N Engl J Med 329:20022012. Demoncheaux, E., T. Higenbottam, P. Foster, C. Borland, A. Smith, H. Marriott, S. Akamine, D. Bee, and M. Davies. 2002. Circulating nitrite anions are a directly acting vasodilator and are donors for nitric oxide. Clin Sci (Lond) 102:77-83. Cremona, G., A. Wood, and L. Hall. 1994. Effects of inhibitors of nitric oxide release and action on vascular tone in isolated lungs of pig, sheep, dog and man. J Physiol (Lond) 481.1:185-195. Cremona, G., T. Higenbottam, M. Takao, E. A. Bower, and L. W. Hall. 1997. Nature and site of action of endogenous nitric oxide in vasculature of isolated pig lungs. JAppl Physiol 82:23-31. Demoncheaux, E., M. Maniscalco, S. Roe, G. Cremona, and T. Higenbottam. 1996. Exhaled nitric oxide: origin and physiological meaning. In Nitric oxide and radicals in the pulmonary vasculature (E. Weir, S. Archer and J. Reeves, editors), Futura Publishing Company, Armonk, NY. 427-445. Cremona, G., T. Higenbottam, M. Takao, L. Hall, and E. A. Bower. 1995. Exhaled nitric oxide in isolated pig lungs. JAppl Physiol 78:59-63. Zweier, J. L., P. H. Wang, A. Samouilov, and P. Kuppusamy. 1995. Enzyme-Independent Formation of Nitric-Oxide in Biological Tissues. Nat Med 1:804-809. Hunt, J., and B. M. Gaston. 2001. Endogenous airway acidification: Implications for asthma pathology Am JResp Crit Care Med 163:293-294. Cremona, G., T. Higenbottam, C. Borland, and B. Mist. 1994. Mixed expired nitric oxide in primary pulmonary hypertension in relation to lung diffusion capacity. QJMed 87:547-551. Dinh-Xuan, A. T, Higenbottam, T. W., Clelland, C. A., Pepke-Zaba, J., Cremona, G., Butt, A. Y., Large, S. R., Wells, F. C., and Wallwork, J. 1991. Impairment of endothelium-dependent pulmonary-artery relaxation in chronic obstructive lung disease. N EnglJ Med 324:1539-47 Cremona, G., T. W. Higenbottam, E. A. Bower, A. M. Wood, and S. Stewart. 1999. Hemodynamic effects of basal and stimulated release of endogenous nitric oxide in isolated human lungs. Circ 100:1316-21. Giaid, A., and D. Saleh. 1995. Reduced expression of endothelial nitric oxide synthase in the lungs of patients with pulmonary hypertension. N EnglJ Med 333:214-21. Forte, P., M. Copland, L. M. Smith, E. Milne, J. Sutherland, and N. Benjamin. 1997. Basal nitric oxide synthesis in essential hypertension. Lancet 349:837-42. Demoncheaux, E., K. Hall, V. Garner, S. Wharton, A. Spivey, and T. Higenbottam. 2001. Whole body nitric oxide production in patients with pulmonary hypertension. Am J Resp Crit Care Med 163: A116 (abstract) Tsoukias, N. M., Z. Tannous, A. F. Wilson, and S. C. George. 1998. Single-exhalation profiles of NO and CO2 in humans: effect of dynamically changing flow rate. JAppl Physiol 85:642-652. Guo, F. H., De Raeve, H. R., Rice, T. W., Stuehr, D. J., Thunnissen, F. B., and Erzurum, S. C. 1995 Continuous nitric oxide synthesis by inducible nitric oxide synthase in normal human airway epithelium in vivo Proc NatlAcadSci USA 1995 92:7809-13. Berlyne, G., and N. Barnes. 2000. No role for NO in asthma? Lancet 355:1029-1030.

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Disease Markers in Exhaled Breath N. Marczin and M.H. Yacoub (Eds.) IOS Press, 2002

Exhaled NO originates in airway epithelium L. Christofer ADDING and Lars E. GUSTAFSSON Department of Physiology and Pharmacology, Karolinska Institute, Stockholm, Sweden. Abstract. Nitric oxide is a highly reactive molecule and binds in the presence of blood rapidly to haemoglobin with subsequent oxidation to nitrate. The alveolar compartment of the lung is constantly perfused with large amounts of blood and therefore nitric oxide produced in the endothelial or alveolar epithelial regions most likely will be absorbed and inactivated by erythrocytes. Literature data is reviewed and a methodology is presented for measuring NO at high precision in intubated patients. The measurements demonstrate that exhaled NO in normal individuals is almost fully of airway origin, with marked breath-hold accumulation , whereas little or no alveolar accumulation of NO occurs during breath-hold. Airway tissue concentrations of NO are far higher than we presume from normal tidal exhalations, underlining the importance of airway NO formation in respiratory system biology.

1. Concentrations of exhaled NO The first measurements of exhaled NO in laboratory animals clearly demonstrated a local enzyme-dependent NO production within the lower respiratory tract: the animals were tracheotomized and exhaled NO could still be detected after circulatory arrest but not in the presence of an inhibitor of NO synthase, the enzyme responsible for NO production (see Gustafsson 1997). Another fundamental indication is that in salt buffer perfused lungs, when blood is no longer present, there is an immediate and more than two-fold increase in exhaled NO concentration [1-5]. In humans the initial oral measurements of exhaled NO [6] were later suggested to be a mixture of NO emanating from the lower respiratory tract, nasally produced NO [7-9], and NO from salivary NO2" and gastric regurgitation [10, 11]. The relative contribution from respective source depends on the measurement technique, especially the exhaled gas sampling procedure. Continuous oral sampling of exhaled NO using e.g. nose clamp may contain up to 90% nasally derived NO [12, 13] due to backward diffusion from the nose [14] whereas oral single-breath exhaled NO measurements, during plateau phase, are not affected by NO derived from nasal airways [15-18]. Studies of awake humans either intubated [8, 13, 19, 20] or with bronchoscopy [21] have also confirmed the excretion of NO from the lower respiratory tract. To the lower airway NO excretion is added a variable oral mainly non-enzymatic NO production [22]. Nevertheless, the anatomic site and main cell type producing the NO molecules detected in exhaled gas from the lower airways are still a matter of debate. Different volumes of dead-space gas have been sampled, reflecting gas emanating from trachea, bronchi.

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bronchioles, and respiratory bronchioles, and analyzed for exhaled NO after breath-holding they show significant contribution from distal airways [23, 24]. However, very low NO concentrations are present in alveolar gas in vivo [15, 24-26] due to the rapid scavenging of NO by erythrocytes in the pulmonary capillaries [27] (see also above). In salt buffer perfused lungs end-tidal NO concentrations are very high [1-5] probably reflecting alveolar (endothelial or macrophage) NO production, otherwise rapidly scavenged by haemoglobin. We have experienced that lung-perfusion experiments using buffered salt solutions after a "grace period" inevitably lead to lung oedema formation and that exhaled NO concentrations then markedly decrease. The fluid that accumulates in interstitial tissue and alveoli is likely to have large numbers of oxygen radicals that degrade NO. Furthermore, the fluid in itself may act as a diffusion barrier. Both mechanisms would reduce the amount of endothelial-derived NO in exhaled air. Along similar lines, Cremona et al. [3] demonstrated a decrease in exhaled NO with increasing weight of lungs perfused with salt solution at high outflow pressure. On the other hand, we have observed increased exhaled NO levels during lung oedema formation in vivo, probably due to an increased diffusion barrier and less NO scavenging by erythrocytes (Adding et al., unpublished observations). However, in a recent study by Duplain et al. there was no increase in exhaled NO in subjects who developed high-altitude pulmonary edema [28]. This might be explained by a reduced NOS activity due to the relative hypoxic conditions as previously discussed. In favour of the idea that endothelial derived NO from alveolar regions does not contribute much to exhaled NO in vivo is the observation that infusion of acetylcholine, which massively stimulates endothelial NO production, when administered in concentrations that do not cause airway effects only marginally increases exhaled NO (unpublished observations). However, in the absence of blood (haemoglobin) in the pulmonary circulation, during salt-buffer perfusion acetylcholine infusions will increase exhaled NO concentrations.

2. Measurement of airway and alveolar NO in single breaths in trachea In order to address the question of origin of exhaled NO in vivo we have measured exhaled NO in anesthetized intubated patients undergoing routine surgery (Schedin and Gustafsson, unpublished). We wanted to explore the initial observations [15] that breathhold can markedly increase single-breath NO concentrations in a peak-plateau pattern reaching maximum during the dead space phase of exhalation as determined by CO, exhalation pattern. In the study by Persson et al. [15], breath-hold concentrations typically reached 50 ppb at 30 s of breath-hold, indicating that airway tissue concentrations are higher by at least a factor of 10 as compared with the concentrations during tidal breathing, not to mention the dilute concentrations obtained in artificial ventilation where exhalation gas flows are even higher. We therefore used the Inspiratory Hold feature of a Siemens 900 ventilator and applied it for 5-30 s followed by sampling of exhaled gas into a rapid exhaled CO2 and NO analysis sytem (SBNO, Aerocrine AB, Solna, Sweden) adjusted to give a continuous aspiration of 300 ml/min. The NO and CO2 recordings were presynchronized in the calibration mode of the SBNO system by means of rapid puncture of a membrane behind which a mixture of known concentrations of NO and CO2 were flowing. The response time (t 10-90%) was 0.15 s for NO and approximately 0.25 s for CO2, and detection limit was less than 1 ppb for NO, allowing also determination of alveolar gas

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concentrations when the Inspiratory Hold mode was released. A typical recording is shown in Figure 1. 20 sec 80.0-

NO, vv ppb

60.040.020.0-

C02, %

10:67.5. 5.02.5-

VOL exhal litres

inn

O.s-

2g;8:

ULAA

Ptrach cmH2O 1 264

Time (min:sec) Figure 1. Anesthetized intubated human subject in propofol anesthesia. Synchronized recordings of, from top, exhaled NO, carbon dioxide, exhaled volume in litres, and insufflation pressure. In the left pan of the recordings the typical pressure pattern of volume controlled ventilation can be seen. In the middle part of the recordings gas sampling for NO and COZ was switched to room air at the first arrow (upwards directed) and subsequently an Inspiratory Hold of 10 s was commenced as indicated by the pressure curve. At the second arrow (downwards directed) airway sampling was reinstituted leading to a slow emptying of lungs during 20 more seconds of Inspiratory Hold, leading to a drop in airway pressure beyond spontaneous drop (spontaneous drop, apart from "relaxation", probably being due to alveolar gas diffusions). During Inspiratory Hold sampling a clearcut airway NO peak can be seen during the dead space phase, followed by an "alveolar CO2 plateau", which however is not the true plateau which however becomes evident when the Inspiratory Hold mode is released, the release being denoted by third arrow (downwards directed). The true alveolar CO2 plateau and the "alveolar" NO can be read at the dashed arrows. During this plateau in CO2 it can clearly be seen that NO falls to very low levels, approximately 5 ppb, and then rises again as airflow essentially stops. This low level is only an estimate of alveolar NO concentration, since NO will be admixed to the expired gas during exhalation, from the sources in the airways. Note very low NO concentrations during exhalation in the controlled ventilation mode, also when resumed directly after the inspiratory hold, and identical to the recorded value at the end of the inspiratory hold, indicating that no accumulation of NO occurs in normal alveoli during brief cessation of ventilation.

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3. Summary Together these observations suggest that nitric oxide in exhaled air mainly emanates from supra-alveolar regions. It can not be argued that true alveolar NO concentrations can be measured directly during single breaths, but an estimate of an upper limit of these can be made. Such estimates should be compared with current methods under development for indirect calculations of alveolar concentrations of NO performed by precise variations in exhalation flow rates [29, 30]. During breath-hold it can be demonstrated in intubated subjects that airway tissue NO concentrations are far higher than determined from single breath exhalation during spontaneous or controlled ventilation. The reproducibility of the exhaled NO concentrations during controlled volume ventilation still suggest that exhalation in the awake state with controlled flow rates gives an excellent estimate of airway NO formation, although it puts a high demand on the flow regulation in the exhaled NO measurement device. In conclusion, exhaled NO is almost exclusively of airway origin. Airway tissue concentrations of NO are far higher than we presume from normal tidal exhalations, confirming our initial observations [1, 15], underlining the importance of airway NO formation in respiratory system biology. Acknowledgement Supported by the Swedish MRC (proj no. 7919} and the Swedish Heart-Lung Foundation. References 1.

2. 3. 4. 5. 6.

7. 8. 9.

10. 11.

Persson MG, Midtvedt T, Leone AM, Gustafsson LE: Ca(2+)-dependent and Ca(2+)-independent exhaled nitric oxide, presence in germ-free animals, and inhibition by arginine analogues. Em J Pharmacol 1994 ;264:13-20. Grimminger F, Spriestersbach R, Weissmann N, Walmrath D, Seeger W: Nitric oxide generation and hypoxic vasoconstriction in buffer-perfused rabbit lungs. J Appl Physiol 1995;78:1509-15. Cremona G, Higenbottam T, Takao M, Hall L, Bower EA: Exhaled nitric oxide in isolated pig lungs. JAppl Physiol 1995;78:59-63. Marczin N, Riedel B, Gal J, Polak J, Yacoub M: Exhaled nitric oxide during lung transplantation [letter]. Lancet 1997;350:1681-2. Adding LC, Agvald P, Persson MG, Gustafsson LE: Regulation of pulmonary nitric oxide by carbon dioxide is intrinsic to the lung. Acta Physiol Scand 1999; 167:167-74. Gustafsson LE, Leone AM, Persson MG, Wiklund NP, Moncada S: Endogenous nitric oxide is present in the exhaled air of rabbits, guinea pigs and humans. Biochem Biophys Res Commun 1991;181:852-7. Alving K, Weitzberg E, Lundberg JM: Increased amount of nitric oxide in exhaled air of asthmatics. Eur Respit-J 1993;6:1368-70. Lundberg JO, Rinder J, Weitzberg E, Lundberg JM, Alving K: Nasally exhaled nitric oxide in humans originates mainly in the paranasal sinuses. Acta Physiol Scand 1994; 152:431 -2. Lundberg JO, Farkas-Szallasi T, Weitzberg E, Rinder J, Lidholm J, Anggaard A, Hokfelt T, Lundberg JM, Alving K: High nitric oxide production in human paranasal sinuses. Nat Med 1995;l:370-3. Zetterquist W, Pedroletti C, Lundberg JO, Alving K: Salivary contribution to exhaled nitric oxide. EurRespirJ 1999; 13:327-33. Lundberg JO, Weitzberg E, Lundberg JM, Alving K: Intragastric nitric oxide production in humans: measurements in expelled air. Gut 1994;35:1543-6.

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12.

13.

14. 15. 16.

17.

18. 19.

20. 21.

22.

23. 24.

25. 26. 27. 28.

29. 30.

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Lundberg JO, Weitzberg E, Nordvall SL, Kuylenstierna R, Lundberg JM, Alving K: Primarily nasal origin of exhaled nitric oxide and absence in Kartagener's syndrome. Ear Respir J 1994;7:1501-4. Schedin U, Frostell C, Persson MG, Jakobsson J, Andersson G, Gustafsson LE: Contribution from upper and lower airways to exhaled endogenous nitric oxide in humans. Ada Anaesthesia! Scand 1995;39:327-32. Kharitonov SA, Barnes PJ: Nasal contribution to exhaled nitric oxide during exhalation against resistance or during breath holding. Thorax 1997;52:540-4. Persson MG, Wiklund NP, Gustafsson LE: Endogenous nitric oxide in single exhalations and the change during exercise. Am Rev Respir Dis 1993; 148:1210-4. Hogman M, Strdmberg S, Schedin U, Frostell C, Hedenstierna G, Gustafsson E: Nitric oxide from the human respiratory tract efficiently quantified by standardized single breath measurements. Acta Physiol Scand 1997; 159:345-6. Silkoff PE, McClean PA, Slutsky AS, Furlott HG, Hoffstein E, Wakita S, Chapman KR, Szalai JP, Zamel N: Marked flow-dependence of exhaled nitric oxide using a new technique to exclude nasal nitric oxide. Am J Respir Crit Care Med 1997;! 55:260-7. Suzuki H, Krasney JA: Nitric oxide in single-breath exhalation in humans. Jpn J Physiol 1997;47:335-9. Tsujino I, Miyamoto K, Nishimura M, Shinano H, Makita H, Saito S, Nakano T, Kawakami Y: Production of nitric oxide (NO) in intrathoracic airways of normal humans. Am J Respir Crit Care Merfl996;154:1370-4. Tsujino I, Miyamoto K, Nishimura M, Shinano H, Kawakami Y: Measurement of exhaled nitric oxide concentration using nasal continuous negative pressure. Respirology 1999;4:155-9. Massaro AF, Mehta S, Lilly CM, Kobzik L, Reilly JJ, Drazen JM: Elevated nitric oxide concentrations in isolated lower airway gas of asthmatic subjects. Am J Respir Crit Care Med 1996;153:1510-4. Palm JP, Graf P, Lundberg JO, Alving K: Characterization of exhaled nitric oxide: introducing a new reproducible method for nasal nitric oxide measurements [In Process Citation]. Eur Respir J 2000; 16:236-41. Dillon WC, Hampl V, Shultz PJ, Rubins JB, Archer SL: Origins of breath nitric oxide in humans [see comments]. Chest 1996;! 10:930-8. DuBois AB, Kelley PM, Douglas JS, Mohsenin V: Nitric oxide production and absorption in trachea, bronchi, bronchioles, and respiratory bronchioles of humans. J Appl Physiol 1999:86:15967. Borland C, Cox Y, Higenbottam T: Measurement of exhaled nitric oxide in man. Thorax 1993;48:1160-2. Byrnes CA, Dinarevic S, Busst C, Bush A, Shinebourne EA: Is nitric oxide in exhaled air produced at airway or alveolar level? Eur Respir J 1997; 10:1021 -5. Rimar S, Gillis CN: Selective pulmonary vasodilation by inhaled nitric oxide is due to hemoglobin inactivation. Circulation 1993;88:2884-7. Duplain H, Sartori C, Lepori M, Egli M, Allemann Y, Nicod P, Scherrer U: Exhaled nitric oxide in high-altitude pulmonary edema: role in the regulation of pulmonary vascular tone and evidence for a role against inflammation. Am J Respir Crit Care Med 2000; 162:221 -4. Tsoukias NM, Tannous Z, Wilson AF, George SC. Single-exhalation profiles of NO and CO2 in humans: effect of dynamically changing flow rate J Appl Physiol. 1998;85:642-52. Hogman M, Holmkvist T, Wegener T, Emtner M, Andersson M, Hedenstrom H, Merilainen P. Extended NO analysis applied to patients with COPD, allergic asthma and allergic rhinitis.Respir Med. 2002;96:24-30

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NO is generated via NOS enzymes Sergei A. KHARITONOV Thoracic Medicine, National Heart and Lung Institute, Imperial College and Royal Brompton Hospital, Dovehouse Street, London SW3 6LY, UK Abstract. The major source of exhaled NO is endogenous NO derived from L-arginine by the enzyme NO synthase (NOS) [1 ].

1. Nitric oxide synthases Two of these enzymes are constitutively expressed and are activated by small rises in intracellular calcium concentration, secondary to cell activation. Neuronal NOS (NOSI, nNOS) is predominantly expressed in neurons and endothelial NOS (NOS3, eNOS) mainly in endothelial cells, although other cell types also express both of these isoforms. A third enzyme is inducible (NOS2, iNOS), has a much greater level of activity and is independent of calcium concentration. NOS2 may be induced by inflammatory cytokines, endotoxin and viral infections and may show increased expression in inflammatory diseases [2-4]. Genetic polymorphisms of all three isoforms of NOS have been detected. Surprisingly, associations have been found between polymorphisms in the NOSI gene and asthma in Caucasian populations [5,6]. In mild asthmatic patients there is a significant association between the length of the AAT repeat polymorphism in intron 20 of the NOSI gene and exhaled NO levels [7]. 2. Cellular sources in airways The cellular source of NO gas in the lower respiratory tract is not yet certain. Studies with perfused porcine lungs suggest that exhaled NO originates at the alveolar surface, rather than from the pulmonary circulation [8], and may be derived from NOS3 expressed in the alveolar walls of normal lungs. Studies in ventilated perfused lungs of guinea pigs show that exhaled NO is reduced during perfusion with calcium-free solutions, suggesting that NO is derived from a constitutive NOS, which is calciumdependent [9]. Airway epithelial cells may express both NOS3 and NOSI and therefore may contribute to NO in the lower respiratory tract [10,11]. There is some expression of NOS2 even in airway epithelial cells from normal subjects [12], and NOS2 appears to be an important isoform contributing to exhaled NO in healthy mice [13]. In inflammatory diseases, such as asthma, it is likely that the increase in exhaled NO reflects further induction of NOS2 in response to inflammatory signals, such as proinflammatory cytokines. Indeed, increased NOS activity has been demonstrated in lung tissue of patients with asthma, cystic fibrosis and obliterative bronchiolitis [14]. In asthmatic patients there is evidence for increased expression of NOS2 in airway epithelial cells [15] and this is likely to be due to increased transcription mediated via the transcription factors STAT-1 and nuclear factor-KB (NF-KB), and increased availability

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of L-arginine [16,17]. Pro-inflammatory cytokines induce the expression of NOS2 in cultured human airway epithelial cells [18,19] and it is likely that these same cytokines are released in asthmatic inflammation. NOS2 may be expressed in other cell types, such as alveolar macrophages, eosinophils and other inflammatory cells [20]. Further evidence that the increase in exhaled NO is derived from increased NOS2 expression is the observation that corticosteroids inhibit inflammatory induction of NOS2 in epithelial cells [16,21], decrease expression in bronchial biopsies of asthmatic patients [20] and also reduce exhaled NO concentrations in asthmatic patients [22]. 3. Non-enzymatic sources of NO NOS is not the only source of NO in exhaled air and exhaled NO is not therefore a direct measure of NOS activity in the lower respiratory tract. NO reacts with thiolcontaining molecules, such as cysteine and glutathione, to form 5-nitrosoproteins and Snitrosothiols [23]. Approximately 70-90% of NO is released by S-nitrosothiols, which therefore provide a major source of NO in tissues [24]. S-nitrosothiols are potent relaxants of human airways and may play an important role in sequestration, releasing and transportation of NO to its site of action [23]. NO in exhaled air may also be derived from nitrite protonation to form nitrous acid, which releases NO gas with acidification [25]. This pH-related pathway has been implicated in acute asthma, when pH in expired condensate is low [26]. 4. Anatomical origin NO is produced along the entire length of human airways. The conducting airways secrete NO into the lumen, which mixes with alveoli NO during exhalation, resulting in the observed expiratory concentration. The levels of NO derived from the upper respiratory tract (200-1000 ppb) [27-29] and sinuses (1000-30000 ppb) [30] are a hundred-fold higher than exhaled NO measured in the lower respiratory tract (1 -9 ppb) [27,27,28,31-36]. Several factors may contribute to high nasal levels. The paranasal sinuses produce a high level of NO [37]. There is a dense innervation with NOS1immunoreactive nerve fibers around nasal blood vessels [38]. Vasculature-derived NO. however, is not the major source of NO in nasal mucosa, as neuropeptide Y, a powerful vasoconstrictor, reduces nasal blood flow by 37%, but NO by only 7% [39]. There appears to be, constitutive expression of NOS2 [40] and the transcription factor NF-KB in nasal mucosa [41]. Interestingly, the NO outputs from the nostrils are significantlylower on the operated side (site with the reduced NO-generating surface) in patients who have undergone unilateral medial maxillectomy [42]. The source of NO in the lower respiratory tract is also of mixed origin and may be derived from airway and alveolar epithelial cells, which express both NOS3 and NOS1. The contribution of endothelial-derived NO is minimal, as inhaled NOS inhibitors are able to reduce exhaled NO by 40% -70% [43-45] without any effect on the systemic circulation. By contrast, L-NMMA infusion modulates blood pressure and heart rate but has only a minimal effect on exhaled NO [43]. Simultaneous measurement of expired COi and NO demonstrate that exhaled NO precedes the peak value of CO2 (end-tidal), suggesting that NO is derived from airways rather than alveoli [27,46]. Direct sampling via fiberoptic bronchoscopy in normal subjects shows a similar levels of NO in trachea and main bronchi to that recorded at the mouth, thus indicating that there is NO derived from the lower airways [27,36]. Exhaled

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NO is therefore most likely to be of epithelial rather than endothelial origin, and most NO is derived from airways rather than alveoli. References [1] S.A.Kharitonov, Barnes PJ. Exhaled markers of pulmonary disease. Am J Respir Crit Care Med 163(2001) 1693-1722. [2] P.J.Barnes, Belvisi MG. Nitric oxide and lung disease. Thorax. 48 (1993) 1034-1043. [3] B.Gaston et aL The biology of nitrogen oxides in the airways. Am. J. Respir. Crit. Care. Med. 149 (1994) 538-551. [4] P.J.Barnes. Transcription factors and inflammatory disease. Hosp. Pract. 31 (1996) 93-96. [5] P.S.Gao et al.. Variants of NOS1, NOS2, and NOS3 genes in asthmatics. Biochem. Biophys. Res. Commun. 267 (2000) 761-763. [6] H.Grasemann et aL A neuronal NO synthase (NOS1) gene polymorphism is associated with asthma. Biochem. Biophys. Res. Commun. 272 (2000) 391-394. [7] M.E.Wechsler et al.. Exhaled nitric oxide in patients with asthma. Association with NOS1 genotype. Am. J. Respir. Crit. Care. Med. 162 (2000) 2043-2047. [8] G.Cremona et al.. Exhaled nitric oxide in isolated pig lungs. J. Appl. Physiol. 78 (1995) 59-63. [9] M.G.Persson et al.. Ca2+-dependent and Ca2+-independent exhaled nitric oxide, presence in germfree animals, and inhibition by arginine analogues. Eur J Pharm 264 (1994) 13-20. [10] P.W.Shaul et al.. Endothelial nitric oxide synthase is expressed in cultured human bronchiolar epithelium. J. Clin. Invest. 94(1994) 2231-2236. [11] J.B.Mannick et al.. Nitric oxide produced by human B lymphocytes inhibits apoptosis and Epstein-Barr virus reactivation. Cell. 79 (1994) 1137-1146. [12] F.H.Guo et al.. Continuous nitric oxide synthesis by inducible nitric oxide synthase in normal human airway epithelium in vivo. Proc. Natl. Acad. Sci. U. S. A. 92 (1995) 7809-7813. [13] W.Steudel et al.. Exhaled nitric oxide production by nitric oxide synthase-deficient mice. Am J Respir Crit Care Med 162 (2000) 1262-1267. [14] M.G.Belvisi et al.. Nitric oxide synthase activity is elevated in inflammatory lung diseases. Eur J Pharmacol 283 (1995) 255-258. [15] Q.Hamid et al.. Induction of nitric oxide synthase in asthma. Lancet 342 (1993) 1510-1513. [16] F.H.Guo et al.. Molecular mechanisms of increased nitric oxide (NO) in asthma: evidence for transcriptional and post-translational regulation of NO synthesis. J. Immunol. 164 (2000) 59705980. [17] Q.Xie et al.. Role of transcription factor NF-kB/Rel in induction of nitric oxide synthase. J. Biol. Chem. 269 (1994) 4705-4708. [18] K.Asano et al.. Constitutive and inducible nitric oxide synthase gene expression, regulation, and activity in human lung epithelial cells. Proc. Natl. Acad. Sci. U. S. A. 91 (1994) 10089-10093. [19] N.A.Chartrain et al.. Molecular cloning, structure, and chromosomal localization of the human inducible nitric oxide synthase gene. J. Biol. Chem. 269 (1994) 6765-6772.

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[20]

D.Saleh et al.. Increased formation of the potent oxidant peroxynitrite in the airways of asthmatic patients is associated with induction of nitric oxide synthase: effect of inhaled glucocorticoid. FASEB.J. 12(1998) 929-937.

[21J R.A.Robbins et al.. Expression of inducible nitric oxide in human lung epithelial cells. Biochem Biophys Res Commun 203 (1994) 209-218. [22]

S.A.Kharitonov et al.. Increased nitric oxide in exhaled air of asthmatic patients. Lancet 343 (1994) 133-135.

[23] J.S.Stamler et al.. S-nitrosylation of proteins with nitric oxide: synthesis and characterization of biologically active compounds. Proc. Natl. Acad. Sci. U. S. A. 89 (1992) 444-448. [24]

F.S.Sheu et al.. Direct observation of trapping and release of NO by glutathione and cysteine with electron paramagnetic resonance spectroscopy. Biophys. J. 78 (2000) 1216-1226.

[25] S.J.Klebanoff. Reactive nitrogen intermediates and antimicrobial activity: role of nitrite. Free Radio. Biol. Med. 14(1993) 351-360. [26] J.F.Hunt et al.. Endogenous airway acidification. Implications for asthma pathophysiology. Am J Respir. Crit. Care. Med. 161 (2000) 694-699. [27] S.A.Kharitonov et al.. The elevated level of exhaled nitric oxide in asthmatic patients is mainly derived from the lower respiratory tract. Am. J. Respir. Crit. Care. Med. 153 (1996) 1773-1780. [28] E.Baraldi et al.. Reference values of exhaled nitric oxide for healthy children 6-15 years old. Pediatr. Pulmonol. 27 (1999) 54-58. [29] S.R.Thomas et al.. Nasal and exhaled nitric oxide is reduced in adult patients with cystic fibrosis and does not correlate with cystic fibrosis genotype. Chest. 117 (2000) 1085-1089. [30] J.O.Lundberg, Weitzberg E. Nasal nitric oxide in man. Thorax. 54 (1999) 947-952. [31] C.Borland et al.. Measurement of exhaled nitric oxide in man. Thorax. 48 (1993) 1160-1162. [32] L.E.Gustafsson et al.. Endogenous nitric oxide is present in the exhaled air of rabbits, guinea pigs and humans. Biochem. Biophys. Res. Commun. 181 (1991) 852-857. [33] K.Alving et al.. Increased amount of nitric oxide in exhaled air of asthmatics. Ear. Respir. J. 6 (1993) 1368-1370. [34] A.F.Massaro et al.. Expired nitric oxide levels during treatment of acute asthma. Am. J. Respir. Crit. Care. Med. 152(1995) 800-803. [35] U.Schedin et al.. Contribution from upper and lower airways to exhaled endogenous nitric oxide in humans. Ada. Physiologica. Scandinavica. 39(1995) 327-332. [36] A.F.Massaro et al.. Elevated nitric oxide concentrations in isolated lower airway gas of asthmatic subjects. Am. J. Respir. Crit. Care. Med. 153 (1996) 1510-1514. [37] J.O.Lundberg et al.. High nitric oxide production in human paranasal sinuses. Nat. Med. \ (1995) 370-373. [38] T.Kondo et al.. Distribution, chemical coding and origin of nitric oxide synthase-con tain ing nerve fibres in the guinea pig nasal mucosa. J. Auton. Nerv. Syst. 80 (2000) 71-79. [39] A.Cervin et al.. Functional effects of neuropeptide Y receptors on blood flow and nitric oxide levels in the human nose. Am. J Respir. Crit. Care. Med. 160 (1999) 1724-1728.

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[40] H.Kawamoto et al.. Localization of nitric oxide synthase in human nasal mucosa with nasal allergy. Acta. Otolaryngol. Suppl. (Stockh. ) 539:65-70 (1998) 65-70. [41] I.Ramis et al.. Constitutive nuclear factor-kappaB activity in human upper airway tissues and nasal epithelial cells. Ear. Respir. J. 15 (2000) 582-589. [42] W.Qian et al.. Unilateral nasal nitric oxide measurement after nasal surgery. Ann Oiol Rhinol Laryngol 109 (2000) 952-957. [43]

C.Sartori et al.. Exhaled nitric oxide does not provide a marker of vascular endothelial function in healthy humans. Am. J. Respir. Crit. Care. Med. 160 (1999) 879-882.

[44]

D.H.Yates et al.. Effect of a nitric oxide synthase inhibitor and a glucocorticosteroid on exhaled nitric oxide. Am. J. Respir. Crit. Care. Med 152 (1995) 892-896.

[45]

D.H.Yates et al.. Endogenous nitric oxide is decreased in asthmatic patients by an inhibitor of inducible nitric oxide synthase. Am. J. Respir. Crit. Care. Med 154 (1996) 247-250.

[46]

M.G.Persson et al.. Endogenous nitric oxide in single exhalations and the change during exercise. Am. Rev. Respir. Dis. 148 (1993) 1210-1214.

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Disease Markers in Exhaled Breath N. Marczin and M.H. Yacoub (Eds.) IOS Press. 2002

Technical aspects of exhaled NO: Investigator point of view Sergei A. KHARITONOV Thoracic Medicine, National Heart and Lung Institute, Imperial College and Royal Brompton Hospital, Dovehouse Street, London SW3 6LY, UK Abstract. There has recently been an explosion of interest in the analysis of breath constituents as a way of monitoring inflammation and oxidative stress in the lungs. Here we review the technical aspects of exhaled nitric oxide measurements (NO) in the diagnosis and monitoring of lung disease.

Expiratory flow, soft palate closure, and dead space air may all influence exhaled NO levels. Therefore, exhaled NO is usually determined during single-breath exhalations against a resistance [1,2] to prevent contamination with nasal NO [3,4], or using reservoir collection with discarding of the dead space [5]. However, this method has proven difficult for some children, who may have trouble maintaining a constant flow, and recently a simple flow-driven method for online NO measurements has been developed that does not require active patient co-operation. Recently, single breath analysis of exhaled NO has been successfully performed in the newborn when exhaled air was sampled from the tip of a thin nasal catheter placed in the hypopharynx [6]. The most commonly used method to measure nasal NO is to sample nasal air directly from one nostril using the intrinsic flow of the chemiluminescence analyzer. A novel method of measuring exhaled NO at several exhalation flow rates has recently been described which can used to approximate alveolar and airway NO production [7]. NO is continuously formed in the airways. Mixing during exhalation between the NO produced by the alveoli and the conducting airways, explains its flow dependency and accumulation during a breath-hold [8]. It is therefore important to register the flow rate if NO is expressed as a concentration The flow rate recommended in 1997 by a Task Force of the European Respiratory Society is 10-15 L/min or 167-250 mL/s [1]. Most authors have used about 100 mL/s, but a more recent recommendation from the American Thoracic Society suggests 50 mL/s [9]. Exhaled and nasal NO in healthy subjects is independent of age, gender and lung function [10]. There is no evidence for significant diurnal variation, and exhaled NO measurements are highly reproducible in normal subjects. Different phases of menstrual cycle may influence exhaled NO [11]. There are several major factors, which may change NO levels in normal subjects [10]. Some routinely used tests can transiently reduce exhaled NO, for example repeated spirometry [12], physical exercise [13], sputum induction [14]. Environmental factors, such as NO ozone, chlorine dioxide, are know to increase exhaled NO levels [15]. Habitual factors, such as smoking [16] and alcohol ingestion [17], reduce exhaled NO. Upper respiratory infection significantly increases exhaled NO [18] and nasal NO [19].

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References [1] S.A.Kharitonov et al.. Exhaled and nasa! nitric oxide measurements: recommendations. Eur. Respir. J. 10(1997) 1683-1693. [2] S.A.Kharitonov et al.. Increased nitric oxide in exhaled air of asthmatic patients. Lancet 343 (1994) 133-135. [3] S.A.Kharitonov, Barnes PJ. Nasal contribution to exhaled nitric oxide during exhalation against resistance or during breath holding. Thorax. 52 (1997) 540-544. [4] P.E.Silkoff et al.. Marked flow-dependence of exhaled nitric oxide using a new technique to exclude nasal nitric oxide. Am. J. Respir. Crit. Care. Med. 155 (1997) 260-267. [5] P.Paredi et al.. Exhalation flow and pressure-controlled reservoir collection of exhaled nitric oxide for remote and delayed analysis. Thorax. 53 (1998) 775-779. [6] A.Artlich et al.. Single Breath Analysis of Endogenous Nitric Oxide in the Newborn. Biol Neonate 79 (2001) 21-26. [7] L.Lehtimaki et al.. Increased bronchial nitric oxide production in patients with asthma measured with a novel method of different exhalation flow rates. Ann Med 32 (2000) 417-423. [8] S.A.Kharitonov et al.. The elevated level of exhaled nitric oxide in asthmatic patients is mainly derived from the lower respiratory tract. Am. J. Respir. Crit. Care. Med. 153 (1996) 1773-1780. [9] Recommendations for Standardized Procedures for the Online and Offline Measurement of Exhaled Lower Respiratory Nitric Oxide and Nasal Nitric Oxide in Adults and Children. Am. J. Respir. Crit. Care. Med. 160(1999) 2104-2117. [10] S.A.Kharitonov, Barnes PJ. Exhaled markers of pulmonary disease. Am J Respir Crit Care Med 163 (2001) 1693-1722. [11] S.A.Kharitonov et al.. Peak expiratory nitric oxide differences in men and women: relation to the menstrual cycle. British. Heart. J. 72 (1994) 243-245. [12] A.Deykin et al.. Exhaled NO following repeated spirometry or repeated plethysmography in healthy individuals. Am. J. Respir. Crit. Care. Med. 161 (2000) 1237-1240. [13] C.R.Phillips et al.. Exhaled nitric oxide during exercise: site of release and modulation by ventilation and blood flow. J. Appl. Physiol. 80 (1996) 1865-1871. [14] G.L.Piacentini et al.. Exhaled nitric oxide is reduced after sputum induction in asthmatic children. Pediatr. Pulmonol. 29 (2000) 430-433. [ 15] J.A.Nightingale et al.. Effect of inhaled ozone on exhaled nitric oxide, pulmonary function, and induced sputum in normal and asthmatic subjects. Thorax. 54 (1999) 1061-1069. [16] S.A.Kharitonov et al.. Acute and chronic effects of cigarette smoking on exhaled nitric oxide. Am. J. Respir. Crit. Care. Med. 152 (1995) 609-612. . [17] D.H.Yates et al.. The effect of alcohol ingestion on exhaled nitric oxide. Eur. Respir. J. 9 (1996) 1130-1133. [ 18] S.A.Kharitonov et al.. Increased nitric oxide in exhaled air of normal human subjects with upper respiratory infections. Eur. Respir. J. (1995) 295-297. [ 19] E.A.Ferguson, Eccles R. Changes in nasal nitric oxide concentration associated with symptoms of common cold and treatment with a topical nasal decongestant. Acta. Otolaryngol. 117(1997) 614617.

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Disease Markers in Exhaled Breath N. Marczin and M.H. Yacoub (Eds.) 1OS Press, 2002

Technical aspects of exhaled Nitric Oxide: Aerocrine point of view Lena KAJLAND-WILEN Marketing Director, Aerocrine AB, Smidesvagen 12, S-171 41 Solna, SWEDEN

Abstract. It is an honour and a great pleasure to present our views on the technical aspects of NO measurements on behalf of my company Aerocrine. We at Aerocrine have spent considerable effort and thoughts into this exiting topic, which has led to the development of our newly introduced NO analyser NIOX®.

Key points to consider when performing NO measurements are: • What shall you use the achieved NO values for? • Electrical safety measures, MOD approval and CE mark • Handling of ambient NO levels • Dead space handling • Exhaled flow accuracy and stability • Response time • Analyser signal drift • Reproducibility of results • Specifications at different ambient conditions • Calibration gas quality and stability • Potential ozone leakage I will go through each of these points more in detail and let you know how we have solved them in our system, the NIOX device. 1. What shall you use the achieved NO values for? At Aerocrine our goal is to establish NO measurements into routine clinical practise as a non-invasive tool to measure and follow the inflammation status in primarily asthma patients.

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Thus we have spent considerable efforts into design a device that will fit into a busy clinic workflow, to make it easy to use for more than just one instrument specialist.

One single unit consists of computer hardware, the NO analyser, pumps and flow controls. A lot of effort has been spent on the smoothness of software-hardware interaction, and the ease of use for both patients and instructor.

2. Electrical safety measures, MDD approval and CE mark NIOX is the only available NO analyser that is CE marked according to the medical device directive and approved for routine clinical use in Europe. NIOX has been checked for emission of electrical signals and how well it resist outside electrical signal interference. The CE mark means that the electrical safety performance to fit into a clinical setting has been scrutinised by an independent notified body. To ensure good and safe performance in a clinical setting, the CE-mark is a prerequisite. 3. Handling of ambient NO levels As ambient NO levels might be very high, in the range of 500 ppb is not unusual, especially in big cities, it is necessary to flush the lungs with NO free air prior to exhalation. One single deep breath of NO free air should be enough, which is also what the ATS standard says. In NIOX, we use a NO filter to ensure NO free inhalation air, also the whole system is flushed with NO free air between measurements. 4. Dead space handling As many of you know the dead space is the air in the beginning of the exhalation that is not originating from the lower parts of the lung. Dead space can be divided into anatomical dead space, i.e. the patient's upper respiratory tract volume and the instrument dead space, i.e. the tubing's etc from the mouth to the analyser.

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In NIOX we have minimized the instrument dead space by putting the sampling port very close to the mouth, use of a unique NIOX filter and by using a small sample tube diameter and with a fast sample tube flow, 5 ml/sec.

The anatomical dead space in NIOX is handled by; exhalation time, e.g. Vz a second, exhalation volume (patient specific) or based on height of the patient. 5. Exhaled flow accuracy and stability As the NO value in exhaled breath is very flow dependent, which is caused by that NO is primarily released from the bronchial walls, it is of extreme importance to keep the exhalation flow constant and tightly measured during the full exhalation, to ensure reproducible results. NIOX is equipped with a patented and calibrated dynamic flow control that keeps the exhalation at an even 50 ml/second regardless of patient skills. Also the flow is tightly measured with accurate, calibrated flow sensors. During exhalation a pressure indicator guides the patient on how hard to blow. Exhalation pressure may vary between 10 and 30 cm H2O without affecting the constant 50 ml/second flow. If patient does not manage to keep the pressure within these borders an indication, "Pressure out of range" will appear, the same will happen if the flow is not kept within the

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set limits of 45 to 55 ml/sec and the reading will not be valid. Thanks to this unique flow control even small children can manage an exhaled NO analysis in NIOX. The attractive balloon view also helps. 6. Response time When scrutinizing the performance of an NO analyser it is very important to consider if tests has been performed on the whole system and with exhaled air, or if the data only refers to data measured on the chemiluminoscence analyser level. As most NO analysers uses the same sensor technology, i.e. chemiluminoscence, the performance on the sensor level is similar. More important, as least we think so at Aerocrine, is the prestanda you get in clinical practise. As NIOX is to be used on real patients in real clinical settings, Aerocrine present total system performance data, i.e. the performance that corresponds to what you will see in your practise. At total system level (exhaled air) NIOX give you a lag time of less than 0.8 seconds, a rise time of less than 0.7 seconds and a total response time of 1.5 seconds. The analyser itself though has a response time of less than 200 mseconds.

7. Analyser signal drift Very important is that your NO analyser give you the same value irrespectively of when you measure your patient or how long time ago the device was calibrated. NIOX has a drift of less than 3 ppb over 14 days. This has been achieved by an automatic self test that checks the zero point drift of the NO signal, the flow and the pressure after each patient measurement. If a signal drift is detected the software automatically compensate for it. If the drift is abnormally high a warning message will appear. NIOX only needs to be calibrated twice a month and in the calibration procedure the NO signal, flow and pressure is simultaneously calibrated in a two point calibration. 8. Reproducibility of results As Dr Kharitonov just presented this reproducibility of NO results in NIOX has shown to hold true also in controlled clinical studies where the pooled standard deviation, was less than 2.5 ppb irrespectively when during the day or when during the week the NO

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measurement was done. This shows that our method and control systems hold true, in real clinical settings.

9. Specifications at different ambient conditions Where is your NO analyser placed? What is the temperature fluctuations? Are you at high altitude? Or maybe in a damp environment? As you are to measure a gas these conditions has a very pronounced effect on your results, and the reproducibility of them. Thus ensure that your NO analyser has been tested and proven to work within the environmental conditions that just your lab has. NIOX prestanda has been proven within 15-30 degrees Celsius, between 860 to 1060 heko-pascal and relative humidity of 30-75%. Also ensure you have a dehumidified sample going into the analyser, as the chemiluminoscence sensor technology is know to be very sensitive to the water content in the sample. 10. Calibration gas quality and stability To ensure good reproducible results you need to calibrate. But what are you calibrating with? If the calibration gas value is not controlled sufficiently and proven not to deteriorate or change over time your whole calibration curve might be wrong and as a consequence your data not correct. Moreover it is important to calibrate within a range that well corresponds to your application, if you are to analyse exhaled NO in asthma patients at 50ml/second flow rate, we have concluded 0 to 200 ppb is optimal as most patients have values within this range. The stability over time of the half-empty calibration cylinder is also of outmost importance, the design of the cylinder, the valve and the regulator are all very important for the end result. We moreover have found that how the calibration cylinder was cleaned and how inert the inner walls of it is, has pronounced effect on the deterioration of the calibration gas during the shelf life. Potential ozone leakage As all NO analysers today used chemiluminescence technology, all has an ozone generator which could potentially leak very high and hazardous levels of ozone. In NIOX we have built in a security system in several levels to ensure this cannot happen. Prior to start up of the ozone generator, the software checks that the vacuum pumps are on and that there is pressure in the system. The pressure and the flow in the system are routinely followed up and if below a certain critical value there is an alarm. Also we have built in an ozone sensor into the cabinet inside NIOX that online checks the level of ozone and that alarms in case of a too high value. If the software should crash there is an automatic system shutdown.

L. Kajland- Wilen / Technical Aspects of Exhaled NO: Aerocrine Point of View

We at Aerocrine have a high interest in finding solutions to the technical challenges of NO measurements and work actively together with many highly respected scientists to continually upgrade and improve our NO analysers. We are dedicated to ensure that, in routine clinical practise doctors shall be able to "See more than your asthma patients can tell you" by supplying accurate and reproducible exhaled NO measurements.

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Section 2. Carbon Monoxide

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Disease Markers in Exhaled Breath N. Marczin andM.H, Yacoub (Eds.) fOS Press, 2002

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Cytoprotection by heme oxygenase / CO in the lung Judit K. SARADY, Leo E. OTTERBEIN and Augustine M. K. CHOI Division of Pulmonary,Allergy and Critical Care Medicine, University of Pittsburgh School of Medicine 3459 5th Avenue, MUH 628, Pittsburgh, PA 15213 , USA

Abstract. The stress inducible gene heme oxygenase-1 (HO-1) provides protection against oxidative stress, although the precise mechanisms by which HO-1 exerts this cytoprotection is not clearly understood. It has been speculated that the by-products of heme catabolism by HO-1, namely bilirubin, ferritin and carbon monoxide (CO) possess important biological functions and may serve as mediators of HO-1 induced cytoprotection. The lung is particularly susceptible to oxidative stress. In this review we summarize the role of HO-1 in oxidant-induced lung injury, and provide evidence that CO may serve as an important mediator of cytoprotection.

It is becoming clear that oxidative stress plays a crucial role in the pathogenesis of many biological processes including aging, atherosclerosis, carcino-genesis, ischemiareperfusion tissue injury as well as many acute and chronic inflammatory diseases [1]. Reactive oxygen species (ROS) including superoxides, hydroxyl radicals and hydrogen peroxide (H2G2), generated from normal cellular respiration, aerobic metabolism and/or respiratory burst of inflammatory and immune cells can cause cellular damage by oxidizing nucleic acids, proteins and membrane lipids [2]. Mammalian systems evolved several enzymatic and non-enzymatic antioxidant defense systems, which eliminate ROS and help to maintain cellular homeostasis. The non-enzymatic systems include the circulative antioxidants, such as glutathione, vitamin E, albumin, transferrin-lactoferrin, bilirubin and ceruloplasmin. Besides the battery of enzymatic mechanisms present to detoxify free radicals including superoxide dismutase (SOD) and glutathione peroxidase, other protective enzymes also exist, designated the stress-response proteins (heat shock protein (HSP)), which include metallothionein (MT) and heme oxygenase-1 (HO-1) [3], 1. Heme oxygenase-1 Heme oxygenase (HO) was originally identified in 1968 by Tenhunen and colleagues [4] as the catalyst of the first and rate-limiting step in the degradation of heme. HO binds with heme, via oxidation cleaves its a-mesocarbon bridge to yield equimolar quantities of

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biliverdin, free iron and CO. Biliverdin is then subsequently converted to bilirubin by biliverdin reductase, while free iron is promptly sequestered into ferritin.(F/g 1)

Ferritin

Biliverdin

Reductase Heme —^«^«^^^ Biliverdin ••••• «•«•«^- Bilirubin

CO Fig 1. Enzymatic reaction of heme oxygenase

Three isoforms of HO have been identified [5,6,7,8]. HO-2 and HO-3 are constitutively synthesized mainly in the brain and testes whilst HO-1 is highly inducible by numerous stimuli [9]. Under physiological conditions HO activity is highest in the spleen where erythrocytes are sequestered and destroyed but its activity has been observed in all organs. Although the main inducer is heme, many non-heme agents including hypoxia [10], hyperoxia [11], endotoxin [12], and ultraviolet A [13] can also induce HO-1 expression. One common feature of these inducers is their capacity to generate ROS. Other studies demonstrated that HO-1 can not only be induced by non-heme agents that cause oxidative stress, but it can function as a cytoprotective molecule against the increased oxidative stress [14,15]. Observations in HO-1 ("} null mice as well as the first HO-1 deficient human [16,17] have also helped to understand the role of HO-1 in maintaining cellular homeostasis.

2. Cytoprotection by HO-1 Although the importance of HO-1 in the host's defense against oxidative stress is well known, the precise mechanisms by which HO-1 provides cytoprotection is still unclear. Recent data however indicate that products of heme catabolism mediate the cytoprotective effects of HO-1. Bilirubin accumulates in the serum of neonates, causing jaundice. In high concentration it is toxic, because it is deposited in the brain resulting in neurotoxicity. Bilirubin however is responsible for the antioxidant activity of human serum [18] and serves as a potent antioxidant in the brain, acting to scavenge peroxyl radicals as efficiently as atocopherol or vitamin E [19]. Free iron with it's two free electrons can generate hydroxyl radicals via the Fenton reaction. Ferritin synthesis is induced by released iron and serves as a reservoir to restrict

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iron from participation in the Fenton reaction. Among others, Balla et al [20] showed that induction of ferritin was cytoprotective in a model of oxidative stress. Carbon monoxide is, after carbon dioxide (CO2) the main atmospheric pollutant, emanating slowly from natural sources as volcanoes or forest fires and quickly as a byproduct of industrial and household activity [9]. Because of numerous lethal accidents caused by CO, for decades it was considered a poisonous gas, a toxic agent. Indeed, by binding to hemoglobin with a greater effect then oxygen, it replaces 62, increases the level of oxyhemoglobin by shifting the dissociation curve to the left and decreases 62 delivery to the cells causing tissue/cell hypoxia or anoxia. It is interesting to note that GO at low concentrations ( less then 0.01 % ) acts as a chemical messenger in neuronal transmission and modulation of vasomotor tone [22, 23] akin to NO. The majority of CO production in the body is derived from HO [22,24]. These effects of CO, just as of NO, are mediated through the activation of guanyl cyclase which cause an increase in the generation of cGMP [25]. CO may also possess anti-inflammatory effects such as the capacity to inhibit platelet activation or aggregation through activation of guanyl cyclase and subsequent generation of cGMP [9]. In recent studies, Otterbein et al. have demonstrated that administration of exogenous HO-1 by gene transfer [26] and exogenous administration of a low concentration of carbon monoxide (CO), protects rats against hyperoxia-induced lung injury [27]. Using a well-established model of LPS-induced endotoxemia both in vivo and in vitro it was also confirmed that HO-1 has potent anti-inflammatory effects which are mediated by CO [28]. Recently investigators [29,30] proposed to use CO as a biological and measurable marker in the exhaled breath of patients as an index of oxidative stress and inflammation.

3. Cytoprotection by HO-l/CO in the lung The lung is a major target organ for injury by ROS. ROS play a vital role in acute and chronic inflammatory lung diseases including ARDS, asthma, interstitial pulmonary fibrosis or emphysema [2]. In addition, patients who require supplemental O2 therapy are even more the "victims" of cellular damage caused by an increased oxidative burden. In order to improve our knowledge about the molecular responses of the lung, several in vitro and in vivo models have been developed to demonstrate the potent cytoprotective effects of HO-1 including endotoxic shock, hypoxia, hyperoxic lung injury or xenotransplantation related tissue rejection [12,26,31] . Studies by Lee and Suttner demonstrated the protective effect of overexpressing HO1 in pulmonary epithelial cells, fibroblasts, macrophages and smooth muscle cells [11] or fetal rat lung cells [32] which showed increased resistance to hyperoxic lung injury. It is interesting to note that high levels of HO-1 expression can be associated with significant cytotoxicity [33], which can be caused by the augmented release of iron from heme. Hyperoxic lung injury represents a model of oxidant-induced lung injury. After hyperoxia, the rat lung exhibits increased level of HO-1 mRNA and protein levels in many cell types, including the bronchoalveolar epithelium, interstitial cells and inflammatory cells [11]. Otterbein et al using exogenous administration of HO-1 by gene transfer could protect the rodents against oxidant-induced tissue injury. Adenoviral gene transfer of HO-1 (Ad5-

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HO-1) into the lungs of rats resulted in increased expression of HO-1 and a marked resistance to hyperoxic lung injury (reduced level of hyperoxia-induced pleural effusion, neutrophil alveolitis and BAL protein leakage) when compared to the untreated controls [26]. Taylor and al. also provided protection from hyperoxic lung injury by intratracheal administration of hemoglobin, a major inducer of HO-1 production [34]. Otterbein examined whether exogenous CO could confer the same cytoprotective effects as exogenous administration of HO-1 via gene delivery [26]. Based on the studies of Stupfel et. al, which showed that rodents and cells tolerated low concentrations (10-500 parts/million) of CO [[36], rats were exposed to hyperoxia in the presence of 250 ppm CO. This dose, which represents one twentieth of the lethal dose of CO in humans and rodents, offered significant protection from hyperoxic lung injury. Lipopolysaccharide (LPS), a constituent of the Gram negative bacterial cell wall can induce many of the pathophysiological changes observed in sepsis following administration to cells or animals. LPS induces high mRNA levels of HO-1 in rat lung tissues which correlate with increased enzyme activity [35]. Pretreatment of rats with hemoglobin resulted in increased HO-1 induction and provided complete protection against subsequent lethal endotoxemia. Tin protoporhyrin, a competitive inhibitor of HO, blocked this protective effect [12]. To test the hypothesis that CO provided cytoprotection against oxidative stress via anti-inflammatory effects, the levels of anti- and pro-inflammatory cytokines were measured. CO inhibited the LPS-induced proinflammatory cytokines tumor necrosis factor (TNF)-a, interleukin (IL)-IP , macrophage inflammatory protein (MlP)-la but augmented the antiinflammatory cytokine IL-10 expression. Examination of the signaling pathways mediating these effects, interesting results have been obtained. In contrast to other model systems such as neuronal and vascular cells, the guanyl cyclase/cGMP pathway was not involved, but the anti-inflammatory effects of CO were dependent on the mitogen activated protein kinase kinase (MKK-3)/ p38 mitogen activated protein kinase pathway [28]. Furthermore, Otterbein et al. , obtained additional supporting evidence for the involvement of the p38 MAPK pathway in the mechanism of CO effects by using a MKK3-deficient mouse model. MKK3 is the major upstream kinase activator of p38. LPS administration resulted in the inhibition of TNF-a in MKK3-deficient mice exposed to air, but no additional inhibition was detected when exposed to CO. At the same time, IL-10 levels were not elevated in the presence of CO as observed in the wildtype littermates. This confirms that inhibition of the MKK3/p38 MAP kinase pathway not only modulated the expression of inflammatory cytokines but also abrogated the COmediated cytoprotection against lung injury [37]. 4. Conclusions HO-1 has been well established as a stress response gene induced by a wide range of agents including heme and non-heme induced cellular stressors such as heavy metals, LPS, hydrogen peroxide, heat shock, cytokines, hypoxia, and hyperoxia. Although the mechanisms by which HO-1 mediates it's cytoprotective effects are still unclear, it seems likely that one or all of the three catalytic by-products of heme catabolism by HO-1, bilirubin, ferritin and CO act to protect the cells and tissues.

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Numerous studies have investigated the cytoprotective effects of HO-1 in the lung, using animal models of sepsis, hypoxia, hyperoxia and xenotransplantation. CO, a poisonous gas when administered at low concentration can mediate the potent cytoprotective effects of HO-1. Otterbein et al demonstrated that CO, when administered exogenously at low concentrations can exert potent anti-inflammatory effects both in vivo and in vitro models of LPS-induced inflammation, hyperoxic lung injury and allergen-induced inflammation [38]. It was also revealed that CO modulated pro- and anti-inflammatory cytokines and the responsible signaling pathways were identified. According to these new discoveries, perhaps in the future, HO-1, either via gene delivery or exogenous administration or CO in low concentration could be considered as a new therapeutic tool.

References [I] Choi AMK, Alam J: Heme oxygenase-1: Function,regulation and implication of a novel stress-inducible protein in oxidant-induced lung injury. Am J RespirCell Mol Biol 15: 9-19, 1996. [2] Kinnula VL, Crapo JD, Raivio KO; Biology of disease.Generation and disposal of reactive oxygen metabolites in the lung. Lab Inv 73: 3-19, 1995. [3] Camhi S, Lee PJ, Choi AMK: The oxidative stress response. New Horizons 3: 170-182, 1995. [4] Tenhunen R, Marver HS, Schmid R : the enzymatic conversion of heme to bilirubin by microsomal heme oxygenase . Proc Natl Acad Sci USA 61: 748-755, 1968. [5] Abraham NG, Lin JH, Dunn MW, Schwartzman ML : Presence of human heme oxygenase and NAPH cytochrome P-450 © reductase in human corneal epithelium. Invest Ophtalmol Vis Sci 28: 1464-1472, 1987. [6] Maines MD: Heme oxygenase:function,multiplicity,regulatory mechanisms and clinical implications. FASEB J 2: 2557-2568, 1988. [7] McCoubrey WK, Ewing JF, Maines MD: Human heme-oxygenase 2: characterization and expression of a full- length cDNA and evidence suggesting that the two HO-2 transcripts may duffer by choice of polyadenylation signal. Arch Biochem Biophys 295: 13-20, 1992. [8] McCoubrey WK, Huang TJ, Maines MD: isolation and characterization of a cDNA from the rat brain that encodes hemoprotein heme oxygenase-3. Eur J Biochem 247: 725-732, 1997. [9] Otterbein LE, Choi AMK: Heme oxygenase: colors of defense against celular stress. Am J Physiol Lung Cell Mol Physiol 279: L1029-L1037, 2000. [10] Carraway MS, Ohio AJ, Carter JD, Piantadosi CA: Expression of heme oxygenase-1 in the lung in chronic hypoxia . AM J Physiol Lung Cell Mol Physiol 278: L806-L812, 2000. [ I I ] Lee PJ, Alam J, Sylvester SL, Inamdar N, Ottrebein L, Choi Am: Regulation of heme oxygenase-1 expression in vivo and in vitro in hyperoxic lung injury . Am J Respir Cell Mol Biol 14: 556-568, 1996. [12] Otterbein LE,Sylvester SL,Choi AM: Hemoglobin provides protection against lethal endotoxaemia in rats: the role of heme oxygenase-1. Am J Respir Cell Mol Biol 13: 595-601, 1995. [13] Vile GF, Tyrrel RM: Oxidativ stress resulting from ultraviolet A irradiation of human skin fibroblasts leads to a heme oxygenase- dependant increase in ferritin . J Biol Chem 268: 14678- 14681, 1993. [14] Keyse SM,Tyrrel RM: Heme oxygenase is the major 32 -kDa stress protein induced in human skin fibroblasts by UVA radiation,hydrogen peroxide and sodium arsenite . Proc,Natl.Acad Sci USA 86: 99-103, 1989. [15] Maeshima H,Sato M, Ishikawa K, Katagata Y, Yoshida T: Participation of altered upstream stimulatory factor in the induction of rat heme oxygenase-1 by cadmium. Nucleid Acids Res 24: 2959-2965, 1996. [16] Poss KD, Tonegawa S: Reduced stress defense in heme oxygenase deficient cells. Proc Natl Acad Sci USA 94, 10925- 10930, 1997. [17] Yachie A, Niida Y, Wada T, Irgarashi N, Kaneda H: Oxidative stress causes enhanced endothelial cell injury in human heme oxygenase-1 defiency. J Clinic Invest 103:129-135, 1999. [18] Gopinathan V, Miller NJ, Milner AD, Rice-Evans CA: Bilirubin and ascorbate antioxidant activity in

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neonatal plasma . FEBS lett 349: 197-200, 1994. [19] Stocker R, Yamamoto Y, McDonagh AF, Glazer AN, Ames BN: Bilirubin is an anti-oxidant of possible physiological importance . Science 235 :1043-1046, 1987. [20] Balla G, Jacob HS, Balla J, Rosenberg M, Nath K, Apple F, Eaton JW, Vercelotti GM: Ferritin: a cytoprotective antioxidant stratagem of endothelium. J Biol Chem 267: 18148- 18153, 1992. [21] Otterbein LE, Chin BY, Otterbein SL, Lowe VC, Fessler HE, Choi AMK: Mechanism of hemoglobininduced protection against endotoxaemia in rats: a ferritin-independent pathway. Am J Physiol Lung Cell Mol Physiol 272: L 268- L275, 1997. [22] Morita T, Perella MA, Lee M, Kourembanas S: Smooth muscle cell -derived carbon monoxide is a regulator of vascualr cGMP. Proc Natl Acad Sci USA 92:1475-1479, 1995. [23] Verma A, Hirsch DJ, Glatt CE, Ronnett GV, Snuder SH: Carbon monoxide, a putatuve neural messenger. Science 259:381-384, 1993. [24] Snyder SH, Jaffrey SR, Zakhary R: Nitric oxide and carbon monoxide: paralell roles as neural messengers. Brain Res Rev 2-3: 167-175, 1998. [25] Cardell LO, Lou YP, Takeyama K, Ueki IF, Lausier J, Nadel JA: Carbon monoxide, a cyclic GMP- related messenger involved in hypoxic bronchodilatation in vivo. Pulm Pharmacol Ther 11: 309-315, 1 998. [26] Otterbein LE, Rolls JK, Mantell LL, Cook JL , Alam J, Choi AMK: Exogenous administration of heme oxigenase-1 by gene transfer provides protection against hyperoxia-induced lung injury. J Clin Invest 103. 1047-1054, 1999. [27] Otterbein LE, Mantell LL, Choi AMK: Carbon monoxide provides protection against hyperoxic lung injury Am J Physiol 276:L688-L694, 1999. [28] Otterbein LE, Bach FH, Alam J, Scares M, Lu HT, Wysk M, Davis RJ, Flavell RA, Choi AMK: Carbon monoxide has anti-inlammatory effects involving the mitogen-activated protein kinase pathway . Nat Med 6 , No 4: 422-428, 2000. [29] Horvath I, Donnelly LE, Kiss A, Paredi P, Kharitonov SA, Barnes PJ: Raised levels of exhaled carbon monoxide are associated with an increased expression of heme-oxygenase-1 in airway macrophages in asthma: a new marker of oxidative stress. Thorax 53: 668-672, 1998. [30] Horvath I, Loukides S, Wodehouse T, Khariatonov SA, Cole PJ, Barnes PJ : Increased levels of exhaled carbon monoxide in bronchiectasis: a new marker of oxidative stress. Thorax 53(10):867-70, 1998. [31] Scares MP, Lin Y, Anrather J, Csizmadia E, Takigami E, Sato K, Grey ST, Colvin RB, Choi AM, Poss KD, Bach FH: Expression of heme oxygenase-1 can determine cardiac xenograft survival. Nat Med 4 : 1073-1077, 1998. [32] Suttner DM, Sridhar K, Lee CS, Tomura T, Hansen TN, Dennery PA: Protective effects of transient HO-1 overexpression on susceptibility to oxygen toxicity in lung cells . Am J Physiol Lung Cell Mol Physiol 274:L443-L451, 1999. [33] Suttner DM, Dennery PA: reversal of HO-1 related cytoprotection with increased expression is due to reactive iron. FASEB J 13: 1800-1809, 1999. [34] Taylor JL, Carraway MS, Piantadosi CA: Lung-specific induction of heme-oxygenase-1 and hyperoxic lung injury . Am J Physiol Lung Cell Mol Physiol 274: L 582-L590, 1998. [35] Camhi SL, Alam J, Wiegand GW, Chin BY, Choi AMK: Transcriptional activation of the HO-1 gene by Hpopolysaccharide is mediate 5' distal enhancers: :role of reactive oxygen intermediates and AP-1. Am J RespirCell Mol Biol 18(2): 226-234, 1998. [36] Stupfel M, Bouley G: Physiological and biochemical effects on rats and mice exposed to small concentrations of carbon monoxide for long periods. Ann NY Acad Sci 174: 342-368, 1970. [37] Otterbein LE, Dyck DE, Otterbein SL, Knickelbein R, Davis RJ, Flavell RA, Choi AMK: MKK3 mitogen activated protein kinase pathway mediates carbon monoxide-induced protection against ox idant-induced lung injury (submitted) [38] Chapman JT, Otterbein LE, Elias JA, Choi AM: Carbon monoxide attenuates aeroallergen-induced inflammation in mice. Am J Physiol Lung Cell Mol Physiol 281(1):L209-16, 2001.

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Role of heme oxygenase in airway smooth muscle contractility Camilla TAILLE, Michel AUBIER and Jorge BOCZKOWSKI Ins ti tut National pour la Sante et la Recherche Medicate (INSERM), Unite 408, Faculte de Medecine Xavier Bichat, Paris, France. Abstract. This chapter summarizes data on the role of HO on smooth muscle contractility. The role of the antioxidant properties of HO are discussed.

1. Introduction Reactive oxygen species (ROS) are produced by all aerobic organisms as a consequence of oxygen consumption and cell respiration. They play the role of intracellular mediators at physiological concentrations. However, in stress situations, increasing production of ROS can lead to cellular injury [1]. One interesting biological relevant effect of oxidants, hydrogen peroxide and superoxide anion in particular, is the regulation of cell functions by controlling level of protein phosphorylation. This is modulated by activation of protein kinases or inactivation of protein phosphatases [1, 2]. In smooth muscle cells, level of phosphorylated proteins is critical for cell contraction : phosphorylation of myosin light chain (MLC) allows the myosin ATPase to be activated by actin and, thus the muscle to contract [3]. Different studies have shown that incubation of tracheal rings with exogenous applied oxidative species or with systems generating ROS, such as xanthine/xanthine-oxydase, induce airway smooth muscle hyperresponsiveness to contractile agonists [4-6]. In the same line, in vitro incubation of guinea pig trachea with superoxide dismutase decreases the contractile response to metacholine [7], suggesting a role of ROS produced by structural cells of the airways. However, the role of ROS in the modulation of MLC phosphorylation is unknown. Ubiquity of ROS production explains the need for strong antioxidant cellular systems. Heme oxygenase, the enzyme responsible for heme degradation, is a powerful antioxidant and protective system [8], Two main isoforms, products of different genes, have been identified: heme oxygenase-1 (HO-1), the inducible form (also known as heat shock protein 32), and HO-2, the constitutive form [8]. Degradation of the tetrapyrrolic ring produces carbon monoxide (CO) and biliverdin, reduced by the biliverdin reductase into bilirubin, one of the most important antioxidant molecule of the organism [9]. Whereas modulation of the HO pathway has shown beneficial effects in different pathological situations where oxidative stress is implicated [10], the role of HO in regulating cellular redox status in normal conditions has not been thoroughly investigated. In this chapter, we will report data from different groups and from our laboratory on the role of HO on smooth muscle contractility. We will discuss the role of the antioxidant properties of HO, including bilirubin, and how this property can modulate MLC phosphorylation.

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2. Heme oxygenase modulates airway smooth muscle contractility Heme oxygenase is strongly expressed in normal and pathologic human airways, especially in epithelial cells [11], but also in smooth muscle cells [12], parasympathetic ganglia, endothelium and macrophages [13]. If HO is a powerful antioxidant, it is also involved in the control of smooth muscle reactivity. HO-induced relaxation of smooth muscle has been well demonstrated in vessels [14], and gastrointestinal tissues [15]. CO release is the mechanism most frequently implied to explain this effect. Indeed, a pro-relaxant effect of CO has been demonstrated in vascular, intestinal and also bronchial smooth muscle [12, 14, 15]. This effect is related to the binding of CO to the heme moiety of the soluble isoform of guanylate cyclase and formation of the second messenger cGMP [14, 16]. Modulation of smooth muscle cell contraction, the other side of smooth muscle reactivity, by the HO pathway has been investigated in different tissues such as vessels [8. 18], myometrium [19], esophageal sphincter [20] and bronchi [21]. Increasing HO-1 activity in myometrium strips by a short time incubation with hemin, in an experimental protocol quite similar to ours, is able to inhibit contractile response to KC1, whereas SnPP, a HO inhibitor [8], has the opposite effect [19]. CO release has been proposed as a mechanism to explain this effect. On the contrary, Canning and coworkers [21] find that neither HO inhibitors nor the guanylate cyclase inhibitor ODQ, had marked effects on vagally-mediated bronchial contractions. We have evaluated the role of HO in the modulation of airway smooth muscle contraction in isolated guinea pig tracheal rings. In rings with an intact epithelium layer, 1 hour incubation with hemin (20 uM), an HO inductor or with tin protoporphyrin IX (SnPP, 10 uM), significantly decreased and increased contractile response to carbachol and histamine, respectively. The effect of hemin was reversed by SnPP. These modulations disappeared in epithelium-denuded tracheal rings, suggesting that epithelial HO modulated the contractility of the underlying smooth muscle. This phenomenon could be related to the low level of expression of HO-1 and HO-2 in tracheal smooth muscle as compared to the epithelium. Airway epithelium is not only exposed to exogenous oxidative damage but is also an important source of oxygen and nitrogen species thus explaining the need for a local strong antioxidant system [22]. Catalase for example, is more strongly expressed in epithelial than in smooth muscle cells [23]. Our results are in agreement with this data, thus confirming the protective role of epithelia against oxidants-induced contraction. We next investigated the role of the CO-cGMP pathway in mediating the contractile effect of HO. This was performed by evaluating the effect of the selective inhibitor of the soluble guanylate cyclase inhibitor ODQ, on the contractile response to carbachol of hemin or vehicle-treated tracheal rings. These experiments showed that ODQ did not interfere with the effect of hemin, suggesting that a role of CO-cGMP was unlikely in the decreased contractility induced by hemin. Although we ruled out the participation of CO-related production of cGMP in airway smooth muscle contraction, we do not exclude that CO can modulate airway reactivity in the present experimental model by a cGMP-independent mechanism. These might involve modulation of potassium channels [24] or kinase activity [25], as demonstrated in other tissues.

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3. Antioxidant properties of heme oxygenase and smooth muscle contractility Since the CO-cGMP pathway is not involved in the effect of HO, a role of the antioxidant properties of this system can be postulated. Indeed, ROS can potentiate smooth muscle contractility by different mechanisms : they are considered as intracellular messengers involved in control of calcium channel sensitivity, mitochondrial respiration or activation of protein-tyrosine kinases and inhibition of protein-tyrosine phosphatases [reviewed in (1), all features involved in muscular contraction. Among them, phosphorylation of the MLC is actually the key event for initiating smooth muscle contraction by modifying the conformation of myosin, and regulation of amount of phosphorylated/dephosphorylated protein is critical for the cellular physiology. Lopez-Ongil and coworkers [26] showed recently that exogenous hydrogen peroxide increased the amount of phosphorylated MLC and increased contraction of endothelial cells. Therefore, we next investigated the role of ROS in mediating the effects of HO on airway smooth muscle contractility. Initially, we evaluated if HO modulated ROS production in our model. This was performed by measuring SOD-inhibable lucigenin-dependent chemilumiscence in epithelium intact trachea! rings. Superoxide anion production was not affected by carbachol or by histamine, but it was significantly increased by SnPP. The effect of SnPP was suppressed by the antioxidants PEG-SOD plus PEG-Catalase. Superoxide anion production was significantly reduced by hemin, and this effect of was reversed by SnPP. In agreement with these results, PEG-SOD plus PEG-Catalase suppressed the increased contractility induced by SnPP, thus showing that the antioxidant properties of HO are involved in its effects on airway smooth muscle contraction. 4. The role of bilirubin Having demonstrated the involvement of the antioxidant properties of HO in its effects on airway smooth muscle contraction, we were interested in the role of bilirubin, since this molecule has been known to possess strong antioxidant properties for a long time. Bilirubin is able to scavenge different oxygen and nitrogen species and to inhibit production of superoxide by the NAD(P)H oxidase [9]. Different studies have shown that bilirubin decreased vascular or gastric smooth muscle contractility [27, 28]. For example, Colpaert and coworkers [27], showed that bilirubin potentiated the relaxant effect of nitric oxide in gastric smooth muscle with a similar potency than other antioxidants such as urate or glutathione. We investigated the effect of bilirubin on ROS production and contractility of guinea pig tracheal rings. We found that 10 nM of bilirubin significantly decreased spontaneous ROS production and suppressed the increased production induced by SnPP. This effect was accompanied by a suppression of the increased contractility induced by SnPP. Moreover, modulating the redox status of airway smooth muscle by hemin, SnPP, antioxidants or bilirubin, modulated the level of phosphorylated MLC. Indeed, Western blot analysis of phosphorylated MLC in airway smooth muscle showed that the basal level was significantly increased by SnPP and significantly reduced by PEG-SOD plus PEG-Catalase, hemin, or bilirubin. Furthermore, PEG-SOD plus PEGCatalase and bilirubin abrogated the increase in phosphorylated MLC induced by SnPP.

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5. Conclusion This chapter reviews evidence that the HO pathway, especially bilirubin, controls smooth muscle reactivity by decreasing ROS-dependent phosphorylation of the MLC. These data support the interest for antioxidant therapy in inflammatory diseases associated with an enhanced contractile response of smooth muscle, such as asthma. References 1. Gabbita SP, Robinson KA, Stewart CA, Floyd RA, Hensley K. Redox regulatory mechanisms of cellular signal transduction. Arch Biochem Biophys. 376: 1-13, 2000 2. Barrett W, DeGnores J, Keng Y, Zhang Z, Yim M and Chock P. Roles of superoxide radical anion in signal transduction mediated by reversible regulation of protein-tyrosine phosphatase IB. J Biol Chem. 274 : 34543-34546, 1999 3. Pfitzer G. Regulation of myosin phosphorylation in smooth muscle. J Appl Physiol. 91:497-503, 2001 4. Szarek J and Schmidt N. Hydrogen peroxide-induced potentiation of contractile responses in isolated rat airways. Am. J. Physiol. 258:L232-L237, 1990 5. Sadeghi-Hashjin G, Henricks PA, Folkerts G, Muis T, Garssen J, Nijkamp FP. Role of the epithelial layer in the generation of superoxide anion by the guinea-pig isolated trachea. Mediators Inflamm. 7:35-40, 1998 6. Katsumata U, Miura M, Ichinose M, Kimura K, Takahashi T, Inoue H and Takishima T. Oxygen radicals produce airway constriction and hyperresponsiveness in anesthetized cats. Am. Rev. Respir. Dis. 141:11581161,1990 7. De Boer J, Pouw F, Zaagsma J and Meurs H. Effects of endogenous superoxide and nitric oxide on cholinergic constriction of normal and hyperreactive guinea pig airways. Am J Respir Crit Care Med. 158:1784-1789, 1998 8. Maines MD. The heme oxygenase system : a regulator of second messenger gases. Anna Rev Pharmacol Toxicol. 37:517-554, 1997 9. Stocker R, Yamamoto Y, McDonagh AF, Glazer AN, Ames BN. Bilirubin is an antioxidant of possible physiological importance. Science. 235:1043-6, 1987 10. Choi AMK, A lam J. Heme oxygenase-1 : function, regulation and implication of a novel stress-inductible protein in oxidant-induced lung injury. Am J Respir Cell Mol Biol. 15:9-19, 1996 11. Lim S, Groneberg D, Fischer A, Gates T, Caramori G, Mattos W, Adcock I, Barnes PJ, Chung KF. Expression of heme oxygenase isoenzymes 1 and 2 in normal and asthmatic airways: effect of inhaled corticosteroids. Am J Respir Crit Care Med. 162:1912-1918, 2000 12. Kinhult J, Uddman R, Cardell LO. The induction of carbon monoxide-mediated airway relaxation by PACAP 38 in isolated guinea pig airways. Lung. 179: 1-8, 2001 13. Horvath I, Donnelly LE, Kiss A, Paredi P, Kharitonov SA, Barnes P. Raised levels of exhaled carbon monoxide are associated with an increased expression of heme oxygenase-1 in airway macrophages in asthma : a new marker of oxidative stress. Thorax. 53:668-672, 1998 14. Motterlini R, Gonzales A, Foresti R, Clark JE, Green CJ, Winslow RM. Heme oxygenase-1-derived carbon monoxide contributes to the suppression of acute hypertensive responses in vivo. Circ Res. 83:568-77, 1998 15. Rattan S, Chakder S. Influence of heme oxygenase inhibitors on the basal tissue enzymatic activity and smooth muscle relaxation of internal anal sphincter. J Pharmacol Exp Ther. 294:1009-10, 2000 16. Rattan S, Chakder S. Inhibitory effect of CO on internal anal sphincter: heme oxygenase inhibitor inhibits NANC relaxation. Am J Physiol. 265:G799-804, 1993 17. Caudill TK, Resta TC, Kanagy NL, Walker BR. Role of endothelial carbon monoxide in attenuated vasoractivity following chronic hypoxia. Am J Physiol. 275:RI025-R1030, 1998 18. Duckers HJ, Boehm M, True AL, Yet SF, San H, Park JL, Clinton Webb R, Lee ME, Nabel GJ, Nabel EG. Heme oxygenase-1 protects against vascular constriction and proliferation. Nat Med. 7:693-8, 2001 19. Acevedo CH, Ahmed A. Hemeoxygenase-1 inhibits human myometrial contractility via carbon monoxide and is upregulated by progesterone during pregnancy. J Clin Invest. 101:949-955, 1998 20. Ny L, Larsson B, Aim P, Ekstrom P, Fahrenkrug J, Hannibal J, Andersson KE. Distribution and effects of pituitary adenylate cyclase activating peptide in cat and human lower oesophageal sphincter. Br J Pharmacol. 116:2873-80, 1995 21. Canning BJ, Fischer A. Localization of heme oxygenase-2 immunoreactivity to parasympathetic ganglia oh human and guinea pig airways. Am J Respir Cell Mol Biol. 18:279-285, 1998

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22. Yamada N, Yamaya M, Okinaga S, Lie R, Suzuki T, Nakayamam K, Yamaguchi T, Itoyama Y, Sekizawa K, Sasaki H. Protective effects of heme oxygenase-1 against oxidant-induced injury in the cultured human tracheal epithelium. Am J Respir Cell Mol Biol. 21 : 428-435, 1999 23. Asano T, Hattori T, Tada T, Kajikuri J, Kamyia T, Saitoh M, Yamada Y, Itoh M, Itoh T. Role of the epithelium in opposing H2O2-induced modulation of acetylcholine-induced contractions in rabbit intrapulmonary bronchiole. Br JPharmacol. 132: 1271-1280,2001 24. Wang R, Wu L, Wang Z. The direct effect of carbon monoxide on KCa channels in vascular smooth muscle cells. Pflugers Arch, 434:285-291, 1997 25. Brouard S, Otterbein LE, Anrather J, Tobiasch E, Bach FH, Choi AM, Scares MP. Carbon monoxide generated by heme oxygenase 1 suppresses endothelial cell apoptosis. J Exp Med. 192:1015-26, 2000 26. Lopez-Ongil S, Torrecillas G, Perez-Sala D, Gonzalez-Santiago L, Rodriguez-Puyol M, Rodriguez-Puyol, D. Mechanisms involved in the contraction of endothelial cells by hydrogen peroxide. Free Radic Biol Med. 26:501-510, 1999 27. Bomzon A, Gali D, Better OS, Blendis LM. Reversible suppression of the vascular contractile response in rats with obstructive jaundice. J Lab Clin Med. 105 :568-572, 1985 28. Colpaert EE, Lefebvre RA. Influence of bilirubin and other antioxidants on nitrergic relaxation in the pig gastric fundus. Br J Pharmacol. 129 :1201-1211, 2000

Disease Markers in Exhaled Breath N. Marczin and M.H. Yacoub(Eds.) IOS Press, 2002

Exhaled Carbon Monoxide is Produced in the Lungs Jigme M. SETHI Division of Pulmonary, Allergy, and Critical Care Medicine, University of Pittsburgh Medical Center, 3459 Fifth Avenue, Pittsburgh, PA 15213 Abstract. Carbon monoxide is produced endogenously by the degradation of heme in the reaction catalyzed by heme oxygenase, and is exhaled by the lungs. Heme oxygenase is highly induced in lung tissue by a variety of stressors, potentially accounting for the increase in exhaled carbon monoxide. In inflammatory diseases of the lung, levels of exhaled carbon monoxide are elevated, and return to normal with topical treatment of the disease, suggesting local production of carbon monoxide in the lung.

1. Introduction Endogenous production of carbon monoxide (CO) in humans was reported in 1949[1], but only in 1967 did it become clear that the gas was produced by the catabolism of hemoglobin [2]. Tenhunen, in 1968, identified heme oxygenase (HO) as the enzyme responsible for this reaction [3], and as might be expected, HO is abundantly present in the liver and spleen. For many years, it was assumed that HO was merely a catabolic enzyme, liberating iron for reutilization in heme synthesis, and biliverdin and bilirubin for excretion via the biliary tract. Similarly, it was believed that CO was also merely a waste product, eliminated almost exclusively by exhalation. The error of this assumption became clear when it was discovered that HO-1, the inducible isoform, is strongly upregulated by a variety of cellular stressors, including heavy metals, oxidizing agents, cytokines, ultraviolet radiation, and inflammatory byproducts [4], and that this upregulation has a cytoprotective, anti-oxidant, and anti-inflammatory role [5]. Admittedly, during basal conditions, the CO derived from heme degradation is definitely eliminated by exhalation, but the more complex issue of the origin of the elevated levels of CO in patients with lung inflammation has not been resolved. Where does this increased CO come from? Does it reflect exaggerated systemic heme turnover during inflammatory states, or is it possible that it is locally produced in inflamed lung? Three lines of evidence support the stance that the exhaled CO in inflammatory states originates from the lung: HO-1 mRNA, protein, and activity are all upregulated in lung tissue in numerous animal models of lung injury, confirming the capacity of the lung to produce CO locally in response to inflammatory injury; CO levels are elevated in the exhaled breath of patients with predominantly pulmonary inflammation; and finally, evidence that CO may actually be added to the systemic circulation by the lungs.

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2. Data from animal studies When rats are given intra-tracheal hemoglobin, increased HO-1 protein can be demonstrated by western blotting of lung homogenates as well as by immunocytochemical staining of alveolar epithelium in intact lung tissue. This increase represents functional HO-1 protein confirmed by assays of HO-1 enzymatic activity [6]. Similarly, progressively increased staining for HO-1 is noted in alveolar epithelium and macrophages over the 24, hours following intra-tracheal instillation of LPS into rat lungs [7]. Peak induction of lung HO-1 protein and enzymatic activity is seen at one day after exposure of rats to hypoxic conditions, and in this model, immunochemical staining for HO-1 is concentrated in alveolar macrophages and smooth muscle cells of the arterioles by day 21 [8]. 3. Data from lung-limited diseases in humans Exhaled CO levels are significantly elevated over non-smoking control subjects in patients suffering from an upper respiratory tract infection, and these levels decline to baseline with recovery from this purely respiratory tract-limited ailment in 3 weeks [9]. Exhaled CO levels are also elevated in patients with bronchiectasis, as compared to healthy, non-smoking subjects [10]. In adult patients with cystic fibrosis, exhaled CO levels are elevated during exacerbations of pulmonary disease and decline to normal after treatment with antibiotics. During these exacerbations, levels of exhaled CO have been noted to be highest in the subset of patients with the most reduction of FEV^ from baseline [11]. Most importantly, exhaled CO levels are lower in the subset of patients with cystic fibrosis who have been treated with inhaled steroids, a class of drugs that exert their anti-inflammatory effects predominantly via local intra-pulmonary deposition [12]. Similar data in asthmatics also suggests intra-pulmonary production of CO. In asthmatics exposed to an inhaled allergen challenge, both immediate and late phase reductions of FEVt are accompanied by temporally linked increases in exhaled CO levels [13]. Untreated asthmatics have higher levels of exhaled CO than control subjects or asthmatics treated with inhaled steroids [14], and indeed, the elevated levels of exhaled CO correspond to the severity and duration of the reduction in peak expiratory flow rates in asthmatic subjects experiencing an exacerbation [15]. After initiation of a course of oral steroids for asthmatic exacerbations, exhaled CO levels return to normal with the same time course as improvement in peak flow rates. Finally, the resolution of sputum eosinophilia in asthmatics treated with inhaled steroids is tightly correlated to simultaneous reduction in elevated levels of CO in the exhaled breath [14]. In light of this clinical data, it would not be surprising to note that sputum macrophages from asthmatic patients, but not normal subjects, show increased HO-1 protein expression by western blotting [16].

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4. Confirmatory studies Confirmatory proof of pulmonary production of CO comes from the observation that exhaled CO concentrations rose dramatically in each of 4 subjects who inhaled 2 mL doses of 1CT4 M hemin, a potent inducer of HO-1 [16]. In a small study of critically ill. mechanically ventilated patients, arterial levels of carboxyhemoglobin were higher than mixed-venous carboxyhemoglobin levels, suggesting net pulmonary addition of CO to the systemic circulation under these conditions [17]. 5. Conclusion While there cannot be any doubt that in basal conditions CO derived from heme breakdown in the reticulo-endothelial system is eliminated from the body by exhalation, the evidence outlined above does confirm the ability of the lung to produce CO locally, in inflammatory conditions characterized by marked up-regulation of heme oxygenase. There can be no doubt that locally produced CO represents a major component of the increased concentration of exhaled CO noted in humans with purely pulmonary diseases, in which there should be no reason for increased delivery of CO to the lungs from the circulation. References 1. 2. 3. 4. 5.

6. 7. 8. 9. 10. 11. 12.

Sjorstrand, T. Endogenous formation of carbon monoxide in man under normal and pathological conditions. Scand. J. Clin. Lab. Invest. 1: 201-214; 1949. Coburn, RF; Williams, WJ; White, P; Kahn, SB. Production of carbon monoxide from hemoglobin in vivo. J. Clin. Invest. 46:346-356; 1967 Tenhunen, R; Marver, HS; Schmid, R. The enzymatic conversion of heme to bilirubin by microsomal heme oxygenase. Proc. Natl. Acad. Sci. USA. 61(2):748-55; 1968. Applegate, LA;Luscher, P;Tyrrell, RM. Induction of heme oxygenase: a general response to oxidant stress in cultured mammalian cells. Cancer Res. 51(3): 974-8; 1991. Sethi, JM; Choi, AMK. Heme oxygenase -1 in Acute Lung Injury, pp 119-132,/w Molecular Biology of Acute Lung Injury. Wong, HR; Shanley, TP (eds.). The Molecular and Cellular Biology of Critical Care Medicine. Clark, RSB; Carcillo, JA (series eds.).KIuwer Academic Publishers. Boston;2001. Taylor, JL;Carraway, MS; Piantadosi, CA. Am. J. Physiol. Lung Cell. Mol. Physiol.274(4):L582590; 1998. Carraway, MS; Ohio, AJ; Taylor, JL; Piantadosi, CA. Am. J. Physiol. Lung Cell. Mol. Physiol.275(3):L583-592;1998 Carraway, MS; Ohio, AJ; Carter, JD; Piantadosi, CA. Am. J. Physiol. Lung Cell. Mol. Physiol. 278(4):L 806-812;2000 Yamaya, M; Sekizawa, K; Ishizuka, S; Monma, M; Mizuta, K; Sasaki, H. Am. J. Respir. Crit. CareMed. 158:311-4; 1998. Horvath, I; Loukides, S; Wodehouse, T; Kharitonov, SA; Cole,PJ; Barnes, PJ. Thorax 53(10):86770; 1998. Antuni, JD; Kharitonov, SA; Hughes, D; Hodson, ME;Margaret, E;Bames PJ. Thorax 55(2)13842; 2000. Paredi,P; Shah, PL; Montuschi,P; Hodson, ME; Kharitonov, SA; Barnes, PJ. Thorax 54(10):91720; 1999.

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13. 14. 15. 16. 17.

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Paredi,P; Leckie, MJ; Horvath, I; Allegra, L; Kharitonov, SA; Barnes, PJ. Eur. Respir. J. 13:4852; 1999. Zayasu, K; Ohrui,T; Sekizawa,K; Yamaya, M; Fukushima, T; Sasaki, H; Takishima,T. Am. J. Respir. Crit. Care Med. 156:1140-3; 1997. Yamara, M; Sekizawa,K; Ishizuka, S; Monma, M;Sasaki,H. Eur. Respir. J. 13:757-60; 1999. Horvath, I; Donnelly, LE; Kiss, A; Paredi, P; Kharitonov, SA; Barnes, PJ.Thorax.53(8):66872;1998. Meyer, J; Prien, T;van Aken, H; Bone, H-G; Waurick, R; Theilmeier, G; Booke, M. Biochem. Biophys. Res. Com. 244:230-2; 1998.

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Disease Markers in Exhaled Breath N. Marczin and M.H. Yacoub(Eds.) IOS Press, 2002

Exhaled Carbon Monoxide is Delivered from Systemic Sources Nandor MARCZIN Department of Anaesthetics, Royal Brompton and Harefield Hospital and Department of Cardiothoracic Surgery, Faculty of Medicine, National Heart and Lung Institute, Imperial College, Heart Science Centre, Harefield Hospital, Harefield, United Kingdom Abstract. Similarly to exhaled nitric oxide (NO), exhaled carbon monoxide (CO) appears to be a promising non-invasive tool to assess lung inflammation in a variety of conditions [1]. However, the origin and determinants of exhaled CO remains controversial. Particularly, the magnitude of local airway production of CO and contribution from delivery from the pulmonary and systemic circulation has not been established. We have addressed these issues by investigating exhaled NO and CO in patients undergoing cardiac surgery. Here, the dynamically changing ventilation and pulmonary blood flow parameters have provided interesting insights into the influence of ventilation and blood flow on gaseous concentrations of CO. The data show that measured concentrations significantly depend on ventilation settings and on pulmonary blood flow. A large proportion of exhaled CO appear to originate from systemic circulation suggesting that this fraction should be taken into consideration before exhaled CO is widely accepted as a marker of pulmonary inflammation.

1. Objectives and study design We have measured exhaled CO and NO in anesthetized intubated patients" undergoing routine open heart surgery, which provides a good opportunity to test the influence of different ventilation maneuvers and dynamically changing pulmonary blood flow on CO and NO concentrations in the gas phase. Since breath holding maneuvers have been particularly useful in delineating characteristics of exhaled gases by showing accumulation of exhaled gases in the airway dead space and by demonstrating changes in concentrations in alveolar gases following breath holding [2,3 and Chapter 8 by Adding and Gustafsson in this volume], we performed these measurements in order to delineate alveolar vs. main airway CO concentrations. In order to investigate influence of pulmonary blood flow on exhaled CO we have studied the influence of PEEP, institution of cardiopulmonary bypass and cardiac arrest. During all these maneuvers, CO was compared to characteristics of CO2, a gas molecule largely derived from systemic sources. 2. Methods Breath to breath measurements of exhaled gas concentrations in the lower airways were performed using a real-time, computer-controlled and integrated system (Logan Research

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Ltd. 2000 and 3000 series) [4]. Inspired and expired samples for analysis of CO, NO and CO2 were continuously withdrawn at a flow rate of 150 ml/minute directly from the main lower airways through a thin Teflon sampling tube positioned within the endotracheal tube. Since detected concentration of exhaled gases depends on both the production rate and ventilation parameters, ventilation was normally standardized for inspired gas (100% 02), tidal volume (5 ml/kg), respiratory rate (10/min) and inspiratory and expiratory ratio (1:2). To eliminate the influence of positive end expiratory pressure on gas phase NO, PEEP was set to zero. 3. Alveolar vs. main airway CO concentrations during breath-hold Figure 1 shows representative traces of gas phase CO, NO and CO2 during normal ventilation cycles and during different types of breath-holding. The traces demonstrate that both end-expiratory and end-inspiratory breath-holding results in accumulation of CO, NO and CO2 in the gas phase. The first respiratory manoeuvre following the release of breath-holding allows us to derive information regarding relative gas concentrations between the alveoli and main airways. Figure 1 left panel shows that following expiratory breath-hold the first inspiration delivered by the Servo 900D ventilator is an inspiration of 100% O2- Since this inspired gas does not contain CO, NO and CO2 , it is expected that this gas dilutes gases accumulated in the main airway during breath-holding and as a consequence, CO, CO2 and NO concentrations would decrease after release of

Figure 1. Characteristics of exhaled gases following end-expiratory (left panel) and end-inspiratory (right panel) breath hold. Downwards arrows above the flow trace indicate the start of breath-hold.

breath hold. Upward arrow in the left panel indicates that this is indeed the case. Interestingly, the first expiration depicts different characteristics of NO and CO. In the case of NO, the trace during the first expiration after breath holding does not appear to be any different from the expiratory pattern of NO before breath-holding. However, exhaled CO and CO2 following breath holding appear to be increased when compared to prior breath holding. Following end-inspiratory breath-holding and during the first spontaneous expiration by the patients, alveolar gases dilute the gases accumulated in the airway dead space during breath-holding. Since alveolar gas is rich in CO2, main airway CO2 levels appear to rise further upon release of inspiratory breath hold (upwards arrow, right hand panel). If concentrations of CO and NO were higher in the alveoli than in the airways, a similar pattern to CO2 should be seen, and if alveolar CO and NO equals airway NO, the

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mixing should not change the detected concentrations. However, it is obvious that NO concentrations rapidly fall during the first exhalation, whereas CO levels appear to rise. This provides clear evidence that despite accumulation of high concentrations of NO in the lower airways, NO concentrations in the alveoli remains very low during breathholding. This suggests the existence of a mechanism that continuously removes NO from the alveoli. Contrary, it appears that the behaviour of CO is similar to CO2 suggesting that alveolar concentrations of CO are high and that measured concentrations during tidal ventilation and breath holding underestimate true alveolar CO concentrations. Thus, there should be mechanisms that continuously enrich alveoli with CO. Pulmonary blood flow can be postulated to fulfil both of these functions ie., delivery of CO to alveoli from systemic sources and uptake of NO delivered from the airways. This might also explain why exhaled CO and CO2 but not NO is higher during the subsequent breaths after the breath holding period. CO and CO2 appear to accumulate in the systemic circulation during apnoea and it might take several breaths for the blood concentrations of these gases to return to baseline. In contrast, pulmonary blood flow has extreme capacity to take up inhaled NO. Thus one single respiratory cycle is likely to be sufficient to restore exhaled NO concentrations to pre-breath holding levels. 4. Influence of pulmonary blood flow The hypothesis that exhaled CO is influenced by pulmonary blood flow was tested by evaluating the influence of a) PEEP, b) cardiopulmonary bypass and c) cardiac arrest on exhaled gases. Influence of PEEP Ventilation with Positive End Expiratory Pressure is frequently employed in anaesthetic practice. Among other physiological changes the principle events during PEEP application are changes in shear forces, recruitment of alveoli and a reduction of capillary' blood flow. Interestingly, PEEP has been shown to dramatically influence exhaled NO concentrations and this argument has been used to support airway origin and the role of shear forces in regulation of airway production of NO [5,6]. We have reasoned that PEEP might similarly affect exhaled CO concentrations and that PEEP induced alterations in exhaled CO might provide insights into origin of exhaled CO. In particular, a PEEP-induced increase on exhaled CO would suggest a local shear stress mediated production in the lungs, whereas a decrease would imply reduced delivery by the decreased pulmonary blood flow. As shown in Figure 2, application of 10cmH20 PEEP produced and immediate change in exhaled gases. Whereas exhaled NO rose dramatically, there was a detectable decrease in expired CO and COi concentrations. Thus, similarly to breath holding, PEEP also produce a differential effect on exhaled CO and NO concentrations and in this respect, too, CO appears to mimic CO2 exhalation profile. These data confirm in humans previous observations on animals regarding the influence of PEEP on exhaled NO [5,6] and are also consistent with CO delivered from systemic sources.

N. Marczin /Exhaled CO is Delivered from Systemic Sources

Figure 2. Influence of PEEP on exhaled CO, NO and CO2 concentrations.

Influence ofCPB We reasoned that upon instrumentation of CPB when the entire output of the right ventricle is diverted away from the pulmonary circulation, the reduction of pulmonary arterial blood flow should affect capillary blood volume despite bronchial arterial blood flow. This is consistent with everyday experience of reduced pulmonary artery pressures during CPB. As shown in figure 3. institution of CPB indeed results in an immediate decline in expired COi as a reflection of decreased pulmonary blood flow. One can appreciate that NO exhibits a different pattern and that gaseous concentration of NO appears to increase following CPB. In addition, CPB is associated with about 50% reduction of expired CO.

Figure 3. Influence of CPB on exhaled concentrations of CO, NO and CO2 Influence of cardiac arrest

In similar experiments during cadaveric organ donor harvesting we had the opportunity to evaluate the influence of cardiac arrest on exhaled CO. In this procedure, during preparation for liver perfusion, the inferior vena cava was drained resulting in progressive decrease in venous return to the heart leading to cardiac arrest. With ventilation parameters maintained we could observe the influence of complete reduction of pulmonary blood flow on exhaled gases. During this procedure we observed that

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similarly to COi, exhaled CO decreased to 10% of its original value, whereas little change in exhaled NO occurred. 5. Conclusions These simple experiments in humans demonstrate that in most experimental situations the behavior of exhaled CO was similar to CO2 and distinct from that of NO. Different lines of evidence suggest that at least in the patients studied a significant amount (up to 90%) of exhaled CO originated from the systemic circulation and was dependent on pulmonary blood flow. These observations might have important implications regarding the use of exhaled CO as a marker of airway inflammation. As discussed in the preceding chapter by Sethi, lung inflammation might contribute to exhaled CO through increased expression of heme oxygenase. However, any local CO production by the lung has to overcome the considerably high CO "noise" from systemic sources before producing a measurable effect on exhaled CO. This high noise obviously lessens the value of exhaled CO as a non-invasive marker in moderate degree of lung inflammation. Another important implication of the presented data is that alterations in exhaled CO may not represent changes in CO production in the lung but might be due to changes in systemic CO production and delivery to the alveoli by pulmonary blood flow. In particular, changes in pulmonary blood flow should be taken into considerations especially since lung inflammation can dramatically alter pulmonary vascular resistance and cardiac output. Acknowledgements This work has been supported by a MRC Clinician Scientist Fellowship to Dr. Nandor Marczin. References [1]

Horvath I, Donnelly LE, Kiss A, Paredi P, Kharitonov SA, Barnes PJ: Raised levels of exhaled carbon monoxide are associated with an increased expression of heme-oxygenase-1 in airway macrophages in asthma: a new marker of oxidative stress. Thorax 53: 668-672, 1998.

[2]

Persson MG, Wiklund NP, Gustafsson LE: Endogenous nitric oxide in single exhalations and the change during exercise. Am Rev Respir Dis 1993; 148:1210-4.

[3]

Marczin N, Riedel B, Royston D, Yacoub M. Exhaled nitric oxide in patients undergoing cardiothoracic surgery: A new diagnostic tool?. In. Matalon S and Sznajder Jl eds. Acute Respiratory Distress Syndrome: Cellular and Molecular Mechanisms and Clinical Management. New York Plenum Press, 1998: 365-374.

[4]

Marczin N, Riedel B, Gal J, Polak J, Yacoub M. Exhaled nitric oxide during lung transplantation. Lancet 1997; 350(9092): 1681-1682.

[5]

Carlin RE, Ferrario L, Boyd JT, Camporesi EM, McGraw DJ, Hakim TS. Determinants of nitric oxide in exhaled gas in the isolated rabbit lung. Am J Respir Crit Care Med 1997; 155:922-927.

[6]

Persson MG, Lonnqvist PA, Gustafsson LE. Positive end-expiratory pressure ventilation elicits increases in endogenously formed nitric oxide as detected in air exhaled by rabbits. Anesthesiology 1995; 82(4):969-974.

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Kinetics of Carbon Monoxide Accumulation in Exhaled Breath Raed A. DWEIK Department of Pulmonary and Critical Care Medicine Cleveland Clinic Foundation 9500 Euclid Avenue Cleveland Ohio 44195 U.S.A. Abstract. CO is an endogenously generated gas that may play important physiological roles. It is synthesized by heme oxygenases which degrade heme to biliverdin and CO. There are several lines of evidence to support an interaction between NO and CO suggesting potential physiologic and pathophysioiogic implications for this interaction. Whether CO in exhaled breath comes from the circulation or is locally produced in the lung remains the subject of considerable controversy. The relationship of CO to airway inflammation is not clear either.

1. Carbon monoxide (CO) CO is an endogenously generated gas that may play important physiological roles. CO is synthesized by heme oxygenases which degrade heme to biliverdin and CO. Two principal isozymes of heme oxygenase have been identified, a constitutive isoform heme oxygenase -2 and an inducible isoform heme oxygenase -1, which is expressed at a low basal level in vascular endothelial and smooth muscle cells and is induced by heavy metals, oxidative stress, inflammatory mediators and oxidized low density lipoproteins. Like NO, CO modulates platelet aggregation, smooth muscle relaxation, and intracellular cGMP levels, but CO has a much lower affinity (by a 100 fold) for soluble guanylyl cyclase than NO [1-4]. 2. CO - NO interaction There are several lines of evidence to support the interaction between NO and CO suggesting potential physiologic and pathophysioiogic implications for this interaction. It is unknown whether the two messengers converge or exhibit reciprocal feedback regulation and this may well depend on the particular organ system [4-12].

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There is dual expression of heme oxygenase and NOS in cells of several organ systems particularly those that require neural (brain, peripheral nerves) or smooth muscle regulation (the liver sinusoids, the intestine, uretheral sphincter, uterus, and pulmonary and systemic vessels). At physiologically and environmentally relevant levels, CO can directly affect endogenous NO levels and NOS activity. While high levels of CO inhibit NOS activity and NO generation, lower concentrations of CO induce release of NO from the intracellular pool and, therefore, increase endogenous NO levels. NO, endogenously produced or exogenously administered, affects heme oxygenase expression in vascular smooth muscles. Both NO and NO donors are capable of inducing heme oxygenase -1 protein expression, in a mechanism that depends on the de novo synthesis of RNA and protein. Molecular oxygen is essential for biosynthesis of both NO and CO. Oxygen levels in the physiologic range affect the endogenous production of both gases. Thus both NO and CO may act as oxygen sensors. Under normoxia, basal levels of NO and CO may act as amplifiers of molecular oxygen and keep the sensory discharge low. During hypoxia, decreased synthesis of NO and CO may contribute in part to the augmentation of sensory discharge. Some CO effects, like apoptotic cell death following exposure to relatively high concentrations of CO, appear to be mediated by nitric oxide [4-14]. 3. CO in exhaled breath CO in exhaled breath may be increased due to airway inflammation and subsequent induction of heme oxygenase in the airway in different lung diseases [3, 1316]. If CO is produced in the inflamed airway by HO-1; we expect that inflammation resulting from an experimental allergen challenge would result in an increase of CO in exhaled breath of atopic asthmatic individuals. Recently the interrelationship of four different exhaled gases [CO, NO, COz, and 02] was evaluated in asthma during an asthmatic response induced by Ag challenge [3]. Baseline exhaled NO is significantly increased in the asthmatics as compared to healthy controls and increases further following Ag challenge. In contrast to NO, however, baseline exhaled CO levels were similar to control individuals. Furthermore, CO levels did not increase over time after Ag challenge. Rather, CO levels decreased immediately after Ag, with levels tending to be lower even 1 h after Ag. Interestingly, there were similarities between the alterations in CO and CO2 a gas that is derived from diffusion into the lung. Exhaled O2 did not change significantly with Ag challenge. If CO is produced primarily within the lung and airways, the decrease of CO immediately after Ag may be related to increased consumption by lung tissue or hemoglobin. Alternatively if exhaled CO is primarily derived by diffusion from the bloodstream, decrease of CO may be due to decreased diffusion into the conducting airway.

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References [I] OtterbeinLE. BachFH. Alam J. Scares M. Tao Lu H. Wysk M. Davis RJ. Flavell RA. Choi AM. Carbon monoxide has anti-inflammatory effects involving the mitogen-activated protein kinase pathway. Nature Medicine. 6(4):422-8, 2000 Apr. [2] Wang R.Resurgence of carbon monoxide: an endogenous gaseous vasorelaxing factor. [Review] [ 102 refs] Canadian Journal of Physiology & Pharmacology. 76(1):1-15, 1998 Jan. [3] Khatri SB, Ozkan M, McCarthy K, Laskowski D, Hammel J, Dweik RA, Erzurum SC. Alterations in Exhaled Gas Profile during Allergen-induced Asthmatic Response. Am J Respir Crit Care Med. 2001; 164:1844-1848. [4] Foresti R. Motterlini R.The heme oxygenase pathway and its interaction with nitric oxide in the control of cellular homeostasis. [Review] [139 refs]Free Radical Research. 31(6):459-75, 1999 Dec, [5] Sharma VS. Magde D. Activation of soluble guanylate cyclase by carbon monoxide and nitric oxide: a mechanistic model.Methods. 19(4):494-505, 1999 Dec. [6] Prabhakar NR.NO and CO as second messengers in oxygen sensing in the carotid body. [Review] [39 refs]Respiration Physiology. 115(2): 161-8, 1999 Apr 1. [7] Snyder SH. Jaffrey SR. Zakhary R.Nitric oxide and carbon monoxide: parallel roles as neural messengers. [Review] [66 refs]Brain Research - Brain Research Reviews. 26(2-3): 167-75, 1998 May. [8] Hartsfield CL. Alam J. Cook JL. Choi AM.Regulation of heme oxygenase-1 gene expression in vascular smooth muscle cells by nitric oxide.American Journal of Physiology. 273(5 Pt 1):L980-8, 1997 Nov. [9] Durante W. Kroll MH. Christodoulides N. Peyton KJ. Schafer AI.Nitric oxide induces herne oxygenase-1 gene expression and carbon monoxide production in vascular smooth muscle cells.Circulation Research. 80(4):557-64, 1997 Apr, [10]Abu-Soud HM. Wu C. Ghosh DK. Stuehr DJ.Stopped-flow analysis of CO and NO binding to inducible nitric oxide synthase.Biochemistry. 37(11):3777-86, 1998 Mar 17. [ I I ] Huang LE. Willmore WG. Gu J. Goldberg MA. Bunn HF.Inhibition of hypoxia-inducible factor 1 activation by carbon monoxide and nitric oxide. Implications for oxygen sensing and signaling. Journal of Biological Chemistry. 274(13):9038-44, 1999 Mar 26. [ 12] Thorn SR. Fisher D. Xu YA. Notarfrancesco K. Ischiropoulos H. Adaptive responses and apoptosis in endothelial cells exposed to carbon monoxide.Proceedings of the National Academy of Sciences of the United States of America. 97(3): 1305-10, 2000 Feb 1. [13] Laskowski D, Ozkan M, Erzurum SC, Dweik RA. Validation of an analyzer to measure exhaled carbon monoxide levels in the part per billion range. Am J Respir Crit Care Med, 2000; 161: A851. [14] Ozkan M, Laskowski D, Dweik RA. High levels of carbon monoxide in patients with Chronic Beryllium Diseases. ChestlQQQ; 118:2535. [15] Ozkan M, Laskowski D, Erzurum SC, Dweik RA. Differences in Exhaled Carbon Monoxide and Nitric Oxide in Alveolar and Airway Inflammation. Am J Respir Crit Care Med2QOl; 163:A714. [16] Acharya SS, Laskowski D, Erzurum SC, Dweik RA. Exhaled Carbon Monoxide Levels in Ventilated Patients. Am J Respir Crit Care Med 2001; A899.

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ETCOc — An Indicator of Hemolysis in Neonatal Hyperbilirubinemia Molly MCCARTHY and Judith HALL Natus Medical Inc., 1501 Industrial Road, San Carlos, CA USA. 94070-4111 Abstract. Of the 4 million neonates born each year in the United States, 60 percent will develop clinical jaundice. The risk factors for normal, or physiologic jaundice, and pathologic jaundice are well-defined and documented. However, it remains difficult to differentiate the etiology of jaundice in routine clinical situations. Therefore, careful monitoring of neonates is important to prevent the potentially toxic effects of bilirubin. Neonatal hyperbilirubinemia results from increased production and/or decreased elimination of bilirubin. Bilirubin production can be exacerbated by several factors, including prematurity, blood group incompatibility, breakdown of extravascular blood, the shorter red cell lifespan associated with newborns, maternal diabetes, and unknown factors associated with ethnicity. Identification of the presence or absence of hemolysis is necessary to establish an effective care plan for each neonate. Understanding the relationship between bilirubin production and carbon monoxide (CO) is important in the management of neonatal jaundice. The catabolism of hemoglobin (Hgb) results in the equimolar formation of CO and bilirubin. End-tidal carbon monoxide corrected for room air (ETCOc) is an indicator of Hemolysis and bilirubin production. An elevated ETCOc identifies the neonate who is a high producer of bilirubin, even before the onset of hyperbilirubinemia, and can aid the clinician in establishing a differential diagnosis of the underlying causes of hyperbilirubinemia. Conversely, the ability to rule out elevated bilirubin production, when applied in conjunction with American Academy of Pediatrics recommendations, may provide the clinician with sufficient information to safely discharge the infant at an earlier hour.

1. Bilirubin Production, Transport and Excretion Catabolism of the red blood cell (RBC) produces bilirubin. The life span of the RBC averages 70 to 90 days in the neonate, a much shorter time period than observed in older children and adults who have a RBC life span of 120 days. Moreover, the rate of bilirubin production in premature infants exceeds that of term neonates [1]. Therefore, in caring for the pre-term or term neonate, it is imperative to consider all parts of the bilirubin pathway, including production, transport, and elimination. Figure 1 illustrates this process. Bilirubin production begins with the formation of RBCs in the bone marrow (Figure 1, Step I). The majority of bilirubin is produced during the catabolism of circulating RBCs, with a small proportion produced from ineffective erythropoiesis [2]. Hgb is released from the RBC during catabolism. The degradation of Hgb by heme oxygenase and biliverdin reductase results in the formation of bilirubin and CO (Figure 1, Step II). This bilirubin, in its unconjugated form, immediately combines with available plasma albumin for transport to the liver (Figure 1, Step III) [2,3]. Because albumin is the mechanism for transporting the bilirubin, neonates who have

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insufficient albumin to bind the bilirubin are at greater risk for hyperbilirubinemia. Once the albumin releases the unconjugated bilirubin into the liver, the hepatic cell membrane absorbs and conjugates the bilirubin (Figure 1, Step IV) [4]. Bilirubin excretion (Figure 1, Step V) begins as the conjugated bilirubin is released from the liver into the bile duct and intestine. In the intestine, the conjugated bilirubin is converted into a highly soluble substance, urobilinogen. Urobilinogen is released in the intestine and excreted as stercobilin in the feces [5]. A small percentage of urobilinogen is transported to the kidneys and excreted in the urine. Some of the conjugated bilirubin may be converted back into unconjugated bilirubin which is absorbed into the blood through the intestinal mucosa and transported to the liver for re-excretion. This pathway constitutes the enterohepatic circulation of bilirubin.

Figure 1: Normal Pathways of Bilirubin Production, Transport and Excretion Some portions of Figure I are adapted with permission from Gartner LM, Hollander M. Disorder s of bilurubin metabolism In Assali NS, editor: Pathophysiology of Gestation, 3rd ed., New York, NY: Academic Press; 1972:273.

2. Hemolysis and Carbon Monoxide Approximately 85 percent of endogenous CO is a byproduct of heme catabolism [3]. Figure 2 illustrates the process of hernolysis and production of endtidal carbon monoxide (ETCO) in detail. The degradation of heme results in the equimolar formation of bilirubin and of CO.I3J CO binds to circulating RBCs and can be measured as carboxyhemoglobin (COHb) in the blood. COHb dissociates in the lungs and CO is excreted in the breath continuously. Thus, total bilirubin production can be estimated from COHb or by ETCO. A fraction

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of ETCO is derived from the ambient CO. Therefore, to accurately reflect the rate of bilirubin production, ETCO should be corrected for room air (ETCOc). This is particularly true in neonatal jaundice where CO levels, measured in parts per million (ppm), are very low. The COHb measurement does not differentiate exogenous CO from breath CO. Therefore it is not possible, to estimate a precise endogenous CO level from a COHb reading.

Hemotysis Breakdown of Red Blood Cells (RBCs)

Environmental Carbon Monoxide (CO)

Hcme CataboUsm Endogenous CO Source

Exposure Exogenous CO Source

Carbon Monoxide (CO)

(COHb) Biliary Excretion

Pulmonary Excretion End-Tidal CO (ETCO)

Figure 2: Hemolysis and Production of End Tidal Carbon Monoxide Some portions of Figure 2 are adapted with permission from V reman HJ. Mahoney JJ, Stevenson DK. Carbon monoxide and carboxyhemoglobin. Adv Pediutr 1995:42:303-334. Si Louis: Mosby, 1995

3. The Impact of Early Discharge The emergence of managed care has resulted in cost containment strategies impacting the care of neonates due to early discharge [6]. The clinical management of neonatal jaundice is especially affected by early discharge (defined as less than 72 hours) because the bilirubin level will continue to rise even after the newborn has left the hospital setting, placing neonates at risk for readmission to the hospital for evaluation and treatment of jaundice [7,8]. The AAP Committee on Fetus and Newborn recommends that a total serum bilirubin (TSB) be performed on any neonate noted to be jaundiced in the first 24 hours following birth. The practice parameter also recommends a follow-up visit on day 2 or 3 of life for the neonate discharged less than 48 hours after birth [9]. This follow-up may involve a TSB. A 1997 study conducted by Stevenson and Vreman demonstrated significant interlaboratory variability of TSB measurements performed at 14 laboratories [10]. Therefore, under-or over-estimation of the serum bilirubin value may occur and can lead to the omission of needed therapy or erroneous clinical information in some neonates [7].

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4. Clinical Management of Jaundice and Hyperbilirubinemia In routine clinical situations, it remains difficult to differentiate the etiology of neonatal jaundice. Neonatal jaundice and hyperbilirubinemia develop in response to the increased production and/or decreased elimination of bilirubin. The identification and incorporation of these causative factors assists in establishing a differential diagnosis and cost-effective plan of care. A study conducted in 1990 at the University of California, San Francisco, estimated that about $25 million is spent annually on routine hyperbilirubinemia evaluations by hospitals in the United States [11]. Tests commonly recommended as part of a bilirubin workup include TSB, blood group and type, direct Coombs' test, reticulocyte count and RBC morphology (blood smearing). Frequent measurement of the TSB level has been the mainstay of anticipatory management of transitional bilirubinemia. However, the TSB level does not differentiate jaundice caused by high bilirubin production (high rate of hemolysis) from that caused by limited elimination. Similarly, transcutaneous bilirubin (TcB) analysis provides an index of the bilirubin level but does not require a blood sample. The intensity of yellow color in the subcutaneous tissue and skin is measured, correlating to the serum bilirubin concentration [12]. Despite the convenience of TcB measurement, the accuracy of such devices is noted to be complicated by skin pigmentation, gestational age, and birth weight [13]. As with the TSB, the TcB measurement is unable to identify the factors contributing to the bilirubin level, i.e., production (hemolysis) versus elimination. The direct Coombs' test determines if the red blood cells are coated with antibodies and may be predisposed to hemolysis, but does not prove the presence of hemolysis or its magnitude [13,14]. The reticulocyte count indicates RBC production by the bone marrow and thus an elevated level may signify that the marrow is releasing an increased number of reticulocytes in the circulation. However, it is not a direct measure of hemolysis and may be a response to anemia from other causes. The blood smear, which reveals variations in erythrocyte size, shape, color and intracellular content, is the least reliable measure of hemolysis and cannot be used to measure the magnitude of red cell breakdown. ETCOc accurately reflects the rate of bilirubin production. Elevated production may be associated with higher risk. In the Nigerian population studied by Slusher et al, neonates with a high rate of bilirubin production were more likely to develop bilirubin toxicity when compared to neonates having a low production rate [15]. The ability to measure ETCOc and apply this information has been limited by the lack of a simple, rapid, non-invasive test to assess the rate of hemolysis [16]. Recently instrumentation became available to measure ETCOc and assist the clinician in detecting the presence or absence of hemolysis. The AAP practice parameter calls upon the clinician to determine the presence or absence of hemolysis in the jaundiced newborn, in order to establish an effective care plan for neonatal hyperbilirubinemia [17]. Despite the value and clinical contribution provided by other tests, only the ETCOc measurement can provide an estimate of the rate of hemolysis. The relationship between ETCOc and TSB is currently under investigation in a multi-center, international clinical trial. Understanding the relationship between bilirubin production and bilirubin excretion is essential in predicting the jaundice trajectory for an individual newborn. Figure 3 was formulated in conjunction with DK

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Figure 3: Potential Diagnoses in the Neonate Using ETCOc and TSB * Ethnicities include Chinese, Korean, Japanese, Aleut, Navajo, Pima. **Related to breast feeding, meconium plug, meconium ileus, Hirschprung's, etc. ***Ifenhanced elimination exists, individual with increased production may not exhibit elevated TSB.

Stevenson, MD, AA Fanaroff, MD, and MJ Maisels, MD (October 1998). It suggests points to consider when predicting the path of jaundice in the newborn.

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5. Conclusions Neonatal hyperbilirubinemia results from increased production and/or decreased elimination of bilirubin. Catabolism of heme results in equimolar parts of bilimbin and CO. Understanding the relationship between bilirubin production and CO is important in the management of neonatal jaundice. ETCOc is an indicator of hemolysis and bilirubin production which can be used to assist clinicians in several ways: • ETCOc identifies high bilirubin producers, even before the onset of jaundice, allowing the clinician to carefully plan in-patient and post-discharge care. • ETCOc enables the clinician to detect a newborn at risk for the development of hyperbilirubinemia due to the overproduction of bilirubin before the onset of jaundice. • ETCOc identifies the rate of hemolysis, assisting the clinician in establishing a differential diagnosis based on a normal or elevated rate of bilirubin production. • ETCOc permits more judicious use of invasive blood tests, phototherapy treatments, extended hospitalizations, home or office visits, and even exchange transfusions. This vital clinical information can be used to devise safe, cost-effective clinical pathways for neonates discharged from the hospital at an early age. References [1] [2] [3] [4]

[5]

[6] [7] [8] [9] [10]

WJ Cashore. Bilirubin metabolism and toxicity in the newborn. In: Pollin RA, Fox WW, eds. Fetal and Neonatal Physiology Isted. Philadelphia, PA: WB Saunders Company; 1992:11601164 CO Frank, BS Turner, et al. Jaundice. In: Frank CG, Merenstein GB, eds. Handbook of Neonatal Intensive Care 3rd ed. Philadelphia, PA: Mosby Year Book; 1993: 272-286 HJ Vreman, JJ Mahoney, DK Stevenson. Carbon monoxide and carboxyhemoglobin. Adv Pediatr. 1995;42:303-334 P Rosenthal. Bilirubin metabolism in the fetus and neonate. In: Pollin RA, Fox WW, eds. Fetal and Neonatal Physiology Isted. Philadelphia, PA: WB Saunders Company; 1992:11541159 Erslev AJ, Beutler E. Production and destruction of erythrocytes. In: Beutler E, Lichtman MA, Coller BS, Kipps TJ, eds. Williams Hematology 5th ed. New York, NY: McGraw-Hill; 1995:425-441 C Catz, JW Hanson, et al. Summary of workshop: early discharge and neonatal hyperbilirubinemia. Pediatrics.. 1995; 96(4):743-745 DK Stevenson, HJ Vreman. Carbon monoxide and bilirubin production in neonates. Pediatrics. 1997;100(2):252-254 D Bratlid. Criteria for treatment of neonatal jaundice. J Perinatal 1996;!6(3)Part 2:S83-S88 American Academy of Pediatrics, Committee on Fetus and Newborn. Practice parameter: management of hyperbilirubinemia in the healthy term newborn. Pediatrics. 1994;94(4):558565 HJ Vreman, J Verier, et al. Interlaboratory variability of bilirubin measurements. Ciin Chem.

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[11] [12] [13]

[14] [15] [16] [17]

1996;42(6):869-873 TB Newman, MJ Easter I ing, et al. Laboratory evaluation of jaundice in newborns. Am J Dis Child. 1990; 144:364-368 Smith DW, Inguillo D, Martin D, et al. Use of noninvasive tests to predict jaundice in fullterm infants: preliminary studies. Pediatrics. 1985;75(2): 278-280 V Bhutan!, L Johnson, G Gourley, et al. Non-invasive measurement of total serum bilirubin by multi-wavelength spectral reflectance by Bilicheck™ in newborn patients. Pediatr Res. 1998;43:167A Y Uetani, H Nakamura, O Okamoto O, et al. Carboxyhemoglobin measurements in the diagnosis of ABO hemolytic disease. Ada Paediatr Jpn. \ 989;31 (2): 172-176 DW Smith, AO Hopper, SM Shahin, et al. Neonatal bilirubin production estimated from endtidal carbon monoxide concentration-/Pediatr Gastroenterol Nutr. 1984;3(1):77-80 TM Slusher, HJ Vreman, DW McLaren, et al. Carboxyhemoglobin predicts bilirubin-related morbidity and mortality in infants. Pediatr Res. 1993;33:237A A Strocchi, S Schwartz, M Ellefson, et al. A simple carbon monoxide breath test to estimate erythrocyte turnover. J Lab Clin Med. 1992;120(3):392-399

Section 3. Volatile Organic Compounds (VOCs)

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Disease Markers in Exhaled Breath N. Marczin and M.H. Yacoub (Eds.) IOS Press, 2002

! 05

Volatile Organic Compounds as Exposure Markers Andrew B. Lindstrom and Joachim D. Pleil U. S. Environmental Protection Agency, National Exposure Research Laboratory, MD-44, Research Triangle Park, NC 27711, USA Abstract. Alveolar breath sampling and analysis can be extremely useful in exposure assessment studies involving volatile organic compounds (VOCs). Over recent years scientists from the US Environmental Protection Agency's National Exposure Research Laboratory have developed and refined an alveolar breath collection and analysis technique called the Single Breath Canister (SBC) method which has been applied in a wide range of investigations. This review covers the development of this breath collection technique in the laboratory and the application of this methodology in a range of field studies. Together these studies show how exhaled breath analysis can be used to clearly demonstrate recent exposures to VOCs, to determine compound-specific uptake and elimination kinetics, and to assess the relative importance of various exposure routes (i.e., dermal, ingestion, inhalation) in multipathway scenarios. Specific investigations covered in this overview include: an assessment of exposures related to the residential use of contaminated groundwater; exposures to gasoline and fuel additives at self-service gas stations; swimmers' exposures to trihalomethanes; and occupational exposures to jet fuel vapors. This work has been funded wholly by the United States Environmental Protection Agency. It has been subjected to Agency review and approved for publication.

1. Introduction Breath collection and analysis has been historically useful as a noninvasive technique to help diagnose illness and determine exposures to xenobiotic compounds. This utility is based on the fact that compounds present in the blood will partition into the breath at the blood/alveolus interface and will be eliminated (like carbon dioxide) during exhalation. Perhaps the best known example of this diagnostic practice is the use of exhaled breath analysis by law enforcement officials to determine whether a driver has been operating a motor vehicle under the influence of ethyl alcohol (i.e., the breathalyzer test). This paper summarizes the development of the Single Breath Canister (SBC) method of exhaled breath collection and analysis that was introduced by the US Environmental Protection Agency (USEPA) in the 1990's. We discuss the utility of this method in the context of human exposure assessment and demonstrate how this technique could be used in clinical settings to evaluate disease, metabolism, or changes in physiology. USEPA's interest in the use of breath sampling as an exposure assessment technique began with the Total Exposure Assessment Methodology (TEAM) studies of the 1980's. The TEAM studies were a series of population-based exposure assessment investigations designed to evaluate the most basic aspects of pollutant exposure. As part of these investigations, breath and blood samples were collected from study subjects to help determine which sampling methodology would be most useful in determining exposures.

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While these early breath collection and analysis techniques were cumbersome, it quickly became apparent that breath sampling was a clean, noninvasive method that could provide more accurate information than traditional blood analysis for many volatile organic compounds (VOC). Given this potential utility we set about to develop a simplified breath collection technique that could be coupled with state of the art laboratory analysis to provide a powerful exposure assessment tool for many VOCs. With the SBC method [1,2], an evacuated 1 L stainless steel canister is outfitted with a small (5 cm length) Teflon tubing stub (see Figure 1). After a normal exhalation (eliminating the deadspace portion of a breath) the sample subject places the end of the Teflon collection tube in his mouth and opens the canister valve to fill it with one liter of the expiratory reserve (Figure 2). Because the canister is initially evacuated (< 50 /^m Hg) the sample is collected until the canister comes to atmospheric pressure. The subject then closes the sample valve and is free to resume normal activities. Unlike other end-tidal breath collection devices, these canisters are extremely durable and no special precautions are needed when shipping samples back to the laboratory. Moreover, most VOCs remain stable in these canisters for periods of 30 days or longer without any appreciable degradation.

Figure 1. Single Breath Canister

Figure 2. Exhaled Breath Collection

Once collected, samples are analyzed using gas chromatography/mass spectrometry (GC/MS) techniques based on the EPA Method TO-14 protocol using a Model 3550A Cryogenic Concentrator (Graseby Nutech, Smyrna, GA). This front end concentrator is also an autosampler for canisters and is used in this mode to perform up to 16 unattended analyses. The analytical system used for most of the work discussed below was an ITS40 (Magnum) GC-MS ion trap instrument (Finigan MAT, San Jose, CA). Specifically, a 50 ml aliquot of sample is cryogenically focused at -165°C in a primary trap, then heated and transferred in a helium stream to a secondary trap and refocused at -190°C on a 0.53 mm precolumn. The precolumn is then rapidly ramped to 150°C and the analytes injected in a sharp plug onto a XTI-5 30 m x 0.25 mm i.d. 1 /*m phase analytical column (Restek Corp., Bellefonte, PA). The oven temperature profile is -50°C 2 min hold, ramp to 220°C at 10°C/min, 8 min hold. Tentative identification of each analyte is made using conventional mass spectral libraries, and conclusive identification is assured by injection of authentic

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standards and verification of both retention times and mass spectra. Because contaminant concentrations in the breath are directly related to levels in the blood and other tissues, breath analysis has been used in an increasing number of exposure assessment studies to: confirm that specific exposures have taken place; help assess the importance of different routes of exposure (e.g., proportion of dermal vs. inhalation routes); to establish uptake and elimination kinetics; and to demonstrate the relationships between particular personal activities and their corresponding body burdens (e.g., smoking and increased levels of benzene and other aromatics in the breath and blood). A model presented in Wallace et al. [3] can be used to help interpret the postexposure breath elimination data. The original model takes the form: C, = A,e- k " + A 2 e k 2 t + / P 1 C a i r

(1)

where C, is the contaminant concentration in the blood at any time t; A, 2 are complex constants, each a function of the contaminant concentrations and other physiological variables in two hypothetical body compartments; k{ is the exponent associated with the ilh term; t is the time in minutes; / is the fraction of the parent compound exhaled at equilibrium; Pj is the unitless blood/breath partition coefficient; and Cair is the ambient air concentration of the contaminant. By dividing this equation through by P! we can estimate the alveolar breath concentration at any time t: CalVeo,ar = M- kl! +

fl2ek2t+/Cair

(2)

where Calveolar is the alveolar breath concentration at any time during the elimination; the coefficients (Q) represent the contributions from both unknown body compartments; the inverse of the exponential term (1/kj) represents the residence time (TJ) of the compound in the ith compartment (i.e., the time that it takes the concentration to fall to 1/e (~ 0.37) of its original value); and finally, the half-life of the compound in compartment i is equal to (In 2)/kj. If the elimination series is collected in an environment where the background level of the contaminant is essentially 0 (e.g., outdoors, in many cases), the last term in equations 1 & 2 becomes 0, simplifying elimination calculations. With the appropriate blood/breath partition coefficient, the alveolar breath concentration can be used to determine the concentration of the contaminant in the blood at any time during the elimination phase: ^--blood

=

^-alveolar X Pj X U

(3)

where Cbtood = the concentration of the contaminant in the bloodstream 0-tg/L); Calveolar = the concentration of the contaminant in the breath (/*g/m3); Pj = to the unitless blood breath partition coefficient; U = units conversion factor (1 m3/1000 L). If the subject is moved to an area with little or no background contaminant levels immediately after the exposure, the modeled breath values at the Y-intercept (i.e., t = 0) can be used with equation (2) to estimate the maximum blood levels of the contaminant resulting from the exposure. Integrating the area under the elimination curve also provides an alternative measure of total absorbed dose: the resulting quantity in jug-min/m3 can be multiplied by the breathing rate (nrVmin) to establish the total mass (jig) of contaminant that leaves the body via exhalation. If it is known what proportion of the compound will be eliminated in this manner (e.g., -10% for trichloroethylene), the unmetabolized mass can be divided by this fraction to provide an indirect estimate of total absorbed dose.

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2. Experimental 2.1 Exposures Via Contaminated Groundwater In the first example of the utility of the SBC method we show how it can be used to confirm and quantify exposures to compounds that would otherwise be extremely difficult to measure directly in blood. The solvent trichloroethylene (TCE) has become a common groundwater contaminant in the US. Once underground it is subject to an iron-mediated reductive dehalogenation process (Figure 3) that leads to the formation of vinyl chloride (VC), a potent human carcinogen.

-c

CI

.CI

H \ C:

cr

CI

Trichloroethylene

H \ Cc

c CI

C CI

Vinyl Chloride

cis-1,2-Dichloroethene

Figure 3. Transformation of Trichloroethylene in Groundwater

If TCE contaminates well water and gives rise to VC, both compounds can often be measured hi residences using the contaminated water. Showering and bathing are of particular concern in such circumstances due to the likelihood of simultaneous inhalation and dermal exposures. Figure 4 is a VC in breath elimination profile collected from an individual after taking a 10 min shower in TCE and VC contaminated water [4]. Because VC has such a low boiling point (-14°C) and high vapor pressure (2,660 mm Hg @ 25°C) it is extremely unlikely that conventional blood-based biomonitoring techniques would be able to confirm exposures to this potent carcinogen. On the other hand, SBC samples collected shortly after the shower coupled with cryogenic preconcentration and GC\MS analysis conclusively demonstrate this exposure.

8-

G) O C

O O

0

A A

j

A""-...

2-

0

A

5

10

15

20

25

30

35

40

Time (min) Calveolar = 6.54 X 6° 38t + 5.14 X e° °5 l Figure 4. Vinyl Chloride in Breath After a 10 min Shower With Contaminated Well Water

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2.2 Exposure to Motor Vehicle Fuels In the next example we show how the SBC method can clearly differentiate between two individuals who were putatively exposed to the same atmosphere. In this experiment two individuals stood side by side while refueling an automotive vehicle at a conventional service station [5]. After refueling, the study subjects walked away from the service station together and breath samples were then colleted from them on a periodic basis for more than 1 hour. The gasoline used in this process contained 15% methyl tertiary butyl ether (MTBE) by volume. MTBE is an oxygenate added to gasoline in the US to help reduce the formation of carbon monoxide in vehicle exhaust. In the elimination profile below (Figure 5) one can see that one individual's exhaled MTBE in breath was more than two times higher than the other subject despite the fact that both stood side by side during the refueling process. These data suggest that even within the same microenvironment, personal differences in uptake and metabolism can greatly affect an individual's ultimate exposure. The SBC method can thus be used to more accurately quantify an internalized dose and therefore reduce exposure misclassification bias in epidemiologic investigations.

1 UUU -

A Pumper D Observer

o"^

o 800- 5, J3-

c jo

A A

600-

A

"(0

•4-*

'A

400-

"A..

o

c o

o

200-

\,

'""""*""---..

°MX^__D_______^^

~~

n.

0

10

20

""

D— 30

A

.— Q 40

50

60

70

Time (min) 0 240 * + 410 e-ao2° * + (0.7 x 12.3) pumper = 443 e '

^observer = 221 e"° 517 l + 1 19 e 0 ' 025 * + (0.7 X 12.3) Figure 5. Elimination of MTBE in Breath Following Motor Vehicle Refueling 2.3 Breath Analysis to Determine the Route of Exposure One important aspect of using breath as a biomarker of exposure is the concept of the/value during uptake. Simply put, if an individual is exposed to a constant level of a VOC in the air he breaths, the concentration of the VOC in his exhaled breath will rise during the exposure and eventually reach an equilibrium with the surrounding atmosphere (Figure 6). The equilibrium concentration of exhaled breath is always a fraction (hence f) of the surrounding atmosphere because the body will always have some ability to metabolize or sequester the external dose. Each individual compound has its own characteristic/value generally ranging between 0.1 to 0.9 depending on the physical

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characteristics of the compound and the human body's ability to internalize it. The concept of the/value can be important because it helps to apportion exposures between various routes (e.g., dermal, inhalation, or ingestion) as illustrated in the example below. Ambient Concentration

f value

c o •J= CO 4= 0)

Exhaled Breath Concentration

o o O

Time (min) Figure 6. Fraction of Ambient Concentration (/) Eliminated at Equilibrium When water supplies are treated with chlorine to reduce or eliminate bacterial contamination a number of potentially hazardous disinfection byproducts are formed (e.g., trihalomethanes). To assess the potential trihalomethane exposures associated with recreational swimming we designed an experiment to measure exhaled chloroform and bromodichloromethane in two individuals engaged in vigorous swim training [6]. Both study subjects provided exhaled breath samples before, during, and after their regular two hour swim training workout while pool water and natatorium air samples were collected for analysis. With long-term indoor chloroform levels measured at 147 Mg/m3 and an/value for chloroform of approximately 0.14, the highest exhaled breath concentration that we would expect from an inhalation only exposure would be - 21 //g/m3 ( = 0.14 x 147 Aig/m3). The post-exposure breath elimination profiles (Figure 7) however indicate concentrations that were much higher than 21 //g/m3, suggesting that inhalation alone did

Male Female

50

100

150

200

Time (Min) C m a i e = m e' 0 5 0 9 t + 107e-° 027t + 45.8e- 0004t C fema .e = 153 e-° 75° ' + 104 e-° °43 ' + 49.7 e'0 °05 '

Figure 7. Elimination of Chloroform in Breath Following Swimming Training

A.B. Lindstrom and J.D. Pleil/ Volatile Organic Compounds

\ 11

not account for the exposure monitored. In fact, if a three compartment model is fitted to these data, breath levels at the very beginning of the elimination phase (the Y axis) are over 260 jug/m3 for both individuals, suggesting that over 90% of the exposure occurred was via the dermal pathway. Considering that blood vessels in the skin dilate during exercise to help dissipate excess heat, it seems likely that the chloroform from the surrounding water was able to diffuse into the skin during this period of intensive physical activity to ultimately give a higher portion of the total dose than the inhalation route. 2.4 Exposure to Complex Mixtures: JP-8 Jet Fuel US and NATO led forces use more than 4.5 billion gallons of JP-8 jet fuel annually. While most is used as jet fuel for the various air forces, JP-8 is also used in heaters, stoves, tanks, generators, and many other types of support equipment. This widespread use leads to a large number of potential exposure scenarios [7], often involving personnel engaged in critical or sensitive operations (e.g., maintenance of sophisticated aircraft and weaponry). Work place protection programs are in place to minimize potential exposures, but anecdotal reports of health effects associated with exposures (including adverse skin reactions) have now raised concerns over the widespread use. Initial studies conducted with laboratory animals suggest potential neurological and immunological effects are associated with high level exposures but effects with humans remain unclear. To obtain a better understanding of the acute health effects associated routine exposure to JP-8 fuel, the US Air Force recently conducted the field phase of an epidemiological investigation involving over 300 personnel exposed to jet fuel at various Air Force bases across the US [8]. Exposures were assessed using a range JP-8 biomarkers measured in blood, breath, and urine samples. Outcome measures of effect included measures of immune and neurological function. Preliminary results indicate that exhaled breath measurements were particularly well suited to determining the exposure of the study subjects. Figure 8 A below shows a typical total ion chromatogram from a breath sample of a maintenance worker before his work shift, while in Figure 8. B we have the same worker's post-shift breath sample. Note that the post shift sample clearly indicates the C9 - C12 alkane signature pattern of JP-8 exposure.

Figure 8. A. Total Ion Chromatogram of Breath Sample Before Exposure to JP-8

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Isoprene

800 0640

1,200 1000

1.600 1320

1000 1640

2,400 2000

2^00 2320

Scans, time (min/min, sec/sec) Figure 8. B. Total Ion Chromatogram of Breath Sample After Exposure to JP-8 3. Conclusions

The SBC method of exhaled breath collection and analysis as outlined above is particularly well suited for exposure assessment studies involving xenobiotic VOCs. The sample collection method is simple and adaptable to a wide range of potential applications. Once collected, samples are stable and easily transported to the laboratory for GC/MS analysis. Two stage cryogenic trapping ensures that even the most volatile VOCs are collected efficiently. When properly applied the SBC method can be used to clearly demonstrate recent exposures to VOCs, link specific activities to a recently incurred body burden of xenobiotic materials, determine compound-specific uptake and elimination kinetics, and to assess the relative importance of various routes (i.e., dermal, ingestion, inhalation) in multipathway exposure scenarios. References [1] J.D. Pleil and A.B. Lindstrom, Collection of a single alveolar exhaled breath for volatile organic compound analysis, American Journal of Industrial Medicine, 28 (1995) 109-121. [2] J.D. Pleil and A.B. Lindstrom, Measurement of volatile organic compounds in exhaled breath as collected in evacuated electropolished canisters, Journal of Chromatography B: Biomedical Applications, 665 (1995) 271-279. [3] L.A. Wallace, E.D. Pellizzari, and S. Gordan, A linear model relating breath concentrations to environmental exposures: application to a chamber study of four volunteers exposed volatile organic chemicals, Journal of Exposure Analysis and Environmental Epidemiology, 3 (1993) 75-102. [4] A.B. Lindstrom and J.D. Pleil, A methodological approach for exposure assessment studies in residences using volatile organic compound contaminated water, The Journal of the Air & Waste Management Association, 48 (1996) 1058-1066. [5] A.B. Lindstrom and J.D. Pleil, Alveolar breath sampling and analysis to assess exposures to methyl tertiary butyl ether (MTBE) during motor vehicle refueling, The Journal of the Air & Waste Management Association, 46 (1996) 676-682. [6] A.B. Lindstrom, J.D. Pleil, and D.C. Berkoff, Alveolar breath sampling and analysis to assess trihalomethane exposures during competitive swimming training, Environmental Health Perspectives, 105(6) (1997) 636-642. [7] J.D. Pleil, L.B. Smith, and S.D. Zelnick, Personal exposure to JP-8 jet fuel vapors and exhaust at air force bases, Environmental Health Perspectives, 108(3) (2000) 183-92. [8] http://sg-www.satx.disa.mil/iera/rsh/IndustrialHygiene/fuels.html

Disease Markers in Exhaled Breath N. Marczin and M.H. Yacoub (Eds.) IOS Press, 2002

113

Volatile Organic Compounds as Markers in Normal and Diseased States Terence H. RISBY Johns Hopkins Medical Institutions Baltimore Maryland 21205, USA

Abstract The concept that blood, urine, and other body fluids and tissues can be sampled and analyzed to yield clinical information for diagnosis of disease states or to monitor therapy is the foundation of modern clinical diagnosis and medical practice. The use of breath as a collectable sample has not received similar clinical use mainly due to the low concentrations (ppm to ppb) of marker molecules in breath. Recent advances in on-line concentration and the development of sensitive analytical techniques have suggested that the use of exhaled breath to study disease processes should now be re-examined. The analysis of exhaled breath has major advantages since it is non-invasive, represents minimal risk to personnel collecting the samples, and can be sampled often. Currently, a number of marker molecules have been identified in breath that can be used to identify disease, disease progression, or to monitor therapeutic intervention. It is expected that this list will soon increase dramatically based upon the available novel analytical instrumentation. Already, the collection and analysis of breath is ideally suited for population-based studies since no special storage conditions are necessary before subsequent analysis in the laboratory. This work was partially supported by NIH Grant P01-HL56091.

1. Introduction The ability to exchange carbon dioxide with oxygen is essential for most life forms. In animals, this gas exchange occurs at the alveolar-capillary membrane in the respiratory tract. Oxygen and carbon dioxide are passively transported from blood to breath or vice versa and the diffusion of these gases is governed by their concentration gradients across the alveolar-capillary membrane. Any additional molecule present in the blood or in the inspiratory air will also pass into the breath or blood respectively. The only requirement for this transport is that the molecule must exhibit a significant vapor pressure. The molecular profile of breath will be the product of the composition of the inspiratory air and volatile molecules that are present in the blood. Cells or tissues in the nose, sinuses, airway and the gastrointestinal tract may also contribute molecules to exhaled breath. The bulk matrix of breath is a mixture of nitrogen, oxygen, carbon dioxide, water vapor and the inert gases. The remainder of breath (<0.000001%) is a mixture of as many as 500 different compounds. These molecules have both endogenous and exogenous sources, however, the concentration is often higher when the origin is exogenous. The concept that breath contains molecules originating from normal or abnormal physiology has its origins in the writings of Hippocrates, the father of medicine. For example, detection of the presence of water vapor in breath has been used as a noninvasive monitor of mortality for thousands of years. Additionally, distinctive breath odors have been used as indications of diseases such as uncontrolled diabetes, liver disease, renal failure or dental disease [for review see 1-3]. However, the use of odors of breath for clinical diagnosis can be confounded by the characteristic odors that result from the ingestion of such materials as garlic, onions, fish, spices, mints, and ethanol.

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The identification and quantification of molecules present at trace concentrations in breath has a much more limited history. Other chapters in this book describe how breath can be used to estimate actual exposure to xenobiotic compounds in environmental or occupational settings or to study the pathophysiology of exhaled nitric oxide or carbon monoxide. This chapter will be limited to the discussion of those molecules that have been found in human breath whose biochemical pathways are known or whose biochemical pathways can be postulated. Molecules of unknown biochemical origins have the potential to be artifactual and using them to identify disease or disease processes could bring the field of breath analysis and human disease into disrepute. 1.1 Definition of exhaled breath Breath can be sampled from any human subject from the neonate to the elderly with relative ease, minimum invasion and multiple times. The easiest way to obtain a representative sample of breath is from a paralyzed human subject whose breathing is supported mechanically. For all human subjects the composition of breath varies extensively over the breathing cycle as a result of normal pulmonary physiology. The initial portion of a breath cycle originates from the conducting airway (anatomic dead-space) and the composition of this gas is determined almost exclusively by the composition of the inhaled gas. The remainder of the breath consists of mixed expired gas and alveolar gas. In human subjects at rest, approximately 80% of the gas that is in contact with the capillaryalveolar membrane is exchanged with each breath. The gas in contact with this membrane will contain about 5% carbon dioxide. If a human subject is breathing normally, then the concentration of carbon dioxide at the end of the breath (end-tidal), the concentration of carbon dioxide in arterial blood (pCO2) and their ratio will remain constant from breath to breath. Changing the tidal volume and/or breathing frequency will increase or decrease these concentrations. The rate of alveolar ventilation is controlled by the arterial blood concentration of carbon dioxide. Hypo- or hyperventilation will, by definition, change the composition of exhaled breath and in turn the arterial blood concentration. Variations in tidal volume or breathing frequencies are particularly important when sampling breath from human subjects breathing spontaneously since any method of breath sampling will make the subject conscious of their breathing patterns and the potential to change breathing patterns is increased. For spontaneously breathing subjects it is preferable to sample multiple breaths in order to ensure that the analysis of the collected breath is representative. 1.2 Real-time monitors versus breath profiles Until recently, only inhalation anesthetics, oxygen and carbon dioxide could be monitored in real-time. Clinically these monitors are used to display trends for the status of the patient and do not provide information on individual breaths. However, advances in monitor technology have changed this limitation and specific breath molecules can now be monitored in real-time. In order to improve the precision to which these novel breath molecules can be measured, protocols have been established whereby the mouth pressure, flow rate or carbon dioxide are monitored and/or controlled during the time of measurement. The major advantage of real-time monitors is that the analysis of a single breath can be repeated to ensure that the measurement is representative of the breath for the study subject. Unfortunately, not all breath molecules can be determined specifically in real-time and therefore most breath analyses are performed on samples of collected breath. There are basically two approaches to breath collection: i.) sampling end-tidal breath, and ii) sampling total breath for a single breath or for a defined period of time (usually 1 minute). i.) Collection of end tidal breath requires that the subject is breathing at rest and that the breath sample is collected when the concentration of carbon dioxide reaches a plateau (end tidal). This approach requires that this portion of breath is representative of all previous and subsequent breaths during resting or relaxed breathing. End-tidal breath sampling will produce samples with the highest concentration of molecules if the molecules of interest are derived from exchange at the alveolar-capillary membrane. Moreover, end-

T.H. Kisby / Volatile Organic Compounds as Markers

115

tidal breath has the lowest concentrations of room air contaminants. The volume of an endtidal breath sample is only a fraction (<30%) of the tidal volume of a single breath. ii.) Collection of total breath for a single breath or for a defined period of time has been the method that has been used most extensively. Our laboratory has collected breath from 500g premature neonates to normal and diseased adults for defined time periods. The implicit assumption of this approach is that if breath is collected over a period of time, concentrated and analyzed, then the analytical result reflects the average concentration of breath molecules in exhaled breath over the period of sampling. By definition, the concentrations of breath molecules in total collected breath are diluted by the physiologic and anatomic dead space ventilation that contains room air and therefore, it is necessary to collect samples of room air and correct for background contamination. However, the sample does represent the average or integration of all the breaths collected during the sampling period and is less susceptible to spurious breaths. Based upon our experience, the coefficient of variation for the analysis of successive timed breath samples for a given individual is less than 3%. Recently a breath-sampling device for adolescent and adult subjects that continuously measures and records the gas flow, concentration of carbon dioxide and mouth pressure as a function of time during breath collection has been developed. After collection, the tidal volume, breathing frequency, end tidal concentration of carbon dioxide for each breath, minute ventilation, and average concentration of exhaled carbon dioxide are calculated for the period of breath collection. Breath collected by either approach must be taken to the laboratory for concentration and analysis. Most published studies have collected total exhaled breath in inert gas sampling bags and analysis is performed as soon as possible after collection. The requirements for gas sampling bags are that they must be inert and not adsorb breath molecules. These bags should have a volume that corresponds to at least twice the amount of breath collected thereby minimizing any back-pressure from the collected gas that could restrict the exhalation of the study subject. Similarly, the inlet to the sampling bag should have sufficient dimensions to minimize any pressure drop across the inlet. Recently, evacuated polished stainless steel cylinders (SUMMA) or thermal desorption tubes packed with adsorbents have been used to collect breath. These devices collect known volumes of the exhaled breath at controlled flow rates. The advantages of these latter devices are that the subject experiences no resistance to breathing and the collected breath samples can be easily transported from the sampling site to the laboratory. Our laboratory has pioneered the use of thermal desorption tubes to collect breath for subsequent automated analysis. This research was performed in collaboration with a commercial manufacturer of specialty carbon adsorbents (Supelco Corporation, Bellefonte, Pennsylvania, USA). A number of thermal desorption tubes were examined and glass thermal desorption tubes packed with equal amounts of a graphitized carbon (Carbopack X), and two carbon molecular sieves (Carboxen-1018, Carboxen-1021) were found to be the optimum tube design for our breath studies to date. The three adsorbents are separated by glass wool. The graphitized carbon has a nitrogen surface area of 240 m2/g, density of 0.41 g/ml, and a pore volume restricted to the meso range (pore diameter 20 nm) of 0.62 ml/g. The carbon molecular sieve, Carboxen-1018, has a nitrogen surface area of 700 m2/g, density of 0.6 g/ml and a pore volume restricted to the micro range (pore diameter 0.6 nm) of 0.352 ml/g. The carbon molecular sieve, Carboxen-1021, has a nitrogen surface area of 650 m /g, density of 0.3 g/ml and a pore volume restricted to micro range (pore diameter 0.5 nm) of 0.35 ml/g. Carbopack X is the weaker adsorbent, Carboxen-1018 is the intermediate adsorbent, and Carboxen 1021 is the stronger adsorbent. When breath is sampled it passes through the bed of graphitized carbon first and then through the two beds of carbon molecular sieves in order of increasing adsorptive strength. The breath samples collected in the thermal desorption tubes are analyzed by twostage thermal desorption capillary gas chromatography with a variety of detection systems. The collected breath is thermally desorbed in the opposite direction to the flow used during breath collection to ensure the removal of the sample. In our laboratory we use a fused silica open tubular column (0.32 mm) wall coated with a thick film (5fj.ni) of cross-linked bonded dimethyl silicone (60 m) to separate molecules found in exhaled breath, with the following temperature protocol: isothermal at 35°C for 10 min., 35-200°C at 5°C/min., isothermal at 200°C for 10 min. If non-selective detectors, such as a flame ionization detector or an electron impact mass spectrometric detector, are used, this separation protocol

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produces a breath molecular profile for breath markers from methane through tetradecane with excellent resolution. Selective breath molecular profiles can also be obtained by the use of detectors such as flame photometric detector (selective to sulfur-containing compounds) or thermionic specific detector (selective to nitrogen-containing compounds). Selective detectors have the advantage of simplifying the resulting separation. 1.3 Expressing breath analysis data The concentration of breath markers can be expressed in concentration units (ppm, ppb or pmol/1) but this information does not permit intersubject comparisons to be made. Therefore, the concentration data are converted to generation rates by the measurement of the minute ventilation (1/min) and subsequently corrected for body weight (pmol/kg min). Data could be corrected by body surface area (nmol/m min) and these are the units that are used clinically by most anesthesiologists. More recently, an additional correction has been made for anatomic, physiologic, and instrumental dead space based upon the concentration of exhaled carbon dioxide. During collection, the end-tidal concentration and average concentration of carbon dioxide over each breath are quantified and recorded. After the breath sample has been collected the average values for these measurements during breath collection can be made. The average end-tidal concentration of carbon dioxide and the average concentration of carbon dioxide during exhalation (mixed expired) can be used to determine the volume that corresponds to the anatomic, physiologic, and instrumental dead space by the following calculation {[end-tidal concentration of carbon dioxide in Torr][average concentration of carbon dioxide during exhalation in Torr]}/{ [end-tidal concentration of carbon dioxide in Torr]x[average tidal volume of each breath]}. This calculation assumes that the subject is breathing at steady-state. The concentration of any marker molecule that is measured in exhaled breath has been diluted by this volume. 1.4 Effects of exercise Discussion to this point has assumed that the subject from whom the breath is collected or sampled is seated and is breathing normally at rest. Under these circumstances steady-state conditions are achieved and pulmonary blood flow and minute ventilation are constant. The instantaneous molecular profile during each breath will vary with inspiration and expiration but the composite molecular profile for each breath will be similar. If the subject is then asked to exercise their ventilation and pulmonary blood flow will change as exercise progresses. Minute ventilation and pulmonary blood flow will no longer be at steady-state and the composite molecular profile of each breath will change. This is particularly evident when the cardiac output reaches a plateau and the minute ventilation continues to increase. Under these conditions, it is possible that molecules are excreted in the breath faster than they are being delivered to the alveolar-capillary membrane or produced in tissues and therefore their concentration in breath and blood will decrease. Similarly, if the lactate threshold (anaerobic threshold) is achieved during exercise, the breath profile will change and the relative abundances of specific breath markers will be different. If breath analysis is to be performed during exercise in order to study exercise physiology, it is preferable to normalize concentrations of breath molecules to oxygen consumption or carbon dioxide production. Using concentration of breath molecules directly or normalizing concentrations of breath molecules to minute ventilation will either over-estimate or under-estimate the production of breath markers, respectively.

2. Breath Markers of Normal and Abnormal Physiology 2.1 Breath hydrocarbons Isoprene (2-methy 1-1,3 -butadiene), the most abundant hydrocarbon in human breath, was first identified in the breath of adults in 1969 [4] and its presence in normal human

T.H. Risby / Volatile Organic Compounds as Markers

117

breath was confirmed subsequently by others [5,6]. However, the existence of detectable isoprene in human breath remained observational until 1984 when the in vivo biosynthesis of isoprene from DL-mevalonate was demonstrated in the cytosolic fraction of rat liver [7]. Subsequently, it was established that isoprene is produced by the decomposition of dimethylallyl pyrophosphate via a carbonium ion mechanism [8]. These studies were confirmed in humans by proving that levels of isoprene in exhaled breath can be lowered by administration of lovastatin, a pharmacological agent that blocks the enzyme 3-hydroxy-3methylglutaryl-CoA reductase (HMGCoA reductase) [9]. HMGCoA reductase catalyzes the production of mevalonic acid, which is the rate limiting intermediate in the pathway of cholesterol biosynthesis. Also, it was shown that feeding the study subjects a cholesterolrich diet caused a reduction in the level of breath isoprene [9]. These studies confirm that breath isoprene can be an excellent non-invasive marker of endogenous cholesterol status in humans. For example, breath isoprehe could be used diagnostically to identify human subjects that have increased risk for coronary artery disease as a result of increased biosynthesis of cholesterol. Breath isoprene has been found to be elevated in the breath of human subjects judged to have familial combined hyperlipidemia (FCH, Type IIA) and familial hypercholesterolemia (FH, Type IIB) as compared to gender, and age-matched subjects with normal lipid profiles (unpublished results). FCH is caused by a defect in the regulation of apolipoprotein B whereas FH is caused by a defect in the cell surface receptor that controls the degradation of plasma LDL. Humans that have these defects are at increased risk for coronary artery disease. One of the potential problems with the use of breath isoprene to identify individuals at risk is that the concentration of breath isoprene has a circadian rhythm. Breath isoprene has been shown to have a maximum concentration at around 6:00 A.M. and reaches a minimum at approximately 6:00 P.M. [10]. Additionally, the concentration of isoprene in breath is age dependent (unpublished results); it is non-detectable in the breath of neonates and increases linearly with age from approximately 6 months until it plateaus in middle age. The concentration of isoprene in breath decreases with old age. A similar age-dependence is known for serum cholesterol. Breath isoprene is also higher in the breath of males as compared to the breath of females but this gender difference disappears after menopause (unpublished results). If breath isoprene is to be used successfully to identify humans at risk for coronary artery disease as a result of high serum cholesterol, then an algorithm must be generated to account for natural variations due to time of day, age and gender. Cholesterol reduction therapy with pharmacological agents could be followed with breath isoprene. Levels of breath isoprene change more rapidly than serum levels of cholesterol and the subject would act as his/her own control. Breath isoprene has been used to follow the repair of cellular damage caused by exposure to ozone [11]. Reduced levels of breath isoprene have also been observed in patients with acute respiratory distress syndrome (ARDS) as compared to ventilation patients without ARDS [12]. In 1974, it was demonstrated that quantifiable increases in the amounts of ethane and ethylene were produced when mouse liver and brain tissue or mice were exposed to carbon tetrachloride [14]. These results lead to the proposition that ethane and ethylene could be used as markers of in vitro and in vivo lipid peroxidation since the metabolism of carbon tetrachloride, a known hepatotoxin, involves the generation of free radicals. Lipid peroxidation is a chain reaction with initiation, propagation, and chain termination steps. It is initiated by a reactive oxygen species, typically the hydroxyl radical, that abstracts an allylic hydrogen atom from an unsaturated lipid to produce a carbon centered radical and water. This radical is conjugated, peroxidized by molecular oxygen, and undergoes a variety of reactions. Their proposition was supported by the observation that the concentrations of ethane and ethylene could be correlated with concentration of malondialdehyde another marker of lipid peroxidation. Evidence of lipid peroxidation was confirmed by the reduction of the concentrations of these products by pretreatment of the tissue or mice with the antioxidant, vitamin E. The net result of lipid peroxidation is extensive damage to unsaturated lipids that can lead to the loss of integrity of cellular membranes. Structural and enzymatic proteins are also susceptible to damage by reactive oxygen species, particularly those proteins that contain thiol and amide functionalities. Reactive oxygen species have been implicated in the pathogenesis of a variety of diseases from cardiovascular, pulmonary.

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autoimmunological, neurological, inflammatory, to connective tissue diseases. Moreover, reactive oxygen species have been proposed to contribute to the aging process. The reactivities of oxygen-centered free radicals demonstrate the need to quantify the presence of excess reactive oxygen species since such determinations would identify humans at risk for disease. Some of the stable end-products of lipid peroxidation such as ethane, ethylene, and 1-pentane are well suited for the estimation of cellular damage because these species are excreted in breath within minutes of their formation in tissues. In many ways, quantifying stable products of lipid peroxidation is superior to the direct measurement of free radicals since the quantification of damage is more relevant to the estimation of adverse effects. The degree of oxidative damage has been assigned the name oxidative stress status (OSS), which is the physiologic balance between the cellular levels of oxidants and antioxidants. Quantifying the concentrations of breath ethane and/or 1-pentane have been used extensively to monitor reactive oxygen mediated-damage in tissues, animals, and humans, for review see [14]. Our laboratory at Johns Hopkins has for more than a decade been investigating the role of oxygen free radicals in normal and diseased humans subjects. Evidence of acute damage to cellular lipids has been demonstrated in the operating room when blood flow was restored to ischemic tissue during organ transplantation, vascular surgery, and coronary artery bypass surgery. Confirmation that this damage was mediated by reactive oxygen species was provided in animal models using selective inhibitors. Additionally, acute damage was shown to occur when tissue was exposed to ionizing radiation during cancer therapy. Moreover, the effects of chronic exposure to reactive oxygen species were demonstrated in neonates, children and adults. The etiologies of these chronic exposures were diet, disease, or life styles. In many instances the progress of therapeutic interventions was followed using the concentrations of breath ethane. A recent review has discussed this body of work [15]. 2.2 Breath oxygen-containing compounds The following oxygen-containing compounds are the major compounds that have been identified and quantified in normal human breath in order of their abundances: acetone, ethanol, acetaldehyde, methanol and 2-propanoI. Breath acetone, which has a concentration comparable to that of isoprene, has been the most widely studied. Acetone is produced by hepatocytes from excess acetyl CoA. Acetoacetate and D-p-hydroxybutyrate are the other species that are also produced concomitantly with acetone, and these species are known collectively as ketone bodies. Ketone bodies diffuse from the hepatocytes into the blood stream and are oxidized via the Krebs cycle in peripheral tissue. Under normal conditions there is a steady-state low concentration of ketone bodies in the blood and hence in the exhaled breath. In times of stress, such as during dieting, fasting or starving (when fat tissue is used as an energy source instead of carbohydrates) the rate of production of ketone bodies exceed the rate of utilization by peripheral tissues and the subject becomes ketonemic. The blood concentrations of ketone bodies are increased with uncontrolled diabetes mellitus or chronic alcoholism. The concentrations of breath acetone have been proposed to be useful in the management of diabetes especially when used in conjunction to the determination of serum glucose [16]. The combination of these two were proposed since breath acetone was a more sensitive indicator of poor control of diabetes than of serum glucose. An interesting application of breath acetone would be to follow people who are dieting to see whether their breath contains high concentrations of ketone bodies. The "Atkins Diet" is a good example of this use. 2-propanol has been found in the breath of normal human subjects although at a lower concentration than acetone [17]. The origin of 2-propanol has been postulated to be the enzyme-mediated reduction of acetone. 2-propanol has been observed in ketonemic subjects who have elevated ratios of NADH/NAD*. The potential uses of breath acetone or 2-propanol are limited since there are many other ways to quantify ketonemia. Ethanol is normally found in human breath albeit at concentrations of orders of magnitude less that the levels of ethanol found in the breath of intoxicated subjects. The source of this ethanol is probably intestinal bacterial flora. Gut bacteria are known to synthesize and metabolize ethanol. In a recent study from this laboratory, the concentration of ethanol in the breath of mice has been related to obesity [18]. Specifically, the

T.H. Risby / Volatile Organic Compounds as Markers

\ 19

concentration of ethanol in the breath of genetically obese ob/ob mice was found to be significantly higher than the ethanol in the breath of their lean littermate controls (C57BL/6). This difference in the concentrations of breath ethanoi was negated when the ob/ob mice were treated orally to a poorly absorbed antibiotic. These results support the proposition that gut flora is producing the ethanol. Genetically obese mice have fatty livers that appear to be similar to the earliest stage of fatty liver disease in human subjects. Obesity has been shown to be a risk factor for the development of fatty liver disease in obese humans. An ob/ob mouse is a good model to study this problem. One feature common to obese humans and genetically obese mice is reduced gut motility. This reduced motility may increase the potential for the gut flora to produce ethanol or may increase gut permeability. Alternatively decreased gut motility may favor bacterial overgrowth. Since obesity-related liver disease has similar histological features to alcohol-induced liver damage it was postulated that endogenous low concentrations of ethanol may be a risk factor in the development of nonalcoholic steatohepatitis (NASH). This hypothesis was tested in a human study with biopsy-proven NASH patients and obese age-matched controls [19]. All subjects had quantifiable levels of breath ethanol, but the patients with NASH did not have elevated levels of breath ethanol compared to their obese controls. None of the study subjects had reported consuming alcohol for at least one year. The severity of liver disease was not influenced by the concentration of breath ethanol. However, there was a relationship between obesity and the concentration of breath ethanol for all study subjects and female subjects had a higher concentration of breath ethanol as compared to male subjects. Gender (female) is a risk factor for the development of NASH in obese subjects. This investigation is being continued with a larger population of normal and diseased subjects and if successful may provide important information on the pathogenesis of NASH, The origin of acetaldehyde found in normal human breath is probably the oxidation of endogenously produced ethanol since the concentration of acetaldehyde is always much lower than ethanol. An alternative reason for the observation that breath acetaldehyde is always much lower than breath ethanol is that low-molecular aldehydes are notoriously susceptible to oxidation to the corresponding acids. There is a report that abstinent men with the low activity polymorphism of alcohol dehydrogenase (ADH) or with low activity polymorphism of aldehyde dehydrogenase (ALDH) were found to have high endogenous concentration of ethanol or acetaldehyde in their blood [20]. Unfortunately, there are no studies reporting the relationship between polymorphisms in alcohol metabolism and breath markers although breath ethanol or acetaldehyde could be a simple method to identify specific polymorphisms. The origin of breath methanol may also be intestinal bacterial flora [21], although there was a report that suggested that methanol is produced in any tissue when the leaving methyl groups are hydrolyzed [22]. Examples are the conversion of S-adenosylmethionine to S-adenosylhomocysteine in various tissues. There are no studies that have reported breath profiles in human subjects with genetic defects in amino acid metabolisms although this is a potential application of a methanol breath test. 2.3 Breath sulfur-containing compounds It has been postulated since the early part of the 20th century that the breath sulfurcontaining compounds, methyl mercaptan, ethyl mercaptan, dimethyl sulfide and dimethyl disulfide, are responsible for the characteristic sweet, musty odor found on the breath of cirrhotic patients. This characteristic odor known as fetor hepaticus was first characterized chemically in 1970 [23]. In this classic paper the origin of these sulfur compounds was proposed to be the incomplete metabolism of methionine and this hypothesis was tested experimentally in humans. Others, who have also improved the analytical methods for the detection of these labile compounds, have confirmed this study. Although the degradation of methionine by liver mitochondria was first observed in 1953 [24,25], the complete transamination pathway in the liver has yet to be completely defined [26]. The production of methyl mercaptan and other volatile sulfur compounds has been shown to require the presence of either glyoxylic acid or 2-oxoglutaric acid and pyruvate [26]. The branched chain 2-oxyacid dehydrogenase complex probably controls the transaminative flux of these compounds. Degradation of methionine can also occur in the gut by the action of bacterial

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T.H. Risby / Volatile Organic Compounds as Markers

methionine gamma-lyase [27], although it has been proposed that the gut is not the source of methyl mercaptan and dimethyl sulfide in liver disease [28]. Mercaptans are easily oxidized to their respective sulfides and that is the reason why breath sulfur-containing compounds are more abundant in breath. The production of methyl mercaptan depresses the synthesis of urea [29] with the result that circulating levels of ammonia, biogenic amines, and shortchain fatty acids are increased, thereby setting up the scenario for hepatic encephalopathy. Animals with compromised livers are demonstrably more sensitive to the CNS depressive effects of methyl mercaptan [30-32], thereby suggesting that the liver is the major organ responsible for the oxidation of methyl mercaptan [33]. It has been demonstrated that methyl mercaptan can be oxidized by erythrocytes to formic acid and sulfite or sulfate [34]. Similar oxidative pathways for the removal of volatile sulfur compounds are probably involved in both liver and erythrocytes. Under normal circumstances there are low concentrations of circulating sulfur-containing compounds present in the blood and breath of humans with normal, healthy livers. However, since impairment of liver function increases the level of reduced sulfur containing compounds, liver disease must affect the hepatic oxidation more than it affects transamination production. Recently the presence of chronic liver failure can be detected with sensitivity and specificity by means of analysis of breath carbonyl sulfide arising from the abnormal metabolism associated with liver diseases [35]. Moreover, monitoring breath carbonyl sulfide appears to distinguish between hepatocellular and biliary tract etiologies, and allows staging for disease severity. This breath marker may provide the clinician with a simple, non-invasive technique for screening large populations and follow-up for patients with chronic liver disease. This same breath marker has been used in other studies to identify lung transplant recipients who were experiencing acute rejection as compared to stable subjects. These preliminary studies suggest an additional diagnostic role for this noninvasive biomarker. The mechanism for the production of this marker in transplant recipients is probably tissue necrosis. Further exploration of breath analysis in lung transplant recipients is warranted with the view to obviate the need for fiberoptic bronchoscopy in some patients [36]. 2.4 Breath nitrogen-containing compounds In normal subjects 20% of the daily production of urea is secreted into the gut where the gut bacterial flora converts the urea to ammonia and carbon dioxide. The ammonia is subsequently reabsorbed from the intestine into the blood stream and converted by the liver back into urea. Under normal circumstances humans reuse most of the ammonia derived from catabolism of amino acids in the urea cycle although some urea, uric acid, and free ammonia is excreted in the urine. In end-stage renal disease, urine excretion is minimal and the concentrations of the catabolites of amino acids increase in the blood. The characteristic odor of uremic breath due toth elevated levels of dimethylamine and trimethylamine has been known for most of the 20 century, although it was not until 1977 that amines were conclusively identified and quantified in breath [37]. Ammonia will only appear in the blood if there is liver damage and the removal of ammonia by conversion to urea is decreased. Until recently the evidence that ammonia was present in breath was observational although it was known that if acidic vapors were inhaled then a portion of these vapors were neutralized in the nasal passage. This neutralization was postulated to be due to endogenously produced ammonia. In 1997 ammonia was positively identified and quantified in the breath of normal and uremic patients. These determinations were only possible as a result of the introduction of a novel mass spectrometer system [38]. Uremic patients had elevated levels of ammonia compared to normal subjects. The concentrations of breath ammonia were found to correlate with their respective plasma urea concentrations for all subjects examined. This correlation supports the hypothesis that hydrolysis of urea is the source for breath ammonia. Both of these studies have reported preliminary investigations on the use of exhaled breath to examine the efficacy of renal dialysis. This is an area where breath analysis has a major potential, particularly if a monitor is available that can quantify breath nitrogen compounds in real-time. Monitors that detect amines or ammonia in breath would be equally suitable for this important application. The development of these detectors will also allow biochemical pathways for the production of nitrogen-containing compounds to be investigated more fully.

T.H. Risby / Volatile Organic Compounds as Markers

\ 21

3. Conclusions Breath markers can be used to study the pathogenesis of a variety of disease states. Acute events are more easily studied since the subject can act as their own control. This approach is ideally suited to pharmacological interventions. Breath markers can also be used to identify individuals with metabolic defects. The ability to determine breath biomarkers in real-time will expand this approaches potential application in the future. This is significant, particularly in the surgical arena when the ability to measure changes in realtime and to ameliorate these changes on the basis of breath analysis could impact the surgical outcome. Such applications will only be possible if real-time monitors are developed for a variety of breath markers. Development of breath markers will expand as novel breath markers are identified that can be relate to the pathophysiology of disease progression. Current technology already allows molecular breath profiles to be generated for both normal and diseased humans subjects. Careful comparisons of these profiles may allow molecules to be identified that are early indicators of disease or disease progression. Once unique marker molecules have been identified then the biochemical pathways that produce these molecules must be proposed. The collection of breath is ideally suited to situations where blood is difficult to sample, e.g., in neonatology. Moreover, breath can be sampled multiple times with minimal invasion. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

G.E. Hayden, Olfactory diagnosis in medicine, Postgraduate Medicine, 67 (1980) 110-115. A. Manolis, The diagnostic potential of breath analysis, Clinical Chemistry, 29 (1983) 5-15. M. Phillips, Breath tests in medicine, Scientific American, July (1992) 74-79. B.O. Jansson and B.T. Larsson, Analysis of organic compounds in human breath by gas chromatographymass spectrometry, Journal of Laboratory & Clinical Medicine, 74, (1969), 961-966. J.P. Conkle et al., Trace composition of human respiratory gas, Archives of Environmental Health, 30, (1975), 290-295. B. Krotoszynski et al., Characterization of human expired air: a promising investigative and diagnostic technique. Journal of Chromatographic Science, 15, (1977), 239-244. E.S. Deneris et al., In vitro biosynthesis of isoprene from mevalonate utilizing a rat liver cytosolic fraction, Biochemical & Biophysical Research Communications, 123, (1984), 691-696. E.S. Deneris et al., Acid-catalyzed formation of isoprene from a mevalonate-derived product using a rat liver cytosolic fraction, Journal of Biological Chemistry, 260, (1985), 1382-1385. B.G. Stone et al,, Effects of regulating cholesterol biosynthesis on breath isoprene excretion in men, L/>w!s, 28, (1993), 705-708. A. Cailleux and P. Allain, Isoprene and sleep, Life Sciences, 441, (1989), 1877-1880. W.M. Foster et al., Breath isoprene: temporal changes in respiratory output after exposure to ozone. Journal of Applied Physiology, 80, (1996), 706-710. J.K. Schubert et al., Application of a new method for the analysis of exhaled gas in critically ill patients, Intensive Care Medicine, 24, (1998), 415-421. C.A. Riely et al., Ethane evolution: a new index of lipid peroxidation, Science, 183, (1974), 208-210. C.M. Kneppkens et al., The potential of the hydrocarbon breath test as a measure of lipid peroxidation. Free Radicals in Biology & Medicine, 17, (1994), 127-160. T.H. Risby and S.S. Sehnert, Clinical application of breath biomarkers of oxidative stress status. Free Radicals in Biology & Medicine, 27, (1999), 1182-1192. D. Barnett et al., Breath acetone and blood sugar measurements in diabetes, Clinical Sciences, 37. (3969), 570. P.L. Davis et al., Endogenous isopropanol: forensic and biochemical implications, Journal of Analytical Toxicology, 8, (1984), 209-212. K. Cope et al., Increased gastrointestinal ethanol production in obese mice: implications for fatty liver disease pathogenesis, Gasteroenterology, 119, (2000), 1340-1347.

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19. S. Nair et al., Obesity and female gender increases breath ethanol concentration: potential implications for the pathogenesis of nonalcoholic steatohepatitis, American Journal ofGasteroenlerology, 96, (2001). 1200-1204. 20. W.F. Borson et al., Genetic polymorphism of human liver alcohol and aldehyde dehydrogenases and their relationships to alcohol metabolism. Hepatology, 6, (1984), 502-510. 21. S. P. Eriksen and A.B. Kulkarni, Methanol in normal human breath, Science, 141, (1963), 639-640. 22. J. Axelrod and J. Daly, Pituitary gland: enzymic formation of methanol from S-adenosylmethionine. Science, 150, (1965), 892-893. 23. S. Chen et al., Mercaptans and dimethyl sulfide in the breath of patients with cirrhosis of the liver. Journal of Laboratory & Clinical Medicine, 75, (1970), 628-635. 24. E.S. Canellakis and H. Tarver, Studies on protein synthesis in vitro. IV. Concerning the apparent uptake of methionine by paniculate preparation from liver, Archives of Biochemistry & Biophysics, 42, (1953), 387-398. 25. E.S. Canellakis and H. Tarver, The metabolism of methyl mercaptan in the intact animal. Archives of Biochemistry & Biophysics, 42, (1953) 446-455. 26. P.W.D. Scislowski and K. Pickard, The regulation of transaminative flux of methionine in rat liver mitochondria, Archives of Biochemistry & Biophysics, 314, (1994), 412-416. 27. M. Johnston el al., Mechanistic studies on reactions of bacterial methionine gamma-lyase with olefinic amino acids, Biochemistry, 20, (1981), 4325-4333. 28. C.J. McClain et al., Blood methanethiol in alcoholic liver disease with and without hepatic encephalopathy. Gut, 21, (1980), 318-323. 29. R.F. Derr and L. Zieve, Methanethiol and fatty acids depress urea synthesis by the isolated perfused rat liver, Journal of Laboratory & Clinical Medicine, 100 (1982), 585-592. 30. A. Finkelstein and N.J. Benevenga, The effect of methanethiol and methionine toxicity on the activities of cytochrome c oxidase and enzymes involved in protection from peroxidative damage. Journal of Nutrition, 116, (1986), 204-215. 31. T. Vahlkamp et al., Inhibition of mitochrondrial electron transfer in rats by ethanethiol and methanethiol, Clinical Sciences, 56, (1979), 147-156. 32. L. Zieve et al., Ammonia, octanoate and a mercaptan depress regeneration of normal rat liver after partial hepatectomy, Hepatology, 5, (1985), 28-31. 33. L. Zieve et al., Effect of hepatic failure toxins on liver thymidine kinase activity and omithine decarboxylase after massive necrosis with acetaminophen in the rat, Journal of Laboratory & Clinical Medicine, 106, (1985), 583-588. 34. H.J. Blom and A. Tangerman, Methanethiol metabolism in whole blood, Journal of Laboratory & Clinical Medicine, 111, (1988), 606-610. 35. S.S. Sehnert et al., Breath biomarkers for detection of human liver diseases: a preliminary study. Biomarkers, in press, 2001. 36. S.M. Studer et al., Patterns and significance of exhaled-breath biomarkers in lung transplant rejection with acute allograft rejection, Journal of Heart Lung Transplantation, in press, 2001. 37. M.L. Simenhoff et al.. Biochemical profile of uremic breath, New England Journal of Medicine, 297. (1977), 132-135. 38. S. Davies et al.. Quantitative analysis of ammonia on the breath of patients in end-stage renal failure. Kidney International, 52, (1997), 223-228.

Section 4. The "Living State"

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Disease Markers in Exhaled Breath N. Marczin and M.H. Yacoub (Eds.) IOS Press, 2002

125

The living state: intimate insights through personal discoveries Miklos KELLERMAYER Department of Clinical Chemistry, Medical School, University of Pecs, Hungary Abstract. Experimental findings on nuclei of living cells may be used as "indicators" of changes of the living state in healthy and in injured cells. Our experimental observations strongly indicate that neither the water molecules, nor the monovalent cations (K+ and Na4 ions) are freely moving, or solubilised inside normal living cells. In other words, the chromatin, the DNA, i.e. the genes are not exposed to a 150-200 mM free electrolyte solution inside the healthy living cells, however, in injured, agonizing and dead cells the freedom of water molecules and K+ - Na+ ions gradually and rapidly increases and DNA progressively becomes more and more exposed to this solute.

Cells of an SV40 virus induced tumor (H-50 cell line) were hybridized with chicken erythrocytes and the nuclei of the heterokaryons were analyzed in the first set of our experiments as herein presented [1]. In order to produce cell fusion UV inactivated Sendai virus extract was used and the tumor specific nuclear protein, the large T-antigen was detected by immune fluorescence microscopy. The first antibody was obtained from blood sera of tumor bearing hamsters. The second antibody was an anti-hamster globulin conjugated with fluorescein isothyocyanate (Progressive Laboratories, Baltimore, USA). The size of the nuclei was determined planimetrically. Dry-mass of the nuclei was measured using the interference microscopic technique (Zeiss, Oberkochen, Germany). The DNA synthetic activity in the nuclei was analyzed autoradiographically after H-thymidine incorporation. In some experiments the heterokaryons were treated with colchicin in order to determine whether the observed changes of the nuclei were or were not dependent upon the integrity of the cytoskeleton or specifically on the integrity of the micro tubular system. The first table summarizes our fluorescence microscopic and autoradiographic observations. Table 1. The effect of colchicin treatment on size, T-antigen positivity and 3Hthymidine incorporation of nuclei of H-50 tumor cells and chicken erythrocyte heterokaryons. Size of nuclei u2

J

T-antigen

H-thymidin inc.

30 h after fusion

H-50

erythr.

n=27

n=25

351.96

46.08

167 ]

-

±13.4

10"4 M colchicin

n=42

n=44

(6 h without and 24 h with colchicin)

384.47

17.91

+ 135.7

+ 3.7

Control

H-50

Erythr.

H-50

erythr.

++++

++++

++++

++++

++++

No

++

no

(no colchicin) ±

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The small, pyknotic nuclei of chicken erythrocytes enlarged inside the heterokaryons were obtained by fusion of H-50 tumor cells and chicken red blood cells. This observation confirmed the original findings of H. Harris [2]. Parallel with the enlargement of these chicken erythrocyte nuclei they became positive for T antigen and incorporated 3Hthymidin. However, when the heterokaryons were treated 6 hours after fusion with colchicin, the chicken erythrocyte nuclei remained small, negative for T-antigen and inactive for DNA synthesis, even if they remained for a period of 24 hours in the cytoplasm of the giant fused cells. These observations confirmed the need for a specific intracellular environment for DNA synthesis which is always present in tumor cells as well as in normal cells processing their generation cycle (G]-S-G2-M phases), but missing in dormant cells like the erythrocytes circulating in chicken blood. The molecular composition (the homogenate) of the rapidly growing cells and dormant (inactive for DNA synthesis) cells are quite similar, which means that the fundamental difference in their DNA synthetic activity most probably originates from the differences in the organization, the arrangement of the molecules; naturally mainly proteins. The essence of the intracellular molecular organization, which creates an environment for genes to be active for DNA synthesis in tumor cells and in rapidly growing normal cells, but the fact that it becomes inhibitory in dormant cells like the nucleated red blood cells of chicken, is practically unknown. On the basis of the most accepted "intracellular free solution - membrane pump" cell view these observations, these experimental facts could never be truly explained. Instead these experimental observations force a change in the hypothetical intracellular free solution view to a more realistic fact-based cell hypothesis. The observed time delay (6 to 24 hours) that was needed to initiate the accumulation of the large T-antigen, the 80 kD size tumor specific protein, into the dormant chicken red blood cell nuclei demands a conceptual system in which a dynamic intracellular molecular rearrangement, recompartmentalization exists and at the same time, denies any kind of random molecular diffusion as the main feature of the interior of the living cell. If the interior of the living cells were a free solution, a cytosol and nuclear sap, as it is assumed in the widely accepted "solution - membrane pump" cell view, the random molecular collisions would be the only way for the intracellular molecular trafficking which is completely contradictory to our observed facts. The dependence of the accumulation of T-antigen into the dormant erythrocyte nuclei on the integrity of cytoskeleton, more specifically on the integrity of the micro tubular system strongly supports a dynamic self-compartmenting, self-rearranging system inside the living cells. Such a system could have its own memory, the so-called spatial memory and could be considered as a three dimensional "programmable" organization of "energized" proteins with the capability of adsorbing water molecules and inorganic ions. Our interference microscopic observations (Table 2) give further insight into such a dynamic protein-waterion co-compartment system. According to our opinion this organization itself can be considered as living state, the state which had been, has been and still being used to specify the life at the cellular level. Table 2. Interference microscopic measurements of nuclei of chicken erythrocytes in the heterokaryons of H-50 and chicken red blood cells.

n 2

Area (n ) 12

Dry mass (grx 10~ )

4h after fusion

24h after fusion

18

17

17.3+4.1

36.2+4.7

18.9 + 4.3

27.1 +3.6

1.10 + 0.08

0.75+0.10

Concentration (grxlO-'2/n2)

M. Kellermayer/ The Living State: Intimate Insights

] 27

In heterokaryons 24 hours after fusion the total dry mass content and the volume (area) of erythrocyte nuclei increased significantly. However, the increase of the nuclear volume was found to be much higher than the increase of the total dry mass content. In other words more water accumulated into the nuclei than mass. (The mass naturally means protein). If the interior of the living cell were a free solution, what is declared in the cytosol and nuclear sap view, our interference microscopic findings would indicate an increased gradient for water molecules inside the enlarged nuclei. Knowing the size of the water molecules and the structure of the nucleus with their large pores, an increasing water gradient between the nucleus and the rest of the cell would be impossible. The only explanation for our interference microscopic observations is an increased water-binding, water-structuring, water-holding capacity of proteins which had migrated into the nuclei and was associated with the different fixed chromatin structures inside the nucleus [3, 4, 5]. Interestingly, the transition of the condensed (inactive) heterochromatin to the loose (more hydrated) genetically active euchromatin (chromatin where the DNA and RNA synthesis occurs) could also only be explained by a higher water-structuring, water-binding capacity of those proteins which were accumulated in these regions and participated in the synthesis of the DNA and RNA. In a second set of the experiments, tissue culture cells (HeLa, Hep2 and human fibroblasts) were used. The cells were stained with Romhanyi's precipitation toluidin blue technique and analyzed with a polarization microscope [6, 7, 8, 9]. The DNA inside the nuclei of the interphase cells was always optically isotrop, while inside the mitotic chromosomes birefringent. Furthermore, it is important to note that the isolated DNA filaments have intensive negative birefringence. When the nuclei of monolayer cultured cells were isolated "in situ" with nonionic detergents (NP40, Triton X100) at 0.1%-0.2% concentration, the DNA inside the isolated nuclei was always birefringent if the cation concentration, i.e. the concentration of the free K+ and/or Na+ ions in the surrounding medium reached or exceeded 70 mmol/1 [6, 10, 11]. On the other hand, if the concentration of K+ and/or Na+ in the solution around the isolated nuclei was lower than 70 mmol/1 the DNA was always isotrop. The structural changes of DNA, i.e. the transition from the isotrop to the anisotrop (birefringent) state and vice versa was always reversible. Consequently, our polarization microscopic studies indicate that the DNA inside the nucleus of an interphase cell can not be exposed to a free 150-200 mmol/1 K+ - Na electrolyte solution, which is the widely accepted intracellular K+ - Na+ ion concentration for the living cells according to the "intracellular solution (cytosol, nuclear sap) membrane pump" cell view. In other words, our polarization microscopic observations are also strongly contradictory to the concept of intracellular free solution, i.e. to the cytosol and nuclear sap view. The reversible birefringence of DNA in mitotic chromosomes proves that the DNA undergoes not only a condensation but also certain structural changes during mitosis. It had been shown that the basic nucleosomal structure of DNA-histones is not broken up in mitotic chromosomes. Consequently, the structural changes of DNA we observed in mitotic chromosomes can only be explained by a local free ion (K+) driven reversible folding-unfolding transition of DNA in the linker region [9]. In other words our polarization microscopic findings on DNA of chromosomes and interphase cell nuclei support the concept of a local free ion regulatory system in the close vicinity of the genes [10, 12]. Naturally, such a local regulatory system can only work if both the water molecules and the inorganic ions are reversible bound to and released from proteins changing their configuration and energy levels in reversible manner. We might state that our polarization microscopic observations confirm our previously presented observations on cell hybrids of H-50 tumor cells and chicken erythrocytes and both of them are very supportive for a dynamic, "programmable", three dimensional spatial memory carrying "energized protein - water - ion co-compartment system" in all living cells.

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The final set of our experiments (Table 3) are also supportive for such a system. HeLa cells growing in monolayers on glass and plastic surfaces were released with mild trypsinization and for the purpose of increasing the number of agonizing and dead cells, they were vigorously shaken for 1 and 2 hours. Parallel samples were stained with nigrosin (viability test) and with Romhanyi's precipitation toluidin blue technique. Ten preparations were analyzed from each sample and 200 cells were counted in every preparation.

Table 3. The nigrosin stainability and nuclear birefringence in mechanically injured HeLa culture cells. % N I"

2"

2000

24.1

38.5

(10x200)

+ 2.4

+ 2.9

Dead cells

2000

23.5

37.9

(nigrosin stained cells)

(10x200)

+ 2.6

+ 3.2

Nuclear birefringent

Comparing the nigrosin-stained cells with cells birefringent DNA in their nuclei, a very close correlation was found. This finding indicated that the cells that were agonizing, dying or already dead permit the nigrosin to diffuse inward and induces the DNA in their pyknotic nuclei to turn from the isotrop to the anisotrop state. On the basis of our previously described observations the change of the DNA from the isotrop to the anisotrop state can only be explained if the DNA in nuclei of agonizing and dead cells were becoming more and more exposed to an increasing (higher then 70 mmol/1) concentration of free K+ - Na+ ions. Therefore, we might conclude that at cell agony and cell death the water molecules and K* and Na"1" ions are gradually or rapidly released from their dynamic co-compartmentalization with proteins. It is important to note that the nuclear pycnosis itself, which is a well-known characteristics of cell agony and cell death at apoptosis and also at cell necrosis is probably caused by the release of water molecules and ions from their dynamic compartments with energized proteins in the close vicinity of DNA. In summary, all of our experimental observations on the nuclei of the living cells are strongly contradictory to the widely accepted free "intracellular solution-membrane pump" cell view. The DNA molecules, the genes within the nuclei of the healthy living cells are not exposed to a 150-200 mmol/1 K* - Na* solution. However, at cell agony and cell death, the DNA is gradually or rapidly becoming more and more exposed to such free electrolyte. All of this indicates that the living state, i.e. the life at cell level must be connected to certain energy driven dynamic associations of proteins with water molecules and inorganic ions. For the question: "What is life?" Szent-Gyorgyi Albert answered this: "The life is the water dancing Jo the tune of solids" [13]. As a corollary, we would answer thus: "The life is water and potassium dancing to the tune of energized proteins" [14, 15]. What are the energized proteins? To answer this question we need to listen to Gilbert N. Ling who speaks about the extended state of proteins with high electron density sites and water structuring capability [16, 17, 18, 19]. We may add to this that our newest strategy, the study of the release kinetics of K+, ATP and proteins from detergent "opened" cells provides promising insights into the major secrete, i.e. the living state [20, 21].

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Acknowledgements: This work was supported by the OTKA T 032043 grant. Critical comments of Carlton F. Hazlewood and Gilbert N. Ling are highly appreciated.

References:

[I]

M. Kellermayer et al., Inhibiton of Intranuclear Transport of SU40-Induced T-antigen in Heterokaryons, Cell Biology International Reports 2(1978) 19-24.

[2]

H. Harris, Nucleus and Cytoplasm. Claredone Press, Oxford, 1967.

[3]

M. Kellermayer et al., Swelling of Nuclei and Decondensation of Chromatin in a Multinuclear Model, European Journal of Cell Biology 23(1980) 204-207.

[4]

M. Kellermayer, Soluble and "Loosely Bound" Nuclear Proteins in Regulation of the Ionic Environment in Living Cell Nuclei. In: H. G. Schweiger (eds.), International Cell Biology 1980-1981. Springer-Verlag, Berlin-Heidelberg, New York, 1981, pp 915-924.

[5]

M. Kellermayer et al., Platelet-derived Growth Factor (PDGF) Induces Intranuclear Protein Accumulation in 3T3 Fibroblats, Experimental Cell Research 152(1984) 255-259.

[6]

M. Kellermayer and K. Jobst, Ion-dependent Anisotropy of Deoxyribonucleoprotein Structures in Tissue Cultures, Experimental Cell Research 63(1970) 204-207.

[7]

M. Kellermayer et al. Polarization Optical Study of the Ultra Structure of Cell Nuclei in Tissue Cultures, Acta Morphologica Acad. Sci. Hung., 18(1970) 131-137.

[8]

K. Jobst and M. Kellermayer, Sodium-induced Changes in the Nuclei of Monolayer HeLa Cultures, Journal Cell Sciences 11(1972) 669-673.

[9]

M. Kellermayer and C. F. Hazlewood, Dynamic Inorganic Ion-Protein Interactions in Structural Organization of DNA of Living Cell Nuclei, Cancer Biochem. Biophys., 3(1979) 181-188. M. Kellermayer and K. Jobst, Isolation of Cell Nuclei in Coverslip Cultures, Beitr. Path 142(1971)321-323.

[10] [II]

M. Kellermayer and K. Jobst, Cytoplasmic Protein Network in HeLa Cells, Histochemistry 44(1975)193-195.

[12]

M. Kellermayer and C. F. Hazlewood, Dynamic Interaction of Water Molecules, Inorganic Ions and Chromatin Structures in Isolated Thymus Nuclei. In: A. Pullman, V. Vasilescu, L. Packer (eds.), Water and Ions in Biological Systems, Plenum Press, New York and London, 1985, pp 57-67.

[13]

A. Szent-Gytfrgyi, The Living State. Academic Press, New York and London, 1972.

[14]

M. Kellermayer, ATP Dependent Cocompartmentation of Proteins and K+ within the Living Cell. In: J. Tigyi, M. Kellermayer, C. F. Hazlewood (eds.), The Physical Aspect of the Living Cell, Akade"miai Kiado, Budapest, 1991, pp 165-180.

[15]

G. H. Pollack, Cells, Gels and the Engines of Life. A New, Unifying Approach to Cell Function. Ebner and Sons Publishers, Seattle, 2001.

[16]

G. N. Ling, A physical theory of the living state. The association-induction hypothesis. Blaisdell Publishing Co. New York and London, 1962.

[17]

G. N. Ling, In Search of the Physical Basis of Life. Plenum Press, New York and London, 1984.

[18]

G. N. Ling, A Revolution in the Physiology of the Living Cell. Krieger Publishing Co. Malabar, Florida, 1992.

[19]

G. N. Ling, Life at the Cell and Below-Cell Level. The Hidden History of a Fundamental Revolution in Biology. Pacific Press, New York, 2001.

[20]

M. Kellermayer et al., Potassium Retention in Membraneless Thymus Lymphocyte Nuclei, Physiol. Chem. Phys. and Med NMR., 16(1984) 503-511.

[21]

M. Kellermayer et al., Cocompartmentation of Proteins and K+ within the Living Cell, Proc. Nat. Acad Sci. USA 83(1986) 1011-1015.

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Part II. Asthma

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Disease Markers in Exhaled Breath N. Marczin andM.H. Yacoub (Eds.) IOS Press, 2002

133

Biology of Asthma Peter J BARNES National Heart and Lung Institute, Imperial College, London, UK

Abstract. Asthma is a chronic inflammatory disease of the airways, characterised by an infiltration of eosinophils, activation of T helper-2 lymphocytes and activation of mast cells. Structural cells of the airway, including epithelial cells and airway smooth muscle participate in the inflammatory process via the production of multiple mediators, including nitric oxide. Many inflammatory mediators, including cytokines and chemokines, are involved in asthma and lead to the characteristic pathophysiology, including contraction of airway smooth muscle, plasma exudation, mucus secretion and activation of sensory nerves. Chronic inflammation may result in structural changes, including proliferation of airway smooth muscle, new vessel formation and fibrosis that may lead to irreversible airway narrowing.

1. Introduction Asthma is a highly complex disease involving many different inflammatory cells, multiple mediators, and complex acute and chronic inflammatory responses in the airways [l](Figure 1). There have been important advances in our understanding of the molecular and cellular pathways involved, partly through the development of new molecular techniques and partly by application of novel inhibitors developed by the pharmaceutical industry [2]. Yet despite these considerable advances many fundamental questions about asthma that remain to be answered.

Figure 1. Many cells and mediators are involved in asthma and lead to several effects on the airways.

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Our understanding of asthma has been transformed by the recognition that chronic inflammation underlies the clinical syndrome. Previously it had commonly been assumed that the basic defect in asthma lay in abnormal contractility of airway smooth muscle, giving rise to variable airflow obstruction, and the common symptoms of intermittent wheeze and shortness of breath. However studies of airway smooth muscle from asthmatic patients have shown no consistent evidence for increased contractile responses to spasmogens such as histamine in vitro, indicating that asthmatic airway smooth muscle is not fundamentally abnormal and suggesting that it is the control of airway calibre in vivo which is abnormal. 2. Airway inflammation in asthma All patients with symptomatic asthma have inflamed airways. This has been confirmed by fibreoptic bronchial biopsies, by bronchoalveolar lavage, by induced sputum and by the use of exhaled markers of inflammation, such as exhaled nitric oxide (NO) and carbon monoxide (CO). This inflammation appears to involve all airways in asthma, from the trachea to terminal bronchioles, but does not extend to the lung parenchyma. The inflammatory pattern in asthma is characteristic, with an increase in activated eosinophils, degranulation of mast cells and activation of T helper (CD4+) lymphocytes. It differs markedly from the inflammation seen in chronic obstructive pulmonary disease and chromic bronchitis. The relationship between inflammation and clinical symptoms of asthma is still not clearly understood. The airway inflammation is related to airway hyperresponsiveness (AHR), as measured by histamine or methacholine challenge, but the degree of inflammation does not clearly correspond to asthma severity. This suggests that other factors, such as structural changes in the airway wall are important. The increased airway responsiveness in asthma is a striking physiological abnormality that is present even when airway function is normal. It is likely that there are several factors that underlie this increased responsiveness to contractor agents, particularly those that act indirectly by releasing bronchoconstrictor mediators form airway cells. Airway hyperresponsiveness may be due to increased release of mediators (such as histamine and leukotrienes from mast cells), abnormal behaviour of airway smooth muscle, thickening of the airway wall by reversible (oedema) and irreversible (airway smooth muscle thickening, fibrosis) elements. Airway sensory nerves may also contribute importantly to symptoms, such as cough and chest tightness, as the nerves become sensitised by the chronic inflammation in the airways. Although most attention has previously focused on the acute inflammatory changes seen in asthmatic airways (bronchoconstriction, plasma exudation, mucus secretion) asthma is a chronic inflammatory disease, with inflammation persisting over many years in most patients. Superimposed on this chronic inflammatory state are acute inflammatory episodes, which correspond to exacerbations of asthma. It is clearly important to understand the mechanisms of acute and chronic inflammation in asthmatic airways and to understand the long-term consequences of this chronic inflammation on airway function. 3. Inflammatory Cells Many different inflammatory cells are involved in asthma, although the precise role of each cell type is not yet certain. No single inflammatory cell is able to account for the complex pathophysiology of asthma, but some cells predominate in asthmatic inflammation. It is likely that there are important interactions between these cells. The pattern of inflammation is similar in all forms of asthma so far investigated with a predominance of activated eosinophils, mast cells and T helper lymphocytes. The inflammation in intrinsic asthma, with no evidence of atopy is identical to that of the common atopic asthma [3j.

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135

Mast cells Mast cells are important in initiating the acute bronchoconstrictor responses to allergen and probably to other indirect stimuli, such as exercise and hyperventilation (via osmolality or thermal changes) and fog. Mast cells also secrete certain cytokines, such as interleukin(IL)-4 that may be involved in maintaining the allergic inflammatory response. However, there are questions about the role of mast cells in more chronic inflammatory events, and it seems more probable that other cells such as macrophages, eosinophils and T-lymphocytes are more important in the chronic inflammatory process, including AHR. Classically mast cells are activated by allergens through an IgE-dependent mechanism and the critical importance of IgE in the pathophysiology of asthma has been highlighted by recent clinical studies with humanised anti-IgE antibodies, which block IgE-mediated effects [4]. Anti-IgE antibody results in a reduction in circulating IgE to undetectable levels and significant clinical improvement even in patients with severe steroid-dependent asthma. Macrophages Macrophages, which are derived from blood monocytes, traffic into the airways in asthma and may be activated by allergen via low affinity IgE receptors (FceRII) and may also be inhibited by anti-IgE therapy. The enormous repertoire of macrophages allows these cells to produce many different products, including a large variety of cytokines, which may orchestrate the inflammatory response. Macrophages have the capacity to initiate a particular type of inflammatory response via the release of certain patterns of inflammatory mediators. Macrophages may both increase and decrease inflammation, depending on the stimulus. Alveolar macrophages normally have a suppressive effect on lymphocyte function, but this may be impaired in asthma after allergen exposure. Macrophages may therefore play an important anti-inflammatory role, preventing the development of allergic inflammation, Macrophages may also act as antigen-presenting cells which process allergen for presentation to T-lymphocytes, although alveolar macrophages are far less effective in this respect than macrophages from other sites, such as the peritoneum. Dendritic cells Dendritic cells are specialised macrophage-like cells in the airway epithelium, that are very effective antigen-presenting cells, and may therefore play a very important role in the initiation of allergen-induced responses in asthma. Dendritic cells take up allergens, process then to peptides and migrate to local lymph nodes where they present the allergenic peptides to uncommitted T-lymphocytes, to programme the production of allergen-specific T cells. Eosinophils Eosinophil infiltration is a characteristic feature of asthmatic airways and differentiates asthma from other inflammatory conditions of the airway. Allergen inhalation results in a marked increase in eosinophils in bronchoalveolar lavage fluid at the time of the late reaction, and there is a close relationship between eosinophil counts in peripheral blood or bronchial lavage and AHR. Eosinophils are linked to the development of AHR through the release of basic proteins and oxygen-derived free radicals. The mechanisms involved in recruitment of eosinophils into asthmatic airways have been carefully investigated, as this may provide a specific target to inhibit inflammation in asthma. Eosinophils are derived from bone marrow precursors and their production is dependent on the cytokine IL-5. After allergen challenge eosinophils appear in BAL fluid during the late response, and this is associated with a decrease in peripheral eosinophil counts and with the appearance of eosinophil progenitors in the circulation. The signal for increased eosinophil production is presumably derived from the inflamed airway. Eosinophil recruitment initially involves adhesion of eosinophils to vascular

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endothelial cells in the airway circulation, their migration into the submucosa and their subsequent activation. The role of individual adhesion molecules, cytokines and mediators in orchestrating these responses has been extensively investigated. Adhesion of eosinophils involves the expression of specific glycoprotein molecules on the surface of eosinophils (integrins) and their expression of such molecules as intercellular adhesion molecule-1 (ICAM-1) on vascular endothelial cells. An antibody directed at ICAM-1 markedly inhibits eosinophil accumulation in the airways after allergen exposure and also blocks the accompanying hyperresponsiveness. However, ICAM-1 is not selective for eosinophils and cannot account for the selective recruitment of eosinophils in allergic inflammation. The adhesion molecules VLA4 expressed on eosinophils and VCAM-1 appear to be more selective for eosinophils and IL-4 increases the expression of VCAM-1 on endothelial cells. Eosinophil migration may be due to the effects of lipid mediators, such as platelet-activating factor (PAF), to the effects of cytokines, such as GM-CSF and IL-5, which may be very important for the survival of eosinophils in the airways, and may "prime" eosinophils to exhibit enhanced responsiveness. Eosinophils from asthmatic patients show exaggerated responses to PAF and phorbol esters, compared to eosinophils from atopic non-asthmatic individuals and this is further increased by allergen challenge, suggesting that they may have been primed by exposure to cytokines in the circulation. There are several mediators involved in the migration of eosinophils from the circulation to the surface of the airway. The most potent and selective agents appear to be CC-chemokines, such as eotaxin. RANTES, and MCP-4, that are expressed in epithelial cells. There appears to be a co-operative interaction between IL-5 and eotaxin, so that both cytokines are necessary for the eosinophilic response in airways. Once recruited to the airways eosinophils require the presence of various growth factors, of which GM-CSF appears to be the most important. In the absence of these growth factors eosinophils undergo programmed cell death (apoptosis). Recently a humanised monoclonal antibody to IL-5 has been administered to asthmatic patients and, as in animal studies, there is a profound and prolonged reduction in circulating eosinophils; the infiltration of eosinophils into the airway after inhaled allergen challenge is completely blocked [5]. However, there is no effect on the response to inhaled allergen and no reduction in AHR, questioning the pivotal role of eosinophils in asthma. Neutrophils The role of neutrophils in asthma is less clear. Neutrophils are found in the airways of patients with COPD and cystic fibrosis, who do not have the degree AHR found in asthma, but are rarely seen in the airways of patients with chronic asthma. However, in patients who die suddenly of asthma large numbers of neutrophils are found in the airways, although this may reflect the rapid kinetics of neutrophil recruitment compared to eosinophil inflammation. Increased numbers of neutrophils are also found in the airway sand induced sputum of patients with severe asthma. Whether these cells are present because of high does of inhaled corticosteroids or are contributing to the pathophysiology of severe asthma is currently unknown. T-lymphocytes T-lymphocytes play a pivotal role in orchestrating the inflammatory response in asthma through the release of specific patterns of cytokines, resulting in the recruitment and survival of eosinophils and in the maintenance of mast cells in the airways. T-lymphocytes are coded to express a distinctive pattern of cytokines, which may be similar to that described in the murine Th2 type of T-lymphocytes, which characteristically express IL-4, IL-5 and IL-9. This programming of T-lymphocytes is presumably due to antigen-presenting cells, such as dendritic cells, which may migrate from the epithelium to regional lymph nodes or which

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137

interact with lymphocytes resident in the airway mucosa. There appears to be an imbalance of Th cells in asthma with the balance tipped away from the normally predominant Thl cells in favour of Th2 cells. The balance between Thl cells and Th2 cells may be determined by locally released cytokines, such as IL-12 derived from dendritic cells and macrophages, which tips the balance in favour of Thl cells and IL-4 which favours Th2 cells. There is some evidence that early infections might promote Thl-mediated responses to predominate and that a lack of infection in childhood may favour Th2 cell expression and thus atopic diseases [6]. Basophils The role of basophils in asthma is uncertain, as these cells have previously been difficult to detect by immunocytochemistry. Using a basophil-specific marker a small increase in basophils has been documented in the airways of asthmatic patients, with an increased number after allergen challenge. However, these cells are far outnumbered by eosinophils. Structural cells Structural cells of the airways, including epithelial cells, fibroblasts and even airway smooth muscle cells may also be an important source of inflammatory mediators, such as cytokines and lipid mediators in asthma. Indeed, because structural cells far outnumber inflammatory cells they may become the major source of mediators driving chronic inflammation in asthmatic airways. In addition, epithelial cells may a key role in translating inhaled environmental signals into an airway inflammatory response and are probably a major target cell for inhaled corticosteroids. 4. Inflammatory Mediators Many different mediators have been implicated in asthma and they may have a variety of effects on the airways and together they account for the pathological features of asthma [7], Mediators such as histamine, prostaglandins, leukotrienes, bradykinin and endothelins contract airway smooth muscle, increase microvascular leakage, increase airway mucus secretion and attract other inflammatory cells. The multiplicity of mediators makes it unlikely that antagonising any single mediator will have a major impact in clinical asthma; this has been confirmed with clinical trials using specific antagonists and synthesis inhibitors of individual mediators. The cysteinyl-leukotrienes LTC4, LTD4 and LTE4 are potent constrictors of human airways and have been reported to increase AHR and may play an important role in asthma. The development of potent anti-leukotrienes has now made it possible to evaluate the role of these mediators in asthma. Potent LTD4 antagonists reduce exercise- and allergen-induced bronchoconstriction and chronic treatment improves lung function and symptoms in asthmatic patients, although the degree of improvement is not nearly as great as seen with an inhaled corticosteroids. It is only through the use of specific antagonists that the role of individual mediators of asthma may be defined. For example, PAF is a potent inflammatory mediator that mimics many of the features of asthma, including eosinophil recruitment and activation and induction of airway hyperresponsiveness, yet even potent PAF antagonists, such as modipafant, do not control asthma symptoms, at least in chronic asthma. Cytokines Cytokines are important in the chronic inflammation of asthma and play a critical role in orchestrating the nature of allergic inflammatory response [8]. Many inflammatory cells (macrophages, mast cells, eosinophils and lymphocytes) are capable of synthesising and releasing these proteins, and structural cells such as airway epithelial, smooth muscle and endothelial cells may also release a variety of cytokines and may therefore participate in the

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chronic inflammatory response. While inflammatory mediators like histamine and leukotrienes are important in the acute and subacute inflammatory responses and in exacerbations of asthma, it is likely that cytokines play a dominant role in chronic inflammation. Almost every cell is capable of producing cytokines under certain conditions. Research in this area is hampered by a lack of specific antagonists, although important observations have been made using specific neutralising antibodies. The cytokines which appear to be of particular importance in asthma include the lymphokines secreted by Th2 cells: IL-4 which is critical in switching B-lymphocytes to produce IgE, for expression of VCAM-1 on endothelial cells and for inducing the differentiation of Th2 cells, IL-13, which acts similarly to IL-4 in IgE switching, and IL-5 which is essential for the differentiation, of eosinophils. The critical role of an IL-5 in eosinophilia has been confirmed by the use of an anti-IL-5 antibody in asthmatic patients. Another Th2 cytokine IL-9 may play a critical role is sensitising responses the the cytokines IL-4 and IL-5. Other cytokines, such as IL-10 and tumour necrosis factor-a (TNF-a), may play an important role in amplifying the inflammatory response in asthma and are released from a variety of cells, including macrophages and epithelial cells. Both cytokines show an increased expression in asthma and activate the proinflammatory transcription factors, nuclear factor-icB (NF-KB) and activator protein-1 (AP-1), which then switch on many inflammatory genes. Inhalation of TNF-a increased airway responsiveness in normal individuals. Oxidative stress As in all inflammatory diseases, there is increased oxidative stress in asthma as activated inflammatory cells, such as macrophages and eosinophils produce reactive oxygen species. Evidence for increased oxidative stress in asthma is provided by the increased concentrations of 8-isoprostane (a product of oxidised arachidonic acid) in exhaled breath condensates and increased ethane (a product of oxidative lipoid peroxidation) in exhaled breath of asthmatic patients. Increased oxidative stress is related to disease severity and may amplify the inflammatory response and reduce responsiveness to corticosteroids. Nitric oxide NO is produced by several cells in the airway by NO synthases. An inducible form of the enzyme (iNOS) is expressed in epithelial cells of asthmatic patients and can be induced by cytokines in airway epithelial cells in vitro. This may account for the increased concentration of NO in the exhaled air of untreated asthmatic patients [9]. NO itself is a potent vasodilator and this may increase plasma exudation in the airways; it may also amplify the Th2lymphocyte mediated response and attract and prolong the survival of eosinophils. The combination of increased oxidative stress and NO may lead to the formation of the potent radical peroxynitrite, that may result in nitrosylation of proteins in the airways. 5. Effects of Inflammation on the Airways The chronic inflammatory response has several effects on the target cells of the airways, resulting in the characteristic pathophysiological changes associated with asthma. Asthma may be regarded as a disease with continuous inflammation and repair proceeding simultaneously. Several structural changes are recognisable in severe asthma and these may not be fully reversible with existing therapies. Airway epithelium Airway epithelial shedding may be important in contributing to AHR and may explain how several different mechanisms, such as ozone exposure, certain virus infections, chemical

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sensitisers and allergen exposure, can lead to its development, since all these stimuli may cause epithelial disruption. Epithelium may be shed as a consequence of inflammatory mediators, such as eosinophil basic proteins and oxygen-derived free radicals, together with various proteases released from inflammatory cells. Epithelial cells are commonly found in clumps in the BAL or sputum (Creola bodies) of asthmatics, suggesting that there has been a loss of attachment to the basal layer or basement membrane. Epithelial damage may contribute to AHR in a number of ways, including loss of its barrier function to allow penetration of allergens, loss of enzymes (such as neutral endopeptidase), which normally degrade inflammatory mediators, loss of a relaxant factor (so called epithelial-derived relaxant factor), and exposure of sensory nerves which may enhance reflex neural effects on the airway. Fibrosis Subepithelial fibrosis is a characteristic of asthmatic airways, and may even be seen in patients with episodic asthma who have no symptoms. The increased thickness is unrelated to asthma severity and its functional significance is uncertain. Type HI and V collagen appear to be laid down, and may be produced by myofibroblasts which are situated under the epithelium. However, there is also evidence for fibrosis deeper within the airway wall and within the airway smooth muscle layer that may have a greater functional significance. The mechanism of fibrosis is not yet clear but several cytokines, including transforming growth factor-p and platelet-derived growth factor may be produced by epithelial cells or macrophages in the inflamed airway. Air-way smooth muscle There is still debate about the role of abnormalities in airway smooth muscle is asthmatic airways. In vitro airway smooth muscle from asthmatic patients usually shows no increased responsiveness to spasmogens. Reduced responsiveness to 6-agonists has been reported in post-mortem or surgically removed bronchi from asthmatics, although the number of B-receptors is not reduced, suggesting that B-receptors have been uncoupled. These abnormalities of airway smooth muscle may be a reflection of the chronic inflammatory process. For example the reduced 13-adrenergic responses in airway smooth muscle could be due to phosphorylation of the stimulatory G-protein coupling fi-receptors to adenylyl cyclase, resulting from the activation of protein kinase C by the stimulation of airway smooth muscle cells by inflammatory mediators, to increased activity of the inhibitory G-protein (Gs) induced by proinflammatory cytokines. In asthmatic airways there is also a characteristic hypertrophy and hyperplasia of airway smooth muscle, which is presumably the result of stimulation of airway smooth muscle cells by various growth factors, such as PDGF, or endothelin-1 released from inflammatory cells. Vascular responses Vasodilatation occurs in inflammation, yet little is known about the role of the airway circulation in asthma, partly because of the difficulties involved in measuring airway blood flow. The bronchial circulation may play an important role in regulating airway calibre, since an increase in the vascular volume may contribute to airway narrowing. Increased airway blood flow may be important in removing inflammatory mediators from the airway, and may play a role in the development of exercise-induced asthma. There may also be an increase in the number of blood vessels in asthmatic airways as a result of angiogenesis in response to growth factors. Microvascular leakage is an essential component of the inflammatory response and many of the inflammatory mediators implicated in asthma produce this exudation of plasma

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from post-capillary venules. Plasma exudation in asthma may have several consequences on airway function, including increased airway secretions, impaired mucociliary clearance, formation of new mediators from plasma precursors (such as kinins) and mucosal oedema which may contribute to airway narrowing and AHR. Mucus hypersecretion Mucus hypersecretion is a common inflammatory response in secretory tissues. Increased mucus secretion contributes to the viscid mucus plugs, which occlude asthmatic airways, particularly in fatal asthma. There is evidence for hyperplasia of submucosal glands, which are confined to large airways and of increased numbers of epithelial goblet cells at all airway levels. This increased secretory response may be due to inflammatory mediators acting on submucosal glands and due to stimulation of neural elements. Little is understood about the control of goblet cells, which are the main source of mucus in peripheral airways, although cholinergic nerves and sensory neuropeptides may be important in stimulating secretion. Epidermal growth factor may also be an important mediator for goblet cell and mucus gland hyperplasia in asthma. Neural effects There has recently been a revival of interest in neural mechanisms in asthma. Autonomic nervous control of the airways is complex, for in addition to classical cholinergic and adrenergic mechanisms, non-adrenergic non-cholinergic (NANC) nerves and several neuropeptides have been identified in the respiratory tract. Several studies have investigated the possibility that defects in autonomic control may contribute to airway hyperresponsiveness and asthma, and abnormalities of autonomic function, such as enhanced cholinergic and ct-adrenergic responses or reduced 6-adrenergic responses, have been proposed. Current thinking suggests that these abnormalities are likely to be secondary to the disease, rather than primary defects. It is possible that airway inflammation may interact with autonomic control by several mechanisms. Cholinergic nerves are the major bronchoconstrictor neural pathway in human airways and abnormalities of cholinergic control of the airways have long been suspected to be involved in the pathophysiology of asthma. Animal studies have demonstrated defective function in M2 muscarinic receptors on cholinergic nerve endings, resulting in increased acetycholine release and increased reflex bronchoconstriction. Inflammatory mediators may act on various pre-junctional receptors on airway nerves to modulate the release of neurotransmitters. Inflammatory products may also sensitise sensory nerve endings in the airway epithelium, so that the nerves become hyperalgesic. Hyperalgesia and pain (dolor) are cardinal signs of inflammation, and in the asthmatic airway may mediate cough and chest tightness, which are such characteristic symptoms of asthma. The precise mechanisms of hyperalgesia are not yet certain, but mediators such as prostaglandins, certain cytokines and neurotrophins may be important. Neurotrophins, which may be released form various cell types in the airways, may cause proliferation and sensitisation of airway sensory nerves [10]. Bronchodilator nerves, which are non-adrenergic, are prominent in human airways, and it has been suggested that these nerves may be defective in asthma. In animal airways vasoactive intestinal peptide (VIP) has been shown to be a neurotransmitter of these nerves but no abnormalities in VIP-immunoreactive nerves have been seen in asthma. In human airways NO appears to be the sole transmitter of bronchodilator nerves. Airway nerves may also release neurotransmitters which have inflammatory effects. Thus neuropeptides such as substance P (SP), neurokinin A and calcitonin-gene related peptide may be released from sensitised inflammatory nerves in the airways which increase

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and extend the ongoing inflammatory response. There is some evidence for an increase in SPimmunoreactive nerves in airways of patients with severe asthma, although this has not been confirmed in milder asthmatic patients. There may also be a reduction in the activity of enzymes, such as neutral endopeptidase, which degrade neuropeptides such as SP. There is also evidence for increased gene expression of the neurokinin receptors (NK,, NK 2 ) that mediates the effects of tachykinins. Thus chronic asthma may be associated with increased neurogenic inflammation, which may provide a mechanism for perpetuating the inflammatory response, even in the absence of initiating inflammatory stimuli. However, there is still no direct evidence for this in asthma and the negative effects of tachykinin antagonists in asthma argue against an important role, at least in mild asthma. Acute and chronic inflammation Asthma is characterised by acute inflammatory episodes, which may occur after upper respiratory tract virus infections or exposure to a large amount of inhaled allergen, resulting in bronchoconstriction, plasma exudation and oedema and mucus secretion. However, asthma is also a chronic inflammatory process, partly driven by exposure to low level environmental allergens, such as house dust mite and moulds, and this may result in structural changes in the airway walls (remodelling) that lead to progressive and largely irreversible narrowing of airways. This may result in permanent structural changes that may underlie the irreversible reduction in airflow that occurs in some patients with asthma. It is likely that genetic factors will influence the extent of remodelling that occurs in individual patients. 6. Transcription Factors The chronic inflammation of asthma is due to increased expression of multiple inflammatory proteins (cytokines, enzymes, receptors, adhesion molecules). In many cases these inflammatory proteins are induced by transcription factors, DNA binding factors that increase the transcription of selected target genes. Several transcription factors, including NFKB, AP-1 and signal transduction activated transcription factors (STATs), are activated by inflammatory stimuli and switch on panels of pro-inflammatory genes [11]. NF-KB may play a critical role in asthma is, as it can be activated by multiple stimuli that exacerbate asthmatic inflammation, including oxidants, proinflammatory cytokines, allergen and rhinovirus infection. There is evidence for increased activation of NF-KB in asthmatic airways, particularly in epithelial cells and macrophages. NF-KB regulates the expression of several key genes that are over-expressed in asthmatic airways, including proinflammatory cytokines (IL-lp, TNF-a, GM-CSF), chemokines (RANTES, MlP-la, eotaxin), adhesion molecules (ICAM-1, VCAM-1) and inflammatory enzymes (cyclo-oxygenase-2 and iNOS). The c-Fos component of AP-1 is also activated in asthmatic airways and often co-operates with NF-KB in switching on inflammatory genes. 7. Anti-inflammatory Mechanisms in Asthma Although most emphasis has been placed on inflammatory mechanisms, there may be important anti-inflammatory mechanisms that may be defective in asthma, resulting increased inflammatory responses in the airways [12]. Various cytokines have anti-inflammatory actions. IL-1 receptor antagonist (IL-lra) inhibits the binding of IL-1 to its receptors and therefore has a potential anti-inflammatory potential in asthma. It is reported to be effective in an animal model of asthma. IL-12 and interferon-y (IFN-y) enhance Thl cells and inhibit Th2 cells and there is some evidence that IL-12 expression may be impaired in asthma. IL-10, which was originally described as cytokine synthesis inhibitory factor, inhibits the expression of multiple inflammatory cytokines (TNF-a, IL-lp, GM-CSF, IL-5), chemokines (eotaxin), and inflammatory enzymes (iNOS, COX-2) and also inhibits antigen presentation. There is

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evidence that IL-10 secretion and gene transcript are defective in macrophages and monocytes form asthmatic patients; this may lead to enhancement of inflammatory effects in asthma and may be a determinant of asthma severity. References 1. Busse WW, Lemanske RF. Asthma. N.EngU.Med. 2001; 344: 350-62. 2. Barnes PJ. Therapeutic strategies for allergic diseases. Nature 1999; 402: B31-B38 3. Humbert M, Menz G, Ying S, et al. The immunopathology of extrinsic (atopic) and intrinsic (non-atopic) asthma: more similarities than differences. Immunol Today 1999; 20: 528-33. 4. Barnes PJ. Anti-IgE therapy in asthma: rationale and therapeutic potential. Int Arch Allergy Immunol 2000;123: 196-204. 5. Leckie MJ, ten Brincke A, Khan J, et al. Effects of an interleukin-5 blocking monoclonal antibody on eosinophils, airway hyperresponsiveness and the late asthmatic response. Lancet 2000; 356: 2144-8. 6. Holt PG, Sly PD. Prevention of adult asthma by early intervention during childhood: potential value of new generation immunomodulatory drugs. Thorax 2000; 55: 700-3. 7. Barnes PJ, Chung KF, Page CP. Inflammatory mediators of asthma: an update. Pharmacol Rev 1998; 50: 515-96. 8. Chung KF, Barnes PJ. Cytokines in asthma. Thorax 1999; 54: 825-57. 9. Kharitonov SA, Barnes PJ. Exhaled markers of pulmonary disease. Am J Respir Crit Care Med 2001; 163: 1693-772. 10. Carr MJ, Hunter DD, Undem BJ. Neurotrophins and asthma. Curr Opin Pulm A/ec/2001; 7: 1-7. 11. Barnes PJ, Adcock IM. Transcription factors and asthma. Eur Respir J 1998; 12: 221-34. 12. Barnes PJ. Endogenous inhibitory mechanisms in asthma. Am J Respir Crit Care Med 2000; 161: S176S181

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Transcriptional Regulation of Airway Inflammation Serpil C. ERZURUM Pulmonary and Critical Care Medicine Cancer Biology Lerner Research Institute Cleveland Clinic Foundation Cleveland Ohio 44195 U.S.A. Abstract. Airway inflammation occurs in response to numerous mediators/cytokines or infectious agents. Increased expression of inducible nitric oxide synthase (iNOS, or NOS2) occurs in association with the airway inflammation of asthma and/or viral infections. Signal transduction to NOS2 in the human airway epithelium occurs through various signaling pathways including signal transducers and activators of transcription 1 (STAT1) and double-stranded RNA dependent protein kinase (PKR). Airway inflammatory signal transduction pathways will be reviewed in the context of NOS2 gene activation.

1. Airway Inflammation Airway inflammation is a defining characteristic of asthma. In recognition of this, recent guidelines for asthma care advocate therapies targeted to decrease inflammation, such as inhaled corticosteroids [1]. Numerous mediators and cytokines contribute to the development and maintenance of inflammation in the asthmatic airway. Asthmatic inflammation has traditionally been associated with Th2 lymphocyte cytokines, such as IL-4. However, interferon gamma (IFNy)(a Thl cytokine) is increasingly implicated in the pathobiology leading to airway inflammation and hyperreactivity in asthma [2 - 5]. For example, animal models of ovalbumin sensitized mice develop airway hyperresponsiveness dependent upon IFNy [2]. Bronchoalveolar cell cultures derived from individuals with asthma spontaneously release increased amounts of IFNy. [3]. Further, adoptive transfer of Thl lymphocytes, which characteristically produce IFNy, increase airway inflammation [5]. Nitric oxides synthase 2 (NOS2) expression is critically dependent upon exposure of cells to IFNy, and may be one mechanism through which it contributes to airway inflammation. Viruses also lead to airway inflammation and are a major cause of asthma exacerbation. Nitric oxide synthase 2 (NOS2) is induced in airway epithelium by common respiratory viruses, including influenza, parainfluenza and respiratory syncytial virus. NOS2 induction occurs in the course of viral infection in part due to IFNy, but

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early in infection gene expression may be induced by the viral replicative intermediate double-stranded RNA (dsRNA) through the dsRNA-activated protein kinase (PKR) [6]. In support of this, NOS2 gene expression in human airway epithelial cells occurs in response to influenza A vims or synthetic dsRNA in vitro. Specifically, dsRNA leads to rapid activation of PKR, followed by activation of signaling components including nuclear factor kappa B (NF-DB) and interferon regulatory factor 1 (IRF-1). Moreover, examination of the transcriptional events leading to NOS2 in the airway are reviewed to reveal how viruses and cytokines lead to airway inflammation.

2. Viral mechanisms of signal transduction to airway inflammation Influenza virus infection causes significant morbidity and mortality in human populations worldwide with a broad spectrum of clinical responses ranging from asymptomatic infection, rhinitis to viral pneumonia [7]. Although factors dictating the severity of virus disease are complex, interaction between inherent viral properties and host cellular response ultimately determines disease outcome [7 - 9]. The first site of viral contact with the host, and main target of infection and inflammation is the airway mucosal epithelium. Epithelial cells at the airway mucosal surface have a variety of inflammatory and immune defense mechanisms to deal with virus, including expression of cytokines with chemoattractant and proinflammatory functions [10 - 13], e.g. NOS2 [12, 14]. Nitric oxide (NO) produced by NOS2 has potent antiviral activity against a number of viruses [15 - 19]. However, NO also contributes to inflammation and injury through formation of toxic reactive nitrogen intermediates [12, 14, 20]. In this context, development of pneumonia in a murine model of influenza infection has been linked to hostNOS2 expression [14, 21]. Viral mechanisms regulating NOS2 expression in human airway epithelial cells IFNy produced by lymphocytes in the airway mucosa, is likely involved in NOS2 induction later in the course of viral infection. However, NOS2 induction by virus early in infection is mediated by proteins responsive to the viral replicative intermediate, double-stranded RNA (dsRNA). Influenza viruses are enveloped viruses with a segmented, single-stranded RNA genome, which generate dsRNA during replication [7, 22]. Intracellular dsRNA formed during viral replication [22], binds to and activates a serine/threonine kinase, dsRNA-activated protein kinase (PKR), which has been implicated in signal transduction [23 - 25]. In this context, PKR is a component of the signaling pathway to NOS2 gene induction following viral infection of human airway epithelium. NOS2 gene expression in human airway epithelial cells occurs after influenza A virus or synthetic dsRNA exposure [6]. Importantly, dsRNA leads to activation of PKR, followed by activation of signal transduction proteins including NFDB and interferon regulatory factor 1 (IRF-1) in airway epithelial cells. A role for PKR in the signal transduction pathway of viral-induced NOS2 expression is conclusively demonstrated in experiments using cells genetically-deficient in PKR (PKR-/-) [6]. Impairment of NOS2 induction in response to LPS is also found in PKR-/-cells, confirming a central role for PKR in the general signaling pathway to NOS2.

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Signal transduction through PKR PKR is important for host antiviral mechanisms, as evidenced by impaired antiviral responses in mice with a homozygous targeted deletion in the PKR gene [24] First identified as a component of interferon-inducible cellular antiviral defenses, PKR exhibits two distinct kinase activities upon activation by dsRNA, autophosphorylation/activation and phosphorylation of substrates [26]. One antiviral effect mediated by PKR is the phosphorylation of eukaryotic initiation factor-2G, effectively restricting viral protein translation and subsequent replication [26]. In addition to effects on translation, PKR regulates transcriptional events by phosphorylation of proteins related to signal transduction pathways. For example, PKR is required for the activation of NF-DB in immortal cell lines in response to different stimuli [23]. NF-KB activation by dsRNA in human airway epithelial cells is most likelydue to PKR activation and phosphorylation of the inhibitor of NF-KB (1KB). Thus, PKR appears to mediate signal transduction in human airway epithelial cells in part through NF-KB. In addition, PKR may impact upon the signaling pathways through transcriptional and/or post-translational effects on the transcription factor interferon regulatory factor 1 (IRF-1) [27]. In fact, expression of IRF-1 protein in cells does not manifest functional DNA binding activity unless a phosphorylation signal is provided [28], potentially by PKR [29, 30]. In support of this concept, IRF-1 protein is induced and activated by dsRNA in human airway epithelial cells. PKR's role in signaling is essential for activation of NOS2, as assessed by experiments in murine embryo fibroblast cells (MEF) derived from mice with homozygous deletions for PKR. PKR contributed significantly to activation of signaling pathways, including NF-KB and IRF-1, which are important for pro-inflammatory gene expression such as NOS2 [27]. Interestingly, PKR is also essential for LPS-induction of NOS2 in murine cells, confirming a central role for PKR in microbial-induced signaling pathway to NOS2. On the other hand, continued NOS2 expression in PKR -/- cells exposed to a combination of IFNy and LPS or poly 1C, albeit at lower levels than in PKR+/+ cells, points out the possibility of inducible alternative signaling pathways to NOS2, which are independent of PKR [6]. Taken together, a model for regulation of NOS2 in the airway early in the course of viral infection is derived. Upon infection of human airway epithelial cells, the viral replicative intermediate dsRNA binds to and activates PKR, which leads to activation of NF-KB. dsRNA also induces de novo synthesis and activation of IRF-1 through PKR activation. IRF-1 in cooperation with other factor(s), such as NF-KB, leads to NOS2 gene induction. Soon, release of IFNy by activated lymphocytes in the virus-infected airway induces prolonged NOS2 gene expression in human airway epithelium. Increases in airway NOS2 expression early in viral infection may be antimicrobial and limit spread of virus. However, IFNy mediated high-level NOS2 expression that persists for days to weeks most likely contributes to airway inflammation and injury, and associated clinical respiratory symptoms.

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3. Signal transduction in the asthmatic airways NOS2 protein is present in healthy airway epithelial cells but is clearly increased in asthmatic airways in vivo. Although translational and post-translational mechanisms are important in the regulation of NO synthesis, NOS2 is substantially regulated at the level of transcription [32 - 34]. As we and others have previously shown, healthy human airway epithelium in vivo expresses the NOS2 gene continuously at abundant mRNA levels [35, 36]. NOS2 mRNA expression in asthmatic airway epithelium is higher than controls in vivo, but not increased in asthmatics receiving inhaled corticosteroid. Inhaled corticosteroids are the most effective therapies for reducing inflammation in asthma. While the use of inhaled corticosteroids as a first-line treatment in asthma has increased, little is known regarding the cellular and molecular mechanisms that contribute to the efficacy of inhaled corticosteroids in vivo. Several studies have shown that inhaled or intravenous corticosteroids reduce exhaled NO [37 - 39]. In situ analysis of the asthmatic airway suggested that NOS2 expression is reduced by corticosteroids [40]. In general, mechanisms by which corticosteroids regulate NOS2 gene expression in vivo are not known. In vitro, glucocorticoids inhibit NOS2 expression at multiple levels including inhibition of gene transcription, reduction of mRNA translation and increased degradation of NOS2 protein [41 - 43]. Increased NOS2 mRNA in asthma, which is downregulated by corticosteroid, supports an association between NOS2 expression and airway inflammation. NOS2 expression is lost in airway epithelial cells upon removal from the airway environment, which substantiates a critical link between airway conditions and/or factors in vivo and NOS2 expression. Induction of NOS2 expression in vitro varies in different cell types, but typically is increased by cytokines [32 - 34, 44]. IFNy is crucial for induction of NOS2 expression in airway epithelial cells in vitro [44]. IFNy signaling to gene expression begins with a specific receptor interaction and oligomerization of receptor chains, causing a tyrosine kinase cascade. STAT1 phosphorylation, dimerization and translocation to the nucleus, is followed by binding to regulatory DNA elements to activate transcription of interferon-stimulated-genes [45, 46]. IFNy leads to STAT1 activation in primary human airway epithelial cells in culture [44, 47]. Recently. STAT1 activation has been demonstrated in the asthmatic airway by nuclear localization of STAT1 in airway epithelial cells, and demonstration of phosphorylation of STAT1 by western analyses of epithelial cell lysates [48]. The STAT1 activation correlated with induction of IFNy/STATl-stimulated-genes, including IRF-1 which has been identified as essential for NOS2 activation in murine macrophages [49]. STAT1 activation quantitated by electrophoretic mobility shift assays is present in controls but increased in asthmatic airway epithelial cell lysates. In contrast to increased STAT1 activation in the asthmatic airway, other cell signaling proteins are not increasingly activated. STAT6 and NFkB activation are not increased in mild asthmatic airway as compared to healthy controls. Previous study has shown that STAT3 and activator protein (AP)-l activation is also not increased in asthma [48]. STAT1 tyrosine phosphorylation and translocation to the nucleus occurs in response to many growth factors and cytokines including IFNy, IL10, IFNa/p, epidermal growth factor, platelet derived growth factor, granulocytemacrophage colony stimulating factor, IL-6, IL-11, leukemia inhibitory factor, ciliary

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neurotrophic factor, Oncostatin M, growth hormone, prolactin, and colony stimulating factor-1 [45, 46]. However, IFNy is higher in asthmatic epithelial lining fluid than control following a segmental bronchoprovocation with antigen [50]. The large number of IFNy/STATl-stimulated-genes, including IRF-1, ICAM-1 and NOS2, are likely involved in the airway inflammatory events of asthma. Collectively, these data provide strong support for STAT1 activation mediating NOS2 gene expression in human airway epithelial cells in vivo. NFkB activation and binding to kB DNA elements in the 5' flanking region of the NOS2 gene plays a role in the cytokine induction of NOS2 in the human lung epithelial cell line A549 in vitro [32 - 34]. However, studies of NFkB activation in asthma are conflicting, perhaps in part due to the types of sample analyzed [48, 51]. Expectorated sputum from asthmatics or pooled biopsies of asthmatic airways have shown increased NFkB activation in comparison to controls [51], while no increase in activation is noted by nuclear localization of NFkB in biopsies and electrophoretic mobility shift assays of whole cell extracts from bronchial brushings of asthmatic airway epithelium [48, 50]. Nevertheless, low level NFkB activation is present in control and asthmatic epithelium, and may contribute to the tonic expression of NOS2 in the airway. In conclusion, multiple mechanisms function coordinately to support high level NO synthesis in the inflamed asthmatic airway. Human airway epithelium has abundant expression of NOS2 due to continuous transcriptional activation of the gene in vivo. Increased NOS2 gene expression in asthmatic airways is related to increased STAT1 activation caused by increased cytokines e.g., IFNy.

4. Signal transduction mechanisms of corticosteroid effect The broad anti-inflammatory and immunomodulatory effects of corticosteroids are effective in treatment of most asthma. The anti-inflammatory mechanisms of corticosteroids have been detailed at the molecular level. At the cellular level, glucocorticoids (GC) freely penetrate the cell plasma membrane and bind to a specific intracellular receptor, the glucocorticoid receptor (OCR). This binding dissociates heatshock proteins and creates an active GC-GCR complex. The GC-GCR complex translocates to the nucleus and binds to specific GCR-responsive elements (GRE) on genomic DNA that induce gene expression, e.g. beta-adrenergic receptors [52]. The GCGCR complex may also suppress gene expression by interfering with the interaction of transcription factor proteins AP-1 and NF-KB with promoter regions of pro-inflammatory cytokines [53]. While AP-1 directly interacts with the glucocorticoid receptor leading to interference of AP-1 -dependent transcription, glucocorticoids prevent NFKB activation by inducing the inhibitor of NFKB (1KB) [53]. Through these mechanisms, glucocorticoids inhibit the production of a wide range of cytokines involved in asthma, including IL-1, IL-3, IL-4, IL-5, IL-6, IL-13, granulocyte monocyte colony stimulating factor (GM-CSF), and tumor necrosis factor-alpha (TNF-a). IL-3, IL-5, and GM-CSF differentiate, activate, and proliferate eosinophils. IL-4 and IL-13 cause the antibody isotype switch to IgE production. IL-1 and TNF-a upregulate adhesion molecules. IL-4, IL-5, and IL-6 promote IgE production and T-helper lymphocyte response [54].

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In addition to inhibiting cytokine production, glucocorticoids also inhibit production of inflammatory leukotrienes and eicosanoids through effects on phospholipase A2 [54]. In contrast, genes for anti-inflammatory or bronchodilatory products are increased by corticocorticosteroids such as beta receptors and lipocortin. Lipocortin, a protein that inhibits phospholipase AI, further dampens inflammation. Neutral endopeptidase, which is expressed on bronchial epithelial cells and enhances degradation of proinflammatory tachykinins, is also increased by corticosteroids [52]. 5. Conclusion Multiple signal transduction pathways are specifically and coordinately activated in the airway in response to inflammation or infection. Identification of the transcription factors activated, such as NF-kB or STAT1, reveal some of the underlying pathogenic mechanisms of inflammation, which may be targets for anti-inflammatory therapy in the future. References [1] Guidelines for the diagnosis and the management of asthma, Expert panel report II. Bethesda, Md.: National Asthma Education and Prevention Program, April 1997. (NIH publication no. 97-4051.) [2] E.M. Hessel, A.J.M. Van Oosterhout, I.V. Ark, B.V. Esch, G. Hofinan, et al. Development of airway hyperresponsiveness is dependent on interferon-y and independent of eosinophil infiltration. Am. J. Respir. Cell Mol. Biol. 16 (1997) 325. [3] M. Cembrzynska-Nowak, E. Szklarz, A.D. Inglot, and J.A. Teodorczyk-Injeyan. Elevated release of tumor necrosis factor-alpha and interferon-gamma by bronchoalveolar leukocytes from patients with bronchial asthma. Am. Rev. Respir. Dis. 147 (1993) 291. [4] X.M. Li, R.K. Chopra, T.Y. Chou, B.H. Schofield, M. Wills-Karp, et al. Mucosal IFN-y gene transfer inhibits pulmonary allergic responses in mice. J. Immunol. 157 (1996) 3216. [5] G. Hansen, G. Berry, R.H. DeKruyff, and D.T. Umetsu. Allergen-specific Thl cells fail to counterbalance Th2 cell-induced airway hyperreactivity but cause severe airway inflammation. J. Clin Invest. 103(1998) 175. [6] K. Uetani, S.D. Der, M. Zamanian-Daryoush, C. de la Motte, B. Lieberman, B.R.G. Williams, and S.C. Erzurum. Central Role of double-stranded RNA-Activated Protein Kinase in Microbial Induction ofNitric Oxide Synthase. J Immunol 165 (2000) 988. [7] R.B. Murphy, and R.G. Webster. 1996. Orthomyxoviruses. In Fields Virology, Vol. 1. B. N. Fields, D. M. Knipe, P. M. Howley, R. M. Chanock, T. P. Monath, J. L. Melnick, B. Roizman, and S. E. Straus, eds. Lippincott-Raven Publishers, Philadelphia, PA, p. 1397. [8] A. Garcia-Sastre, R.K. Durbin, H. Zheng, P. Palese, R. Gertner, D. E. Levy, and J. E. Durbin.. The role of interferon in influenza virus tissue tropism. J. Virol. 72 (1998) 8550. [9] M. F. Kagnoff, and L. Eckmann.. Epithelial cells as sensors for microbial infection. J. Clin. Invest. 100 (1997)6. [10]S. Matsukura, F. Kokubu, H. Noda, H. Tokunaga, and M. Adachi. Expression of IL-6, IL-8, and RANTES on human bronchial epithelial cells, NCI-H292, induced by influenza virus A. J Allergy. Clin. Immunol. 98 (1996) 1080. [11]Z. Zhu, W. Tang, A. Ray, Y. Wu, O. Einarsson, M. L. Landry, J. Gwaltney, Jr., and J. A. Elias. Rhinovirus stimulation of interleukin-6 in vivo and in vitro. Evidence for nuclear factor kappa Bdependent transcriptional activation. J. Clin. Invest. 97 (1996) 421. [12]K. Tanaka, H. Nakazawa, K. Okada, K. Umezawa, N. Fukuyama, and Y. Koga. Nitric oxide mediates murine cytomegalovirus-associated pneumonitis in lungs that are free of the virus. J. Clin. Invest. 100 (1997) 1822.

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Molecular Mechanisms of Steroid Actions Ian M. ADCOCK and Kazuhiro ITO Thoracic Medicine, National Heart & Lung Institute, Imperial College of Science, Technology and Medicine, Dovehouse Street, London SW3 6LY Abstract. Steroids are the most effective treatment for chronic inflammatory diseases such as asthma. They act through binding to a cytosolic receptor (GR) which undergoes nuclear trans location. Within the nucleus, GR represses inflammatory gene induction by inhibiting the action of pro-inflammatory DMA binding proteins (transcription factors) such as AP-1 and NF-tcB. This occurs through alterations in the chromatin structure associated with changes in the histone acetylation status. This effect can be demonstrated both at the whole cell level but more importantly at specific promoters and is associated with specific gene induction. Under conditions of oxidative and nitrosyl stress GR function is impaired possibly as a result of nitrosylation and/or phosphorylation of GR-associated proteins.

1. Introduction Inflammation is a central feature of many lung diseases, including asthma and chronic obstructive pulmonary disease (COPD). The specific characteristics of the inflammatory response and the site of inflammation differ between these diseases, but all involve the recruitment and activation of inflammatory cells and changes in the structural cells of the lung. These diseases are characterised by an increased expression of many proteins involved in the complex inflammatory cascade. These inflammatory proteins include cytokines, chemokines, growth factors, enzymes, receptors and adhesion molecules. The increased expression of most of these proteins is the result of enhanced gene transcription since many of the genes are not expressed in normal cells under resting conditions but are induced in a cell-specific manner.

2. Over-expression of mediators Using in-situ hybridisation and immunohistochemistry increased gene expression of cytokines, chemokines, receptors and adhesion molecules have been reported [Ijand references therein). Included amongst these induced mediators are the Th2 cytokines interleukin (IL-)5 and GM-CSF [1]. IL-5 and GM-CSF are important cytokines involved in eosinophil survival with GM-CSF appearing to be the most important contributor, and is predominantly expressed in airway epithelium and macrophages [2]. IL-5 and GM-CSF can prime eosinophils such as to increase the release of granule-associated proteins such as eosinophil cationic protein (ECP) from stimulated eosinophils [2]. GM-CSF can also enhance the production of leukotrienes from eosinophils [2].

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3. Transcription Factors Changes in gene transcription are regulated by transcription factors. Transcription factors are proteins that bind to DNA-regulatory sequences (enhancers and silencers), usually localised in the 5' upstream region of target genes, to increase (or sometimes decrease) the rate of gene transcription. This may result in increased or decreased protein synthesis and subsequent altered cellular function. Many transcription factors are common to several cell types (ubiquitous) and may play a general role in the regulation of inflammatory genes, whereas others are cell specific and may determine the phenotypic characteristics of a cell [3]. Transcription factor activation is complex and may involve multiple intracellular signal transduction pathways, including kinases (such as tyrosine kinases of the mitogen activated protein kinases (MAPKs) and Janus kinases (JAKs) families) and the protein kinase C family (PKCs), stimulated by cell-surface receptors. Activation of MAPK pathways by inflammatory stimuli leads to activation of a number of ubiquitous transcription factors such C/EBPP, Elk-1, c-Fos, c-Jun, c-Myc, NF-KB and STATs. Transcription factors may therefore convert transient environmental signals at the cell surface into long-term changes in gene transcription, thus acting as "nuclear messengers". 3.1 NF-KB Activation of NF-KB leads to the co-ordinated induction of multiple genes that are expressed in inflammatory and immune responses. Many of these genes are induced in inflammatory and structural cells and play an important role in the inflammatory process [4]. While NF-KB is not the only transcription factor involved in regulation of the expression of these genes it often appears to have a decisive regulatory role [4]. NF-KB often functions in co-operation with other transcription factors, such as AP-1 and C/EBPp, which are also involved in regulation of inflammatory and immune genes. NF-KB is activated by many of the stimuli that exacerbate asthmatic inflammation (e.g rhinovirus infection, allergen exposure proinflammatory cytokines, oxidants)[5]. There is also evidence for activation of NF-KB in the bronchial epithelial cells of patients with asthma [6]. The role of NF-KB should be seen as an amplifying and perpetuating mechanism that will exaggerate the disease-specific inflammatory process through the co-ordinated activation of multiple inflammatory genes. One of the most important concepts to have emerged is the demonstration that maximal transcription factor activation is associated with co-incident activation of several intracellular pathways and other transcription factors. This may explain how transcription factors that are ubiquitous may regulate particular genes in certain types of cells. The complexity of the activation pathways and their ability to engage in cross-talk enables cells to overcome inhibition of one pathway and retain a capacity to activate specific transcription factors. 3.2 Histone acetylation Binding of transcription factors to their specific binding motifs in the promoter region may alter transcription by interacting directly with components of the basal transcription apparatus or via co-factors that link the specific transcription factor to the basal transcription apparatus [7]. Large proteins that bind to the basal transcription apparatus may bind many transcription factors and thus act as integrators of gene transcription. These co-activator

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molecules include CREB-binding protein (CBP), and the related p300, thus allowing complex interactions between different signalling pathways. DNA is wound around histone proteins to form nucleosomes and the chromatin fibre in chromosomes [7]. It has long been recognised at a microscopic level that chromatin may become dense or opaque due to the winding or unwinding of DNA around the histone core [8]. CBP and p300 have histone acetylase activity (HAT) which is activated by the binding of transcription factors, such as AP-1, NF-icB and STATs. The central role of CBP and associated proteins in controlling the inflammatory response may explain synergy between transcription factors such as NFKB and AP-1. Numerous pro-inflammatory transcription factors interact with, and activate, CBP-associated HAT activity including CREB, AP-1, NF-KB, SV40 promoter-1 (Spl), E26 (Ets), nuclear factor of activated T-cells (NF-AT) and STATs [9] (Figure 1).

Figure 1. The co-activator molecule CBP integrates transcription factor activity and regulates histone acetylation. Stimulation of intracellular signalling pathways (JNKs, JAKs and IKK) activates transcription factors (CREB, c-Jun, STATs and NF-icB). These can bind to CBP, singly or in combinations, to enhance CBPassociated histone acetylase (HAT) activity leading to histone acetylation (Ac") within the inflammatory gene promoter, chromatin modification, loosening of the nucleosome structure and recruitment of RNA polymerase II (RNA pol II) and enhanced inflammatory gene transcription.

Acetylation of histone residues results in unwinding of DNA coiled around the histone core, thus opening up the chromatin structure, which allows other transcription factors, accesory molecules (e.g. TAFs) and RNA polymerase II to bind more readily, thereby increasing transcription [7]. Acetylation is an active process that is held in balance by the action of histone deacetylases (HDACs). Deacetylation of histone, increases the winding of DNA round histone residues, resulting in dense chromatin structure and reduced access of transcription factors to their binding sites, thereby leading to repressed transcription of genes [7]. We have recently shown that IL-lp stimulates histone acetyltransferase activity in a time- and concentration-dependent manner [10]. This acetylation occurs predominantly on histone H4 rather than other histones H2A, H2B or H3. Histone H4 has four potential aceylation sites, lysine residues 5, 8, 12 and 16. IL-lp-activated NF-KB induces acetylation on K8 and K12 only [10]. These changes do not occur across the genome but are targeted to the promoter sites of inflammatory genes transcription factors such as NF-KB. Thus we have recently shown that IL-lp-activated NF-KB is directed to the GM-CSF promoter where histone acetylation on lysines 8 and 12, but not lysine 5, occur [10].

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4. Repression of Inflammatory Gene expression by Glucocorticoids Glucocorticoids are the most effective therapy in the long-term control of asthma and appear to reduce inflammation in asthmatic airways largely by inhibiting abnormal gene expression [9]. Although it is not yet possible to be certain of the most critical aspects of glucocorticoid action in suppressing inflammation, it is likely that their inhibitory effects on cytokine synthesis are particularly important. Glucocorticoids inhibit the transcription of several cytokines and chemokines that are relevant in inflammatory lung diseases, including IL-lp, TNFa, GM-CSF, IL-4, IL-5, IL-8 and eotaxin [9] Glucocorticoids may not only block the synthesis of cytokines, but may also block their effects by inhibiting the synthesis of cytokine receptors, such as the IL-2 receptor [9] (Table 1). Glucocorticoids markedly reduce the survival of eosinophils and T-lymphocytes. Eosinophil survival is dependent upon the cytokines GM-CSF and IL-5 whose actions are blocked by glucocorticoids leading to programmed cell death or apoptosis [9]. Glucocorticoids also promote T-cell apoptosis although the mechanism of action is unclear it is thought to involve gene induction [9], Increased gene transcription Lipocortin-1 (annexin-1) p2-adrenoceptor Secretory Leucocyte Inhibitory Protein IL-RII Decreased gene transcription Cytokines IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8. IL-11, IL-13, TNfiz, GM-CSF Chemokines RANTES, Eotaxin, NHP1a.MCP-1, MCP-3 Enzymes iNOS, COX-2, cPLA, Adhesion Molecules ICAM-1.VCAM-1 Receptors IL-2R, NK,R

Table 1. Target genes for glucocorticoid action.

4.1 The glucocorticoid receptor Glucocorticoids exert their effects by binding to a single cytoplasmic receptor (GR) [11]. The inactive GR is bound to a protein complex that includes two subunits of the heat shock protein hsp90 which thus act as molecular chaperones preventing the nuclear localisation of unoccupied GR [11]. Once the ligand binds to GR, hsp90 dissociates allowing the nuclear localisation of the activated GR-steroid complex and its binding to specific DNA sequences (GREs, GGTACAnnnTGTTCT) or interaction with co-activator complexes [11]. 4.2 Induction of gene transcription Binding of the GR homodimer to GREs changes the rate of transcription, resulting in either induction of steroid-responsive genes. An increased number of GREs and proximity to the TATA box increases the inducibility of a gene [11]. Other transcription factors binding in the vicinity of GRE may also have a powerful influence on steroid inducibility and the relative abundance of different transcription factors may contribute to the responsiveness of a particular cell type. GR-DNA interactions changes DNAsel sensitivity, indicating that there may be a local change in DNA or chromatin configuration, which may expose previously masked areas, resulting in increased binding of other transcription factors and the formation of a more stable transcription initiation complex [11]. Recent data has

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suggested that GR interactions with DNA are very dynamic such that once a GR molecule binds to DNA, it quickly dissociates and allows another to replace it. Thus although the DNA footprint will be present it is a result of many distinct GR proteins binding to the specific ORE [12]. Glucocorticoids also increase the synthesis of anti-inflammatory proteins, including lipocortin-1, serum leukoprotease inhibitor, Clara Cell protein 10 (CC-10) and IL-1 receptor antagonist (IL-lra) and these effects are presumably mediated via GREs in the promoter regions of these genes [9]. Glucocorticosteroids have also been reported to increase the expression of iKB-a in lymphocytes and thus to inhibit NF-KB, but this has not been seen in other cell types [9]. GRs, as with other transcription factors increase gene transcription through an action on chromatin remodelling and recruitment of RNA polymerase II to the site of local DNA unwinding as described above for pro-inflammatory transcription factors. GR interacts with CBP and other coactivator proteins, including steroid receptor coactivator-1 (SRC-1), transcription factor intermediary factor-2 (TIF2), p300/CBP co-integrator-associated protein (p/CIP) or glucocorticoid receptor interacting protein-1 (GRIP-1) which enhance local HAT activity [7]. High concentrations of dexamethasone (>10~8M) in A549 cells results in binding of activated GR to CBP and/or associated co-activators resulting in histone acetylation on ly sines 5 and 16 of histone H4 and increased gene transcription [10]. 5. Cross-talk between GR and other Transcription Factors In spite of the ability of glucocorticosteroids to induce gene transcription, the major antiinflammatory effects of glucocorticosteroids are through repression of inflammatory and immune genes. The inhibitory effect of glucocorticosteroids appears to be due largely to a protein-protein interaction between activated GR and transcription factors, such as AP-1 and NF-KB [13]. Direct protein-protein interactions have been demonstrated between GR and AP-1 and between GR and the p65 component of NF-KB [13],

Figure 2. Glucocorticoid receptor inhibition of NF-icB-induced histone acetylation. NF-KB (p65/p50 dimer) activates histone acetylation (HAT activity) at the NF-icB responsive site in inflammatory gene promoters. Activated glucocorticoid receptors (GR) inhibit NF-icB-activated HAT activity by a combination of direct inhibition of HAT activity and by recruitment of histone deacerylase (HDAC2) to the NFicB/HAT complex.

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The interplay between pro-inflammatory transcription factors and GR may reflect differing effects on histone acetylation/deacetylation. This may occur through one of several mechanisms that are probably not exclusive. The repressive action of glucocorticosteroids may be due to competition between GR and the binding sites on CBP for other transcription factors, including AP-1, NF-KB and STATs. Alternatively, activated GR may bind to one of several transcription co-repressor molecules, such as HDACs, RIP 140 NCoRl and GRIP1, which associate with proteins that have differing histone deacetylase activity [14]. Thus, IL-lp and TNFa can cause histone acetylation on lysines 8 and 12 of histone H4 and low concentrations of dexamethasone (>10~9 M) can represses this IL-lp-stimulated histone acetylation. This occurs partly by a direct inhibition of NF-icBassociated HAT activity and partly by active recruitment of HDAC proteins to the NF-icBactivated transcriptional complex [10]. In addition, high concentrations of dexamethasone can induce HDAC expression over longer time periods [10]. Overall, this results in the deacetylation of histones at the site of inflammatory gene transcription, increased tightening of DNA round histone residues and reduced access of transcription factors such as AP-1 and NF-KB to their binding sites and of RNA polymerase II to the activation complex. This results in repression of inflammatory genes [10] (Figure 2). Other models propose that GR interferes with p65 association with the TATA box environment thus inhibiting p65 actions [15] Recent data has suggested another mechanism for GR action. Serine phosphorylation of RNA polymerase II induced by NF-icB activity at the IL-8 promoter is reduced by GR without affecting the assembly of the pre-initiation complex. This suggests a downstream action for GR/p65 interactions [16]. 6. Failure to respond to glucocorticoids A small proportion of asthmatic patients are glucocorticoid-resistant and fail to respond to even high doses of oral steroids [17]. This impaired response appears to be specific to monocytes and T-lymphocytes. This resistance is seen at the site of inflammation where cytokines are produced - i.e. in the airways of asthmatic patients, but not at non-inflamed sites. GR function may be regulated by several factors acting on different components of the GR pathway including nuclear translocation and interaction with, or recruitment of. transcription complexes. Steroid-insensitive asthmatic subjects have a reduced number of activated GR (GRE binding) within the nucleus of mononuclear cells compared to controls after glucocorticoid treatment in vitro [18]. This suggests a reduction in GR nuclear translocation or modification of the GR DNA binding complex occurs. In a majority of asthmatic subjects there is a direct correlation between the GR nuclear translocation and induction of histone acetylation. In most severe steroid-dependent and -resistant asthmatics there is reduced nuclear translocation implying a specific defect in GR dissociation from the hsp90 chaperonine complex or passage through the nuclear membrane. As GR does not appear to redistribute into the nuclear membrane dissociation from the hsp90 complex is the more likely explanation. A similar effect has been reported in COS? and CHO cells by Okamoto and colleagues [19]. Here, oxidative stress (lOOuM HiO2) reduced GR nuclear transport. Nitrosyl stress induced by the NO donor S-nitroso-DL-penicillamine has also been shown to prevent GR dissociation from the hsp90 complex and a reduction in ligand binding [20]. Other potential causes of reduced GR function involve nuclear events. Since HDACs are important in GR function we have investigated HDAC expression and activity in the presence of oxidative stress. HDAC activity is decreased in BAL macrophages and biopsies of smokers and patients with COPD and this correlates with increased inflammatory gene

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expression and reduced steroid-responsiveness [21]. This effect can also be mimicked by pre-treatment of U937 cells with I^hOi (lOOuM) and parallels the effects seen with the HDAC inhibitor trichostatin A [21]. This suggests that oxidative stress by repression of HDAC activity can modulate GR function in BAL macrophages and U937 cells. In order to examine the mechanism for this effect we investigated the action of H2O2 and SIN-1, an NO donor, on HDAC2 phosphorylation and nitration status and activity. H2O2 did not affect IL-1B stimulated NF-KB nuclear translocation but enhanced the ability of DNA bound NF-KB to cause histone acetylation. This suggests either activation of a specific HAT has occurred or repression of an HDAC activity. We have preliminary evidence to suggest that HiO2 induced tyrosine nitration of HDAC2 and that this is associated with a loss of HDAC2 enzymic activity [22]. Therefore, tyrosine nitration can modulate HDAC2 activity and thereby enhance inflammatory gene transcription. Furthermore, this will result in reduced steroid sensitivity as HDAC2 recruited to the NF-KB activation complex will also have reduced activity.

Figure 3. Pro-inflammatory cytokine production is induced by inflammatory stimuli following activation of kinase cascades. These same cascades act in a negative feedback manner to switch off cytokine mRNA production after an initial burst of production. This is due, at least in part, to phosphorylation of tyrosine residues in histone deacetylases (HDAC) increasing their activity and thus inducing a silenced chromatin structure. Oxidative stress overcomes this feedback activity of HDAC activity by inducing nitration of distinct tyrosine residues and repressing HDAC activity. Similar effects may occur on GR itself to modify its actions in response to oxidative stress.

In summary, histone acetylation is important in the control of inflammatory gene transcription and recruitment of HDAC2 to the activated NF-KB complex by GR mediates at least 50% of dexamethasone-induced repression of these genes. Furthermore, phosphorylation and nitration can modify GR function either directly by targeting GR or indirectly by targeting GR-associated proteins such as HDACs. Whether these effects occur within the cytoplasm or the nucleus the net effect is a reduction in GR function.

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Acknowledgements This work was funded by the British Lung Foundation, The Clinical Research Committee (Brompton Hospital) and GlaxoSmithKline (UK). References [1] Barnes, P.J., Chung, K.F., & Page, C.P. (1998) Inflammatory mediators of asthma: an update. Pharmacot Rev, 50, 515-596. [2] Giembycz, M.A. & Lindsay, M.A. (1999) Pharmacology of the eosinophil. Pharmacol. Rev., 51,213-340. [3] Barnes, P.J. & Adcock, I.M. (1998) Transcription factors and asthma. Ear Respir J, 12,221-234. [4] Baldwin, A.S., Jr. (1996) The NF-kappa B and I kappa B proteins: new discoveries and insights. Annu Rev. Immunol., 14,649-683. [5] Barnes, P.J. & Karin, M. (1997) Nuclear factor-kappaB: a pivotal transcription factor in chronic inflammatory diseases. N Engl J Med, 336, 1066-1071. [6] Hart, L.A., Krishnan, V.L., Adcock, I.M., Barnes, P.J., & Chung, K.F. (1998) Activation and localization of transcription factor, nuclear factor-kappaB, in asthma. Am J Respir Crit Care Med, 158, 1585-1592 [7] Imhof, A. & Wolffe, A.P. (1998) Transcription: gene control by targeted historic acetylation. Curr Biol, 8, R422-R424. [8] Allfrey, V.G., Faulkner, R., & Mirsky, A.E. (1964) Acetylation and methylation of histones and their possible role in the regulation of RNA synthesis. Proc. Natl. Acad. Sci. U. S. A., SI, 786-794. [9] Adcock, I.M. & Ito, K. (2000) Molecular mechanisms of corticosteroid actions. Monaldi Arch Chest Dis, 55, 256-266. [10]Ito, K., Barnes, P.J., & Adcock, I.M. (2000) Glucocorticoid receptor recruitment of histone deacetylase 2 inhibits interleukin-1 beta-induced histone H4 acetylation on lysines 8 and 12. Mol Cell Biol, 20, 68916903. [1 IJBeato, M., Truss, M., & Chavez, S. (1996) Control of transcription by steroid hormones. Ann. N. Y. Acad. Sci. ,784,-1996. [12]McNally, J.G., Muller, W.G., Walker, D., Wolford, R., & Hager, G.L. (2000) The glucocorticoid receptor: rapid exchange with regulatory sites in living cells. Science, 287, 1262-1265. [13] Karin, M. (1998) New twists in gene regulation by glucocorticoid receptor: is DNA binding dispensable? Cell, 93,487-490. [14] Ding, X.F., Anderson, C.M., Ma, H., Hong, H., Uht, R.M., Kushner, P.J., & Stallcup, M.R. (1998) Nuclear receptor-binding sites of coactivators glucocorticoid receptor interacting protein 1 (GRIP1) and steroid receptor coactivator 1 (SRC-1): multiple motifs with different binding specificities. Mol Endocrinoi, 12,302-313. [15]Vanden Berghe, W., Vermeulen, L., De Wilde, G., De Bosscher, K., Boone, E., & Haegeman, G. (20"00) Signal transduction by tumor necrosis factor and gene regulation of the inflammatory cytokine interleukin-6. Biochem. Pharmacol., 60, 1185-1195. [16]Nissen, R.M. & Yamamoto, K.R. (2000) The glucocorticoid receptor inhibits NFkappaB by interfering with serine-2 phosphorylation of the RNA polymerase II carboxy-terminal domain. Genes Dev. 14, 2314-2329. [17] Leung, D.Y., de Castro, M., Szefler, S.J., & Chrousos, G.P. (1998) Mechanisms of glucocorticoidresistant asthma. Ann. N. Y. Acad. Sci., 840, 735-746. [18]Adcock, I.M. (1996) Steroid resistance in asthma. Molecular mechanisms. Am J Respir Crit Care Med. 154,S58-S61. [19]Okamoto, K., Tanaka, H., Ogawa, H., Makino, Y., Eguchi, H., Hayashi, S., Yoshikawa, N., Poellinger, L., Umesono, K., & Makino, I. (1999) Redox-dependent regulation of nuclear import of the glucocorticoid receptor. J. Biol. Chem.,274, 10363-10371. [20]Galigniana, M.D., Piwien-Pilipuk, G., & Assreuy, J. (1999) Inhibition of glucocorticoid receptor binding by nitric oxide. Mol Pharmacol, 55, 317-323. [21] Ito, K., Lim, S., Caramon, G., Chung, K.F., Barnes, P.J., & Adcock, I.M. (2001) Cigarette smoking reduces histone deacetylase 2 expression, enhances cytokine expression, and inhibits glucocorticoid actions in alveolar macrophages. FASEBJ, 15, 1110-1112. [22] Ito, K., Hanazawa, T., Kharitonov, S.A., Tomita, K., Barnes, P.J., & Adcock, I.M. Tyrosine nitration and oxidative stress inhibit histone deactylase 2 activity and function. American Journal of Respiratory and Critical Care Medicine 163, A575. 2001.

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Nitric Oxide Reactions in the Asthmatic Airway Raed A. DWEIK Department of Pulmonary and Critical Care Medicine Cleveland Clinic Foundation 9500 Euclid Avenue Cleveland Ohio 44195 U.S.A. Abstract. The dynamics of nitric oxide metabolism during an asthmatic response reveal multiple and sequential reactions, and suggest a multifunctional role for NO in the airway. In comparison to healthy controls, mild well-controlled atopic asthmatics tend to have increased NO, NO3", and nitrotyrosine but undetectable SNO in the lower airways. Within 10 min of antigen-induced asthmatic response, NO3" increases markedly in all asthmatics, while NO2" or SNO do not change, and NO tends to decrease. By 48 h after antigen-induced asthmatic response, NO, SNO and NO3" are strikingly increased. Despite notable changes in asthmatic airways, healthy control individuals have no changes in levels of NO or NO reaction products. Thus, these changes are distinct to atopic asthmatic individuals. Careful evaluation of the temporal sequence of NO reactions in the lower airway of asthmatics reveals that while NO may have some harmful effects, it also serves a protective role in the asthmatic response.

1. Nitric Oxide (NO) in Asthma The exhaled air of asthmatic individuals contains higher levels of NO than found in exhalate of healthy non-smoking individuals (1-7). The role of NO in asthma is unclear but studies suggest that NO relaxes bronchial smooth muscle leading to bronchodilatation, inhibits pro-inflammatory signaling events (8, 9) or conversely contributes to airway inflammation and injury through formation of toxic reactive nitrogen species (RNS) (10). Ultimately, the functional role of NO, as any molecule, will depend on both its concentration and association with other biomolecules and proteins (11). In this context, NO is a highly reactive molecule and exhaled NO likely represents only a fraction of the total NO in the lung. NO reacts with oxygen or reactive oxygen species (ROS) to form oxidation products, such as NO2", NO3~, and RNS (8, 10-13). NO or RNS may lead to nitration of tyrosine residues in proteins, or nitrosylation of biologic constituents to form S-nitrosothiols (SNO); 100% of NO reaction products are partitioned in the liquid phase of the lung (8, 10, 11, 13). Studying the temporal sequence of these

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downstream reaction products within airways during an asthmatic attack has greatly improved our understanding of the role(s) played by NO in asthma. 2. Antigen Challenge in the Study of Asthma Airway antigen (Ag) challenge has been used in atopic individuals as an experimental model to study mechanisms/mediators that lead to asthmatic responses and airway inflammation (14-19). Exposure of asthmatic individuals to appropriate Ag results in both an immediate asthmatic response occurring within minutes and a similar but prolonged late response after many hours. The immediate response has been associated with release of bronchoconstrictor mediators and ROS, and the late response with thickening of the airway mucosa by edema and inflammatory cell influx (14-19).

3. NO and NO Metabolite Levels Before and After Antigen Challenge NO in exhaled and intrapulmonary gases Intrapulmonary NO levels in the gas phase achieve a steady-state plateau which can be quantitated. Baseline intrapulmonary (plateau) NO in asthmatic airways are higher than controls. NO at 10 min after Ag challenge are similar to baseline levels, with only a small tendency to decrease NO in asthmatic airways. In contrast, intrapulmonary NO at 48 h after Ag challenge increases 2-fold in asthma but not in controls (Table). Exhaled NO at the mouth underestimates intrapulmonary NO in asthma but it clearly reflects the changes in intrapulmonary NO.

Table: Nitric oxide reaction products in the lower airway in asthma before and after antigen challenge. (t increased, «-> unchanged, I decreased) Compared to Compared to asthma baseline controls (before challenge) Baseline

10 minutes

48 hrs

NO

t

i

ttt

NO2"

<->

«•

<^

NO3"

t

t

tt

SNO

44

«+

tt

R.A. Dweik/Nitric Oxide Reactions in the Asthmatic Airway

NO2, NOi, and SNO in Bronchoalveolar Lavage (BAL) Fluid Total NO reaction products in BAL fluid tend to be higher in asthma at baseline. NO reaction products increase in asthmatics but not controls, after Ag challenge. The major NO reaction product in the healthy control is NOz", while NOs" is predominant in the asthmatic BAL fluid. Levels of NOi~ are remarkably similar in asthmatic and controls, with no changes during Ag challenge. In contrast, NOa" in asthmatic BAL fluid increases at 10 min of Ag challenge with further increases at 48 h, whereas control NOs" does not change (Table) (3). In contrast to healthy controls, SNO are undetectable in BAL fluid from asthmatics at baseline or 10 minutes after Ag challenge. However 48 h after Ag challenge, SNO in asthmatic BAL fluid increases and to levels similar to control levels (Table) (3). Nitrotyrosine in Endobronchial Biopsies Biopsies of asthmatic airways demonstrate thickened basement membranes, epithelial shedding and increased numbers of goblet cells at baseline. Immunoreactivity for nitrotyrosine is positive in epithelial cells of the asthmatic airways as compared to healthy control epithelial cells (4, 22, 23). At 48 h after Ag challenge, epithelial cells are lost, with areas of denuded basement membrane. Furthermore, prominent influx of eosinophils is noted in Ag challenged segments. Epithelial cells and eosinophils stain positive for nitrotyrosine. Due to marked loss of epithelial cells with Ag challenge, quantitation of nitrotyrosine at 48 hr Ag challenge was not possible. Semiquantitative assessment of the basal cells in epithelial biopsies at baseline revealed a tendency to more intense staining for nitrotyrosine in asthmatic as compared to control epithelium (3).

4. Role of NO Metabolism in the Pathophysiology of Asthma The key difference between asthmatics and controls is that NOa" in the asthmatic lung is higher than in controls at baseline and increases further within minutes of an antigen challenge (Table). Based upon the known chemistry of NO and the known (oxidizing and acidic) chemistry in the lower airway of asthmatic individuals (19, 25), we can construct a model of the events in the asthmatic airway and determine the pathways that may account for the enhanced formation of MV in asthma (Figure). Role of Oxyhemoglobin In the presence of the rich supply of oxyhemoglobin in the lung, reaction of NO with oxyhemoglobin results in NO3" (reaction A in the figure) (26). Although oxyhemoglobin concentration is the same in well-controlled asthmatic and healthy control lungs, enhanced formation of NOs" in asthmatic lungs at baseline may result from the increased NO generated in asthma (4). The lack of any significant increase of NO

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R.A. Dweik/Nitric Oxide Reactions in the Asthmatic Airway

during the immediate asthmatic response indicates that this pathway (reaction A) is not the etiology of the rapid rise in NOs" within minutes of Ag challenge. Indeed the lack of immediate increase of intrapulmonary NO, but rather a tendency to decrease, suggests that NO is being consumed by a more rapid reaction. Given the assumption that the amount of oxyhemoglobin does not change in the asthmatic lung after a challenge, then the increased formation of NOs" likely results from a different reaction.

02

NOS

H

ONOOCC

NO|-^ONOQ02

RSNO

ONOOH

Fe-O

Figure 1. Model of NO reactions in the lung during an antigen-induced asthmatic response (from reference 3 with permission)

Role ofSuperoxide and Peroxynitrite A more likely reaction is the rapid generation of peroxynitrite (ONOO") via reaction of superoxide and NO (reaction B) (27, 28). Indeed, spontaneous asthma attacks and models of experimental antigen challenge are both associated with immediate release of superoxide (Or') which persists throughout the late asthmatic response (15-18). As early as 10 min following local instillation of antigen into airways of atopic individuals, Or is generated by airspace cells, with uM levels of superoxide at sites of Ag challenge (15-18). The oxidative environment in the airway during asthmatic response is confirmed by a decrease in reduced glutathione and increase in oxidized glutathione at 10 min following Ag challenge (19). Notably, NO undergoes a direct bimolecular reaction with O2-~ yielding ONOO" at almost diffusion-limited rates (28, 31, 32). The rate constant is over 3.5 times faster than the dismutation of O2'~ by superoxide dismutases (SOD). Taken together with the rapid loss of SOD activity during the immediate antigeninduced asthmatic response (19), this reaction (B) is overwhelmingly favored in the asthmatic airway.

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163

Following NO reaction with superoxide and generation of ONOO", stopped flow experiments demonstrate peroxynitrite decay depends upon both COi and pH (reactions C and D) (29-31). At physiologic CO2 and pH, the CC>2 catalyzed decomposition of ONOO" is faster than the proton catalyzed decomposition/isomerization to NOs" (28, 29, 31). COi levels are relatively high in the lung (1.76 mM), and the reaction rate with ONOO" is one of the fastest for ONOO" that likely occurs in biologic systems (29, 31, 33). ONOO" and CO2 lead to nitrosoperoxycarbonate adduct (ONOOCO2~), which has a lifetime of < 3 ms (28, 31). On the basis of kinetics, ONOOCO2~ has enhanced nitrating capabilities as compared to ONOO" or ONOOH, such that nonenzymatic nitration of tyrosines becomes a physiologically significant process in cells (29, 31). In the absence of target molecules, decomposition of ONOOCO2" to NOs" occurs rapidly with regeneration of CX>2 (3). Protonation of ONOO" (reaction D) is markedly enhanced by acidification (27, 28, 31), and increased acidity occurs in asthmatic airway during an attack (25). The H+ in the asthmatic airway may reach levels of 6.3 uM, as opposed to H+ levels in the healthy airway of 22 nM (25). Thus, enhanced NOa" formation in asthmatic response is likely due to increased ROS and acidity in the asthmatic airway (3). NO{ is not involved Importantly, NO2~ is not predicted to increase in this proposed reaction scheme (3). The third order reaction of NO and O2 is relatively very slow in comparison to the rapid consumption of -NO by superoxide (27). Depending on the reactions available for NO, some fraction of the -NO in vivo reacts with molecular oxygen to yield NOi, and subsequently NO2~. While MV can be produced from dimerization of NO2 to yield N2O4 followed by nitrosation of water to give NO2~ and NOs", this is an unlikely route in biological systems since -NO2 is formed slowly at -NO concentrations found in vivo (3, 27). SNO and NO in the late Asthmatic Response While studies have shown that humans with severe asthma have low airway SNO (25), this is the first report that SNO is undetectable in airways of mild well-controlled asthmatic individuals. Low levels of SNO in asthma have been attributed to increased catabolic processes (34). Two soluble protein fractions have been identified from homogenates of rodent lung which specifically breakdown SNO and prevent airway smooth muscle relaxation in vitro (34). Furthermore, increased catabolic activity has been identified in rodent lung homogenates following ovalbumin sensitization. While enzymatic processes for SNO catabolism exist in neutrophils (34), neutrophils are <1% of cells in asthmatic and control lungs at baseline, and do not increase significantly at 10 min after allergen challenge (24). Thus, neutrophils are a less likely mechanism for low SNO in the asthmatic lung. Nevertheless, these findings indicate that SNO catabolism is

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accelerated in mild stable asthma as well as in severe asthma. Interestingly, SNO increases to "normal" during the late asthmatic response. SNO may be formed in the lung by organic or inorganic reactions. NO, present at high levels in asthmatic airways (4), may produce SNO under conditions in which glutathione levels are high, as found in the airway (8, 16, 34). Specifically, ONOO" may be scavenged by high thiol concentrations producing a source of SNO formation by NOS (35). Reactions between oxygen, superoxide and NO may also form nitrosating species in the oxidative environment of the asthmatic airway. Nitrosylation of thiols has also been proposed to occur via reaction with ONOOCO2~ or by formation of thiyl radicals (13, 27-29, 31). Oxygen dependent pathways for SNO formation through thiol autooxidation or NO2 formation may also be relevant in the oxidizing environment of the asthmatic lung (13). However, the fact that SNO increase during the late asthmatic response, and not in the immediate response, implies a mechanism of formation that may depend upon an influx of inflammatory cells and/or enzymatic processes, e.g. peroxidases (24). Overall, these findings support that the ongoing inflammation upregulates SNO catabolism in mild asthma, but that SNO formation overwhelms catabolism in the late asthmatic response, perhaps through enzymatic acceleration of reactions or acidification, which favor SNO synthesis and stability. NO increases only during the late asthmatic response (6, 7, 14). NO is endogenously produced in the lung by NO synthases (NOS) 1-3 (4, 36-38). Of the three enzymes, NOS2 expression is regulated primarily at the transcriptional level by cytokines (4, 36). Specifically, upregulation of NOS2 gene expression in airway epithelial cells occurs in a delayed fashion at 8 to 24 h following exposure to interferon-gamma in vitro (36, 37). The increase of NO during the late asthmatic response is consistent with a mechanism that may include upregulation of NOS2 gene expression by cytokines generated by cells recruited into Ag-challenged airways.

5. Conclusions NO reactions in the asthmatic airway suggest several relevant biologic functions for NO in the airway. NO rapidly consumes cytotoxic reactive oxygen species produced during the immediate asthmatic response. The reaction product ONOO7ONOOH is far less reactive than superoxide and leads to the accumulation of the innocuous product NOa". This immediate detoxification role for NO is supported by animal models of asthma in which inhibition of NO synthesis leads to worsening toxic reaction to an antigen challenge (6, 39, 40). Nitrosylation reactions predominate during the late asthmatic response with accumulation of SNO, which have been proposed as safe reservoirs for removal of toxic NO derivatives (13). While NO may have some harmful effects in the airways, the temporal sequence of NO participation in asthmatic airway chemical events suggests that NO may also serve a protective role in the asthmatic response.

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References [I] Persson, M.G., Zetterstrom, O., Agrenius, V., Ihre E., Gustafsson, L.E. (1994) Lancet 343, 146-147. [2] Kharitonov, S. A., Yates, D., Robbins, R.A., Logan-Sinclair, R., Shinebourne, E.A., Barnes, P.J. (1994) Lancet 343, 133-135. [3] Dweik RA, Comhair SAA, Gaston B, Thunnissen FBJM, Farver C, Thomassen MJ, Kavuru M, Hammel J, Abu-Soud HM, Erzurum SC (2001) Proceedings of the National Academy of Sciences (USA) 98, 2622-27. [4] Guo. F.H., Comhair, S.A.A., Zheng, S., Dweik, R.A., Eissa, N.T., Thomassen, M.J., Calhoun, W., Erzurum, S.C. (2000) J. Immunol. 164, 5970-5980. [5] Silkoff, P., Sylvester, J., Zamel, N., Permutt, S. (2000) Am. J. Respir. Crit. Care Med. 161, 1218-1228. [6] Mehta, S., Lilly, C., rotlenhagen, J., Haley, K., Asano, K., Drazen, J. (1997) Am. J. Physiol. 272, L124L131. [7] Kharitonov, S., O'connor B., Evans D., Barnes, P. (1995) Am. J. Respir. Crit. Care Med. 151, 18941899. Sanders, S. P. (1999). ,4m. J. Respir. Cell Mol. Biol. 21, 147-149. Raychaudhuri, B., Dweik, R., Connors, M.J., Buhrow, L.T., Malur, A., Drazba, J., Erzurum, S.C.. Kavuru, M.S., Thomassen, M.J. (1999) Am. J. Respir. Cell Mol. Biol. 21, 311-316. [10] van der Vliet, A., Elserich, J. P., Shigenaga, M. K., Cross, C. E.. (1999) Am. J. Respir. Crit. Care. Med. 160, 1-9. [ I I ] Dweik, R.A., Laskowski, D., Abu-Soud, H.M., Kaneko, F.T., Hutte, R., Stuehr, D.J., Erzurum, S.C, (1998)J. Clin. Inv. 101,660-666. [12] Wink, D. A., I. Hanbauer, M. B. Grisham, F. Laval, R. W. Nims, et al. (1996) Curr. Top. Cell. Regul. 34, 159-187. [13] Gaston, B., Sears, S., Woods, J., Hunt, J., Ponaman, M., McMahon, T., and Stamler, J.S. (1998) Lancet S S I , 1317-1319, [14] Thomassen, M.J., Raychaudhuri, B., Dweik, R.A., Farver, C., Buhrow, L.T., Malur, A., Hammel, J., Erzurum, S.C., Kavuru, M.S. (1999) J. Allergy Clin. Immunol. 104, 1174-1182. [ 15] Calhoun, W.J., Reed H.E., Moest, D.R., Stevens, C.A. (1992) Am. Rev. Respir Dis. 145, 317-325. [16] Liu, M.C., Hubbard, W.C., Proud, D., Stealey B, Galli, S, Gagey-Sobotka, A., Bieeker, E, Lichtrenstein, L. (1991) Am. Rev. Respir Dis. 144, 51-58. [17]Jourjour, N., Calhoun, W., Kelley, E.A., Gleich, G., Schwartz, L., Busse, W. (1997) Am. J. Respir. Crit. Care Med. 155, 1515-1521. [18] Sanders, S., Zweier, J., Harrison, S., Trush, M., Rembish, S., Liu, M. (1995) Am. J. Respir. Crit. Care Med. 151, 1725-1733. [19]Comhair, S.A.A., Bhathena, P.R., Dweik, R.A., Kavuru, M., Erzurum, S.C. (2000) Lancet 355, 624. [20] Guidelines for the diagnosis and the management of asthma, Expert panel report II. Bethesda, Md.: National Asthma Education and Prevention Program, April 1997. (NIH publication no. 97-4051.) [21] Fang, K., Ragsdale, N.V., Carey, R.M., Macdonald, T., and Gaston, B. (1998) Biochem. Biophys. Rex. Commun. 252, 535-540, [22] Kaminsky, D., Mitchell, J., Carroll N., James, A, Soultanakis, R., Janssen, Y. (1999) J. Allergy Clin. Immunol. 104, 747-754. [23]Saleh, D., Ernst, P., Lim, S., Barnes, P.J., Giaid, A. (1998) FASEB Journal. 12, 929-937. [24]Wu, W., Samoszuk, M., Comhair, S., Thomassen, M.J., Farver, C., Dweik, R.A., Kavuru, M.. Erzurum, S.C., Hazen, S.L. (2000) J. Clin. Inv. 105, 1455-1463. [25] Hunt, J., Fang, K., Malik, R., Snyder, A. Malhotra, N., Platts-Mills, T., Gaston, B. (2000) Am. ./., Respir. Crit. Care Med. 161, 694-699. [26]Fukuto J.M. (1995) in Nitric Oxide: Biochemistry, Molecular Biology, and Therapeutic Implications. eds. Ignarro L & Murad F (Academic Press), pp. 1-15. [27] Crow J.P., and Beckman J.S. (1995) in Nitric Oxide: Biochemistry, Molecular Biology, and Therapeutic Implications, eds. Ignarro, L & Murad, F. (Academic Press), pp. 17-43.

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[28] Freeman B.A, White C.R., Gutierrez H., Paler-Martinez A, Trapey M.M., Rubbo H. (1995) in Nitric Oxide: Biochemistry, Molecular Biology, and Therapeutic Implications, eds. Ignarro, L. & Murad. F. (Academic Press), pp. 45-69. [29]Radi R., Denicola A, Freeman B. (1999) Methods Enzymol. 301, 353-367. [30]Padmaja, S., Kissner, R., Bounds, P, Koppenol, W. (1998> Helv. Chim. Ada 7, 1201-1206. [31]Koppenol, W. (1999) in Metal tons In Biological Systems, eds. Sigel, A. & Sigel, H. (Marcel Dekker. New York), pp. 597-619. [32]Rochelle, L., Fischer, B., Adler, K. (1998) Free Radio. Biol. Med. 24, 863-868. [33]Pryor, W.A., Lemercier, J.N., Zhang, H., Uppu, R.M., Squadrito, G.L. (1997) Free Radic. Biol. Med. 23,331-338. [34] Fang, K., Johns, R., Macdonald, T., Kinter, M., Gaston, B. (2000> Am J Physiol. Lung Cell Mol. Physiol.23,Lll6-L72\. [35] Schmidt, H.H.H.W., Hofrnan, H., Schindler, U., Shutenko, Z.S., Cunningham, D.D., Feeelisch, M. Proc. Natl. Acad. Sci. USA. 93, 14492-14497. [36]Guo, F.H., Uetani, K., Haque, J., Williams, B.R.G., Dweik, R.A., Thunnissen, F.B.J.M., Calhoun, W.. Erzurum, S.C. (1997) J. Clin. Inv. 100, 829-838. [37] Uetani, K., Der, S.D., Zamanian-Daryoush, M., de La Motte, C., Lieberman, B.Y., Williams, B.R.. Erzurum, S.C. (2000) J. Immunol. 165, 988-996. [38] De Sanctis, G.T., MacLean, J.A., Hamada, K., Mehta, S., Scott, J.A., Jiao, A., Yandava, C.N., Kobzik, L., Wolyniec, W.W., Fabian, A.J., Venugopal, C.S., Grasemann, H., Huang, P.L., Drazen, J.M. (1999) J. Exp. Med. 189, 1621-1630. [39] Schuiling, M., Meurs, H., Zuidhof, A.B., Venema, N., Zaagsma, J. (1998) Am. J. Respir.Crit Care. Med.158, 1442-1449. [40] Ricciardolo, F.L., Geppetti, P., Mistretta, A., Nadel, J.A., Sapienza, M.A., Belloflore, S., Di Maria, G.U. (1996) Lancet. 348, 374-377.

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Regulation of pH in the Human Airway: Mechanisms and Monitoring John F. HUNT Division ofPediatric Respiratory Medicine, Box 800386 The University of Virginia Health System, Charlottesville, VA 22908, USA Phone: 804-924-1820; Fax: 804-243-5392. Email: jfh2m @ Virginia, edu Abstract. The pH of exhaled breath condensate (EBC) is measurable and stable after deaeration with Argon and is found to be moderately alkaline in healthy subjects. In contrast, in patients with asthma, EBC is prominently acidic and normalizes during corticosteroid therapy. Depletion of ammonia (NH3) in exhaled air is necessary but not sufficient for EBC acidification. Human airway epithelial cells respond to extracellular acidic challenge with upregulation of glutaminase activity and resultant release of ammonia. This process serves a pH homeostatic role that may be dysregulated by pro-inflammatory cytokines, allowing airway pH disturbance in inflammatory lung diseases. The measurement of EBC pH and NH3 allows simple non-invasive assays of biochemical processes relevant to lung disease in humans.

1. Introduction Nebulized acidic challenge to the airway is used in the research setting to induce bronchoconstriction, cough and pulmonary function changes[l-7] . Gastro-esophageal reflux disease is causally associated with chronic asthma symptoms by a mechanism attributable to microaspiration of acidic gastric fluid[8]. Inhalation of acid fog has been reported to be a risk factor for asthma hospitalization, bronchial hyperreactivity and decreased pulmonary function[9-15]. In the process of clarifying potential adverse effects of environmental acidic insult, investigators have identified that acidic airway pH (below 6.8) diminishes ciliary function[16], causes epithelial damage and sloughing[17], and alters mucous viscosity[18]. Eosinophils subjected to acidic stress in vitro undergo necrosis and release of inflammatory granule products [19]. Importantly, the chemistry of diverse chemical reactions—including those involving nitrogen oxides[20, 21]—is altered by protonation of the reactants. These observations are consistent with recent findings concerning airway pathophysiology in several lung diseases—most specifically asthma—and led to the hypothesis that airway acidification might also occur endogenously within the lung. In this regard, multiple biochemical processes release acids in the airway, especially so jn the setting of inflammation [22-25]. The condensable portion of exhaled breath (EBC) can be assayed for pH, and in addition, specific acids and bases can be measured that together contribute to final fluid pH.

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9 i 8 -

7 '

Exhaled Breath Condensate pH

fr I. — m

-

•

6 -

,

j-

5 • A

-

***

f

r B

C

Figure 1. Exhaled breath condensate pH is low in acute asthma. After EBC collection, samples were deaerated with Argon until pH stabilized. Control EBC pH (A) was 2-3 orders of magnitude higher than the EBC pH of acutely ill asthma patients (B) but similar to asthma patients treated for > 48 hours with systemic corticosteroids. Amer. J. Respir. Crit Care Med. 2000: 161(3): 694-9.

These measurements have identified prominent changes in condensate chemistry in various disease states. In particular, we have reported that the pH of EBC is prominently acidic during acute exacerbations of asthma—with a mean pH of 5.2—and rises to normal (mean pH of 7.7) during resolution of symptoms with systemic corticosteroid treatment [Figure 1][26]. Subjects with stable asthma have normal to subtly low EBC pH. We fortuitously identified two asthmatic patients whose EBC pH was prominently low while they were asymptomatic, but each of these patients developed symptomatic airway obstruction 48-72 hours later, while maintaining the low EBC pH. The large effect size of these findings (change in hydrogen ion concentration of up to 3 log orders), the possibility that a simple pH assay might be suitable to predict asthma exacerbations, and the potential that airway acidbase disturbance might contribute to asthma pathophysiology has prompted continued investigations into the factors underlying EBC acidity. 2. Methods of Collecting and Processing Exhaled Breath Condensate for pH Assay Our initial method of collecting EBC—using home built devices consisting of Tygon® (Norton, USA) tubing surrounded by a frozen high specific heat gel—relied on gravity and exhaled airflow to propel condensed fluid down the collecting surface wall to an attached test tube[26, 27]. This system was functional for small studies but was encumbered by size, some difficulty with portability, and the necessity for 10-minute collections to be able to obtain sample. In order to ease collections from hospitalized patients, enable use in the clinic, home and work environment, and allow for more automated assays of pH, we developed systems (RTube™, Respiratory Research, Inc, USA, along with a version modified for rapid pH measurement: the pHTube™) that are readily portable and incorporate a one-way exhalation valve that serves also as a syringe-style plunger used to collect fluid off the condenser walls for quick aggregation of sample. These devices have allowed simple collections of sufficient EBC volumes in as little as one minute. Using this system, we have collected samples from

J. F. Hunt / Regulation ofpH in the Human Airway

1 69

spontaneously breathing patients through the mouth and through tracheostomies. Additionally, we have collected multiple samples from intubated patients with the RTube connected directly to endotracheal tubes. Samples have also been collected unsupervised in subjects' homes and at school-based clinic. The pH of an EEC sample is affected by carbon dioxide (CO2) diffusing in and out of solution. Thus, pH measured during or immediately after collection in healthy individuals has a pH of between 6.3 and 7.2, and tends to rise slowly as dissolved CC»2 is spontaneously evolved from the fluid. To obtain assay stability, we employ a system that evolves COi rapidly out of EBC samples by deaeration. This is accomplished by bubbling Argon gas at 700 ml/min (in the pHTube system) or 350 ml/min in 300 uL of EBC in a 2 ml microcentrifuge tube. As CO2 is evolved from the EBC, protons are consumed and pH rises until no further CC»2 evolves and pH becomes stable. We have found in multiple comparisons that this final, deaerated pH is essentially identical to the pH of secretions suctioned from the airway in matched samples (although very limited data exist from matched tracheobronchial secretions from patients with low pH). Beyond the simplicity of the assay, pH measurements in EBC are also robust. The assay is highly reproducible with a coefficient of variation of 4% among healthy subjects. Duration of sample collection does not significantly affect EBC pH. Samples can be left capped at room temperature for months with no change in pH (healthy subjects), and minimal rise in pH (acute asthmatic subjects). The minimal rise that occurs with asthmatic samples at room temperature amounts to less than 0.3 log order, and may relate to loss of protons to nitrite, forming nitrous acid, and subsequent inorganic generation of nitric oxide. We find that samples frozen at -80° C maintain reproducible pH even after years of storage. Transportation or storage of unfrozen samples is not a barrier to reproducible and useful pH assays. Some small effect of salivary pH on EBC pH is to be expected since there is airflow through the mouth during collection. However, salivary pH in matched samples does not significantly correlate with EBC pH[26], and pronounced experimental acidification of saliva with citric acid only minimally effects EBC pH (0.2 log order) [28]. As others have shown, we have been unable to detect salivary amylase in our samples, suggesting that gross contamination is not a concern. Nonetheless it is conceivable that certain foods/beverages ingested just prior to EBC collection, if containing a highly volatile acid, could affect EBC pH, and therefore caution needs to be exercised when performing collections until such issues are more clearly defined. 3. A Review of pH as it Relates to Airway and Exhaled Breath Condensate Chemistry The concentration of hydrogen ions (protons) can be measured by proton-selective electrodes, colorimetric indicator dyes, and solid-state devices. It has become accepted to present these concentrations as the negative log of the hydrogen concentration, or 'pH.' A pH of 7 therefore represents a hydrogen ion concentration of 100 nanomolar (1 x 10 ~ ), a pH of 6 being 1 micromolar (1 x 10 ~6), etc. Although it is functional and simple to consider protons in solution, the reality is that hydrogen ions are mixed with hydronium ions and other higher order complexes of H2O. In this chapter, the terms "hydrogen ions" or "protons" are used, and will be assumed to include these higher order complexes with K^O. A solution with a pH of 5 by definition has a 10 micromolar concentration of protons, but that provides little indication of the availability of protons for reactions. For depending on the amount and type of buffers in the solution, as protons are used up in reactions, additional protons can be released from buffering compounds in large numbers. At pH 5 the available protons in a solution of a strong acid, such as hydrochloric acid, is smaller than the

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available protons of a weak acid, such as acetic acid, at the same pH. This is because the concentration of acetic acid needed to achieve a pH of 5 is much greater than that of hydrochloric acid. This represents the concept of titratable acidity: it's the available inventory of protons that is important, not just the number that are in solution at a given moment. A discussion of the carbonic acid (^COs) buffer system is warranted because of its potential role in condensate and airway lining fluid chemistry. Buffers maintain pH stability best within 0.8 log order of the pKa of the relevant proton. The FhCOa buffer system (including its components HCOa" and COj2") is the most important buffer in the human blood system, helping to maintain the pH at 7.4. However, the two protons of HzCOs have pKa's of 6.1 and 10.3, seemingly making this system poorly suitable to buffer at pH 7.4. However, this buffer system is not a classic buffer. First, when equilibrium is perturbed by addition of a proton to a solution containing HCOs", H2CO3 is formed that is then directly dehydrated to CO2 [Figure 2]. If the CO2 molecule formed is removed from solution—as would occur in the blood as the respiratory system responds with increased ventilation—the proton is successfully neutralized. This process can continue to neutralize protons as long as there is HCOs" present and this compound therefore serves as a neutralizing base, not a buffer system. Likewise, if the deviation in equilibrium is caused by continuous removal of CC>2 from solution, the reaction continues to the right until 1) CC>2 is at sufficiently low concentrations no longer to be volatile from the solution (and therefore equilibrium is achieved), or 2) the HCOs" is completely consumed. The consumption of HCOs" requires the de novo formation of protons from water, with concomitant release of hydroxide anion (OH") in a process that is alkalinizing. The reaction to form H2CO3 of course slows as the concentrations of reactants (HCO3~ and H+) falls. The key element of the bicarbonate buffer system in blood is that the human senses the concentrations of CC>2 and protons, and responds with a physiologic change (by altering respiratory rate or renal ammonia, phosphate or bicarbonate excretion) to maintain blood pH at 7.4. The utility of H2CC)3 as a pH buffering system in human blood depends on these abilities to maintain homeostasis of the individual components of the system. In a compartment that does not sense and respond to alterations in the system's components, the H2CC>3 system has limited utility as a buffer. For the bicarbonate system to be pH regulatory in the airway lining fluid requires not only an excretion mechanism, but one that is responsive to pH aberration. In the human airway, much controversy exists as to the mechanisms of HCCV excretion, and its concentration and availability to neutralize acid loads in airway lining fluid are uncertain.

pKa=10.3

pKa = 6.1

HCO3 +rf
T

'

Figure 2. The carbonic acid buffer system.

During the deaeration process in EBC, as HCCh" and H+ concentrations decline (as CO2 evolves), the process slows and pH rises progressively slowly and generally no longer changes after ten minutes. At this point, either all HCCV has evolved, the remaining CC>2 is below the level of volatility from a fluid (EBC), or another buffer system dominates the system allowing pH assay stability to be achieved.

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171

Although temperature does affect pH measurements in general[29], the measures of EEC pH are performed in liquid phase at or below ambient, at which temperature-dependent pH effects are insignificant compared to the changes in EBC pH seen in different lung diseases. 4. Role of Ammonia in Neutralizing Airway Acids In contrast to HCCV, there are data supporting pH-regulated formation of ammonia (NHs) to buffer acid in the human airway. It was previously known that airway NH;? functions to neutralize inhaled acid fogs[30] and thereby prevents acid-induced decrements in lung functional]. If the source of the acid is endogenous, NHs should function similarly. The anatomic source of the ammonia in the airways is under investigation, but it is clear that both the upper and lower airways contribute to exhaled ammonia. The alveoli appear to contribute to airway NHs by supplying gaseous NHa by diffusion from plasma[32]. Ammonia is formed by bacterial urease enzymes in the oral cavity[33], and we have presented evidence that glutaminase activity is robust in airway epithelial cells and releases substantial amounts of NHs (American Journal of Respiratory and Critical Care Medicine, in press}. This glutaminase pathway in airway epithelial cells is essentially identical to the mechanism employed in the renal tubular epithelium to secrete NHs to prevent excessive urinary acidification while eliminating metabolic acid loads[34, 35]. Indeed, in airway epithelium in vitro, glutaminase expression and activity with resultant NHs production is upregulated by acidic stress in a pH homeostatic process that appears to be relevant in vivo as well [Figure 3]. In vitro, glutaminase is downregulated by a combination of the inflammatory cytokines interferon y and tumor necrosis factor a. As evidenced by immunohistochemistry, glutaminase appears to be highly expressed in the airway in vivo of healthy subjects, poorly expressed in patients with acute asthma exacerbations, and as in other systems, appears to be upregulated by systemic corticosteroids[36].

AIRWAY LUMEN NOx 1 1

Antimicrobial cytotoxic and other effects / TT+ N. \ \

^" sT s

x; GLUTAMINE ^~ N S

GLUTAMINASE

/

/NW?

BASOLATERATERAL ASPECT OF AIRWAY EPITHELIAL CELL

S S IbylFNyamlTNFa S t by coriicosteroids, acidity v^

X or

-^ ^\

•I- S-nitrosoglutathione

X X X

> (

NH3 + GLUTAMATE + H+ L

A

NH4+

Figure 3. Glutaminase pathway in airway epithelial cells. NH 3 : ammonia, NOx: nitrogen oxides.

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J.F, Hunt / Regulation ofpH in the Human Airway

Ammonia can be measured in exhaled air in gas phase by selective-ion flow spectroscopy and other methods, but caution needs to be taken to assure no condensation of water vapor occurs prior to NHs assay, because NHs is highly soluble in water and is sumped into the condensate in the circuit[37]. We have instead taken advantage of this high water solubility and used

Figure 4. Ammonia in EBC is low in acute asthma (Am J Respir Crit Care Med vol 165, in press)

EBC to trap exhaled NHa for liquid phase measurement. Assaying EBC for NHs allows for simple determination of exhaled levels, not only of trapped gaseous NHs but also of NHs and NH4+ in solution in particles evolved from the airway wall. Ammonia concentration is determined by reaction with NADPH in the presence of Lglutamate dehydrogenase, which reductively aminates added 2-oxoglutarate. The decrease in absorbance at 340 nm reflects NADPH oxidation and is proportional to ammonia concentration[38]. The assay is available as a kit from Sigma Diagnostics. Although we do not use them for our studies, NHa test strips are available that can be dipped into the EBC and provide a colorimetric readout that is in the appropriate range (St. JON Laboratories, USA). Such test strips may prove useful as a simple method of monitoring exhaled NJ-^. We have identified pronounced deficiencies in EBC NHs levels occurring in association with acute exacerbations of asthma. Median breath condensate NH3 concentration was ten-fold lower in specimens from acutely ill asthmatic subjects (30 uM {range: 0-233}; n=18, age 23 ± 2.5 years) than from controls (327 u.M {14-1220}, n=24, age 24 ± 2.4 years. pO.OOl) [Figure 4]. In many acutely ill asthmatic patients, EBC NHa is undetectable. Logarithmically transformed NHa concentrations in EBC correlate, but incompletely so. with same-sample pH measurements (r2 = 0.34, pO.OOl). This assay, like pH, is simple, and is reproducible even after long-term storage at room temperature. Importantly, the effect size between healthy and ill patients is over a log. Given the inherent variability in EBC levels of biomarkers, the large effect size for NFb, as for pH, make these assays appealing. We have also identified that failure of airway epithelial cells in culture to respond to acidic challenge with enhanced glutaminase activity and NHs production leads to failure to neutralize media pH, prolonged exposure to an acid environment, and rapid epithelial cell death. In contrast, successful upregulation of glutaminase expression and activity in response to acidic challenge is cytoprotective. One reason why EBC NHs is low in samples from

J. F. Hunt f Regulation ofpH in the Human Airway

5 73

acutely ill asthma patients may be failure of the glutaminase pH homeostatic activity in the asthmatic airway. The relative contribution to EEC NHs of pathways of formation other than glutaminase is uncertain. Specifically, salivary NI-L? levels are in the low millimolar range, and bacterial urease contributes to these concentrations. However, glutaminase is highly expressed in lymphoid tissues[39] and so it is likely that tonsil and adenoid tissue could contribute to salivary NHs levels. It is important to note that during inhalation, the lower airway can be exposed to NHa formed in the pharynx. Orally formed NHs in addition to airway epithelial NHs could serve to neutralize endogenous acids. The opposite affect is also likely: experimental acidification of the oral cavity, such as with citric acid or other acid rinses, is known to trap gas-phase airway NHs, which subsequently fails to be exhaled[40]. Such an intervention may impressively lower EEC NHs concentrations transiently, although pH only has an insignificant trend to declining by 0.2 log order[28]. This effect needs to be considered both for the planning of clinical studies and for the interpretation of collected data. 5. The roles of HCOs" and CO2 in Exhaled Breath Condensate pH Chemistry Carbon dioxide and HCOs" are present in EEC and, as all acids and bases do, they contribute to the pH. Carbon dioxide can diffuse in and out of EEC depending on temperature, pH, surface tension, and ambient CC>2 tension. In developing these assays, wre found it difficult to obtain stable readings of pH in the modestly buffered EEC without controlling CO2 levels. Argon deaeration allowed for removal of CO2 to a concentration where it was no longer volatile, prevention of new exposure of the sample to ambient CO2, and stabilization of the pH readings. Most CO2 is removed from EEC during the deaeration process. Small amounts may remain likely due to loss of volatility at these low concentrations. In the lab, the pH of EEC from a healthy subject rises from the mid-6 range to approximately 8.0 during deaeration. An asthmatic patient may have an initial pH of 4.7 with only a 0.1 log order increase during deaeration. This minimal change in pH results from removal of roughly the same amount of CC>2 as in the healthy sample, but the log pH scale disguises this. Addition of 5% CO2 to water can acidify it to the 4 range, but the same is not true for the EEC from healthy subjects, in which addition of 5% CC>2 can lower pH to only around 6.3. Exhaled breath condensate clearly contains buffers or bases that prevent more pronounced acidification by COi. One of the compounds involved is NHa. However, simple absence of NHj is also insufficient to acidify EEC. There is clearly the presence of one or more acids present in the EEC of asthmatic patients, but not controls, that contributes to this effect. Indeed, our data suggest that it is only in the relative absence of NH3 and the presence of production of acid other than carbonic acid, that EEC is able to acidify. Is the prominent acidity of EEC seen in asthmatic samples the result of the tendency of ill asthma patients to mildly hyperventilate? An argument has been presented that a hyperventilating patient will have less CO2 in their EEC as it condenses, and therefore a higher pH (because CO2 functions as an acid). The higher EEC pH would then trap less exhaled NHs (a base), and when deaeration is undertaken, the CO2 is evolved off, leaving less ammonia, with a final result being a low pH in the EEC of asthmatic patients[41]. This argument fails on theoretic grounds as well as when empirically investigated. On theoretic grounds, the degree of hyperventilation in acute asthma tends to be mild (with an end-tidal pCO2 falling by perhaps 5 torr), and therefore pH changes before deaeration based on the small difference in CO2 will be small. Second, although end-tidal CO2 is commonly low in acute asthma, these patients exhale more CO2 per minute, not less, because of increased metabolic production from increased work of breathing[42]. Third, an absence of NHs can

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not by itself cause acidification of EBC—which still requires the presence of an acid other than CC»2. Finally, we have empirically examined this issue rigorously and found no significant changes from baseline in pH or NHj concentration from subjects either hyperventilating or hypo ventilating. Asthmatic hyperventilatory response is not responsible for the prominent alterations in EBC pH that others and we have reported. 6. Conclusions Measurement of exhaled breath condensate pH and ammonia levels provide easy assessments of relevant acid-base characteristics of the airway that show marked and readily apparent deviations during acute asthma. These assays are easy to perform, and stable over time during storage. Although exhaled NHa can be prominently affected by the saliva pH, EBC pH is not significantly affected. The low levels found during asthma exacerbations cannot be explained by ventilatory parameters, but may result from failure of airway pH homeostatic mechanisms—such as the glutaminase pathway—to respond to an endogenous acid load. Abnormal levels of EBC pH, bases and acids are providing new insights into airway biochemistry that appear to be particularly relevant to inflammatory lung conditions such as asthma. 7. Acknowledgements. Supported by the American Academy of Allergy and Immunology Education and Research Trust, GlaxoSmithKline, NIH Asthma Center Grant 1U19-A134607, Respiratory Research, Inc., and the University of Virginia Child Health Research Center. JH is a Parker B. Francis Fellow in Pulmonary Research.

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8. References 1. Winther FO. Experimentally induced cough in man by citric acid aerosol. An evaluation of a method. Acta Pharmacol Toxicol 1970; 28(2): 108-12. 2. Fine JM, Gordon T, Thompson JE, Sheppard D. The role of titratable acidity in acid aerosolinduced bronchoconstriction. Am Rev Respir Dis 1987; 135(4):826-30. 3. Avol EL, Linn WS, Whynot JD, Anderson KR, Shamoo DA, Valencia LM, Little DE, Hackney JD. Respiratory dose-response study of normal and asthmatic volunteers exposed to sulfuric acid aerosol in the submicrometer size range. Toxicol IndHealth 1988; 4(2):173-84. 4. Balmes JR, Fine JM, Christian D, Gordon T, Sheppard D. Acidity potentiates bronchoconstriction induced by hypoosmolar aerosols. Am Rev Respir- Dis 1988; 138(1 ):35-9. 5. Koenig JQ, Covert DS, Pierson WE. Effects of inhalation of acidic compounds on pulmonary function in allergic adolescent subjects. Environ Health Perspect 1989; 79:173-8. 6. Balmes JR, Fine JM, Gordon T, Sheppard D. Potential bronchoconstrictor stimuli in acid fog. Environ Health Perspect 1989; 79:163-6. 7. Zuskin E, Mustajbegovic J, Schachter EN, Pavicic D, Budak A. A follow-up study of respiratory function in workers exposed to acid aerosols in a food-processing industry, fnt Arch Occup Environ Health 1997;'70(6):413-8. 8. Jack CI, Calverley PM, Donnelly RJ, Tran J, Russell G, Hind CR, Evans CC. Simultaneous trachea! and oesophageal pH measurements in asthmatic patients with gastro-oesophageal reflux. Thorax 1995; 50(2):201-4. 9. Tanaka H, Honma S, Nishi M, Igarashi T, Teramoto S, Nishio F, Abe S. Acid fog and hospital visits for asthma: an epidemiologica! study. Eur Respir J 1998; 11(6):1301-6. 10. Kopferschmitt-Kubler MC, Blaumeiser-Kapps M, Millet M, Wortham H, Mirabel P, Nobelis P, Pauli G. [Study by questionnaire of the influence of weather conditions, particularly fog, on the symptomatology of asthmatic subjects]. Rev Mai Respir 1996; 13(4):421-7. 11. Thurston GD, Ito K, Hayes CG, Bates DV, Lippmann M. Respiratory hospital admissions and summertime haze air pollution in Toronto, Ontario: consideration of the role of acid aerosols. Environ Res 1994; 65(2):271-90. 12. Linn WS, Avol EL, Anderson KR, Shamoo DA, Peng RC, Hackney JD. Effect of droplet size on respiratory responses to inhaled sulfuric acid in normal and asthmatic volunteers. Am Rev Respir Dis 1989; 140(1): 161-6. 13. Raizenne ME, Burnett RT, Stern B, Franklin CA, Spengler JD. Acute lung function responses to ambient acid aerosol exposures in children. Environ Health Perspect 1989; 79:179-85. 14. Dockery DW, Cunningham J, Damokosh AI, Neas LM, Spengler JD, Koutrakis P, Ware JH, Raizenne M, Speizer FE. Health effects of acid aerosols on North American children: respiratory symptoms. Environ Health Perspect 1996; 104(5):500-5. 15. Neas LM, Dockery DW, Koutrakis P, Tollerud DJ, Speizer FE. The association of ambient air pollution with twice daily peak expiratory flow rate measurements in children. Am J Epidemiol 1995; 141(2): 111-22. 16. Luk CK, Dulfano MJ. Effect of pH, viscosity and ionic-strength changes on ciliary beating frequency of human bronchial explants. Clin Sci 1983; 64(4):449-51. 17. Holma B, Lindegren M, Andersen JM. pH effects on ciliomotility and morphology of respiratory mucosa. Arch Environ Health 1977; 32(5):216-26. 18. Holma B, Hegg PO. pH- and protein-dependent buffer capacity and viscosity of respiratory mucus. Their interrelationships and influence on health. Sci Total Environ 1989; 84:71-82. 19. Hunt J, Fang K, Platts-Mills T, Gaston B. Nitrogen oxide redox balance in asthma [abstr]. American Journal of Respiratory and Critical Care Medicine 1999; 159(3):A860. 20. Gaston B, Stamler JS. Nitrogen Oxides. In: Crystal RG, ed. The Lung: Scientific Foundations (2nd edition). Philadelphia: Lippincott-Raven, 1997; 239-253. 21. Crow JP, Spruell C, Chen J, Gunn C, Ischiropoulos H, Tsai M, Smith CD, Radi R, Koppenol WH, Beckman JS. On the pH-dependent yield of hydroxyl radical products from peroxynitrite. Free Radic Bio/ Med 1994; 16(3):33l-8. 22. Acevedo M, Steele LW. Na(+)-H+ exchanger in isolated epithelial tracheal cells from sheep. Involvement in tracheal proton secretion. Exp Physiol 1993; 78(3):383-94. 23. Pacheco G, Lippo de Becemberg 1, Gonzalez de Alfonzo R, Alfonzo MJ. Biochemical characterization of a V-ATPase of tracheal smooth muscle plasma membrane fraction. Biochim Biophyx Acta 1996; 1282(2): 182-92. 24. Wadsworth SJ, Spitzer AR, Chander A. Ionic regulation of proton chemical (pH) and electrical gradients in lung lamellar bodies. Am J Physiol 1997; 273(2 Pt l):L427-36.

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25. Swenson ER, Robertson HT, Hlastala MP. Effects of carbonic anhydrase inhibition on vent Mat ionperfusion matching in the dog lung. JClin Invest 1993; 92(2):702-9. 26. Hunt JF, Fang K, Malik R, Snyder A, Malhotra N, Platts-Mills TA, Gaston B. Endogenous airway acidification. Implications for asthma pathophysiology. Am J RespirCrit Care Med 2000; 161(3 Pt l):694-9. 27. Hunt J, Byms RE, Ignarro LJ, Gaston B. Condensed expirate nitrite as a home marker for acute asthma [letter]. Lancet 1995; 346(8984): 1235-6. 28. Vaughan J, Hunt J. Stability of exhaled breath precipitate pH during experimental salivary acidification. Am J Respir Crit Care Med 2001; 163(3):A408. 29. Beynon RJ, Easterby JS. Buffer Solutions. Oxford, UK: Oxford University Press, 1996. 30. Larson T, Frank R, Covert D, Holub D, Morgan M. The chemical neutralization of inhaled sulfuric acid aerosol. AmJIndMed 1980; l(3-4):449-52. 31. Utell MJ, Frampton MW, Morrow PE. Air pollution and asthma: clinical studies with sulfuric acid aerosols. Allergy Proc 1991; 12(6):385-8. 32. Ament W, Huizenga JR, Kort E, van der Mark TW, Grevink RG, Verkerke GJ. Respiratory ammonia output and blood ammonia concentration during incremental exercise. Int J Sports Med 1999; 20(2):71-7. 33. Burne RA, Marquis RE. Alkali production by oral bacteria and protection against dental caries. FEMS Microbiol Lett 2000; 193( 1): 1 -6. 34. Welbourne TC, Phromphetcharat V. Renal glutamine metabolism and hydrogen ion homeostasis. In: Haussinger D, Sies H, eds. Glutamine metabolism in mammalian tissues. New York: Springer-Verlag, 1984; 161-177. 35. Laterza OF, Hansen WR, Taylor L, Curthoys NP. Identification of an mRNA-binding protein and the specific elements that may mediate the pH-responsive induction of renal glutaminase mRNA. J Biol Chem 1997;272(36):22481-8. 36. Sarantos P, Abouhamze Z, Copeland EM, Souba WW. Glucocorticoids regulate glutaminase gene expression in human intestinal epithelial cells. JSurgRes 1994; 57(1):227-31. 37. Spanel P, Davies S, Smith D. Quantification of ammonia in human breath by the selected ion flow tube analytical method using H30+ and 02+ precursor ions. Rapid Commun Mass Spectrom 1998; 12(12):76366. 38. Neeley WE, Phillipson J. Automated enxymatic method for determining ammonia in plasma with 14 day reagent stability'. Clin Chem 1968; 34:1868. 39. Newsholme EA, Calder PC. The proposed role of glutamine in some cells of the immune system and speculative consequences for the whole animal. Nutrition 1997; 13(7-8):728-30. 40. Norwood DM, Wainman T, Lioy PJ, Waldman JM. Breath ammonia depletion and its relevance to acidic aerosol exposure studies. Arch Environ Health 1992; 47(4):309-13. 41. Effros RM. Endogenous airway acidification: implications for asthma pathology [letter]. Am J Respir Crit Care Med 2001; 163( 1 ):293-4. 42. Freedman AR, Lavietes MH. Energy requirements of the respiratory musculature in asthma. Am J Med\ 986; 80(2):215-22.

Disease Markers in Exhaled Breath N. Marczin and M.H. Yacoub (Eds.) IOS Press, 2002

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Markers in Exhaled Air and Condensate to Monitor Treatment in Asthma Sergei A. KHARITONOV Thoracic Medicine, National Heart and Lung Institute, Imperial College and Royal Brompton Hospital, Dovehouse Street, London SW3 6LY, UK Abstract. NO is the most extensively studied exhaled marker and abnormalities in exhaled NO have been documented in several lung diseases, particularly asthma[l,2]. Exhaled breath condensate is collected by cooling or freezing exhaled air and is totally non-invasive. The collection procedure has no influence on airway function or inflammation, and there is accumulating evidence that abnormalities in condensate composition may reflect biochemical changes of airway lining fluid. Several non-volatile chemicals, including proteins, have now been detected in breath condensates, which may be useful to monitor treatment in asthma.

1. Asthma monitoring. It is difficult to monitor the response of different classes of anti-inflammatory drugs in asthma, as there is no single test that can be used to quantify airway inflammation. Peripheral blood markers are unlikely to be adequate as the most important mediator and cellular responses occur locally within airways. Eosinophils in induced sputum originate from more proximal rather than small airway [3]. It is clear that different markers of airway inflammation should be considered together to monitor asthma [4]. Exhaled NO has been used to monitor the effect of anti-inflammatory treatment in asthma [2,5] and asthma exacerbations, both spontaneous [6] and induced by steroid reduction [7,8]. There is a lack of long-term serial studies of exhaled NO, together with other markers of airway inflammation in sputum and exhaled condensate, lung function and symptoms. Exhaled NO behaves as a "rapid response" marker, which is extremely sensitive to steroid treatment, as it may be significantly reduced even after 6 hours following a single treatment with a nebulized corticosteroid steroid [9], or within 2-3 days after inhaled corticosteroids [5], reaching maximal effect after 2-4 weeks of treatment [5,7,10-14]. An important issue in asthma management is to prevent over-treatment of patients with steroids. The high sensitivity of exhaled NO to corticosteroid treatment is an advantage, as higher doses of inhaled steroids are not necessary to improve asthma control, e.g. in mild persistent asthma [4]. We have demonstrated a dose-dependent reduction in exhaled NO and improvement in asthma symptoms in mild asthmatics following treatment with low doses of inhaled corticosteroids [14], whereas the reduction in sputum eosinophils and similar improvement in symptoms was observed only after the higher dose of steroids [11]. This suggests that exhaled NO levels may be too sensitive to determine whether inflammation is adequately controlled [4]. Although exhaled NO levels are normal in patients with moderate asthma treated with corticosteroids [15], increased levels have been observed in patients with severe asthma, despite treatment with oral corticosteroids [16,17]. Individual NO values, like individual peak expiratory flows, should be established and monitored, and when the levels are

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above or below a certain reference level, steroid treatment should be either reduced or increased. A considerable advantage of exhaled NO is that NO levels may increase before any significant changes in other parameters, such as lung function and sputum eosinophils and may therefore and serve as an early warning of loss of control [18]. Thus, exhaled NO levels increase by 40% and 100% after 2 and 4 weeks, respectively, following the reduction in steroid treatment [8]. This increase in exhaled NO levels is accompanied by lung function deterioration and asthma symptoms. Although the baseline high number of eosinophils in sputum of patients who eventually develop exacerbations is a good predictor of asthma deterioration, the changes in eosinophils following the steroid reduction are slow [8]. Prospective studies, which look at asthma outcomes over a prolonged period of time, where NO is used as a decision point for modifying inhaled corticosteroid treatment will be needed to evaluate of the value of exhaled NO as a useful way of monitoring asthma. 2. Responsiveness to asthma therapy 2.1. Corticosteroids. Systemic corticosteroids have no effect on exhaled NO in normal subjects, but decease its levels in patients with asthma [6,19]. Oral dexamethasone (4 mg/day for 2 days) similarly has no effect on exhaled NO or on serum concentrations of interferon-y and IL-lp in normal subjects [20]. A large dose (Img/kg/day for 5 days) of oral prednisolone normalized exhaled NO in infants and young children with wheezing exacerbations [21], whereas the same dose in more severe asthmatic children only shifted their exhaled NO down to the levels of mildto-moderate asthma, in spite of the improvement in lung function [22]. A cumulative dose of methylprednisolone (180-500 mg) causes 36% reduction within 50 h in the majority of severe adult patients with acute asthma [6], and a combination of oral prednisolone and inhaled steroids reduces exhaled NO by 65% in children with acute asthma [23]. Recently, it has been shown that NO levels correlate with the percentage improvement in FEVj from baseline to the post-steroid (30 mg prednisolone/day for 14 days) postbronchodilator value. A NO level of >10 ppb at baseline has a positive predictive value of 83% for an improvement in FEVi of >15%, and therefore may be useful in predicting the response to a trial of oral steroid in asthma [24]. A key question is why has it been so difficult to show a dose-dependent effect of inhaled corticosteroids in the treatment of asthma? First, it is possible that the small change in doses makes it difficult to detect changes in asthma symptoms and lung function (FEVi). Secondly, the currently recommended doses may be at the upper end of the dose-response curve, making it difficult to detect a relatively small change in dose. In view of concerns about systemic effects and the better effects of adding an inhaled longacting P2~agonist compared to doubling the dose of inhaled steroid, there is now a trend towards use of lower doses of inhaled corticosteroids. Exhaled NO as an inflammatory marker sensitive to corticosteroids may be the ideal tool to demonstrate a dose-response effect and to adjust the dose in clinical practice. It may also be useful in patients using a fixed combination inhalers (corticosteroids and long acting Pi-agonist) to ensure that inflammation is controlled, as this may be difficult to assess from symptoms when a longacting bronchodilator is taken.

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179

In fact, inhaled corticosteroids reduce exhaled NO in asthmatic patients [5] and this effect is dose-related [11]. However, a plateau effect on exhaled NO measured after 6 to 12 h since the last treatment may be seen at a dose of 400 u,g budesonide and higher [11,25] in contrast to dose-related improvements in adenosine monophosphate and metacholine reactivity up to 1600 [ig in patients with mild-to-moderate asthma [14,26]. The effect of inhaled steroids on exhaled NO is very rapid and may occur within 6 hours after a single high-dose (8 mg) budesonide (Pulmicort Respules®) in symptomatic moderate asthma [9]. Therefore, chronic and acute reduction in exhaled NO may be of a different magnitude. Recently, it has been shown that the onset of action of inhaled BUD on exhaled NO and the time to reach the maximal reduction were also dose-dependent [14]. A gradual reduction in exhaled NO is seen during the first week of regular treatment [5,13,14] with maximal effect between 3 [5,12] or 4 [10,11] weeks. It is still uncertain whether exhaled NO is useful to direct changes in asthma therapy. Recently, it has been shown that exhaled NO values above 13 ppb had a sensitivity of 0.67 and a specificity of 0.65 to predict a step up in therapy [27], but clearly more studies needed using exhaled NO to direct therapy. Corticosteroids may reduce exhaled NO by directly inhibiting the induction of NOS2 [28] or by suppressing the proinflammatory cytokines that induce NOS2. There is inhibition of NOS2 immunoreactivity with inhaled corticosteroid treatment in asthmatic patients and a parallel reduction in immunoreactivity for nitrotyrosine, which may reflect local production of peroxynitrite from an interaction of NO and superoxide anions [29].

2.2. p2-agonists. Neither short-acting [5,22,30-33] nor long-acting [22,25,30,32,34] Pi-agonists reduce exhaled NO. This is consistent with the fact that they do not have any anti-inflammatory effects in asthma, although it has been shown that regular treatment with inhaled formoterol reduces inflammatory cells in the mucosa of asthmatic patients [35]. There may even be a short-term increase in exhaled NO after Pi-agonists, which may be due to opening up of airways with higher local NO concentrations [36]. 2.3. Anti-Ieukotrienes. The leukotriene receptor antagonist pranlukast blocks the increase in exhaled NO when inhaled corticosteroids are withdrawn [37], and montelukast rapidly reduce exhaled NO by 15-30% in children with asthma [38]. Anti-leukotrienes have a moderate effect in patients with asthma and seasonal allergic rhinitis [39,40], Both formoterol and zafirlukast were equally effective in maintaing asthma control, and zafirlukast caused a significant reduction in exhaled NO [31]. 2.4 NOS inhibitors. Nebulized L-NMMA and L-NAME, which are non-selective inhibitors of NOS, both reduce exhaled NO in asthmatic patients, although this is not accompanied by any changes in lung function [19,41]. Aminoguanidine, a more selective inhibitor of NOS2, reduces exhaled NO in asthmatic patients, but has little effect in normal subjects, indicating that NOS2 is an important source of the increased exhaled NO in asthma [42].

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2.5. Prostaglandins. Prostaglandin (PG)E2 down-regulates NOS2 expression [43] and inhaled PGE2 and PGF2a decrease exhaled NO in normal and asthmatic subjects [44].

2.6. Other drugs. The immunosuppressive drugs, cyclosporin and rapamycin, inhibit NOS2 expression [45], suggesting that exhaled NO can be used to monitor their effect. Ibuprofen, a cyclooxygenase inhibitor, reduces the elevated levels of exhaled NO in normal subjects after i.v. administration of endotoxin [46], and indomethacin partially prevents an increase in exhaled NO and asthma symptoms in patients whose dose of steroids was reduced [47] A low dose of theophylline has no effect on exhaled NO levels in asthmatic patients [48]. Nebulized IL-4 receptor (altrakincept) reduces exhaled NO in patients with moderate asthma [49]. 3. Breath condensate Several non-volatile chemicals, including proteins, have now been detected in breath condensates (Figure 1).

Figure 1. Exhaled breath condensate: Panel A: diagram of the apparatus (EcoScreen, Jaeger, Germany);

3.1. Hydrogen peroxide H202 has been detected in exhaled condensate in healthy adults and children with increased concentrations in asthma [50-53]. There is no correlation between the levels of exhaled H2O2 and age, gender, or lung function in healthy children [53]. However, exhaled H2O2 concentration is related to the number of sputum eosinophils and airway hyperresponsiveness in asthma of different severity, and is elevated in severe unstable asthmatics, although exhaled NO is significantly reduced by the treatment with corticosteroids [51]. This may be related to the fact that neutrophils, prevalent in severe asthma [17], generate higher amounts of superoxide radicals and therefore H2O2 [54].

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181

Asthmatic patients also exhale significantly higher levels of thiobarbituric acid-reactive products (TBARs), which indirectly reflect increased oxidative stress [50].

3.2. Leukotriens Leukotrienes (LTs), a family of lipid mediators derived from arachidonic acid via the 5lipoxygenase pathways, are potent constrictor and pro-inflammatory mediators that contribute to pathophysiology of asthma. Detectable levels of LTB4, C4, D4, E4 and F4 have been reported in exhaled condensate of asthmatic and normal subjects [55,56]. Elevated exhaled condensate levels of LTB4 have been found in healthy calves during an experimental chest infection [57]. There have been attempts to measure leukotrienes in urine and increased levels of LTE4 have been reported in some asthmatic patients, but they are not consistently increased after allergen challenge [58]. Allergen provocation increases LTC4 and LTE4 concentrations in BAL and in urine during early and late asthmatic responses [59]. However, measurement of airway mediators in urine is problematic because of dilution of the lung-derived signal and delay in excretion. Increased levels of LTE4 have also been found in induced sputum during the late response to allergen in patients with mild asthma [60]. In mild asthmatic patients levels of LTE4, LTC4, LTD4 levels in exhaled condensate are increased during the late asthmatic response to allergen challenge [61]. The levels of leukotrienes LTE4,C4,D4 in breath condensate are elevated significantly in patients with moderate and severe asthma [56], and steroid withdrawal in moderate asthma leads to worsening of asthma and further increase in exhaled NO and the concentration of LTB4, LTE4, LTC4, LTD4 in exhaled condensate [61] (Figure 2)-

80-,

r800

Stable asthma Unstable asthma p<0.05

Nitrotyrosine

Leukotrienes LTE4/C4/D4

LTB4

Figure 2. Exhaled nitrotyrosine and leukoteienes before and after steroid withdrawal in patients with moderate asthma (from Ref 381);

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3.3. Isoprostanes Isoprostanes are a novel class of prostanoids formed by free radical-catalyzed lipid peroxidation of arachidonic acid [62]. They are formed initially esterified in membrane phospholipids, from which they are cleaved by a phospholipase AI, circulate in plasma, and are excreted in urine and can be detected in exhaled breath condensate and BAL. Their formation is largely independent of COX-1 and COX-2. They can be detected by ELISA [63,64] and by GC/MS analysis [62]. p2-isoprostanes are the major candidates for clinical measurement of oxidative stress in vivo. They are stable compounds, detectable in all normal biological fluids and tissues [65], and their formation is increased by systemic oxidative stress, for example in patients with diabetes [66], or ARDS [67]. Fiisoprostanes are reduced by antioxidants, for example by alpha-lipoic acid in normal subjects [68]. They are not simply markers of lipid peroxidation but also possess biological activity, and could be mediators of the cellular effects of oxidant stress and a reflection of complex interactions between the RNS and ROS. Indeed, peroxynitrite is capable of activating biosynthesis of endoperoxide synthase and thromboxanes in inflammatory cells [69], and oxidizing arachidonic acid to form p2-isoprostanes. The most prevalent isoprostane in humans in 8-epi-PGP2a, also known as 8-isoprostane. p2-isoprostanes are increased in plasma [70] and BAL fluid of asthmatic patients and further increased after allergen challenge [71]. 8-isoprostane levels are approximately doubled in mild asthma compared with normal subjects, and increased by about 3-fold in those with severe asthma, irrespective of their treatment with corticosteroids [63]. The relationship to asthma severity is a useful aspect of this marker, in contrast to exhaled NO. The relative lack of effect of corticosteroids on exhaled 8-isoprostane has been confirmed in a placebo-controlled study with the two different doses of inhaled steroids [14]. This provides evidence that inhaled corticosteroids may not be very effective in reducing oxidative stress. Exhaled isoprostanes may a better means of reflecting disease activity than exhaled NO. 4. References [ 1 ] S. A.Kharitonov, Barnes PJ. Clinical aspects of exhaled nitric oxide. Eur Respir. J. \ 6 (2000) 781 -792. [2] L.E.Gustafsson. Exhaled nitric oxide as a marker in asthma. Eur. Respir. J. Suppl. 26 (1998) 49S-52S. [3] E.Pizzichini et al.. Induced sputum, bronchoalveolar lavage and blood from mild asthmatics: inflammatory cells, lymphocyte subsets and soluble markers compared. Eur Respir J 11 (1998) 828834. [4] S.A.Kharitonov, Barnes PJ. Clinical aspects of exhaled nitric oxide. Eur Respir J 16 (2000) 781 -792. [5] S.A.Kharitonov et al.. Inhaled glucocorticoids decrease nitric oxide in exhaled air of asthmatic patients. Am. J. Respir. Crit. Care. Med. 153 (1996) 454-457. [6] A.F.Massaro et al.. Expired nitric oxide levels during treatment of acute asthma. Am. J. Respir Crit Care. Med. 152 (1995) 800-803. [7] S.A.Kharitonov et al.. Changes in the dose of inhaled steroid affect exhaled nitric oxide levels in asthmatic patients. Eur. Respir. J. 9 (1996) 196-201. [8] A.Jatakanon et al.. Changes in sputum eosinophils predict loss of asthma control. Am. J. Respir Crit Care. Med. 161 (2000) 64-72.

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[9] S.A.Kharitonov et al.. Reduction in exhaled nitric oxide after a single dose of nebulised budesonide in patients with asthma. Am. J, Respir, Crit. Care. Med 153 (1996) A799. [10]

S.Lim et al.. Effect of inhaled budesonide on lung function and airway inflammation. Am. J. Respir. Crit. Care. Med. 159(1999) 22-30.

[11] A.Jatakanon et al.. Effect of differing doses of inhaled budesonide on markers of airway inflammation in patients with mild asthma. Thorax. 54 (1999) 108-114. [12] E.L.van Rensen et al.. Effect of inhaled steroids on airway hyperresponsiveness, sputum eosinophils, and exhaled nitric oxide levels in patients with asthma. Thorax. 54 (1999) 403-408. [13]

P.E.Silkoff et al.. Exhaled nitric oxide and bronchial reactivity during and after inhaled beclomethasone in mild asthma. J. Asthma. 35 (1998) 473-479.

[14] S.A.Kharitonov et al.. Dose-dependent onset and duration of action of 100/400 meg budesonide on exhaled nitric oxide and related changes in other potential markers of airway inflammation in mild asthma. Am. J. Respir. Crit. Care. Med. 161 (2000) A186. [15] S.A.Kharitonov et al.. Increased nitric oxide in exhaled air of asthmatic patients. Lancet 343 (1994) 133-135. [16] R.G.Stirling et al.. Exhaled NO is elevated in difficult asthma and correlates with symptoms and disease severity despite treatment with oral and inhaled corticosteroids. Thorax. 53 (1998) 1030-1034. [17] A.Jatakanon et al.. Neutrophilic inflammation in severe persistent asthma. Am. J. Respir. Crit. Care. Med. 160(1999) 1532-1539. [18] S.A.Kharitonov. Exhaled nitric oxide and carbon monoxide in asthma. Eur. Respir. J. 9 (1999) 212218. [19] D.H.Yates et al.. Effect of a nitric oxide synthase inhibitor and a glucocorticosteroid on exhaled nitric oxide. Am. J. Respir. Crit. Care. Med. 152 (1995) 892-896. [20] K.Sato et al.. Lack of inhibitory effect of dexamethasone on exhalation of nitric oxide by healthy humans. Intern. Med. 35 (1996) 356-361. [21] E.Baraldi et al.. Exhaled nitric oxide concentrations during treatment of wheezing exacerbation in infants and young children. Am. J. Respir. Crit. Care. Med. 159 (1999) 1284-1288. [22] E.Baraldi et al.. Corticosteroids decrease exhaled nitric oxide in children with acute asthma. J. Pediatr. 131 (1997) 381-385. [23] MJ.Lanz et al.. Comparison of exhaled nitric oxide to spirometry during emergency treatment of asthma exacerbations with glucocorticosteroids in children. Ann. Allergy. Asthma. Immunol. 82 (1999) 161-164. [24] S.A.Little et al.. Non-invasive markers of airway inflammation as predictors of oral steroid responsiveness in asthma. Thorax. 55 (2000) 232-234. [25]

I.Aziz et al.. Effects of once-daily formoterol and budesonide given alone or in combination on surrogate inflammatory markers in asthmatic adults. Chest 118 (2000) 1049-1058.

[26]

A.M.Wilson, Lipworth BJ. Dose-response evaluation of the therapeutic index for inhaled budesonide in patients with mild-to-moderate asthma. Am J Med 108 (2000) 269-275.

[27]

M.Griese et al.. Asthma severity, recommended changes of inhaled therapy and exhaled nitric oxide in children: a prospective, blinded trial. Eur J Med Res 5 (2000) 334-340.

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[28] F.H.Guo et al.. Molecular mechanisms of increased nitric oxide (NO) in asthma: evidence for transcriptional and post-translational regulation of NO synthesis. J. Immunol. 164 (2000) 5970-5980. [29] D.Saleh et al.. Increased formation of the potent oxidant peroxynitrite in the airways of asthmatic patients is associated with induction of nitric oxide synthase: effect of inhaled glucocorticoid. FASEB J. 12(1998) 929-937. [30] D.H.Yates et al.. Effect of short- and long-acting inhaled beta2-agonists on exhaled nitric oxide in asthmatic patients. Eur. Respir. J. 10(1997) 1483-1488. [31] B.J.Lipworth et al.. Effects of adding a leukotriene antagonist or a long-acting beta(2)-agonist in asthmatic patients with the glycine-16 beta(2>adrenoceptor genotype. Am J Med 109 (2000) 114-121. [32] D.H.Yates et al.. Effect of short- and long-acting inhaled beta2-agonists on exhaled nitric oxide in asthmatic patients. Eur. Respir. J. 10(1997) 1483-1488. [33] P.Garnier et al.. Exhaled nitric oxide during acute changes of airways calibre in asthma. Eur. Respir J. 9(1996) 1134-1138. [34] G.Fuglsang et at.. Effect of salmeterol treatment on nitric oxide level in exhaled air and dose-response to terbutaline in children with mild asthma. Pediatr. Pulmonol. 25(1998) 314-321. [35] A.Wallin et al.. The effects of regular inhaled formoterol, budesonide, and placebo on mucosal inflammation and clinical indices in mild asthma. Am J Respir Crit Care Med \ 59 (1999) 79-86. [36] L.P.Ho et al.. The current single exhalation method of measuring exhales nitric oxide is affected by airway calibre. Eur. Respir. J. 15 (2000) 1009-1013. [37] H.Kobayashi et al.. Decreased exhaled nitric oxide in mild persistent asthma patients treated with a leukotriene receptor antagonist, pranlukast. Jpn. J. Physiol. 49 (1999) 541-544. [38] H.Bisgaard et al.. NO in exhaled air of asthmatic children is reduced by the leukotriene receptor antagonist montelukast. Am. J. Respir. Crit. Care. Med. 160(1999) 1227-1231. [39] A.M.Wilson et al.. Antiasthmatic effects of mediator blockade versus topical corticosteroids in allergic rhinitis and asthma. Am J Respir Crit Care Med 162 (2000) 1297-1301. [40] D.L.Bratton et al.. Exhaled nitric oxide before and after montelukast sodium therapy in school-age children with chronic asthma: A preliminary study. Pediatr. Pulmonol. 28 (1999) 402-407. [41] F.P.Gomez et al.. Effect of nitric oxide synthesis inhibition with nebulized L-NAME on ventilationperfusion distributions in bronchial asthma. Eur Respir J 12 (1998) 865-871. [42] D.H.Yates et al.. Endogenous nitric oxide is decreased in asthmatic patients by an inhibitor of inducible nitric oxide synthase. Am. J. Respir. Crit. Care. Med. 154 (1996) 247-250. [43]

F.D'Acquisto et al.. Prostaglandins prevent inducible nitric oxide synthase protein expression by inhibiting nuclear factor-kappaB activation in J774 macrophages. FEBS. Lett. 440 (1998) 76-80.

[44]

S.A.Kharitonov et al.. Prostaglandins E 2 and F2a reduce exhaled nitric oxide in normal and asthmatic subjects irrespective of airway calibre changes. Am. J. Respir. Crit. Care. Med. 158 (1998) 1374-1378.

[45] M.G.Attur et al.. Differential anti-inflammatory effects of immunosuppressive drugs: cyclosporin, rapamycin and FK-506 on inducible nitric oxide synthase, nitric oxide, cyclooxygenase-2 and PGE2 production. Inflamm. Res. 49 (2000) 20-26. [46]

R. W. Vandivier et al.. Down-regulation of nitric oxide production by ibuprofen in human volunteers. J. Pharmacol. Exp. Ther. 289(1999) 1398-1403.

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[47] J.Tamaoki et al.. Effect of inhaled indomethacin in asthmatic patients taking high doses of inhaled corticosteroids. J Allergy Clin Immunol 105 (2000) 1134-1139. [48] B.Oliver et al.. The effect of low dose theophylline on cytokine production in alveolar macrophages in patients with mild asthma. Am. J. Respir. Crit. Care. Med. 161 (2000) A614. [49] L.C.Borish et al.. Interleukin-4 Receptor in Moderate Atopic Asthma. A phase i/ii randomized, placebocontrolled trial. Am. J. Respir. Crit. Care. Med. 160 (1999) 1816-1823. [50] A.Antczak et al.. Increased hydrogen peroxide and thiobarbituric acid-reactive products in expired breath condensate of asthmatic patients. Eur. Respir. J. 10 (1997) 1235-1241. [51] I.Horvath et al.. Combined use of exhaled hydrogen peroxide and nitric oxide in monitoring asthma. Am. J. Respir. Crit. Care. Med. 158(1998) 1042-1046. [52] A.W.Dohlman et al.. Expired breath hydrogen peroxide is a marker of acute airway inflammation in pediatric patients with asthma. Am. Rev. Respir. Dis. 148 (1993) 955-960. [53] Q.Jobsis et al.. Hydrogen peroxide in exhaled air of healthy children: reference values. Eur. Respir. J. 12(1998) 483-485. [54] A.Antczak et al.. Hydrogen peroxide in expired air condensate correlates positively with early steps of peripheral neutrophil activation in asthmatic patients. Arch. Immunol. Ther. Exp. (Warsz, ). 47 (1999) 119-126. [55] G.Becher et al.. Breath condensate as a method of noninvasive assessment of inflammation mediators from the lower airways. Pneumologie. 51 Suppl 2:456-9 (1997) 456-459. [56] T.Hanazawa et al.. Increased Nitrotyrosine in Exhaled Breath Condensate of Patients with Asthma. Am J Respir Crit Care Med 162 (2000) 1273-1276. [57] P.Reinhold et al.. Breath condensate--a medium obtained by a noninvasive method for the detection of inflammation mediators of the lung. Berl. Munch. Tierarztl. Wochenschr. 112(1999) 254-259. [58] R.Dworski, Sheller JR. Urinary mediators and asthma. Clin. Exp. Allergy. 28 (1998) 1309-1312. [59] S.O'Sullivan et al.. Urinary excretion of inflammatory mediators during allergen-induced early and late phase asthmatic reactions. Clin. Exp. Allergy. 28 (1998) 1332-1339. [60] A.J.Macfarlane et al.. Sputum cysteinyl leukotrienes increase 24 hours after allergen inhalation in atopic asthmatics. Am. J. Respir. Crit. Care. Med. 161 (2000) 1553-1558. [61] T.Hanazawa et al.. Nitrotyrosine and cystenyl leukotrienes in breath condensates are increased after withdrawal of steroid treatment in patients with asthma. Am. J. Respir. Crit. Care. Med. 161 (2000) A919. [62] J.D.Morrow, Roberts LJ. The isoprostanes: unique bioactive products of lipid peroxidation. Prog. Lipid Res. 36(1997) 1-21. [63] P.Montuschi et al.. Increased 8-Isoprostane, a Marker of Oxidative Stress, in Exhaled Condensate of Asthma Patients. Am. J. Respir. Crit. Care. Med 160 (1999) 216-220. [64] P.Montuschi et al.. 8-Isoprostane as a biomarker of oxidative stress in interstitial lung diseases. Am. J. Respir. Crit. Care. Med 158 (1998) 1524-1527. [65] L.J.Roberts, Morrow JD. Measurement of F(2)-isoprostanes as an index of oxidative stress in vivo. Free. Radio. Biol. Med. 28 (2000) 505-513. [66] T.A.Mori et al.. Effect of dietary fish and exercise training on urinary F2-isoprostane excretion in noninsulin-dependent diabetic patients. Metabolism. 48 (1999) 1402-1408.

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[67] C.T.Carpenter et al.. Exhaled breath condensate isoprostanes are elevated in patients with acute lung injury or ARDS. Chest. 114 (1998) 1653-1659. [68]

K.Marangon et al.. Comparison of the effect of alpha-lipoic acid and alpha-tocopherol supplementation on measures of oxidative stress. Free. Radic. Biol. Med. 27 (1999) 1114-1121.

[69] L.M.Landino et al.. Peroxynitrite, the coupling product of nitric oxide and superoxide, activates prostaglandin biosynthesis. Proc. Natl. Acad. Sci. U. S. A. 93 (1996) 15069-15074. [70]

L.G.Wood et al.. Lipid peroxidation as determined by plasma isoprostanes is related to disease severity in mild asthma. Lipids 35 (2000) 967-974.

[71] R.Dworski et al.. Allergen-induced synthesis of F(2)-isoprostanes in atopic asthmatics. Evidence for oxidant stress. Am J Respir Crit Care Med 160 (1999) 1947-1951.

Disease Markers in Exhaled Breath N. Marczin and M.H. Yacoub (Eds.) /OS Press, 2002

I 87

Extended NO analysis applied to patients with known altered values of exhaled NO Marieann HOGMAN Department of Medical Cell Biology; Section of Integrative Physiology, Uppsala University, Box 571,SE 75123 Uppsala, SWEDEN Abstract. If exhaled NO is measured at multiple flow rates it is possible to partition NO into airway and alveolar NO-levels. A further step can be taken by dividing the airway NO into wall concentration and diffusion rate of NO when an iterative NO analysis is applied. With this analysis it is shown that allergic asthmatics as well as patients with allergic rhinitis have increased diffusion rates of NO. Patients with COPD have increased alveolar levels of NO and these patients can be divided into two groups with regards to NO diffusion rate. A quality control feature is also introduced in order to gain acceptance for these measurements in clinical practice.

1. Introduction A new research field opened up when nitric oxide (NO) was discovered in exhaled breath [ 1 ]. It became even more interesting when NO was found in increased levels in inflammatory diseases such as asthma [2], allergic rhinitis [3], and chronic obstructive pulmonary disease (COPD) [4]. Scientists stared to argue about the source of the NO production. The NO measurements only reflected the whole respiratory system like a black box. One step forward was taken with the discovery of the flow dependence of the NO signal [5,6]. This led to the standardisation of the NO measurements, first in Europe [7] then in North America [8]. However, these recommendations did not spread any light on the source of NO in the respiratory system.

2. Extended NO analysis - theory Several research groups started to apply different models to the exhaled NO measurements [9,10]. An attempt to distinguish between the NO generated in the alveolar region and in the airways was made by Tsoukias & George [11]. A plot of the exhaled volume of NO versus the expiratory flow rate was used, where the slope of the curve represents the alveolar concentration and the intercept the airway NO-flux. The mechanism behind the flow dependency of the NO output from the lower airways has been suggested to be diffusion-related [11]. Applying a simplified model (Figure 1) and mathematics based on the classical Pick's 1st law of diffusion [12] to the concept of Tsoukias and George, we derived an equation for the NO concentration as a function of exhaled flow The model has three unknown variables FAA NO, FawNO and Daw NO. Here FAA NO L[13]. J represents the alveolar NO concentration, FawNO the NO concentration in the airway wall

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tissue and DawNO the airway transfer rate of NO. If enough data points of NO concentration at various flows are measured, it is possible to find the value of these three variables by a recursive least square fitting (LSF) algorithm [13-15]. However, LSF method can give erroneous values quite frequently. We therefore wanted to develop a simple and robust solution algorithm as an alternative for the LSF. Since the NO-measurements are meant to be a clinical tool we also wanted to introduce a data quality control feature. The model consists of an alveolar compartment, and a conductive airway compartment modelled as a cylindrical tube with a diffusion barrier layer between the tissue and airway (Figure 1). For the algorithms and the validation of the iteration NO analysis the reader is referred to reference [19]. The iteration algorithms can be run from a simple spreadsheet.

r

FANO - * • - > - > 4 4 4

-+•

-+

FENO

4

Figure 1. The model consists of an alveolar compartment with the NO fraction FANO. The airways are brought together into one big tube with an airway wall fraction, F,WNO with a diffusion rate D1WNO.

3. Extended NO analysis - practice When the slope-intercept method [11] was applied to exhaled NO values in patients with allergic asthma it was found that the increase in exhaled NO was generated in the airways and that the alveolar region had normal NO levels [16]. This is not surprising since asthma is an airway disease. In smokers, airway NO is decreased [17] and in alveolitis the alveolar NO is increased [18]. For the iteration NO analysis the guidelines for NO measurements [7] were followed, except for using three flows (0.005, 0.1 and 0.5 L-s"1) and no vital capacity manoeuvre since a deep breath with slow inhalation was found sufficient [13]. The analysis was applied to patients with known alterations in exhaled NO, e.g. asthma, allergic rhinitis, COPD, smokers and healthy subjects [19]. Patients with Sjogren's syndrome were also investigated. From Table 1 it can be seen that the reason for an increase in exhaled NO at an expiratory flow of 0.1 L-s"1 (FeNOo i) in asthma is due to an increased fraction in the airway wall together with an increased diffusion rate. Interestingly, the diffusion rate in allergic rhinitis was also increased but the airway wall showed normal values. Hence the increase in exhaled NO that Henriksen et al. found in patients with allergic rhinitis [3] might be explained by an altered diffusion rate.

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Table 1. Extended NO analysis applied to patients with known altered exhaled NO values. Data are from reference [19], except for the SjOgren's patients, and given in meaniSEM. Controls n=40 FENO0.i ppb FANO ppb DawNO nL'S" 1 FawNO ppb Quality value

Allergic rhinitis n=15

9±1

1312

21 1

211

8±0

12± 1* 98+ 10 8± 1

98 ±7 12± 1

allergic asthma n=15 18±3*

2± 1 12± 1* 144121*

5± 1

Sjdgren's syndrome n=5

12±3 4±1* 71 1 121 ±34 13± 1

ANOVA, different from control * p<0.05

Whether the bronchial hyperreactivity for indirect and direct stimuli is characterised by an increase in diffusion rate is not jet known. The allergic asthmatics were known to have an increase in bronchial hyperreactivity and many of the patients with allergic rhinitis have a hyperreactive airway. They were found to have increased diffusion rate. However, patients with Sjogren's syndrome have also bronchial hyperreactivity, but in the limited numbers of patients investigated the diffusion rate was normal. Sjogren's syndrome is a systemic disease and circulating cytokines might be the cause for an up-regulation of inducible NO-synthase in alveolar and airway tissue. The most interesting feature in the patients with COPD was a clear-cut difference in diffusion rate (Table 2). None of the COPD patients fell into the 95% confidence interval of the healthy controls. One could speculate that COPD could be divided into to two diseases like in asthma, e.g., atopic and non-atopic asthma. The patients with a high diffusion rate might have an asthmatic component to their disease. Further studies have to address this. These patients also had a significant increase in alveolar NO, unrelated to the diffusion rate. This might be a sign of ventilation/perfusion mismatch of the lung, known to be present in this disease. Other explanations must also be put forward, namely that NO can be upregulated in the alveolar region or that ther is a chemical interplay between NO and human haemoglobin, i.e., S-nitrosylation, that hypothetically can release NO into the gas phase [20], Further studies have to address these different possibilities. The decease in FawNO seen in the smokers is possibly due to a decrease in NOS activity [21]. Table 2. Extended NO analysis applied to patients COPD and divided according to diffusion rate, group A and B. NO-values for smokers are also given. For reference values see Table 1. Data are from reference [19] and given in mean±SEM. Smokers n=20 F E NO 0 , ppb FANO ppb DawNO nL-s"' FawNO ppb Quality value

6±1 2±1 8± 1 56 ±8 13 ± 1

COPD (A) n=10 19±5*

5± 1* 1612* 87119 51 1

COPD (B) n=10

81 It 31 l*t 41 l*t 135124

81 1

ANOVA, different from control * p<0.05 Table 1, different from group A t p<0.05

4. Summary By using an extended NO analysis on the exhaled NO measurements, for example the slope-intercept method or the iteration NO analysis, we can obtain knowledge from where in the respiratory system NO is produced. The drawback of the model based on Pick's first law of diffusion is that the airways cannot be divided up into small and large airways. However, the knowledge we can gain from the extended NO analysis, a non-invasive measurement,

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may justify the model. Utilizing our new iteration NO analysis we also obtain a quality control of the NO measurements which will come in useful when the method is applied in clinical practice. However, first we have to elucidate the relevance of an increase in diffusion rate, airway wall and alveolar NO concentration. We also need to study if disease symptoms or pharmaceutical treatment can affect these values. References [1] L.E. Gustafsson, A. Leone, M. Persson, N. Wiklund, S. Moncada, Endogenous nitric oxide is present in the exhaled air of rabbits, guinea pigs and humans, Biochem Biophys Res Comm 181 (1991) 852-857. [2] K. Alving, E. Weitzberg, J.M. Lundberg, Increased amount of nitric oxide in exhaled air of asthmatics. Eur Respir J 6(1993) 1368-1370. [3] A.H. Henriksen, M. Sue-Chu, T. Lingaas Holmen, A. Langhammar, L. Bjermer, Exhaled and nasal NO levels in allergic rhinitis: relation to sensitization, pollen season and bronchial hyperresponsiveness. Eur /tapir 713 (1999) 301-306. [4] M. Corradi, M. Majori, G.C. Cacciani, G.F. Consigli, E. deMunari, A. Pesci, Increased exhaled nitric oxide in patients with stable chronic obstructive pulmonary disease, Thorax 54 (1999) 572-575. [5] M.HOgman, S. Stromberg, U. Schedin, C. Frostell, G. Hedenstierna, L.E. Gustafsson, Nitric oxide from the human respiratory tract efficiently quantified by standardized single breath measurements, Ada PhysiolScand 159 (1997) 345-346. [6] P.E. Silkoff, P.A. McClean, A.S.Slutsky, H.G. Furlott, E. Hoffstein, S. Wakita, K.R. Chapman, J.P. Szalai, N. Zamel, Marked flow-dependence of exhaled nitric oxide using a new technique to exclude nasal nitric oxide, Am J Respir Crit Care Med 155 (1997) 260-267. [7] S.A. Kharitonov, K. Alving, P.J. Barnes, Exhaled and nasal nitric oxide measurements: recommendations, Eur Respir J10 (1997) 1683-1693. [8] A.S. Slutsky el at, Recommendations for standardized procedures for the online and offline measurement of exhaled lower respiratory nitric oxide and nasal nitric oxide in adults and children - 1999, Am J Respir Crit Care Med 160 (1999) 2104-2117. [9] R.A. Jorres, H. Sonnemann, A.M. Kirsten, K. Richter, H. Magnussen, Effective mucosal nitric oxide concentrations derived from exhaled air in asthmatic patients with and without steroids, Eur Respir J 10 (1997) 159s. [10] R.W. Hyde, E.J. Geigel, A.J. Olszowka, J.A. Krasney, R.E. Forster II, M.J. Uteli, M.W. Frampton, Determination of production of nitric oxide by lower airways of humans- theory, J Appl Physiol 82. (1997)1290-1296. [11] N.M. Tsoukias, S.C. George, A two-compartment model of pulmonary nitric oxide exchange dynamics, J Appl Physiol 85 (1998) 653-666. [12] A. Pick, Die Medizinische Physik, Druck und Verlag von Friedrich Vieweg und son, Braunschweig. 1856, pp. 19-52. [13] M. HOgman, N. Drca, C. Ehrstedt, P. Merilainen, Exhaled nitric oxide partitioned into alveolar, lower airways and nasal contributions, Respir Med 94 (2000) 985-991. [14] P.E. Silkoff, J.T. Sylvester, N. Zamel, S. Permutt, Airway nitric oxide diffusion in asthma. Role in pulmonary function and bronchial responsiveness, Am J Respir Crit Care Med 161 (2000) 1218-1228. [15] R.A. J6rres, Modelling the production of nitric oxide within the human airways, Eur Respir J 16 (2000) 555-560. [16] M. Hogman, S.D. Anderson, L. Hakansson, D. Ludviksdottir, P. Merilainen, S.C. George, Increased airway production of nitric oxide in asthmatics determined by elimination rate flow diagram, Am J Respir Crit Care Med 159 (1999) A862. [17] M. HSgman, T. Holmkvist, P. Merilainen, R. Walinder, D. Ludviksdottir, L. Hakansson, H. HedenstrSm, Smokers increase the NO-flux from the airways to normal levels after four weeks of tobacco cessation. Eur Respir J\4(\999) 445. [18] L. LehtimaJd, H. Kankaanranta, S. Saarelainen, P. Hahtola, R. Jarvenpaa, T. Koivula, V. Turjanmaa, E. Moilanen, Extended exhaled NO measurement differentiates between alveolar and bronchial inflammation, Am J Respir Crit Care Med 163 (2001) 1557-1561. [19] M. Hogman, T. Holmkvist, T. Wegener, M. Emtner, M. Andersson, H. HedenstrOm, P. Merilainen, Iteration NO analysis. Extended NO analysis applied to patients with COPD, allergic asthma and allergic rhinitis, Resp Med; 96 (2002) 24-30. [20] A.J. Gow, B.P. Luchsinger, J.R. Pawloski, D.J. Singel, J.S. Stamler, The oxyhemoglobin reaction of nitric oxide, Proc NatlAcadSci USA % (1999) 9027-9032. [21] J. Assreuy, F.Q. Cunha, F.Y. Liew, S. Moncada, Feedback inhibition of nitric oxide synthase activity by nitric oxide, BrJPharmacol 108 (1993) 833-837.

Disease Markers in Exhaled Breath N. Marczin and M.H. Yacoub (Eds.) 1OS Press, 2002

Exhaled Nitric Oxide and Atopy Christina GRATZIOU Head of Asthma and Allergy Centre Pulmonary and Critical Care Department, Medical School of Athens University, Greece.

Abstract. Exhaled NO appears a useful noninvasive marker of airway inflammation that offers complementary information for monitoring asthma or allergic rhinitis. However, recent data support the view that exhaled NO mainly reflects the allergic origin of an inflammation of the airways, since it is elevated only in atopic patients

Endogenous Nitric oxide (NO) is detectable in the exhaled air of normal individuals [1,2]. Several recent studies have demonstrated that the concentration of exhaled NO is increased in patients with inflammatory disorders of the airways, such as asthma [3-6], bronchiectasis [7] and upper or lower respiratory tract infection [8]. NO is generated from L-arginine by a family of enzymes, the nitric oxide synthases (NOS) [9]. Three distinct isoforms of human NO-synthase have been cloned. The isoforms of endothelial and neuronal NO synthase are the constitutively expressed forms (cNOS) and changes in their activity regulate vascular tone, platelet activation and neurotransmission. The third isoform, the inducible NO synthase (iNOS, is only expressed after induction by certain inflammatory cytokines or by bacterial lipopolysaccharide by epithelial and infiltrative inflammatory cells [9,10]. Inducible NO synthase has been localized in the airway and alveolar epithelium, the vascular endothelium, the smooth muscle and alveolar macrophages. The lungs of healthy human subjects produce low but detectable levels of NO which presumably originate from cNOS activity. In asthmatic patients, the increase in exhaled NO may reflect increased expression of iNOS in response to inflammatory stimuli, such as cytokines [11]. Indeed, iNOS has been detected immunocytochemically in the airway epithelium of asthmatic patients but not in normal individuals [12]. Corticosteroids, which control inflammation in asthmatic airways, have also been shown to inhibit the expression of iNOS but not of cNOS [13,14,15], directly and/or indirectly by reduction in the levels of stimulatory cytokines. Treatment with corticosteroids decreases exhaled NO in a dose dependent manner in patients with stable asthma and in those suffering an acute exacerbation i but not in normal subjectst [16,17]. The above findings suggest that exhaled NO may be used as a surrogate marker of airway inflammation In patients with difficult asthma and moderate to severe symptoms, despite high doses of anti-inflammatory treatment, increased levels of exhaled NO are found. A possible explanation for this finding is that there is a persistent airway inflammation in this group of patients with more severe disease and exhaled NO reflects the intensity of inflammation and the non-response to steroid treatment. Increased levels of nasal and exhaled NO have also been observed by some investigators in symptomatic allergic rhinitis, which may reflect an inflammation of the lower airways [18,19]. This increase can be modulated by local treatment with nasal steroids [20,21].

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Figure 1 : Exhaled NO in atopic and non-atopic patients with asthma and /or rhinitis and in normal subjects, (mean value in ppb). * P<0.05

All these findings suggest that NO in exhaled air may be used as a marker of airway inflammation in asthma or rhinitis and as a useful index for monitoring anti-inflammatory treatment [22]. However, in these previous studies, either atopic asthmatics were mainly included or the atopic condition was not defined and, consequently, no conclusion could be drawn about the exhaled NO levels in non-atopic asthmatics. In a recently presented paper by our group [23] increased exhaled NO levels have been observed only in atopic patients with stable asthma and/or rhinitis and not in non-atopic patients (Figure 1). Non-atopic asthmatics had statistically significant lower levels of NO detected in exhaled air, reaching the levels found in normal subjects.Contrary to our findings, Pearson et al. found no difference in a small group of subjects in peak NO concentration between allergic and non-allergic asthmatics [4]. However, the results our study were based on a large population and there was a clear distinction of patients with asthma in atopies and non-atopies. Higher doses of exhaled NO are found also in children with asthma. However, these higher levels seem to be more correlated to the existence of the atopic condition. According to the results of an epidemiological study in children population exhaled NO levels were higher in atopic children with asthma than in non atopic children with upper or lower airways disease (Figure 2) [24]. Based on our findings, one could hypothesize that it is the atopic condition per se that increases the NO production. However, no difference was found in NO detected in exhaled air between atopic and non-atopic normal subjects. Thus, the results of the present study indicate that both atopy and upper or lower airways disease are necessary conditions that may cause higher NO in exhaled air. The findings of this study may suggest that it is rather the allergic nature of an inflammation of the airways that is mainly responsible for the higher NO production in the lower airways. Previous studies also provide evidence that allergic inflammation increases NO production. The experimental allergen challenge in asthmatic subjects causes a further increase of the already elevated exhaled NO concentration during the late asthmatic reaction [25]. Furthermore, increased exhaled and nasal nitric oxide were also found in subjects

Ch. Gmtziou / Exhaled Nitric Oxide and Atopy

I93

Figure 2. Exhaled NO in atopic and non-atopic children with respiratory symptoms (mean value in ppb). * PO.05

working in animal laboratories with early sensitization to animal allergens without clinical evidence of asthma [26], suggesting the important role of allergen exposure and sensitization in the pathophysiology of NO production. Undoubtedly, the important question is what the underlying mechanism of our findings is. Our experimental design does not allow us to figure out why solely atopic and not non-atopic patients with asthma and/or rhinitis under stable conditions have elevated exhaled NO levels. It has been suggested that the increase of nitric oxide levels in atopic asthmatics and in patients with allergic rhinitis above those found in controls, would be consistent with the induction of inducible nitric oxide synthase found in association with mucosal mast cell, eosinophil, and T-lymphocyte activation, that characterize allergic rhinitis and asthma [27,28]. An upregulation of expression of inducible nitric oxide synthase has been observed in bronchial biopsies in asthmatics [12,29] and immunocytochemical studies have also demonstrated expression of NO synthases in human nasal mucosa [30]. According to our results, it is tempting to speculate that the inflammatory process in the non-atopic patients does not induce iNOS efficiently, in contrast to the inflammatory milieu of atopic asthmatics. In fact, there is conflicting data regarding the differences in the immunopathogenesis of these two clinically distinct types of intrinsic and extrinsic asthma. Thus, there are some investigators who found a lot of similarities in the pathophysiological profile between the two types of the disease [31,32], based on bronchoalveolar lavage or biopsies data, looking at Tcells phenotype, differential expression of cytokines, (such as interleukin-8, Interleukin-5, interleukin-4, interferon gamma), and chemokines, (such as RANTES, MCP3). On the other hand, there are studies which present differences in the pathophysiological inflammatory process [33,34,35] supporting our theory. In addition, no studies have concentrated on the role of the epithelium in atopic and non-atopic asthma that may be very important and thus require investigation. In addition, further investigation regarding the iNOS expression in bronchial tissue in atopic and non-atopic patients with asthma or rhinitis is, of course, needed to clarify the pathophysiological mechanism that might explain our findings. In conclusion, exhaled NO is a useful noninvasive marker of airways inflammation that is useful in clinical practice and offers complementary informations for monitoring asthma or allergic rhinitis (picture 1) However recent data support the view that exhaled NO mainly reflects the allergic origin of an inflammation of the airways, since it is elevated only in atopic patients. It is therefore suggested that atopy should be defined before using NO measurements as an index for monitoring airway inflammation, in patients with asthma and/or

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ASTHMA Acute A Chronic [j Airway InflammatJonV Inflammation V Remodelling

{

Physiological parameters }

Airway ^_ narrowing

BHR

4 Fixed

airway obstruction

Clinical parameters Inflammatory markers (eNO , sputum eosinophils) rhinitis under stable conditions. Further studies are needed to delineate the molecular mechanisms of this different NO production in the lower airways between atopic and nonatopic subjects with airway diseases like asthma or rhinitis.

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 13. 14. 15.

GustafFson LE, Leone AM, Persson M, Wiklund NP, Moncada S. Endogenous nitric oxide is present in the exhaled air of rabbits, guinea pigs and humans. Biochem Biophys Res Commun 1991; 181:852-7. Borland C, Cox Y, Higenbottam T. Measurement of exhaled nitric oxide in man. Thorax 1993; 4:11602. Alving K, Weitzberg E, Lundberg JM. Increased amount of nitric oxide in exhaled air of asthmatics. Eur Respir J 1993; 6:1368-70. Pearson MG, Zetterstrom O, Argenius V, Ihre E, Gustafsson LE. Single breath oxide measurements in asthmatic patients and smokers. Lancet 1994; 343:146-147. Kharitonov SA, Yates D, Robbins RA, Logan-Sinclair R, Shineboume EA, Barnes PJ. Increased nitric oxide in exhaled air of asthmatic patients. Lancet 1994- 343.133-5. Barnes P, Belvisi MG. Nitric oxide and lung disease. Thorax 1993; 4:1034-1043. Kharitonov SA, Wells AU, O'Connor BJ, Hansell DM, Cole PJ, Barnes PJ. Elevated levels of exhaled nitric oxide in bronchiectasis. Am J Respir Crit Care Med 1995; 151:1889-93. Kharitonov SA, Yates DH, Barnes PJ. Increased nitric oxide in exhaled air of normal human subjects with upper respiratory tract infections. Eur.Respir.J 1995; 8:295-297. Jorens PG, Vermeire PA, Herman AG. L-arginine dependent nitric oxide synthase: a new metabolic pathway in the lung and airways. Eur.Respir.J. 1993; 6:258-266. Gaston B, Drazen JM, Loscalzo J, Stamler JS. The biology of nitrogen oxides in the airways. Am JRespir Crit Care Med 1994; 149:538-551. Moncada S, Highs EA The L-arginine nitric oxide pathway. N Eng J Med 1993; 329 (27):2002-12. Ham id Q, Springall DR, Riveros-Moreno V, et al. Induction of nitric oxide synthase in asthma. Lancet 1993; 342:1510-3. Massaro AF, Gaston B., Kita D, Fanta C, Stamler JS, and Drazen JM. Expired nitric oxide levels during treatment of acute asthma. Am J Respir Crit Care Med 1995;152:800-803. Di Rosa M., Radomski M., Carnuccio R.,Moncada S. Glucocorticoids inhibit the induction of nitric oxide synthase in macrophages. Biochem Biophys Res Commun 1990; 172:1246-1252. Yates DH, Kharitonov SA, Robbins A, Thomas PS, Barnes PJ. Effect of a nitric oxide synthase inhibitor and a glycocorticosteroid on exhaled nitric oxide. Am J Respir Crit Care Med 1995; 152:892896.

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16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31.

32.

33. 34. 35.

i 95

Kharitonov SA, Yates DH, Barnes PJ. Inhaled glucocorticoids decrease nitric oxide. Am J Respir Crit CareMed 1996; 153:454-457. Kharitonov SA, Yates DH, Barnes PJ. Changes in the dose if inhaled steroid affect exhaled nitric oxide levels in asthmatic patients. Eur J Respir Dis 1996;9:196-201 Martin U, Bryden K, Devoy M, Howarth P. Increased levels of exhaled nitric oxide during nasal and oral breathing in subjects with seasonal rhinitis. J Allergy Clin Immunol 1996;97(3):768-772. Arnal JF, Didier Abram J, M'Rini C., Chcarlet JP, Serrano E., Bescombes JP Nasal nitric oxide is increased in allergic rhinitis. Clin Experim Allergy 1997;358-63. Kharitonov SA, Rajakulasingam K, O'Connor B, Durham SR, Barnes P. Nasal nitric oxide is increased in patients with asthma and allergic rhinitis and may be modulated by nasal glucocorticoids. J Allergy Clin Immunol 1997;9(l):58-64. Gratziou Ch, Rovina N, Lignos M, Vogiatzis I and Roussos Ch. Exhaled Nitric oxide in seasonal Allergic Rhinitis: Influence of pollen season and therapy. Clin. Exp. Allergy 2000;31:1-9. Barnes PJ, and Liew 1995, Nitric oxide and asthmatic inflammation. Immunol Today 1995; 16:OSISO. Gratziou Ch, Lignos M, Dassiou M, Roussos Ch. Influence of atopy on exhaled nitric oxide in patients with stable asthma and rhinitis. Eur Respir J 1999; 14:897-901. Gratziou Ch, Katsardis Ch, Lignos M, Rovina N, Dassiou M, Alexandri A, Tsakiris A, Roussos Ch. Exhaled nitric oxide in children:Relationship with chronic respiratory problems and atopy. Eur Resp J.1999;14:Suppl30:450s. Kharitonov SA, O'Connor BJ, Evans DJ, Barnes PJ. Allergen-induced late asthmatic reactions are associated with elevation of exhaled nitric oxide. Am J Respir Crit Care Med 1995;151:1894-9. Adisesh LA, Kharitonov SA, Yates DH, Snashell DC, Newman- Taylor AJ, and Barnes PJ. Exhaled and nasal nitric oxide is increased in laboratory animal allergy. Clin Exp Allergy 1998,28:876-880. Howarth PH, Wilson J, Djukanovic R, et al. Airway inflammation and atopic asthma: a comparative bronchoscopic investigation. Int Arch Allergy Appl Immunol 1991 ;4:266-9. Jatakanon A, Lim S, Kharitonov SA, Chung KF, Barnes PJ. Correlation betwee exhaled nitric oxide, sputum eosinophils, and metacholine responsiveness in patients with mild asthma. Thorax 1998;53:9195. Robbins RA, Barnes PJ, Springall DR, et al. Expression of inducible nitric oxide synthase in human bronchial epithelial cells. Biochem Biophys Res Commun 1994; 203:209-18. Furukawa K, Harrison D, Saleh D, Shennib H, Chagnon F, Giaid A. Expression of nitric oxide synthase in human nasal mucosa Am J Respir Crit Care Med 1996; 153:847-850. Humbert M., Durham SR., Ying S., Kimmit p. , Barkans. Assoufi B., Pfister R., Menz G., Robinson DS., Kay BA., Corrigan C.IL-4 and IL-5 mRNA and protein in bronchial biopsies from atopic and nonatopic asthma:evidence againsit "intrinsic" asthma being a distinct immunopathologiacal disease. Am J Respir Crit Care Med 1996;154:1497-1504. Humbert M., Ying S., Corrigan C., Menz G., Barkans J, Pfister R., Meng Q., Van Damme J., Opdenakker G., Durham SR., Kay BA. Bronchial mucosal expression of the genes encoding chemokines Rantes and MCP-3 in symtomatic atopic and non-atopic asthmatics: relationship to the eosinophil-active cytokines interleukin-5, granulocyte macrophage-colony-stimulating factor, and IL-3. Am J Respire Cell Moll Biol 1997;16:l-8. Walker C, Bode E., Boer L., Hansell TT, Blaser K., Virchow JC. Allergic and non-allergic asthma have distinct patterns of T -cell activation and cytokine production in peripheral blood and BAL. Am Rev Respir Dis 1992;146: 109-115. Folkard SG, Westwick J., Millar AB. Production of interleukin-8, Rantes and MCP-1 in intrinsic and extrinsic asthmatics. Eur Respir J 1997;10:2097 -2104. Tang C, Rolland LM, Ward C, Quan B and Walters EH. 11-5 production by bronchoalveolar lavage and peripheral blood mononuclear cells in asthma and atopy. Eur Respir J 1997; 10:624-632.

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Disease Markers in Exhaled Breath N. Marczin and M.H. Yacoub (Eds.) IOS Press, 2002

Bradykinin and exhaled nitric oxide

in asthma Fabio L.M. RICCIARDOLOU and Peter J. STERK2 Dept. of Respiratory Disease, Ospedali Riuniti di Bergamo, Italy; 2 Dept. of Pulmonology, Leiden University Medical Center, The Netherlands !

Abstract Nitric oxide (NO) is formed by the enzyme NO synthase (NOS), functionally differentiated in constitutive (cNOS) and inducible (iNOS) isoforms. cNOS is activated by calcium (Ca2+>dependent mechanisms upon receptor stimulation by several agonists, including bradykinin. iNOS is induced at pretranslational level by pro-inflammatory cytokines, such as IL-IB and TNFa, provoking a Ca2+-independent release of high levels of NO inhibited by corticosteroids. NO is detectable in the exhaled air of healthy humans and is increased in atopic asthma. Recently, it has been shown that increased levels of exhaled NO in asthmatics are related to the increased expression of iNOS. Bradykinin (BK) is a mediator formed in airway mucosa from plasma kininogens and is a potent bronchoconstrictor in asthma. Recently, the capability of inhaled BK has been shown to rapidly release NO by activation of cNOS reducing its own bronchoconstriction in asthmatics (bronchoprotective effect). Furthermore, BK is also able to rapidly reduce exhaled NO, an index of iNOS expression, in stable asthma. iNOS is upregulated by the cytokines released in allergic inflammation, and allergen exposure produces long-term increase of exhaled NO in atopic asthma. The increased exhaled NO production in allergen-induced asthma exacerbation is also inhibited by aerosolized bradykinin. We conclude that bradykinin has dual effects on NO pathways in the airways of asthmatic subjects: activation of cNOS with subsequent bronchoprotective role and inhibition of excessive exhaled NO with over-expressed iNOS. This provides new opportunities for successful interventions in asthma by manipulating endogenous and physiological modulatory mechanisms.

1. Introduction Nitric oxide (NO) has been detected in the exhaled breath of humans by means of chemiluminescence method. Increased levels of exhaled NO have been described in patients with asthma as compared to healthy controls [1]. In asthma the increased levels of exhaled NO are related to the presence of atopy and airway eosinophilic inflammation. It has been reported that different physiological (acute airways obstruction and ventilatory level), proinflammatory (allergen and viruses) and other (smoking and ethanol) stimuli influence exhaled NO concentrations. This short review points to summarize the capability of bradykinin, a pro-inflammatory peptide, to modulate exhaled NO in asthma.

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197

2. Nitric oxide and bradykinin in the airways NO is formed by the enzyme NO synthase (NOS), of which constitutive (cNOS) and inducible (iNOS) isoforms have been described functionally in the airways [2]. cNOS rapidly releases small amounts of NO in response to increases in cytosolic Ca + upon membrane receptor stimulation by several agonists, including bradykinin [2]. The expression of iNOS is induced by pro-inflammatory cytokines, a process that results in a protracted and Ca2"independent release of high levels of NO [2], which is inhibited by corticosteroids [3]. Increased levels of NO in the exhaled air of asthmatics seem to be associated with expression of iNOS [3]. Bradykinin is an autacoid formed in the airway mucosa from plasma kininogens and is involved in the pathophysiology of asthma [2,4]. Bradykinin inhalation in asthmatics resembles an asthma attack producing bronchoconstriction, cough and chest tightness. Bradykinin-induced bronchoconstriction results from indirect (cholinergic and tachykininergic) mechanisms. Recently, it has been shown that bradykinin also activates epithelium-dependent bronchorelaxant mechanisms [2]. In addition to the release of the bronchodilator prostaglandin £2 (PGEi), another relaxant pathway activated by bradykinin is the release of cNOS-dependent NO from the airway epithelium [2]. In mild asthma bradykinin not only provokes indirect bronchoconstriction, but also releases bronchoprotective NO [2,4]. 3. Bradykinin and exhaled nitric oxide in asthma It is most likely that exhaled NO in asthma is related to the control of the disease rather than to the severity. In fact, inducers (allergen and viruses) of asthma exacerbation increase exhaled NO. Interestingly, exhaled NO levels are significantly reduced by 20-40% immediately after a 20% fall in FEV) by histamine, adenosine 5'-monophosphate, or hypertonic saline challenge in steroid-naive asthmatics [5]. Most importantly, during recovery, the changes in airway calibre and those in exhaled NO appeared to be positively correlated, suggesting that exhaled NO is modulated by the levels of airway obstruction. Additionaly, in stable mild asthma inhalation of bradykinin, prostaglandin £2 or p2 has been shown to reduce levels of exhaled NO irrespective of the level of bronchoconstriction [6], suggesting an inhibitor role of such molecules on NO production in the airways of asthmatics in the absence of disease activity. The late asthmatic response (LAR) after allergen exposure is characterized by bronchoconstriction associated with airway cellular inflammation [7]. Interestingly, it has been shown that the LAR is associated with elevated NO concentrations in the exhaled air [7], suggesting that exhaled NO may reflect allergic inflammation in asthmatic airways. The longterm increase in exhaled NO during the LAR could be explained by the action of proinflammatory cytokines, released from inflammatory cells, on iNOS expression. Increased exhaled NO during LAR is also inhibited by aerosolized bradykinin [8]. It has been hypothesized that bradykinin stimulates PGE2 release from airway epithelial cells, and that PGEz negatively modulates the induction of iNOS expression at pre- or post-translational level. An alternative explanation is that bradykinin, a potent vasodilator of the airway microcirculation, increases blood flow in the bronchial mucosal vessels, thereby enhancing the NO trapping by hemoglobin with subsequent reduction in external diffusion of NO.

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4. Conclusions Bradykinin is a well-known pro-inflammatory peptide with acute (detrimental as well as protective) effects in the airways. New evidence suggests a potential role for bradykinin in reducing excessive NO production with the subsequent inhibition of NO-derived inflammatory change in asthmatic airways. We may conclude that bradykinin has dual effects on NO pathways in the airways of asthmatic subjects: activation of cNOS with subsequent bronchoprotective role and inhibition of excessive exhaled NO with over-expressed iNOS. Such balanced, endogenous modulation of disease mechanisms in asthma may provide new possibilities for successful interventions. References [1] S.A. Kharitonov et ai, Increased nitric oxide in exhaled air of asthmatic patients, Lancet 343 (1994) 133135. [2] F.L.M. Ricciardolo et al., Allergen-induced impairment of bronchoprotective nitric oxide synthesis in asthma, J Allergy Clin Immunol 108 (2001) 198-204. [3] D. Saleh et ai, Increaesd formation of the potent oxidant peroxynitrite in the airways of asthmatic patients is associated with induction of nitric oxide synthase: effect of inhaled glucocorticoid, FASEBJ 12 (1998) 929937. [4] F.L.M. Ricciardolo et al.. Randomised double-blind placebo-controlled study of the effect of inhibition of nitric oxide synthesis in bradykinin-induced asthma, Lancet 348 (1996) 374-377. [5] H.W.F.M. de Gouw el al, Exhaled nitric oxide (NO) is reduced shortly after bronchoconstriction to direct and indirect stimuli in asthma, AmJRespir Crit Care Med 158 (1998) 315-319. [6] S.A. Kharitonov et al., Prostaglandins mediate bradykinin-induced reduction of exhaled nitric oxide in asthma, Ear Respir J 14(1999) 1023-1027. [7] S.A. Kharitonov et ai, Allergen-induced late asthmatic reactions are associated with elevation of exhaled nitric oxide, AmJRespir Crit Care Med 151 (1995) 1894-1899. [8] F.L.M. Ricciardolo et ai, Effects of inhaled bradykinin and allergen on exhaled nitric oxide in relation to airway caliber in atopic asthma, Am J Respir Crit Care Med 163 (2001) A435

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Exhaled NO is an Optimal Marker of Severity and Responsiveness to Therapy in Asthma Serpil C. ERZURUM Pulmonary and Critical Care Medicine Cancer Biology Lerner Research Institute Cleveland Clinic Foundation Cleveland Ohio 44195 U.S.A. Abstract. Exhaled NO is derived from endogenous NO synthesis in the healthy lung by nitric oxide synthases (NOS) which convert L-arginine to L-citrulline and NO. Asthmatic individuals have higher than normal exhaled NO related to upregulation of expression of the NOS 2. Many studies support that exhaled NO is a sensitive marker of the effect of anti-inflammatory treatment and asthma deterioration.

1. Importance of monitoring inflammation and oxidative stress in asthma Airway inflammation, a defining characteristic of asthma, is the primary target in recent guidelines for asthma care. Thus, assessment of degree of airway inflammation is useful in designing asthma treatment plans. Standard diagnosis and monitoring of asthma are based upon airflow measures, including home peak expiratory flow rate diaries, spirometry, and bronchial challenge, which indirectly and often inconsistently reflect changes in airway inflammation. The gold standard for assessment of airway inflammation, especially in research studies, is sampling inflammatory cells or lung tissue via bronchoscopy. However, invasive approaches carry risk and are high cost, making this unacceptable for widespread application to patients. Because exhaled NO is easy to measure, safe, reproducible and relatively simple to perform, it has been proposed as an ideal test to monitor lower airway inflammation, and in determining response to anti-inflammatory therapy [1-4]. 2. Measurement of exhaled NO Exhaled NO is usually measured by chemiluminescent analyzers which utilize a photochemical reaction between NO and ozone generated in the analyzer. The technique is accurate and specific as confirmed by gas chromatography mass spectrometry. Both

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online and offline methods have been standardized, guidelines published, and are widely used [5]. Standardization of methods allows measures to be reproducible in clinical labs between different centers, making this a useful test for monitoring asthma severity and response to therapy. 3. Relationship of NO levels to asthma severity and response to therapy Exhaled NO is increased in asthma [1 - 4, 6]. NO correlates with bronchial reactivity, e.g. the provocative concentration that causes a 20% decrease in FEV| (PCio) for histamine or methacholine. Furthermore, exercise induced bronchoconstriction is associated with higher levels of NO. Other studies have shown that exhaled NO correlates with sputum eosinophils, a certain marker of inflammation in the airway. Invasive studies using bronchoalveolar lavage, demonstrate that NO is correlated to inflammatory markers in bronchoalveolar lavage fluid, including eosinophils and cytokines. Inhaled corticosteroids reduce eosinophils and NO levels. In this context, anti-inflammatory therapy with leukotriene pathway modifiers or the soluble IL-4 receptor also reduce exhaled NO, while lung function improves. On the other hand, withdrawal of corticosteroid leads to increase of exhaled NO in those patients that have deterioration of lung function [1-4].

4. Molecular regulation of NO synthesis NO is increased in asthma due to increased expression of NOS2 in the asthmatic airway epithelial cells. NOS 2 is subject to predominantly transcriptional regulation by proinflammatory cytokines [6]. NOS 2 mRNA expression in asthmatic airway epithelium is higher than controls in vivo, but not increased in asthmatics receiving inhaled corticosteroid. Several studies have shown that inhaled or intravenous corticosteroids reduce exhaled NO. In situ analysis of the asthmatic airway suggests that NOS 2 expression is reduced by corticosteroids, concomitant with reduction of exhaled NO. Glucocorticoids inhibit NOS 2 expression at multiple levels including inhibition of gene transcription, reduction of mRNA translation and increased degradation of NOS 2 protein. Increased NOS 2 mRNA in asthma, which is down regulated by corticosteroid, supports that NOS 2 expression and subsequent generation of NO is a sensitive meter of airway inflammation.

5. Conclusions NO is a sensitive marker of airway inflammation. It is a potentially useful test for monitoring severity of asthma, response to therapy, or clinical deterioration with withdrawal of anti-inflammatory therapy.

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References

[1] P. E. Silkoff. Noninvasive measurement of airway inflammation using exhaled nitric oxide and induced sputum. Clinics in Chest Medicine 21 (2000) 345. [2] S. A. Kharitonov, and P. J. Barnes. Clinical aspects of exhaled nitric oxide. Eur Resp J16 (2000) 781. [3] S. P. Sanders. Nitric oxide in asthma: Pathogenic, therapeutic or diagnostic? Am JRespir Cell Mol Biol 21(1999)147. [4] S. A. Kharitonov, and P. J. Barnes. Exhaled markers of pulmonary disease. Am J Resp Crit Care Med 163(2001) 1693. [5] Recommendations for a standardized procedure for the online measurement of exhaled nitric oxide in adults. In: The official statement of the American Thoracic Society Recommendations for the standardized procedures for the online and offline measurement of exhaled lower respiratory nitric oxide and nasal nitric oxide in asults and children-1999. Am J Respir Crit Care Med 160 (1999) 2104. [6] F.H. Guo, and S.C. Erzurum. Regulation of inducible nitric oxide synthase in human airway epithelium. Environmental Health Perspectives 106 (1998) 1119.

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Superoxide-NO Interactions in Paranasal Sinus Inflammatory Diseases Jacques KAMI Service d'Explorations Fonctionnelles Physiologqucs-Exploration FonctionneUe Rfspiratom, CHU Rangueil, 31403 Toulouse Cedex, France

The cavities that constitute the para-nasal sinuses are lined with a pseudostrarified ciliated epithelium covered by a film of mucus, which open in the nasal cavity by ostia. In spite of the proximity with a highly coloni2ed cavity, sinuses are free of bacterial colonisation and are considered sterile. The traditional mechanisms of defense of the paranasal sinuses associate [1]: (1) the epithelial barrier and the mucodliary system whose function seems stimulated by nitric oxide (NO), allowing mucus to be continuously removed towards nasal cavities via the ostia; (2) the acquired defense annexed to the nasal mucous membrane widi numerous immune cells in the corion, responsible for immunoglobulin secretion, in particular IgA; (3) non-specific innate defense. Three large mechanisms of non-specific innate defense are concerned in response to an aggression. These defenses consist in the rush of cells (monocyte-macrophage and polymorphonuclears) producing highly toxic reactive derivatives involving the following enzymatic reactions: (1) through N ADPH-oxidase, allowing the synthesis of reactive oxygen species (ROS), including superoxide anion O2~; the O2" half-life is very short (about a microsecond). In an inflammatory context, the NADPH-oxidase of the macrophages and polynuclear neutrophils and eosinophils produce large amounts of O2''. To defend against the oxidising stress rising from the generation of O2~', cells express several enzyme families as the superoxide dismutases (SOD), which catalyse the dismutation of O2" into hydrogen peroxide (HjOj); (2) through myeloperoxidase, which generates HOC1 after interaction between nitrite and CINO2, highly reactive [2]; (3) through NO-synthases, which generates NO. NO-synthases (NOS) constitute a multigenic enzyme family, which are either "constitutive" or "inducible". The first are expressed permanently in the endothelium (type III) and various types of neurons (type I). The monocyte-macrophage and polymorphonuclears express a third isoforme, which is known as "inducible" (type II), in response to pro-inflammatory cytokines. Several interactions can occur between these reactive oxygen species. Some components, originating from NO and O2~ interaction, have oxidizing, mutagenic and cytotoxic properties contrasting with antioxidant, anti-inflammatory and cyto-protective properties of NO. The reaction between NO and O2"' is very fast, and is three times higher than the O2" dismutation by SOD [3]. In addition to the reciprocal inactivation of the two molecules, the

/. Rami / Superoxide-NO Interactions in Paranasal Sinus Inflammatory Diseases

Nonallergic polyposis

Allergic polyposis

Chronic sinusitis

Kartagener's syndrome

Figure 1. Nasal nitric oxide concentration (parts per billion (ppb)) in control subjects (C) and in patients with nasal nonallergic and allergic polyposis, chronic sinusitis and Kartagener's syndrome. *: p<0.05 versus control; #: p<0.05 versus nonallergic polyposis (from Reference 5),

interaction between NO and O2~" lead to, if stoichiometric conditions are favorable (ratio NO/O2"' — 1), peroxynitrite (ONOO") which can break up in its turn and generate the radical hydroxyl (OH). In man, type II NO-synthase is mainly expressed in the epithelium of the paranasal sinuses. The type II NO-synthase term is better than inducible NO-synthase that can lead to confusion because it has been clearly demonstrated that this enzyme is "constitutively" expressed in the normal sinus epithelium. The activity of this NO-synthase is significant, and NO concentrations of 10-20 ppm have been measured in normal maxillary sinuses [4]. These NO concentrations are sufficient to inhibit the growth of several bacteria, suggesting that NO plays a primordial role in the preservation of paranasal sinus sterility [6]. In the majority of the cavities (pulmonary alveolis, peritoneal cavity ), the first line of defense is represented by resident macrophages. These macrophages generate reactive species of oxygen which kill the pathogenic agents that they phagocyte [7]. Any inflammatory stimuli leads macrophages to express type II NO-synthase (inducible). So, NO seems to be a second line of defense against hard-to-kill pathogenic agents. A strictly opposite situation could characterize para-nasal sinus cavities. The first line of host defense is represented by NO produced continuously by para-nasal sinus epithelium, while resident macrophages are absent under normal conditions. However, chronic sinusitis is characterized by the recruitment of phagocytic cells, in particular polynuclear eosinophils [1, 5]. These latter express very strongly the NADPH-oxydase, and can generate large amounts of O2"° [7]. We showed that the nasal concentrations of NO is decreased during chronic sinusitis and significantly in non-allergic patients with nasal polyposis (Fig. 1). The drop in nasal NO in some patients could involve a decreased production (fall of the local synthesis: local vascularisation, NOS, substrate modifications) and/or an increased breakdown of nitric oxide by inflammation-derived reactive oxygen species [3]. It has been shown that the NO concentrations found in normal sinuses are sufficient to inhibit the growth of several bacteria [6, 8]. This reinforces the idea that in normal conditions, NO represents the first line of defense of the paranasal sinuses. Indirect evidence is supported by the dramatic decrease of nasal NO concentration in patients with Kartagener's syndrome (referred to as "an immobile cilia syndrome" and characterized by situs inversus, sinusitis, and bronchiectasis[5, 9] or by the evaluation of the relationship between NO concentrations and

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120

s

•s (0

o.

no TPA

+ SOD

+DPI

+cat

+LNAME

TPA Figure 2. Characterisation of lucigenin-elicited chemiluminescent signals from nasal polyps. SOD - superoxide dismutase (100 U/ml), DPI = diphenyleneiodonium (100 uM), cat = catalase (2,000 U/ml), L-NAME = Nwnitro-L-arginine methyl ester (100 uM). Data are means of triplicate incubations and representative of two separate experiments, (from Reference 10).

nasal polyp production of O2" [10]. In this last work we studied eosinophil infiltration, fibrosis and O2~" production by lucigenin-enhanced chemiluminescence in polyp fragments from patients (fig. 2). Basal and phorbol ester-stimulated O2~ production varied largely among patients, but both were highly related to eosinophilic infiltration. A slow releasing NO donor DETA NONOate (DETA/NO NOC-18) dose dependently inhibited lucigeninenhanced chemiluminescence from phorbol ester-stimulated polyp fragments (EC^ 1.5 mM.). The NO concentration present in normal maxillary sinuses was estimated about 10 ppm (i.e., 0.5 uM in aqueous phase) [4]. Calculations revealed that the DETA NONOate 0.75 mM and 1.5 mM generate steady-state concentrations of NO of 0.5 uM and 2.5 uM respectively [11]. The NO concentration present in paranasal sinus appears to partially suppress (approximately 20-40%) O2 production from polyp eosinophils (Fig. 3)

Figure 3. The dose-response effect of DETA/NO NOC-18 was studied on the lucigenin-enhanced chemiluminescence generated by TPA-stimulated polyp fragments. Indicated concentrations of DETA NONOate were added 5 min before the addition of luM TPA. Data are expressed from TPA-stimukted polyp fragments obtained in the absence of NO donor (from Reference 10).

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However, the sensitivity of the various pathogens to NO, ROS, peroxynitrite, and the synergism and/or antagonism between these two mechanisms of defense require further study [8], and understanding of their respective roles in the pathophysiology of paranasal sinuses probably requires refined modeling. Finally, myeloperoxidase of eosinophils represents an alternative and potentially prominent mechanism of protein nitration [12]. This important question about the link between these mechanisms and the pathobiology of the paranasal sinuses also deserves specific treatment. In conclusion, it appears that the paranasal sinuses are an unusual anatomic site where NO accounts for the first line of nonspecific host defense, which could interfere and counteract the second line of defense constituted by the phagocytic cells. The NO concentrations found in paranasal sinuses appear to be within the critical range to inactivate the production of O2"' by phagocytes. In viwy this scheme could be complicated by additional events such as: (1) allergic rhinitis increases in nasal NO concentration [5, 13], which can be normalized upon glucocorticoid treatment [14]; (2) the potential generation of peroxynitrite, as the respective amounts of NO and O2 reach stoichiometry near 1 : 1, a condition favorable for the generation of peroxynitrite [15] ; and (3) the inhibition of apoptosis of certain phagocytic cells such as eosinophils by NO [16]. All three mechanisms could contribute to the chronicity of the inflammatory process and thus to the pathophysiology of polyposis. All these events should be analyzed in future studies as the evaluation of NO, O2~' and peroxynitrite production in nosocomial maxillary sinusitis.

References [I] M. Kaliner ttaL, Sinusitis: bench to bedside,] Allergy Clin ImmunolW (1997) S829-S848. [2] M. Trujillo et aL, Peroxynitrite biochemistry: formation, reactions and detection, Analusis 28 (2000) 518527. [3] H. Rubbo et aL, Nitric oxide regulation of tissue free radical injury, Chem Res ToxicoW (1996) 809-820. [4] J. Lundberg et aL, High nitric oxide production in human paranasal sinus, Nature Med 1 (1995) 370-373 [5] JF. Arnal et aL, Nasal nitric oxide concentration in paranasal sinus inflammatory diseases, Eur RespirJ., 13 (1999) 307-312. [6] R. Mancinelli and C. McKay, Effects of nitric oxide and nitrogen dioxide on bacterial growth, ApfilEnviron MicroM46 (1983) 198-202. P] B. Babior, Phagocytes and oxidative stress, Am] Med 109 (2000) 33-44. [8] S. Kaplan et aL, Effect of nitric oxide on staphylococcal killing and interactive effect with superoxide, Infect Immun. 64 (1996) 64-76. [9] J. Lundberg et aL, Primarily nasal origin of exhaled nitric oxide and absence in kartagener's syndrome, Eur RefirJ 7 (1994) 1501 -1504. [10] M. Pasto et aL, Nasal polyp-derived superoxide anion. Dose-dependent inhibition by nitric oxide and pathophysiological implications 163 (2001) 145-151. [II] V. Kharatinov et aL, Kinetics of nitric oxide autoxidation in aqueous solution, J EiolCbem 269 (1994) 58815883. [12] A. Van Der Vliet et aL, Reactive nitrogen species and tyrosine nitration in respiratory tinct,Ata]RespirCrit Can Med 160 (1999) 1-9. [13] JF. Arnal. etaL, Nasal nitric oxide is increased in allergic rhinitis, C&n Exf) Allergy 27 (1997) 358-362. [14] S Kharatinov. et aL, Nasal nitric oxide is increased in patients with asthma and allergic rhinitis and may be modulated by nasal glucocorticoids,/v4/£/gy C£ti Immunol99 (1997) 58-64. [15] S. Beckman and WH. Koppenol, Nitric oxide, superoxide and peroxynitrite: the good, the bad and the ug\j,AffiJPhysiolZ1\ (1996) C1424-C1437. [16] H. Hebestreit et aL; Disruption of fas receptor signaling by nitric oxide in oesinophils, / Exp Med 187 (1998)415-425

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Part III.

Chronic Lung Diseases

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Biology, Diagnosis and Management of COPD N. M. SIAFAKAS, G. CHRYSOFAKIS and N. TZANAKIS Department of Thoracic Medicine University General Hospital 71110 Heraklion, Greece Abstract. Chronic obstructive pulmonary disease (COPD) constitutes an enormous and growing health problem, the treatment of which has been less than satisfactory so far. It is a disease state characterized by an abnormal inflammatory process in the airway wall of the large and peripheral airways as well as in the parenchyma as a response to inhalation of several noxious agents including mainly the smoking. This inflammatory response is characterized by an increase in macrophages, neutrophils and T-lymphocytes with a predominance of CD8+ cells. Several inflammatory mediators are likely to play a role in COPD including LTB4, IL-8 and TNF-a. The inflammatory process in COPD is markedly different from that in asthma and requires different therapy. A diagnosis of COPD should be considered in any patient who has cough, sputum production, dyspnea and a history of current or ex-smoking or exposure to several risk factors. The diagnosis should be confirmed by an obstructive ventilatory disorder with spirometry. The effective management of COPD includes the assessment and monitoring of the disease, reducing risk factors with emphasis to smoking cessation, managing stable COPD and effective treatment of the exacerbations. Improved methods for early detection, new medication through targeted pharmacotherapy and possible means to identify the susceptible smokers could be new therapy and to prevent modalities against this epidemic.

1. Introduction Chronic obstructive pulmonary disease is a major public health problem. It is the fourth leading cause of chronic morbidity and mortality in the United States1 and is projected to rank fifth in 2020 as a worldwide burden of disease according to a study published by the World Bank/World Health Organization [1]. However COPD does not seem to receive enough attention from health care communities and government officials. Under these circumstances the U.S. National Heart, Lung and Blood Institute and the World Health Organization formed the Global Initiative for Chronic Obstructive Lung Disease (GOLD). 2. Epidemiology and risk factors of COPD The majority of the data available on the prevalence morbidity and mortality of COPD comes from developed countries. However, epidemiological information on COPD is difficult and expensive to gather. Prevalence and morbidity data underestimate the total burden of COPD since the disease is usually not diagnosed until it is clinically apparent and moderately advanced.

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Prevalence. In the Global Burden of Disease Study conducted under the auspices of the WHO and the World Bank [2,3], the worldwide prevalence of COPD in 1990 was estimated to be 9.34/1000 in men and 7.33/1000 in women. The prevalence of COPD is higher in those countries where cigarette smoking is very common, whereas it is lower in countries with less common smoking. Apart form the impact of COPD, as a burden of disease, there is also an economic burden related to COPD. Medical expenditures amounted to an estimated $ 14.7 billion in the U.S.A. Morbidity. The information available on COPD morbidity suggests that the disease tends to increase with age and is greater in men than women [4]. COPD is responsible for a major part of physician visits, emergency department visits and hospitalization. Between 1985 and 1995 the number of physician visits for COPD in the United States increased from 9.3 million to 16 million. The number of hospitalizations for COPD in 1995 was estimated to be 500000. Mortality. COPD is the fourth leading cause of death in the world, behind heart disease, cancer and cerebrovascular disease [4j. In the year two thousand, the World Health Organization estimated that 2.74 million deaths had occurred worldwide from chronic obstructive pulmonary disease. Also, in 1990 COPD occupied the 12th position as a burden of disease although it is estimated that by the year 2020 it will occupy the 5th position. Risk factors for COPD can include both host factors and environmental exposures. Usually the disease emerges from an interaction between these two kinds of factors. The host factor best recorded is a rare hereditary deficiency of al-antitrypsin. The major environmental factors include tobacco smoke, heavy exposure to occupational dusts and chemicals and indoor /outdoor air pollution. It should be noted that only a 25% of smokers tend to develop airflow limitation. The role of gender as a risk factor for COPD remains unclear. In the past the majority of studies showed that COPD prevalence and mortality were greater among men than women. However more recent studies from developed countries show that the prevalence of the disease is almost equal in men and women probably suggesting changing patterns of tobacco smoking [5]. 3. Definition of COPD and classification of severity. COPD is a disease state, characterized by airflow limitation that is not completely reversible. The airflow limitation is usually progressive and also linked to an abnormal inflammatory response of the lungs to noxious particles or gases. A diagnosis of COPD should be taken into consideration when a patient has manifested symptoms of cough, sputum production or dyspnea and/or a history of exposure to various risk factors for the disease. The diagnosis is confirmed by the application of spirometry. The presence of a postbronchodilator FEV1<80% of the predicted value combined with an FEVi/FVC < 70% confirms the presence of airflow limitation that is not fully reversible [6]. Four stages have been recommended for the classification of COPD. Stage 0: at risk. This stage features chronic cough and sputum production. The pulmonary function test in terms of spirometric values is normal. Stage I: mild COPD. This stage is characterized by mild airflow limitation (FEVi/FVC<70% but FEV]>80% predicted) and sometimes, although not always, by chronic cough and sputum production. Stage II: moderate COPD. At this stage, worsening airflow limitation is present (30%
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21

Patients may have severe COPD even if FEVj is larger than 30% predicted whenever these complications manifest themselves [6]. 4. Inflammation and COPD. Nowadays it is well established that inflammation is apparent in COPD. Chronic inflammation is present in the airways and lung parenchyma of patients with COPD although the complete event of this inflammation is currently uncertain. The inflammatory process in COPD appears to be different in most aspects from that in asthma concerning inflammatory cells, inflammatory mediators and inflammatory responses. Cigarette smoke alveolar macrophage neutrophil Proteinase

Alveolar wall destruction (Emphysema)

Mucus hypersecretion (Chronic bronchitis)

Figure 1. Cellular mechanism in COPD. Activation of macrophages leads to recruitment of neutrophils and both cells release proteinases, which may result in emphysema and chronic bronchitis

Cigarette smoking induces an inflammatory response in the airways and lung parenchyma of the smokers [11] (Figure 1). Thus the inflammatory changes described in COPD could be an exaggeration of this response. COPD is characterized by chronic inflammation in the airways, lung parenchyma and pulmonary vessels [7,8,9,10], Macrophages, T-lymphocytes and neutrophils are increased in various parts of the lung. The activated inflammatory cells release a number of mediators -including leukotriene B4 (LTB4), interleukine-8 (IL-8), tumor necrosis factor-a (TNF-a) and others- able to damage lung structures or to maintain neutrophilic inflammation. Apart from the inflammation there are two other processes considered to be important in the pathogenesis of COPD: i). an imbalance of proteinases and antiproteinases in the lungs and ii) oxidative stress. The inflammatory process in COPD differs from the process in asthma in terms of inflammatory cells, inflammatory mediators, inflammatory responses and response to corticosteroid therapy. Asthma is a clinical syndrome characterized by allergic inflammation of bronchi and bronchioli in which CD4+ T lymphocytes and eosinophils predominate [12]. There is an increased production and release of IL-4 and IL-5. A tracheobronchial responsiveness is observed to a variety of stimuli and the condition is usually manifested as a

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variable airflow obstruction. Although the differences between COPD and asthma have been recorded, the new data from various studies suggest that there may also be similarities. 4.1. Approaches towards documenting the inflammatory response in COPD. A number of approaches have been employed to document COPD inflammatory response. Lung biopsies. Various histological examinations of pathological specimens of the lung have demonstrated inflammatory changes in the airways and lung parenchyma of COPD patients [13,14]. In the airway mucosa of large and small airways, there is an increase in macrophages and T-lymphocytes, particularly CD8+ T-cells. Bronchoalveolar lavage. BAL may reflect inflammation in the lung periphery and is characterized by a marked increase in the numbers of macrophages and neutrophils [9]. Bronchial biopsy. Bronchial biopsies show an increase of macrophages and T-cells, especially CD8+ cells [15]. Also there is an increase of the number of eosinophils in the airways of some patients with COPD particularly in those with acute exacerbations. Induced sputum. Sputum obtained with nebulized hypertonic saline has shown an increase in total cell numbers particularly indicating an increase in macrophages and neutrophils [16]. There is an inverse correlation between the proportion of neutrophils and FEVi. Although eosinophil numbers are not increased in induced sputum there is a notable increase in eosinophil cationic protein (ECP) and eosinophil peroxidase (EPO), suggesting that eosinophils may have been degranulated. Inflammatory markers in the breath. Exhaled nitric oxide (NO) has been thoroughly investigated and its levels have been reported to be slightly increased or normal in stable COPD and increased during exacerbations. Levels of exhaled carbon monoxide (CO) are increased in COPD patients, although such measurements are affected by cigarette smoking [17]. Markers of oxidative stress, including hydrogen peroxide (H2O2) and 8-isoprostane are increased in expired condensates of patients with COPD. 4.2. Inflammatory cells and COPD. Many inflammatory cells are present and/or activated in COPD although their role to the progression of the disease is unknown. Neutrophils. Increased numbers of activated neutrophils are found in sputum and BAL fluid of COPD patients and they are slightly increased in the airways or lung parenchyma [8,16,18]. Neutrophils secret several proteinases including neutrophil elastase (NE), cathepsine G and proteinase 3, which may contribute to parenchymal destruction. Circulating neutrophils show up regulation of MAC-1 (GDI lb/ CD 18) in stable COPD patients. Adherent neutrophils then move towards the respiratory tract affected by neutrophil chemotactic factors such as chemokine interleukine-8 (IL-8) and leukotriene B4 (LTB-4). As a result, activated neutrophils cause mucus hypersecretion and may be involved in elastolysis. Macrophages. There is an increase in the numbers of macrophages in airways, lung parenchyma, BAL fluid and sputum in patients with COPD. Macrophages may be activated

N.M. Siafakas et al. /Biology, Diagnosis and Management of COP D

by cigarette smoke to release inflammatory mediators, including tumor necrosis factor a (TNF-a), IL-8 and LTB4. Macrophages are likely to play an important orchestrating role in the inflammation of COPD. T-lymhocytes. There is an increase in the total numbers of T-lymphocytes in lung parenchyma, peripheral and central airways of patients with COPD, with the greatest increase in CD8+ (cytotoxic) cells [15,19], There is a correlation between the number of T-cells, the amount of alveolar destruction and the severity of airflow obstruction. In our department we have studied the role of CD8+ cells and their subpopulations (Tc 1 and Tc2) in induced sputum of current smokers with COPD. Our results have shown that smokers with COPD have a higher number of CD8+ cells in induced sputum compared to non -COPD smokers and healthy individuals. Also CD8+ producing -IFN-y cells (Tel) were found to be decreased in COPD smokers than smokers without airflow limitation [20]. (Figure 2) According to our data differences in T cells subpopulations occur between smokers with COPD and those without. 100

p=O.OO1-

80 60 4O 20

O

COPD smokers

Non- COPD smokers

Non-smokers healthy

Figure 2. Tel cells were found to be decreased in COPD smokers compared with smokers without COPD.

4.3. Inflammatory mediators in COPD. The available information on the production and role of mediators in COPD is still not much. However many mediators are involved and mediator antagonists have potential as new therapies for COPD. Reactive oxygen species. Cigarette smoke reveals a high concentration of reactive oxygen species (ROS>10' moles/puff) with the contribution of inflammatory cells such as activated macrophages and neutrophils. There are increased concentrations of H2O2 in expired condensates especially during exacerbations thus providing evidence for increased oxidative stress in COPD. The oxidative stress can have various effects such as oxidation of antiproteinases (al-antitrypsin) and secretory leukopretease inhibitor (SLPI). Oxidants also activate the transcription factor -KB (NF-KB) which orchestrates the expression of multiple inflammatory genes like IL-8 and TNF-a [21]. Lipid mediators. LTB4 is chemoattractant of neutrophils and is increased in the sputum of patients with COPD [7]. It is probably coming from alveolar macrophages since there are

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more amounts of LTB4 in COPD patients. The role of prostaglandins in COPD is unclear. 8isoprostane is a constrictor of human airways. Platelet activating factor (PAF) enhances the release of LTB4 from activated neutrophils suggesting that it may have an amplifying effect on neutrophilic inflammation [22]. It is not established yet if PAF is released from alveolar macrophages in COPD patients. Chemokines. IL-8 is chemoattractant to neutrophils and exists in high concentrations in induced sputum of COPD patients [8,23]. IL-8 concentrations are elevated in BAL fluid in COPD patients showing a correlation with neutrophil counts. Macrophages, neutrophils and airway epithelial cells may produce IL-8. Other chemokines include macrophage inflammatory protein-1 p (MIP-lp) and macrophage chemotactic peptide -1 (MCP-1). MIPlp is increased in COPD compared to normal subjects and healthy smokers. MCP-1 is increased in BAL fluid of both COPD patients and healthy smokers. Cytokines. TNF-a activates the transcription factor nuclear factor-KB (NF-K.B), which activates the transcription of the IL-8 gene in epithelial cells and macrophages. Other cytokines include the GM-CSF and transforming growth factor-b (TGF-b) and epidermal growth factor (EOF). GM-CSF is increased in stable COPD but more elevated during exacerbations. GM-CSF is important for neutrophil survival and can play an important role in neutrophilic inflammation. TGF-b and EGF have increased expression in epithelial and submucosal cells in COPD patients and may be involved in airway structural changes in COPD. Endothelins. Several studies have reported that endotheline-1 (ET-1) exists in an increased concentration in induced sputum of COPD patients. Also the increased expression of ET-1 in pulmonary endothelial cells of COPD patients, suggest that ET-1 may contribute to the vascular remodeling associated with hypoxic pulmonary hypertension. Neuropeptides. Two neuropeptides among several have been studied in COPD patients. Substance P (SP) and vasoactive intestinal peptide (VIP). SP is detected in sputum of COPD patients with increased concentration. VIP is found in bronchial biopsies obtained from patients with COPD. 4.4. Proteinase-Antiproteinase imbalance. It has been proposed long time ago that there is an imbalance between proteases and endogenous anti-proteinases in COPD. Serine anti-proteinase includes al-AT which is the major antiptoteinase in plasma and lung parenchyma and SLPI which is the major antiptotease in airways. Oxidative stress may oxidize sulfydryl groups on methionine in these antiproteinases and harm their efficiency, or there may be genetic polymorphisms that reduce the production of anti-proteinases. A number of studies suggest that enzymes (proteinases) are released in emphysema and appear to cause alveolar damage through the decretation of proteins. A combination of proteinases released in COPD can degrade collagen and elastin thus damaging alveolar walls. These enzymes particularly attack elastin and create in this way less elasticity in the lung parenchyma in patients with emphysema. Also the fragmented pieces of elastin may act as chemotactic agents for macrophages and neutrophils thus perpetuating inflammation. Among the various proteinases, important ones include neutrophil elastase (NE) and the group of matrix metalloproteinases (MMP) [24]. In the MMP group twenty closely related endoproteinases are included.

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5. Diagnosis of COPD. The diagnosis of COPD should take place after the assessment of symptoms, the patient medical history, the physical examination and the measurement of airflow limitation [6]. Assessment of symptoms. Chronic productive cough is usually the first symptom to develop. Dyspnea is also a symptom associated with the disease and is a major cause of disability and the reason most patients seek medical attention. When there is more deterioration of lung function breathlessness is present. Wheezing and chest tightness are nonspecific symptoms. Medical history. The obtaining of detailed medical history of a possible COPD patient should be assessed. A possible exposure to risk factors, a past medical history, a family history of COPD or relevant chronic respiratory disease, a history of exacerbations or previous hospitalization for respiratory disorder and the social and family support provided to the patient can also be applied. Physical examination. Although important in other diseases it is rarely diagnostic in COPD. Physical signs of airflow limitation do not usually become manifest until major damage of lung function has occurred. Measurement of airflow limitation. To identify patients earlier in the course of the disease, spirometry should be performed for those patients who have chronic cough, sputum production and a history of exposure to risk factors even if they do not have dyspnea. Spirometry should measure the FVC and FEVi values and their ratio. COPD patients have a decreased FEVi and FVC. The presence of a postbronchodilator FEVi<80% predicted combined with an FEV]/FVC < 70% suggest the presence of airflow limitation not fully reversible.

6. Management of COPD Four components can be included for an effective COPD management plan: (1) assess and monitor disease (2) reduce risk factor (3) manage stable COPD and (4) manage exacerbations. The goals of effective COPD management are to prevent disease progression, relieve symptoms, improve exercise tolerance, improve health status, prevent and treat complications and exacerbations and reduce mortality. These goals should be achieved with a minimum of treatment side effects [6]. Assessing and monitoring of disease. Concerning this component it should be noted that COPD diagnosis is based on a history of exposure to risk factors and the presence of airflow limitation not fully reversible, with or without symptoms. Also those patients with chronic productive cough and previous exposure to risk factors should be tested for possible airflow limitation. Spirometry is considered as a standard way to measure airflow limitation. In the presence of FEVi/FVC < 70% and a postbronchodilator FEVi<80% predicted evidence speaks of a not fully reversible airflow limitation. The measurement of arterial blood gas tension is indicative in patients with FEV1< 40% predicted or when clinical signs point to respiratory failure or right heart failure. Reducing risk factor. Considering this component, a reduction of personal exposure to tobacco smoke, occupational dust and chemicals and indoor or outdoor air pollutants are fundamental in order to prevent the beginning and progression of COPD. Abstaining from

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smoking is the most effective way of reducing the risk to develop COPD. The patient should receive social support and counseling inside and outside the treatment. Tobacco- depended therapies can be administered if no contraindications appear. Management of stable COPD. In this field a step-by-step increase in treatment should occur based on the severity of the disease. Health education can assist COPD patients in an attempt to cope with illness and health status. Of the available medications for COPD, none has succeeded in altering the long-term decline in lung function. Thus pharmacotherapy is useful to decrease symptoms and complications. Bronchodilators are essential to the symptomatic management of COPD. Main bronchodilators treatments include |32 -agonists, anticholinergics, theophylline and a combination of one or more of these. Inhaled glucocorticosteroids should be administered to patients with a documented spirometric response to glucocorticosteroids or for those with FEVi < 50% predicted and repeated exacerbations which demand treatment with antibiotics or oral glucocosteroids. The long-term administration of oxygen (> 15h per day) to patients with chronic respiratory failure has been shown to increase survival. Other pharmacologic treatments for the management of stable COPD include vaccines, therapy with antitrypsin al, antibiotics, mucolitic agents, antioxidant agents, immunoregulators, antitussives, vasodilators, respiratory stimulants, narcotics and others. Apart from pharmacologic treatments nonpharmacologic approaches are available. These include rehabilitation, oxygen therapy and ventilatory support. Surgical treatments such as bullectomy, lung volume reduction surgery (LVRS) and lung transplantation can be used to carefully selected patients. Management of COPD exacerbations. Exacerbations of symptoms in COPD are important clinical events. Common causes of exacerbations are infection of the tracheobronchial tree and air pollution. The cause of about one third of severe exacerbations however cannot be established. Effective treatments for acute exacerbations include inhaled bronchodilators, theophyline and systemic glucocorticosteroids. Patients with exacerbations from airway infection may use antibiotic treatment. Noninvasive positive pressure ventilation (NIPPV) in acute exacerbations improves blood gases and pH reduces in- hospital mortality, decreases the need for invasive mechanical ventilation and intubation and also decreases the length of hospital stay.

References [1] Pauwels, R. A., Buist, A. S., Calverley, P. M., Jenkins, C. R., Hurd, S. S., on behalf of the GOLD scientific committee. Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease. NHLBI/WHO Global Initiative for Chronic Obstructive Lung Disease (GOLD) Workshop summary. Am J Respir Crit Care Med, 2001. 163(5): p. 1256-76. [2]. Murray CJL, Lopez AD, Evidence-based health policy-lessons from the Global Burden of Disease Study. Science 1996; 274:740-743. [3]. Murray CJL, Lopez AD. The Global Burden of disease: A Comprehensive Assessment of Mortality and Disability from Diseases, Injuries and Risks factors in 1990 and Projected to 2000. Harvard University Press, Cambridge, MA. [4]. National Heart, lung and Blood Institute. Morbidity and Mortality: Chartbook on Cardiovascular, Lung and Blood Diseases. U.S. Department of Health and Human Services, Public health Service, National Institutes of Health. Bethesda. MD. 1998. [5].Xu X, Weiss ST, Rijcken B, Schouten JP. Smoking, changes in smoking habits, and rate of decline in FEVI: new insight into gender differences. Ear Respir J 1994:7:1056-1061.

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[6].Romain A. Pauwels, A. Sonia Buist, Peter M.A. Calverley et al. Global Strategy for the diagnosis, management, and prevention of Chronic Obstructive Pulmonary Disease. NHLBI/WHO Global Initiative for Chronic Obstructive Lung Disease (GOLD) Workshop Summary. Am J Respir Crit Care Med, Vol 163.pp 12561276,2001. [7]. Hill AT, Bayley D, Stockey RA. The interrelation ship of sputum inflammatory markers in patients with chronic bronchitis. Am J Respir Crit Care Med 1996; 153:530-534. [Sj.Keatings VM, Collins PD, Scott DM, Barnes PJ. Differences in interleukin -8 and tumor necrosis factoralpha in induced sputum from patients with chronic pulmonary disease or athma. Am J Respir Crit Care Med 1996; 153:530-534. [9]. Pesci A, Balbi B, Majori M, Cacciani G, et al. Inflammatory cells and mediators in bronchial lavage of patients with chronic obstructive pulmonary disease. Eur Respir J 1998: 12:380-386. [10].Yahamoto C, Yoneda T, Yoshikawa M et al. Airway inflammation in COPD assessed by sputum levels of interleukin -8. Chest 1997; 112:505-510. [1 l].Niewoehner DE, Kleinerman J, Rice DB. Pathologic changes in the peripheral airways of young cigarette smokers. NEnglJMed 1974; 291:755-758. [12]. Giles F.Filey Lecture. Comparison of the structural and inflammatory Features of COPD and Asthma. Chest /117/5/May, 2000 Supplement. [13]. Jeffery PK. Structural and inflammatory changes in COPD: a comparison with asthma. Thorax 1998; 53:129-136. [14]. Saetta M, Di Stefano A, Maestrelli P et al. Activated T-lymphocytes and macrophages in bronchial mucosa of subjects with chronic bronchitis. Am J Respir Crit Care Med 1993: 147:301-306. [15]. O'Shaugnessy TC, Ansari TW, Barnes NC, Jeffery PK. Inflammation in bronchial biopsies of subjects with chronic bronchitis: inverse relationship of CD8+ T lymphocytes with FEV1. Am J Respir Crit Care Med 1997; 155:852-857. [16]. Peleman RA, Rytila PH, Kips JC et al. The cellular composition of induced sputum in chronic obstructive pulmonary disease. Eur Respir J 1999; 13:839-843. [17]. Maziak W, Loukides S, Culpitt S et al. Exhaled nitric oxide in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 1998; 157:998-1002. [18]. Pesci A, Maiori M, Cuomo A, Borciani N, Bertacco S, Cacciani G, Gabrielli M. Neutrophils infiltrating bronchial epithelium and chronic obstructive pulmonary disease. J Allergy Clin Immunol 1993; 92:537-548. [19].Finkelstein R, fraser RS, Ghezzo H, Cosio MG. Alveolar inflammation and its relation to emphysema in smokers. Am J Respir Crit Care Med 1995; 152:1666-1672. [20]. N.Tzanakis, G.Chrysofakis, D.Kyriakou, I.Tsiligianni, M.Froudarakis, D.Bouros, N.M.Siafakas. Subpopulations of CD8+ cells (Tel, Tc2) in induced sputum of COPD patients and smokers without COPD. ATS 2001. American Journal of respiratory and critical care medicine, vol. 163, April 2 001 (abstract). [21 ].Repine JE, Bast A, Lankhorst I. Oxidative stress in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 1997; 156:341-357. [22]. Shindo K, Koide K, Fukumura M. Enhancement of leukotriene B4 release in stimulated asthmatic neutrophils by platelet activating factor. Thorax 1997; 52:505-510. [23]. Capelli A, Di Stefano A, Gnemmi I, Balbo P, Cerutti CG et al. Increased MCP-land MlP-Ib in bronchoalveolar lavage fluid of chronic bronchitis. Eur Respir J 1999; 14:160-165. [24].FiInay GA, O'Driscoll LR, Russell KJ et al. Matrix metalloproteinases expression and production by alveolar macrophages in emphysema. Am J Respir Crit Care Med 1997; 156:240-247.

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Disease Markers in Exhaled Breath N. Marczin and M.H. Yacoub (Eds.) fOS Press, 2002

Disease markers in COPD: exhaled breath vs. exhaled condensate Sergei A. KHARITONOV Thoracic Medicine, National Heart and Lung Institute, Imperial College and Royal Brompton Hospital, Dovehouse Street, London SW3 6LY, UK Abstract. Exhaled breath analysis has enormous potential as a non-invasive means of monitoring of airway and inflammation, oxidative stress in COPD. The technique is simple for patients to perform and may be applied in neonates and patients with severe disease. Because the techniques are non-invasive it is possible to make repeated measurements without disturbing the system, in contrast to the invasive procedures currently used [1]. At the moment single exhaled markers are usually evaluated in isolation, but as indicated above markers are affected differently in different diseases, and different markers vary in their sensitivity to certain maneuvers, such as the effect of therapy. For example, asthma is characterized by a large increase in exhaled NO, a modest increase in CO and a moderate increase in exhaled 8-isoprostane, whereas COPD is characterized by little or no increase in exhaled NO, and by larger increases in exhaled CO and 8-isoprostane.

1. Exhaled gases 1.1. Nitric oxide Exhaled NO levels in stable COPD [2-4] and chronic bronchitis [5] are lower than in either smoking or non-smoking asthmatics [6] and are not different from normal subjects. This reduction in exhaled NO is due to the effect of tobacco smoking, which down-regulates eNOS [7] and reduces exhaled NO [2] suggesting that this may contribute to the high risk of pulmonary and cardiovascular disease in cigarette smokers.. In addition to the effects of cigarette smoking, a relatively low value of exhaled NO in COPD may reflect more peripheral inflammation than in asthma, low NOS2 expression [4] and increased oxidative stress that may consume NO in the formation of peroxynitrite [8]. Patients with unstable COPD, however, have high NO levels compared with stable smokers or ex-smokers with COPD [9], which may be explained by increased neutrophilic inflammation and oxidant/antioxidant imbalance. Eosinophils that are capable of expressing NOS2 and producing NO are present in exacerbations of COPD [10]. Acidosis, which is frequently associated with exacerbations of COPD, may increase the release of NO [11]. Pulmonary hypertension has the opposite effect, as COPD patients with Cor Pulmonale have low exhaled NO levels [12], which may reflect their impaired endothelial NO release. A small proportion of patients with COPD appear to response to corticosteroids and these patients, who are likely to have coexistent asthma, have an increased proportion of eosinophils in induced sputum [13]. These patients also have an increased in exhaled NO[14]. This suggests that exhaled NO may be useful in predicting which COPD patients will respond to long-term inhaled corticosteroid treatment.

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1.2. Carbon monoxide (CO) A major limitation of exhaled CO in COPD is the marked effects of cigarette smoking, which masks any increase that may occur due to the disease process. There is no difference in exhaled CO in patients with chronic bronchitis (without airflow obstruction) when compared with normal subjects [15]. However, exhaled CO levels are elevated in ex-smoking COPD patients [16], suggesting ongoing oxidative stress or inflammation. HO is induced in fibroblasts exposed to cigarette smoke [17]. There is an increase in exhaled CO during acute exacerbations of COPD, with a decline after recovery [18]. 2. Exhaled condensate 2.1. Hydrogen peroxide Activation of inflammatory cells, including neutrophils, macrophages and eosinophils, result in an increased production of Q{ which by undergoing spontaneous or enzyme-catalyzed dismutation lead to formation H2O2- As HI^I is less reactive than other reactive oxygen species, it has the propensity to cross biological membranes and enter other compartments [19]. Because it is soluble, increased H2Oi in the airway equilibrates with air [20]. Compared with the cellular antioxidant scavenging systems, the extracellular space and airways have significantly less ability to scavenge reactive oxygen species [21,22]. Catalase is the major enzyme involved in removing HaO2 and is preset in low concentrations in the respiratory tract. Thus exhaled H2O2 has potential as a marker of oxidative stress in the lungs. Cigarette smoking causes an influx of neutrophils and other inflammatory cells into the lower airways and five-fold higher levels of H2O2 have been found in exhaled breath condensate of smokers than in non-smokers [23]. Levels of exhaled H2Oa are increased compared to normal subjects in patients with stable COPD and are further increased during exacerbations [24,25]. Cigarette smoking is by far the commonest cause of COPD, but only 10-20% of smokers develop symptomatic COPD. No significant differences have been found between H2O2 levels in current smokers with COPD and COPD subjects who have never smoked, and there is no correlation between expired HiO2 concentration and daily cigarette consumption [25], Thus oxidative stress is a characteristic feature of COPD and presumably related to airway inflammation and cannot be explained entirely by the oxidants present in tobacco smoke.

2.2. Leukotrienes Leukotrienes (LTs), a family of lipid mediators derived from arachidonic acid via the 5-lipoxygenase pathways, are potent constrictor and pro-inflammatory mediators that contribute to pathophysiology of asthma. The cysteinyl-leukotrienes (cys-LTs) LTC4, LTD4 and LTE4 are generated predominantly by mast cells and eosinophils, and are able to contract airway smooth muscle, cause plasma exudation and stimulate mucus secretion, as well as recruiting eosinophils [26]. By contrast, LTB4 has potent chemotactic activity towards neutrophils [27]. Detectable levels of LTB4, C4, 04, £4 and F4 have been reported in exhaled condensate of asthmatic and normal subjects [28,29]. LTB4 concentrations are increased in exhaled breath condensate of patients with COPD [1]. This suggests that

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LTB4 may be involved in exacerbations of asthma and may contribute towards neutrophils recruitment.

2.3. Isoprostanes Isoprostanes are a novel class of prostanoids formed by free radical-catalyzed lipid peroxidation of arachidonic acid [30]. They are formed initially esterified in membrane phospholipids, from which they are cleaved by a phospholipase A2, circulate in plasma, and are excreted in urine and can be detected in exhaled breath condensate and BAL. Their formation is largely independent of COX-1 and COX-2. They can be detected by ELISA [31,32] and by GC/MS analysis [30]. p2-isoprostanes are the major candidates for clinical measurement of oxidative stress in vivo. Urinary levels of isoprostanes, in particular 8-isoprostane, are increased in COPD, and declined in patients with acute exacerbation as their clinical condition improves [33]. Aspirin treatment fails to decrease urinary levels of isoprostanes, whereas TxB2 were significantly reduced, confirming that cyclo-oxygenases are not involved in their formation. The concentration of 8-isoprostane in exhaled condensate is also increased in normal cigarette smokers, but to a much greater extent in COPD patients [34]. Interestingly, exhaled 8-isoprostane is increased to a similar extent in COPD patients who are ex-smokers as in smoking COPD patients, indicating that the exhaled isoprostanes in COPD are largely derived from oxidative stress from airway inflammation, rather than from cigarette smoking.

2.4. S-nitrosothiols Habitual smokers have unusually high antioxidant concentrations in the epithelial lining fluid and higher resistance to oxidative pulmonary damage. NO can be trapped in the epithelial lining fluid of the respiratory tract in the form of S-nitrosothiols or peroxynitrite and released thereafter, leading to transient elevation of exhaled NO after smoking of a cigarette [35]. Chronic oxidative stress presented to the lung by cigarette smoke may decrease the availability of thiol compounds and may increase decomposition of nitrosothiols, explaining elevated levels of S-nitrosothiols in exhaled condensate in healthy smokers, which are related to smoking history [36]. Levels of exhaled nitrite/nitrate are increased in COPD (unpublished observation). A significant negative correlation between FEVi and the amount of nitrotyrosine formation has been demonstrated in patients with COPD, but not in those with asthma and normal subjects [37], suggesting that NO produced in the airways is consumed by its reaction with superoxide anion and/or peroxidase-dependent mechanisms and reactive nitrogen species play an important role in the pathobiology of the airway inflammatory and obstructive process in COPD.

References [1] S.A.Kharitonov, Barnes PJ. Exhaled markers of pulmonary disease. Am J Respir Crii Care Med 163(2001) 1693-1722.

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[2] S.A.Kharitonov et al.. Acute and chronic effects of cigarette smoking on exhaled nitric oxide. Am. J. Respir. Crit. Care. Med. 152(1995) 609-612. [3] R.A.Robbins et al.. Measurement of exhaled nitric oxide by three different techniques. Am. J. Respir. Crit. Care. Med. 153 (1996) 1631-1635. [4] S.R.Rutgers et al.. Markers of nitric oxide metabolism in sputum and exhaled air are not increased in chronic obstructive pulmonary disease. Thorax. 54 (1999) 576-580. [5] S.G.Von Essen et al.. Respiratory tract inflammation in swine confinement workers studied using induced sputum and exhaled nitric oxide. J. Clin. Toxicol. 36 (1998) 557-565. [6] G.M.Verleden et al.. The effect of cigarette smoking on exhaled nitric oxide in mild steroid- naive asthmatics. Chest. 116(1999) 59-64. [7] Y.Su et al.. Effect of cigarette smoke extract on nitric oxide synthase in pulmonary artery endothelial cells. Am. J. Respir. Crit. Care. Med. 19 (1998) 819-825. [8] J.P.Eisehch et al.. Formation of nitric oxide-derived inflammatory oxidants by myeloperoxidase in neutrophils. Nature. 391 (1998) 393-397. [9] W.Maziak et al.. Exhaled nitric oxide in chronic obstructive pulmonary disease. Am. J. Respir. Crit. Care. Med. 157(1998) 998-1002. [10] M.Saetta et al.. Airway eosinophilia in chronic bronchitis during exacerbations. Am. J. Respir. Crit. Care. Med. 150(1994) 1646-1652. [11] J.F.Hunt et al.. Endogenous airway acidification. Implications for asthma pathophysiology. Am. J. Respir. Crit. Care. Med. 161 (2000) 694-699. [12] E.Clini et al.. Production of endogenous nitric oxide in chronic obstructive pulmonary disease and patients with cor pulmonale. Correlates with echo-Doppler assessment. Am J Respir Crit Care Med 162 (2000) 446-450. [13] K.Fujimoto et al.. Eosinophilic inflammation in the airway is related to glucocorticoid reversibility in patients with pulmonary emphysema. Chest 11.5 (1999) 697-702. [14] A.Papi et al.. Partial reversibility of airflow limitation and increased exhaled NO and sputum eosinophilia in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 162 (2000) 1773-1777. [15] F.M.Delen et al.. Increased exhaled nitric oxide in chronic bronchitis. Comparison with asthma and COPD. Chest. 117 (2000) 695-701. [16] S.V.Culpitt et al.. Exhaled carbon monoxide is increased in COPD patients regardless of their smoking habit. Am. J. Respir. Crit. Care. Med. 157 (1998) A787. [17] T.Muller, Gebel S. The cellular stress response induced by aqueous extracts of cigarette smoke is critically dependent on the intracellular glutathione concentration. Carcinogenesis. 19 (1998) 797801. [18] W.Biernacki et al.. Carbon monoxide in exhaled air in patients with lower respiratory tract infection. Eur. Respir. J. 12 (1998) 345S. [19] B.A.Freeman, Crapo JD. Biology of disease: free radicals and tissue injury. Lab. Invest. 47 (1982) 412-426. [20] A. W.Dohlman et al.. Expired breath hydrogen peroxide is a marker of acute airway inflammation in pediatric patients with asthma. Am. Rev. Respir. Dis. 148 (1993) 955-960.

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[21] J.E.Heffher, Repine JE. Pulmonary strategies of antioxidant defense. Am. Rev. Respir. Dis. 140 (1989) 531-554. [22] J.E.Godwin, Hefrher JE. Platelet prevention of oxidant lung oedema is not mediated through scavenging of hydrogen peroxide. Blood. Coagul. Fibrinolysis. 3 (1992) 531-539. [23] D.Nowak et al.. Increased content of hydrogen peroxide in the expired breath of cigarette smokers. Eur. Respir. J. 9 (1996) 652-657. [24] P.N.Dekhuijzen et al.. Increased exhalation of hydrogen peroxide in patients with stable and unstable chronic obstructive pulmonary disease. Am. J. Respir. Crit. Care. Med. 154 (1996) 813816. [25] D.Nowak et al.. Cigarette smoking does not increase hydrogen peroxide levels in expired breath condensate of patients with stable COPD. Monaldi. Arch. Chest. Dis. 53 (1998) 268-273. [26] A.R.Leff. Role of leukotrienes in bronchial hyperresponsiveness and cellular responses in airways. Am. J. Respir. Crit. Care. Med. 161 (2000) S125-S132. [27] G.Larfars et al.. Activation of nitric oxide release and oxidative metabolism by leukotrienes B4. C4, and D4 in human polymorphonuclear leukocytes. Blood. 93 (1999) 1399-1405. [28]

G.Becher et al.. Breath condensate as a method of noninvasive assessment of inflammation mediators from the lower airways. Pneumologie. 51 Suppl 2:456-9 (1997) 456-459.

[29]

T.Hanazawa et al.. Increased Nitrotyrosine in Exhaled Breath Condensate of Patients with Asthma. Am J Respir Crit Care Med 162 (2000) 1273-1276.

[30] J.D.Morrow, Roberts LJ. The isoprostanes: unique bioactive products of lipid peroxidation. Prog. Lipid. Res. 36(1997) 1-21. [31] P.Montuschi et al.. Increased 8-Isoprostane, a Marker of Oxidative Stress, in Exhaled Condensate of Asthma Patients. Am. J. Respir. Crit. Care. Med. 160(1999) 216-220. [32] P.Montuschi et al.. 8-lsoprostane as a biomarker of oxidative stress in interstitial lung diseases. Am. J. Respir. Crit. Care. Med. 158(1998) 1524-1527. [33]

D.Pratico et al.. Chronic obstructive pulmonary disease is associated with an increase in urinary levels of isoprostane F2alpha-III, an index of oxidant stress. Am. J. Respir. Crit. Care. Med. \ 58 (1998) 1709-1714.

[34]

P.Montuschi et al.. Exhaled 8-isoprostane as an in vivo biomarker of lung oxidative stress in patients with COPD and healthy smokers. Am J Respir Crit Care Med 162 (2000) 1175-1177.

[35]

D.C.Chambers et al.. Acute inhalation of cigarette smoke increases lower respiratory tract nitric oxide concentrations. Thorax. 53 (1998) 677-679.

[36]

M.Corradi et al.. Elevated levels of nitrosothiols in breath condensate of healthy smokers. Am. J. Respir. Crit. Care. Med. 161 (2000) A857.

[37]

M.Ichinose et al.. Increase in reactive nitrogen species production in chronic obstructive pulmonary disease airways. Am. J. Respir. Crit. Care. Med. 162 (2000) 701-706.

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The Biology of Cystic Fibrosis Tamas JILLING Department of Pediatrics, Evanston Hospital, Northwestern University Medical School, 2650 Ridge Ave., Evanston, IL 60201, USA Abstract: Cystic fibrosis is the most common lethal, autosomal recessive hereditary disease among Caucasians. The disease affects approximately 1:2000 live births in the general Caucasian population, but its frequency is considerably higher among families of northern European origin, among Mormons and in the Ashkenazi Jewish population. The disease is caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a protein that is essential for mucosal homeostasis. The cellular roles of CFTR, and the possible mechanisms connecting CFTR mutations with disease pathology are discussed.

1. Time line of cystic fibrosis research. The first description of a clinical syndrome connecting the symptoms of airway disease and the cystic fibrosis of the pancreas was authored in 1936 by the Swiss pediatrician Guido Fanconi [1]. In 1938 the basic pathology of CF was described, and the name of the disease (Cystic Fibrosis of the Pancreas) was coined by Dorothy H. Andersen [2]. Subsequently, the inheritance pattern of CF was described in 1946. Another major milestone in our understanding of CF was the discovery of elevated sweat chloride in CF patients in 1953 [3], which established the gold standard in early CF diagnosis, as well as directed the attention of basic scientists to the investigation of Na+ and Cl" transport in CF. The description of the pilocarpine iontophoresis method for the rapid determination of Cl" from sweat enabled early diagnosis of CF in the clinic [4]. The abnormality of Cl- transport regulation at the cellular level was first demonstrated in 1983 [5,6], followed by a flourishing array of discoveries along the lines of Cl" channel regulation in normal and CF cells. In fact, much of our current understanding of the physiology, cell and molecular biology of Cl" channel regulation has been driven by CF-related research. Prior to the cloning of the CFTR, there were numerous publications describing a lack of cAMPstimulated Cl" transport in CF sweat duct, airway and intestinal epithelial cells. The CFTR gene was discovered in 1989 [7], and allowed a detailed molecular level investigation of CFTR function in cells and in animal models. Genetic complementation of the Cl" secretory defect in CF cells provided direct evidence for a role of CFTR in Cl" transport [8-10]. Shortly thereafter, using recombinant CFTR in planar lipid bilayers allowed the bone fide demonstration that CFTR can function as a cAMP-regulated Cl" channel [11]. Following the identification of the murine CFTR homolog, several transgenic CFTR knock out mouse strains have been developed [12]. These murine CF models express an intestinal phenotype resembling meconium ileus in humans, but the pulmonary phenotype of the CF mice is nearly normal. Since these first steps, a plethora of information has been generated regarding the molecular aspects of CFTR function, and regarding the various roles that CFTR plays in cells in addition to its primary function as a Cl" channel.

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2. The pathology of CF The disease is characterized by impaired mucociliary clearance in the lung, recurrent respiratory infections, intestinal obstruction, male and female infertility, and, in most cases, pancreatic insufficiency. The life-limiting aspect of CF is a loss of pulmonary function as a result of recurrent respiratory infections and inflammation. However, other aspects of the disease such as male infertility are becoming more relevant as improvements in clinical treatments have prolonged the mean life expectancy of CF patients from 10 years in the 1960s to 28 years in 1990, and to the mid 30-s by today.. This improvement has been due primarily to the refinement of CF treatment methods that focus on alleviating symptoms such as pancreatic enzyme replacement therapy, treatment of recurrent infections with antibiotics, and reducing inflammation and airway obstruction with pharmacological and physical methods. Novel therapeutic approaches that attempt to correct or bypass the cellular defect caused by CFTR mutations including in vivo gene therapy are currently under development, but so far have not lived up to the expectations. Viruses and Streptococcus and Haemophilus bacterial species are the predominant lung pathogens in the first 2 years of life [13]. Subsequent colonization of airways by Pseudomonas aeruginosa provides the major clinical challenge. Recent data indicate that, in addition to bacterial infections, an exaggerated inflammatory response also contributes to the deterioration of pulmonary tissue in CF (see section 7.). 3. CFTR The CFTR gene encodes a protein that consists of 1480 amino acids [7]. Various CFTR splice variants have been reported without known functional significance. The five-domain model of CFTR protein which was proposed at the time of discovery is now generally well accepted (figure 1). CFTR is arranged in two symmetrical halves; each containing a set of

Figure 1 Functional domains of CFTR.

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six membrane-spanning a helices and a cytoplasmic nucleotide binding domain (NBD). The two halves are separated by a cytoplasmic regulatory (R) domain. The R domain includes multiple phosphorylation sites for a number of protein kinases including cyclic AMP-dependent protein kinase (PKA) and protein kinase C. This architecture places CFTR in the family of ATP binding cassette (ABC) transporters. The R domain distinguishes CFTR from other ABC transporters, which generally lack a similar feature. Interestingly, most other members of the ABC transporter family are active transporters that transport solutes at the expense of ATP hydrolysis. Such active transport function has not been firmly linked to CFTR. In addition to the classical five domains, several new functional domains have been discovered on CFTR during the last few years. The cytoplasmic C terminus contains multiple internalization signaling domains [14,15], FLVI (AA 1413-1416), YDSI (AA 1424-1427) and LL (AA 1430-1431), of which the YDSI motif has been shown to directly interact with the D2 subunit of the AP2 endocytic adaptor complex [16,17]. The carboxy terminal tail of CFTR also contains a PDZ binding motif, DTRL (AA 1477-1480), which interacts with a variety of molecules, such as the Na+/H+ exchanger regulatory factor (NHERF), the ezrin-radixin-moesin phosphoprotein 50 (EBP50), protein kinase A and others [18]. These interactions bear significance both in terms of CFTR localization to the apical domain of epithelial cells, and in terms of channel gating and regulation. The amino terminus of CFTR interacts with syntaxin 1A [19], and also interacts with the junction of NBD1 and the R domain of CFTR [20]. These interactions have been shown to participate in CFTR conductance regulation. Another recently identified domain of CFTR, is a putative LPS core oligosaccharide binding domain (AA 103-117) on the first extracellular loop of CFTR, that is thought to participate in Pseudomonas aeruginosa binding and internalization in airway and corneal epithelial cells [21]. 4. CFTR, the cAMP-regulated Cl" channel in the apical plasma membranes of epithelial cells: CFTR has been recognized mainly as the cAMP regulated Cl" channel in the apical plasma membranes of epithelial cells. In order to fulfill its role as a regulated Cl" channel CFTR has to conduct Cl" in a specific, and regulated manner. The Cl" channel function of CFTR is very well characterized at the single channel level based on studies in planar lipid bilayer and patch clamp studies [11]. CFTR can be characterized by the following parameters: (a) Halide selectivity of Br">Cl">r>F", (b) a single channel conductance of 5-10 pS, (c) linear current/voltage relationship under symmetrical ion concentrations, (d) regulation by the phosphorylation of the R domain by protein kinase A, (e) requirement for the presence of ATP at the cytoplasmic side for channel activity, (f) unlike several other Cl" channels, CFTR conductance is not blocked by disulfonic stilbenes. 5. CFTR functions other than apical Cl" channel in epithelial cells CFTR protein is expressed predominantly by epithelial cells lining the airways, gastrointestinal tract and the urogenital tract. The best characterized, and therefore the one fully accepted cellular function of CFTR is a cAMP-regulated Cl" channel function in the apical plasma membranes of epithelial cells. Additional proposed functions of CFTR can be divided into three groups: (a) Conductance of other molecules in addition to chloride, (b) regulation of transepithelial ion transport via interaction with other ion channels, (c) Cl" channel function in intracellular membranes, and (d) apparently ion transport-independent functions.

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Conductance of bicarbonate, ATP and glutathione: CFTR has been shown to conduct HCO3~, in addition to Cl" [22]. The HCCV conductance has been shown to manifest in transepithelial HC(V secretion in airway epithelial cells [23]. This finding suggests a potential physiological role for CFTR in the regulation of pH on epithelial surfaces, such as the airway lining fluid. Given that pH affects many physiological functions, including the regulation of mucus viscosity, ciliary beat frequency and the regulation of phagocytes to internalize and destroy pathogens, this observation might have further implications regarding the pathology of CF. Bicarbonate conductance by CFTR also has an effect on the regulation of intracellular pH, and this might have implications in the regulation of apoptosis in CFTR-expressing cells [24]. CFTR expression has been linked to the ability of cells to secrete ATP, perhaps via an interaction with another transporter [25]. CFTR also conducts glutathione [26], CF cells appear to be defective of glutathione secretion [27], and the use of a synthetic Cl" channel peptide can overcome this defect at the cellular level [28]. The absence of this function of CFTR might have implications in the inflammatory state of the CF mucosa. Regulation of transepithelial ion transport via interactions with other channels: CFTR has been shown to regulate amiloride-sensitive Na* channels [29], and the outwardly rectifying chloride channel (ORCC) [30]. The regulation of Na+ channels by CFTR appears to involve an interaction with the actin cytoskeleton [29]. The inhibitory regulation of Na" channels by CFTR is thought to have pathological significance, however despite apparent success in an early pilot study [31], clinical trials aimed at reducing the increased activity of Na+ channels in the CF airways using amiloride failed to demonstrate any clinically significant improvement in lung function [32-34]. The physiological, or pathological significance of ORCC regulation by CFTR is yet to be determined. CFTR Cl~ channel function in intracellular membranes: Several studies indicated that a substantial pool [35-37], in some cells as much as 50% [36,37] of all fully matured (i.e., fully glycosylated; band C) CFTR is localized in intracellular membrane compartments, and that CFTR is rapidly recycles through the endocytic recycling compartment [17,35,38,39]. There are at least two frequently voiced hypotheses regarding a potential role of intracellular CFTR: 1) The intracellular pool of CFTR could provides a reserve, from which CFTR can be recruited to the cell surface upon stimulation by cAMP [37,40] and 2) CFTR plays a functional role in intracellular membranes [37,40-45]. Such proposed intracellular functions for CFTR include a role in endosomal and TON acidification, sialylation of glycoproteins, regulation of endocytosis and membrane recycling. The hypothesis that CFTR has intracellular functions is surrounded by controversy, since many of the observations that were made in one model system could not be reproduced using other model systems. A major part of this controversy might be due to the diversity of cellular systems that were used for studies addressing the role of intracellular CFTR. Studies that investigated the intracellular functions of CFTR in epithelial cells usually found a correlation between CFTR expression, TON acidification or the regulation of endocytosis and membrane recycling by cAMP [37,40-42,46], while studies addressing the same issues using heterologous expression of CFTR in fibroblasts, or other unpolarized cells found no role for CFTR in either endosomal acidification or in the regulation of membrane traffic [47-50]. Therefore, it is possible that CFTR-dependent regulation of organelle acidification exists only in the context of a polarized epithelial cell. Further complication is that the bicarbonate status of cells, and the expression of other Cl" channels in certain cell types might have an effect on endosome, or TON pH. In order to resolve the controversy over the role of CFTR in pH regulation of intracellular organelles, more carefully designed expression systems in polarized epithelial cells, and a better accounting for the role of CFTR bicarbonate conductance will be required. A CFTRdependent regulation of endosome fusion was also reported [51]. The regulation of

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endosome fusion was shown to be dependent on CFTR Cl" channel function, however, without any dependence on endosomal acidification. Ion transport-independent functions of CFTR: CFTR has been proposed to function as a receptor for certain bacteria, including Pseudomonas aeruginosa [21]. This function has been proposed to directly underlie the susceptibility of CF airways to Pseudomonas colonization. 6. CFTR Mutations Disease-causing mutations in CFTR that have been reported enumerate almost 1,000, although only a small fraction of these have been functionally characterized. Up-to-date information on all mutations can be found at the Cystic Fibrosis Mutation Database (http://www.genet.sickkids.on.ca/cftr/)) which is maintained by the Cystic Fibrosis Genetic Analysis Consortium. CFTR mutations can be categorized into four groups based on their functional consequences. Class I mutants are nonsense, splice, and frameshift mutants that encode truncated or aberrant forms of CFTR (e.g., G542X). These mutants in general associate with greatly reduced mRNA expression and protein levels. Many of these mutants cause severe pathology including pancreatic insufficiency. Interestingly, some premature stop mutations in the CFTR coding region (G542X and R553X) can be overcome by low doses of the aminoglycoside antibiotics neomycin and gentamicin [52]. Treatment of cells harboring these alieles with aminoglycosides results in read-through of the inefficient stop codons generated by these mutations. For the limited number of CF patients affected by these mutations, chronic low dose aminoglycoside therapy might provide an efficient therapy [53]. Class II mutants, which are defective in their posttranslational folding, and/or processing constitute the most prevalent disease causing alieles including the DF508 mutation, accounting for approximately two-thirds of all CF alieles. This class of mutations was discovered, similar to many other discoveries, as a consequence of a controversy. While initial attempts failed to detect functional DF508 CFTR in mammalian expression systems [10], there were several reports indicating that DF508 CFTR is functional based on experiments conducted in insect cells [54] and xenopus oocytes [55]. This controversy was resolved by the realization that insect cell cultures and oocytes are maintained at, or near ambient temperature, and mammalian cells had to be cooled to similar temperatures to observe functional DF508 CFTR [56]. Based on these results, and on in vitro findings indicating a folding defect in DF508 CFTR NBD synthetic peptide, the notion emerged that the temperature sensitive CFTR mutants represent a folding defect in the endoplasmic reticulum [57]. DF508, and other similar mutants obtain their final and stable conformation slower, and therefore, are recognized by the ER quality control machinery, become polyubiquitinated, and targeted for degradation in the proteasome [58]. Class II mutations mostly, but not exclusively represent single amino acid substitutions or deletions in one of the two NBDs. CFTR is an inefficiently processed protein. Only approximately 25% of wild type CFTR nascent chain is folded and processed completely, the rest is degraded during the process. Various class two mutants decrease this low efficiency even further, but to varying degrees. Consequently, disease severity within this class correlates with the amount of mutant protein that can be released from the ER. Almost none of DF508 mutant CFTR processes completely, thus, this mutation is associated with severe disease. Some other calls II mutants, such as A455E and P574H process more efficiently, and are associated with milder disease.

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Class III and class IV mutants are fully glycosylated and are targeted to the plasma membrane but either exhibit defective channel regulation (class HI) or ion conduction (class IV). Class III mutants generally exhibit mutations in the NBDs that lead to compromised channel activation. Class IV mutants represent a small group of amino acid substitutions in the membrane-spanning domains (e.g., R117H and R347P). The consequence of these mutations is reduced single channel conductance. 7. CFTR mutations as therapeutic targets: The discovery of CFTR gene in 1989 generated great anticipation of the forthcoming gene therapy for CF. Although it is still likely that such therapy eventually will be available in the future, research targeting this problem has shown us that accomplishing clinically useful CF gene therapy faces extraordinary challenges. However based on the emerging details of the cell biology and physiology of CFTR's cellular functions, and based on the understanding how the various CFTR mutations cause disease several alternative therapeutic approaches have been proposed. Developing some of these novel therapeutic concepts into practical therapies will be useful even if gene therapy becomes available, as they might allow the alleviation of airway obstruction that might be a prerequisite for the access of gene therapy vehicle to the lower airways. The discovery of the folding defect of Class II mutations, and the realization that these mutants once folded are functional, fueled the development of several therapeutic concepts. Since a miniscule proportion of DF508 CFTR is processed to functional form, boosting its expression level using butyrate [59] promises to have at least partial correction of the defect. Moreover, several observations indicated that the use of chaotropic agents such as DMSO [60] can increase the efficiency of DF508 CFTR processing in the ER, perhaps by not allowing the misfolded conformation to stabilize. Based on these in vitro data, finding a biologically safe and clinically usable combination of chemicals that boost expression levels and facilitate processing could provide a novel therapy for CF. As mentioned previously, certain class I mutants represent inefficient stop codons generated by single base substitutions in the CFTR gene. Aminoglycoside antibiotics can facilitate read through these stop codons, allowing the translation of full length, functional CFTR in cells where at least one of the mutant alleles falls into this category [61]. Furthermore, there is clinical evidence that such treatment strategy can alleviate the Cl" transport abnormality in the airways of CF patients as evidenced by a partial correction of the abnormal nasal transepithelial potential difference following parenteral gentamicin treatment for 7 days [53]. In addition to therapies aimed at increasing the amount of functional CFTR, various additional therapeutic approaches have been considered. Since the majority of CF scientists agree that a correction of the ion transport defect in the CF lung would alleviate the CF lung pathology, pharmacological manipulation of airway epithelial cell ion transport pathways is considered as therapy. These approaches include (a) CFTR openers that might increase the amount of Cl" transported by the available small amount of CFTR, (b) openers of alternative Cl" conductance pathways, such as synthetic channels and openers of Ca+ activated Cl" channels, (c) activators of K+ channels that increase the driving force for Cl- across the airway epithelial monolayer [62] and (d) inhibitors of Na+ channels [31].

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8. Inflammation in CF It has been known that the CF lung is continuously in an exaggerated state of inflammation [63]. There are two aspects of this inflammatory state that will be discussed here: (a) Inflammation might be a direct consequence of the CF defect, and not only a reaction to the infections that are present in the lung, and (b) inflammation reduces CFTR expression and function via multiple mechanisms. (a) Inflammation might be a direct consequence of the CF defect: Inflammation is present in airways of CF infants even under conditions when infectious agents could not be detected [64,65]. Additionally, an increased accumulation of mast cells and macrophages can be observed during fetal development in CF [66]. Furthermore, it has been shown that sustained dosage of Ibuprofen lowered the rate of lung function deterioration of CF patients over the period of 4 years [67]. These findings suggest that an exaggerated inflammatory response to pathogens is directly related to the CF defect, and might underlie the deterioration of lung function during the progress of the disease. There have been several proposed mechanisms that might explain the connection between mutant CFTR and inflammation at the cellular and molecular level. The expression of trafficking mutants of CFTR in cells has been correlated with an activation of the nuclear factor DB (NFDB) [68], NFDB activation has been generally considered to be an underlying mechanism of inflammatory processes at a cellular level. Cultured CF airway epithelial cells exhibit an exaggerated production of the inflammatory cytokines IL-6 and IL-8 upon stimulation with TNFD, when compared to normal, or corrected CF cells [69]. The mechanism of this abnormal response is not known. As discussed earlier, CFTR can also conduct glutathione. In CF airways the relative absence of glutathione might contribute to the lack of protection from inflammatory insults [70]. The exact contribution of epithelial secretory products to the inflammatory balance of the airway mucosa is not completely understood, but it is known that epithelial cells have the capacity to secrete a wide variety of molecules with inflammatory or antiinflammatory activity such as cytokines, surfactants, antiproteases. We have shown that in a CFTR-expressing epithelial HT29-CL19A cell line the regulation of the apical constitutive secretory pathway by cAMP is Cl"-dependent, and it likely involves the regulation of TGN acidification [45]. One of the main secretory products of HT29CL19A cells is D1 antitrypsin, an important antiinflammatory protein on mucosal surfaces. In the same cell line the inhibition of CFTR expression by the inducible expression of antisense CFTR mRNA [71], or by the cytokines IL-4 and IFND dramatically changes the secretion profile of apically secreted proteins, and eliminates the regulation of this apical secretion by cAMP [72]. These findings suggest that the regulation of the epithelial apical macromolecule secretion machinery might be affected in CF, and that this effect might have an impact on the inflammatory balance on mucosal surfaces. (b) Inflammation reduces CFTR expression and function via multiple mechanisms: Epithelial CFTR expression has been shown to be inhibited by various inflammatory mediators, including IFND, IL-4, and TNFD. [73-76]. Interestingly, IL-1D increases CFTR expression via a mechanism that is dependent on NFkB activation [77]. Furthermore, exposure of stably transduced cells to exogenous nitric oxide, dramatically reduces heterologously expressed CFTR [78]. On the other hand, treatment with Snitrosoglutathione, which promotes S-nitrosylation of proteins, as opposed to tyrosine nitration by nitric oxide in the presence of superoxide, increases DF508 CFTR expression and improves processing [79]. Understanding the exact relationship between CF mutations and the inflammatory response in the lung might aid the design of better therapies for CF. Additionally, in vitro data regarding inflammatory regulation of CFTR expression suggest that temporary downregulation of CFTR expression might have pathological implications in lung diseases other than CF when inflammation is present.

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9. Summary Since the first description of cystic fibrosis in 1936 an enormous amount of knowledge accumulated regarding the pathology of cystic fibrosis. The discovery of CFTR in 1989 has accelerated the studies aimed at the understanding of cellular level CF defects. However, the direct relationship between the various cellular level defects and disease pathology needs clarification. Most agree that ultimately CF will be cured by gene replacement therapy, but its practical application appears to be far in the future. A better understanding between the cellular and molecular CF defects, and the resulting clinical disease should aid the design of better pharmacotherapy for CF.

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[68JA.J. Weber, G. Soong, R. Bryan, S. Saba and A. Prince Activation of NF-kappaB in airway epithelial cells is dependent on CFTR trafficking and Cl- channel function, Am J Physiol Lung Cell Mol Physio! 281 (2001 )L71-78. [69JA.A. Stecenko, G. King, K, Torii, R.M. Breyer, R. Dworski, T.S. Blackwell, J.W. Christman and K.L. Brigham Dysregulated cytokine production in human cystic fibrosis bronchial epithelial cells, Inflammation 25 (2001)145-155. [70] V.M. Hudson Rethinking cystic fibrosis pathology: the critical role of abnormal reduced glutathione (GSH) transport caused by CFTR mutation, Free Radic Biol Med 30 (2001) 1440-1461. [71]T. Jilling, E. Weber, E.J. Sorscher and K.L. Kirk Modulation of CFTR expression in HT29-CL19A colonic cells via the regulated expression of antisense mRNA, Fed. Pulm. Supplement 13, A#71 (1996) 229. [72] T. Jilling, C.J. Venglarik, E.J. Sorscher and K.L. Kirk CFTR expression correlates with the regulation of apical constitutive protein secretion in polarized epithelial cells, Fed. Pulm. Supplement 13, A#68 (1996) 228. [73] T. Jilling, C.J. Venglarik, E.J. Sorscher and K.L. Kirk IFN gamma and IL-4 regulate CFTR expression in HT29-CL19A colonic epithelial cells, Fed. Pulm. Supplement 13, A#70 (1996) 228. [74] H. Nakamura, K. Yoshimura, G. Bajocchi, B.C. Trapnell, A. Pavirani and R.G. Crystal Tumor necrosis factor modulation of expression of the cystic fibrosis transmembrane conductance regulator gene, FEBS Lett 314 (1992) 366-370. [75] F. Besancon, G. Przewlocki, I. Baro, A.S. Hongre, D. Escande and A. Edelman Interferon-gamma downregulates CFTR gene expression in epithelial cells, Am J Physiol 267 (1994) C1398-1404. [76JS.M. Fish, R. Proujansky and W.W. Reenstra Synergistic effects of interferon gamma and tumour necrosis factor alpha on T84 cell function, Gut 45 (1999) 191-198. [77] E.G. Cafferata, A.M. Guerrico, O.H. Pivetta and T.A. Santa-Coloma NF-kappaB activation is involved in regulation of cystic fibrosis transmembrane conductance regulator (CFTR) by interleukin-lbeta, J Biol Chem 276 (2001) 15441-15444. [78] T. Jilling, I.Y. Haddad, S.H. Cheng and S. Matalon Nitric oxide inhibits heterologous CFTR expression in polarized epithelial cells, Am J Physiol 111 (1999) L89-96. [79] K. Zaman, M. McPherson, J. Vaughan, J. Hunt, F. Mendes, B. Gaston and L.A. Palmer Snitrosoglutathione increases cystic fibrosis transmembrane regulator maturation, Biochem Biophys Res Commun 284 (2001)65-70.

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Disease Markers in Exhaled Breath N. Marczin and M.H. Yacoub{Eds.) IOS Press, 2002

Exhaled markers in cystic fibrosis Beatrix BALINT1, Sergei A. KHARITONOV3, Ildiko HORVATH2, Peter J. BARNES3 Dept. of Thoracic Medicine, Medical Faculty of University of Szeged, Hungary, 2Dept. of Pathophysiology at the National Kordnyi Institute, Budapest, Hungary, 3Dept. of Thoracic Medicine, Imperial College School of Medicine at National Heart and Lung Institute, London, United Kingdom 1. Introduction Cystic fibrosis (CF) is a genetic disorder caused by mutation of CFTR gene leading a life-long chronic airway inflammation. Lung disease in CF is the primary cause of morbidity and mortality due to recurrent airway infection. The diagnosis of acute respiratory infection in individuals with CF is difficult because conventional measures of acute infection such as fever, raised leukocyte count, deterioration in lung function, and positive sputum culture are not always helpful [1]. The chronicity of lung disease in CF and the tendency for acute respiratory infections to present in an atypical fashion, poses problems for research design. Sputum is readily available in CF and does not require the patient to undergo a moderately invasive procedure, although measurement of cytokines and inflammatory mediators in CF sputum did not prove to be helpful for identifying acute exacerbations [2]. Invasive diagnostic method, such as bronchoscopic examinations and sampling via bronchoalveolar lavage (BAL) require a high deal of expenditure, cause strain to the patients and modify the samples to be taken as they irritate the airways [3,4]. It would be useful to have some other indicators of infection and inflammation as a diagnostic tool and as a way to monitor disease and guide therapy. Analysis of exhaled breath constituents may be a non-invasive method of monitoring inflammation and oxidative stress in the lungs. Measurement of exhaled nitric oxide (FENO) is the most widely investigated method for monitoring airway inflammation in several inflammatory lung diseases, such as asthma [5, 6], bronchiectasis [7], unstable chronic obstructive lung diseases (COPD) [8], viral and bacterial airway infection [9]. Surprisingly, in CF patients FENO and nasal nitric oxide (NO) are significantly lower than in normal subjects, despite the intense neutrophilic inflammation in the airways [10-14]. Carbon monoxide (CO), another exhaled marker may be more useful in CF, because it is elevated significantly in CF patients and increases further during acute exacerbation [15, 16]. Analysis of exhaled breath condensate is a new non-invasive method for detection of changes in lung metabolism and the inflammatory status of the lung. Several compounds of exhaled breath condensate have been recently investigated in CF, such as hydrogen peroxide (fyOi) [17, 18], NO-related products [11, 19-21], eicosanoids [22] and cytokines [23]. The aim of this publication to overview data regarding exhaled breath and exhaled breath condensate analysis in CF. We will discuss our experience regarding NO metabolites (nitrotyrosine) and cytokine (IL-8) in exhaled breath condensate from CF patients with and without acute exacerbation.

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2. Measurement of exhaled breath in CF Exhaled Nitric Oxide NO is produced in the respiratory system and is detectable by chemiluminescence analyser in airway gas. Concentrations are higher in the upper than the lower airways. Several different tissues in the lung have been identified as capable of NOS expression and NO formation. Generally FENO is increased in inflammatory condition such as asthma [5, 6], bronchiectasis [7], unstable COPD [8], upper airway infection [9], probably due to iNOS induction. In asthma, measurement of FENO may serve as a marker of airway inflammation [8, 24]. Surprisingly, despite chronic inflammation in CF lungs, exhaled and nasal NO levels are significantly lower in patients with CF either being stable or having acute exacerbation [1014]. The level of FENO in CF is not influenced by the age of patients [13, 14, 17], use of antibiotics [17], use of corticosteroids [11] as well as CF genotype [14]. Besides some technical issues, the possible explanations for the paradoxical reduction in FENO may be complex. On one hand, a reduced expression of iNOS may occur in CF lung [25], which can contribute to the decreased concentration of FENO. In inflammatory lung diseases expression of iNOS is induced by inflammatory signals such as cytokines and lipid polysacharide (LPS) [26, 27]. In contrast, CF epithelial cells when stimulated by cytokine mix and co-cultured with activated neutrophils, have reduced iNOS expression compared to normal epithelial cells [27]. On the other hand, an increased metabolism of NO to reactive nitrogen intermediates would account for the low levels of nasal and exhaled NO. NO can be oxidized rapidly to form reactive nitrogen intermediates such as nitrite (NOi"), nitrate (NOs") and peroxynitrite (ONOO") [25]. The microenvironment of CF lung - viscous mucus secretion and increased reactive oxygen species release from inflammatory cells - may facilitate the reaction of NO with inflammatory oxidants causing an increased formation of reactive NO metabolites. In CF airways NO in its different oxidative states can be trapped in the mucus. Measurement of free NO could therefore be misleading and may not reflect to the total NO production in CF airways. Exhaled Carbon Monoxide In contrast to FENO exhaled CO levels are markedly increased in patients with CF [15]. CO is a product of heme degradation by heme-oxygenase (HO). Two isoforms of HO have been described: the constitutive HO-2 and the inducible HO-1, which is ubiquitously distributed. HO-2 can be upregulated by oxidative stress and proinflammatory cytokines [27, 28] and is part of the protective response to oxidative stress [29], HO is present in the pulmonary vascular endothelium and alveolar macrophages [30, 31]. CF is characterized by increased oxidative stress in the airways, so probably elevated level of CO in exhaled breath reflects the increased HO-1 expression by the pulmonary vascular endothelium and alveolar macrophages. Furthermore high level of CO may inhibit iNOS activity [32] and therefore reduce the level of FENO. Exhaled CO elevates further during acute exacerbation suggesting that exhaled CO not only a marker of oxidative stress but is also a marker of disease activity [16]. It has been observed that exhaled CO can be decreased by corticosteroid therapy [15] and patients homozygous for delta508 mutation have higher exhaled CO concentration, than heterozygous patients [15].

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Taken together, measurement of exhaled CO seems to be a useful tool of detecting and monitoring cytokine mediated inflammation and oxidant stress in the lower respiratory tract and of assessing the efficacy of therapy. CO measurement is simple and non-invasive, so it can be repeated as needed, and is suitable for using at patients with advanced diseases as well as at children. Exhaled Hydrocarbons Hydrocarbons are non-specific markers of lipid peroxidation, which is one of the consequences of the constant and inevitable formation of oxygen radicals in the body. During the process of peroxidation of polyunsaturated fatty acids hydrocarbons are distributed in the body, partly metabolized and excreted in the breath. Ethane is produced from lipid peroxidation and can be measured in the exhaled breath. Exhaled ethane can be collected into a reservoir and can be analyzed by chromatograpy. It is elevated in CF compared to normal subjects and reduced by steroid-treated patients. The level of exhaled ethane correlates with exhaled CO and lung function parameter (RVYTLC) [33]. It may be a useful noninvasive marker of oxidative stress. 3. Measurement of exhaled breath condensate in CF NO-related products NO reacts with superoxide to yield peroxynitrite and it can be trapped by thiolcontaining molecules such as cysteine and glutathione, to form S-nitrosothiols or can be oxidized to nitrite (NO2~) and nitrate (NOs") [34]. NO metabolites, such as NOi", and NOs", can be detected in airway aspirates, BAL fluid and exhaled breath condensate from normal subjects [12]. Inflammatory conditions are associated with enhanced NO formation reflecting to the increased concentrations of its metabolites. NO metabolites have been measured is sputum from CF patients [35] as well as in exhaled breath condensate [11] and probably reflect better to NO metabolism than FENO in CF [11 ]. Nitrite, S-nitrosothiols and Nitrotyrosine Nitrite levels in exhaled breath condensate are significantly higher in patients with stable CF compared to normal subjects [11, 20, 23]. NO2~ concentration in breath condensate correlates positively with circulating plasma leucocytes and neutrophils, and does not correlate with FENO and lung function parameters. It supports the hypothesis, that in inflamed airways, a significant proportion of NO from the lower airways may have been degraded by oxidation to NO2" and/or NO3". S-nitrosothiols are formed by interaction of NO with glutathione and may limit the detrimental effect of NO. It can be detected in exhaled breath condensate in several inflammatory diseases such as asthma, COPD and cystic fibrosis [20]. It has been published that S-nitrosothiols values are elevated in adult patients with more severe CF during both stable period and acute exacerbations [20]. Nitrotyrosine is a marker of protein nitration and can be detected by using a specific nitrotyrosine antibody. Tyrosine nitration can be mediated by multiple pathways under different conditions, suggesting that nitrotyrosine may be considered as a collective indicator for the involvement of reactive nitrogen species [36]. Nitration of tyrosine could impact deleteriously on cellular function and viability because this specific modification is known to alter protein function in vitro [36].

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Peroxynitrite is a potent oxidant, formed by the rapid reaction of the free radicals NO and Oi" and causes tyrosine nitration in lung tissue [37]. The toxicity of peroxynitrite is due to the direct reactions of the anion (ONOO"), as well as the reactivity of the acid (ONOOH). Activation of inflammatory cells, such as neutrophils, eosinophils and macrophages induces a marked production of superoxide facilitating the formation of peroxynitrite [38]. In chronic inflammation, or other inflammatory cell-mediated process, the myeloperoxidase (MPO)dependent pathways must be considered. Large numbers of polymorphonuclear neutrophils (PMN) accumulate in airways of CF patients, and lead to increased MPO activity [39]. Activated human PMNs can convert NOi" into inflammatory oxidants through MPO pathway [36]. It has been suggested that MPO-catalysed nitration in the presence of IHhOi to form nitrating intermediates from NO2~, a main end-product of NO, is an alternative mechanism of protein nitration, which is independent of peroxynitrite [36]. The other pathways of the formation of nitrotyrosine which are detected in vitro, including direct oxidation of NO2~ by HiOi or hypochlorous acid or reaction of NO or nitrogen dioxide with tyrosyl radicals in vivo have not been completely elucidated [37]. We found increased levels of nitrotyrosine in exhaled breath condensate from stable CF patients compared to normal subjects [21]. It has overlapped with the finding of elevated level of nitrotyrosine in CF sputa [23]. In our study there was no correlation between FENO and nitrotyrosine concentration, although there was an inverse correlation between nitrotyrosine levels and disease severity measured by lung function. There was no significant difference in nitrotyrosine levels in breath condensate between patients treated or not treated with steroids. In this study we have provided evidence that oxidative stress induced by inflammation produces nitrotyrosine, which presumably reflects increased direct nitration by granulocyte peroxidases. Nitration of proteins by MPO is, perhaps a major source of nitrotyrosine in patients with CF who have, a low NO production. p < 0.001

p < 0.05

50 ! IS) 40 c

g 30-

|,j |j 10 0J

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steroid Treated

Normal Subjects

CF steroid Untreated Treated

Figure 1. FENO in stable CF patient and normal subjects (right panel). Nitrotyrosine in exhaled breath condensate from stable CF patient and normal subjects (left panel)

Hydrogen peroxide Activation of inflammatory cells, including neutrophils, macrophages and eosinophils, results in an increased production of superoxid, which by undergoing spontaneous or enzymcatalyzed dismutation leads to formation of H2O2. As HzOz is less reactive than other reactive oxygen species, furthermore it is soluble, increased H2O2 in the airway equilibrates with air [40]. In CF breath condensate H2O2 level proved to be not elevated in patients with stable CF [18], although it has been reported being increased in CF with acute exacerbation, and

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decreased during intravenous antibiotic treatment [17]. Measurement of HaC^ in exhaled breath condensate may serve as a useful noninvasive parameter of airway inflammation. Eicosanoids Eicosanoids are inflammatory mediators, can cause vasodilatation or vasoconstriction, plasma exudation, mucus secretion, bronchoconstriction or bronchodilatation and inflammatory recruitment. Several types of biomolecules with either opposite effects belong to this group. All of them derive from the arachidonic acid cascade and include prostaglandins, thromboxane, isoprostanes and leukotrienes. Several of them can be detected in exhaled breath condensate providing an opportunity to assess the eicosanoid profile in lung diseases. Isoprostanes are a novel class of prostanoids formed by free radical-catalyzed lipid peroxidation of arachidonic acid [41]. 8-Isoprostane is elevated in plasma as well as in exhaled breath condensate in stable CF. Its level negatively correlates with FEV] and positively correlates with exhaled CO [22]. Leukotrienes, prostaglandines in exhaled breath condensate have been investigated in asthma and COPD, but there are no published data available regarding their levels in CF. Cytokines Chronic and acute inflammation is associated with activation of pro-inflammatory cytokine network. Several pro- and anti-inflammatory cytokines can be detected in sputum [42] as well as in BAL fluid in different inflammatory diseases including CF [43]. Interleukin-8 (IL-8) is a neutrophil-activating peptide associated with acute and chronic inflammation is produced by a wide variety of cells, including bronchial epithelial cells, monocytes, alveolar macophages, endothelial cells, fibroblasts and PMNs. IL-8 levels are elevated in sputum and BAL fluid in CF [42, 44], promoting the destructive inflammatory process in the lung. It has been reported to play a major role in the early inflammatory pathogenesis in the airways of CF patients before the manifestation of bacterial infection [45]. Recent publication has shown that IL-8 could be detected in exhaled breath condensate of children with CF [23]. We assessed the level of IL-8 in exhaled breath condensate from clinically stable and unstable adult CF patients. Furthermore, we investigated the alteration of IL-8 level in exhaled breath condensate in unstable CF after recovery from acute respiratory tract infection. We compared the levels of IL-8 in breath condensate from CF patients to other non-invasive inflammatory marker like FENO, as well as lung function and blood test. Subjects and Methods: 18 clinically stable and 12 unstable CF patients were recruited into the study along with 11 healthy normal subjects. 7 CF patients with acute exacerbation were followed after 10-14 days of complex treatment and IL-8 was detected again after the recovery from the acute symptoms. For all subjects FENO, lung function measurement and collection of exhaled breath condensate as well as blood test were performed. IL-8 levels in exhaled breath condensate were measured by sandwich ELISA (R & D Systems Europe, Abingdon, UK). Initially, all samples from the breath condensate were concentrated fourfold, using a freeze dryer (Modulyo, Edwards, Crawley, UK), and then analyzed according to the manufacturer's instructions. Detection was performed with tetramethylbenzidine (R & D Systems Europe. Abingdon, UK) following R & D instructions. The lower limit of detection for this assay was 16pg/ml.

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Results: FENO was significantly lower in stable CF patients than in normal subjects (3.9 ±0.5 vs 6.1 ± 1.0 ppb, p < 0.05) and did not alter significantly during the acute exacerbation. There was no significant difference in the levels of IL-8 in exhaled breath condensate between normal subjects and stable CF patients, although it was significantly elevated in unstable CF compared with normal subjects (36.1 ± 5.1 vs 17.4 ± 2.8 pg/mL, p < 0.01). 7 patients were followed after 10-14 days of antibiotic treatment and IL-8 decreased significantly to the baseline level after recovery (34.3 ± 2.0 vs 21.8 ± 3.9 pg/ml, p < 0.05). Discussion: Our study demonstrated that IL-8 can be detected in exhaled breath condensate of normal subjects as well as in adult CF patients. IL-8 levels were not different in exhaled breath of stable CF patients from normal subjects, but it was significantly higher in CF with acute exacerbation than in non-smoking healthy controls. IL-8 is an important proinflammatory cytokine, which has an outstanding role in CF pathogenesis. In CF patients endogenous signal may be generated, leading to an intense inflammatory response with the production of factors, which could damage the airway surface and favor infection and bacterial colonization. This signal may be directly linked to the abnormal CFTR and may be associated with a dysregulated inflammatory response. Conclusion: Our data indicate that IL-8 in exhaled breath condensate may be useful noninvasive marker of airway inflammation in CF exacerbation. p < 0.01 ns

p < 0.05

ns

50-

50-

25-

25-

0-

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Stable Unstable CF

Admission

Day 10

Figure 2. IL-8 in exhaled breath condensate from CF patient with and without acute exacerbation (right panel). IL-8 in exhaled breath condensate from CF patient with acute exacerbation at the onset of exacerbation and at the time of recovery (left panel)

4. Summary This chapter has overviewed the recent developments of assessment of exhaled breath and exhaled breath condensate in CF patient with and without acute exacerbation. NO concentrations in the expired air of patients with stable CFare significantly decreased compared to normal subjects. Furthermore there is no significant alteration in FENO during acute exacerbation. This is likely to be on one hand due to the decreased iNOS expression by the epithelial cells, and on the other hand due to the increased consumption of NO by superoxide in CF airways. Measurement of exhaled CO may be promising non-invasive tool in CF, because its level is significantly increased in stable CF and elevates further during exacerbation. Analysis of exhaled breath condensate is a new, currently studied, non-invasive research procedure, which may have an important place in the diagnosis and management of inflammatory lung diseases such as CF. NO-related products in exhaled breath increased

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significantly in CF patients reflecting the increased oxidative metabolism of NO in CF airways. Eicosanoids, proteins and cytokines, electrolytes and pH of breath condensate in CF have been investigated, or the topic of the present research, which may offer a possibility to us to monitor the airway inflammation and to assess the efficacy of therapy. Acknowledgements This study was supported by the Hungarian Respiratory Society, Foundation for Patients with Lung & Heart Diseases (Hungary) and the British Lung Foundation (NHLI, UK). References 1. A. L. Smith, G. Redding, C. Doershuk, D. Goldmann, E. Gore, B. Hilman, M. Marks, R. Moss, B. Ramsey, T. Rubio, et al. Sputum changes associated with therapy for endobronchial exacerbations in cystic fibrosis, JPediatr 112 (1988) 547-554. 2. J. M. Wolter, R.L. Rodwell, S. D. Bowler, J. G. McCormack. Cytokines and inflammatory mediators do not indicate acute infection in cystic fibrosis, Clin and Diagnostic Lab Immunol 6 (1999) 260-265. 3. M. W. Konstan, K. A. Milliard, T. M. Norvell, M. Berger. Broncoalveolar lavage findings in cystic fibrosis patients with stable, clinically mild lung disease suggest ongoing infection and inflammation, Am J Respir Crit Care Med. 150(1994) 448-54 4. K. Balough, M. McCubbin, M. Weinnnberger, W. Smits, R. Ahrens, R Pick, The relationship between infection and inflammation in the early stages of lung disease from cystic fibrosis, Pediatr Pulmonol 20 (1995)63-70. 5. K. Alving, E. Weitzberg and J. M. Lundberg, Increased amount of nitric oxide in exhaled air of asthmatics. Ear Respir J. 6(1993) 1368-1370. 6. S. A. Kharitonov, D. Yates, R. A. Robbins, R. LoganSinclair, E. A. Shineboume and P. J. Barnes, Increased nitric oxide in exhaled air of asthmatic patients, Lancet343 (1994) 133-135. 7. S. A. Kharitonov, D. A.U Wells, B. J. Oconnor P. J. Cole, D. M. Hansell, LoganSinclair, P. J. Barnes. Elevated levels of exhaled nitric oxide in bronchiectasis, Am J Respir Crit Care Med 151 (1995) 18891893. 8. W. Mazaik, S. Loukides, S. V. Culpitt, P. Sullivan, S. A. Kharitonov and P. J. Barnes, Exhaled nitric oxide in chronic obstructive pulmonary disease, Am J Respir Crit Care Med 157 (1998) 998-1002. 9. S. A. Kharitonov, D. H. Yates, P. J. Barnes, Increased nitric oxide in exhaled air of normal human subjects with upper respiratory infections, Eur Respir J. 12 (1995) 295-297. 10. H. Grasemann, E. Michler, M. Wallot, F. Ratjen. Decreased concentrations of exhaled nitric oxide (NO) in patients with cystic fibrosis. Pediatr Pulmonol. 24 (1997) 173-177. 11. Ho LP, Innes JA, Greening AP. Nitrite levels in breath condensate of patients with cystic fibrosis is elevated in contrast to exhaled nitric oxide. Thorax 53 (1998) 680-684. 12. H. Grasemann, F. Ratjen. Cystic fibrosis lung disease: The role of nitric oxide. Pediatr Pulmonol 28 (1999)442-448. 13. H. E. Elpick, E. A. Demoncheaux, S. Ritson, T. W. Higenbottam, M. L. Everard, Exhaled njtric oxide is reduced in infants with cystic fibrosis, Thorax 56 (2001) 151-152. 14. S. R. Thomas, S. A. Kharitonov, S. F. Scott, M. E. Hodson, P. J. Barnes, Nasal and exhaled nitric oxide is reduced in adult patients with cystic fibrosis and does not correlate with cystic fibrosis genotype, Chest 117(2000)1085-1089. 15. P. Paredi, P. L. Shah, P. Montuschi, P. Sullivan, M. E. Hodson, S. A. Kharitonov, P. J. Barnes, Increased carbon monoxide in exhaled air of patients with cystic fibrosis, Thorax 54 (1999) 917-920. 16. J. D. Antuni, S. A. Kharitonov, D. Hughes, M. E. Hodson, P. J. Barnes, Increase in exhaled carbon monoxide during exacerbations of cystic fibrosis, Thorax 55 (2000) 138-142. 17. Q. Jobsis, H. C. Raatgeep, S. L. Schhellekens, A. Kroesbergen, W. C. Hop, J. C. de Jongste, Hydrogen peroxide and nitric oxide in exhaled air of children with cystic fibrosis during antibiotic treatment, Eur /?esp .716 (2000) 95-100. 18. L P Ho, J. Faccenda, J. A. Innes, A. P. Greening, Expired hydrogen peroxide in breath condensate of cystic fibrosis, Eur Respir J 13 (1999) 103-106. 19. Grasemann H, loannidis I, Tomkiewicz RP, de Groot H, Rubin BK, Ratjen F. Nitric oxide metabolites in cystic fibrosis lung disease. Arch Dis Child 78 (1998) 49-53.

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Disease Markers in Exhaled Breath N. Marczin and M.H. Yacoub (Eds.) 1OS Press, 2002

Lung Cancer Screening by Breath Analysis Michael J. Berry Quadrivium, L.L.C., P. O. Box 1421, Pebble Beach, CA 93953 USA Abstract. Laser photoacoustic detection of biomarkers in exhaled breath samples may be suitable for lung cancer screening in the general population.

1. Background Lung cancer (LC) is a major cause of death worldwide, accounting for more than 1.1 million deaths (ca. 2.1% of all causes) annuallyf'!. In the United States (US), the five-year survival rate following LC diagnosis is only 14% for all patients!2), but improves to 60% or better for patients diagnosed with early-stage (Stage 0 - carcinoma in situ or Stage IA small localized tumor) disease!3). A simple, inexpensive LC screening test that can identify early-stage LC patients with high sensitivity (i.e., a low percentage of false negatives) and high specificity (i.e., a low percentage of false positives) is needed to provide the basis for a significant reduction in LC deaths. 2. Previous Studies Over 1700 endogenous volatile organic compounds (VOCs) have been identified in human exhaled breatW4!; these VOCs are primarily excreted metabolic products. Malignant cells exhibit differences in metabolism compared to normal cellsl5'; these differences may produce VOC biomarkers. Candidate VOC biomarkers have been identified by several groups using analyses of LC patient and control breath samples by gas chromatography/ mass spectrometry (GC/MS^12!. Results of these studies are summarized as follows. Gordon, et al. (1985)l6); In a retrospective study involving w=12 LC patients and m=9 control subjects, statistical analysis of GC/MS data by a discriminant model yielded 93% classification (LC vs. control) accuracy using relative concentrations of only three VOCs (acetone, 2-butanone, and 1-propanol). Preti, et aL (1988)171: In a retrospective study involving w=10 LC patients and m=16 controls (in two subgroups: age-matched and younger subjects), statistically significant (p<0.05) increases of o-toluidine, and in some cases aniline, were identified in LC patient samples vs. both controls. Biomarkers previously identified in the Gordon, et al. studyl6! could not be confirmed due to inadequate GC separations of these VOCs. O'Neill, et aL (1988)[81: In a retrospective study involving four breath samples from each of n=8 LC patients, 28 VOCs occurred in >90% of the samples; this small group (of the total 386 VOCs observed) represents the best candidates for high sensitivity LC biomarkers. Two of these high-occurrence rate VOCs (acetone and 2-butanone) were the same as previously-identified LC biomarkersl6). O'Neill (1990)191: Several other retrospective studies by this group were reported 1) In a replication study involving four breath samples from each of n=10 LC patients, the investigators concluded that a) differences among the four samples from each patient were not significant, but b) the 2nd morning (AM) aliquot (2nd 20 I aliquot from the AM 50 i collection bag) should be used for clinical studies. However, systematic differences in both number and concentration of VOCs between 1st and 2nd aliquots from AM and afternoon samples suggest that there was a systematic sampling error of undetermined cause. 2) In a comparison study involving pre- and postsurgical breath samples from w=13 LC patients, candidate LC biomarkers were identified for use in discriminant tests on two independent groups: p=25 LC patients and m=25 smoker controls. One or more of a subset

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of 4 VOCs (tolualdehyde, e-caprolactone, and two unknowns) was present in 68% of the p-25 LC patients, but in only 4% of the m=25 smoker controls; however, these VOCs were not the same as previously-identified LC biomarkers'6-7]. 3) In an expanded study (compared to the initial study by the same group!6!) involving «=30 matched (in terms of age, gender, smoking history, etc.) presurgical LC patients and w=30 matched smoker controls, as well as ^=14 unmatched presurgical LC patients, q=\0 subjects with lung diseases other than LC, and r=10 subjects with cancers other than LC, 73 VOCs (of a total of 418 observed) were not present in n and m groups in homogeneous proportions (at the a<0.05 confidence level). Several discriminant functions using subsets of these 73 candidate LC biomarkers were tested for their utility in identifying LC cases in all groups («, m, p, q and r). For example, a discriminant function using a subset of only 5 VOCs achieved perfect classification of all subjects in groups n, m and p as well as classification of all subjects in group r as LC patients and classification of most of the subjects (7 of 10) in group q as controls. However, in only one case (e-caprolactone) were any of the candidate VOC biomarkers (in any of the discriminant functions) the same as previously-identified LC biomarkers from either earlier studies!6'7! or from the comparison study by the same group on w=13 pre- and postsurgical LC patients. Phillips, et al. (1999)tn-'21: in a retrospective study involving 5=108 subjects with abnormal chest radiographs, LC was confirmed histologically in w=60 LC patients and excluded in /=48 subjects. Two separate analyses identifying candidate LC biomarkers were reported: 1) a discriminant model using 22 candidate VOCs (of a total of 1124 observed) with a post-test probability of 0.46 had 100% sensitivity and 81.3% specificity in identifying Stage 1 LC patients (but only 80.4% sensitivity for later-stage LC patients)!1'1, and 2) a different statistical model using 44 candidate VOCs had 93.3% sensitivity and 91.7% specificity for classifying all .s=108 subjects as LC patients vs. non-LC subjects!12'. However, only 4 VOCs were the same in the two sets(1M2J of candidate LC biomarkers and these were not the same as previously-identified LC biomarkers from earlier studies!6-9]. In all the studies summarized above, each GC/MS analysis typically required the collection of at least 10 i of exhaled breath sample, concentration of the VOCs in the breath sample on an adsorbent, thermal desorption into a GC column, MS detection of the column elutants, and matching of MS data to library spectral data; in only one studyt7! were authentic samples used to confirm a few VOC identities. Experimental pitfalls contribute to uncertainties in VOC identification, so it is perhaps not surprising that various GC/MS studies differ in their conclusions about key LC biomarkers. A definitive GC/MS study should be performed, in part to provide a "gold standard" of analysis, but even when that study is completed, it seems unlikely that GC/MS methodology will be routinely suitable for LC screening. GC/MS analyses on complex, multicomponent breath samples are inherently time-consuming and relatively expensive compared to, for example, the prostatespecific antigen (PSA) blood test that is recommended for annual prostate cancer screening in males!13'. The PSA test is inexpensive (ca. US$ 50-80), simple, and minimally invasive, requiring collection of only a small blood sample without separations, followed by irnmunoassay. A low cost, simple, non- or minimally invasive test of this kind is needed for LC screening, which may then be followed by more extensive diagnostic procedures (e.g., bronchoscopy and histologic evaluation) in subjects with positive tests. One non-VOC LC biomarker [nitric oxide (NO)] in exhaled breath should also be considered. Liu, et al. (1998)!14!; In a retrospective study involving «=28 LC patients and m=20 control subjects, statistically significant (p<0.001) increases of NO were identified in LC patient samples vs. controls. Although increased NO excretion has been observed in other inflammatory lung diseases such as asthma!15! (leading to concern about the specificity of LC screening by NO detection), additional LC clinical studies including NO detection are certainly desirable.

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3. Laser Photoacoustic Detection VOCs and NO have characteristic infrared (IR) absorption spectra that provide a basis for LC screening of exhaled breath samples. Since these molecules are present at part-perbillion by volume (ppbV) or lower concentrations, an ultrasensitive IR absorption detection method is required. "Listening" to molecules being excited [photoacoustic detection (PAD)!16-'7]] using a high-power IR laser (modulated at audio frequency) for excitation combined with a sensitive microphone for detection of the resulting sound waves (i.e., pressure pulses following vibration-to-translation collisional energy transfer from exerted molecules to the gas mixture) provides the required sensitivity. PAD using a low-power, line-tunable (in the 5.0-6.5 urn spectral region) carbon monoxide (CO) laser has been demonstrated to be suitable for NO detection at the 10 ppbV level and VOC detection down to the 1-5 ppbV level for acrolein and other VOCs with intense IR absorptions within the (restricted) spectral range covered'16]. Improvements such as laser intracavity cell operation!17) and the use of higher power, broadly-tunable CO lasers!'*-19! extend the PAD sensitivity for NO and most VOCs well below 1 ppbV. Interferences by H2O and CO2 (the major IR absorbers within breath samples) can be overcome by tuning the laser to interference-free spectral regions and/or by using matched sample and reference cells!'6!. Laser PAD offers many advantages compared to GC/MS analysis: 1) only small sample volumes (<100 m£) are required so single breath collection methods!20! are feasible, 2) no concentration and/or separation steps are required (as demonstrated by IR absorption spectral simulations using relative concentrations of the most abundant VOCs observed in LC patients vs. controls!9! - not shown) so sample contamination, loss, and pre-reaction artifacts are either removed or minimized, and 3) the detection and analysis procedure is computer-automated and fast (less than 5 min per sample). 4. Current Program We are in an early stage of program development: 1) a CO laser PAD system has been constructed and operated, 2) a cell culture study (involving comparative analysis of VOCs from normal vs. malignant bronchial epithelial cells) has been planned, and 3) a human clinical trial has been planned. The planned cell culture study is useful to identify candidate LC biomarkers in an in vitro model by conventional GC analyses (similar to previous excreted alkane analyses for isolated liver cells and even perfused liversl2'!), without interference from VOCs produced by liver metabolism or other confounding sources. The planned human clinical trial has several phases that are designed to satisfy US Food and Drug Administration (FDA) safety and efficacy requirements for Pre-Market Approval (PMA) of the laser PAD device for LC screening: a) Phase I - a pilot clinical study involving n=\2 LC patients and m=24 control subjects (in two groups of 12: nonsmokers and heavy smokers with no known pulmonary disease), b) Phase II - a retrospective clinical study involving w=120 LC patients and /w=480 controls (in four groups of 120: nonsmokers, heavy smokers with no known pulmonary disease, subjects with pulmonary disease other than LC, and subjects with cancer other than LC), and c) Phase III - a prospective clinical study involving x=1200 high-risk subjects (e.g., heavy smokers at least 50 years old with a family history of LC). Note that the numbers of LC patients and control subjects in our planned clilnical trial are much larger than those enrolled in previous GC/MS studies!6-12l; after consuhation with present and former FDA staff members, it is anticipated that this large enrollment is required in order to have sufficient statistical power to assess the safety and efficacy (including the sensitivity, specificity, and predictive power) of the laser PAD LC screening device and procedure. Although all or most of the clinical trial will be completed in the US, it is also highly desirable to complete several non-US human clinical studies prior to, and in parallel with, US studies in order to develop successful protocols.

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Acknowledgments The author thanks the sponsor of this work (Edward Q. Yavitz, MD) as well as co-workers and colleagues (Sina Bahmanyar, MD; Richard P. Chiacchierini, PhD; Walter F. Coulson, MD; Kim M. Kerr, MD; Andrew B. Lindstrom, MS; Richard F. Menefee, AAS; Clifton F. Mountain, MD; Dennis L. Myers, MD; Hugh J. O'Neill, PhD; Terence H. Risby, PhD; Giuseppe L. Valderrama, PhD) for stimulating discussions and experimental help. References [I] World Health Organization, World Health Report 2000, Annex Table 3 (Deaths by Cause, Sex and Mortality Stratrum in WHO Regions, Estimates for 1999 - statistical data for lung, bronchus, and trachea cancers are grouped together). Available online at http://www.who.int/whr/2000/en/statistics.htm. [2] R. T. Greenlee, M. B. Hill-Harmon, T. Murray and M. Thun, Cancer Statistics 2001, CA Cancer JClin 51 (2001) 15-36. Survival rates are relative to the age-adjusted population. [3] C. F. Mountain, Revisions in the International System for Staging Lung Cancer, Chest 111 (1997) 1710-17. [4] M. Phillips, J. Herrera, S. Krishnan, M. Zain, J. Greenberg and R N. Cataneo, Variation in Volatile Organic Compounds in the Breath of Normal Humans, J Chromatogr B 729 (1999) 75-88. [5] D. M. Goldberg and E. P. Diamandis, Models of Neoplasia and Their Diagnostic Implications: A Historical Perspective, Clin Chem 39 (1993) 2360-2374. [6] S. M. Gordon, J. P. Szidon, B.K. Krotoszynski R. D. Gibbons and H. J. O'Neill, Volative Organic Compounds in Exhaled Air from Patients with Lung Cancer, Clin Chem 31 (1985) 1278-1282. [7] G. Preti, J. N. Labows, J. G. Kostelc, S. Aldinger and R. Daniele, Analysis of Lung Air from Patients with Bronchogenic Carcinoma and Controls Using Gas Chromatography-Mass Spectrometry, J Chromatogr 432(1988) 1-11. [8] H. J. O'Neill, M.H. O'Neill, R. D. Gibbons and J. P. Szidon, A Computerized Classification Technique for Screening for the Presence of Breath Biomarkers in Lung Cancer, Clin Chem 34 (1988) 1613-18. [9] H. J. O'Neill, Non-Invasive Approach for Detection of Lung Cancer, Final Report, National Institutes of Health Grant Number CA37056 (1990). {10] N. Rizvi and D. F. Hayes, A Breathalyser for Lung Cancer?, Lancet 353 (1999) 1897-8. [ I I ] M. Phillips, K. Gleeson, J. M. B. Hughes, J. Greenberg, R. N. Cataneo, L. Baker and W. P. McVay, Volatile Organic Compounds in Breath as Markers of Lung Cancer: A Cross-Sectional Study, Lancet 353 (1999) 1930-33. [12] M. Phillips, Breath Test for Detection of Lung Cancer, US Patent Number 5,996,586 (1999). [13] R. A. Smith, A. C. von Eschenbach, R. Wender, B. Levin, T. Byers, D. Rothenberger, D. Brooks, W. Creasman, C.Cohen, C. Runowicz, D. Saslow, V. Cokkinides and H. Eyre, American Cancer Society Guidelines for the Early Detection of Cancer: Update of Early Detection Guidelines for Prostate, Colorectal, and Endometrial Cancers, CA Cancer JClin 51 (2001) 38-75. The new guideline includes the recommendation that: "The PSA test and the DRE (digital rectal examination) should be offered annually beginning at age 50 to men who have a life expectancy of at least 10 years. Men at high risk should begin testing at age 45." This type of guideline is a model for LC screening, which should be offered to both men and women beginning at age 50 (or earlier for high-risk individuals). [14] C-Y Liu, C-H Wang, T-C Chen, H-C Lin, C-T Yu and H-P Kuo, Increased Level of Nitric Oxide and Up-Regulation of Inducible Nitric Oxide Synthase in Patients with Primary Lung Cancer, BrJCancer 78 (1998)534-541. [15] S.A. Kharitonov and P. J. Barnes, Exhaled Markers of Pulmonary Disease, AmJRespir Crit Care Med 163(2001) 1693-1722, [16] M. W. Sigrist, Environmental and Trace Gas Analysis by Photoacoustic Methods. In: A. Mandelis (ed.), Progress in Photothermal and Photoacoustic Science and Technology, Vol. I. Elsevier, New York, 1992, pp. 369-427. [17] R. G. Bray and M. J. Berry, Intramolecular Rate Processes in Highly Vibrationally Excited Benzene, J Chem Phys 71 (1979) 4909-4922. Intracavity laser PAD increases detection sensitivity by up to 100-fold. [18] S. Buscher, O. Schulz, A. Dax, H. Kath and W. Urban, Improvement of the Performance of cw CO Lasers by Using Externally Ribbed Wall Cooled Discharge Tubes, Appl Phys B 64 (1997) 307-309. [19] J. Puerta, W. Herrmann, G. Bourauel and W. Urban, Extended Spectral Distribution of Lasing Transitions in a Liquid-Nitrogen Cooled CO Laser, Appl Phys B 19 (1979) 439-440. [20] J. D. Pleil and A. B. Lindstrom, Collection of a Single Alveolar Exhaled Breath for Volatile Organic Compounds Analysis, AmJInd Med 28 (1995) 109-121. [21] A. Miiller and H. Sies, Assay of Ethane and Pentane from Isolated Organs and Cells, Meth Enzymol 105 (1984)311-319.

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Nitric Oxide and Rheumatic Diseases Giovanni ROLLA Universita di Torino (Italy) - Immunologia Clinica e Allergologia Ospedale Mauriziano Umberto I di Torino largo Turati 62, 10128 Torino (Italy) Abstract. Excessive nitric oxide production has been reported during the course of many rheumatic diseases (systemic lupus erythematosus, rheumatoid arthritis, Sjogren's syndrome, systemic sclerosis). The finding of an increased production of NO also by the respiratory tract in some of these diseases, through its measurement in the exhaled air, may represent an easy-to obtain marker of inflammation, the clinical value of which is currently under evaluation.

1. Introduction Experimental and clinical observations have been accumulated in the last few years pointing to an excessive NO production during the course of many rheumatic diseases. The studies pertinent to exhaled NO in systemic lupus erythematosus (SLE), systemic sclerosis (SSc) and Sjogren's syndrome (SS) will be reviewed here. The role NO might play in rheumatic diseases appears to be dual, that is pro-inflammatory and antiinflammatory, depending on the amount produced, the site of production and the local environment. A dual effect of NO on lymphocyte functions and on apoptosis might be relevant to the pathogenesis and clinical course of rheumatic diseases In a murine model it has been demonstrated that Th-1 T cell clones produce NO following activation and that NO inhibits the cytokine production of Th-1, but not of Th-2 T cell clones upon Tcell receptor activation (1) . Bauer et al., however, reported that cytokine production by purified T-cell subpopulations (Th-1 and Th-2) was equally impaired by NO donors, that is NO did not preferentially inhibit Th-1 cytokine secretion of activated human T cells in vitro (2). Apoptosis is another important mechanism through which NO may contribute to the pathogenesis of rheumatic diseases. An increase in apoptosis rate due to the release of cytoplasmic and/or nuclear antigens (e.g. Rp/La, dsDNA) may play a central role in promoting and exacerbating autoimmune diseases, such as SLE. Also a decrease in apoptosis may play an important immunoregulatory role. A decrease in eosinophil apoptosis may be crucial in eosinophil-mediated diseases, such as bronchial asthma and Churg-Strauss vasculitis, and defective apoptosis may cause survival of autoreactive T cell clones, which are normally deleted from the host repertoire. NO has been shown to display both pro-apoptotic and anti-apoptotic actions. Low concentration of NO inhibits apoptosis in human B lymphocytes (3), while high concentration of NO may induce apoptosis through the formation of peroxynitrite (4). Possible pathogenetic role of increased NO production in autoimmune diseases is synthesized in Fig.l.

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1

1 l^\J

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direct cytotoxlclty

1f Release of intracellular antigens

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Fig.l Role of NO in autoimmune diseases. Intracellular antigens may be released as a result of direct cytotoxicity and increased apoptosis in target organs. A decrease in autoreactive lymphocytes apoptosis, combined with an expansion of CD4+ Th2 lymphocytes lead to B cells activation and autoantibodies production. The immunocomplexes may eventually upregulate NO synthase in macrophages and in endothelial cells with resulting increased NO production.

2. Systemic Lupus Erythematosus (SLE). SLE is a disease of unknown etiology in which tissues and cells are damaged by pathogenic autoantibodies and immune complexes. The disease is generally multisystemic, even if at onset SLE may involve only one organ and additional manifestations occur over time. Most patients experience exacerbations interspersed with periods of relative quiescence. A murine model of lupus-like disease is accompanied by increased urinary excretion of nitrite, enhanced macrophage NO production, an increased immuno reactive NO synthase in spleen and renal tissue. In the same murine model, the oral administration of the NOS inhibitor L-NG monomethyl arginine (LNMMA) after the onset of disease, reduced proteinuria and the addition of dietary arginine restriction to L-NMMA also significantly improved renal pathology scores (5). An increased production of NO has been observed in patients with SLE in comparison to normals by Wanchu et al. (6), who also observed highest values of nitrites in patients with more active disease. The activity of disease is calculated on the basis of a score which takes into account clinical, laboratory, instrumental activity indexes referred to the various organs and systems (SLE Disease Activity Index or European Consensus on Lupus Activity Measurement). The clinical manifestations of activity in Wanchu series were mainly articular and dermatologic. Similar results have been obtained by Gilkesson et al , who measured also serum levels of nitrotyrosine (a metabolite not affected by diet) (7). Belmont et al. (8) have demonstrated that SLE is characterized by an increased production of NO, as reflected by elevation of serum nitrites (37 ± 6 uM/1 in 46 patients with SLE compared to 15 ± 7 uM/1 in controls, p< 0.01). The same authors showed that patients with active disease had more elevated serum nitrites compared with those with inactive disease. Serum nitrite levels correlated with disease activity (r = 0.47, p= 0.04), evaluated by SLE disease activity index (SLEDAI), and with level of

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antibodies to dsDNA (r = 0.35, p= 0.02). Endothelial and keratinocyte expression of iNOS were significantly elevated in SLE patients compared with controls, and were higher in patients with active disease as compared to those with inactive disease. Exhaled NO. Higher than normal expired NO concentrations have been reported in SLE patients (9). Mean values of peak concentration of NO in exhaled air were 64.8 ± 27.5 ppb in 27 SLE patients compared to 31.6 ± 7.7 ppb in controls, p< 0.001. (see Fig.2)

Fig.2. Mean +/- SE values of exhaled NO in patients with SLE and in normal controls.

In the same patients NO concentration was directly related to ECLAM activity score (r = 0.42, p< 0.05) . No patient had a major respiratory problem (pleurisy, pneumonia, radiologic evidence of interstitial lung disease), but 15/27 and 17/27 respectively had a significant decrease in lung diffusing capacity and hi MEF25, a test that reflects small airway function, and a significant inverse correlation was observed between eNO and MEF25 (r = -0.44, p< 0.05). Even in the absence of overt respiratory disease, the studied patients had a high prevalence of functional lung impairment, in agreement with literature data. A mixed pattern of functional lung damage, characterized by mild restrictive and diffusion abnormality, along with a decrease of MEF25, is commonly reported in SLE (10). Respiratory function abnormalities in patients with SLE have been related to inflammatory reactions. Alveolar lymphocytosis has been correlated with lung diffusion decrease (11), while peribronchial and bronchial wall inflammation, together with interstitial mononuclear cell infiltrate, have been suggested as the pathologic basis for small airway obstruction (12). The inverse correlation between eNO and MEF25 may support the hypothesis that respiratory tract inflammation causes increased eNO in patients with SLE, but direct investigation (i.e. BAL, lung biopsies) is needed to clarify this point.

Origin and Significance of NO in SLE. The identity of cells producing NO in SLE and the role of NO in the pathogenesis of the disease are important questions. An emerging concept is that in active SLE there is a

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widespread activation of the vascular endothelium. Activated endothelial cells may release excessive amounts of NO, correlated with evidence of increased disease activity, based on SLEDAI, anti-DNA levels and C3a levels (13). Activated endothelial cells have also been shown to increase the expression of adhesion molecules like E-selectin, intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM1), the scenario for tissue injury in the disease (13). Immune stimuli which may account for endothelial cell activation in SLE include immune complexes, complement components, antiphospholipid antibodies (13). The correlation between exhaled NO and ECLAM score of disease activity (9) may thus depend either upon respiratory tract inflammation or upon inflammatory cytokines and other circulating factors released in sites other than the respiratory system. In conclusion, NO production is increased in patients with SLE proportionally to the activity of the disease. The ultimate clinical significance of this observation is presently undefined. The measurement of NO in the exhaled air is the simplest and more rapid test which may document the increased NO production in SLE. Whether eNO measurement is merely one of the many available nonspecific markers of inflammation or whether it may help in the follow up of patients, particularly those with lung involvement, remains to be assessed. 3. Rheumatoid arthritis (RA). Rheumatoid arthritis is a chronic disease characterized by joint swelling, synovial inflammation and cartilage destruction. The importance of NO in the development of RA is increasingly recognized. Nitric oxide is thought to be a critical mediator of the inflammatory cascade which leads to synovial proliferation and mononuclear cell infiltration, which are the characteristics of involved joints in RA. Adjuvant-induced arthritis is a murine model of human RA, whose symptoms are preceded by elevated production of nitrates and nitrites A significant increase of urinary nitrate excretion was found in rats 20 days after induction of adjuvant arthritis compared with non-arthritic rats and L-NAME decreased NO biosynthesis, paw swelling and histopathologic changes in ankle joints (14). These data suggest that in adjuvant arthritis endogenous NO formation is clearly enhanced and related to joint inflammation. Parrel et al. (15) first reported an increased concentration of nitrite in synovial fluid and in serum of patients with RA, compared to serum nitrite concentration of healthy controls. Nitrite concentration in synovial fluid were higher than in serum samples. This finding is consistent with the presence, within inflamed synoviurn, of NO generating cells, such as fibroblasts, chondrocytes, macrophages, endothelial cells and granulocytes. Widespread synovial inflammation might increase serum nitrite by equilibration with the vascular compartment, but this may not entirely account for the whole serum nitrite concentration. A possible source of increased nitrite is the systemic vasculature, where the induction of NO syntehsis may occur, as an effect of circulating cytokines (16). Stichtenoth et al. (17) found the urinary nitrate excretion of 10 patients with RA to be 2.7-fold greater (p< 0.001) than that of healthy controls. After 2-week therapy with prednisolone (0.5 mg/Kg) , when inflammatory activity (as indicated by C reactive protein, ESR, swelled joint count, early morning stiffness) was significantly reduced, urinary nitrate excretion decreased significantly by 28%. By chemiluminescence after nitrite reduction, Ueki et al. (18) found that the serum concentration of NO was significantly higher in patients with RA as compared with patients with osteoarthritis and healthy subjects (291.4± 108.5 33.4 + 4 and 35.9 + 4.5 nM/1 respectively, p< 0.01).

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Furthermore, the serum concentration of NO was significantly higher in patients with active RA than in patients with inactive disease (670.9 ± 44.7 vs 97.7 ± 11 nM/1, p< 0.001) and serum NO levels were significantly correlated with the duration of morning stiffness, the number of tender or swollen joints, CRP, serum TNF-a and IL-6 levels. Peripheral mononuclear blood cells (PMBC) from RA patients had increased NOS activity and increased iNOS antigen content compared to those from normal subjects, and responded to interferon-y with increased NOS expression and nitrite/nitrate production in vitro (19). NOS activity of freshly isolated blood mononuclear cells correlated significantly with disease activity, as assessed by tender and swelled joint counts. These findings demonstrate that patients with RA have systemic activation of iNOS expression, and that the degree of activation correlates with disease activity. Clinical trials of TNF-a neutralizing antibodies or a soluble recombinant TNF-a receptor fusion protein (p75) in RA patients demonstrate that TNF-a blockade leads to reduction in disease activity (20, 21). Since TNF-a can promote iNOS expression and increased NO production, Perkins et al. (22) investigated if a chimeric monoclonal antibody against TNF-a might decrease iNOS protein expression and NOS enzyme activity in PBMC from RA patients. They found that the elevated levels of baseline iNOS protein and NOS enzyme activity in PBMC from RA patients were significantly reduced by the anti- TNF-a treatment. Changes in NOS activity following treatment correlated significantly with changes in the number of tender joints. These findings clearly indicate that the anti-inflammatory affects of anti-TNF-a treatment may be mediated by a reduction of NO overproduction. Role of NO in Rheumatoid Arthritis. The evidence that NO play a proinflammatory role in RA may be summarized as it follows. 1- Elevated NO levels in the serum and synovial fluid of patients with RA have been found. 2- Increased production in synovial tissue and overexpression of iNOS antigen and NOS activity of PBMC have been shown in RA patients. 3- The production of NO and the NOS activity have been shown to be related to clinical activity of the disease. 4- Therapeutic interventions (corticosteroids, anti- TNF-a) which decrease clinical activity of the disease, also decrease NO production and NOS activity. NO from synovial fibroblasts has been suggested to enhance proinflammatory cytokine production by macrophages which in turn may upregulate iNOS expression, thereby generating a positive feedback loop (23). Additionally, NO may upregulate matrix metalloprotease production (24) and is implicated in IL-1B mediated inhibition of proteoglycan synthesis (25).

4. Sjogren's Syndrome (SS). Sjogren's syndrome is a chronic, slowly progressive, polysystemic disease, characterized by lymphocyte infiltration of the exocrine glands, which results in diminished or absent glandular secretion and mucosal dry ness, particularly of mouth and eyes (sicca

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syndrome). SS can occurr alone (primary SS) or in association with other rheumatic diseases such as RA, SLE and SSc . NO and Sicca Syndrome. NO has been measured in human saliva (26) demonstrating that it is released from salivary glands rather than derived from the activity of oral bacteria. NO may influence parasympathetic vasodilation and salivary flow by regulating peptide release and second messenger system for both VIP and acetylcholine, as reported in pigs and rats (27). Physiologically NO is produced in normal salivary glands mainly by constitutively expressed neural and endothelial NOS isoforms, and may contribute to the many events that regulate vascular reactivity, salivary flow, and neuropeptide release. Increased concentration of nitrites (mean ± SEM 307+51 /xM vs 97±16 /ttM, p< 0.05) and output (166±46 nmoles/min vs 37 ±7 nmoles/min) have been reported in SS patients compared with healthy control subjects (28). The increased salivary NO production in SS patients has been found to be mediated by iNOS upregulation, induced by cytokines, such as TNF-a, IL-1 and IFN-y , which are produced by the salivary gland epithelial cells and/or activated T lymphocytes (29, 30). In conclusion, salivary NO production is greatly increased in SS patients and may contribute to the diminished salivary flow, characteristic of sicca syndrome, either through cytotoxic mechanism and increased rate of apoptosis (31, 32) or through functional impairment of secretion. Exhaled NO. Pulmonary involvement has been reported both in primary and secondary SS, the most common respiratory symptoms being chronic dry cough and dyspnea (33). Evidence of airway obstruction has been shown in patients with SS by several authors (34, 35) and bronchial hyperresponsiveness to methacholine has been observed in 60 % of the patients. An increased production of exhaled NO has been reported in patients with SS compared to healthy controls (147 ± 82 vs 88 ± 52 nL/min, p = 0.04) by the Swedish BHR study group (36). Exhaled NO was correlated to age and serum human neutrophil lipocalin, a newly recognized secretory protein of the secondary granules of neutrophil granulocytes (37). Exhaled NO was not found to be correlated with bronchial responsiveness, respiratory symptoms or lung function. The mechanisms underlying the increased exhaled NO in SS patients were not investigated. The elevated NO levels may derive from inflammatory cells in the bronchial mucosa and/or from epithelial cells or macrophages activated by cytokines released from lymphocytes. An increased number of CD4+ T lymphocytes has been shown in the bronchial mucosa of patients with SS (38). The possibility of a contribution to exhaled NO from nonenzymatically formed NO derived from nitrites in saliva cannot be excluded in SS patients (39).

5. Systemic Sclerosis (SSc). Systemic sclerosis is a multisystemic disease characterized by Raynaud's phenomenon,

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vascular lesions and widespread fibrosis of the skin and visceral organs. The accumulation of inflammatory mononuclear cells is frequently detected in early cutaneous and visceral SSc lesions (40), followed by excessive collagen deposition. Yamamoto et al. (41) found that serum of patients with SSc contains increased amount of nitrites compared with controls (47.8 ± 17 uM vs 25.6 ± 10.3 fiM respectively, p< 0.01). The same authors using immunohistochemical techniques found positive staining for iNOS in infiltrating mononuclear cells and in fibroblasts of patients with SSc, while fibroblasts in normal skin showed only faint staining. Inducible NOS messenger RNA expression was detected in scleroderma derived culture fibroblasts, while negative expression was detected on cultured normal fibroblasts. These results suggest that SSc fibroblasts are already activated by exposure to inflammatory cytokines. Actually patients with SSc are exposed to the stimulation of various cytokines, such as IL-1, TNF-a, platelet-derived growth factor (PDGF) and TGF-B. While IL-1 and TNF-a increase NO production in a wide variety of cells (42), both PDGF and TGF-B decrease NO production, by inhibiting iNOS gene expression and by suppressing IL-1 induced NO production in macrophages by post-transcriptional mechanism (43). Peripheral blood mononuclear cells from patients with SSc have been shown to produce higher levels of NO spontaneously and in response to IL-1 stimulation (44). In conclusion, the majority of studies show that scleroderma mononuclear cells and fibroblast may escape the downregulation of NO production, and the resulting excessive NO or its reaction products may be cytotoxic to host cells. Exhaled NO. Pulmonary involvement is common in SSc and pulmonary hypertension is the most frequent cause of death (45). More than 30% of SSc patients may develop pulmonary hypertension (46), either in association with extensive interstitial lung disease or as a consequence of prominent vascular lesions of the pulmonary arterioles. In 23 patients with SSc Kharitonov et al. (47) found peak eNO levels significantly higher than in normal controls. They also found that the six patients with pulmonary hypertension had eNO values significantly lower than SSc patients without pulmonary hypertension (20 ± 6 ppb vs 149 ± 19 ppb respectively, p< 0.001) and normal controls (80 ± 7 ppb, p< 0.002). There was no relationship between eNO and lung function tests, while a negative correlation was observed between PaO2 and peak eNO in patients with SSc without pulmonary hypertension. In 47 patients with SSc, 19 with limited and 28 with diffuse form of disease, higher levels of eNO (plateau value) than in controls (16.6 ± 9 . 1 ppb vs 9.9 ± 2.9 ppb respectively, p< 0.0001) were observed (48). Patients with lung involvement (defined on the basis of a CT score for interstitial lung disease > 5, associated with a TLC or VC < 80% of predicted) had the highest value of eNO, while patients with isolated decreased DLco and patients without lung involvement had mean eNO not significantly different from controls. In agreement with previous observation by Kharitonov et al (47), eNO was significantly lower in the 16 patients with pulmonary hypertension (mean PAP 40.7 ± 15.4 mmHg, range 30-93 mmHg) than in patients without pulmonary hypertension (10.7 ± 5 ppb vs 19.6 ± 9 ppb, p< 0.001) and there was a significant inverse correlation between PAP and eNO in all the patients (r = -0.53, p = 0.004, Fig.3). In 17 patients with fibrosing alveolitis associated with SSc (FASSc), Paredi et al.

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(49) found eNO levels higher than in controls (9.8 ± 1 ppb vs 6.9 ± 0.5 ppb, p< 0.05) and the eight patients who had active BAL (defined as either lymphocyte > 14%, neutrophils > 4% or eosinophils > 3%) had significantly higher NO levels than patients who had inactive BAL ( 13.2 ± 1.8 ppb and 6.7 ± 1.2 ppb respectively, p< 0.05). There was a significant correlation between eNO and lymphocyte cell count in patients with FASSc (r = 0.58, p< 0.05). Recently, Moodley and coll. (50) found that patients with SSc and associated ILD had normal value of exhaled NO, but all their patients had pulmonary hypertension (PAP > 30 rnmHg). Origin and Significance of eNO in SSc. All studies concerning eNO measurement in SSc patients report values higher than in controls. The inflammatory process underlying interstitial lung disease in SSc is the most likely explanation for the increased NO production by respiratory tract. On the other hand patients with pulmonary hypertension associated with SSc have been shown to have normal or low values of exhaled NO (47, 48, 51). In SSc pulmonary hypertension may develop either in association with extensive pulmonary fibrosis or as a consequence of prominent vascular lesions of the pulmonary arterioles with minor parenchymal fibrosis.. Damage of endothelial cells either primary or linked to substances circulating in the serum of patients with SSc (52) may explain the low levels of exhaled NO in patients with pulmonary hypertension not associated with interstitial lung disease. These patients have been shown to have eNO values even lower than patients with pulmonary hypertension associated with parenchymal lung involvement (48). The diminished production of NO could favour the unopposed action of vasoconstrictor substances such as endothelin-1 and lead to obstruction and proliferation changes in the pulmonary arteries (53). The capability to increase NO production by respiratory system after Larginine administration has been proposed as a test of endothelial health, which could anticipate the response of pulmonary artery pressure to vasodilating drugs in patients with SSc (51, 54) (Fig.3). 2005/3 JD

160-

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120-

V)

o~ z: x O£ V.

80-

r = 0.76, p = 0.078

40-

O

z:

0-

)

5

10

15

20

25

30

A PAP before and after iloprost (mmHq) Fig.3. Relationship between the increase in exhaled NO after i.v. L-arginine administration and the decrease in PAP after inhaled iloprost in patients with SSc.

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In conclusion, the measurement of exhaled NO may be a useful non-invasive marker of activity in patients with SSc and interstitial lung disease as well as an useful test to identify SSc patients with pulmonary hypertension. References I- Taylor Robinson AW, Liew FY, Severn A et al. Regulation of the immune response by nitric oxide differentially produced by T helper type 1 and T helper type 2 cells. Eur J Immunol 1994; 24: 980-984 2-Bauer H, Jung T, Tsikas D, Stichtenoth DO, Frolich JC, Neumann C. Nitric oxide inhibits the secretion of T-helper 1- and T-helper 2-associated cytokines in activated human T cells. Immunology 1997; 90: 205211 3- Mannick JB, Asano K, Izumi K, Kieff E, Stamler JS. Nitric oxide produced by human B lymphocytes inhibits apoptosis and Epstein-Barr virus reactivation. Cell 1994; 79: 1137-1146 4- Geller DA, Billiar TR. Molecular biology of nitric oxide synthases. Cancer Metastasis Rev 1998; 17: 723 5- Gates JC, Ruiz P, Alexander A, Pippen AM, Gilkeson GS. Effect of late modulation of nitric oxide production on murine lupus. Clin Immunol Immunopathol 1997; 83: 86-92 6- Wanchu A, Khullar M, Deodhar SD, Bambery P, Sud A. Nitric oxide synthesis is increased in patients with systemic lupus erythematosus. Rheumatol Int 1998; 18: 41-3 7- Gilkeson G, Cannon C, Goldman D, Petri M. Correlation of a serum measure of nitric oxide production with lupus disease activity measures (abstract). Arthritis Rheum 1996; 39 Suppl 9: S251 8- Belmont HM, Levartovsky D, Goel A, Amin A, Giorno R, Rediske J, Skovron ML, Abramson SB. Increased nitric oxide production accompanied by the up-regulation of inducible nitric oxide synthase in vascular endothelium from patients with systemic lupus erythematosus. Arthritis Rheum 1997; 40: 1810-16 9- Rolla G, Brussino L, Bertero MT, Colagrande P, Converse M, Bucca C, Polizzi S, Caligaris-Cappio F. Increased nitric oxide in exhaled air of patients with systemic lupus erythematosus. J Rheumatol 1997; 24: 1066-71 10- Rolla G, Brussino L, Bertero MT, Bucca C, Converse M, Caligaris-Cappio F. Respiratory function in systemic lupus erythematosus: relation with activity and severity. Lupus 19%; 5: 38-43 II- Groen H, Aslander M, Bootsma H, van der Mark ThW, Kallenberg CGM, Postma DS. Bronchoalveolar lavage cell analysis and lung function impairment in patients with ststemic lupus erythematosus. Clin Exp Immunol 1993; 94: 127-33 12- Grennan DM, Howie AD, Moran F, Buchanan WW. Pulmonary involvement in systemic lupus erythematosus. Ann Rheum Dis 1987; 37: 536-9 1997; 403: 273-8 13- Belmont HM, Abramson SB, Lie JT. Pathology and pathogenesis of vascular injury in systemic lupus erythematosus: interactions of inflammatory cells and activated endothelium. Arthritis Rheum 19%; 39: 922 14- Stefanovic-Racic M, Meyers K, Meschter C, Coffey JW, Hoffman RA, Evans CH. N-monomethylarginine, an inhibitor of nitric oxide synthase, suppresses the development of adjuvant arthritis in rats. Arthritis Rheum 1994; 37: 1062-69 15- Farrell AJ, Blake DR, Palmer RMJ, Moncada S. Increased concentrations of nitrite in synovial fluid and serum samples suggest increased nitric oxide synthesis in rheumatic diseases. Ann Rheum Dis 1992; 51: 1219-22 16- Kilbourn RG, Belloni P. Endothelial cell production of nitrogen oxides in response to interferon gamma in combination with tumour necrosis factor, interleukin-1 or endotoxin. J Natl Cancer Inst 1990; 82: 772-6 17- Stichtenoth DO, Fauler J, Zeidler H, Frolich JC. Urinary nitrate excretion is increased in patients with rheumatoid arthritis and reduced by prednisolone. Ann Rheum Dis 1995; 54: 820-4 18- Ueki Y, Miyake S, Tominaga Y, Eguchi K. Increased nitric oxide levels in patients with rheumatoid arthritis. J Rheumatol 1996; 23: 230-6

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19- St. Clair EW, Wilkinson WE, Lang T, Sanders L, Misukonis MA, Gilkeson GS, Pisetsky DS, Granger DL, Weinberg JB. Increased expression of blood mononuclear cell nitric oxide synthase type 2 in rheumatoid arthritis patients. J Exp Med 1996; 184: 1173-78 20- Elliott MJ, Maini RN, Feldmann M, Kalden JR, Antoni C, Smolen JS, et al. Randomised double-blind comparison of chimeric monoclonal antibody to tumour necrosis factor alpha (cA2) versus placebo in rheumatoid arthritis. Lancet 1994; 344: 1105-10 21- Weinblatt ME, Kremer JM, Bankhurst AD, Bulpitt KJ, Fleischmann RM, Fox RI, Jackson CG, Lange M, Surge DJ. A trial of etanercept, a recombinant tumor necrosis factor receptor: Fc fusion protein, in patients with rheumatoid arthritis receiving methotrexate. N Engl J Med 1999; 340: 253-9 22- Perkins DJ, St Clair EW, Misukonis MA, Weinberg JB. Reduction of NOS2 overexpression in rheumatoid arthritis patients treated with anti-tumor necrosis factor ### monoclonal antibody (cA2). Arthritis Rheum 1998; 41: 2205-2210 23- Mclnnes IB, Leung BP, Field M, Wei XQ, Huang FP, Sturrock RD, Kinninmonth A, Weidner J, Mumford R, Liew F. Production of nitric oxide in the synovial membrane of rheumatoid and osteoarthritis patients. J Exp Med 1996; 184: 1519-24 24- Murrell GAC, Jang D, Williams RJ. Nitric oxide activates metalloprotease enzymes in articular cartilage. Biochem Biophys Res Commun 1995; 206: 15-21 25- Hauselmann HJ, Oppliger L, Michel BA, Stefanovic-Racic M, Evans CH. Nitric oxide and proteoglycan biosynthesis by human articular chondrocytes in alginate culture. FEES Lett 1994; 352: 631364 26-Bodis S, Haregewoin A. Evidence for the release and possible neural regulation of nitric oxide in human saliva. Biochem Biophys Res Commun 1993; 194: 347-350 27- Modin A, Weitzberg E, Lundberg JM. Nitric oxide regulates peptide release from parasympathetic nerves and vascular reactivity to vasoactive intestinal polypeptide in vivo. Eur J Pharmacol 1994; 261: 185197 28- Konttinen YT, Platts LAM, Tuominen S, Eklund KK, Santavirta N, Tornwall J, Sorsa T, Hukkanen M, Polak JM. Role of nitric oxide in Sjogren's syndrome. Arthritis Rheum 1997; 40: 875-883 29- Fox RI, Kang HI, Ando D, Abrams J, Pisa E. Cytokine mRNA expression in salivary gland biopsies of Sjogren's syndrome. J Immunol 1994; 152: 5532-39 30- Boumba D, Skopouli FN, Moutsopoulos HM. Cytokine mRNA expression in the labial salivary gland tissues from patients with primary Sjogren's syndrome. Br J Rheumatol 1995; 34: 326-33 31- Zeher M, Szodoray P, Gyimesi E, Szondy Z. Correlation of increased susceptibility to apoptosis of CD4+ T cells with lymphocyte activation and activity of disease in patients with primary Sjogren's syndrome. Arthritis Rheum 1999; 42: 1673-81 32- Matsamura R, Umeniya K, Kagami M, Tamioka H, Tanabe E, Sugiyama T, et al. Glandular and extraglandular expression of Fas-FasL and apoptosis in patients with primary Sjogren's syndrome. Clin Exp Rheumatol 1998; 16: 561-98 33- Gudbjornsson B, Hedenstrom H, Stalenheim G, Hallgren R. Bronchial hyperresponsiveness to methacholine in patients with primary Sjogren's syndrome. Ann Rheum Dis 1991; 50: 36-40 34- Potena A, La Corte R, Fabbri LM, Papi A, Trotta F, Ciaccia A. Increased bronchial responsiveness in primary and secondary Sjogren's syndrome. Eur Respir J 1990; 3: 548-553 35- Constantopoulos SH, Papadimitriou CS, Moutsopoulos HM. Respiratory manifestations in primary Sjogren's syndrome: a clinical, functional and histologic study. Chest 1985; 88: 226-229 36- Ludviksdottir D, Janson C, Hogman M, Gudbjornsson B, Bjornsson E, Valtysdottir S, Hedenstrom H, Venge P, Boman G, on behalf of the BHR study group. Increased nitric oxide in expired air in patients with Sjogren's syndrome. Eur Respir J 1999; 13: 739-743 37- Xu SY, Peterson C, Carlson M, Venge P. The development of an assay for neutrophil lipocalin to be used as a specific marker of neutrophil activity in vitro and in vivo. J Immunol Meth 1994; 171: 245-252 38- Papiris SA, Saetta M, Turato G, La Corte R, Trevisani L, Mapp CE, Maestrelli P, Fabbri LM, Potena A. CD4-positive T-lymphocytes infiltrate the bronchial mucosa of patients with Sjogren's syndrome. Am J Respir Crit Care Med 1997; 156: 637-641 39- Zetterquist W, Pedroletti C, Lundberg JON, Alving K. Salivary contribution to exhaled nitric oxide. Eur Respir J 1999; 13: 327-333 40- Prescott RJ, Freemont AJ, Jones CJ, Hoyland J, Fielding P. Sequential dermal miscrovascular and perivascular changes in the development of scleroderma. J Pathol 1992; 166: 255-63 41- Yamamoto T, Katayama I, Nishioka K. Nitric oxide production and inducible nitric oxide synthase expression in systemic sclerosis. J Rheumatol 1998; 25: 314-17

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42- Kovacs EJ, Di Pietro LA. Fibrogenic cytokines and connective tissue production. FASEB J 1994; 8: 854-861 43- Vodovotz Y, Bogdan C, Paik J, Xie Q, Nathan C. Mechanisms of suppression of macrophage nitric oxide release by transforming growth factor ###. J Exper Med 1993; 178: 605-13 44- Yamamoto T, Sawada Y, Katajama I, Nishioka K. Increased production of nitric oxide stimulated by interleukin-l### in peripheral blood mononuclear cells in patients with systemic sclerosis. Br J Rheumatol 1998; 37: 1123-25 45- Lee P, Langevitz P, Alderdice CA, Aubrey M, Baer PA, Baron M, Buskila D, Dutz JP, Khostant I, Piper S. Mortality in systemic sclerosis (scleroderma). Quart J Med 1992; 82: 139-48 46- Ungerer RG, Tashkin DP, Furst D, Clements PhJ, Gong H, Bein M, Smith JW, Roberts N, Cabeen W. Prevalence and clinical correlates of pulmonary artery hypertension in progressive systemic sclerosis. Am J Med 1983; 75: 65-74 47- Kharitonov SA, Cailes JB, Black CM, duBois RM, Barnes PJ. Decreased nitric oxide in the exhaled air of patients with systemic sclerosis with pulmonary hypertension. Thorax 1997; 52: 1051-55 48- Rolla G, Colagrande P, Scappaticci E, Chiavassa G, Dutto L, Cannizzo S, Bucca C, Morello M, Bergerone S, Bardini D, Zaccagna A, Puiatti P, Fava C, Cortese G. Exhaled nitric oxide in systemic sclerosis: relationship with lung involvement and pulmonary hypertension. J Rheumatol 2000 (in press) 59- Paredi P, Kharitonov SA, Loukides S, Pantelidis P, du Bois RM, Barnes PJ. Exhaled nitric oxide is increased in active fibrosing alveolitis. Chest 1999; 115: 1352-56 50- Moodley YP, Lalloo UG. Exhaled nitric oxide is elevated in patients with progressive systemic sclerosis without interstitial lung disease. Chest 2001; 119: 1449-54 51- Rolla G, Colagrande P, Brussino L, Bucca C, Bertero MT, Caligaris-Cappio F. Exhaled nitric oxide and pulmonary response to iloprost in systemic sclerosis with pulmonary hypertension. Lancet 1998; 351: 1491-92 52- Etoh T, Igararhi A, lozunii K, Ishibashi Y, Takehara K. The effects of scleroderma sera on endothelial cell survival in vitro. Arch Dermatol Res 1990; 282: 516-19 53- Rolla G, Caligaris-Cappio F. Nitric oxide in systemic sclerosis lung: controversies and expectations. Clin Exper Rheumatol 1998; 16: 522-24 54- Rolla G, Colagrande P, Bucca C, Dutto L, Audano G, Caligaris-Cappio F. Exhaled nitric oxide (NO) after L-arginine may predict the effect of aerosolized iloprost in pulmonary hypertension associated with systemic sclerosis (SSc). Eur J Clin Invest 1999; 29 (Suppl 1): 82 (abstract)

Disease Markers in Exhaled Breath N. Marczin and M.H. Yacoub (Eds.) [OS Press, 2002

Nitric Oxide in Hepatopulmonary Syndrome Giovanni ROLLA Universita di Torino (Italy) - Immunologia Clinica e AllergologiaOspedale Mauriziano Umberto I di Torino, Largo Turati 62, 10128 Torino (Italy) Abstract. Hepatopulmonary syndrome is characterised by severe hypoxemia due to intrapulmonary vascular dilatations in patients with liver disease. An imbalance between vasodilating and vasocostricting substances is supposed to lead to oxygenation abnormalities in patients with liver disease. Experimental and clinical data support the hypothesis that increased production of nitric oxide plays a key role in determining hypoxemia in hepatopulmonary syndrome.

Impaired arterial oxygenation, particularly increased alveolar-arterial oxygen gradient (AaO2), is frequent in patients with liver cirrhosis, even if the true frequency has not been established. However, studies of patients referred for liver transplantation indicate that as many as 20 % are hypoxaemic at rest, with 3-7 % having severe hypoxaemia (PaO2 < 60 mrnHg) (1), while a widened AaO2 gradient is reported in up to 60% (2). In 1977 Kennedy et al. (3) first used the term hepatopulmonary syndrome (HPS) to characterise the association of severe hypoxaemia with intrapulmonary vascular dilatations in hepatic failure. 1. Definition The syndrome can be defined as a clinical triad of: a) liver disease b) increased AaO2 gradient (> 15 mmHg) while breathing room air c) evidence of intrapulmonary vascular dilatations (commonly by contrast-enhance echocardiography, see below). 2. Clinical findings Liver diseases that have been associated with HPS include most commonly cirrhosis (cryptogenertic, alcoholic, post-viral hepatitis and biliary), but also noncirrhotic portal hypertension (4). No relationship has been found between HPS and biochemical indexes of hepatic function, ascites or gastrointestinal bleeding. On the other hand, the number of cutaneous spider naevi have a strong association with HPS (5). Dyspnea is a common symptom, and it may be the presenting symptom in 18 % of patients (4). Platypnea, defined as dyspnea induced by the upright position and releived by recumbency and orthodeoxia, defined as a decrease (> 10 %) of PaO2 when changing from the supine to the standing position (6), are characteristic, but not unique, of the HPS and may be seen in 5% of patients with cirrhosis. Platypnea and orthodeoxia may also be found in other clinical contexts such as intracardiac shunts, post-pneumonectomy, recurrent pulmonary emboli, chronic lung disease (7). A hyperdynamic circulation characterised by systemic

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vasodilatation and elevated cardiac output (often > 7 1/min), associated with low pulmonary vascular resistance is the haemodynamic pattern more commonly observed in patients with HPS (8). 3. Oxygenation impairment Impaired oxygenation is a hallmark of the HPS, ranging from a widened AaO2 gradient to severe hypoxaemia. The most important mechanism underlying impaired gas exchange in HPS include ventilation/perfusion inequality (9), alveolar-capillary diffusion limitation and intrapulmonary shunts (10). Precapillary pulmonary vascular dilatations and direct arterio-venous communications (11) are the pathological bases for oxygenation impairment. These intrapulmonary vascular dilatations range from 15 to 500 fim in diameter. As supplemental oxygen enhances oxygenation more than it would be expected with "true anatomic" shunts, a new mechanism, called diffusion-perfusion impairment, has been hypothesised to explain the hypoxaemia associated with HPS. As the capillary is dilated, O, molecules from adjacent alveoli cannot diffuse to the centre of the dilated vessel to oxygenate the erythrocytes at the central stream of venous blood (12). Supplemental oxygen provides enough driving pressure to overcome the relative diffusion defect. Intrapulmonary vascular dilatations include two types of vascular abnormalities: vascular dilatations at the pre-capillary level which cause oxygenation impairment responsive to supplemental oxygen (PaO2 > 500 mmHg when breathing 100% oxygen for 15 min) and larger arterio-venous communications, which cause hypoxaemia, poorly responsive to 100 % oxygen breathing (4). Contrast-enhanced (CE) echocardiography is the most frequently used technique to detect intrapulmonary vascular dilatations. It is based on the observation that microbubbles (agitated saline solution injected through a peripheral vein) can pass through dilated pulmonary vessels (> 15 (im) and can be detected as echogenicity in the left heart chambers (11). 4. NO theory NO is a powerful local vasodilator, which contributes to the normally low pulmonary vascular tone. Vallance et al (12) postulated that an increased NO production may account for the hyperdynamic circulation of liver cirrhosis. An increased production of pulmonary NO may contribute to oxygenation abnormalities through abnormal pulmonary vasodilatation as well as through the inhibition of hypoxic vasocostriction. It has been suggested that patients with cirrhosis may have a continuous stimulation of endothelial NO-synthase by circulating endotoxins and/or circulating cytokines,such as TNF-a (13 ). In a rat model of HPS, Fallen and coll. (14) showed that the endothelial NO synthase content of lung homogenates progressively increased and the increase was closely correlated with the development of hypoxemia. Increased NO output in exhaled air has been reported in patients with advanced cirrhosis, in whom exhaled NO was associated with systemic circulatory disturbances (15). Exhaled NO was reported to be raised almost threefold in three patients with HPS, compared with normal volunteers and with normoxemic cirrhotic patients (16). In one case of severe HPS we reported that i.v. methylene blue (a dye that inhibits the effect of NO on soluble guanylate cyclase and thereby

G. Rolla /NO in Hepatopulmonary Syndrome

prevents the cascade of events leading to vasodilation) acutely improved oxygenation, through a marked decrease in pulmonary shunting (17). After the patient had been breathing 100 percent oxygen, PaO2 was 325 mmHg (normal value > 500 mmHg) while she was in the supine position, and it decreased to 115 mmHg while she was standing. Twenty minutes after the i.v. administration of methylene blue (3 nig/Kg) PaO2 during oxygen breathing was 480 mmHg in the supine position and 390 mmHg in the standing position. Very recently (18) Schenk and coll showed that i.v. methylene blue improved hypoxemia and hyperdynamic circulation in 7 patients with liver cirrhosis and severe HPS. We hypothesised that NO locally produced in the lung may play an important role in determining oxygenation abnormalities in patients with cirrhosis. To this aim, we investigated the relationship between NO production in the lung, assessed by exhaled NO measurement, and oxygenation abnormalities in patients with advanced cirrhosis (19), In 45 cirrhotic patients we showed mean values of exhaled NO output and serum NO2V NO3 significantly higher than in normal controls (252 + /117 vs 75.5 +/- 19 nL/min/m2, p< 0.0001 and 47.5 +/- 29.4 vs 32.9 +/- 10.1 ^imol/L, p< 0.02, respectively). In all patients there was a significant correlation between exhaled NO and arterial-alveolar oxygen gradient (r= .78, p< 0.001). The nine patients who met the criteria for the diagnosis of HPS had also the highest values of exhaled NO (331 +/- 73.2 vs 223 +/- 118.4 nL/min/m2, p< 0.05). To further investigate the association between NO produced in the lung and oxygenation abnormalities in patients with cirrhosis, we measured exhaled NO and oxygenation measures before and after liver transplantation in a selected group of 18 patients with cirrhosis who did not have obvious cardiorespiratory diseases (20). Before transplantation, the mean exhaled NO was higher in patients than in normal controls (13 +/- 4.9 ppb compare with 5.75 +/- 1.9 ppb, p< 0.001). After transplantation, the AaO2 gradient significantly decreased (from 17.3 +/- 7.1 mmHg to 9 +/- 5.2 mmHg, p< 0.001), as did the exhaled NO concentration (from 13 +/- 4.9 ppb to 6.2 +/- 2.8 ppb, p< 0.001). The decrease in exhaled NO was significantly correlated with the decrease in AaO2 gradient (r = 0.56, p = 0.014). Five patients met the criteria for the diagnosis of HPS before transplantation and the syndrome was cured by transplantation. The correlation between the decrease in exhaled NO after liver transplantation and the improvement in oxygenation reinforces the hypothesis that NO is an important mediator of impaired oxygenation in patients with cirrhosis. In a murine model, chronic inhibition of lung NO production , by L-NAME administration, could prevent the development of HPS and associated hemodynamic alterations (21). Very recently, we reported that smoking, by decreasing respiratory NO, apparently contributed to improve oxygenation in a 44-year-old man with cirrhosis, complicated by severe HPS, who underwent liver transplantation (22). In conclusion, experimental and clinical data support the theory that NO plays a major role in oxygenation abnormalities of patients with liver cirrhosis, complicated by hepatopulmonary syndrome. References [1] Krowka MJ, Cortese DA. Pulmonary aspects of chronic liver disease and liver transplantation. Mayo Clin Proc 1985; 60: 407-18

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[2] Hourani JM, Bellamy PE, Tashkin DP, Batra P, Simmons MS. Pulmonary dysfunction in advanced liver disease: frequent occurrence of an abnormal diffusing capacity. Am J Med 1991; 90: 693-700 [3] Kennedy TC et al. Exercise-aggravated hypoxemia and orthodeoxia in cirrhosis. Chest 1977; 72: 305-9 [4] Krowka MJ, Cortese DA. Hepatopulmonary syndrome. Current concepts in diagnostic and therapeutic considerations. Chest 1994; 105: 1528-37 [5] Agusti AGN et al. The lung in patients with cirrhosis. J Hepatol 1990; 12: 262-3 [6] Robin ED et al. Platypnea related to orthodeoxia caused by true vascular lung shunts. N Engl J Med 1976; 294:941-3 [7] Seward JB. Platypnea-Orthodeoxia: clinical profile, diagnostic workup, management, and report of seven cases. Mayo Clin Proc 1984; 59: 221-3 [8] Naeije R et al. Pulmonary hemodynamics in liver cirrhosis. Semin Respir Med 1985; 7: 164-170 [9] Hedenstiema G, SOderman C, Eriksson LS, Wahren J. Ventilation-perfusion inequality in patients with non-alcoholic liver cirrhosis. Eur Respir J 1991; 4: 711-7 [10] Edell ES, Cortese DA, Krowka MJ, Rehder K. Severe hypoxemia and liver disease. Am Rev Respir Dis 1989; 140: 1631-35 [11] Krowka MJ, Tajik J, Dickson ER, Wiesner RH, Cortese DA. Intrapulmonary vascular dilatations (IPVD) in liver transplant candidates. Screening by two-dimensional contrast-enhanced echocardiography. Chest 1990; 97: 1165-70 [12] Vallance P, Moncada S. Hyperdynamic circulation in cirrhosis: a role for nitric oxide ? Lancet 1991;337:776-778 [13] Khoruts A, Stahnke L, McClain CJ, Logan G, Allen JI. Circulating tumor necrosis factor, interleukin-1 and interleukin-6 concentrations in chronic alcoholic patients. Hepatology 1991; 13: 267-276 [14] Fallen MB, Abrams GA, Luo B, Hou Z, Dai J, Ku DD. The role of endothelial nitric oxide synthase in the pathogenesis of a rat model of hepatopulmonary syndrome. Gasstroenterology 1997; 113:606-614 [15] Matsumoto A, Ogura , Hirata Y, Kakoki M, Watanabe F, Takenaka K, Shiratory Y, et al. Increased nitric oxide in the exhaled air of patients with decompensated liver cirrhosis. Ann Intern Med 1995; 123: 110-113 [16] Cremona G, Higenbottam TW, Mayoral V, Alexander G, Demoncheaux E, Borland C, et al. Elevated exhaled nitric oxide in patients with hepatopulmonary syndrome. Eur Respir J 1995; 8: 1883-1885 [17] Rolla G, Bucca C, Brussino L. Methylene blue in the hepatopulmonary sindrome. N Engl J Med 1994; 331: 1098 [18] Schenk P, Madl C, Rezaie-Majd S, Lehr S, MUlier C. Methylene blue improves the hepatopulmonary syndrome. Ann Intern Med 2000; 133: 701-706 [19] Rolla G, Brussino L, Colagrande P, Dutto L, Polizzi S, Scappaticci E, Bergerone S, et al.Exhaled nitric oxide and oxygenation abnormalities in hepatic cirrhosis. Hepatology 1997; 26: 842-847 [20] Rolla G, Brussino L, Colagrande P, Scappaticci E, Morello M, Bergerone S, Ottobrelli A,-et al. Exhaled nitric oxide and impaired oxygenation in cirrhotic patients bifore and after liver transplantation. Ann Intern Med 1998; 129: 375-378 [21] Nunes H, Lebrec D, Heller Y, Mazmanian M, Zerbib E, Herve P. Prevention of hepatopulmonary syndrome by inhibition of nitric oxide synthase. Am J Respir Crit Care Med 1999; 159: A523 (abstract) [22] Rolla G, Brussino L, Dutto L, Ottobrelli A, Bucca C. Smoking and hypoxemia caused by hepatopulmonary sindrome before and after liver transplantation. Hepatology 2001; 34: 430-431

Disease Markers in Exhaled Breath N. Marczin and MM. Yacoub (Eds.) 1OS Press, 2002

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Pathological Changes in the Airways Epithelium of Liquidators of the Chernobyl Catastrophe

Victoria POLYAKOVA Institute of Human Ecological Pathology, Vasylkivska Street 45, Kyiv, 03022, Ukraine Abstract. By method of transmission electron microscopy (TEM) bronchial biopsies of liquidators of the Chernobyl catastrophe and persons of control nosological group were studied. Statistically significant distinctions in the manifestation degree of pathological changes of the bronchial epithelium were found in the main groups in comparison to control parameters. Obtained results testify stereotype reactions in the epithelium of airways on the effect of the "Chernobyl factor".

1. Introduction In spite of recently closing of the Chernobyl Nuclear Power Station a lot of medical and biological problems caused by the accident of 1986 still remained. Among them the prevalence of chronic obstructive pulmonary diseases in persons which had taken part in the liquidation of the Chernobyl disaster consequences is of a great importance. The main subject of our research was superficial bronchial epithelium as it plays the leading role in the pathogenesis of lung diseases. 2. Material and methods Bronchial biopsies obtained in 1989-1999 from persons with chronic bronchitis: 110 liquidators of the Chernobyl accident and from 23 patients of control nosological group without radiation factor in an anamnesis were studied. All patients were not older than 40 years old. A chronic bronchitis was diagnosed to all of them after 1986. Liquidators were in region with high radiation in 1986-87 (irradiation doses mainly under 10 cSv). Samples were proceed with routine technique for TEM and observed in JEM 100CX electron microscope. All investigated cases of liquidators were divided into two periods of observations: 1989-1992- I group (48 persons) and 1994-1999 - II group (62 persons).

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3. Results and Discussion In the control group sites of normal unchanged bronchial epithelium were more often determined (52%) whereas in the main groups a percentage of such places was extremely decreased (27% - I group, 20% - II group). Similarly in relation to control observations (48%) the number of epithelial loci with normal ratio of main cell types was reduced (32%-I group, 15% - II group). The decrease of bronchial endocrine cell number was also observed. If in the control material presence of APUD-cells were found out in 68% of observation in the main groups they were seen quite seldom (11% - I group and 14% - II group). Besides most of endocrine cells of liquidators had dystrophic changes: dilatation of perinuclear space, vacuolation of cytoplasm,

essential decrease of specific granules and intracellular

organoides quantity. High frequency of bronchial basal cell hyperplasia was a typical feature of a researched material but it was more significantly marked in the main groups (61% - control group, 77% - I group, 92% - II group). Alongside in liquidators reliable increase of basal cells rows number was revealed, whereas thickness of an epithelial layer remained practically constant. It allows to assume the activization of proliferative processes and acceleration of cellular population turnover in bronchial epithelium, probably, as a result of contraction of a cell cycle growth phase. In the II group squamous metaplasia of bronchial epithelium was found more often (43%) than in the control group (30%). Besides in the control group squamous metaplasia was detected only in elderly and senile persons. In the main groups such changes were revealed even in young people that can indicate the acceleration of involution. Dystrophic changes of bronchial ciliated cells such as: a vacuolation of cytoplasm, accumulation of a large number of myelin-like structures, appearance of swollen mitochondria with the clarified matrix and paniculate or complete cristal degeneration and often loss of cilia were observed more often in the I group (69%) in spite of control parameters (43%). Numerous ciliary damages like disorientation of cilia, winding ciliary membrane, their swelling, pathology of microvilis were found. The percentage of ciliary defects significantly grew up in liquidators of II group (66%) as against nosological indices (30%). In addition, the increased number of microtubular ciliary disturbances was observed. Besides in the second group different pathological changes of basal bodies such as disturbances of their location and structure were observed. Probably, the increased cellular

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turnover leads to the acceleration of the ciliogenesis processes and causes the formation of pathological basal bodies that give rise to abnormal cilia. The changes found in bronchial basal cells deserve our special interest. These cells in early terms were characterized by a hypertophy of intracellular organoids with the subsequent progressing development of dystrophic changes. The features of basal cells population, probably, made for fenotype changes of mature epithelial ceils such as disturbances of polarity in goblet cells with accumulation of mucous granules in both apical and basal loci of cytoplasm (fig.l) and appearance of epithelial cells that had morphological signs of ciliated cells and II type pneurnocytes (fig.2), Such transformation must testify the realization of basal cells genome injuries and its turn are important in carcinogenesis respect. Our subsequent investigations of liquidators upper airways indicate similar pathological changes in the nasal epithelium with the only difference in their manifestation degree.

Figure 1. Disturbance of polarity in goblet cells of bronchial epithelium of II group liquidators. There are mucous granules both in apical and basal loci of cytoplasm. X 3,600.

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Figure 2. The lamellar bodies of II type pneumocytes in cytoplasm of bronchial ciliated cell of II group liquidator. X 7,200.

Thus, obtained results testify there is certain dynamics in the development of pathological reactions in the epithelium of airways. More expressed in comparison with the control data epithelial pathology in liquidators was caused by the effect of stronger than in usual inflammation pathogenic inductor. The permanent impact of the "chernobyl factor" promote the intensification of structural and functional systems of the airways epithelium that leads to exhaustion of compensatory and adaptive mechanisms and to deterioration of the protective properties of the epithelium. These changes are stereotype for both upper and lower airways with the different expression and have an important diagnostic and prognostic significance.

Part IV. Transplantation

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Heme Oxygenase-1 and/or Carbon Monoxide can Promote Organ Graft Survival Miguel P. SCARES12, Lukas GUENTHER, Pascal BERBERAT and Fritz H.BACH1 ' Immunobiology Research Center,Beth Israel Deaconess Medical Center, Department of Surgery, Harvard Medical School, Boston, MA 02215 ^Instituto Gulbenkian de Ciencia, Apartado 14, 2781-901 Oeiras, Portugal Abstract. Endothelial cells (EC) play a pivotal role in regulating inflammatory reactions such as those involved in the rejection of transplanted organs. This occurs through the expression of a series of pro- and anti-inflammatory genes that are associated with the activation of these cells. Expression of pro-inflammatory genes promotes events that lead to graft rejection while expression of anti-inflammatory (protective) genes suppresses those events and thus contribute to sustain graft survival. Understanding how the expression of these genes is regulated and their mechanism of action are important issues for the development of new therapeutic strategies to suppress graft rejection. The main thesis of our work is to exploit the mechanisms that are physiologically used by an organ to contribute to its own survival. This concept expands our previous vision that the main, if not only, way of sustaining graft survival is to suppress the anti-graft immune response of the recipient. We have studied protective genes and molecules using experimental models of transplantation in rats. We discuss here data that supports the concept that grafts can express " protective genes" and their products that are both anti-apoptotic and antiinflammatory (protective). The anti-inflammatory response mitigates inflammatory reactions leading to graft rejection. The data reviewed focus on the role of one of such genes, i.e. heme oxygenase-1 (HO-1), a stress responsive gene. One product of HO-1 action on heme is the production of carbon monoxide (CO), which can suppress graft rejection and lead to long-term graft survival.

1. Introduction The success of organ transplantation is largely due to the development of potent but largely non-specific immunosuppressive drugs that block T cell mediated events involved in graft rejection. However, we have found that the fate of a transplanted organ depends not only on the immune response against the graft but also on the ability of the graft to protect itself from immune mediated injury. We have suggested that the survival of such grafts depends in a critical manner on the ability of the graft vascular endothelium to protect itself against injury. The data reviewed here supports the concept that the vascular endothelium of a graft can express a series of anti-apoptotic, anti-inflammatory (protective) genes that act to help overcome graft rejection.

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2. Grafts can protect themselves from rejection The combination of anti-organ graft antibodies that deposit on the graft endothelium plus activated complement normally leads to rapid rejection of the organ. In some situations, however, the graft can survive despite the presence of the antibodies plus the complement. We refer to such situations as "graft accommodation", i.e. the survival despite the presence of the anti-graft antibodies and complement. We have tested specifically whether immediately-vascularized transplants could develop "resistance" to rejection caused by anti-graft antibodies and complement. To do so, we have used as an experimental model the transplantation of mouse or hamster hearts into rats, a model of xenotransplantation (transplantation between different species). Naive grafts undergo acute vascular rejection, which occurs around day 3 to 4 after transplantation, when elicited anti-graft antibodies are synthesized in the presence of complement [1-3]. This type of rejection is characterized by a series of inflammatory lesions involving EC activation [4], graft infiltration by activated host monocyte/macrophages and NK cells [5], coagulation and platelet aggregation associated with vascular occlusion and tissue necrosis [4]. We have shown that accommodated grafts express one or more protective genes in their vascular endothelium and smooth muscle cells [6]. That accommodated grafts are protected from rejection was formally demonstrated by the observation that a naive graft transplanted into a recipient carrying a first "accommodating" graft for 10 days undergoes rejection in few minutes while the first graft survives long-term [7. 8]. In addition, we found that the expression of a single protective gene on the vascular endothelium of these grafts, i.e. heme oxygenase-1 (HO-1), acted directly to suppress rejection [9]. Normal mouse hearts transplanted into immunosuppressed rats up-regulate the expression of HO-1 within hours after transplantation and survive indefinitely while HO-1"" deficient mouse hearts transplanted under the same immunosuppressive regimen are rejected in 3-7 days [9]. These results provide direct evidence that a single protective gene, i.e. HO-1, expressed in the vasculature of a graft can function to prevent graft rejection and to allow the development of accommodation. While the mechanism(s) underlying this effect of HO-1 is still not entirely clear, we believe that it is related to the ability of HO-1 to protect EC from apoptosis and from (immune-mediated) injury leading to graft rejection.

3. Protective Genes in Prevennting Rejection There are several genes that are expressed in endothelial cells and smooth muscle cells of grafts that accommodate. These include A20, a gene actively studies by our colleague, Dr. Christiane Ferran, and other genes that are both anti-apoptotic and anti-inflammatory. It is our purpose here to discuss heme oxygenase-1 (HO-1) and especially one of the products generated when HO-1 acts on heme: carbon monoxide as protective gene/molecules.

4. Heme oxygenases The heme oxygenase system is composed of three proteins referred to as heme oxygenases-1 (HO-1), -2 (HO-2) and -3 (HO-3) (reviewed in [10-12]). Among these only HO-1 and -2 are thought to act as enzymes that catabolize heme into biliverdin, free iron and the gas carbon monoxide (CO) [13]. Biliverdin is subsequently

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catabolyzed into bilirubin by biliverdin reductase (reviewed in [14]) and free iron induces the expression of ferritin [15, 16]. HO-2 is expressed constitutively in most cell types, including EC, while HO-1 is expressed at low or undetectable levels but is rapidly up-regulated under stress conditions such as upon UV radiation [17] or exposure to hydrogen peroxide, heavy metals, cytokines (IL-1, IL-6 or TNF-a), bacterial lipopolysaccharide, shear stress or heat shock (reviewed in [I I, 14]). Expression of HO-1 is rapidly up-regulated in the vascular endothelium of transplanted organs [9, 18]. In this situation the primary stimuli leading to HO-1 expression is not clear but this is likely to occur through the exposure of EC to circulating heme, a potent activator of HO-1 in most cell types [16]. The reason for this is that endothelial cells in the vasculature of a graft are probably exposed to high levels of free heme following transplantation. There are at least two potential "reservoirs" of pre-synthesized heme that can contribute to this effect. These are heme derived from hemoglobin and myoglobin when these proteins are released from red blood cells or myocytes through hemolysis or necrosis, respectively [19]. Once released, free heme can intercalate into EC cytoplasmic membranes and by this route become incorporated into the intracellular compartments of EC where it acts as a potent pro-oxidant [14, 16, 19]. A number of studies have suggested that acute exposure to heme is a highly cytotoxic [16, 20], probably related to the fact that Fe2" in the core of the heme becomes available to participate in the generation of free radicals through the Fenton reaction [14]. Generation of free radicals in this manner initiates a variety of signal transduction pathways that can induce both the expression of pro-inflammatory genes associated with EC activation as well as EC apoptosis. These events are highly deleterious and presumably can contribute to initiate the rejection of transplanted organs. Under these circumstances the only known mechanism by which EC can clear pro-oxidant free heme is to up-regulate the expression of HO-1. Once expressed at sufficiently high levels, HO-1 acts as the ratelimiting enzyme in the clearance of pro-oxidant heme and generates bilirubin as well as CO that suppress the pro-oxidant effects of heme [14]. In addition, HO-1 action on heme releases free iron, which up-regulates the expression of the iron sequestering protein ferritin (reviewed in [21]). The combined action of bilirubin, CO and ferritin are thought to contribute in a crucial manner to the cytoprotective response of EC exposed to extracellular heme.

5. HO-1 derived CO can prevent graft rejection We have recently shown that CO can account in large or full measure for the protective effects of HO-1 in terms of preventing graft rejection [18]. As described above, mouse to rat cardiac transplants survive long-term under transient complement depletion in combination with T cell immunosuppression [2]. HO-1 expression by the graft vasculature is critical to achieve long-term graft survival in this experimental model since HO-1 deficient mouse hearts transplanted under the same conditions are rapidly rejected [9]. We showed that the HO-1 protective effect was attributable to CO [18]. Under the same immunosuppressive regimen that allows mouse to rat cardiac transplants to survive long-term, inhibition of HO-1 activity by the specific HO inhibitor tin protoporphyrin (SnPPIX), precipitates graft rejection in 3-7 days [18], a model similar to using the HO-1 deficient mouse heart. Graft rejection under inhibition of HO activity by SnPPIX is associated, with microvessel platelet

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sequestration, thrombosis of coronary arterioles, myocardial infarction and apoptosis of EC as well as cardiac myocytes. Under inhibition of HO-1, exogenous CO suppresses graft rejection and restores long-term graft survival [18]. This effect of CO is associated with inhibition of platelet aggregation, thrombosis, myocardial infarction and apoptosis [18]. CO appears to bring the situation back to the normal. The exact mechanism by which HO-1 derived CO prevents graft rejection is not clear. CO is a signaling molecule that exerts a large spectrum of biological functions in several cell types, including in EC. The biological functions attributed to CO are thought to result directly or indirectly from binding of CO to iron in various proteins. Presumably this accounts for the ability of CO to modulate the activation of several signal transduction pathways, including guanylyl cyclase/cGMP [22], p38 mitogen activated protein kinase (MAPK) [23, 24], p21cipl [25] and thus regulate the expression of vasoconstrictor [25, 26], pro-inflammatory [23] as well as procoagulant molecules [27]. This broad action of CO is thought to account for its ability to promote vasodilation [25, 28] as well as to inhibit inflammation [23], apoptosis [24, 29], cell cycle progression [25, 30] and thrombosis [31, 32]. As for NO, another gaseous signaling molecule, CO also inhibits platelet activation/aggregation through activation of guanylyl cyclase and subsequent generation of cGMP [31, 33]. This is also thought to contribute in a critical manner to the ability of CO to suppress graft rejection. There are additional features of CO that may contribute to its protective function in terms of suppressing graft rejection. CO inhibits the pro-inflammatory phenotype associated with the activation of monocyte/macrophages (M0) [23, 34]: CO inhibits the generation of M0 derived proinflammatory molecules such as TNF-a while increasing that of anti-inflammatory molecules such as IL-10 [23]. The mechanism by which CO acts to modulate M0 activation in this manner is not dependent on activation of the guanylyl cyclase signal transduction pathway. Rather CO acts through a signaling pathway that involves activation of the p38 MAPK [23]. CO can also act through the p38 MAPK signal transduction pathway. The ability of CO to suppress EC apoptosis likely also contributes to suppressing graft rejection [24].

6. EC Apoptosis in graft rejection Under acute inflammatory conditions such as those associated with the rejection of a transplanted organ, widespread EC apoptosis can occur and thus enhance the pro-inflammatory environment leading to graft rejection [35]. EC apoptosis can lead to disruption of the integrity of the vascular endothelium with exposure of the pro-coagulant sub-endothelial matrix and subsequent induction of thrombosis, hypoxia, and tissue necrosis. Apoptosis of EC can also promote thrombosis directly through the exposure of pro-coagulant apoptotic bodies [36] as well as through the activation of the classical pathway of complement [37] and the activation of circulating platelets [38].

7. HO-1 derived CO suppresses EC apoptosis When cultured in vitro EC can be induced to undergo apoptosis when exposed to TNF-a in the presence of the transcription inhibitor Actinomycin D (Act.D). We have used this well-established experimental system to analyze whether expression of

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HO-1 in EC could suppress TNF-ct mediated apoptosis. When exposed to extra-cellular heme, EC up-regulate the expression HO-1 and as a result of that become resistant to TNF-ct mediated apoptosis [24]. Similar results are observed when HO-1 is overexpressed in EC [9, 24]. The protective effect of HO-1 requires its enzymatic activity, indicating that this effect involves the generation of one or several end-products of heme catabolism by HO-1, i.e. Fe2\ bilirubin and/or CO [24]. Since heme derived Fe2+ up-regulates the expression of the iron sequestering protein ferritin [15, 16], we tested if Fe2+ sequestration/elimination could account for the antiapoptotic effect of HO-1 in EC. Our data [24] as well as that of others [39] indicates that this is the case: the anti-apoptotic effect of HO-1 can be mimicked by the exogenously-administered iron chelator Desferoxarnine [24] and/or by overexpressing the heavy chain of ferritin (P. Berberat et al., manuscript submitted). Our data, however, suggests that in addition to the induction of systems that lead to iron sequestration [39], HO-1 generates physiological levels of CO that act to suppress EC apoptosis [24]. This notion is supported by the observation that exposure of EC to exogenous CO suppresses EC apoptosis [24]. At least in EC, the anti-apoptotic action of CO is strictly dependent on the activation of the p38 MAPK signal transduction pathway: HO-1 derived CO enhances p38 MAPK activation in EC and inhibition of p38 MAPK activation abrogates the anti-apoptotic effect of HO-1 and/or CO in these cells [24]. How HO-1 derived CO acts to modulate the activation of this specific signal transduction pathway and how this acts to suppress EC apoptosis remains to be fully elucidated.

8. Other actions of CO in transplant related models We have evaluated the role of CO in ischemia-reperfusion injury by transplanting a rat heart to a syngeneic recipient after the heart was held under ischemic conditions for 24 hours. Control hearts all failed within 24 hours. Hearts treated with CO had various rates of survival after 1, 7 or 14 days. Treatment of the donor alone led to 50% 1 day survival and 33% at 7 and 14 days; treatment of the heart while ex vivo alone had a similar effect as treating the donor; the combination of these two treatments led to 100% 1 day, 83% 7 day 66% 14 day; and the combination of these two treatments plus treatment of the recipient led to 100% survival on day 1 and 83% on days 7 and 14. Clearly the CO treatment improves the function of a heart not susceptible to immune rejection but subject to ischemia-reperfusion injury without CO treatment.

9. Concluding remarks The findings reviewed hereby show that transplanted organs can express a series of protective genes in their vasculature that may contribute to promote the survival of such organ. We found that one of these protective genes, HO-1, acts in such a manner. The mechanism by which HO-1 suppresses graft rejection is still not clear. Our data suggests that this protective effect relies in large measure on the ability of HO-1 to catabolize pro-oxidant heme, as it accumulates following transplantation, into the gas CO. HO-1 derived CO has a series of biological effects that can contribute to suppress graft rejection. These include its anti-apoptotic function that has yet to be fully elucidated in terms of its molecular basis. Our data suggests that

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HO-1 derived CO suppresses EC apoptosis through a mechanism that is dependent on the activation of p38 MAPK and the transcription factor NF-icB. These findings raise important questions related not only the mechanism of action of CO in terms of preventing graft rejection but also to the development of new strategies aimed to promote graft survival. Based on the observation that inhibition of NF-icB impairs the anti-apoptotic effect of CO, it is possible that the protective effect of CO in terms of suppressing graft rejection may also be impaired upon inhibition of NF-icB activation. Given that inhibition of NF-KB activation is perceived as a potential therapeutic tool to suppress the expression of pro-inflammatory genes that promote graft rejection; our present data raises significant questions about the relative therapeutic values of the two approaches. Footnotes This paper was taken in large part from a recent review written from our laboratories. The work from our laboratories was supported by a grant from the Roche Organ Transplantation Research Foundation (ROTRF; 998521355) awarded to MPS, NIH grants (HL67040) awarded to MPS and (HL58688) awarded to FHB. Fritz H. Bach is the Lewis Thomas Professor at Harvard Medical School and is a paid consultant to Novartis Pharma, Basel, Switzerland. Abbreviations ActD: Actinomycin-D; CO: Carbon monoxide; EC: Endothelial cell; HO-1: Heme oxygenase-l; IicBa: Inhibitor nuclear factor-tcBa; MAPK: Mitogen-activated Protein Kinases; Ma; Monocyte macrophage. MnSOD: Manganese Superoxide Dismutase; NF-KB: Nuclear Factor-icB; TNF: Tumor Necrosis Factor.

References 1. Bach, F. H., Winkler, H., Ferran, C., Hancock, W. W. & Robson, S. C. (1996) Immunology Today 17,379-84. 2. Koyamada, N., Miyatake, T., Candinas, D., Mark, W., Hechenleitner, P., Hancock, W. W., Scares, M. P. & Bach, F. H. (1998) Transplantation 65,1210-5. 3. Scares, M. P., Lin, Y., Sato, K., Takigami, K., Anrather, J., Ferran, C., Robson, S. C. & F.H.Bach (1999) Current opinion in organ transplantation 4, 80-89. 4. Bach, F. H., Robson, S. C., Ferran, C., Winkler, H., Millan, M. T., Stuhlmeier, K. M., Vanhove, B., Blakely, M. L., van, der, Werf, Wj, Hofer, E. & et, a. 1. (1994) Immunological Reviews 141, 5-30. 5. Fryer, J. P., Leventhal, J. R., Dalmasso, A. P., Chen, S., Simone, P. A., Jessurun, J., Sun, L. H., Reinsmoen, N. L. & Matas, A. J. (1994) Transplant Immunology 2, 87-93. 6. Bach, F. H., Ferran, C., Hechenleitner, P., Mark, W., Koyamada, N., Miyatake, T., Winkler, H., Badrichani, A., Candinas, D. & Hancock, W. W. (1997) Nature Medicine 3, 196-204. 7. Lin, Y., Soares, M. P., Sato, K., Takigami, K., Csizmadia, E., Smith, N. & Bach, F. H. (1999) Journal of Immunology 163,2850-2857. 8. Soares, M. P., Lin, Y., Sato, K., Stuhlmeier, K. M. & Bach, F. H. (1999) Immunology Today 20,434-437. 9. Soares, M. P., Lin, Y., Anrather, J., Csizmadia, E., Takigami, K., Sato, K., Grey, S. T., Colvin, R. B., Choi, A. M., Poss, K. D. & Bach, F. H. (1998) Nature Medicine 4, 1073-1077. 10. Willis, D. (1999) in Inducible enzymes in the inflammatory response, eds. Willoughby, D. A. & Tomlinson, A. (Birkhauser, Basel), pp. 55-96. 11. Choi, A. M. & Alam, J. (1996) American Journal of Respiratory Cell & Molecular Biology 15,9-19. 12. Maines, M. D. (1997) Annual Review of Pharmacology & Toxicology 37,517-54. 13. Kutty, R. K., Daniel, R. F., Ryan, D. E., Levin, W. & Maines, M. D. (1988) Archives of Biochemistry & Biophysics 260,638-44. 14. Ryter, S. W. & Tyrrell, R. M. (2000) Free Radical Biology & Medicine 28, 289-309.

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Mechanisms of and Clinical Efforts to Minimise Perioperative Lung Injury I. Gavin WRIGHT and Nandor MARCZIN Department of Anaesthesia and Critical Care Royal Brompton and Harefield NHS Trust Harefield Hospital Harefield, Middlesex. UB9 6JH Abstract The success of clinical lung transplantation is constrained by a shortage of suitable donor organs. In addition, even suitable donor lung grafts exhibit significant perioperative dysfunction and are at risk to develop lung injury throughout the procedure. Significant recent progress into the cellular and molecular mechanisms of pulmonary ischemia-reperfusion injury provides a good opportunity for implementation of this understanding into clinical practice. On the basis of recent evidence that the endothelium plays an essential role in regulating the dynamic interaction between pulmonary vasodNatation and vasoconstriction and is a major target during lung injury due to enhanced leukocyte-endothelial interactions, we at Harefield Hospital intend to implement protective strategies to limit perioperative lung injury during lung transplantation. This review summarises the major mechanisms potentially contributing to lung injury and the currently available surgical and anaesthetic protective strategies with a focus on clinical applicability.

1. Introduction Lung transplantation has become an established therapeutic modality for end-stage lung diseases worldwide due to improvement in surgical technique, advances in the anaesthetic management and pharmacological repertoire including immunosuppression. Although lung transplantation now offers a realistic opportunity for mid term survival in selected patients with end stage pulmonary disease, the practice is constrained by alarming shortage of suitable donor organs, significant primary graft failure and chronic rejection in the form of obliterative bronchiolitis. This is illustrated in the recent survival figures reported by the International Society for Heart and Lung Transplantation (ISHLT) [1] and can be summarised as follows 1) Heart transplantation has achieved good long term survival with 5 year survival rates in the 70% range, whereas combined heart lung transplantation is 40%. Isolated Ltx is also in the 40-50% range, suggesting that lung transplantation at the moment can only offer mid term survival. The most important factor adversely affecting long-term survival after lung transplantation is bronchiolitis obliterans syndrome, which is generally considered to be a complex end result of a chronic rejection process. 2) At least 50 % of the 5 year mortality occurs during the 1st year post transplant in both heart and lung transplantation with a significant number of death taking place perioperatively.

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3) Although there is a small but significant improvement in late mortality presumably due to better immunosuppression, no improvement in perioperative mortality can be seen when compared to data during the early 80's. 4) Analysis of the causes of perioperative death reveal that the majority of death is due to primary organ failure. The high incidence of loss or compromise of transplanted organs in the immediate posttransplantation period due to primary graft dysfunction prompted many research groups to study mechanisms of lung injury and transplant centres to re-evaluate donor management, and preservation/reperfusion protocols. Lung injury can occur at any stage of this complex procedure with distinct mechanisms that might provide a number of opportunities for surgical and anaesthetic efforts to minimise lung damage. 2. Organ damage during transplantation Much recent evidence indicates that components of the alveolocapillary unit are the major targets during acute lung injury with the micro vascular endothelium being the most susceptible element. During the injury process, endothelial cells become activated and more permeable with characteristic loss of function it plays as an essential regulator of pulmonary vasoreactivity, intravascular coagulation, inflammatory response and gas exchange [2]. In addition, the composition, function, and metabolism of pulmonary surfactant produced by alveolar type II epithelial cells are increasingly being recognised as important factors in acute lung injury. Although Type I epithelial cells appear to be more resistant to inflammatory response and injury and their barrier and metabolic function might remain intact even in high permeability pulmonary oedema, long-term outcome may depend upon Type I epithelial cell survival. Thus donor evaluation should include cellular and molecular assessment of endothelial and epithelial cell integrity and organ management, rescue and preservation should provide strategies to maintain structural and functional integrity of these cells in the face of multiple insults to the alveolocapillary unit. Donor lung injury Many donor lungs exhibit severe hypoxemia and diffuse infiltrates on chest roentgenograms owing to oedema, infection, aspiration, contusion, or ventilator-induced injury. The highpermeability pulmonary oedema that is prevalent in many lung donors often results from neurogenic causes, in addition to iatrogenic overhydration during the resuscitation attempts. Neurogenic pulmonary oedema occurs after a sudden, massive sympathetic neural discharge, which engenders a "blast injury" to the pulmonary circulation and may disrupt the anatomic integrity of the pulmonary capillaries. Recent studies have confirmed that in the hours after brain death there is significant increase in right ventricular hydraulic power and pulmonary artery blood flow These changes, along with increases in systemic and pulmonary vascular pressures, may lead to pulmonary endothelial cell injury, impaired lymphatic drainage, and an increase in pulmonary extravascular lung water [2]. Even in donors who do not have overt lung injury by standard criteria, measurements of intrapulmonary shunt may be highly abnormal and fiberoptic bronchoscopy may reveal aspiration of a significant volume of gastric contents or blood.

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In addition to injury during the hyperdynamic phase of brain death, the following period of haemodynamic instability and restoration manoeuvres might also contribute to lung damage. Hypovolemic shock might result in microvascular pulmonary occlusion by platelet and leukocyte aggregates, pulmonary endothelial injury and pulmonary parenchymal neutrophil sequestration. These changes are further aggravated by ventilator induced injury. It is now widely accepted that improper ventilator management has greatly compromising effects on the lung in the setting of an impending injury, through a mechanism which is related to biochemical upregulation of an inflammatory response mediated by a pro-and anti-inflammatory cytokine imbalance within the lung. This mechanism in concert with a systemic inflammatory response resulting from polytrauma with increased levels of cytokines might underlie the upregulation of vascular adhesion molecules even before harvesting, which might have implications for post ischaemic lung injury. Thus it can be argued that a significant number of cases of severe ischaemia-reperfusion injury probably result from evolving or subclinical donor lung dysfunction that was not readily apparent during the preharvest assessment [2]. Ischaemia-reperfusion Survival as the balance of microvascular vasoactive and cytotoxic/protective bioactivities Under basal conditions as well as during evolving lung injury, the function of the pulmonary endothelium depends on a dynamic balance between endothelial protective and injurious substances. Three important protective agents produced by endothelial cells are prostacyclin, nitric oxide, and adenosine all primarily acting by modulating cellular cyclic nucleotide (cAMP and cGMP) levels. Prostacyclin is a potent eicosanoid that causes vasodilation, prevents neutrophil adherence, inhibits platelet aggregation, and stabilises lysosomal membranes. Nitric oxide is produced in endothelial cells by nitric oxide synthase, a calcium- and calmodulin-dependent enzyme. The effects of nitric oxide are mediated by activation of guanylate cyclase, resulting in the formation of cyclic guanosine monophosphate (GMP). Nitric oxide has a shorter biological half-life than prostacyclin (10 to 20 seconds), is able to quench superoxide radicals produced by endothelial cells, and causes vasodilatation, decreased neutrophil adherence, and inhibition of platelet aggregation. A third key protective agent formed by the endothelium is adenosine, which produces its effects by activation of purinergic receptors coupled to adenylate cyclase by a guanosine triphosphate-dependent mechanism. Adenosine is rapidly metabolized by the enzymes adenosine deaminase and adenosine kinase. Like prostacyclin and nitric oxide, adenosine is a potent vasodilator, decreases neutrophil adherence to endothelial cells, and diminishes neutrophil cytotoxicity . Adenosine also inhibits superoxide radical production by neutrophils, and like prostacyclin and nitric oxide has produced beneficial effects in a variety of models of ischaemia-reperfusion injury. In addition to cytoprotective agents, the endothelium generates substances that have a marked vasoconstrictor and thrombogenic effect, thus promoting cell injury. These include endothelin-1, the phospholipid platelet activating factor, which causes the release of various leukotrienes and thromboxanes, all of which are vasoconstrictors. Oxidativc stress During the past few years many of the cellular and molecular events modulating the inflammatory response to ischaemia-reperfusion injury have been elucidated. Although

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changes in the energy homeostasis within the lung has received much initial attention, recent studies have confirmed that lung dysfunction after storage and reperfusion is associated with significant direct oxidative injury to endothelial cell constituents including lipids, protein, and DNA [3-4]. While many components of the oxidative stress are initiated during the ischaemic and hypoxic phase, the critical step mediating this injury appears to be the burst in the production of oxygen and nitrogen derived oxidative species at the onset of uncontrolled reperfusion [5-7]. Role of neutrophils Although in some models lung injury occurred even after neutrophil depletion, most studies suggest that endothelial and lung injury depends upon enhanced interaction between white cells particularly neutrophils and the endothelium [8,9]. During reperfusion after pulmonary ischaemia, neutrophils may contribute to lung injury in several ways. 1) Neutrophils are the major source of reactive oxygen metabolites such as the superoxide anion and hydrogen peroxide, which can damage pulmonary endothelial cells. 2) Due to cytoskeletal reorganisation, activated neutrophils are stiffer than quiescent cells, making them less able to undergo deformation as they circulate through the pulmonary capillaries and thus more likely to induce adherence to endothelial cells and capillary plugging. 3) Activated neutrophils can produce TNF, IL-1, IL-6, and IL-8 contributing to cytokine imbalance. 4) In addition, neutrophils can generate leukotriene 84, a potent chemotactic agent that activates neutrophils and promotes their adherence to the endothelium. 5) Finally, upon degranulation neutrophils release elastase and other proteases, which directly injure pulmonary endothelial and parenchymal cells. A recent study has identified heparin binding protein as one of the major neutrophil product causing changes in endothelial permeability [10]. Neutrophil protease inhibitors have been shown to attenuate experimental lung injury. The potential role of neutrophil-endothelial interactions in the clinical setting is important because many pharmacological and mechanical strategies can target these interactions that have been shown to be beneficial in many animal models with clinical applicability. However, in order to devise optimal strategies to reduce the injurious effects of neutrophils, one has to consider the origin of the harmful neutrophils and the consequence of total body elimination of neutrophil activity in the globally immunosuppressed patient. a) The number of neutrophils in the normal lung is approximately three times the circulating pool of neutrophils and due to their marginalisation, a significant number of neutrophils may reside in close proximity to endothelial cells even after flushing [2,11]. b) Postoperative infections are important factors in early mortality following transplantation, therefore systemic attenuation of neutrophil function is undesirable. This makes it difficult to adopt techniques aimed at depleting the circulating neutrophil pool or preventing neutrophil activation and migration through the endothelium in the long run. However recent data suggest that temporary attenuation of neutrophil function for as short as 15-30 minutes during reperfusion offers considerable benefit. This might be achievable with pharmacological therapy such as antioxidants, and agents modulating neutrophil

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function such as cAMP elevating agents and NO. In addition, an interesting model has been recently proposed to selectively perfuse the pulmonary circulation with a separate circuit, which includes a neutrophil depleting filter. This strategy can be easily considered on lung transplants performed utilising cardiopulmonary bypass. Cytokine network It is now evident that many of the cellular and molecular processes in the lung are controlled by a vast network of cytokines, which function as extracellular signalling proteins. Cytokines such as IL-1 and TNF play critical roles in the inflammatory response to ischaemia-reperfusion injury. Both of these cytokines cause endothelial cell activation, a phenotype which is markedly adhesive for neutrophils and other white cells. These cytokines induce cytoskeletal reorganisation, expression of leukocyte adhesion molecules and vasoactive and prothrombotic factors. They also induce neutrophil activation characterised by increased neutrophil phagocytosis, respiratory burst activity, and degranulation. All these events can cause pulmonary vascular endothelial cell injury both in vitro and in vivo. Studies utilising bronchoalveolar lavages in animal lung transplant models have demonstrated early release of pro-inflammatory cytokines. Similarly, IL-6 has been detected in the clinical situation, and peak serum IL-6 level has been correlated with the severity of preservation injury [16]. However the levels of other cytokines in bronchoalveolar lavage and serum during the first few days after clinical lung transplantation remains unknown. However, despite the postulation of their role in lung injury, no single cytokine has been identified to explain the onset, extent and outcome of acute lung injury. It is becoming evident that in addition to the pro-inflammatory, and pro-apoptotic cytokines there is a phased anti-inflammatory and cytoprotective response. Current efforts are increasingly focused on the characterisation of the actual net imbalance in these activities by utilising a number of bioassay s . It is thus evident that pulmonary ischaemia-reperfusion injury has multifaceted effects on the complex cytokine network and balance within the lungs which remains to be established. Although administration of anti-inflammatory cytokines such as IL-10 or monoclonal antibodies directed against selected cytokines involved in the early stages of this injury may ultimately prove useful clinically, enthusiasm is diminished in light of the complexity of the system and recent clinical trials in other forms of sepsis and lung injury. It is more convincing that a global strategy to interfere with cytokine response would be more attractive and simpler for example in the form of administration of high dose methylprednisolone to the donors prior to organ harvest. Epithelial injury and protection, surfactant The pulmonary alveolar epithelium is composed of two cell types, elongated type I cells, which cover most of the alveolar surface, and cuboidal type II cells, which predominate in the alveolar corners. Both type I and II cells exist in close proximity to pulmonary capillary endothelial cells and play important roles in pulmonary surfactant production and in modulating host defense in the alveolar space [2]. It is evident that the endogenous surfactant system undergoes profound alterations after pulmonary ischaemia and transplantation that are qualitatively similar to the changes in surfactant that occur in nontransplant lung injury models. Also there are many studies showing that exogenous surfactant therapy can mitigate pulmonary dysfunction in a wide array of experimental models. Although case reports show beneficial effects of surfactant

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after reperfusion, most studies suggest that for surfactant to have a maximum impact on lung injury after prolonged storage, donor surfactant therapy is probably essential [2]. The role of Type I function has received considerably less attention. However, recently it is suggested that these cells have a paramount role not only in modulating permeability but also in resolution of pulmonary oedema after lung injury in humdns. By monitoring alveolar fluid clearance in transplant patients through determination of alveolar lining fluid and plasma protein concentrations, a recent study established the capacity of the alveolar epithelium to reabsorb fluid from the alveolar space. This appeared to be a marker of less severe reperfusion injury on the basis of histological and clinical criteria and outcome [15]. 3. Strategies to limit lung injury Hypothermic preservation Flush perfusion of pulmonary grafts with cold modified EuroCollins solution supplemented by prostaglandin treatment was introduced clinically 10 years ago. During the last decade, much experimental work has led to reports of alternative storage solutions, differing storage conditions, and pharmacologic interventions that improve pulmonary graft performance. A world-wide survey of the 125 centres performing lung transplantation revealed that most centres continue to use EuroCollins solution of whom 69% include prostaglandin therapy and 32% donor steroid treatment [16]. University of Wisconsin solution (UW) is used by 15 centres (13.5%), of which 10 (67%) use prostaglandin and seven (47%) use donor steroids. Nine centres use Papworth solution and one uses donor core cooling. This report suggests that there has been a trend toward the use of UW solution and a slightly warmer storage temperature. However, for most centres, graft storage techniques have changed little over the last decade. Laboratory work showing that high-potassium storage solutions impair vascular endothelial cell function prompted studies to evaluate low-potassium-dextran pulmonary flush solution (LPD). It has been shown that LPD provides excellent 12-hour lung preservation in different animal models of experimental lung transplantation and exhibits favourable profile on alveolar cells injury and survival. The Toronto Program has now adopted LPD preservation solution into clinical practice after approval was obtained for the use of LPD in clinical lung transplantation in 1998. In a recent article they have reported their initial experience with the use of LPD in comparison with EC in 94 lung clinical lung transplant procedures [17]. Vasodilator and protective strategies These agents are used in selected centres as part of donor management and in the preservation flush solution Prostagiandins Similarly to many studies showing endothelial cytoprotection by prostaglandins, recent animal work has demonstrated that a continuous prostaglandin Ej infusion in the recipient, starting before reperfusion, was associated with improved oxygenation and less lung oedema after transplantation. Although clinical studies have not addressed this organ protection, there is evidence for efficient haemodynarnic effects of aerosolized prostacyclin

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in human lung transplantation. Also there is increasing numbers of centres using prostacyclin as part of their preservation protocol. Nitric oxide donors, nitroglycerin, inhaled NO As discussed above, NO is one of the major endogenous protective modalities of the lung. It exerts the majority of its effects via activation of guanylate cyclase and the resultant formation of cyclic GMP. A number of studies have documented characteristic changes in NO and NO bioactivity in the setting of ischaemia reperfusion. Most cellular studies including our own suggests that NO is consumed during oxidative stress. This has been demonstrated in animal models showing that both nitric oxide and cyclic GMP levels decreased markedly during ischaemia and at the onset of reperfusion . Finally this has been observed in ARDS and by us in lung transplantation [18]. Thus, decrease in NO levels might be a sensitive index of lung dysfunction and prevention of the decrease in endogenous NO or supplementation with exogenous NO should be a major aspect of lung protection strategies. Stimulation of guanylate cyclase activity can be achieved by exogenous nitric oxide, nitroglycerin, or nitroprusside. The ability of these agents to increase cyclic GMP levels was first noted almost 20 years ago. Recently, inhaled nitric oxide has been used as a potent and selective pulmonary vasodilator in many pathological situations including ARDS. In the setting of lung transplantation, nitric oxide is theoretically attractive because of its ability to quench superoxide radicals and to protect pulmonary endothelial cell function. Nonetheless, the use of nitric oxide can actually contribute to lung injury as a result of its combination with superoxide to form peroxynitrite. Peroxynitrite can generate the highly toxic hydroxyl anion and is a strong oxidant in its own right; it readily catalyses membrane lipid peroxidation, reacts with metals to form toxic nitrosylating species, and oxidises sulfhydryl groups on cellular proteins. Thus inhaled NO can only be recommended in the presence of strong antioxidant supplementation where the potential for peroxynitrite toxicity is reduced. Phosphodiesterase inhibitors Levels of cAMP and cGMP not only depend on stimulated production but also by the rate of their degradation. Inhibition of the degradation pathways through inhibition of phosphodiesterase enzymes is one of the major developments in haemodynamic management and organ preservation. These agents exhibit beneficial haemodynamic profile in heart failure, can reduce cathecolamine requirement and offer cytoprotection in many systems. Pentoxifylline is a methylxanthine derivative that has been used as a hemorrheologic agent for the treatment of peripheral vascular disease. In vitro studies have demonstrated that pentoxifylline has a marked inhibitory effect on neutrophils, particularly neutrophils activated by inflammatory cytokines. In view of its myriad antineutrophil effects and beneficial hemorrheologic properties, as well as its long track record of use in humans, pentoxifylline has clearly been recommended in the preservation of clinical lung grafts for transplantation [2]. Antioxidants In addition to a vast literature in animal models, the compromised antioxidant status of lung transplant recipients before and after transplantation is being increasingly recognised. Before surgery, the antioxidant status of patients was poor as serum ascorbate and total thiol concentrations were significantly lower man control subjects [19]. Two weeks post-surgery.

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ascorbate and total thiol concentrations were still low and urate concentrations had fallen compared to control subjects. These data provide solid support for perioperative antioxidant administration to these patients. One problem, however, is that there are distinct reactive species and it is likely that an antioxidant mixture including different components would be more effective than single antioxidants. Ascorbic acid Numerous studies have confirmed the beneficial effect of ascorbic acid in multiple oxidative stress conditions both in animal models and humans. Regarding lung transplantation ascorbic acid was able to weaken reperfusion injury in an in situ autotransplantation model in sheep [20]. N-acety 1-1 -cysteine (NAC), an oxidant scavenger, promotes glutathione in its reduced form (GSH) that is depleted during ischaemia. Numerous studies have recently demonstrated its efficacy in protecting lungs in animal reperfusion injury. In addition NAC has been shown to modulate inflammatory gene transcription and cytokine secretion in many cell types. Allopurinol: It is the only inhibitor of the enzyme xanthine oxidase, whose activation is involved in free radical generation. In addition to intracellular mechanisms, circulating xanthine oxidase activity has been linked to lung reperfusion injury. This circulating enzyme could be an important target of allopurinol. Its efficacy is well established in reducing the inflammatory components in gout. Superoxide dismutase: Recombinant human superoxide dismutase (rh-SOD) has been shown the potential to mitigate free radical-mediated reperfusion injury-induced acute endothelial cell damage that potentially may contribute to the process of chronic obliterative rejection arteriosclerosis. In a prospective randomized double-blind placebo-controlled trial, the effect of rhSOD, given in a dose of 200 mg intravenously during surgery to cyclosporine-treated recipients of cadaveric renal allografts, on both acute and chronic rejection events as well as patient and graft survival was investigated. The results obtained show that rh-SOD exerts a beneficial effect on acute rejection events and significant improvement of the actual 4-year graft survival rate in rh-SOD-treated patients [21]. Anticytokine therapy Corticosteroids are theoretically attractive drugs to control many inflammatory events in a wide number of pathological situations. This is likely related to inhibition of proinflammatory cytokines and pro-apoptotic activities. In addition to this theoretical advantage, the clinical benefit of steroid administration has recently been confirmed in donor lung viability. A retrospective study of all thoracic organ donors procured by the California Transplant Donor Network investigated which donor management factors were associated with an increased likelihood of successful lung procurement. Corticosteroid usage and initially clear breath sounds were independent predictors of successful procurement by multivariate analysis [22]. In addition to contribution of donor lung viability, corticosteroids might modulate the inflammatory response during ischaemia/reprefusion. A number of clinical studies have demonstrated that administration of methylprednisolone attenuated the inflammatory response to cardiopulmonary bypass, although the clinical benefit remains to be established [23]

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Complement attenuation Activation of the contact as well as the complement system is regulated by a common inhibitor, Cl-esterase inhibitor (Cl-INH). In addition to case reports where severe uncontrollable capillary leak syndrome after lung transplantation was successfully managed by Cl inhibition, there are data from a randomised, double-blinded, placebocontrolled multicenter trial at five North American centres regarding the influence of TP10 (soluble C receptor 1 inhibitor). TP10 led to a significant increase in early extubation of patients. Although there was no difference in PaO2/fraction of inspired oxygen between groups, total time receiving mechanical ventilation both tended to be shorter in the TP10 group but did not achieve statistical significance [24]. Serine protease inhibition: aprotinin The addition of aprotinin to EC and UW solutions increases endothelial cell viability in hypoxic cold storage conditions. In terms of whole organ function, aprotinin has been shown to improve lung preservation as demonstrated by increased oxygenation and compliance, and decreased capillary permeability. These observations are clinically applicable as there is already extensive experience with the use of aprotinin in heart and lung transplant recipients, in addition to its routine use in conventional cardiac operations in reducing perioperative blood loss [25]. The risk to develop lung injury continues in the postoperative period and thus lung protective strategies also have to be extended to this period. It is now well accepted that subclinical injury can be augmented with improper ventilation strategy. Thus currently proven protective ventilation strategies including low tidal volume and PEEP above lower inflection point are desired if tolerated in this condition. However during this period donor/recipient size mismatch, differential lung compliance especially during single lung transplantion remains a clinical challenge which might recuire sophisticated strategies including differential lung ventilation. References. [ 1 ] http://www.ishlt.org/regist_heart-lung_main.htm [2]

Novick RJ, Gehman KE, AH IS, Lee J. Lung preservation: the importance of endothelial and alveolar type II cell integrity. Ann Thorac Surg 1996; 62:302-314.

[3]

Granger DN. Ischemia-reperfusion: mechanisms of microvascular dysfunction and the influence of risk factors for cardiovascular disease. Microcirculation 1999; 6:167-178.

[4]

Garden DL, Granger DN. Pathophysiology of ischaemia-reperfusion injury. J Pathol 2000;

[5]

Verrier ED, Morgan EN. Endothelial response to cardiopulmonary bypass surgery. Ann Thorac Surg 1998;66:S17-S19.

[6]

Boyle EM, Jr., Pohlman TH, Cornejo CJ, Verrier ED. Endothelial cell injury in cardiovascular surgery: ischemia-reperfusion. Ann Thorac Surg 1996; 62(6): 1868-1875.

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Boyle EM, Jr., Canty TG, Jr., Morgan EN, Yun W, Pohlman TH, Verrier ED. Treating myocardial ischemia-reperfusion injury by targeting endothelial cell transcription. Ann Thorac Surg 1999; 68(5): 1949-1953.

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[8]

Bando K, Pillai R, Cameron DE, Brawn JD, Winkelstein JA, Hutchins GM et al. Leukocyte depletion ameliorates free radical-mediated lung injury after cardiopulmonary bypass. J Thorac Cardiovasc Surg 1990;99(5):873-877.

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Eppinger MJ, Deeb GM, Boiling SF, Ward PA. Mediators of ischemia-reperfusion injury of rat lung. Am J Pathol 1997; 150:1773-1784.

[10]

Gautam N, Olofsson AM, Herwald H, Iversen LF, Lundgren-Akerlund E, Hedqvist P, Arfors KE, Flodgaard H, Lindbom L. Heparin-binding protein (HBP/CAP37): a missing link in neutrophil-evoked alteration of vascular permeability. Nat Med. 2001 Oct;7(10):l 123-7.

[11]

Gee MH, Albertine KH. Neutrophil-endothelial cell interactions in the lung. Annu Rev Physiol 1993;55:227-48

[12]

Halldorsson AO, Kronen M, Allen BS, Rahman S, Wang T, Layland M, Sidle D. Controlled reperfusion prevents pulmonary injury after 24 hours of lung preservation. Ann Thorac Surg. 1998 Sep;66(3):877-84

[13]

Pham SM, Yoshida Y, Aeba R, et al. Interleukin-6, a marker of preservation injury in clinical lung transplantation. J Heart Lung Transplant 1992;! 1:1017-24.

[14]

Martin TR. Cytokines and the acute respiratory distress syndrome (ARDS): a question of balance. Nat Med. 1997Mar;3(3):272-3.

[15]

Ware, L. B., Golden, J. A., Finkbeiner, W. E., Matthay, M. A. Alveolar Epithelial Fluid Transport Capacity in Reperfusion Lung Injury after Lung Transplantation. Am J Respir Crit Care Med . 1999; 159:980-988

[16]

Hopkinson DN, Bhabra MS, Hooper TL. Pulmonary graft preservation: a worldwide survey of current clinical practice. J Heart Lung Transplant. 1998 May;17(5):525-31.

[17]

Fischer S, Matte-Martyn A, De Perrot M, Waddell TK, Sekine Y, Hutcheon M, Keshavjee S. Lowpotassium dextran preservation solution improves lung function after human lung transplantation. J Thorac Cardiovasc Surg. 2001 Mar;121(3):594-6.

[18]

Marczin N, Riedel B, Gal J, Polak J, Yacoub M. Exhaled nitric oxide during lung transplantation [letter]. Lancet 1997; 350(9092):1681-1682

[19]

Williams A, Riise GC, Anderson BA, Kjellstrom C, Schersten H, Kelly FJ. Compromised antioxidant status and persistent oxidative stress in lung transplant recipients. Free Radic Res. 1999;30(5):383-93

[20]

Demertzis S, Scherer M, Langer F, Dwenger A, Hausen B, Schafers HJ. Ascorbic acid for amelioration of reperfusion injury in a lung autotransplantation model in sheep. Ann Thorac Surg. 2000 Nov;70(5): 1684-9.

[21]

Land W, Schneeberger H, Schleibner S, Illner WD, Abendroth D, Rutili G, Arfors KE, Messmer K. The beneficial effect of human recombinant superoxide dismutase on acute and chronic rejection events in recipients of cadaveric renal transplants. Transplantation. 1994 Jan;57(2):211-7.

[22]

McElhinney DB, Khan JH, Babcock WD, Hall TS. Thoracic organ donor characteristics associated with successful lung procurement. Clin Transplant. 2001 Feb;15(l):68-71.

[23]

Chaney MA. Corticosteroids and cardiopulmonary bypass : a review of clinical investigations. Chest. 2002Mar;121(3):921-31.

[24]

Martin R. Zamora et al. Complement inhibition attenuates human lung transplant reperfusion injury Chest 1999;116:46S

[25]

Royston D. Preventing the inflammatory response to open-heart surgery: the role of aprotinin and other protease inhibitors. Int J Cardiol. 1996 Apr 26;53 Suppl:Sl 1-37

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Disease Markers in Exhaled Breath N. Marczin and M.H. Yacoub (Eds.). IOS Press, 2002

Condensate Inflammatory Markers in Lung Transplantation Karen McRAE Director of Anesthesia for Thoracic Surgery and Lung Transplantation 585 University Avenue Toronto, Ontario Canada M5G 2C4 Abstract. In human lung transplantation, ischemia-reperfusion (IR) injury occurs in 15-30% of recipient patients after lung engraftment. IR injury is the primary cause of death in the early postoperative period. Additionally, there is increasing evidence indicating a relationship between early IR injury and chronic graft dysfunction. Traditional respiratory monitoring techniques have failed to predict the occurrence of IR injury in the transplanted lung. The collection of breath condensate has been suggested as a technique to acquire samples representative of the interstitial fluid compartment of the lung parenchyma, and has been shown to contain protein macromolecules including cytokines. Cytokines play a critical role in modulating inflammatory processes and in enhancing cellular infiltration in injured tissue, including ischemia-reperfusion.

1. Introduction In clinical lung transplantation, the kinetics of cytokine release at the time of lung reperfusion, and the resulting clinical implications are the focus of ongoing investigation. In animal models of a variety lung injuries, plasma cytokine levels have failed to predict severity of injury. Tissue cytokines, specifically increased levels of the pro-inflammatory cytokine IL-8, have been demonstrated to correlate to worse clinical outcome in human lung transplantation [1], but tissue cannot be serially sampled. In this pilot study, three cytokines (TNF-a, IL-8 both pro-inflammatory and the anti-inflammatory IL-10) were measured in lung tissue, plasma and breath condensate in a porcine single lung transplant model throughout four hours of reperfusion, unmodified by immunosuppression. The goals of the study were: 1. To determine the relative amounts of cytokines measurable in samples taken from the three compartments of interest: lung parenchymal tissue, arterial plasma, and breath condensate. 2. To determine the kinetics of cytokines release throughout the transplant procedure and reperfusion, in each compartment. 3. To compare the amounts of cytokines measured in each compartment of the transplanted lung (allograft) and the native lung.

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2. Methods Left single lung transplantation was performed in five 60-70kg Yucatan swine. Allografts were harvested from donors after perfusion with a low potassium dextran (LPD) preservation solution and maintenance at 4°C for 24 hours. Anesthesia was induced and maintained with intravenous agents. Recipient swine were intubated with a single lumen endotracheal tube and ventilated with 50% oxygen, PEEP of 5cmH2O, to maintain peak airway pressure of less than 30cmH2O. Respiratory rate was adjusted to maintain PaCO2 of 37±5mmHg. Norepinephrine infusion was used as needed to maintain mean blood pressure of 60 mmHg throughout the reperfusion period of four hours. Lung parenchymal tissue was sampled from the allograft after cold ischemia, prior to and hourly after reperfusion, and from the native lung hourly after reperfusion. Plasma was sampled prior to and after thoracotomy, 10 and 30 minutes after reperfusion and hourly thereafter from arterial blood. Breath condensate was collected from exhaled gases of both lungs and each lung separately hourly. Lung separation was achieved by positioning of a bronchial blocker in the contralateral bronchus. Breath condensate was collected by the diversion of exhaled gas through a 20cm length of 1 cm diameter Tygon tubing encased in ice. Analysis for cytokines was performed in tissue, plasma and breath condensate using ELISA kits (Biosource, Camarillo California). For tissue samples, a supranatant was created and protein content was determined by the Bradford method [2]. Results of cytokine analysis in tissue are expressed in picograms per milligram of protein. For all data mean ± standard deviation are presented in graphical form.

3. Results Transplanted lungs appeared progressively hyperemic throughout reperfusion and a moderate amount of pulmonary edema was observed in several but not all recipient animals. Arterial oxygenation was relatively stable after the first hour of reperfusion, as seen in Figure 1. Fluid resuscitation and norepinephrine infusion was required in all animals, reflecting a systemic inflammatory response.

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3.1. TNF-a Tissue TNF-a in the left lung (allograft) was highest at the end of cold ischemic time (CIT), prior to engraftment, and steadily decreased throughout the four hour reperfusion period (Figure 2). Tissue TNF-a in the right (native) lung was in a similar range to the allograft (not shown). Plasma TNF-a exhibited two peaks (Figure 3). The first peak occurred after thoracotomy incision. Plasma TNF-a then significantly increased over the four hour reperfusion period long after tissue levels had reached a plateau, suggesting extrapulmonary production. TNF-a measured in the breath condensate collected from both lungs simultaneously increased after thoracotomy, then remained essentially stable during reperfusion Figure 4). TNF-a measured in breath condensate from the left lung showed a trend to being higher that levels obtained from the right lung (not shown). It must be noted that in breath condensate the range of TNF-a observed was close to the reported sensitivity of the ELISA assay.

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3.2. IL-8 IL-8 increased in both tissue (Figure 5) and plasma (Figure 6) toward a plateau after reperfusion. Unfortunately IL-8 was not detectable in the breath condensate samples. 70 60

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3.3. IL-10

Tissue IL-10 was highest at the end of CIT in the left lung, and steadily decreased (Figure 7). This may represent a washout of the cytokine from tissue. Tissue IL-10 in the native right lung was in a similar range to the allograft and appeared to decrease initially after reperfusion, but the trends are not significant. Thoracotomy alone produced a striking increase in plasma IL-10 measured in arterial blood, a decrease during lung engraftment with no significant alteration during reperfusion (Figure 8). In contrast, IL-10 measured in the exhaled breath condensate collected from both lungs simultaneously increased after thoracotomy, and appeared to peak one hour after reperfusion (Figure 9). Analysis of the condensate collected separately from each lung revealed the same trend in the breath condensate from the left lung (allograft). IL-10 in the breath condensate from the right (native) lung remained in the range of baseline measurements.

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TIME Figure 9. IL-10 in breath condensate from left and right lungs

4. Discussion 1) This pilot study presents the time course of the cytokines TNF-a, IL-8 and IL-10 detected in various sample compartments of a porcine model of single lung transplantation; some but not all cytokines are detected in breath condensate. 2) The time courses of cytokines measured in tissue, plasma and breath condensate vary considerably. Cytokine levels detected in breath condensate were unlike those in plasma or tissue samples in magnitude and trend, Thoracotomy alone produced a significant increase in the TNF-a and IL-10 detected in plasma, the degree to which plasma levels of cytokines influenced measurements in breath condensate is unclear. 3) Animal studies may be limited by a lack of species specific ultrasensitive ELISA kits, particularly for measurement of low concentrations of cytokines in breath condensate. 4) Separate measurement of IL-10 in breath condensate from each lung in a single lung transplant model suggests that the IL-10 from the transplanted lung most influences the measurement for both lungs together. This may reflect increased droplet formation from injured small airways of the allograft, as compared to the native lung. 5) The clinical usefulness of the analysis of breath condensate for inflammatory mediators in lung transplantation requires further study.

Acknowledgement. Many thanks to collaborators Marc De Perrot, Stefan Fischer and Shaf Keshavjee of the Thoracic Surgery Research Laboratory, Toronto General Hospital and the University of Toronto

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References 1. De Perrot M, Sekine Y, Fischer S, Waddell T, McRae K, Wigle D, Keshavjee S. IL-8 release during early reperfusion predicts outcoe in human lung trapslantation. In press Am J Resp Crit Care Med 2002. 2. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248-54, 1976.

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Exhaled Nitric Oxide (NO) in Human Lung Ischeniia-Reperfusion (I/R) Nandor MARCZIN Department of Anaesthetics, Royal Brompton and Harefield Hospital and Department of Cardiothoracic Surgery, Faculty of Medicine, National Heart and Lung Institute, Imperial College, Heart Science Centre, Harefield Hospital, Harefield, United Kingdom, Abstract. This review emphasizes recent progress in the direct evaluation of endogenous NO through bedside monitoring of NO concentrations in the expired air of patients subjected to ischaemia and reperfusion during cardiothoracic surgery. There has been recent progress in our understanding of the determinants of exhaled NO, the anatomical, cellular and molecular origin of NO in the expired air. The scientific community has widely accepted that NO levels in the gas phase reflect in an accurate and qualitative manner the dynamics of NO production and consumption in the airways, especially in the microenvironment of epithelial cells. The contribution of vascular compartments to exhaled NO has been debated and it appears that changes negatively affecting NO metabolism in the microvasculature remain largely undetectable with exhaled NO. We have provided evidence that augmented vascular NO from endogenous metabolism of GTN can be detected in the expired air in humans and have postulated that this phenomenon could be used to assess vascular NO consumption in ALL In the setting of ischaemia and reperfusion related ALI, we have obtained intriguing data, which might have implications to mechanisms, extent and management of ALI associated with cardiothoracic surgery.

1. Introduction: lung ischaemia-reperfusion in cardiothoracic surgery Complete and prolonged lung ischaemia up to several hours is unavoidable during lung transplantation with dire consequences. Transbronchial biopsies performed after lung transplantation showed characteristic histologic features of diffuse alveolar damage in 35 % of patients, even when implantation was performed without CPB [1], This is associated with severe graft dysfunction in about 20% of lung transplant recipients with the clinical manifestation of progressive hypoxemia, decreased pulmonary compliance, high permeability pulmonary edema, and widespread alveolar densities on chest radiographs [24], The early lung allograft dysfunction remains the primary cause of early mortality in lung transplantation. Although severe graft dysfunction can be reversible, it is often associated with the need for prolonged mechanical ventilation, intensive care and hospital stay and compromised recovery among survivals [5]. In addition to this morbidity, ischaemiareperfusion injury may also predispose grafts to acute and chronic rejection via upregulation of class II major histocompatibility complex antigens, release of endothelial cell antigens potentially triggering antiendothelial antibody production and via generation of proinflammatory mediators including cytokines and growth factors [6],

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2. Mechanisms of ischemia-reperfusion injury Role ofmicrovascular endothelial and epithelial cells and neutrophils in I/R Significant progress has been made in understanding the complex cellular and molecular events that mediate and modulate ischaemia-reperfusion lung injury [2;7;8]. Much recent evidence indicates that in addition to arteriolar and postcapillary venular alterations, components of the alveolo-capillary unit are the major targets during acute lung injury with the microvascular endothelium being the most susceptible element. During the injury process, endothelial cells become activated and more permeable with characteristic loss of their normal function as essential regulators of pulmonary vasoreactivity, intravascular coagulation, and inflammatory response and gas exchange. In addition, the composition, function, and metabolism of pulmonary surfactant produced by alveolar type II epithelial cells are increasingly being recognized as important factors in lung injury [2;9]. Although Type I epithelial cells appear to be more resistant to inflammatory response and injury, and their barrier and metabolic function might remain intact even in high permeability pulmonary edema, long-term outcome might depend upon Type I epithelial cell survival. It is now established that human endothelial cells are substantially altered either during hypoxia associated with ischaemia or during reestablishment of blood flow and oxygen (reperfusion and reoxygenation) or in response to inflammatory mediators resulting in an activated phenotype [10]. This includes changes in the profile of vasoregulatory endothelium dependent factors, and the expression of activities that initiate and amplify inflammation and coagulation. Prolonged hypoxia may lead to severe depletion of energy stores causing cellular energetic failure. Immediately after reperfusion, there appears to be a burst of oxidant production within the hypoxic endothelial cells. This together with complement fragment activation on the surface of these cells cause transient expression of preformed proteins and release of mediators that promote leukocyte-endothelial cell interactions, coagulation and cytoskeletal rearrangement which might underlie transient increase in permeability [11]. Alternatively, this oxidative stress can initiate signal transduction events to activate a delayed transcriptional program of several genes resulting in the translation and prolonged surface expression of leukocyte adhesion molecules and cytokines that mediate further recruitment of neutrophils to sites of inflammation [12]. There has been considerable circumstantial evidence both from animal and clinical studies implicating the neutrophil as a potentially important mediator of the early changes in lung endothelial and epithelial permeability following ischaemia reperfusion. The enhanced neutrophil-endothelial interactions might promote microvascular injury by multiple mechanisms. First, activation of neutrophils in the close proximity of endothelial cells might accentuate and prolong oxidative stress resulting in oxidative stress signaling in endothelial cells to further enhance pro-inflammatory phenotype and sustain cytoskeletal reorganization [13]. Neutrophils can produce significant quantities of a number of pro-inflammatory cytokines contributing to cytokine imbalance, potent chemotactic agents that further promote their adherence to the endothelium. Finally, they can release elastase and other proteases, which might contribute to direct pulmonary cell injury [14]. Role of NO in I/R Beyond oxygen centered free radicals and oxidants, nitric oxide appears to play a multifaceted role in ischaemia-reperfusion and to modulate the biological effects of reactive oxygen species. Under normal conditions, there is a considerable release of NO both in the

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microvasculature and airways to elicit a number of bioactivities through either direct signaling or via a guanylate cyclase and cGMP dependent process. In the normal lung these include 1) regulation of pulmonary arteriolar and bronchial tone by relaxing smooth muscle; 2) prevention of platelet aggregation and thrombus formation; 3) modulation of multiple aspects of lung inflammation through attenuation of the adhesive interactions between leukocytes and the endothelial or epithelial cell surface, leukocyte trafficking and reduction of oxidative stress by effectively scavenging the low intracellular levels of superoxide anion (8). NO production and bioactivity is subjected to great alterations during hypoxia, ischaemia and reperfusion. Enzymatic NO production exhibits a characteristic O2 dependence and thus hypoxia reduces enzyme activity to synthesize NO. This phenomenon has been demonstrated in both cells in culture and animal and human lungs [15; 16] . Hypoxia however might increase NO generation from non-enzymatic sources. This involves non-enzymatic reduction of inorganic nitrite to NO, a reaction that takes place predominantly during acidic/reducing conditions [17]. Thus hypoxia and ischaemia might alter NO concentrations and bioactivity by multiple and sometimes opposing mechanisms. Nevertheless, animal studies suggest that under conditions of hypoxia and ischaemia of the lung the predominant effect appears to be reduction in NO concentrations. In an orthotopic rat model of lung transplantation, NO release at the surface of the lung was diminished during hypothermia storage [18]. In addition to changes in NO availability during ischaemia, reperfusion can cause further consumption of NO through interactions with superoxide. In this situation NO undergoes radical-radical reactions with superoxide at near diffusion-limited rates to yield peroxynitrite, a potent oxidizing agent to lipids, aromatic amino acid residues, protein sulfhydryls and DNA [19]. Peroxynitrite has been shown to initiate lipid peroxidation in biological membranes at rates that are a thousand-fold higher than for hydrogen peroxide. However, NO displays a dual action with lipids: in addition to pro-oxidant characteristics through peroxynitrite mediated oxidation reactions it has potent capability to inhibit lipid radical chain propagation [20]. Thus although NO can serve both as antioxidant (by inhibiting lipid free radicals) or an oxidant (by contributing to peroxinitrite formation), both of these reactions will lead to consumption of NO and reduced levels of bioactivity to elicit normal signaling and biological functions in the lung. Following the acute phase of NO-superoxide interactions, the redox milieu is further complicated by transcriptional induction of iNOS and various antioxidant enzymes [21 j. The resulting reactions will again depend on relative quantities of NO and superoxide and the local redox microenvironment. It is conceivable to believe that in case of continuous ongoing superoxide production, increased NO synthesis may contribute to further peroxynitrite formation, however increased NO may attenuate the extent of cellular injury through inhibition of apoptosis or may restore endothelial function if concomitant superoxide generation had subsided. All these considerations predict that I/R will be associated with a complicated picture of NOS expression, NO generation and consumption. Actual NO concentrations will be different accordingly to the dynamically changing cytokine environment, nature of microvascular and airway inflammation, neutrophil activation, concomitant production of reactive oxygen species and acidity in the immediate environment of endothelial and airway epithelial cells. Understanding of these reactions and their consequences in lung microvascular or airway damage or protection may provide a more rational basis for new therapeutic strategies towards better preservation of organ viability and function during and following I/R.

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Given the pivotal importance of all of these fluid phase reactions in acute lung injury, monitoring changes in the production and bioavailability of NO in the lung would be extremely desirable. However due to the short half life of NO and peroxynitrite and the nature of the fluid phase reactions analytical repertoire has been limited to detect stable end products of NO metabolism such as nitrite and nitrate and footprints of peroxynitrite such as nitrotyrosine. The mindful discovery of Gustafsson and colleagues, followed by recent technological developments allowing direct measurements of NO in the expired air, however, have provided an exciting opportunity to evaluate changes in NO production and consumption in the clinical setting [22]. 3. Physiology of exhaled NO Although measurements of exhaled NO provide important direct information regarding NO concentrations in the gas phase, interpolation of these data to in vivo NO metabolism is far from straightforward. The biological reactions of NO are likely related to local NO concentrations in the fluid phase which is influenced by many processes including generation rate, fluid phase reactions such as autooxidation, and consumption by a variety of mechanisms including interaction with hem-iron groups, proteins and scavenging by hemoglobin and interactions with superoxide. Since many of these processes appear to be anatomical site-dependent within the lung and they are likely to be differentially altered by dynamically changing pathological processes, it is of crucial importance to consider the implications of the anatomical origin of NO in exhaled air to molecular pathology of acute lung injury (ALI). As discussed in details elsewhere in the book, the anatomic site and the type of cells responsible for the release of NO into the gas phase remains a matter of debate. On one hand, there is evidence that under certain conditions vascular mechanisms could contribute to exhaled NO. In particular, infusion of endothelium-dependent vasodilators increased exhaled NO in isolated perfused lung models suggesting that a fraction of microvascular NO may diffuse into the alveolar compartment contributing to exhaled NO. However, elegant studies by Sartori et al utilizing an inhaled or infused NO synthase inhibitor suggest that exhaled NO is mostly of airway epithelial rather than of vascular endothelial origin [23]. On the basis of these considerations they have concluded that exhaled NO may not be used as a marker for vascular NO production and/or endothelial function in healthy humans. These observations and conclusions provide a solid basis for current promotion to use exhaled NO as a diagnostic tool to monitor inflammatory responses affecting primarily the conducting airways in asthma [24]. In contrast, the same considerations indicate major limitations of exhaled NO as a marker of ALI, which is primarily characterised by microvascular and alveolar dysfunction. The major implications are that changes in vascular NO metabolism in ALI likely remain undetectable by exhaled NO measurements and detected changes in exhaled NO would probably reflect altered epithelial NO generation and consumption. We have recently suggested a potential solution to this problem by utilizing exhaled NO responses following intravenous administration of nitroglycerin (GTN), which elicits its biological effect by NO release mediated by thiol-dependent enzymatic biotransformation. Shortly after the original observations of Persson et al in animal models regarding increased exhaled NO levels following vascular metabolism of intravenous nitric oxide donors [25], we have established and characterized this phenomenon in humans [26,27]. We concluded that a fraction of nitroglycerin is metabolized in the pulmonary microvasculature to NO,

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which then diffuses into the alveolar space, giving rise to exhaled NO. On the basis of these considerations, we have suggested that GTN-induced exhaled NO might be a useful tool to monitor metabolic function of the pulmonary microvasculature. 4. Exhaled NO following CPB and lung transplantation At Harefield Hospital, we have studied these issues in the setting of clinical ischaemiareperfusion [28]. We have measured endogenous NO in the expired air as a means to assess bronchial epithelial function and we have examined GTN-induced exhaled NO indicating vascular and alveolar metabolic function. We have performed these studies in the setting of complete and prolonged lung ischaemia and reperfusion during lung transplantation and compared them to those occurring with transient and incomplete lung ischaemia during routine open-heart surgery for coronary artery bypass grafting (CABG) utilizing CPB. Breath to breath measurements of NO concentrations in the lower airways were performed using a real-time, computer-controlled and integrated system (Logan Research Ltd. 2000 and 3000 series). Inspired and expired samples for analysis of NO and COi were continuously withdrawn directly from the main lower airways through a thin Teflon sampling tube at a flow rate of 150 ml/minute. Since detected concentration of exhaled gases depends on both the production rate and ventilation parameters, ventilation was standardized for inspired gas (100% 02), tidal volume (5 ml/kg), respiratory rate (10/min) and inspiratory and expiratory ratio (1:2). To eliminate the influence of positive end expiratory pressure on gas phase NO, PEEP was set to zero. Baseline measurements were performed prior to CPB to evaluate endogenous levels of exhaled NO. After the baseline measurements, three increasing boluses of 1, 2 and 3 fig/kg GTN were administered to the patient via the central venous catheter with exhaled NO and haemodynamic response recorded. Between each boluses of GTN a short period of time was allowed for both the haemodynamic and exhaled gas parameters to return to the baseline values. The similar protocol was repeated 1, 3 and 6 hours after CPB. Arterial blood was simultaneously collected for haemoglobin, blood gas, electrolytes and full blood count analysis. In all 12 patients undergoing myocardial revascularisation involving cardiopulmonary bypass, NO was detectable in the exhaled air before CPB as a characteristic oscillating signal which appeared to increase with expiration as judged by the COi. Intravenous bolus administration of 1, 2 and 3 fig/kg GTN resulted in a rapid, transient and dose-dependent increase in exhaled levels of NO. Associated with the transient increase in exhaled NO following administration of GTN boluses, systolic arterial blood pressure transiently and concomitantly decreased. Endogenous exhaled NO levels remained unchanged 1 and 3 hours after CPB in these patients. Although measurements were performed in the intubated patients 6 hours after the operation, at this time point the majority of patients already made some spontaneous breathing efforts. There were characteristic changes in GTN-induced response in exhaled NO after CPB. The dose-dependent increases in exhaled NO by GTN were significantly smaller at 1 hour and 3 hours after CPB when compared to levels measured before CPB. There was no characteristic exhaled NO signal, such as seen with CABG patients in the majority of the lung transplant recipients during the post CPB period. This was either due to reperfusion induced loss of a detectable signal during the ischaemia period in two patients, who exhibited characteristic NO signals before reperfusion of their lungs (measured during CPB after completion of airway anastomoses. In most of the patients, however this was unrelated to reperfusion, since no detectable signals were obtained during ischaemia. A

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comparable NO signal to CABG patients was only seen in two out of the 10 lung transplant recipients during the post reperfusion period. When considered as a group and compared to exhaled NO levels in CABG patients, both peak expired NO and NO output were lower in lung transplant recipients after reperfusion. GTN-induced increases in exhaled NO were generally absent or appeared very small in lung transplant recipients after reperfusion. Furthermore, total NO output over 30 seconds was also profoundly reduced. Interestingly, GTN-induced exhaled NO was attenuated even in those patients whose endogenous exhaled NO was preserved. In addition GTN-induced exhaled NO recovered slowly in the postoperative period (> 24 hours) despite earlier normalisation of endogenous exhaled NO.

Figure 1. Representative traces of GTN-induced exhaled NO in routine heart surgery patient (top panel) and in lung transplant recipient following reperfusion (lower panel). Please note the absence of detectable basal and GTN-induced exhaled NO in the transplant recipient.

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This series of investigations aimed to clarify the influence of open-heart surgery and/or CPB on NO concentration in the expired air. Currently there are contradicting published results showing no change, increase or decrease in NO concentrations by different groups of investigators using a variety of methodological approaches [29-32]. The two principal observations of this part of the study are the unchanged basal concentrations of expired NO during the immediate postoperative period and decreased GTN-induced exhaled NO after surgery. Our interpretation of these data is that basal and GTN-induced exhaled NO represents distinct anatomical compartments and physiological mechanisms contributing to exhaled NO and that these mechanisms are differently affected by CPB and heart surgery. Our conclusion is that in the clinical setting of routine open-heart surgery, CPB-induced inflammatory response and ischaemia-reperfusion injury do not reach sufficient levels to compromise endogenous NO mechanisms to produce exhaled NO, (events that likely reflect airway epithelial processes). Similarly, the pulmonary and systemic haemodynamic response to a challenge with a bolus of GTN is also preserved, yet the characteristic increase in evolution of NO into expired air from GTN (which likely reflects lung microvascular events) is impaired in the early post reperfusion period. This might have clinical implications to heart surgery and CPB induced pulmonary microvascular injury and support our original idea to further evaluate this response as a bedside test of the metabolic function of the lung. Although routine CPB and open heart surgery is associated with a degree of clinically significant pulmonary dysfunction this rarely fulfills the criteria of ALL This might be related to the transient and incomplete nature of ischaemia and reperfusion. In contrast, lung transplantation is frequently associated with perioperative ALI, which might be related to prolonged and complete lung ischaemia. In accordance with the greater potential to ischaemia-reperfusion injury, lung transplantation was associated with a profound loss of GTN metabolism to produce exhaled NO. In addition and in contrast to open-heart surgery we found a variable decrease in endogenous exhaled NO levels. The ability to measure exhaled NO levels during ischaemia, reperfusion and after operation allows the elucidation of distinct mechanisms contributing to loss of exhaled NO. In conclusion, our findings provide additional evidence that during even in clinically successful lung transplantation ischaemia-reperfusion injury may reach sufficient levels to routinely compromise vascular mechanisms at least those responsible for pulmonary metabolism of organic nitrates, transport and release of NO to the air space. In addition, there is evidence of epithelial dysfunction in releasing NO into the gas phase. In light of recent observations suggesting the critical role of epithelial cells in the resolution of acute lung injury [33], exhaled NO might be a useful bedside tool to monitor the onset, extent and resolution of vascular and epithelial injury and the involvement of the NO pathways.

Acknowledgements. This work has been supported by a MRC Clinician Scientist Fellowship to Dr. Nandor Marczin. Magdi Yacoub is a British Heart Foundation Professor of Cardiothoracic Surgery. The contribution of the Julia Polak Transplant Fund is greatly appreciated.

References

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Gammie JS, Cheul LJ, Pham SM, Keenan RJ, Weyant RJ, Hattler BG et al. Cardiopulmonary bypass is associated with early allograft dysfunction but not death after double-lung transplantation. J Thorac Cardiovasc Surg 1998; 115:990-997.

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Novick RJ, Gehman KE, AH IS, Lee J. Lung preservation: the importance of endothelial and alveolar type II cell integrity. Ann Thorac Surg 1996; 62:302-314.

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Khan SU, Salloum J, O'Donovan PB, Mascha EJ, Mehta AC, Matthay MA et al. Acute pulmonary edema after lung transplantation: the pulmonary reimplantation response. Chest 1999; 116:187-194.

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Kundu S, Herman SJ, Winton TL. Reperfusion edema after lung transplantation: radiographic manifestations. Radiology 1998; 206:75-80.

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Christie JD, Bavaria JE, Palevsky HI, Litzky L, Blumenthal NP, Kaiser LR et al. Primary graft failure following lung transplantation. Chest 1998; 114(1):51-60.

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Serrick C, Giaid A, Reis A, Shennib H. Prolonged ischemia is associated with more pronounced rejection in the lung allograft. Ann Thorac Surg 1997; 63:202-208.

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Granger DN. Ischemia-reperfusion: mechanisms of micro vascular dysfunction and the influence of risk factors for cardiovascular disease. Microcirculation 1999; 6:167-178.

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Garden DL, Granger DN. Pathophysiology of ischaemia-reperfusion injury. J Pathol 2000; 190(3):255266.

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Freeman BA, Panus PC, Matalon S, Buckley BJ, Baker RR. Oxidant injury to the alveolar epithelium: biochemical and pharmacologic studies. Res Rep Health Efflnst 1993; 1-30.

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Verrier ED, Morgan EN. Endothelial response to cardiopulmonary bypass surgery. Ann Thorac Surg 1998;66:S17-S19.

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Boyle EM, Jr., Pohlman TH, Comejo CJ, Verrier ED. Endothelial cell injury in cardiovascular surgery: ischemia-reperfusion. Ann Thorac Surg 1996; 62(6): 1868-1875.

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Boyle EM, Jr., Canty TG, Jr., Morgan EN, Yun W, Pohlman TH, Verrier ED. Treating myocardial ischemia-reperfusion injury by targeting endothelial cell transcription. Ann Thorac Surg 1999; 68(5): 1949-1953.

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Bando K, Pillai R, Cameron DE, Brawn JD, Winkelstein JA, Hutchins GM et al. Leukocyte depletion ameliorates free radical-mediated lung injury after cardiopulmonary bypass. J Thorac Cardiovasc Surg 1990; 99(5):873-877.

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Eppinger MJ, Deeb GM, Boiling SF, Ward PA. Mediators of ischemia-reperfusion injury of rat lung. Am J Pathol 1997; 150:1773-1784.

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Dweik RA, Laskowski D, Abu-Soud HM, Kaneko F, Hutte R, Stuehr DJ et al. Nitric oxide synthesis in the lung. Regulation by oxygen through a kinetic mechanism. J Clin Invest 1998; 101(3):660-666.

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Phelan MW, Faller DV. Hypoxia decreases constitutive nitric oxide synthase transcript and protein in cultured endothelial cells. J Cell Physiol 1996; 167(3):469-476.

(17) Zweier JL, Samouilov A, Kuppusamy P. Non-enzymatic nitric oxide synthesis in biological systems. Biochim Biophys Acta 1999; 1411(2-3):250-262. (18) Pinsky DJ, Naka Y, Chowdhury NC, Liao H, Oz MC, Michler RE et al. The nitric oxide/cyclic GMP pathway in organ transplantation: critical role in successful lung preservation. Proc Natl Acad Sci U S A 1994; 91(25): 12086-12090. (19) Freeman BA, White CR, Gutierrez H, Paler-Martinez A, Tarpey MM, Rubbo H et al. Oxygen radicalnitric oxide reactions in vascular diseases. Peroxynitrite-induced membrane lipid peroxidation: the cytotoxic potential of superoxide and nitric oxide. Adv Pharmacol 1995; 34:45-69.

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(20) O'Donnell VB, Freeman BA. Interactions between nitric oxide and lipid oxidation pathways: implications for vascular disease. Circ Res 2001; 88:12-21. (21) Liu M, Tremblay L, Cassivi SD, Bai XH, Mourgeon E, Pierre AF et al. Alterations of nitric oxide synthase expression and activity during rat lung transplantation. Am J Physiol Lung Cell Mol Physiol 2000;278(5):L1071-L1081. (22) Gustafsson LE, Leone AM, Persson MG, Wiklund NP, Moncada S. Endogenous nitric oxide is present in the exhaled air of rabbits, guinea pigs and humans. Biochem Biophys Res Commun 1991; 181(2):852857. (23) Sartori C, Lepori M, Busch T, Duplain H, Hildebrandt W, Bartsch P et al. Exhaled nitric oxide does not provide a marker of vascular endothelial function in healthy humans [see comments]. Am J Respir Crit Care Med 1999; 160:879-882. (24)

Kharitonov SA, Barnes PJ. Nitric oxide in exhaled air is a new marker of airway inflammation. Monaldi Arch Chest Dis 1996; 51(6):533-537.

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Persson MG, Agvald P, Gustafsson LE. Detection of nitric oxide in exhaled air during administration of nitroglycerin in vivo. Br J Pharmacol 1994; 111:825-828.

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Marczin N, Riedel B, Royston D, Yacoub M. Intravenous nitrate vasodilators and exhaled nitric oxide Lancet 1997; 349:1742-1742.

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Marczin N, Riedel B, Royston D, Yacoub M. Exhaled nitric oxide and pulmonary response to iloprost in systemic sclerosis [letter; comment]. Lancet 1998; 352(9125):405-406.

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Marczin N, Riedel B, Gal J, Polak J, Yacoub M. Exhaled nitric oxide during lung transplantation [letter]. Lancet 1997; 350(9092): 1681-1682.

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Beghetti M, Silkoff PE, Caramori M, Holtby HM, Slutsky AS, Adatia I. Decreased exhaled nitric oxide may be a marker of cardiopulmonary bypass-induced injury. Ann Thorac Surg 1998; 66:532-534.

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Hill GE, Snider S, Galbraith TA, Forst S, Robbins RA. Glucocorticoid reduction of bronchial epithelial inflammation during cardiopulmonary bypass. Am J Respir Crit Care Med 1995; 152:1791-1795.

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Brett SJ, Quinlan GJ, Mitchell J, Pepper JR, Evans TW. Production of nitric oxide during surgery involving cardiopulmonary bypass [see comments]. Crit Care Med 1998; 26(2):272-278.

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Ishibe Y, Liu R, Hirosawa J, Kawamura K, Yamasaki K, Saito N. Exhaled nitric oxide level decreases after cardiopulmonary bypass in adult patients. Crit Care Med 2000; 28:3823-3827.

(33) Ware LB, Golden JA, Finkbeiner WE, Matthay MA. Alveolar epithelial fluid transport capacity in reperfusion lung injury after lung transplantation. Am J Respir Crit Care Med 1999; 159(3):980-988.

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In Situ Lung Autotransplantation Model in Pigs E. N. KOLETSISl, A. CHATZIMICHALIS1, K. KOKINIS^, V. FOTOPOULOS 2,1. BELLENIS1, D. DOUGENIS2. 1

Dept. of Thoracic Vascular Surgery, Evagelismos Medical Center, 2 Dept. of CardioThoracic Surgery & 3 Dept of Anesthesiology and Intensive Care Unit University ofPatras. Abstract. Lung transplantation is a well accepted treatment for patients with end stage pulmonary disease [1]. Early graft dysfunction remains one of the major causes of early morbidity and mortality, with reperfusion injury (RI) being the most responsible mechanism [2, 8}. The exact pathophysiology of RI in lung transplantation has not been fully evaluated and understood [2]. Experimental transplantation after cold storage has been so far unable to duplicate the complete clinical picture of RI, such as, hypoxia, severe impairment of endothelial permeability, and frank alveolar oedema. On the basis of our previous experimental works with pigs, this paper describes a single lung transplantation model [3,4,5], which might be useful in assessing ischaemia and reperfusion injury without the potential interference of acute rejection.

1. Introduction. Our aim was to create a steady and reproducible experimental protocol that could demonstrate several parameters associated with the mechanisms of reperfusion injury, including impaired gas exchange, elevated pulmonary vascular resistant, local [6,7] and systemic aspects of the reperfusion syndrome, but without the interference of the pathology concerning acute graft rejection. [2] 2. Methods Female Pigs with a body weight between 25 and 30 kg were used for the experiments. All animals received humane care in compliance with the word wide accepted quidlines for care and use of laboratory animals. [3] The experimental protocol was approved by the Patras University Ethical Committee. Anaesthesia The animals were premedicated with midazolam 5mg and atropine sulfate 0.5 mg both given by intramuscular (IM) injection. A venous line was established by puncturing an auricular vein. Induction of anesthesia was performed with sodium thiopental 250 - 400 mg intravenously. The animals were initially intubated with an 6.5 mm internal diameter

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orotracheal tube followed by a proper tracheostomy to gain better access to the tracheobronchial tree and facilitate bronchoscopy. Volume-controlled mechanical ventilation was instituted (Siemens Servo 900 C respirator). Initially tidal volume was set to 10 mL/kg body weight, respiratory rate to 14 breaths per minute with a FiO2of 0.5. The respirator settings were subsequently adjusted to achieve a Pco2 of 40 to 45 mm Hg and arterial oxygen saturation of more than 90%. Anaesthesia was maintained with 300 mg sodium thiopental and 0.2 mg fentanyl [9] both given as IV bolus injections every 15 to 30 minutes. For muscular relaxation 4 mg pancouronium bromide was added intravenously as appropriate. Perioperative antibiotic medication consisted of cefuroxime IV 750 mg bolus injection. Central and core temperatures were recorded. [3,4,5,9]. Surgical Technique Following dissection of the right femoral artery and vein, a central venous catheter was inserted in the right femoral vein using the Seldinger technique and a Swan-Ganz catheter was placed subsequently. An arterial catheter for invasive blood pressure monitoring was placed in the in right femoral artery in the same fashion. A Foley catheter was introduced by cystostomy. [3]. The animal was placed in the right lateral position. A posterolateral thoracotomy incision was performed in the left fifth intercostal space. The incision was extended anteriorly toward the sternum. Meticulous control of bleeding was used all through the surgical procedure. A thoracoplasty type procedure was used by removing 2 or 3 ribs, so as to achieve a wide opening exposure for careful and safe manipulation of the left lung. With the lung retracted inferiorly, the pleura overlying the left pulmonary artery is opened with careful dissection of the left pulmonary artery. The hemiazygos was liquated and all lymphatic nodes of the hilum, paratracheal and aortopulmonary window space were removed. The left main pulmonary artery and bronchus were isolated in the pulmonary hilum. The lung was elevated superiorly, exposing and dividing the inferior pulmonary ligament. The inferior pulmonary vein was isolated. The pericardium was opened and the origin of the left pulmonary vein was dissected and isolated. A tape was passed around the left main bronchus, which was stripped by all bronchial arteries in order to ablate the bronchial circulation.The pulmonary veins were further dissected at their entrance into the left atrium and the hemiazygos was dissected intrapericardialy. During these steps of the surgical procedure particular care was taken to minimize manipulation of the prone to damage and particular fragile ventilated left lung. A Swan-Ganz catheter was inserted through the inferior vena cava and placed in the right pulmonary artery, the proper position of the Swan-Ganz catheter in to the right pulmonary artery was verified by palpating the left pulmonary artery, and it is notable that the pulmonary catheter had been positioned in the right place all through the experiments. Heparin was given intravenously (300 lU/kg). A purse-string stitch 6-0 Prolene suture (Ethicon, Hamburg, Germany) was placed in the pulmonary artery; taking care not to penetrate into the lumen. An artery cannula 18 G was carefully inserted in the pulmonary artery. The artery was proximally occluded by a tourniquet. A side-biting clamp was placed on the left atrium central to the left pulmonary veins and a minor incision was made for fluid drainage (vent). The left lung was then flushed with cold modified Euro-Collins solution (60 mL/kg). Pulmonary artery pressure during flushing was kept at 15 mm Hg approximately. (Fig 1). Ventilation was continued during flush perfusion. After completion of perfusion, the main left bronchus was crossclamped with bronchial clamp and the lung kept semi-inflated. The lung was left in situ and covered with cold swabs inside an isotherm and waterproof bug. The temperature was measured continually in the left interlobar space and when exceeded 8°C, additional cold normal saline and ice was applied to the towels over the isotherm bag, which isolated the lung from the pleural space. Warm normal saline at 38,5°C was added into the pleural

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space with a control way, when ever the central temperature dropped to less than 36.5°C so that the central temperature was kept between 37 and 38.5°C. Additional measurements were used to maintain animal's normal temperature according to the core temperature. Total ischemic time of the left lung was set at 3 hours. Reperfusion was initiated by removing the tourniquet from the pulmonary artery. Vent from the pulmonary veins was kept until fresh red blood was coming out ensuring that all Euro-Collins lung preservation fluids had been flushed out by the pulmonary circulation. Following that, the incision of the left atrium vent was repaired with a 5-0 Prolene suture (Ethicon, Hamburg, Germany) and subsequently the left atrium and the bronchial clamp were removed and ventilation to the left lung restored.

Fig 1. Surgical field after complete preparation. LIPV: Left Inferior Pulmonary Vein, LSPV Left Superior Pulmonary Vein, LA: Left Atrium as well as LPA Left Pulmonary Artery is isolated. * Vent, ** artery cannula

Cardiopulmonary assessment. Cardiopulmonary function assessment consisted of the following measurements: heart rate, cardiac output by thermodilution, pulmonary artery pressure, wedge capillary pressure, central venous pressure, arterial pressure, continues SVO2 measurement, arterial blood gases, urine output [3,7,11].

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Subsequently the arterio-alveolar oxygen difference (AaDO2 ) and the pulmonary vascular resistance (PVR) were calculated according to the following formulas: Effective AaD(>2 : Fio2 x (Pbar - P H2o ) - Paco2 - Pao2 where Pbar is barometric pressure, P H2o is partial pressure of water vapour, Fio2 is inspiratory oxygen fraction, and Paoi and Paco2 are arterial partial pressures for O2 and CO2 ). PVR: (MPAP - LAP)/CO x 80 where MPAP is pulmonary artery mean pressure, LAP is left atrial pres-sure, and CO is cardiac output. [9] We also assessed static compliance (Cst) using the following formula: Cst =Vt / (PinsPexp) where Vt is tidal volume, Pins is pressure at inspiratory hold and Pexp is pressure at expiratory hold. The evaluation time points were at the start of the experiment, after completion of instrumentation and hilar preparation, 60, 120, and 180 minutes after ischemia and 60, 120. and 180 minutes after reperfusion. 3. Conclusion This experimental model can offer a steady and reproducible environment that could be used to assess the ischaemia and reperfusion injury without the potential interference of acute rejection mechanism of graft failure. In this model, various pharmaceuticals manipulations could be used to evaluate reperfusion injury in lung transplantation with the view to reducing local and systemic complication[l,12]. References: 1. 2. 3. 4.

5. 6. 7. 8. 9. 10. 11. 12.

Meyers B, Patterson A. Lung transplantation: Current status and future prospects. World J. Surg. 23,1156-1162,1999 Palazzo R, Hamvas A, Shuman T, Kaiser L, Cooper J, Schuster DP. Injury in nonischemic lung after unilateral pulmonary ischemia with reperfusion. J Appl Physiol 1992; 72:612-20 Dougenis D, Tzorakoelefterakis E., KS Filos, et al. Experimental single lung transplantation in pigs. Intern J Artif Organ 1992; 15(9): 533 Koletsis E, Melachrinou M, Filos K, Dougenis D, Androulakis J. Experimental Lung Transplantation. Initial results. 3rd Pan-Hellenic Scientific Medical Students Congress; Thessaloniki March 1993 Dougenis D, Tzorakoelefterakis E, Filos K, Goudas L, Melachrinou M, Moschos S, Kyriakopoulou T, Poulopoulou M, Koletsis E, Androulakis J. Single lung transplantation: An experimental study in pigs. Acta Chirurgica Hellenica 1993; 65(1): 50-6 Novick RJ, Gehman KE, Ali IS, Lee J. Lung preservation: the importance of endothelial and alveolar type II cell integrity. Ann Thorac Surg 1996; 62:302-14. Novick RJ, Menkis AH, McKenzie FN. New trends in lung preservation: a collective review. J Heart Lung Transplant 1992; 11:377 King CR, Binns O, Rodriguez F et al. Reperfusion injury significantly impacts clinical outcome after pulmonary transplantation. Ann. Thorac Surg 2000; 69:1681-5 Swindell MM, Anesthetic and perioperative techniques in swine. Wilmington, MA: Charles River Laboratories, CRL Tech. Bull, (winter) 1991 Merin RG, Verdouw PD, Jong JW. Myocardial functional and metabolic responses to ischemia in swine during halothane and fentanyl anesthesia. Anesthesiology 56:84-92,1982 Filos K, Dougenis D, et al. The prognostic value of continuous SvO2 monitoring by fiberoptic oxymetry in single lung transplantation. A pilot study. Intens Care Med 1992; 18 (suppl): 117. Cooper JV, Vreim CE. Biology of lung preservation for transplantation. Am Rev Resp Dis 1992; 146:803

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Disease Markers in Exhaled Breath N. Marczin and M.H. Yacoub {Eds.) IOS Press, 2002

Pathogenetic Mechanisms Leading to Obliterative Bronchiolitis Erkki A. KALLIO*, Jussi M. TIKKANEN*, Petri K. KOSKINEN*, Karl B. LEMSTR6M*, and Magdi YACOUBf ^Transplantation Laboratory, University of Helsinki and Helsinki University Central Hospital. Hartmaninkatu 3, FIN-00014 University of Helsinki and f Department ofCardiothoracic Surgery, Imperial College, Faculty of Medicine, Heart Science Centre, Harefield, United Kingdom

Abstract Obliterative bronchiolitis causes significant mortality and morbidity among lung transplant recipients. Obliterative bronchiolitis presents as gradual deterioration of graft function in the absence of any other cause such as infection. The clinical diagnosis of Obliterative bronchiolitis syndrome is based on decline in forced expiratory volume in 1 second. Obliterative bronchiolitis syndrome manifests as obstructive ventilatory defect, leading to shortness of breath, wheezing, and gradually to hypoxia, hypercapnia, and ultimately, to death. Pathologically, Obliterative bronchiolitis is characterized by dense eosinophilic hyaline fibrous plaques in the submucosa of small airways resulting in partial or complete luminal compromise of membranous and respiratory bronchioles. This bronchiolar scar tissue may be concentric or eccentric, and may extend through smooth muscle wall to peribronchial interstitium. Currently, Obliterative bronchiolitis is considered as a form of chronic rejection of lung allografts. Although clinical and experimental studies have provided significant insight into risk factors and pathophysiology of Obliterative bronchiolitis, the underlying pathogenetic mechanisms are largely unknown. In this communication, we review our experience of the pathogenesis of experimental Obliterative bronchiolitis in rat heterotopic tracheal allografts.

1. Rat heterotopic tracheal transplantation as a model for Obliterative bronchiolitis Rat heterotopic tracheal allografts develop similar histological changes as seen in human obliterative bronchiolitis (OB) [1]. These changes are not observed in syngeneic grafts, indicating that they are not due to ischemia or interruption of blood vessels and lymphatics. Tracheal epithelium strongly expresses major histocompatibility complex (MHC) class II enabling direct antigen presentation, also observed in human lung allografts developing OB [2, 3]. Submucosal and peritracheal inflammation resembles that seen in lymphocytic bronchitis/bronchiolitis (LBB) and acute cellular rejection [4], with submucosal inflammatory cell infiltrates, focal epithelial necrosis and neutrophil infiltration. There is progressive loss of normal respiratory epithelium, first replaced by cuboidal and squamous epithelium, progressing to complete loss of epithelium. Similar sequence of events has been observed also by others using heterotopic tracheal transplantation model [5, 6]. The inflammatory cell subsets in tracheal allografts are similar to those in human lung allografts

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consisting first of CD4+ T cells and later, in increasing numbers, of CD8+ T cells and macrophages, which have been suggested to be required for the development of OB [7]. Epithelial injury and airway inflammation lead to ingrowth of granulation tissue consisting of mononuclear inflammatory cells and intense proliferation of a-smooth muscle actin positive myofibroblast-like cells causing gradual occlusion of airway lumen. Airway inflammation and epithelial injury have been suggested to initiate the development of OB in human lung allografts [8, 9] and prevention of epithelial injury has been shown to ameliorate OB in experimental models [10]. Heteropic tracheal transplantation may be considered as a model for human OB which has its benefits, but also limitations. Tracheal transplantation is technically simple and reproducible. In addition, no baseline immunosuppression is required, making it possible to investigate different drugs without drug interactions. On the other hand, the anatomy of trachea and bronchioli differ considerably, and pathological changes of OB seen in bronchioli are not observed in large airways in human. There is no airflow, which may affect epithelial function and pathology. The blood supply comes from systemic circulation, in contrast to lung allografts which receive their blood mainly from pulmonary circulation. Further, tracheal allograft is not a vascularized organ, but is revascularized as capillary network infiltrates the graft, making it difficult to investigate the very early events associated with ischemia and reperfusion, and possibly affecting the infiltration of inflammatory cells. The immunogenecity of tracheal allografts may be lower than in orthotopic lung transplants as tracheal allografts contain less lymphoid tissue. On the other hand, tracheal allografts develop obliterative changes in accelerated fashion. 2. Alloimmune response causes graft injury and the development of OB Cyclosporine A (CsA) inhibits the development of experimental OB in dose-dependent fashion, and inhibition of OB correlates with CsA trough levels [11, 12]. Similar results have been obtained in other experimental studies using mouse and rat tracheal allografts [13, 14]. Also other immunosuppressive drugs which block the proximal events leading to acute rejection, i.e., IL-2R activation and cytokine transcription, such as tacrolimus, leflunomide, and rapamycin, have been effective in inhibition of experimental OB [13-15], demonstrating that inhibition of alloimmune induced graft injury prevents the development of experimental OB. However, the level of immunosuppresssion needed to prevent experimental OB has been relatively high, and drugs affecting later steps of alloimmune response, such as mycophenolate mofetil and 15-deoxyspergualin, have been relatively ineffective in preventing OB. Role of the early events of alloimmune activation in the development of OB, is further emphasized by the effect of blockade of T cell costimulation. Blockade of CD28/B7-1 and CD28/B7-2 T cell costimulatory pathways with CTLA4Ig fusion protein reduces intragraft expression of tumor necrosis factor (TNF)-ct, interleukin (IL)-2, and interferon (IFN)-y as well as epithelial injury and graft occlusion [16]. Interestingly, selective blockade of CD28/B7-1 pathway does not affect graft cytokine profiles or the development of experimental OB [16]. Clinical studies demonstrate that rejection, especially multiple acute rejection episodes, late acute rejection episodes, and LBB are most important risk factors for OB and bronchiolitis obliterans syndrome (BOS) [17-19]. In addition, low level of immunosuppression has been associated with the development of BOS [19]. Improved immunosuppression with new immunosuppressive drugs has delayed the onset of BOS and

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augmentation of immunosuppression stops or delays the progression of BOS [20, 21]. Taken together, both clinical and experimental studies demonstrate the important role of alloimmune induced graft injury in the pathogenesis of OB, and that sufficient immunosuppression may partially prevent or delay the development of OB.

3. Cytomegalovirus infection enhances the development of OB Cytomegalovirus (CMV) infection has been identified as a risk factor for OB in man [18, 22, 23], In our studies, both chronic (latent) and acute recipient rat Cytomegalovirus (RCMV) infection significantly enhanced the development of experimental OB. Enhancing effect of RCMV infection has been demonstrated also in aortic, heart, and kidney allograft models of chronic rejection [24-26] and OB [27]. CMV may enhance OB by several mechanisms. CMV pneumonitis may contribute to epithelial injury and thereby enhance the development of OB. However, CMV seropositivity, without clinical infection, has also been associated with enhanced OB [22]. It is likely that CMV infection modulates the alloimmune response to facilitate chronic rejection and OB. In vitro studies have demonstrated that CMV infection directly induces MHC class I expression [28, 29]. CMV encodes a protein with sequence homology and cross-reactivity with MHC class II. Expression of MHC class II may also be indirectly induced by CMV activated CD4+ T cells [30, 31]. CMV infection induces endothelial and epithelial ICAM-1 expression in vitro and in vivo [32, 33] and causes endothelialitis in allograft vascular wall [34]. In addition, CMV infection may upregulate IL-2 and IL-2 receptor gene expression thus inhibiting the effect of CsA [35, 36]. CMV may also induce TNF-ct [37] as well as IFN-y [38] expression by monocytes and macrophages. These events may enhance acute rejection, and thus facilitate OB development. On the other hand, cytokines, especially TNF-a, produced during acute rejection and released during antibody treatments, may activate latent CMV infection [39], promoting alloreactivity that leads to a vicious circle. In addition, CMV may directly induce mesenchymal cell proliferation by inhibiting cell growth suppressor genes, leading to manifestations of chronic rejection by inducing transcription of different growth factors and inactivating growth suppressors [4042]. In rat tracheal allografts, both acute and latent rat CMV infection enhances the development of experimental OB. RCMV infection induces epithelial MHC class II expression, the number of graft infiltrating CD4+ T cells, and macrophages, as well as alloimmune activation of graft infiltrating inflammatory cells. RCMV infection upregulates epithelial platelet-derived growth factor (PDGF)-AA and alfa-receptor expression [43]. Increased inflammation leads to epithelial injury, inducesing the reparative prosesses culminating to myofibroproliferation and enhanced graft occlusion. In RCMV-infected grafts, treated prophylactically with ganciclovir or anti-CMV hyperimmune serum, the degree of obliteration is similar to that of non-infected controls [44]. On the other hand, anti-RCMV treatment, initiated a few days after infection, is not effective in inhibiting the RCMV-enhanced experimental OB. Interestingly, increased immunosuppression is similarly effective in inhibiting RCMV-enhanced OB in rat tracheal allografts. Our observations indicate, that in order to prevent the enhancing effects of CMV infection on the development of OB, CMV infection should be treated prophylactically, or at least preemptively. In addition, CMV infection is associated with induction of various proinflammatory cytokines, which may be prevented by increasing the level of immunosuppression during CMV infection [43, 44].

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The effect of CMV on the development of OB may be summarized as bidirectional and biphasic. Bidirectional because CMV infection augments alloimmune responses to the graft leading to OB, and, on the other hand, alloimmune activation may activate latent CMV infection. Biphasic because CMV infection affects alloimmune responses in both acute and chronic rejection [45]. 4. Protective role of nitric oxide in the pathogenesis of OB Nitric oxide (NO) may have different roles in various stages of transplantation in regard of pathophysiological effects and therapeutic implications. During reperfusion NO functions as a scavenger of free radicals [46], and NO supplementation improves graft preservation [47, 48], In addition, inhaled NO has been effective in treatment of early graft dysfunction after lung transplantation [49]. We investigated the role of NO in the pathogenesis of OB, by treating tracheal allograft recipients with either a inducible nitric oxide synthase (iNOS) inhibitor, aminoguanidine, or by supplementing NO production with L-arginine, the substrate for iNOS [50]. We observed, that epithelial iNOS expression and NO production were decreased during the development of OB. Inhibition of NO production enhanced graft inflammation and occlusion, whereas suplementation of NO induced a switch towards Th2 type immune response and reduced graft obliteration [50]. During acute rejection, macrophage- and T cell-derived cytokines, such as TNF-a, IL-1, IL-2, and IFN-y may induce iNOS expression in macrophages producing of large quantities of NO. Macrophage-derived NO and superoxide anion (O2-) may form peroxynitrite anion (ONOO-), a free radical, which may cause tissue damage [46]. Myocyte death by apoptosis in acute cardiac allograft rejection has been associated with induction of iNOS expression and NO production [51 ] and inhibition of NO improves rat cardiac allograft survival by downregulation of apoptosis [52]. Inhibition of iNOS with aminoguanidine ameliorates acute lung allograft rejection in the rat [53]. Functional studies using iNOS knockout mice heart transplantation model show that reduced NO production inhibits inflammation and cell injury during acute rejection [54] suggesting a damaging role for NO in acute rejection. In chronic rejection, iNOS is upregulated in rat [55] and human [56] cardiac allografts, and rat aortic allografts [57]. In human lung allografts, induction of iNOS and peroxynitrite are associated with epithelial damage and the development of OB [58]. Similarly in our study, iNOS expression was induced, but NO production was reduced by the loss of continuously expressed epithelial iNOS as epithelium was damaged during the development of experimental OB. Our results demonstrate, that inhibition of iNOS accelerates and supplementation of iNOS inhibits the development of OB [50]. In rat aortic allografts, inhibition of NO production accelerated allograft arteriosclerosis [59], whereas, transduction of iNOS expression with adenoviral vector suppressed the development of allograft arteriosclerosis [59]. In iNOS knockout mice, lack of iNOS enhanced the development of chronic cardiac allograft rejection [54]. In addition, supplementation of iNOS pathway with L-arginine prevented allograft arteriosclerosis in rabbit heart allografts [60]. NO may modulate immune responses and graft homeostasis in several ways that may downregulate OB. NO inhibits leukocyte adhesion [61] and platelet aggregation [62]. NO prevents proliferation Thl cells, but not Th2 cells [63]. We observed a switch from Thl towards Th2 type alloimmune response with inhibition of IL-2 expression and induction of IL-10 expression in rat tracheal allografts [50]. Th2 type responses have been associated

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with enhanced graft survival and inhibition of experimental OB [64, 65]. L-arginine treatment inhibited myofibroproliferation, which may be mediated by NO directly by increasing intracellular cyclic-GMP levels [66, 67], and indirectly by inhibiting growth factor release [68, 69]. Further, NO induces apoptosis of smooth muscle cells, which may reduce proliferative responses in chronic rejection [70], In conclusion, NO may have different roles in acute and chronic rejection. While NO behaves as a cytotoxic effector molecule contributing to graft injury during acute rejection, it seems to be protective in chronic rejection and OB.

5. Inhibition of complement activation prevents epithelial injury and graft occlusion Our studies demonstrate that complement activation takes place during the development of experimental OB, and that inhibition of complement activation with human recombinant soluble complement receptor type 1 (sCRl) reduces epithelial injury and myofibroproliferation, leading to ameloriation of OB. In the transplant, complement activation may occur via both classical and alternative pathways [71]. Complement mediates much of the tissue damage occuring in ischemia-reperfusion injury [72, 73]. In addition to early complement induced graft injury leading to enhancement of reparative processes and myofibroproliferation, complement components may facilitate chronic rejection and OB by several other mechanisms. Complement may enhance antigen presentation and T cell proliferation promoting cell-mediated rejection [74, 75]. Complement activation components increase leukocyte chemotaxis by upregulating expression of endothelial adhesion molecules [76, 77] and production of chemoattractants monocyte chemoattractant protein, macrophage inflammatory protein-la and IL-8 by monocytes and macrophages [78, 79]. In addition, complement may induce release of IL-1 and TNF-a from monocytes and macrophages [80, 81], and induce release of bFGF and PDGF from endothelial cells [82]. All these factors have been implemented to contribute to alloimmune induced graft injury ultimately leading to OB. Complement receptor type 1 (CR1) is an endogenous regulatory protein of complement, which inhibits C3 and C5 convertases, thus inhibiting activation of both classical and alternative pathways [83]. Recombinant human soluble CR1 (sCRl) has the same capacity to inhibit complement activation as CR1 [84]. sCRl effectively protects against complement mediated tissue damage in ischemia/reperfusion injury as well as in immune complex, thermally and cobra venom factor induced injury models [84, 85]. In addition, sCRl effectively inhibits hyperacute rejection in xenograft models [86] and in allografts [87], as well as vascular injury and inflammation in acute rat renal allograft rejection [88]. Although complement is an important mediator of acute lung injury [73], little data exists on its role in acute lung allograft rejection or OB. In our study, sCRl-treatment reduced epithelial injury, inflammatory cell proliferation, and graft occlusion, that are the hallmarks of OB. sCRl inhibited ICAM-1 and IL-8 expression and neutrophil infiltration, previously associated with the development of OB [89]. In addition, sCRl upregulated IL10 expression, which was recently shown to prevent experimental OB [65]. 6. Regulatory role of platelet-derived growth factor in the pathogenesis of OB In our study, platelet-derived growth factor (PDGF)-AA and platelet-derived growth factor receptor-a (PDGF-Ra) were identified as key regulatory molecules in the

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pathogenesis of experimental OB. Upregulation of PDGF-AA, PDGF-Ra, and PDGF-Rp protein expression was demonstrated during the early phases of the development of OB, while PDGF-BB expression was downregulated in allografts compared to syngeneic grafts. Inhibition of signaling downstream of PDGF receptors with CGP 53716, protein-tyrosine kinase inhibitor selective for PDGF receptors [90], markedly inhibited the development of experimental OB. In human lung allografts with OB, increased concentrations of PDGF in BAL fluid, upregulation of PDGF-AB protein expression in epithelial cells and mononuclear cells, and PDGF-B mRNA expression in macrophages have been observed [91]. Locally applied PDGF induces OB-like changes in heterotopic tracheal syngeneic grafts [92]. In experimental models of lung fibrosis and inflammation, overexpression of PDGF-B gene increases fibroproliferation and collagen deposition [93] and exogenous PDGF-BB induces mesenchymal cell and epithelial cell proliferation [94]. In vitro, PDGF-BB and -AB are more potent chemoattractants and mitogens for non-stimulated lung fibroblasts than PDGFAA [95, 96]. However, fibroblasts stimulated with cytokines such as IL-1|3 and TNF-a, express more PDGF-AA and PDGF-Ra [97, 98]. Expression of PDGF-Ra, to which PDGF-AA binds, appears to be required for maximal chemotaxis and proliferation of lung fibroblasts [99]. In this study, upregulation of PDGF-AA and -Ra occured concomitantly with the peak of inflammation and myofibroproliferation. A specific role for cytokine activated expression of long chain PDGF-AA, which is required for full activation of PDGF-Ra protein-tyrosine kinase, has been suggested in the pathogenesis of chronic heart allograft rejection in human [100]. These findings suggest that although PDGF-BB may have a significant role in normal physiology and in the pathogenesis of several fibroproliferative disorders, PDGF-AA and PDGF-Ra may be more important in mediating myofibroproliferation in chronic rejection and OB. Inhibition of signal transmission of PDGF-R by CGP 53716 effectively attenuated neointimal formation in carotid denudation model of atherosclerosis [101] and chronic rejection in rat heart allografts [102]. In our study, the compound inhibited myofibroproliferation and airway occlusion but had no effect on airway wall inflammation indicating that it is not immunosuppressive, but rather that its effects are mediated by inhibition of myofibroproliferation. However, the inhibition of OB by CGP 53716 was not complete, indicating that also other growth factors in addition to PDGF, operate in the disease process. 7. "Response-to-injury" hypothesis of the pathogenesis of obliterative bronchiolitis Accumulating body of evidence derived from both clinical and experimental studies support the the hypothesis that epithelial injury and airway wall inflammatory responses activate reparative processes leading to OB. Graft injury is initiated already at donor brain death, and enhanced by ischemia and reperfusion, where neutrophils, free radicals, and complement cause the tissue damage. Acute rejection, mediated by CTLs, T helper cells and macrophages, augmented by antibody induced responses and NK cells, further damages epithelium. CMV infection may contribute to the development of OB by inducing epithelial injury and promoting acute rejection. As acute rejection subsides, alloimmune activation may be maintained by activated T cells and macrophages. Macrophages release cytokines and growth factors which induce myofibroproliferation. Graft injury and alloimmune activation induce growth factor release in several cell types in the graft. Growth factors released by epithelial cells, endothelial cells, smooth muscle cells, and fibroblasts may act

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in autocrine and paracrine fashion and induce phenotypic changes in fibroblasts favoring proliferation. Growth factors, especially PDGF, enhance migration and proliferation of myofibroblasts leading to gradual occlusion of the airway lumen. Our studies suggests several sites for intervention in order to prevent the development of OB, starting with reducing early graft injury caused by reperfusion and acute rejection, further with diminishing the constant alloimmune activation and inflammation by effective immunosuppression and CMV prophylaxis, and finally by inhibiting mesenchymal cell proliferation.

References [1] S. A. Yousem, J. B. Berry, P. T. Cagle, et al. 1996. Revision of the 1990 working formulation for the classification of pulmonary allograft rejection: lung rejection study group. J. Heart Lung Transplant. 15:1-15. [2] P. M. Taylor, M. L. Rose, and M. H. Yacoub. 1989. Expression of MHC antigens in normal human lungs and transplanted lungs with obliterative bronchiolitis. Transplantation. 48:506-510. [3] S. A. Yousem, L. Ray, I. L. Paradis, et al. 1990. Potentential role of dendritic cells in bronchiolitis obliterans in human heart-lung transplantation. Ann. Thorac. Surg. 49:424-428. [4] S. A. Yousem. 1993. Lymphocytic bronchitis/bronchiolitis in lung allograft recipients. Am. J. Surg Pathol. 17:491-496. [5] M. I. Hertz, J. Jessurun, M. B. King, et al. 1993. Reproduction of the obliterative bronchiolitis after heterotopic transplantation of mouse airways. Am. J. Pathol. 142:1945-1951. [6] A. Boehler, D. Chamberlain, S. Kesten, et al. 1997. Lymphocytic airway infiltration as a precursor to fibrous obliteration in a rat model of bronchiolitis obliterans. Transplantation. 64:311 -317. [7] K. E. Kelly, M. I. Hertz, and D. L. Mueller. 1998. T-cell and major histocompatibility complex requirements for obliterative airway disease in heterotopically transplanted murine tracheas. Transplantation. 66:764-77'1. [8] I. Paradis, S. Yousem, and B. Griffith. 1993. Airway obstruction and bronchiolitis obliterans after lung transplantation. Clin. Chest Med. 14:751-763. [9] H. Levrey, and M. I. Hertz. 1998. Chronic lung allograft dysfunction. Transplant. Rev. 12:183-202. [10] H. D. Tazelaar, J. Prop, P. Nieuwenhuis, et al. 1988. Airway pathology in the transplanted rat lung. Transplantation. 45:864-869. [11] E. A. Kallio. 2000. Pathogenesis of obliterative bronchiolitis in rat heterotopic tracheal allogfafts. An experimental approach to chronic lung allograft rejection. In Transplantation Laboratory. University of Helsinki, Helsinki. [12] P. K. Koskinen, E. A. Kallio, R. Krebs, et al. 1997. A dose-dependent inhibitory effect of cyclosporine A on obliterative bronchiolitis of rat tracheal allografts. Am. J. Respir. Crit. Care Med 155:303-312. [13] R. E. Morris, X. Huang, C. R. Gregory, et al. 1995. Studies in experimental models of chronic rejection: use of rapamycin (sirolimus) and isoxazole derivates (Leflunomide and its analogue) for suppression of graft vascular disease and obliterative bronchiolitis. Transplant. Proc. 27 (3):2068-2069. [14] N. A. Yonan, P. Bishop, A. El-Gamel, et al. 1998. Tracheal allograft tranplantation in rats: the role of immunosuppressive agents in development of obliterative airway disease. Tanplant. Proc. 30:2207-2209. [15] J. A. Fahmi, G. J. Berry, R. E. Morris, et al. 1997. Rapamycin inhibits development of obliterative airway disease in a murine heterotopic airway transplant model. Transplantation. 63:533-537. [16] J. M. Tikkanen, K. B. LemstrOm, and P. K. Koskinen. 2001. Blockade of CD28/B7-2 costimulation inhibits experimental obliterative bronchiolitis in rat tracheal allografts: A shift towards Th2-dominated immune response. Am. J. Resp. Crit. Care Med. in press.

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[17] S. A. Yousem, J. A, Dauber, R. Keenan, et al. 1991. Does histological acute rejection in lung allografts predict the development of bronchiolitis obliterans? Transplantation. 52:306-309. [18] T. J. Kroshus, V. R. Kshettery, K. Savik, et al. 1997. Risk factors for the development of bronchiolitis obliterans syndrome after lung transplantation. J. Thorac. Cardiovasc. Surg. 114:195-202. [19] A. N. Husain, M. T. Siddiqui, E. W. Holmes, et al. 1999. Analysis of risk factors for the development of bronchiolitis obliterans syndrome. Am. J. Respir. Crit, Care Med. 159:829-833. [20] A. R. Glanville, J. C. Baldwin, C. M. Burke, et al. 1987. Obliterative bronchiolitis after heart-lung transplantation: apparent arrest by augmented immunosuppression. Ann. Intern. Med. 107:300-304. [21] R. Speich, A. Boehler, R. Thurnheer, et al. 1997. Salvage therapy with mycophenolate mofetil for lung transplant bronchiolitis obliterans. Importance of dosage. Transplantation. 64:533-535. [22] R. J. Keenan, M. E. Lega, J. S. Dummer, et al. 1991. Cytomegalovirus serologic status and postoperative infection correlated with risk of developing chronic rejection after pulmonary transplantation. Transplantation. 51:433-438. [23] D. Heng, L. D. Sharpies, K. McNeil, et al. 1998. Bronchiolitis obliterans syndrome: Incidence, natural history, prognosis, and risk factors. J. Heart Lung Transplant. 17:1255-1263. [24] K. B. Lemstrom, J. H. Bruning, C. A. Bruggeman, et al. 1993. Cytomegalovirus infection enhances smooth muscle cell proliferation and intimal thichening of rat aortic allografts. J. Clin. Invest. 92:549-558. [25] K. Lemstrom, P. Koskinen, L. Krogerus, et al. 1995. Cytomegalovirus antigen expression, endothelial cell proliferation, and intimal thickening in rat cardiac allografts after Cytomegalovirus infection. Circulation. 92:2594-2604. [26] S. Yilmaz, P. K. Koskinen, E. A. Kallio, et al. 1996. Cytomegalovirus infection-enhanced chronic kidney allograft rejection is linked with vascular endothelial and tubular epithelial intercellular adhesion molecule-1 expression. Kidney Int. 50:526-537. [27] H, Reichenspurner, V. Soni, M. Nitschke, et al. 1998. Enhancement of Obliterative airway disease in rat tracheal allografts infected with recombinant rat Cytomegalovirus. J. Heart Lung Transplant. \ 7:439-451. [28] W. T. van Dorp, E. Jonges, C. A. Bruggeman, et al. 1989. Direct induction of MHC class I, but not class II, expression on endothelial cells by Cytomegalovirus infection. Transplantation. 48:469-472. [29] L. Ibrahim, M. Dominguez, and M. Yacoub. 1993. Primary adult lung epithelial cells in vitro: response to interferon-y and Cytomegalovirus. Immunology. 79:119-124. [30] R. S. Fujinami, J. A. Nelson, L. Walker, et al. 1988. Sequence homology and immunologic crossreactivity of human cytomegalovrus with HLA-DR (3 chain: a means for graft rejection and immunosuppression../ Virol. 62:100-105. [31] W. J. Waldman, D. A. Knight, P. W. Adams, et al. 1993. In vitro induction of endothelial HLA class II antigen expression by cytomegalovirus-activated CD4-t- T cells. Transplantation. 56:1504-1512. [32] W. T. van Dorp, P. A. M. van Wieringen, E. Marselis-Jonges, et al. 1993. Cytomegalovirus directly enhaces MHC class I and intercellular adhesion molecule-1 expression on cultured proximal epithelial cells. Transplantation. 55:1367-1371. [33] G. Steinhoff, X.-M. You, C. Steinmtiller, et al. 1995. Induction of endothelial adhesion molecules by rat Cytomegalovirus in allogeneic lung transplantation in the rat. Scand. J. Infect. Dis. 899:58-60. [34] P. Koskinen, K. Lemstrom, C. Bruggeman, et al. 1994. Acute Cytomegalovirus infection induces a subendothelial inflammation (endothelialitis) in the allograft vascular wall. A possible linkage with enhanced allograft arteriosclerosis. Am. J. Pathol. 144:41-50. [35] L. J. Geist, M. M. Monick, M. F. Stinski, et al. 1991. The immediate early genes of human Cytomegalovirus upregulate expression of the interleukin-2 and interleukin-2 receptor genes. Am. J. Respir. CellMol. Biol. 5:292-296. [36] L. J. Geist, M. M. Monick, M. F. Stinski, et al. 1992. Cytomegalovirus immediate early genes prevent the inhibitory effect of cyclosporin A on interleukin 2 gene transcription. J. Clin. Invest. 90:2136-2140. [37] P. D. Smith, S, S. Saini, M. Raffeld, et al. 1992. Cytomegalovirus induction of tumor necrosis factor-a by human monocytes and mucosal macrophages. J. Clin. Invest. 90:1642-1648.

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Part V. ARDS and Oxidative Stress

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Disease Markers in Exhaled Breath N. Marczin and M.H. Yacoub (Eds.) IOS Press, 2002

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Clinical Pathology of ARDS Stylianos E. ORFANOS, Antonia KOUTSOUKOU, Irene MAVROMMATI, Ekaterini PSEVDI, loanna KOROVESI, Anastasia KOTANIDOU, and Chans ROUSSOS Department of Critical Care and Pulmonary Medicine, University of Athens Medical School, Evangelismos Hospital, Athens, Greece Abstract. Acute lung injury (ALI) is an acute, diffuse and severe alteration of lung structure and function that occurs after exposure to noxious endogenous or exogenous agents, ALI represents a pathologic continuum characterized by impairment of arterial oxygenation and diffuse chest x-ray abnormalities. The most severe extreme of this continuum is the acute respiratory distress syndrome (ARDS) an overt non-cardiogenic pulmonary edema that carries high morbidity and mortality. ALI/ARDS is associated with a variety of etiologies and, although its pathogenesis is still partly understood, pulmonary endothelium appears to play a major role in the syndrome development. Numerous biological markers of ARDS have been investigated in an effort to identify pathogenic and prognostic significance. Among them, pulmonary endothelium-bound angiotensin converting enzyme activity, assessed at the bedside by means of indicatordilution techniques: i. offers a direct and quantifiable index of pulmonary endothelial dysfunction, ii. is altered early in ALI, and iii. correlates with the severity of lung injury. ARDS treatment remains mainly supportive, with positive-pressure mechanical ventilation always playing a major role in the treating process. Studies on lung mechanics have offered new insights into the clinical pathology of the syndrome, allowing the clinician to apply appropriate ventilatory management in an effort to increase survival.

1, Definitions Acute lung injury (ALI) is a pathologic continuum characterized by acute respiratory distress, severe impairment of oxygenation, and noncardiogenic pulmonary edema. ALI varies in severity and acute respiratory distress syndrome (ARDS) is a term applied to patients with more severe manifestations of ALI. Both terms are used to reflect a relatively specific form of lung injury to the lung occurring from a wide variety of causes or associated conditions. This acute injury involves the alveolar epithelium, the alveolar capillary endothelium and the pulmonary interstitium, and it is caused by an acute inflammatory response, usually with a tremendous influx of neutrophils, leading to a breakdown of the lung barrier and gas exchange functions. Initially this results in flooding of the alveolar spaces with protein-rich edema fluid, leading to severe gas exchange and lung compliance abnormalities. If the process is sustained, fibroproliferation occurs with collagen deposition and lung remodeling. ALI and ARDS are defined in terms of the associated clinical, physiologic, and radiologic manifestations. The current definitions are those introduced by the AmericanEuropean Consensus Conference (AECC) on ARDS published in 1994 [1]. This international group limited the criteria for ALI to: i. oxygenation abnormality with an

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arterial partial pressure of oxygen to inspired oxygen fraction ratio (PaO2/FiO2) < 300 mm Hg, ii. bilateral opacities on chest radiograph, and iii. pulmonary arterial occlusion pressure (PAOP) < 18 mm Hg, or no clinical evidence of left heart failure. An additional criterion is the acute onset of the syndrome. ARDS is the most severe part of ALI, with PaO2/FiO2 being < 200 mm Hg. 2. Epidemiology The incidence of ALI/ARDS is not clear. A National Institutes of Health (NIH) panel in 1972 estimated the incidence of ARDS to be approximately 75cases/100000 population/year [2]. This number has been widely used since that time, without confirmation from epidemiological studies. Data on the incidence of either ARDS or ALI using AECC criteria have only recently been published. One such study screened patients for an 8-week period in most intensive care units in Sweden, Denmark and Iceland, and identified incidences of 17.9 cases/100000 population/year for ALI and 13.Scases/100000 population/year for ARDS [3]. In contrast, a 3-year study in one center in Finland, using the AECC definition for ARDS plus the requirement of a known triggering cause, found an incidence of 4.9 cases ofARDS per 100000 population per year [4]. Clinical risk factors can be categorized mechanistically as causing either direct (primary) or indirect (secondary) injury. The latter refers to an extrapulmonary injury that affects the lungs through activation of systemic inflammatory pathways, mainly cytokines and other biochemical and cellular mediators [1,5]. Direct risk factors include pneumonia, aspiration of gastric contents, pulmonary contusion, smoke inhalation, and near drowning. Indirect risk factors include sepsis, trauma, pancreatitis, multiple transfusions, and shock. Factors shown to increase ARDS risk, following predisposing conditions include: age, severity of illness, cigarette smoking, chronic alcohol abuse, and combination of risk factors [6]. Attempts to predict which patients at risk will develop ARDS are mostly based on biochemical measurements in blood or bronchoalveolar lavage (BAL) fluid. Obtaining BAL fluid in all patients at risk for ARDS, in order to identify those with early or mild lung injury, is not a practical approach and is limited essentially to research studies. Measurement of blood biochemical markers, including cytokines reputedly associated with lung injury, has not been very successful. The prevalence of any given risk condition varies considerably by geography, and by the clinical population seen at a given institution. In general however sepsis is the commonest, with aspiration of gastric contents being relatively common, and trauma less common but still important. Diffuse pneumonia also appears to be a relatively common risk condition, regardless of being classified as pneumonia or as sepsis originating from the lungs [6]. Sepsis as a risk for ARDS is generally associated with a considerably higher mortality rate than most other common risks, including trauma and aspiration of gastric contents [5]. Older patients (> 65 years of age), have an increased mortality rate when compared to younger patients [7]. ARDS severity at the time of first diagnosis, given by the degree of hypoxemia, has not been constantly associated with poor outcome, except perhaps at extreme abnormalities [8]. Montgomery et al [9] found that patients dying with ARDS appeared to die primarily of multiple organ failure and sepsis, rather than respiratory causes such as hypoxia or uncontrollable respiratory acidosis. Recent studies have confirmed the above observations [4,10]. Through most of the 1980s, ARDS-related mortality was in the range of 60% or higher. Since that time, mortality appears to be decreased. Abel et al [11] found that mortality in the years 1993-96 was decreased as compared to 1990-93 (30 to 60%. respectively). More recent studies have reported ARDS mortalities of 37% and 41.2% [12]. The exact reasons for this improvement in survival remain unclear, but they may relate to

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improved treatment of the underlying cause(s) and improved supportive care of patients with ALI/ARDS; the latter includes changes in ventilatory management. 3. Pathogenesis ofARDS The pathophysiologic hallmark of ALI/ARDS is a progressive injury of the alveolar-capillary membrane that leads to increased vascular permeability and pulmonary edema. Although the pathogenesis of ALI/ARDS is still partly understood, extensive basic and clinical research performed during the last 25 years revealed the existence of an inflammatory syndrome, where numerous mediators and cell types participate in multiple cascades that interact with each other, either promoting or suppressing inflammation. The way this "inflammation-balance" will finally move, will determine patients' outcome. A detailed analysis of all the known pathogenic mechanisms is not within the scope of this chapter. Briefly, pro-inflammatory cytokines are released following pulmonary and/or extra-pulmonary stimuli, such as endotoxin [13]. They amplify the inflammatory process by releasing additional mediators from macrophages and other cell-types, and by activating the complement system, neutrophils and other blood components that aggregate in the pulmonary circulation. Activated neutrophils adhere to the pulmonary endothelium and release proteases and oxidative products that injure the vascular wall and increase permeability. The release of several vasoactive substances, lipids and peptides is either induced or altered, while the endothelial vascular layer becomes thrombogenic. Ongoing endothelial injury appears to be a key component of the pathogenic process. Studying ALI/ARDS pathogenesis has an additional important role: identify potential biological markers that could predict either ARDS development in high-risk patients, or outcome from the already established syndrome. Such markers would allow better triage and better patient care.

4. Inflammatory mediators Among the earliest events of the inflammatory process is the release of tumor necrosis factor-alpha (TNF-a), interleukin-1 (IL-1), as well as interleukins-6 and -8 (IL-6, IL-8), following pulmonary or extra-pulmonary insults. All these mediators are involved in inflammation, their production and release are stimulated by multiple relevant mediators, including endotoxin [14], and they are regulated by nuclear factor kappa B (NFicB) [15]. They have all been shown to be implicated in the development of ALI in animal models, and they are all present in patients at risk for and with established ARDS. TNF-a was the first cytokine to be extensively studied in patients at risk for and with ARDS. TNF-a is increased in BAL and pulmonary edema fluid from patients with ARDS, but it is not specific for ARDS and does not correlate with morbidity and mortality [16]. The likely source of TNF-a in the lung is the alveolar macrophage, although there could be leak from the circulation as well. IL-1, IL-6, and IL-8 have been measured in both the circulation and lung fluid from patients at risk for and with ARDS. IL-1 levels are increased in the BAL fluid obtained from patients with ARDS [17]. Alveolar macrophages from these patients release higher IL-1 levels, both at the baseline and following lipopolysaccharide (LPS) stimulation, than alveolar macrophages obtained from either normal subjects or patients with other lung diseases [18]. There is evidence that IL-1 likely accounts for the majority of lung proinflammatory cytokine activity in ARDS [19]. Both TNF-a and IL-1 stimulate the production of IL-8, which is a potent neutrophil chemoattractant. In light of its chemoattractant activity, it is likely that IL-8 contributes more to the acute inflammatory

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process in the lung than in the circulation. IL-8 levels are increased in both BAL and pulmonary edema fluid in ARDS [19] and correlate with neutrophil concentrations within the lung. This supports further the hypothesis that IL-8 contributes to ALI, at least in part, through its neutrophil chemoattractant capacity. IL-8 levels are higher in the lungs of patients at risk who ultimately develop ARDS [20], while higher IL-8 levels may be associated with higher mortality [21]. IL-6 has both pro-inflammatory and anti-inflammatory properties [22]. Plasma IL-6 appears to be the best predictor of ARDS morbidity and mortality, from all the proinflammatory cytokines measured to date. Several studies have found that IL-6 levels are significantly increased in patients at risk for ARDS, while they are higher and persist longer in patients who die [23]. However, there is no specific IL-6 level that predicts which patient will die. Consequently, measuring IL-6 is more likely to be useful in identifying groups of patients at high risk for mortality. Recent investigations have focused on the role of a more proximal common regulatory factor, namely NFicB. NFicB is the regulatory transcription factor for TNF-a, IL1, IL-6, and IL-8, as well as for many other protein compounds that could be involved in ARDS development [15]. In humans, NFicB activation is significantly increased in alveolar macrophages obtained from patients with ARDS, as compared to macrophages of patients without lung injury [24]. Further studies should determine the role of NF-icB in ALI/ARDS development, and its utility as a target for novel therapeutic options.

5. Neutrophils The focus on the role of neutrophils in ALI arose from early observations that in ARDS, neutrophils accumulate in the airspaces and the lung interstitium [25]. In addition, BAL fluid from these patients is characterized by neutrophilia [26]. Moreover, several studies suggest that circulating neutrophils are activated in ARDS [27], while after ARDS has been developed the severity of lung injury, estimated by gas exchange abnormalities, correlates with the extent of neutrophil influx into the airspaces [28]. In some patients, the persistence of the initial neutrophilic inflammatory response is associated with higher mortality [29]. Neutrophils injure the lung by at least two potential mechanisms: the release of proteases (elastase, collagenase, gelatinase), and the production of reactive oxygen species (hydrogen peroxide, hydroxyl radicals and superoxide anions) [30]. Neutrophil adherence to endothelium is thought to be an early event that leads to subsequent infiltration of lung parenchyma. Several cell surface adhesion molecules are involved in neutrophil adhesion and migration into the extravascular spaces [31]. Such molecules include the selectin family, which mediates the rolling of neutrophils on endothelial cells, and the beta 2 integrins (GDI 1/CD18) that mediate the firm adherence to the endothelium via interaction with endothelial ligands such as intercellular adhesion molecule-1 (ICAM-1) [31]. The mechanisms for regulating neutrophil sequestration and adherence to the lung are somewhat different from those reported in the systemic circulation, and include physical factors such as neutrophil deformability, as well as several specific adhesion molecules [32]. 6. Pulmonary Endothelium Pulmonary endothelium possesses numerous physiologic and pharmacokinetic functions. These functions may be altered early in ALI and further contribute to ARDS development, mainly through the promotion of cell-cell adhesion and endothelial

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permeability, and through endothelial metabolic alterations [33]. The pulmonary endothelium is a major metabolic organ that synthesizes, releases and degrades vasoactive peptides, and possesses antithrombogenic-thrombolytic activities [34]. Ectoenzymes responsible for many of these functions are located on the luminal endothelial cell surface, with their catalytic sites exposed to the blood stream; they are directly accessible to bloodborne substrates, and their activities may be measured in vivo by means of indicatordilution techniques [35]. One such ectoenzyme is angiotensin converting enzyme (ACE) [36]. Several investigations on endothelium-related mediators of ALI/ARDS have provided insights into the pathogenesis of the syndrome, and have examined potential predictors of either ARDS development or outcome [33]. In this respect, Morel et al, [37] have shown that the pulmonary endothelium-mediated extraction of serotonin in humans with ARDS correlates with the severity of the syndrome, while pulmonary propranolol extraction is decreased in patients at risk. More recently, the net balance between pulmonary clearance and release of endothelin-1 was found to be decreased early in ALL while it reversed in patients who recovered [38]. Other endothelium-related markers altered in patients with ALI/ARDS include plasma levels of von Willebrand factor antigen [39], soluble endothelial-derived adhesion molecules, such as E- and P-selectins and ICAM-1 [40,41], as well as plasma soluble ACE (sACE) activity [42]. Endothelial markers in plasma, however, are surrogate indices of endothelial function and may not be directly linked to pulmonary endothelial dysfunction. In addition, they have not been useful to predict ARDS development in multiple at-risk patients [33]. Consequently, the need for methods that will directly assess pulmonary endothelial function in humans is still present. Pulmonary capillary endothelium-bound (PCEB) ACE activity Estimating pulmonary endothelial ectoenzyme activity in vivo or in situ, by means of indicator-dilution techniques, offers direct assessment and quantification of endothelial function [34,35]. Applying indicator-dilution techniques we and others have studied pulmonary endothelium-bound ACE activity in different animal models, by measuring, under first order reaction conditions, the single pass transpulmonary hydrolysis of synthetic substrates highly specific for ACE [35,36]. The most widely used synthetic ACE substrate is benzoyl-Phe-Ala-Pro (BPAP), radiolabeled with 3H [36]. This technique allows estimations of very rapid interactions between substrate and the endothelium-bound enzyme, thus minimizing the contribution of sACE. Furthermore, ACE located on alveolar capillaries with a diameter < 20 um appears to be responsible for the great majority of the product formed, due to the very high local enzyme concentration. Consequently in this type of studies, monitoring pulmonary endothelial ACE activity is in practical terms equal to monitoring PCEB ACE activity [36]. Substrate hydrolysis is expressed as either percent metabolism (%M) or v (=enzyme concentration x capillary transit time x k^/K^), where kcat is the catalytic rate constant and K,n is the Michaelis-Menten constant [43]. The modified kinetic parameter A^/K™ (=enzyme mass x kcat/K,,,) may be additionally calculated [44]. Under normal conditions Amax/Km is an index of dynamically perfused capillary surface area (DPCSA) in animals [34,35,45] and humans [46,47], while under pathological conditions Amax/Km is an index of functional capillary surface area (PCS A) related to both enzyme mass (available for reaction) and functional integrity [46]. Both v and %M are reflections of ACE activity per capillary, while A^/K™ reflects ACE activity per vascular bed. An additional advantage of this method is that it may help distinguish between abnormalities secondary to endothelial dysfunction per se and decreased pulmonary vascular surface area [46,48].

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Figure 1. Top: PCEB-ACE activity expressed as BPAP hydrolysis (v) and A „„/!(,,, in 33 mechanicallyventilated patients belonging to high risk groups for ARDS development, divided in those who have no acute lung injury (NoALI) and those who have ALI/ARDS according to the American-European Consensus Criteria [1]; "p<0.01, with Student's t-test. Middle: BPAP hydrolysis (v) and A.^,/!^ in the above-mentioned patients divided according to Murray et al [49] as having: no lung injury (LIS=0), mild-to-moderate lung injury (LIS=0.1-2.5) and severe lung injury (LIS>2.5); *p<0.05, "p<0.01 from L1S=0 with ANOVA and NewmanKeuls' test. Bottom: BPAP hydrolysis (v) and A^,/!^ in 11 from the above-mentioned patients who underwent two additional PCEB-ACE activity estimations 48 hours apart from each other (+2 & +4 days); 'p<0.05 from initial LIS with ANOVA for repeated measures and Newman-Keuls' test, "p<0.01 between L1S and BPAP hydrolysis with ANCOVA for repeated measures. In all panels values are means ± SEM. Modified from [50] and [51] with permission.

PCEB ACE activity in the critically-ill We have recently assessed PCEB ACE activity in 33 critically-ill, mechanically ventilated patients belonging in high risk-groups for ARDS development and suffering from various degrees of ALI/ARDS, with lung injury scores (LIS according to Murray et al [49]; a scoring system that quantifies lung injury) ranging from 0 to 3.7 (i.e. from no lung injury to severe lung injury) [50]. Both BPAP transpulmonary hydrolysis and A^/K,,,

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decreased early during the ALI/ARDS continuum (Figure 1; top and middle) and were inversely related to LIS, implying that the clinical severity of the syndrome is related to the degree of PCEB ACE activity depression, and consequently to the underlying pulmonary endothelial dysfunction. This was further supported by the sustained negative correlation of v with LIS over time in 11 subjects who underwent sequential PCEB ACE estimations (Fig 1; bottom); no sustained correlation occurred between A^/K^ and LIS, probably due to variations of FCSA during the course of the disease [51]. Interestingly, in this population high A^/K™ values were associated with better survival, raising the intriguing possibility that this kinetic parameter might be of value as an outcome predictor in ARDS [50]. Pulmonary endothelial injury may begin as a subtle metabolic dysfunction, while PCEB ACE activity reduction appears to be among the earliest features of experimental lung injury, preceding changes in all commonly measured parameters [36]. These facts let us hypothesize that PCEB ACE dysfunction might be present in critically-ill subjects who maintain normal chest radiographs and arterial oxygenation during their disease course (i.e. they have no ALI [1]), but suffer from conditions predisposing to endothelial injury. Sequential PCEB ACE activity determinations performed over 6 days in six mechanicallyventilated trauma patients with no ALI revealed a negative relation of both substrate hydrolysis and FCSA with time [52]. These preliminary data imply that sub-clinical pulmonary endothelial dysfunction: i. was already present in this population, ii. deteriorated with time, and iii. should be attributed to noxious mediators related either to disease per se (i.e. trauma), or to ventilator-associated lung injury, or a combination of the two. In a similar respect, twelve brain-dead subjects with no ALI had reduced PCEB ACE activity when compared to seven controls with similar mechanical-ventilation duration and settings, denoting subtle pulmonary endothelial injury probably related to cytokines produced in brain-death [53]. Monitoring PCEB ACE activity in humans by means of indicator dilution techniques offers a safe, relatively simple, and highly reproducible quantification of pulmonary endothelial function [50]. Future studies should determine the utility of this method in a variety of conditions related to the critically-ill, such as predicting either the onset or the outcome from ARDS, or improving the selection of potential donor lungs among brain-dead subjects.

7. Pulmonary Epithelium The alveolar epithelium plays a key role in ARDS development; it may be more resistant than the lung endothelium to injury, and may thus function as a barrier preventing edema fluid from flooding into the airspace [54]. In addition, 30 to 40% of ALI patients appear to have a significant fraction of the alveolar epithelial barrier sufficiently intact, so as to efficiently remove excess alveolar fluid. This is associated with a more rapid recovery and perhaps a lower mortality rate [55]. Finally, there is experimental evidence that the lung epithelium removes excess fluid from the airspaces at a supranormal rate in animal models of septic and hemorrhagic shock [56,57]. The lack of specific markers of lung epithelial injury has made it difficult to study it in ALI/ARDS patients. Several reports have shown alterations of the endogenous surfactant system in ALI [58,59]. Surfactant abnormalities were already detected in the earliest reports ofARDS [60]. Recently, the concentration of surfactant-associated proteins SP-A and SP-B was reported to decrease in BAL samples obtained from patients with ALI [61]. BAL concentrations of SP-A decreased in trauma patients with ALI; SP-A decreased early post-trauma, normalized simultaneously with recovery, but remained low in patients with persistent severe injury [62]. This is consistent with the hypothesis that SP-A and

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other surfactant-associated proteins in BAL may be markers of alveolar epithelial type II cell dysfunction, in patients with acute lung injury.

8. Pulmonary pathology The pathologic features of the lung in ARDS derive from severe injury to the alveolar-capillary unit. Extravasation of intravascular fluid dominates the onset of the disease. As the process unfolds however, edema is overshadowed by cellular necrosis, epithelial hyperplasia, inflammation, and fibrosis. The morphologic picture of the lung in ARDS has been labeled diffuse alveolar damage (DAD) [63]. The pathologic appearance of this damage is temporal and can be divided into three interrelated and overlapping phases, that correlate with the clinical evolution of the disease: (1) the exudative phase of edema, hemorrhage, and hyaline membrane formation (< 6 days), (2) the proliferative phase of organization and repair, when interstitial fibrosis starts ( 4 - 1 0 days), and (3) the fibrotic phase of end-stage prominent fibrosis (from 8 days onward) [64,65]. The pathologic features correlate more with the time frame than with the initiating cause.

9. Lung mechanics Changes in lung mechanics are of paramount importance in the pathophysiology of ARDS. Although ARDS is characterized by extensive bilateral lung involvement, alveolar damage can also affect the lung in a localized fashion. The American-European Consensus Conference on ARDS, Part 2 [66] cancelled recently any mention of "diffuse" from the definition of ARDS, recognizing the heterogeneous characteristics of the syndrome. These characteristics combine flooded areas with abnormal permeability of the alveolar-capillary barrier, and normally aerated alveoli with the ability to exchange gas through the alveolarcapillary membrane. A third "group" of airspaces can be "recruited" or "derecruited'" secondary to ventilation, by modifying body position and the level of inflating pressure and volume, and by applying positive end-expiratory pressure (PEEP). In fact, studies using computerized axial tomography, revealed a non-homogeneous process of parenchymal consolidation, mainly distributed into the most dependent lung zones [67,68]. The structural alterations of ARDS serve as a basis for understanding its major clinical and functional manifestations. Hypoxia is a hallmark of ARDS and a major therapeutic goal is to improve arterial oxygen content. The mechanism underling hypoxia appears to be true intrapulmonary shunt due to non-ventilated perfused alveoli [69], with little contribution of ventilation-perfusion mismatching. Increasing inspiratory oxygen concentration is not enough to correct hypoxia (i.e. refractory hypoxia). Three principal reasons account for alveolar exclusion from ventilation: i. the filling of airspaces with exudates, pseudomembranes, cellular debris and inflammatory cells, related to the structural damage of the alveolar-capillary unit, ii. the collapse of small airways, as a consequence of reduced lung volume and loss of lining surfactant [70], and iii. atelectasis due to compression of dependent lung units, by either increased weight of the overlying edematous lung parenchyma [71] or high inspired O2 concentrations [72]. The wide range of obliterative pulmonary vascular lesions contribute to the creation of pulmonary hypertension, and accounts for an increased physiologic dead space due to the ventilation of poorly perfused lung regions. Hypoxia acts on the vascular bed, causing vasoconstriction and increasing vascular resistance.

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Respiratory compliance and resistance in ARDS A marked reduction in the compliance of the respiratory system was already reported in the original ARDS description [60]. This reduction is mainly due to low lung compliance, which may be explained by a decrease in ventilated lung volume. The latter appears to be the result of a "competition" between extravascular lung water and air, for the occupation of alveolar spaces. In this respect, Gattinoni et al [73] showed that specific compliance (i.e. compliance corrected for the absolute volume of the aerated lung) is within the normal limits. Therefore, low compliance in ARDS appears secondary to "baby lung" rather than to "stiff lung". Initially, chest wall compliance (Cw) in ARDS was considered to be basically normal. However, when respiratory compliance components were individually examined, it became apparent that Cw might also be decreased. Although factors affecting Cw have not been fully identified, a few theories have been proposed, including the abnormal distension and accumulation of fluid in the thoracic and abdominal tissues and cavities. According to Gattinoni et al, Cw might be normal in patients suffering from ARDS of pulmonary origin, but abnormal in patients with ARDS associated with extrapulmonary diseases [74]. Computed values for airway and tissue resistance are elevated in ARDS [75]. Although the pathophysiology underlying this phenomenon has not been fully elucidated, three major mechanisms may account for this elevation: i. the presence of fluid in the airways, ii. the observed reduction of lung volume, and iii. the increased bronchomotor tone, secondary to neurogenic reflexes and inflammatory mediators.

10. Clinical implications of lung mechanics Positive pressure mechanical ventilation has remained a mainstay for the support of the ARDS patient, allowing time for both the treatment of the underlying cause and lung healing. More recently, it has been recognized that mechanical ventilation may by itself induce lung injury clinically indistinguishable from ARDS [76]. Ventilator associated lung injury (VALI) occurs in 5% to 38% of all mechanically ventilated patients and carries a mortality of 13% to 35%. Furthermore, it is possible that VALI and/or ventilator induced lung injury (VILI) may exacerbate and perpetuate the systemic inflammatory response that underlies ARDS, thus promoting multiple system organ failure [77]. Consequently, the particular mode of ventilation is probably less important than the "lung protection" goals and principles, which should guide ventilatory management. VALI prevention strategies should be targeted at reducing over-distension shearing injury while, at the same time, ensuring sufficient oxygenation (i.e. arterial hemoglobin saturation > 90%). On this basis, pressure limited lung protective strategies of mechanical ventilation have been proposed [66], emphasizing the need to "open the lung and keep it open" [78]. Refractory hypoxemia can be attenuated by supplementing inspired O2 and/or by raising mean alveolar pressure and PEEP. Each of these interventions, however, carries risks and benefits. Selecting optimal plateau pressure and PEEP values has been commonly based on the assessment of the static pressure-volume (V-P) curve of the respiratory system. Usually, in patients with ARDS, the inflation limb of the aforementioned V-P curve has a sigmoidal shape, with lower (LIP) and upper (UIP) inflection points [79]. It has been proposed that LIP and UIP pressures (PLIP & PUIP) identify a mechanical-ventilation safe range. In this respect, animal and human studies have shown that ventilation within this pressure range may reduce VALI and improve outcome [80]. Thus, determining LIP and UIP in mechanically ventilated ARDS patients should be a major point of the lung

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protective strategy. However two, at least, reservations apply: i. The aforementioned assumption considers PLn> to be the pressure value above which "massive" alveolar recruitment occurs, while this might not be always the case [81,82], and ii. PLn> is usually estimated from the static V-P curve of the total respiratory system and is thus affected by the mechanical properties of the chest wall, the compliance of which is reduced in extrapulmonary ARDS [74]. In addition, in an effort to avoid large inflation pressures (> Pun.) and volumes, and subsequently high lung volume injury, current recommendations include the use of the smallest tidal volume compatible with adequate PaO2 (without necessarily attempting to normalize PaCO2 - i.e. permissive hypercapnia). However, this might promote alveolar derecruitment and atelectasis. Consequently, the need for new techniques that will identify optimal ventilatory management is always present. Flow limitation in ARDS and clinical significance Utilizing the negative expiratory pressure technique (NEP; [83]), we have recently shown that at zero end-expiratory pressure (ZEEP) many ARDS patients exhibit expiratory flow limitation (FL), with concurrent intrinsic PEEP (PEEPi) up to 12 cm H2O [82,84]. The presence of FL implies cyclic dynamic compression and re-expansion of the peripheral airways, with concurrent inhomogeneous filling of airspaces. In non-homogeneous ARDS lungs, this should entail development of high shear forces currying the risk of low lung volume injury [85,86,87]. To avoid this condition, external PEEP has to be applied in order

-100 VOLUME

[

0.5 L

Figure 2. Flow-volume loops of control and NEP test breaths of an ARDS patient on ZEEP (left) and on PEEP of 10 cm H2O (right; thin lines = control breams, heavy lines = NEP). On ZEEP, NEP application did not induce an increase in expiratory flow over 37% of tidal volume (VT), indicating presence of flow-limitation over this tidal volume range. With PEEP no FL occurred, since flow with NEP exceeded the reference flow throughout expiration From [83] with permission.

to increase the end-expiratory lung volume above the "FL volume". Such therapeutic levels of PEEP can be readily determined by on-line inspection of the NEP effect on expiratory flow-volume loops: PEEP should be increased until tidal FL disappears. Figure 2 presents the expiratory flow-volume loops of an ARDS patient during ventilation on ZEEP (including the NEP test breath), and on 10 cm H2O of PEEP. On ZEEP the patient was flow-limited (Figure 2, left), while on PEEP flow limitation disappeared (Figure 2, right).

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We feel that FL estimation in ARDS, by means of the NEP technique: i. provides a first objective assessment of the cyclic compression and re-expansion of the peripheral airways that has been advocated as a potential hazard for low lung volume injury, and ii. assists the clinician in applying adequate PEEP, thus improving ventilatory management. References [I]

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S.E. Orfanos, D. Langleben, J. Khoury, R.D. Schlesinger, L. Dragatakis, Ch. Roussos, J.W. Ryan, J.D. Catravas Pulmonary capillary endothelium-bound angiotensin converting enzyme activity in humans Circulation 99 (1999) 1593-1599 S.E. Orfanos, D. Langleben, A. Artnaganidis, J. Khoury, P. Sarafidou, C. Glynos, R.D. Schlesinger, L. Dragatakis, J.D. Catravas, Ch. Roussos, Patterns of an angiotensin converting enzyme (ACE) substrate hydrolysis by pulmonary capillary endothelium-bound ACE in critically ill patients In: J.D. Catravas, A.D. Callow, U.S. Ryan (eds) Vascular Endothelium Pharmacologic and Genetic Manipulations, Plenum Press, New York, 1998, pp. 269-271 S.E. Orfanos, A. Kotanidou, Ch. Roussos, Pulmonary endothelial angiotensin converting enzyme activity in lung injury In: J.L. Vincent (ed) 2002 Yearbook of Intensive Care and Emergency Medicine , Springer, Berlin, 2002, In Press J.F. Murray, M.A. Matthay, J.M. Luce, M.R. Flick, An expanded definition of the adult respiratory distress syndrome. Am Rev Resp Dis 138 (1988) 720-723 S.E. Orfanos, A. Armaganidis, C. Glynos, E. Psevdi, P. Kaltsas, P. Sarafidou, J.D. Catravas, U.G, Dafni, D. Langleben, Ch. Roussos, Pulmonary capillary endothelium-bound angiotensin converting enzyme activity in acute lung injury Circulation 102 (2000) 2011-2018 S.E. Orfanos, A. Armaganidis, C. Glynos, E. Psevdi, P. Kaltsas, P. Sarafidou, J.D. Catravas, U.G. Dafni, D. Langleben, Ch. Roussos, Pulmonary capillary endothelium-bound angiotensin converting enzyme activity in acute lung injury and the acute respiratory distress syndrome In: J.D. Catravas, A.D. Callow, U.S. Ryan, M. Simionescou (eds) Vascular Endothelium Source and Target of Inflammatory Mediator, IOS Press, Amsterdam, 2001, pp. 322-331 P. Kaltsas, S.E. Orfanos, U. Dafni, E. Psevdi, C. Sotiropoulou, Ch. Roussos, A. Armaganidis, Early pulmonary capillary endothelial dysfunction in mechanically-ventilated patients in the absence of acute lung injury. Int Care Meet 26 suppl.3 (2000) S353 I. Mavrommati, E. Psevdi, I. Korovesi, P. Kaltsas, C. Glynos, Ch. Roussos, S.E. Orfanos, Pulmonary endothelial dysfunction is present in brain-dead subjects in the absence of acute lung injury. Int Care Med21 suppl.2 (2001) SI57 M.A. Matthay, H.G. Folkesson, A. Campagna, F. Kheradmand, Alveolar epithelial barrier and acute lung injury, New Horizons 1 (1993) 613-622. M.A. Matthay, J.P. Wiener-Kronish, Intact epithelial barrier function is crucial for the resolution of alveolar edema in humans, Am Rev Respir Dis 142 (1990) 1250-1257. J.F. Pittet, J.P. Wiener-Kronish, M.C. McElroy, H.G. Folkesson, M.A. Matthay, Stimulation of alveolar epithelial clearance by endogenous release of catecholamines in septic shock, J Clin Invest 70(1994)663-671. J.F. Pittet, T.J. Brenner, K. Modelsra, M.A. Matthay, Alveolar liquid clearance is increased by endogenous catecholamines in hemorrhagic shock in rats, JAppl Physiol 81 (1996) 830-837. M. Hallman, R. Spragg, G.H. Harrell, K.M. Moser, L. Gluck, Evidence of lung surfactant abnormality in respiratory failure, J Clin Invest 70 (1982) 673-683. J.F. Lewis, A. Jobe, Surfactant and the adult respiratory distress syndrome, Am Rev Respir Dis 147 (1993)218-233. D.G. Ashbaugh, D.B. Bigelow, T.L. Petty, B.E. Levine, Acute respiratory distress syndrome, Lance! 2(1967)319-323. T.J. Gregory, W.J. Longmore, M.A. Moxley, J.A. Whitsett, C.R. Reed, A.A. Fowler, L.H. Hudson, R.J. Maunder, C. Crim, T.M. Hyers, Surfactant chemical composition and biophysical activity in acute respiratory distress syndrome, J Clin Invest 88 (1991) 1976-1981. K.E. Greene, J.R. Wright, W.B. Wong, K.P. Steinberg, J.T. Ruzinski, L.D. Hudson, T.R. Martin, Serial SP-A levels in BAL and serum of patients with ARDS (abstract), Am J Respir Crit Care Med 153(1996) A587. A.A. Katzenstein, C.M. Bloor, A.A. Liebow, Diffuse alveolar damage: the role of oxygen, shock and related factors, AmJPathol 85 (1976) 210-222. G. Nash, J.B. Blennerhassett, H. Pontoppidan, Pulmonary lesions associated with oxygen therapy and artificial ventilation, NEnglJMed276 (1967) 368-374. P.C. Pratt, R.T. Vollmer, J.D. Shelbume, Pulmonary morphology in a multihospital collaborative extracorporeal membrane oxygenation project I: Light microscopy, AmJPathol 95 (1979) 191-208. A. Artigas, G.R. Bernard, J. Carlet, D. Dreyfuss, L. Gattinoni, L. Hudson, M. Lamy, J.J. Marini, M.A. Matthay, M.R. Pinsky, R. Spragg, P.M. Suter, Consensus Committee, The AmericanEuropean Consensus Conference on ARDS, Part 2, Int Care Med 24 (1998) 378-398 R.J. Maunder, W.P. Shuman, J.W. McHugh, S.I. Marglin, J. Butler, Preservation of normal lung regions in the adult respiratory distress syndrome. Analysis by computed tomography, JAMA 255 (1986)2463-2465.

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L. Gattinoni, D. Mascheroni, A. Torresin, Morphological response to positive end-expiratory pressure in acute respiratory failure. Computerized tomography study, Intensive Care Med 12 (1986) 137-142. D.R. Dantzker, C.J. Brook, P. Dehart, J.P. Lynch, J.G. Weg, Ventilation-perfiision distribution in the adult respiratory distress syndrome, Am Rev Respir Dis 120 (1979) 1039-1052. H. Pontoppidan, B. Geffm, E. Lowenstein, Acute respiratory failure in the adult. 1, N Engl J Med 287(1972)690-698. P. Pelosi, L. Dandrea, G. Vitale, A. Pesenti, L. Gattinoni, Vertical gradient of regional lung inflation in adult respiratory distress syndrome, Am J Respir Crit Care Med 149 (1994) 8-13. G. Hedenstierna, The recording of FRC- Is it of importance and can it be made simple? Intensive Care Med 19 (1993) 365-366. L. Gattinoni, L. Dandrea, P. Pelosi, G. Vitale, A. Pesenti, R. Fumagalli, Regional effects and mechanism of positive end-expiratory pressure in early adult respiratory distress syndrome, JAMA 269(1993)2122-2127. L. Gattinoni, P. Pelosi, P.M. Suter, A. Pedoto, P. Vercesi, A. Lissoni, Acute respiratory distress syndrome caused by pulmonary and extrapulmonary disease. Different syndromes? Am J Respir Crit Care Med 158 (1998) 3-11. A. Pesenti, P. Pelosi, A. Rossi, The effects of positive end-expiratory resistance in patients with the adult respiratory distress syndrome and in normal anesthetized subjects, Am Rev Respir Dis 144 (1991)101-107. American Thoracic Society. International Consensus Conferences in Intensive Care Medicine: Ventilator-associated Lung Injury in ARDS, Am J Respir Crit Care Med 160 (1999) 2118-2124. V.M. Ranieri, F. Giunto, P.M. Suter, A.S. Slutsky, Mechanical ventilation as a mediator of multisystem organ failure in acute respiratory distress syndrome, JAMA 284 (2000) 43-44 B. Lanchmann, Open up the lung and keep the lung open, Int Care Med 24 (1992) 319-321 D. Matamis, F. Lemire, A. Harf, C. Brun-Buisson, J.C. Ansqer, G. Allan, Total respiratory system pressure-volume curves in the adult respiratory distress syndrome Chest 86 (1984) 58-66 The acute respiratory distress syndrome network, Ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome. A' Engl J Med 342 (2000) 1301-1308 S. Crotti, D. Mascheroni, P. Caironi, P. Pelosi, G. Ronzoni, M. Mondino, J. Marini, L. Gattinoni, Recruitment and derecruitment during acute respiratory failure Am J Respir Crit Care Med 164 (2001) 131-140 A. Koutsoukou, V. Bekos, Ch Sotiropoulou, N. Koulouris, A. Armaganidis, Ch. Roussos, J. MilicEmili, Effect of PEEP in mechanically ventilated ARDS patients Eur Respir J 18 suppl. 33 (2001) 480s A. Koutsoukou, J. Milic-Emili, Ch. Roussos, Effect of PEEP and targets during mechanical ventilation in ARDS. In: J.L. Vincent (ed) 2002 Yearbook of Intensive Care and Emergency Medicine , Springer, Berlin, 2002, In Press A. Koutsoukou, A. Armaganidis, C. Stavrakaki-Kallergi, Th. Vassilakopoulos, A. Limberis, C. Roussos, J. Milic-Emili, Expiratory Flow Limitation and Intrinsic Positive End-Expiratory Pressure in patients with adult respiratory distress syndrome, Am J Respir Crit Care Med 161 (2000) 15901596 J.G. Muscedere, J.B. Mullen, K. Gan, A.S. Slutsky, Tidal ventilation at low airway pressures can augment lung injury Am J Respir Crit Care Med 149 (1994) 1327-1334 L.R. Blanch, R. Fernadez, J. Valles, C. Solle, Ch, Roussos, A. Artigas, Effect of two tidal volumes on oxygenation and respiratory system mechanics during the early stage of the adult respiratory distress syndrome J Crit Care 9 (\ 994) 151-158 V.M. Ranieri, H. Zhang, L. Mascia, M. Aubin, C.Y. Lin, J.B. Mullen, S. Grasso, M. Binnie, G.A. Valgyesi, P. Emy, A.S. Slutsky,, Pressure time curve predicts minimally injurious ventilatory strategy in an isolated rat lung model Anesthesiology 93 (2000) 1833-1859

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Markers of Oxidative Stress in Exhaled Breath Condensate Adam ANTCZAK Department ofPneumology & Allergology, Medical University of Lodz Kopcinskiego Str. 22, 90-153 Lodz Poland

Abstract. Exhaled breath condensate (EBC) is simple, non-invasive means to sample the lower respiratory tract in humans. Markers of oxidative stress were the first molecules to be assessed by using EBC. Oxidant stress is purported mechanism of several lung diseases including chronic obstructive pulmonary disease (COPD), adult respiratory distress syndrome (ARDS), acute lung injury (ALI), asthma, cystic fibrosis (CF, bronchiectasis and lung cancer. It is overall part of inflammatory processes in the airways. An increased content of H2C>2 in EBC has been described in asthma, COPD, ARDS, CF and lung cancer and in healthy smokers. Levels of ^2®"! m ^BC were further increased in exacerbation of COPD and asthma and decreased after steroid treatment and long-term treatment with N-acetylcysteine in COPD patients. Products of lipid peroxidation (thiobarbituric acid -reactive substances and 8-isoprostane) have been increased in asthma, COPD and smokers. Nitrosothiols are elevated in asthma, CF and COPD and nitrotyrosine in asthma, CF and smokers. Increased total nitrite/nitrate levels have been described in asthma. As concentrations of markers of oxidative stress in EBC vary in the different airway diseases and increase with the severity of some and decrease during the treatment, their measurement may have some clinical relevance as a non-invasive biomarker of inflammation.

1. Oxidative stress Reactive oxygen species (ROS) are produced by several inflammatory and structural cells within the airways. An imbalance of oxidant/antioxidant in favour of oxidants, oxidative stress, has been implicated in the pathophysiology of a number of respiratory disorders such as asthma, chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF) [1]. Activation of inflammatory cells such as eosinophils, neutrophils, and macrophages induces a respiratory burst resulting in the production of ROS. When liberated ROS cause tissue damage and cell death or apoptosis. Lung cells, in particular alveolar epithelial type II cells, are particularly susceptible to the injurious effects of oxidants. Therefore oxidative stress and overall inflammatory response are fundamental processes involved in the pathogenesis of many lung diseases [2]. New techniques such as exhaled breath condensate (EBC) might be utilized in detecting and assessing oxidative stress non-invasively and might offer an opportunity to gain insights into the local processes in the airways. EBC provides an easy to perform method to study the airways, without the need to undertake invasive procedures, such as bronchoscopy. It is very well tolerated by patients and no adverse events have been reported. Growing body of evidence suggests that it is a useful way to monitor markers of inflammation and oxidative stress in various respiratory tract diseases, such as asthma, COPD and cryptogenic fibrosing alveolitis.

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2. Markers of oxidative stress 2.1. H2O2 An important source of H2®2 1S me phagocytes. Activated neutrophils, eosinophils and macrophages generate Oi.", which is converted to H2Oz by superoxide dismutase. and hydroxyl radical, fonned non-ezymatically in the presence of Fe2*. ROS are highly reactive and, when close to cell membranes, oxidize membrane lipids (lipid peroxidation), which continue as a chain reaction [3]. This potentially leads to membrane damage by disruption of its function or cell death. An intense airway inflammation can be caused either by H2O2 alone or newly generated hydroxyl radical [3]. H2O2 is released in extracellular fluid and in the airways a part of H2C>2, which has not been decomposed by antioxidant enzymes, can be exhaled with exhaled breath. H2O2 is elevated in EBC in various inflammatory lung disorders such as asthma [4,5], cystic fibrosis (CF) [6], bronchiectasis [7], ARDS and acute hypoxemic respiratory failure [8], cigarette smoking [9], and COPD [10]. Cigarette smoking is a strong pro-inflammatory and prooxidant factor and therefore high levels of FhOi have been found in EBC from smokers compared to non-smoking subject [11]. Dohlman described an increased H2O2 level in EBC in a relatively small group of asthmatic children, mainly in those with acute disease, however elevated H2O2 in stable asthmatics has been also observed [12]. As postulated by Horvath and co-workers, measurement of H2O2 in EBC and exhaled NO in asthmatic patients provides complementary data for monitoring the disease activity [13]. Not only as a diagnostic test, but exhaled H2O2 may be used to guide anti-inflammatory treatment. Inhaled beclomethasone dipropionate in low dose has been shown to decrease H2O2 in EBC following a 2-week treatment [14]. This has also been observed in children with stable asthma, some of them receiving inhaled cortiosteroids in a daily regimen [5]. There was significant difference in median H2C»2 concentration between asthmatics without antiinflammatory treatment and healthy controls. A study in ARDS patients treated with corticosteroids showed a tendency towards lower levels of H2O2 in the expired air condensate as compared to steroid-naive ARDS patients [15]. In a recent study it has been observed that long-term treatment of N-acetylcysteine (600 mg daily) decreases FbO2 exhalation in subject with COPD [16]. Both, asthma and CF patients with an acute pulmonary exacerbation have abnormally high levels of exhaled FhOi, which decrease during antibiotic treatment [17]. 2.2. Products of lipid peroxidation 2. 2. 7. Thiobarbituric acid - reactive substances Measurement of thiobarbituric acid - reactive substances (TEARS) seems to be the most simple, but non-specific method to assess lipid peroxidation damage in tissue, cells and body fluids. Levels of TBARS are increased in exhaled breath condensate in asthma [4] and COPD [18] and they increase during exacerbations [17]. These results are consistent with those of Ignatova who found increased levels of conjugated dienes in exhaled breath condensate and bronchial biopsies from patients with COPD and simple chronic bronchitis [19].

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2.2.2. 8-isoprostane 8-isoprostane, a stable prostaglandin-like arachidonate product formed on membrane phospholipids by the action of reactive oxygen species is postulated to be a reliable biomarker of lipid peroxidation caused by oxygen reactive species and represents a quantitative measure of oxidant stress in vivo [20]. 8-isoprostane appears to reflect oxidative stress in EBC and is progressively increased with the severity of asthma and appears to be very high in aspirininduced asthma [21,22]. It is also reported to be increased in EBC in COPD patients [23] and further increases are observed with exacerbation of COPD [24]. There was also a positive correlation between exhaled H2O2 and 8-isoprostane levels, which might reflect the causeand-effect relationship (A. Antczak - not published data). 8-isoprostane has also been detected in EBC from patients with ARDS [25]. 3. Nitrogen-reactive species 3.1. Nitrotyrosine Nitrotyrosine formation in EBC may be a marker of oxidative stress in airways. The reaction of nitric oxide (NO) and superoxide anions in the airways leads to the formation of peroxynitrite, a highly reactive oxidant species. Peroxynitrite in reaction with tyrosine residues in proteins forms the stable product nitrotyrosine. Nitrotyrosine concentrations have been detected in EBC of normal subjects, and were increased significantly in patients with mild asthma [26]. However, the levels of nitrotyrosine in EBC have been lower in patients with moderate and severe asthma. Moreover, there was a significant correlation between nitrotyrosine in EBC and exhaled NO in patients with mild asthma [26]. Exhaled NO is decreased in cystic fibrosis (CF) patients, perhaps because it is metabolised to oxidative end products. 3-nitrotyrosine may indicate local formation of reactive nitrogen species. As shown by Balint and co-workers nitrotyrosine levels in EBC were increased significantly in stable CF patients, compared with normal subjects. There was an inverse correlation between the levels of nitrotyrosine and the severity of lung disease [27]. Production of nitrotyrosine may reflect increased formation of reactive nitrogen species such as peroxynitrite or direct nitration by granulocyte peroxidases, indicating increased oxidative stress in airways of cystic fibrosis patients [27]. 3.2. Nitrosothiols Nitrosothiols (RS-NOs) are formed by interaction of nitric oxide (NO) with glutathione and may limit the detrimental effect of NO. RS-NOs are detectable in EBC of healthy subjects and are increased in patients with inflammatory airway diseases. RS-NOs in EBC were higher in subjects with severe asthma compared with normal control subjects and with subjects with mild asthma [28]. Elevated RS-NOs were also found in CF patients and in smokers. In current smokers a correlation between RS-NOs values and smoking history (pack/year) was observed. As RS-NOs concentrations in EBC vary in the different airway diseases and increase with the severity of asthma, it is postulated that their measurement may have clinical relevance as a non-invasive biomarker of oxidative-nitrosative stress. 3.3. Nitrites/nitrates NO may be oxidized to nitrite (NO2) and nitrate (NOs), both of which are end products of NO metabolism. Total EBC NO2/NO3 concentrations were significantly higher in CF patients and

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smokers compared to healthy controls [29, 30]. They were also higher in patients with asthma than in normal subjects [31]. Moreover, patients who were on inhaled steroid therapy had significantly lower values compared to steroid-naive subjects. There was a significant positive correlation between NO2/NO3 levels and H2C>2 concentration in EBC [31]. Measurement of expired NC^/NOa levels and F^Oz may be clinically useful in the management of oxidation and inflammation mediated lung injury. 4. Conclusions There is accumulating evidence that abnormalities in exhaled breath condensate composition may reflect biochemical changes of airway lining fluid. Detecting and monitoring of biomarkers in exhaled breath condensate may be helpful in diagnosis and follow-up of patients with various pulmonary diseases. References 1) 2) 3) 4)

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Rahman, D. Morrison, K. Donaldson, W. McNee. Systemic oxidative stress in asthma, COPD, and smokers. American Journal of Critical Care and Respiratory Medicine 154 (1996) 1055-1060. P. J. Barnes PJ. Reactive oxygen species and airway inflammation. Free Radical Biology and Medicine 9 (1990) 235-243. B. Z. Joseph, J.M. Routes, L. Borish. Activities of superoxide dismutase and NADPH oxidase in neutrophils obtained from asthmatic and normal donors. Inflammation 17 (1993) 361-370. A. Antczak, D. Nowak, M. Krol, B. Shariati, Z. Kurmanowska, Increased hydrogen peroxide and thiobarbituric acid-reactive products in expired breath condensate of asthmatic patients, European Respiratory Journal 10(1997) 1231 -1241. Q. Jobsis, H.C.Raatgeep, P.W.M. Hermans, J.C. de Jongste, Hydrogen peroxide in exhaled air is increased in stable asthmatic children, European Respiratory Journal 10 (1997) 519-521. Q. Jobsis, H.C. Raatgeep, S.L. Schellekens, A. Kroesbergen, W.C. Hop, J.C. de Jongste, Hydrogen peroxide and nitric oxide in exhaled air of children with cystic fibrosis during antibiotic treatment. European Respiratory Journal 16 (2000) 95-100. S. Loukides, I. Horvath, T. Wodehouse, P.J. Cole, P.J. Barnes PJ, Elevated levels of expired breath hydrogen peroxide in bronchiectasis, American Journal of Critical Care and Respiratory Medicine 158(1998)991-994. J.I. Sznajder, A. Fraiman, J.B. Hall, W. Sanders, G. Schmidt, G. Crawford, A. Nahum, P. Factor, L.D.H. Wood, Increased hydrogen peroxide in the expired breath condensate of patients with acute hypoxemic respiratory failure, Chest 96 (1989) 606-612. D. Nowak, A. Antczak, M. Kr6l, T. Pietras, B. Shariati, P. Bialasiewicz, K. Jeczkowski, P. Kula, Increased content of hydrogen peroxide in expired breath of cigarette smokers European Respiratory Journal 9 (1996) 652-657. P.N. Dekhuijzen, K.K. Aben, I. Dekker, L.P. Aarts, P.L. Wielders, C.L. van Herwaarden, A. Bast, Increased exhalation of hydrogen peroxide in patients with stable and unstable chronic obstructive pulmonary disease. American Journal of Critical Care and Respiratory Medicine 154 (1996) 813816. M. Kasielski, D. Nowak, A. Antczak, T. Pietras, M. Krol, Increased content of hydrogen peroxide in the expired breath condensate of patients with chronic obstructive pulmonary disease. Current Pneumology\(\991)47-5\. A.W. Dohlman, H. W. Black, J.A. Royall, Expired breath hydrogen peroxide is a marker of acute airway inflammation in pediatric patients with asthma, American Review of Respiratory Diseases 148(19930955-960. I. Horvath, L. Donnelly, A. Kiss, S. A. Kharitonov, S. Lim, K. F. Chung and P.J. Barnes, Combined use of exhaled hydrogen peroxide and nitric oxide in monitoring asthma, American Journal of Critical Care and Respiratory Medicine 158(1998) 1042-1046. A. Antczak, Z. Kurmanowska, M. Kasielski, D. Nowak, Inhaled glucocorticosteroids decrease hydrogen peroxide in expired air condensate in asthmatic children, Respiratory Medicine 94 (2000) 416-421.

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Volatile Organic Compounds as Prognostic Markers in ARDS JK Schubert, W Miekisch, GFE Noldge-Schomburg Department of Anaesthesia and Intensive Care, University of Rostock, Schillingallee 35, 18057 Rostock Germany Abstract. Exhaled volatile substances in ARDS can be linked to different pathways of inflammation and to membrane metabolism. As concentrations of such markers show a specific pattern along the course of ARDS, diagnosis of different stages of the disease should be possible by means of breath analysis. Future progress of analytical techniques and a better understanding of the relationship between expired substance concentrations and the clinical status will certainly help to improve diagnosis, to stimulate basic research, and to guide and evaluate the therapy of ARDS.

1. ARDS: Definition, underlying diseases, lung pathology, clinical aspects First described by Ashbough et al [1] in 1967, the acute respiratory distress syndrome (ARDS) represents a uniform response of the lung to direct or indirect injury. According to the AmericanEuropean consensus conference it is characterised by an acute onset of respiratory failure, a PaO2/FiO2 < 200 mmHg regardless of positive end expiratory pressure (PEEP), bilateral infiltrates on frontal chest radiograph, and pulmonary artery wedge pressure (PC WP) < 18 mmHg or lack of clinical evidence of left atrial hypertension [2]. The incidence is about 75 cases/100,000 population, meaning 150,000 cases annually in the United States [3]. Various clinical conditions may precipitate the onset of ARDS. ARDS may arise from mechanical injury to lung parenchyma, from bacterial, viral or mycotic pulmonary infection or may result from inhalation of toxic gases such as nitrous oxide (NC>2), hydrochloric acid (HC1) or sulphuric oxide (SO2). Indirect lung injury may be elicited by multiple trauma without lung injury, by sepsis, by ischemia/reperfusion or by mass transfusion. Pathophysiology of ARDS is closely related to inflammatory responses including cellular and humoral components. The early stage of ARDS is characterised by neutrophil sequestration into the lung. Activation of these neutrophils causes liberation of oxygen derived free radicals and numerous mediators. Increased concentrations of superoxide, hydrogen peroxide or hypochlorous acid [4] have been found in the blood of ARDS patients. Inflammatory mediators such as complement fragments, thromboxane, leukotrienes, proteases, cytokines or platelet activating factor (PAF) are variably present in both patients at risk for, as well as those with ARDS. Recent work has shown that at the same time hydrogen peroxide, serum catalase. manganese-superoxide dismutase and ceruloplasmine are increased, glutathione is decreased. Findings such as these suggest that the balance between oxidants and anti-oxidant capacity must be important [5]. Reactive oxygen species and inflammatory mediators released from neutrophils have a direct effect on endothelial and alveolar cells and may simultaneously precipitate a general inflammatory response (SIRS = systemic inflammatory response syndrome) in the body. These remote effects contribute significantly to the mortality of ARDS. In fact, many patients die because of multiple organ failure (MOF) rather than of respiratory failure. Direct injury to endothelial and alveolar cells results in increased alveolar-capillary permeability, lung oedema.

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pulmonary hypertension and respiratory failure. Hypoxic pulmonary vasoconstriction is deranged and shunt fraction is consecutively elevated contributing to refractory hypoxemia. During later stages of ARDS fibrosis of the lung tissue determines the course and outcome of the disease. Despite new methods in the treatment such as inhalation of NO, instillation of surfactant, ventilation in prone position or extracorporeal membrane oxygenation (ECMO) mortality of ARDS remains high. Many of the mechanisms in the pathogenesis of ARDS have not yet been elucidated. As a result, a specific therapy of ARDS is lacking. Actual therapy consists of controlling the underlying disease, avoiding hypoxemia by means of lung protective ventilatory support and supportive care. Diagnostic parameters indicating risk or onset of the disease and characterising the course of ARDS are lacking. The association between lung injury and the systemic inflammatory response (SIRS) very often triggered by an ARDS has not been completely understood. Specific prognostic criteria are still to be defined. In ARDS inflammatory processes take place near or in the alveoli. Elevated concentrations of volatile substances generated through the effect of radicals or cytokines on cellular structures should, therefore, be found in the alveolar gas. If these substances can be identified and linked to a specific event in the course of ARDS, analysis of exhaled air will provide valuable information for diagnosis, treatment control and prognosis in ARDS. 2. Analytical procedures in mechanically ventilated patients Many ARDS patients require mechanical ventilation because of respiratory insufficiency and refractory hypoxia. Breath sampling in mechanically ventilated patients requires techniques which are different from those that can be used in spontaneously breathing individuals. Gas sampling in those patients is affected by the high water content of the samples, the respiratory circuit and possible contaminants coming out from tubings or the gas supply. Therefore, substance concentrations have to be corrected for inspiratory values. Special attention has to be paid to the mode of gas sampling. Only alveolar concentrations reflect concentrations in blood, whereas mixed expired concentrations may be diluted and contaminated by dead space gas [6], Due to the very low substance concentrations in the exhaled air preconcentration is mandatory. This can be done by adsorption onto activated charcoal [7], organic polymers, e.g. Tenax [8,9] or via solid phase micro extraction (SPME) [10]. Desorption from the adsorbents takes place through microwave energy or some sort of thermal treatment. Substances are separated by gas chromatography and detected by flame ionisation and identified by mass spectrometry. 3. Marker substances in ARDS A considerable number of substances have been described that could be related to some aspect of acute lung injury (ALI)/ARDS. Hydrogen peroxide levels in expired air condensate were increased in patients with respiratory failure and were highest in ARDS patients [11]. Concentrations decreased after clinical improvement. NO from alveolar macrophages and superoxide from membrane bound NADPH oxidase can combine to form peroxynitrite a potent oxidant capable of damaging cell structures by peroxidation. Peroxynitrite has to be determined via nitrotyrosine in lung sections or breath condensate [12, 13]. Toxic levels of peroxynitrite have been found in ALI/ARDS.

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Isoprostanes, prostanoid compounds formed nonenzymatically via lipid peroxidation are markers of in vivo oxidant stress, and can be determined in breath condensate. Expired isoprostane levels were elevated in patients with ALI or ARDS when compared to healthy controls [14]. Elevated isoprostane concentrations in plasma were predictive of increased mortality in ARDS [15]. A correlation between isoprostane concentrations in breath condensate and the severity of lung injury may, therefore, be assumed. Since many ARDS patients require mechanical ventilation sampling of breath condensate is affected by the respiratory circuit and the mode of gas humidification. Hence quantitative analysis in breath condensate of mechanically ventilated patients is difficult and prone to error, standards of sampling are still to be defined. Exhaled NO concentrations are elevated in patients with inflammation of the airways [16,17]. Inflammatory insults induce an increase in the expression of the inducible isoform of nitric oxide synthase (iNOS) in pulmonary tissue [18]. Since inflammatory processes play an important role in ARDS, elevated exhaled NO concentrations are expected in those patients. Surprisingly, Brett et al [19] found lower exhaled NO concentrations in ARDS patients when compared to mechanically ventilated patients without lung injury. Changes in diffusion capacity of NO or formation of peroxynitrite from NO and superoxide are considered the cause of this finding. Alkanes such as ethane and n-pentane are believed to be markers of lipid peroxidation [20 - 23] and have been demonstrated in a variety of pathological conditions. Pentane arises from peroxidation of co-6 polyunsaturated fatty acids [24], ethane from peroxidation of co-3 fatty acids (e.g., linolenic acid) [25]. Elevated ethane concentrations that could be reduced by radical scavengers were found in ARDS patients. [26]. Since pentane is believed to be a marker of lipid peroxidation elevated exhaled pentane concentrations are expected in ARDS. However, no difference in the pentane production could be observed between ARDS and non-ARDS patients in our first studies (Table 1) [27, 28]. Table 1: Substance exhalation (nmol/m2/min) in patients with and without ARDS Substance Acetone Pentane Isoprene

ARDS Median (95% CI) 119(52-270) 5.1 (1.4- 18.6) 21.8(13.9-41.4)

Non-ARDS Median (95% CI) 149(113-485) 4.15(3.7-9.3) 9.8(8.2-21.6)

P 0.25 0.37 0.04

CI confidence interval, ARDS: Acute Respiratory Distress Syndrome

This finding may be due to the fact that the analysis of air was not performed at the very beginning of ALI/ARDS where inflammatory activity is highest. Furthermore, patients in the non-ARDS group covering a wide range of diagnoses were not completely free of inflammation. In a group of patients who developed pneumonia during their ICU stay, it could be shown that pentane concentrations did increase at the very beginning of an inflammatory process [27]. In order to get a better understanding of the role of pentane in ARDS exhaled breath markers were determined in healthy volunteers, in patients with and being at risk for ARDS and in patients with head injury (Table 2). Pentane concentrations were significantly increased in ARDS patients and in those being at risk to develop ARDS when compared with healthy volunteers or with patients with cranio-facial trauma without lung injury. Isoprene, (2-methylbutadiene-l,3) is always present in human breath, and is thought to be formed along the mevalonic pathway of cholesterol synthesis [28]. This reaction involves mevalonate, isopentenyl pyrophospate and dimethylallyl pyrophosphate (DMPP).

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Table 2: Exhaled breath markers in 65 critically ill patients and 10 healthy volunteers Control group (1)

Substance Acetone Median 25-75% P [nmol/1] Isoprene Mean 95%CI [nmol/1] n-Pentane Median 25-75% P [nmol/1]

Head Injury

(ID

ARDS (III)

At risk (IV)

33.2 20.8 - 38.6

51.4 14.5-76.6

50.0 19.6-72.3

26.0 14.1-54.4

5.99f 3.53 - 8.45

7.53* 5.02- 10.05

2.18*st 1.1 -3.89

6.44§ 5.04-7.85

0.12*s 0.10-0.16

0.33 0.11-0.83

01.00s 0.26- 1.72

0.49* 0.30 - 0.99

Values are given as medians and 25% - 75% percentiles or as mean and 95% confidence interval, respectively. *, §, t indicate results that are significantly different ( p< 0.05).

The rate limiting step of sterol synthesis, i.e. the formation of mevalonate is catalysed by hydroxymethylglutaryl(HMG)-CoA. Although some details of isoprene formation in humans are still unknown there is experimental evidence that isoprene exhalation may be related to oxidative damage to fluid lining of the lung [30] and the body [31]. Surprisingly, isoprene concentrations in the breath of laboratory animals are considerably lower than in human breath [32]. In our studies [27] ARDS patients produced over 50 % less isoprene than those without ARDS (Table 2). In 151 mechanically ventilated patients with various diagnoses a negative correlation between exhaled isoprene concentrations and the severity of lung injury (Murray score) [33] was found (Fig. 1). Decreasing isoprene concentrations in ALI/ARDS may be due to a reduction of cholesterol synthesis in the lung, to reactions of isoprene with other reactive species like peroxynitrite or to a decreased exhalation because of ventilation/perfusion mismatch. Fig. 1 Exhaled isoprene concentration vs. Murray score

§ 15,00 I

10,00

TT t-i 0,00 i 0,00

i « 1,50

2,00

2,50

Murray Score

Correlation coefficient = - 0.300, p < 0.01, N = 151

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J.K. Schubert el al. / Volatile Organic Compounds as Prognostic Markers

There were no correlations between exhaled concentrations of acetone, sulphur containing compounds like dimethylsulfide or €82 [34] or aldehydes like hexanal or nonenal and ALI/ARDS. As for breath condensate, standards of quantitative analysis in exhaled air are lacking and very often results show a wide variation. Some of the very volatile substances, such as ethane require sophisticated analytical techniques [9]. Other substances, such as pentane may be present in ambient air. When standardised alveolar sampling techniques are used as described above (including correction for inspiratory concentrations) analysis of exhaled volatile organic compounds (VOCs) seems to be more reliable than other methods. 4. Requirements to define prognostic markers Due to the complex pathophysiology of ARDS and a lack of knowledge on origin and physiological meaning of exhaled substances for the time being it is not possible to define a single prognostic breath marker in ALI/ARDS. But different markers are known that are specifically linked to single aspects of airway inflammation and lung injury. Isoprene is linked to cholesterol metabolism, ethane and pentane are linked to lipid peroxidation, NO is linked to airway inflammation in a very complex way and isoprostanes are linked to the arachidonic acid metabolism. In order to obtain information on prognosis of ALI/ARDS a set of breath markers has to be used. These parameters have to be chosen in a way that the different aspects of inflammation are taken into account. The upcoming progress in analytical procedures will enlarge our knowledge on exhaled breath markers concerning physiology, mechanisms and kinetics of exhalation. Thus, in the near future it should be possible to define a set of parameters characterising onset, course and prognosis of ALI/ARDS. References 1. Ashbaugh DG, Biglow DB, Petty TL et al. Acute respiratory distress syndrome in adults. Lancet 1967;ii:319323 2. Bernard GR, Artigas A, Brigham K.L, Carlet J, Falke K, Hudson L et al. The American-European consensus conference on ARDS. Definitions, mechanisms, relevant outcomes, and clinical trial coordination. Am J Respir Crit Care Med 1994; 149:818-824 3. American Lung Program. Respiratory Diseases. Task force report on problems, research approaches, needs. The Lung Program. National Heart and Lung Institute. Washington DC, US Government Printing Office, 1972. DHEW Publication No. (NIH) 73-432, pp 165-180 4. Tate RM, Regine JE. Neutrophils and the adult respiratory distress syndrome. Am Rev Respir Dis 1983; 125:552-559 5. Brigham KL. Role of free radicals in lung injury. Chest 1986; 89:859-863 6. Schubert JK, Spittler K-H, Braun G, Geiger K, Guttmann J. COyControlled Sampling of Alveolar Gas in Mechanically Ventilated Patients. J Appl Physiol 2001; 90:486-492 7. Muller WPE,. Schubert JK, Benzing A, Geiger K. Method for the analysis of exhaled air by microwave energy desorption coupled with gas chromatography -. flame ionisation detection - mass spectrometry. J. Chromatogr. B 1998; 716:27-38 8. Miekisch W, Schubert JK, Mailer WPE, Geiger K: Analysis of Exhaled Air As a New Means of Critical Care Testing. In "Advances in Critical Care Testing", W.F. List, M.M. MQller, M.J. McQueen (Eds.), Springer Verlag 1999, p 202 9. Risby TH, Sehnert SS. Clinical application of breath biomarkers of oxidative stress status. Free Rad Biol Med 1999; 27:1182-1192 10. Grote C, Pawliszyn J: Solid-phase micro extraction for the analysis of human breath. Anal Chemistry 1997; 69:587-596 11. Kietzmann D, Kahl R, MQller M, Burchardi H, Kettler D. Hydrogen peroxide in expired breath condensate of patients with acute respiratory failure and with ARDS. Intensive Care Med 1993; 19:78-81. 12. Haddad IY, Pataki G, Hu P, Galliani C, Beckman JS, Matalon S. Quantitation of nitrotyrosine levels in lung section of patients and animals with acute lung injury. J Clin Invest 1994; 94:2407-13

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13. Haddad IY, Ischiropoulos H, Holm BA, Beckman JS, Baker JR, Matalon S. Mechanisms of peroxynitriteinduced injury to pulmonary surfactants. Am J Physiol 1993; 265:L555-L564. 14. Carpenter CT, Price PV, Christman BW. Exhaled breath condensate isoprostanes are elevated in patients with acute lung injury or ARDS. Chest 1998; 114:1653-1659 15. Delanty N, Reilly M. 8-epi-PGF2 a: specific analysis of an isoeicosanoid as an index of oxidant stress in vivo. Br J Clin Pharmacol 1996; 42:15-19 16. Olopade CO, Christon JA, Zakkar M, Hua C, Swedler I, Scheff PA, Rubinstein I: Exhaled pentane and nitric oxide levels in patients with obstructive sleep apnea. Chest 1997; 111:1500-1504 17. Kharitonov SA, Yates D, Robbins RA, Logan-Sinclair R, Shinebourne EA, Barnes PJ. Increased nitric oxide in exhaled air of asthmatics. Lancet 1994; 343:13-135 18. Liu SF, Barnes PJ, Evans TW. Time course of lipopolysaccharide-induced inducible nitric oxide synthase mRNA expression in the rat in vivo. Am J Respir Crit Care Med 1996; 153:A186 19. Brett SJ, Evans TW. Measurement of endogenous nitric oxide in the lungs of patients with the acute respiratory distress syndrome. Am J Respir Crit Care Med 1997; 156:993-7 20. Phillips M, Greenberg J: Ion-trap detection of volatile organic compounds in alveolar breath. Clin Chem 1992; 38:60-5 21. Van Gossum A, Decuyper J: Breath alkanes as an index of lipid peroxidation. Eur Respir J 1989; 2:787-91 22. Van-Rij AM, Wade CR: In vivo lipid peroxidation in man as measured by the respiratory excretion of ethane, pentane, and other low-molecular-weight hydrocarbons. Anal Biochem 1985; 150:1-7 23. Morita S, Snider MT, Inada Y: Increased n-pentane excretion in humans: A consequence of pulmonary oxygen exposure. Anesthesiology 1986; 64:730-33 24. Frankel EN. Volatile lipid oxidation products. Prog Lipid Res 1982; 22:1-33 25. Do BQ, Harinder BS, Garewal S, Clements Jr NC, Peng Y, Habib MP. Exhaled Ethane and antioxidant vitamin supplements in active smokers. Chest 1996; 110:159-64 26. Ortolani O, Conti A. Protective effects of N-acetylcysteine and rutin on the lipid peroxidation of the lung epithelium during the adult respiratory distress syndrome. Shock 2000; 13:14-18 27. Schubert JK, Muller WPE, Benzing A, Geiger K. Application of a new method for analysis of exhaled gas in critically ill patients. Intensive Care Med 1998; 24: 415-421 28. Miekisch W, Schubert JK, Miiller WPE, Geiger K. Analysis of Exhaled Air As a New Means of Critical Care Testing. Clin Chem Lab Med 1999; 37:S347 29. Stone BG, Besse TJ, Duane WC, Evans CD, DeMaster EG: Effect of regulating cholesterol biosynthesis on breath isoprene excretion in men. Lipids 1993;28:705-8 30. Foster MW, Jiang L, Stetkiewicz PT, Risby TH: Breath isoprene: Temporal changes in respiratory output after exposure to ozone. J Appl Physiol 1996; 80:706-10 31. Mendis S, Sobotka PA, Euler DE: Expired hydrocarbons in patients with acute myocardial infarction. Free Radical Res 1995; 23:117-22 32. Cailleux A, Cogny M, Allain P: Blood isoprene concentrations in humans and some animal species. Biochem Med Metabol Biol 1992; 47:157-160 33. Murray JF, Matthay MA, Luce JM, Flick MR. An expanded definition of the adult respiratory distress syndrome. Am Rev Respir Dis 1988; 138:720-723 34. Phillips M, Saba M, Greenberg J: Increased pentane and carbon disulfide in the breath of patients with schizophrenia. Clin Pathol 1994; 46:861-864

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Disease Markers in Exhaled Breath N. Marczin and M.H. Yacoub (Eds.) IOS Press. 2002

Do Reactive Oxygen-Nitrogen Intermediates Contribute to the Pathogenesis of ARDS? Judy M. HICKMAN-DAVIS1, Ian C. DAVIS2, Phillip O'REILLY3, Philip IV^ARDLE1 and Sadis MATALON1*2'4 'Departments of Anesthesiology, 2Genomics and Pathobiology, 3Pulmonary and Critical Care Medicine and 4Physiology and Biophysics, University of Alabama at Birmingham, 619 South 19th Street, Birmingham, AL 35233-6810 Abstract: Inhaled nitric oxide (NO) has been proven effective in lowering pulmonary arterial pressure and improving gas exchange in patients with pulmonary hypertension. Furthermore, because of its potential anti-inflammatory properties, inhaled NO* therapy can be a potentially useful adjunct in the treatment of a number of inflammatory conditions, including acute lung injury (ALI) and the acute respiratory distress syndrome (ARDS). However, NO is also a free radical and can therefore combine with reactive oxygen species to form highly reactive oxygen-nitrogen intermediates, which may overwhelm antioxidant defenses and cause significant damage to alveolar epithelial cells. Nitrotyrosine, a stable end product of reactive oxygen-nitrogen species interactions, is commonly detected in infectious and inflammatory diseases. Nitration and oxidation of a variety of crucial proteins present in the alveolar space has been shown to be associated with diminished function in vitro and has also been identified ex vivo in proteins sampled from patients with ALI/ARDS. Oxidant-mediated tissue injury is therefore likely to be important to the pathogenesis of ARDS. The purpose of this chapter is to review the results from various studies that demonstrate increased levels of NO* and its reactive intermediates in the alveolar spaces of patients with ALI/ARDS, or which show that various proteins are nitrated and or oxidized, and discuss the physiological consequences of protein nitration. 1. Introduction Inhaled nitric oxide (NO») has been proven effective in lowering pulmonary arterial pressure and improving gas exchange in patients with pulmonary hypertension [1]. Furthermore, because of its potential anti-inflammatory properties, inhaled NO* therapy can be a potentially useful adjunct in the treatment of a number of inflammatory conditions, including acute lung injury (ALI) and the acute respiratory distress syndrome (ARDS). However, NO* is also a free radical and can therefore combine with reactive oxygen species (such as superoxide and peroxyl radicals) to form highly

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reactive oxygen-nitrogen intermediates which may overwhelm antioxidant defenses and cause significant damage to alveolar epithelial cells, resulting in functional and structural abnormalities. In addition, S-nitrosothiols, formed by the interaction of NO intermediates with thiols present in hemoglobin, albumin or other proteins, may be capable of producing systemic vasodilation [2]. Potential sources of NO in the lungs include activated alveolar macrophages (AMs) [3;4], neutrophils [5], alveolar type II cells [6;7], endothelial cells, and airway cells [8]. Both neuronal nitric oxide synthase (nNOS) and endothelial NOS (eNOS) have been detected in human lungs [8]. nNOS is localized to nerve terminals that likely contribute to nonadrenergic/noncholinergic airway innervation, and is also present in human and rat airway epithelial cells [8]. eNOS is localized to pulmonary endothelium and bronchial epithelium [9]. Studies have suggested that inducible NOS (iNOS) is constitutively expressed in human upper airway epithelium [10] and occasional AMs [8], but this may be a result of chronic exposure of these cells to inhaled pollutants and microbes [11], Expression of iNOS in other regions of the normal lung is believed to be minimal. However, iNOS has been immunolocalized to airway cells and human lung tissue obtained from patients with ARDS [12;13], bacterial pneumonia [14], lung cancer [15], pulmonary sarcoidosis [16], idiopathic pulmonary fibrosis [17], asthma [18] and tuberculosis [19]. These findings raise the possibility that during lung inflammation, an increased amount of NO' may be released into the epithelial lining fluid, where it may have both beneficial (antimicrobial) and detrimental (tissue-damaging) effects. 2.

Reactive oxygen-nitrogen intermediates contribute to lung injury in experimental models of ARDS

There is now substantial experimental evidence that reactive oxygen-nitrogen species (RNS) may be involved in pulmonary epithelial injury in a variety of pathological situations. Induction of immune complex alveolitis in rat lungs results in increased alveolar epithelial permeability, which is associated with the presence of elevated concentrations of NO* decomposition products in bronchoalveolar lavage (BAL) fluid [20]. Alveolar instillation of the NOS inhibitor L-NMMA ameliorates NO* production and alveolar epithelial injury. Similarly, both paraquat-induced [21] and ischemia-reperfusion-induced [22] lung injury are associated with stimulation of NO* synthesis, and are abrogated by NOS inhibitors. Tracheal epithelial cytopathology induced by Bordetella pertussis is associated with the induction of NO* synthesis, and is remarkably attenuated by inhibition of NOS [23]. Likewise, influenza virus-induced lung pathology in mice results from increased expression of iNOS and increased generation of NO* [24]. Administration of NOS inhibitors significantly improves survival of influenza-infected mice. Additional evidence that RNS play a role in pulmonary inflammation is derived from studies utilizing transgenic Nos2~'~ mice. Lung damage induced by either injection of lipopolysaccharide (LPS) [25], influenza virus infection [26], or hemorrhage and resuscitation [27], is markedly reduced in these mutant mice. Likewise, in an experimental murine model of allergic airway disease, deletion of the Nos2 gene results in a significant decrease in eosinophil infiltration into the lungs [28]. However, levels of NO* produced by inflammatory cells vary widely among species; thus extrapolation of animal data to human disease states is not warranted. Herein we will: (1) review the results from various studies demonstrating increased levels of NO* and its reactive intermediates in the alveolar spaces of patients with ALI/ARDS; (2) show that various proteins are nitrated and or oxidized and (3) discuss the physiological consequences of protein nitration.

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3. Increased levels of NO* in the BAL and edema fluid of patients with ARDS ARDS is a disease process characterized by diffuse inflammation in the lung parenchyma. Nitrate and nitrite (NOX), the stable breakdown by-products of NO, can be measured in biological fluids using the Greiss reaction. While this assay is relatively simple to perform, it is important that any nitrate present is first chemically or enzymatically reduced to nitrite (in most biological fluids, nitrite is usually converted to nitrate because of slow oxidation by hemoglobin). If this reduction step is not performed, levels of NOX production are likely to be grossly underestimated. For example, NOX concentrations were significantly higher than normal in the BAL fluid from patients at risk for developing ARDS as well as those with ARDS [12] and remained elevated throughout the course of ARDS. In all cases, the majority of the products detected were in the form of nitrate (>90% nitrate, and <10% nitrite). NOX was barely above background in BAL fluid from normal subjects (range 2.5-4.3 uM, median 2.5 |iM ). In patients at-risk for ARDS, the NOX concentration in BAL fluid from days 1 and day 3 after onset of the risk factors (such as multiple trauma, sepsis or multiple transfusions; see Table 1) was significantly higher than in normal subjects (Figure 1).

[NOJin BAL 1UU

*

2 *

_l

•

*

m c ® 10 .t: Z

:

T

:

E^3?

iz

(10)

1 Days 0

T T i

(19)

(14)

1

3

Risk

* *

T T

_L

1 -3C- -J-

(36)

(41)

1

3

(30)

7

(16)

14

T (11)

21

ARDS

Figure 1. Nitrate and nitrite concentration (NOX) in BAL from normal volunteers (NL), patients at-risk for ARDS (RISK) and patients with established ARDS (ARDS) studied at sequential times. The horizontal axis shows the patient group and the day on which the BAL was performed, (n) = number of subjects in each group. The data are presented as box plots showing the 10*, 25*, 75* and 90* percentiles and the median. */> < 0.005 vs. normal subjects. Reprinted with permission from reference [12].

In comparing patients at-risk for ARDS and patients with established ARDS, there was no statistically significant difference in BAL NOX concentration, either at the onset of ARDS or at any subsequent time. However, the patients studied on day 21 after the onset of ARDS, a time when the course of ARDS was waning, had the lowest concentrations of NOX in BAL fluid. While there were no statistically significant differences in the BAL NOX concentrations from patients at-risk for the development of ARDS, the NOX concentrations in the BAL of patients with ARDS who subsequently died were significantly higher on days 3 and 7.

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Levels of NOX in the epithelial lining fluid of these patients cannot be easily estimated since they are diluted considerably (more than 50 fold) by the BAL fluid. To address this issue, we measured NOX levels in pulmonary edema fluid (EF) and plasma samples from patients with ALI/ARDS and for comparison, in samples from patients with hydrostatic pulmonary edema. All of these patients were admitted to the intensive care units at the University of California at San Francisco (UCSF) or San Francisco General Hospital between 1985 and 1998. Pulmonary EF was collected from each patient within 30 min after endotracheal intubation by passing a standard 14 Fr tracheal suction catheter through the endotracheal tube into a wedged position in a distal airway as described previously [29]. Pulmonary EF from patients with ALI had significantly higher levels of NOX compared to pulmonary EF from patients with hydrostatic pulmonary edema (108 ± 13 uM vs. 66 + 9 uJVI; Means ± SEM; p < 0.05). In addition, patients with shock had higher plasma NOX levels than those without shock (79 ± 11 uM vs. 53 ± 12 uM, p < 0.05). The ratios of nitrite to nitrate in 11 edema and 9 plasma samples were 0.01 ± 0.005 vs. 0.008 ± 0.004, indicating that more than 90% of NOX were present as nitrate, in agreement with our BAL data (see above). Acidemia and increased anion gap, markers of systemic hypoperfusion, were also associated with two fold higher plasma NOX levels. TABLE 1. Patient population, (reprinted from reference [12] with permission) Characteristic

N Age, yr.

Gender (% male)

- —At Risk—

—

-

--ARDS

Day 21

Day 1

Day 3

Day 1

Day 3

Day 7

Day 14

19

14

36

41

30

16

11

48.0

48.2

42.5

45.1

46.3

44.6

42.6

58

64

67

59

63

50

46 3 (27.3)

Primary Risk Sepsis, n (%)

8(42.1)

4 (28.6)

10(27.8)

15(36.6)

10(33.3)

4 (25.0)

Trauma, n (%)

1 1 (57.9)

10(71.4)

15(41.7)

15(36.6)

14(46.7)

8 (50.0)

5 (45.5)

0(0)

0

1 1 (30.6)

1 1 (26.8)

6 (20.0)

4 (25.0)

3 (27.3)

Other, n (%) Apache II Score

21.8+1.4

14.6 + 2.1

21.1 + 1.1

21.0+1.1

18.8+ 1.4

18.9+1.3

15.5+ 1.8

PO,/Fi02 ratio

209.6 + 23.8*

243.8 + 30.0

152.5 + 8.3*

162.7 + 8.8

203.1 +14.0

197.2+13.1

207.7 + 18.7

2(10)

1(7)

8(22)

8(20)

3(10)

1(7)

0

Mortality, n (%)

Data are the mean + SE P02/Fi02 ratio. Ratio of arterial oxygen tension to inspired fraction of oxygen. * p = 0.03 for the comparison of Day 1 At Risk vs. Day 1 ARDS.

An additional benefit of sampling undiluted pulmonary EF rather than diluted BAL fluid is the opportunity to measure alveolar fluid clearance by following serial changes in the protein concentration in the pulmonary EF. Patients with ALI who were able to concentrate alveolar protein (as a result of active sodium reabsorption) had a better prognosis than those that did not [29;30]. Our results indicate that increased levels of NOX in EF samples were associated with slower rates of alveolar fluid clearance (Figure 2). One possible explanation for this finding is that the generation of reactive oxygen and nitrogen species in the alveolar compartment leads to nitration, oxidation and inactivation of proteins important in alveolar epithelial sodium transport, such as the epithelial sodium channel. Indeed, intra-tracheal instillation of DETANONOate, an NO- donor, decreased amiloride-sensitive fluid clearance in rabbit lungs (31).

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J.M. Hickman-Davis el al. /Pathogenesis ofARDS 250

200 -

150 -

i 100 -\ c 0}

5

50 H

Submaximal/lmpaired Maximal Alveolar Fluid Clearance (%/hr)

Figure 2. Box-plot summary of mean interval EF nitrate and nitrite concentration versus two categories of alveolar fluid clearance. Maximal alveolar fluid clearance is > 14%/h. Impaired/Sub-maximal alveolar fluid clearance is < 14%/h. The horizontal line represents the median, the box encompasses the 25* to 75* percentile and the error bars encompass the 10* to 90* percentile. *p < 0.05 by Mann Whitney U-test. Reprinted with permission from reference [30].

4. Evidence for the existence of nitrated proteins in vivo Several studies have provided evidence that nitration reactions occur in vivo during inflammatory processes. 3-nitrotyrosine residues, products of the addition of a nitro-group (NOi) to the ortho position of the hydroxyl group of tyrosine, are stable end products of RNS mediated reactions. They therefore serve as footprints of RNS action, which are readily detectable by immunohistochemistry, ELISA or high-pressure liquid chromatography (HPLC) [32]. Nitrotyrosine is commonly detected in tissues infiltrated by neutrophils and monocytes during infectious and inflammatory processes [12;33]. In vitro, proteins can be nitrated either by peroxynitrite or by reactive intermediates generated by the myeloperoxidase-catalyzed reaction of reactive species released from activated neutrophils [34;35]. Irnmunohistochemical studies showing evidence of nitrotyrosine residue formation on proteins in cells taken from lung tissues of pediatric patients with ALI, were first reported by Haddad et al. [33]. Oxidant mediated tissue injury is likely to be important in the pathogenesis ofARDS [36;37]. Protein nitration and oxidation by reactive oxygen nitrogen species in vitro has been associated with diminished function of a variety of crucial proteins present in the alveolar space, including ai-proteinase inhibitor and surfactant protein A (SP-A) [38;39]. Gole el al. [40] reported the presence of nitrated ceruloplasmin, transferrin, a i-protease inhibitor, ai-anti-chymotrypsin and p-chain fibrinogen in the plasma of patients with ALI/ARDS. Using quantitative ELISA and HPLC, we detected significant levels of protein-associated nitrotyrosine (~400 - 500 pmol/mg protein) in EF samples from both ALI/ARDS and hydrostatic edema patients [41] and in the BAL from patients with ARDS [12]. These levels of nitrotyrosine are at least one order of magnitude higher than those found in proteins in normal human BAL fluid (28 pmol/mg protein) [42], or normal rat lung tissue (-30 pmol/mg protein) [43], or than those found in normal human serum albumin (-30 pmol/mg protein) [44], and normal human plasma low-density lipoprotein (-85 pmol/mg protein) [44]. Lamb et al. [45] also measured nitrotyrosine content in the BAL fluid of

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patients with severe ARDS and healthy volunteers using HPLC, although their values were considerably higher than those reported by Sittipunt et al. [12] and Zhu et al. [41]. Nitrated pulmonary SP-A was also detected in the EF but not in the plasma of patients with ALI after immunoprecipitation with a specific antibody against this protein (Figure 3). Although we previously demonstrated that SP-A is nitrated and oxidized in vitro, using LPS-stimulated rat AMs as the source of reactive species [46] (see below), this is the first in vivo evidence for nitration of a specific protein in the alveolar spaces of human lung. Results of previous in vitro studies indicated that nitrated SP-A loses its ability to enhance the adherence of Pneumocystis carinii to rat AMs [39] and inhibits killing of Mycoplasma pulmonis by mouse AMs (Hickman-Davis et al.; unpublished observations). Also, nitration of human SP-A by peroxynitrite or tetranitromethane inhibited its lipid aggregation and mannose binding activities [47]. Finally, SP-A isolated from the lungs of lambs exposed to high concentrations of inhaled NO* had decreased ability to aggregate lipids. Thus, nitration of SP-A may be one of the factors responsible for increased susceptibility of patients with ARDS to nosocomial infections [48]. Interestingly, despite being present at high concentrations in the epithelial lining fluid of patients with ARDS, albumin was nitrated to a much lesser degree than SP-A [41].

Figure 3. Nitration of SP-A in pulmonary EF samples from ALI/ ARDS patients. SP-A was immunoprecipitated and Western blotting used to identify SP-A (A) and nitrotyrosine (B). SP-A was detected in the pulmonary EF but not in the plasma of all patients. E!-E5: pulmonary EF samples from 5 different ALI/ARDS patients; Pi-Pa: plasma samples from 3 different ALI/ARDS patients; C: purified human SP-A from a patient with alveolar proteinosis. Notice the lack of nitration in the control sample. (Reprinted with permission from reference [41].

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5. Nitration of SP-A is sufficient to inhibit function In addition to being a nitrating agent, peroxynitrite is a strong oxidant. The inactivation of al-antitrypsin inhibitor by peroxynitrite has been attributed to the oxidation of a methionine in its active site (38). Likewise, SP-A isolated from the epithelial lining fluid of patients with ARDS was shown to be oxidized as well as nitrated [41]. How then can one be sure that tyrosine nitration, instead of oxidation, was responsible for the observed loss of SP-A function? Several pieces of evidence support the hypothesis of nitration-mediated protein inactivation: first, exposure of SP-A to tetranitromethane at pH 6.5, at which pH it acts as an oxidizing agent, did not decrease the ability of SP-A to aggregate lipids or bind to mannose [49], while exposure of SP-A to TNM at pH 7.5, when it functions as a nitrating agent, decreased its function. Second, carbon dioxide (CC^) (0 - 1.2 mM) augmented peroxynitrite (0.5 mM)-induced SP-A nitration and decreased oxidation in a dose-dependent fashion, as assessed by western blotting. Peroxynitrite also decreased the ability of SP-A to aggregate lipids and this inhibition was augmented by 1.2 mM CO2, in spite of the CO2-mediated decrease in oxidation [46]. Finally, exposure of SP-A to generators of reactive oxygen intermediates (such as xanthine and xanthine oxidase), did not decrease SP-A function [50]. 6. Conclusions Data presented herein indicate that stable decomposition products of both NO* and intermediated generated by its reaction with reactive oxygen species, are detected in high concentrations in both the BAL and EF of patients who are at risk of developing ARDS or who have established ARDS. Levels of reactive species correlate both with the outcome of the disease and the severity of injury to the alveolar epithelium. Finally, significant levels of nitrated SP-A and fibrinogen are detected in the EF and plasma of patients with ARDS. Although in vitro studies indicate that nitration of both proteins leads to diminished function it still needs to be established whether there are sufficient levels of nitration in vivo to contribute to the pathogenesis ofARDS. Acknowledgements This work was supported by grants RR00149 (J.M.H.-D.), HL31197 and HL51173 (S.M.) from the National Institutes of Health. Dr. Ian Davis is a Parker B. Francis Foundation Fellow.

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Pendino,K.J., LaskinJ.D., ShulerJR.L., Punjabi,C.J., and Laskin,D.L. 1993. Enhanced production of nitric oxide by rat alveolar macrophages after inhalation of a pulmonary irritant is associated with increased expression of nitric oxide synthase. J.Immunol. 151:7196-7205.

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21. Berisha,H.I., Pakbaz,H., Absood,A., and Said,S.I. 1994. Nitric oxide as a mediator of oxidant lung injury due to paraquat. Proc.Natl.Acad.Sci.USA 91:7445-7449. 22. Ischiropoulos,H., al-Mehdi,A.B., and Fisher.A.B. 1995. Reactive species in ischemic rat lung injury: contribution of peroxynitrite. Am.J.Physiol. 269:LI58-64. 23. Heiss,L.N., Lancaster,J.R., Jr., Corbett,J.A., and Goldman,W.E. 1994. Epithelial autotoxicity of nitric oxide: role in the respiratory cytopathology of pertussis. Proc.Natl.Acad.Sci.USA 91:267270. 24. Akaike.T., Noguchi,Y., Ijiri,S., Setoguchi,K., Suga,M., Zheng,Y.M., Dietzschold,B., and Maeda,H. 1996. Pathogenesis of influenza virus-induced pneumonia: involvement of both nitric oxide and oxygen radicals. Proc.Natl.Acad.Sci.USA 93:2448-2453. 25. Kristof,A.S., Goldberg,?., Laubach.V., and Hussain.S.N. 1998. Role of inducible nitric oxide synthase in endotoxin-induced acute lung injury. Am.J.Respir.Crit Care Med.1998.Dec. 158:18831889. 26. Karupiah,G., Chen,J.H., Mahalingam.S., Nathan,C.F., and MacMicking,J.D. 1998. Rapid interferon gamma-dependent clearance of influenza A virus and protection from consolidating pneumonitis in nitric oxide synthase 2-deficient mice. J.Exp.Med. 188:1541 -1546. 27. Szabo,C. and Billiar,T.R. 1999. Novel roles of nitric oxide in hemorrhagic shock. Shock 12:1-9. 28. Xiong,Y., Karupiah,G., Hogan,S.P., Foster,P.S., and Ramsay,A.J. 1999. Inhibition of allergic airway inflammation in mice lacking nitric oxide synthase 2. J.lmmunol. 162:445-452. 29. Matthay.M.A. and Wiener-Kronish,J.P. 1990. Intact epithelial barrier function is critical for the resolution of alveolar edema in humans. Am. Rev. Respir. Dis. 142:1250-1257. 30. Ware,L.B. and Matthay,M.A. 2001. Alveolar fluid clearance is impaired in the majority of patients with acute lung injury and the acute respiratory distress syndrome. Am.J.Respir.Crit Care Med. 163:1376-1383. 31. Nielsen,V.G., Baird,M.S., Chen.L., and Matalon.S. 2000. DETANONOate, a nitric oxide donor, decreases amiloride-sensitive alveolar fluid clearance in rabbits. Am.J.Respir.Crit Care Med. 161:1154-1160. 32. Beckman,J.S., Ye,Y.Z., Anderson.P.G., Chen.J., Accavitti.M.A., Tarpey.M.M., and White,C.R. 1994. Extensive nitration of protein tyrosines in human atherosclerosis detected by immunohistochemistry. Biol.Chem.HoppeSeyler 375:81-88. 33. Haddad,I.Y., Pataki.G., Hu,P., Galliani.C, Beckman,J.S., and Matalon,S. 1994. Quantitation of nitrotyrosine levels in lung sections of patients and animals with acute lung injury. J.Clin.Invest. 94:2407-2413. 34. Beckman.J.S., Beckman.T.W., Chen,J., Marshall,?.A., and Freeman.B.A. 1990. Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. Proc.Natl.Acad.Sci.USA 87:1620-1624. 35. van der Vliet,A., Eiserich,J.P., Halliwell,B., and Cross.C.E. 1997. Formation of reactive nitrogen species during peroxidase-catalyzed oxidation of nitrite. A potential additional mechanism of nitric oxide-dependent toxicity. J.Biol.Chem. 212:''617'-7625. 36. HaddadJ.Y., Pitt,B.R., and Matalon,S. 1996. Nitric oxide and lung injury. In Pulmonary Diseases and Disorders. A.P.Fishman, editor. McGraw-Hill, 337-346. 37. Fukuto,J.M., Hobbs,A.J., and Ignarro.L.J. 1993. Conversion of nitroxyl (HNO) to nitric oxide (NO) in biological systems: the role of physiological oxidants and relevance to the biological activity of HNO. Biochem. Biophys. Res. Commun. \ 96:707-713.

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38. Moreno,].J. and Pryor,W.A. 1992. Inactivation of alpha 1-proteinase inhibitor by peroxynitrite. Chem.Res. Toxicol. 5:425-431. 39. Zhu,S., Kachel,D.L., Martin,W.J., and Matalon,S. 1998. Nitrated SP-A does not enhance adherence of Pneumocystis carinii to alveolar macrophages. Am.J.Physiol. 275:L1031-L1039. 40. Gole.M.D., SouzaJ.M., Choi,!., Hertkorn,C., Malcolm,S., Foust,R,F., Ill, Finkel,B.( Lanken,P.N., and Ischiropoulos,H, 2000. Plasma proteins modified by tyrosine nitration in acute respiratory distress syndrome. Am.J.Physiol Lung Cell Mol.Physiol 278:L961-L967. 41.

Zhu,S., Ware,L.B., Geiser,T., Matthay,M.A., and Matalon,S. 2001. Increased levels of nitrate and surfactant protein A nitration in the pulmonary edema fluid of patients with acute lung injury. Am.J.Respir.Crit Care Med. 163:166-172.

42. de AndradeJ.A., Crow,J.P., Viera,L., Bruce,A.C., Randall,Y.K., McGiffin,D.C., Zorn,G.L., Zhu,S., Matalon,S.( and Jackson,R,M. 2000. Protein nitration, metabolites of reactive nitrogen species, and inflammation in lung allografts. Am.J.Respir.Crit Care Med. 161:2035-2042. 43. Tanaka S, Choe,N., Hemenway D.R, Zhu,S., Matalon,S., and Kagan,E. 1998. Asbestos inhalation induces reactive nitrogen species and nitrotyrosine formation in the lungs and pleura of the rat. J.Clin.lnvest. 102:445-454. 44. Khan,J., Brennand,D.M, Bradley,N., Gao,B., Bruckdorfer,R., Jacobs,M., and Brennan,D.M. 1998. 3-Nitrotyrosine in the proteins of human plasma determined by an ELISA method. BiochemJ. 330:795-801. 45. Lamb,N.J., Gutteridge,J.M., Baker,C., Evans,T.W., and Quinlan,G.J. 1999. Oxidative damage to proteins of bronchoalveolar lavage fluid in patients with acute respiratory distress syndrome: evidence for neutrophil-mediated hydroxylation, nitration, and chlorination. Crit Care Med 27:1738-1744. 46. Zhu,S., Basiouny,K.F., Crow.J.P., and Matalon,S. 2000. Carbon dioxide enhances nitration of surfactant protein A by activated alveolar macrophages. Am.J.Physiol Lung Cell Mol.Physiol 278-.L1025-L1031. 47. Zhu,S., HaddadJ.Y., and Matalon,S. 1996. Nitration of surfactant protein A (SP-A) tyrosine residues results in decreased mannose binding ability. Arch.Biochem.Biophys. 333:282-290. 48. Fagon.J.Y., Chastre,J., Domart,Y., TrouilletJ.L., Pierre,J., Dame,C., and Gibert,C. 1989. Nosocomial pneumonia in patients receiving continuous mechanical ventilation. Prospective analysis of 52 episodes with use of a protected specimen brush and quantitative culture techniques. Am.Rev.Respir.Dis. 139:877-884. 49. HaddadJ.Y., Zhu,S., lschiropoulos,H., and Matalon,S. 1996. Nitration of surfactant protein A results in decreased ability to aggregate lipids. Am.J.Physiol. 270:L281-8. 50. Haddad,I.Y., Crow,J.P., Hu,P., Ye,Y., Beckman.J., and Matalon,S. 1994. Concurrent generation of nitric oxide and superoxide damages surfactant protein A. Am.J.Physiol. 267:L242-9.

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Disease Markers in Exhaled Breath N. Marczin andM.H. Yacoub (Eds.) IOS Press. 2002

Modulation of Active Alveolar Na+ Transport by Reactive Oxygen-Nitrogen Intermediates Karin M. HARDIMAN1, Ahmed LAZRAK2, Vance NIELSEN2 and Sadis MATALON1*2 1 Departments of Physiology and Biophysics and 2Anesthesiology, University of Alabama at Birmingham, 619 South 19th Street, Birmingham, AL 35233-6810 Abstract. Active sodium transport across the alveolar epithelium creates an osmotic force, which contributes to the reabsorption of edema fluid across the alveolar epithelium. Various studies in animals and humans have demonstrated the importance of active Na* transport in limiting alveolar flooding in acute lung injury and the reabsorption of fetal lung fluid shortly after birth. Macroscopic measurements of Na* transport across alveolar have shown that Na* ions enter the cytoplasm of alveolar cells mainly through amiloride-inhibitable Na* channels. Molecular biology studies have shown the existence of three Na* channel subunit mRNAs and proteins (a, ji and y-rENaC) in adult alveolar type II (ATII) cells. Patch clamp studies have demonstrated the existence of various types of amilorideinhibitable Na+ channels, located in the apical membranes of ATII cells. The activities of these channels are modulated by a variety of agents including cAMP, glucocorticoids, mineralocorticoids and oxygen. Increased levels of reactive oxygen-nitrogen intermediates, secreted in the alveolar space by activated inflammatory cells, down regulate the activity of these channels and decrease Na transport in vivo, by increasing cGMP, and/or interacting with key residues of channel proteins.

1. Introduction In order for gas exchange to occur optimally, the alveoli must remain open and free from fluid. In utero, the fetal lung is filled with fluid which is removed shortly after birth, mainly because active transport of sodium ions (Na+) across the alveolar epithelium, starting shortly before birth, creates an osmotic force favoring reabsorption of alveolar fluid [1,2]. Studies showing reabsorption of intratracheally instilled isotonic fluid or plasma across the alveolar spaces of adult anesthetized animals and resected human lungs, and the partial inhibition of this process by amiloride and ouabain, indicate that adult alveolar epithelial cells are also capable of actively transporting sodium (Na*) ions [reviewed in [3]]. Whether or not active Na+ transport plays an important role in maintaining the normal alveoli free of fluid, remains to be established. On the other hand, a variety of studies have clearly established that active Na+ transport limits the degree of alveolar edema in pathological conditions in which the alveolar epithelium has been damaged. For example, intratracheal instillation of a Na* channel blocker in rats exposed to hyperoxia, increased the amount of extravascular lung water [4]. Conversely, intratracheal instillation of adenoviral vectors containing copies of the Na+,K+-ATPase genes increased survival of rats exposed to hyperoxia [5]. Patients with Acute Lung Injury (ALI) who were able to concentrate alveolar protein (as a result of fluid efflux secondary to active Na+ reabsorption) had a better prognosis than those that did not [6,7]. In addition, p-agonists, which upregulate Na* transport across the alveolar epithelium

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of animal and resected human lungs [8] by increasing cAMP levels, reversed the decrease in amiloride-sensitive alveolar fluid clearance in rats exposed to hypoxia [9]. Thus, p-agonists may prove useful in the amelioration of high altitude pulmonary edema. 2. ATII cells contain functional Na+ transporters. Additional insight into the nature and regulation of transport pathways has been derived from electrophysiological studies in freshly isolated and cultured alveolar type II (ATII) ceils. These cells, which make up 67% of the alveolar epithelial cells but constitute only 3% of the alveolar surface area in the adult lung, can be isolated at high purity from rats and grown on permeable supports to form confluent monolayers [10,11]. Based on the results of a variety of biophysical studies we know that Na+ ions diffuse passively into ATII cells mainly through apically located amiloride-sensitive, amiloride-insensitive and cGMP-gated cation channels with conductances of 4-25 pS [12],[13,14] and are extruded across the basolateral membranes by the ouabain-sensitive Na+,K+-ATPase [15]. To preserve neutrality, chloride (Cl") ions move from the apical to the basolateral compartments either through the paracellular junctions and/or through chloride channels located in alveolar epithelial cells [16,17]. The three subunits of the epithelial Na+ channel (aENaC, fiENaC, and yENaC) have been found in freshly isolated and cultured ATII cells using western blotting and RT-PRC, and anti-sense oligonucleotides directed against aENaC mRNA caused a decrease in ATII cell channel activity [13]. In situ hybridization studies identified the presence aENaC and yENaC but not pENaC in the alveolar region of both fetal and adult lungs [18]. Recently alveolar type I (ATI) cells have been isolated from adult rat lungs, and like ATII and FDLE cells, have been shown to contain aquaporins and possess very high permeability to water [19-21]. Preliminary (currently unpublished) data from a number of laboratories indicate that ATI cells contain proteins antigenically related to ENaC and Na,K-ATPase subunits. Thus, it seems likely that ATI cells are also capable of vectorial Na+ transport, although no direct evidence exists at present. 3. Sources of Reactive Oxygen and Nitrogen Species in the Lung. Because of their location, alveolar epithelial cells are often exposed to increased intracellular and extracellular concentrations of reactive oxygen and nitrogen species (RONS) present in cigarette smoke, environmental pollutants, oxidant gases or generated by activated inflammatory cells. Reactive oxygen species are formed as intermediates of the incomplete reduction of oxygen in mitochondrial electron-transport systems, by microsomal metabolism of endogenous compounds and xenobiotics, or by various enzymatic generators, such as xanthine oxidase. Neutrophils and other inflammatory cells generate and release reactive oxygen species via an NADPH oxidase-dependent mechanism which is mediated by membrane receptor activation of protein kinase C and phospholipase C[22]. Nitric oxide («NO) synthesis involves the five-electron oxidation of the guanidino nitrogen of L-arginine by nitric oxide synthases (NOS) [23]. The three enzymes that make •NO are endothelial nitric oxide synthase (eNOS), neuronal nitric oxide synthase (nNOS), and inducible nitric oxide synthase (iNOS). Potential sources of -NO include both rat and human activated alveolar macrophages [24,25], neutrophils [26], alveolar type II ceils [27,28], and airway cells [29], Increase iNOS levels have been found in airway cells and human lung tissue obtained from patients with ARDS [30-32] and other inflammatory lung diseases. The biological effects of *NO depend on its concentration, the biochemical composition of the target, and the presence of other radicals. Nitric oxide may bind to the heme group of soluble guanylate cyclase resulting in increased cellular cGMP levels [33]; it

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may react with superoxide ((V) at diffusion limited rates (6.7 x 109 M"1 x s"1) to produce peroxynitrite (ONOO") [34] and higher oxides of nitrogen; or, in the presence of an electron acceptor, it may react with thiols to form nitrosothiols (RS-NO) [35]. RONS may cause extensive cellular injury by initiating iron-independent lipid peroxidatation, sulfhydryl oxidation, DNA strand scission, tyrosine nitration, apoptosis and cellular necrosis [36]. Herein we will review the results of various studies indicating that over-production of reactive oxygen-nitrogen intermediates damage ATII cell ion channel function, and decrease vectorial Na+ transport across alveolar epithelial cells both in vitro and in vivo. 4. Assessment of Na+ transport across ATII cells in vitro. There are various methods of showing the presence of active vectorial Na+ transport in freshly isolated and cultured ATII cells, including: (1) detection of whole-cell and single channel currents across single ATII cells by patch clamp; (2) measurements of 22Na+ and 86 Rb* fluxes across ATII cells, and the extent to which these fluxes are inhibited by amiloride and ouabain respectively; and (3) quantitation of short circuit current (Isc) across confluent monolayers of ATII cells mounted in Ussing chambers. The effects of reactive oxygennitrogen intermediates are then assessed by measuring changes in these variables following exposure of ATII cells to -NO, ONOO'. Results of studies from various laboratories are discussed below. a. Patch clamp studies: This technique relies on the formation of an electrically tight seal between a glass micropipette and the membrane, with a resistance around 5-50 Gft (1 gigaohm = 109 Q). Jain et al [37] isolated rat ATII cells to high purity, grew them on transparent filters and patched them in the cell attached mode. The authors found that most ATII cells expressed cation channels, equally selective to Na* and K* ions with a conductance of 20 pS. Addition of S-nitrosoglutathione (GSNO) and S-nitroso-Nacetylpenicillamine (SNAP), agents that generate oxides of nitrogen, into the bath, increased ATII cell cGMP content and significantly reduced the open probability (Po) this channel; pretreatment of ATII cells with a PKG inhibitor prevented the inhibitory effects of GSNO on this channel; incubation of ATII cells with a cell-permeable analogue of cGMP (8-BrcCMP) also decreased the Po. They concluded that »NO decreased the activity of this channel by activating a cGMP-dependent protein kinase. It should be noted however that GSNO has been shown to alter ion channel function by nitrosating channel proteins[38]. Only a small fraction of ATII cells patched in the cell-attached mode have single channel activity. Furthermore, the contribution of various types of channels with different conductances and densities to overall Na+ transport is difficult to ascertain. Additional information can be obtained from measurements of current-voltage relationships across the entire ATII cell membranes. In this case, after the cell is patched in the cell attached mode, an electrical pulse and or suction is applied rupturing the cell membrane and allowing equilibration between the contents of the pipette and cytoplasm. Movements of ions into or out of the cytoplasm, in response to altering the membrane potential, create inward and outward currents respectively. Lazrak et al. [39] reported that A459 cells, an adenocarcinoma cell line which has some properties similar to those of ATII cells, when patched in the whole cell mode, exhibited significant levels of amiloride-sensitive Na* currents. Perfusion of A549 cells with PAPANONOate (a «NO donor) decreased the amiloride-sensitive component of the Na+ whole cell current in a rapid and reversible fashion (Figure 1).

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A. 100 MM PAPANONOATE

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Figure 1. (A) Time course recording of whole-cell inward (Na+) current, evoked by -100 mV voltage pulses every 10 s across an A549 cell. Initially, the cell was perfused with a solution containing (in mM): NaCl 145, KC1 2.7, CaCl2 1.8, MgCl2 2, Glucose 5.5, N- [2-HydroxyEthyl] Piperazine-N'- [2-EthaneSulfonic acid] (HEPES) 10, pH 7.4, 323 milliosmoles). At the interval indicated by the horizontal bar, the solution was changed to one containing 100 uM PAPANONOate. The pipette was filled with a solution containing: (mM): K-methylsulphonic acid 135, KC1 10, NaCl 6, Mg2ATP 1, Na3ATP 2, glucose 5.5, HEPES 10, EGTA 0.5. Whole-cell I-V relations before (B) and five min post PAPANONOate perfusion, when the steady state currents were seen (C) are shown. The whole-cell current inhibited by NO (NO sensitive) was calculated by digitally subtracting the currents at the steady state effect of NO (as shown in C) from the current before the perfusion with NO containing SES (as shown in B). Mean I-V relationships for the total and NO-sensitive currents are shown in panel D. For panel D, values are means ± 1 SEM (n=6). (from reference [39] by permission).

Significant inhibition was seen with as low as 300 nM »NO, well below the levels expected to be present in the alveolar epithelial space during lung inflammation. Pre-incubation of A549 cells with 3 uM ODQ (a potent inhibitor of soluble guanyl cyclase) for 30 min prior to perfusion with NO donors, totally prevented the reduction of the Na+ currents. As in the studies of Jain et al. [37], these authors also found that perfusion of A549 cells with 100 uM 8-Br-cGMP markedly inhibited the inward (Na+) but not the outward (K+) currents. In additional studies, they also demonstrated that -NO release by PAPANONOate markedly decreased single channel activity in the cell-attached patches of A549 cells [39]. These results are in agreement with in vivo data showing that intratracheal instillation of «NO donors in rabbits decreases levels of amiloride-sensitive transport in vivo [40] (see below). However, intratracheal instillation of dibutyryl-cGMP (1 mM) in anesthetized rats increased the amiloride-insensitive fraction of Na+ transport across the alveolar epithelium [41]. Thus •NO may be inhibiting Na+ transport via cGMP-independent pathways. Duvall et al. [42] reported that peroxynitrite, but not »NO, decreased amiloride sensitive currents across Xenopits oocytes injected with cDNA's of a, p and y ENaC, the three subunits of the amiloride sensitive sodium channel. Thus, RONS may damage either Na+ channel proteins directly or cytoskeletal proteins (such as actin and fondrin) which are required for proper Na+ channel function [43,44]. b. Short-circuit current measurements: Guo et al. [45] examined the mechanisms by which »NO decreased vectorial Na+ transport across confluent monolayers of rat ATII cells grown on permeable supports. Two different sets of experiments were performed: In the first, spontaneous potential difference (PD) and transepithelial resistance

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(Rt) across ATII cell monolayers were measured daily. Forty-five minutes following addition of either of two »NO donors (spermine NONOate (200 uM) or PAPANONOate (100 uM) in the apical and basolateral media bathing the monolayers, the equivalent current (leq; calculated from the Ohm's law under open-circuit conditions) was decreased by -80% whereas the Rt increased by -30%, consistent with the inhibition of a conductive pathway across the epithelium. Under these conditions, the effect of -NO on the Ieq was concentration dependent (ICso = 0.4 uM), well within the concentrations of »NO likely to be present in the alveolar epithelial lining fluid during inflammation. In the presence of the »NO scavenger oxy-hemoglobin (50 uM), -NO donors did not increase »NO levels above baseline and Ieq remained at baseline. Moreover, the Ieq recovered when monolayers were treated with oxyhemoglobin 45 min after administration of the »NO donors (Figure 2). These findings demonstrated that »NO specifically inhibited transepithelial Na+ transport across ATII monolayers.

100

.2

75

MO

50

* 25

0

20

40

60

80

100

120

Time (min) Figure 2. Rat ATII cells were cultured untill they formed confluent monolayers. Equivalent short circuit currents (7e
In the second series of experiments, monolayers were mounted in Ussing chambers, and the current necessary to maintain the potential difference to zero [short circuit current (Isc)] was measured continuously as previously described [45]. Addition of PAPANONOate, caused IK to decrease by -60%. The residual Ix was subsequently inhibited to -10% of the basal level by amiloride added to the apical bath. »NO donors did not increase intracellular cGMP and, in contrast to the findings of Jain et al. [37] the lsc was not affected when monolayers were treated with 8-bromo-GMP (400 uM), suggesting that the effects of «NO on ATII cell Na"1" transport were not dependent on cGMP [45]. However, addition of the ONOO" generator SIN-1, also decreased the lsc. This suggests that the decreased transport following »NO generation may be due, at least in part, to the reaction of •NO with O2»" to form ONOO". Moreover, this is consistent with our finding that ONOO" inhibits the amiloride-sensitive whole-cell conductance in oocytes that heterologously expressed the three ENaC subunits [42]. One possible explanation for the differences

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between the results of Guo et al and Jain et al. is that culturing ATII cells may have resulted in downregulation of PKG activity which may explain the lack of cGMP effect. To obtain additional insights as to which transporters were affected by »NO, Guo et al. [45] permeabilized either the apical or the basolateral membranes of ATII cells grown on monolayers and mounted in Ussing chamber by the addition of amphotericin B in either compartments and measured Isc before and after addition of -NO donors. To examine the effects of »NO on the apical membrane Na+ conductance, experiments were performed in the presence of a Na+ concentration gradient ([Na+]mucosai:[Na+]serosai = 145:25 mM). Following permeabilization of the basolateral membrane but in the absence of "NO, the amiloride-sensitive Isc (AJscam'1') was -10 uA/cm2 following. By contrast, the AIscam'1 was decreased -60% in monolayers pretreated with papa NONOate (Figure 3). Figure 3. Rat ATII cells were cultured till they formed confluent monolayers and mounted in Ussing chambers. In this experiment (pane A), the composition of the apical and basolateral compartments were: Apical: 145 mM; basolateral: 25 mM. Short circuit current (/vc) was recorded continuously as mentioned in the text. Addition of 100 u_M PAPA NONOate into both the apical and basolateral compartments decreased /„ as shown. Subsequently, amphotericin B (10 uM) was added into the basolateral compartment permeabilizing the basolateral membrane, causing /„. to increase. Under these conditions, in the absence of Na,K-ATPase, the Isc was driven by the existing apical:basolateral gradient. Addition of amiloride (10 uM) into the apical compartment completely eliminated /«.. Mean values of amiloride-sensitive Na+ currents across apical membranes in the presence or absence of NO are shown in panel B. Values are means±SEM; * significantly different from control (student t-test; n=4 for each group) (from reference [45].

The effects of »NO on Na+,K+-ATPase were examined with symmetrical Na+ concentrations (145 mM) and amiloride in the apical bath. Permeabilization of the apical membrane produced an ouabain-sensitive Isc (AIscpump) which was inhibited by -65% in the presence of papa NONOate. In conjunction with these findings, they also found that «NO inhibited intracellular ATP, which might have accounted for the decreased Na+,K+-ATPase activity. However, 2-deoxy-D-glucose, which produced a comparable decrease in cellular ATP (~ 45% of control value) did not alter AIscpump. These data indicate that »NO, at non-cytotoxic concentrations, decreased Na+ absorption across cultured ATII monolayers by inhibiting both the amiloride-sensitive Na+ transporters and the Na+,K+-ATPase, through cGMP-independent mechanisms [45]. In additional studies, Compeau et al [46] assessed changes in sodium transport across monolayers of rat distal fetal epithelial cells following incubation of these

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cells with macrophages, stimulated with endotoxin for 16 h. They reported a 75% decline in Rt and a selective 60% reduction in amiloride-sensitive Isc. The products of activated macrophages exerted their effect by expression of inducible »NO synthase in the lung epithelium [47]. In additional studies, Hu et al [48] showed that addition of boluses of ONOO" (0.5 or 1 mM) into suspensions of freshly isolated rabbit ATII cells, decreased amiloride-inhibitable 22 Na+ uptake, to 68 and 56 % of their control values respectively without affecting cell viability. Peroxynitrite, but not reactive oxygen species, also decreased 22Na+ uptake into membrane vesicles of colonic cells of dexamethasone-treated rats known to contain Na"channels [49]. At physiological pH values, ONOO" decomposes spontaneously with a halftime of about 1 s [34]. Thus, a bolus instillation of 1 mM ONOO" resulted in a steady-state concentration of 2.6 u,M during a ten min period [48,49], Significantly higher concentrations may be generated in the epithelial lining fluid [50]. Taken as whole, these data indicate that reactive oxygen-nitrogen intermediates may decrease the ability of alveolar epithelial cells to transport Na+ ions in vivo. 5. Effects of RONS on Na* transport across the alveolar epithelium in vivo. Several studies have investigated the possible association between reactive oxygen nitrogen species and Na+ transport across the alveolar epithelium in both animals with ARDS-type injury and patients with patients with cardiogenic edema or ARDS. In anesthetized animals, a solution isoosmotic with plasma (usually NaCl with 5% bovine albumin) is instilled in the right lung lobe. At some time later (usually at 15, 30 or 60 min) the fluid is aspirated and its protein concentration is measured. Since albumin crosses the alveolar epithelium very slowly, changes in albumin concentration reflects solvent flux. Alveolar fluid clearance (AFC) is then calculated by the change in albumin concentration and expressed as % of instilled volume per unit time. This method, was developed by Dr. Matthay [51] and has been used successfully in rabbits, sheep, hamsters, and mice during the last 20 years [see [3] for a comprehensive review]. Modelska et al. [52] showed that reabsorption of isotonic fluid, secondary to Na+ reabsorption across the alveolar space, was inhibited followed prolonged hemorrhagic shock. Moreover, instillation of aminoguanidine, an inhibitor of iNOS, restored fluid reabsorption to normal levels. Similar results were reported by Pittet et al. [53] who found that inhibition of «NO production by iNOS upregulation in a model of hemorrhagic shock restored the ability of p-adrenergic agonists to increase Na* dependant alveolar fluid clearance in rats. Finally, Nielsen et al. showed that intratracheal instillation of DETANONOate, a «NO donor, decreased the amiloride-sensitive fraction of alveolar fluid clearance but did not alter total AFC [40]. This is an important finding since the amiloridesensitive and insensitive components of AFC are regulated differently. For example alveolar hypoxia results in upregulation of the amiloride-sensitive but not of the amiloride-insensitive component of AFC. In patients with pulmonary edema secondary to alveolar epithelial injury, AFC is calculated by measuring changes in albumin concentration in aspirated edema fluid [7]. Large amount of nitrite and nitrate were detected both in the edema fluid and plasma of patients with acute lung injury ALI [50]. Furthermore these patients had significant levels of nitrotyrosine in their edema fluid and others have shown that nitrotyrosine in the BAL increases during inhalation of -NO [54]. Zhu et al. [50] showed that increased levels of nitrate and nitrite in edema fluid samples of patients with ALI were associated with slower rates of alveolar fluid clearance across the lungs of patients with ALI. As mentioned previously, the data of Guo et al.[45] indicate that co-incubation of confluent monolayers of ATII cells with nitric oxide donors decreased both ATP and vectorial Na+ transport, as

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assessed by a decrease in lsc. However, an equivalent decrease in ATII cell ATP levels, following incubation of ATII cells with 2-deoxy-D-glucose did not decrease Isc. Thus, it seems likely that generation of reactive oxygen and nitrogen species in the alveolar compartment leads to nitration, oxidation and inactivation of proteins important in alveolar epithelial sodium transport. 6. Regulation of AFC by endogenous levels of NO in vivo. There is recent evidence to support a role for regulation of amiloride-sensitive AFC by endogenous -NO from constitutively active iNOS. Hardiman et al. [55]showed that iNOS(-/) mice lack amiloride-sensitive and forskolin stimulated AFC (Figure 4) and amiloridesensitive nasal potential difference when compared to control C57BL/6 mice. These iNOS(-/) mice had the same AFC and NPD without amiloride as the control animals. AFC was eliminated in both control and iNOS(-/-) mice when Na+ was removed from the instilled fluid. Furthermore, iNOS (+/+) mice had significantly higher levels of nitrate and nitrite in their BAL compared to iNOS (-/-) mice. Thus in contrast to aforementioned results showing that large levels of »NO decrease Na+ transport by down regulating both apical and basolateral transporters [45], the studies by Hardiman et al. imply that constitutive production of "NO by iNOS[40] may be needed for these transporters to function properly. The mechanisms involved are currently under investigation. Figure 4. Measurements of alveolar fluid clearance (AFC) in anesthetized, ventilated iNOS (+/+) and iNOS (-/-) mice. AFC was calculated from changes in instilled BSA concentration during a 30 min period. Data are means ±1 SD (n > 6 for each group). In all cases, the instillate contained 5% fat free BSA in NaCl or NMDG+C1' as noted. The osmolarity was adjusted to that of the plasma for the appropriate group. Amiloride (1.5 mM), and or forskolin (50 uM) were also added in the instillate as noted. * p< 0.05 compared with the corresponding control iNOS(+/+) value. # p< 0.05 compared with the corresponding iNOS(+/+) value for the same experimental conditions (from reference [55]).

7. Conclusions. Amiloride-sensitive Na+ channels play an essential role in removal of fluid from the airways under pathologic conditions when RONS are present, therefore understanding the interaction between RONS and ENaC is of tremendous importance. Very little is known about the mechanisms of the effects of RONS on vectorial Na+ transport. Clearly, additional work is needed to understand the cellular and molecular mechanisms by which reactive species regulate both the epithelial Na"1" channels and the Na,K-ATPase.

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Disease Markers in Exhaled Breath N. Marczin and M.H. Yacoub (Eds.) IOS Press, 2002

Luteolin Reduces LPS-Induced TNF-a Production and Protects Mice against LPS Toxicity Angeliki XAGORARI, Anastasia KOTANIDOU, Chans ROUSSOS and Andreas PAPAPETROPOULOS "George P. Livanos " Laboratory, Department of Critical Care and Pulmonary Services, University of Athens, Greece Abstract. We have compared the ability of several flavonoids to modulate the production of TNF-a both in vitro and in vivo and have investigated their mechanism(s) of action. From the flavonoids tested luteolin was the most potent and efficacious in inhibiting TNF-a production through a mechanism involving inhibition of protein tyrosine phosphorylation, ERK1/2 and p38 activation as well as inhibition of NF-icB.

Flavonoids are naturally occurring polyphenolic compounds with a wide distribution in the plant kingdom. The average daily consumption of flavonoids in western diet is estimated to be Ig [1]. This class of compounds numbers more than 4,000 members and can be divided into five subcategories flavones, flavanols, flavanones, flavonols, and anthocyanidines. Flavonoids possess anti-oxidant, anti-tumor, antiangiogenic, anti-inflammatory, anti-allergic and anti-viral properties [2]. In addition, flavonoids inhibit tyrosine and serine/threonine kinases by competing with ATP binding [3]. As agents with tyrosine kinase blocking activity inhibit both LPS-stimulated TNF-a production and LPS-induced lethality in mice [4], we tested whether flavonoids reduce pro-inflammatory cytokine production and investigated the mechanism of action for the most potent of these compounds. To investigate the effects of flavonoids on LPS-induced TNF-a release cultured murine macrophages were pretreated with flavonoids prior to the LPS exposure [5]. Myricetin and catechin showed no effect on LPS-induced TNF-a release, whereas hesperetin, luteolin-7-glucoside and eriodictyol reduced TNF-a release approximately by 50%. Quercetin and luteolin were the two most efficacious inhibitors allowing only for minimal LPS-induced TNF-a release [5]. Our findings are in line with the observations that the flavonoids quercetin and resveratrol inhibit TNF-a production [6,7], To extend our observations in vivo we treated mice with LPS and determined serum TNF-a levels in the presence and absence of luteolin. Control animals had undetectable levels of circulating TNF-a. Pretreatment of mice with luteolin (0.2mg/kg i.p. for 30min) attenuated the increase in serum TNF-a levels elicited by LPS (11.0+2.5ng/ml vs l.l+0.2ng/ml). To test if luteolin can also prevent the LPS-induced lethal toxicity in a mouse model of sepsis, 10-12 week-old animals were injected with DMSO or luteolin (0.2mg/kg) prior to LPS (32mg/kg i.p. S. enteriditis). Mice receiving DMSO or luteolin alone exhibited no lethality, whereas only 4.1% of the mice receiving LPS survived after 7 days. Pretreatment with luteolin increased survival rate throughout the time of observation with 48% of the mice pretreated with luteolin remaining alive after 7 days. To study the mechanism of action of flavonoids we tested the ability of luteolin, to inhibit tyrosine phosphorylation. Exposure of RAW 264.7 cells to LPS led to a time-

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dependent increase in tyrosine phosphorylation. Pretreatment of the cells with luteolin, attenuated LPS-induced tyrosine phosphorylation of many discrete proteins covering a molecular weight size from 40-120kDa [5]. The action of luteolin on tyrosine phosphorylation was comparable to that of genistein, a known tyrosine kinase inhibitor that also blocked the LPS-induced TNF-a release in RAW 264.7 by 75%. Exposure of macrophages to LPS is known to active most of the components of the MAPK signaling cascade [8]. Indeed, exposure of RAW 264.7 to LPS stimulated the phosphorylation of ERK1/2, JNK1/2 and p38 in a time-dependent manner. Activation of the different MAPK signaling cascades is believed to control different steps in the TNFa production process. Activation of ERK1/2 has been reported to stimulate TNF-a transcription and control the transport of TNF-a mRNA from the nucleus to the cytoplasm [9, 10]. On the other hand, p38 and JNK control TNF-a mRNA stability and TNF-a translation [11, 12]. To test the ability of luteolin to interfere with LPS signaling,

Fig.l . Effects of luteolin on NF-KB activation. Top: phospho- and total IicBa levels were determined in RAW 264.7 lysates. Bottom left: immunoblotting of p50 and p65 in nuclear extracts. Bottom right: luciferase activities in transiently transfected cells.

cells were exposed to luteolin prior to the LPS challenge. Luteolin pretreatment blockaded phosphorylation of both ERK1/2 and p38, without affecting the total levels of these kinases. Moreover, pretreatment of cells with a combination of lOuM PD98059 (MEK1/2 inhibitor) and 2.5uM SB203580 (p38 inhibitor) blocked the LPS-induced TNF-a release by 67.7%, suggesting that the effects of luteolin on TNF-a release might be mediated by its ability to block activation of ERK1/2 and p38 pathways. Activation of NF-KB plays a key role in the LPS-induced pro-inflammatory gene expression [13]. To determine if luteolin affects NF-KB activation, RAW 264.7 cells were treated with LPS and p50/p65 translocation to the nucleus was determined (Fig.l). Pretreatment of cells with luteolin blocked translocation of both NF-KB subunits to the nucleus. In addition, treatment of cells with LPS increased iKB-a phosphorylation, leading to a reduction in IicB-a levels; pretreatment of cells with luteolin abolished the effects of LPS on IicB-a. To test if the phosphorylation and translocation events are functionally relevant we tested the luciferase activity in cells transiently transfected with a reporter gene expressed under the control of six KB m-acting elements. Incubation of transfected RAW 264.7 cells with LPS increased luciferase activity in a luteolin-

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sensitive manner indicating that inhibition of pro-inflammatory cytokine expression correlates with decreased NF-icB-stimulated promoter activity. In summary, we have screened a number of flavonoids and have identified luteolin as a potent inhibitor of LPS-stimulated TNF-a release. The mechanism by which luteolin blocks the pro-inflammatory gene expression correlates with inhibition of LPSinduced tyrosine phosphorylation, ERK1/2, p38 and NF-icB activation. Inhibition of the various cascades involved in pro-inflammatory molecule expression by luteolin could be useful in preventing cytokine expression in pathophysiological conditions. References [I] Kuhnau J (1976) World Rev. Nutr. Diet 24: 117-191 [2] Middleton EJ, Kandaswami C and Theoharides TC (2001) Pharmacol. Rev. 52:673-751. [3] Graziani Y, Erikson E, and Erikson RL (1983) Eur. J. Biochem. 135: 583-589 [4] Novogrodsky A, Vanichkin A, Patya M, Gazit A, Osherov N, and Levitzki A (1994) Science 264 (5163): 1319-22 [5] Xagorari A, Papapetropoulos A, Mauromatis A, Economou M, Fotsis T and Roussos C (2001) J. Pharmacol. Exp. Ther. 2%: 181-187. [6] Wadsworth TL, and Koop DR (1999) Biochem.Pharmacol. 57: 941-949 [7] Kawada N, Seki S, Inoue M, and Kuroki T (1998) Hepatology 27: 1265-1274 [8] Feng GJ, Goodridge HS, Harnett MM, Wei XQ, Nikolaev AV, Higson AP and Liew FY (1999) J Immunol. 163:6403-6412. [9] Dumitru CD, Ceci JD, Tsatsanis C, Kontoyiannis D, Stamatakis K, Lin JH, Patriotis C, Jenkins NA, Copeland NG, Kollias G and Tsichlis PN (2000) Cell 103:1071-1083. [10] Geppert TD, Whitehurst CE, Thompson P and Beutler B (1994) Mol. Med 1:93-103. [II] Kontoyiannis D, Pasparakis M, Pizarro TT, Cominelli F and Kollias G (1999) Immunity 10:387-398. [12] Swantek JL, Cobb MH and Geppert TD (1997) Mol.Cell. Bio. 17:6274-6282.[13] Baeuerle PA, and Henkel T(1994)/fwii/. Rev. Immunol. 12: 141-179 [13] Baeuerle PA, and Henkel T (1994). Anna. Rev. Immunol. 12: 141-179

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Acute Lung Injury in the Setting of Cardiopulmonary Bypass David ROYSTON Department of Anaesthesia and Critical Care Royal Brompton and Harefield NHS Trust Harefield Hospital Harefield, Middlesex. UB9 6JH

Abstract. The pivotal part of the innate defense system is to recognize injury and "non self. Once recognized, hemostatic and immune systems integrate to conduct a process whereby the insult is walled off from the rest of the body (using the haemostatic or thrombotic process) and the injury or invasion is subsequently repaired or consumed (typically by phagocytic cells). The innate immune and hemostatic processes involved have much in common with each other [1]. This chapter will concentrate on the pulmonary microvasculature as a target for injury associated with heart surgery. The two questions addressed are 1. Is there incontrovertible evidence for a pulmonary microvascular inflammatory response in humans having heart surgery with cardiopulmonary bypass? 2. Can the use of expired breath markers contribute to our understanding of abnormal lung functions?

1. Lung injury after heart surgery Introduction Pulmonary dysfunction is one part of the so-called postperfusion syndrome. Fever of non-infective origin, renal dysfunction, leucocytosis and abnormal bleeding are other components. In the early years of heart surgery tissue injury and organ dysfunction were largely attributed to micro-emboli and protein denaturation, as there was no unifying hypothesis to explain the complex humoral and cellular events occurring around the time of surgery. Since the 1980s the expansion of methods for measuring aspects of the inflammatory process has lead to post perfusion tissue injury being interpreted as a 'whole body inflammatory response' or systemic inflammatory response syndrome (SIRS). The term systemic implies that the normally localized innate response to trauma or invasion has become unleashed on the whole body. The extracorporeal system, and its driver usually take the blame for this! The mantra of the 1980s was that the contact of blood with the foreign surface of the oxygenator released phlogistic agents that stimulated both the endothelium and the white cell to produce abnormal retention of these cells in the tissues. Once retained the cells would either directly, or following a second challenge, release cytotoxic products to inflict injury on self tissues rather than on the invading hordes. Continuing study and experience suggest that this model is not appropriate to all facets of the abnormal lung functions that follow heart surgery. It is obvious that the lung is in a unique position and therefore may be particularly susceptible, directly or indirectly to different methods and mechanisms of injury. For example, there may be a direct ischaemia-reperfusion injury as the lung is taken from the circuit during the period of bypass. There is an increased incidence of acute pulmonary oedema following revascularisation of the lung after a prolonged time period such as

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following lung transplantation. This effect is also a recognized complication following reestablishment of pulmonary blood flow [2], Lung function is also altered as a consequence of ischaemia-reperfusion of a distant organ. The best categorized example is the increase in protein flux and lung water observed following reperfusion of the ischaemic lower limb [3]. Returning to the model of tissue injury it is apparent that a rise in concentration of a single putative phlogistic agent is not necessarily associated with a significant biological effect. An obvious example of this is the effect of modifications to the surface characteristics of the oxygenator. Heparin bonded or coated perfusion systems have been shown to reduce the activation of certain inflammatory mediators [4, 5]without any physiological benefit or improvement in lung functions [6]. This may be because there are a system of checks and balances in nature. As a general rule for each activator there is a cognate inhibitor. This complex balance is most apparent with severe sepsis. Epidemiological and genetic studies have shown that patients with meningococcal septicaemia are more likely to survive if they have a large TNF and /or a low IL-10 response [7]. Preliminary studies have extended this concept of achieving a balance to human heart surgery. Using NF KB activation [8] or cytotoxicity [9] in human cell lines as the end-point for cell activation, these studies showed no significant effect of addition of plasma separated from the blood of patients after heart surgery. The system showed significant upregulation when the plasma from whole blood stimulated with endotoxin (LPS) was applied to the cell line. Most interestingly this effect of LPS was significantly downregulated when tested in the whole blood collected after heart surgery, implying that the overall balance was now "anti-inflammatory". Mechanisms in pulmonary injury are not the same as in the systemic circulation. Continuing the discussion of the currently held model highlights two problems. These are related to the fact that the lung microcirculation does not follow the pattern of the peripheral circulation with regard to phagocytic cells and blood flow response to injury. The model for an inflammatory reaction, categorized in studies of the peripheral circulation, is that a stimulus will induce an increase in microvascular flow together with an influx of phagocytic cells. This leads to the Galenic response of rubor, calor, dolor, tumor to which loss of function can be added. The physiology and architecture of the pulmonary circulation is completely different to that found in the skin or other microvascular beds. In addition, the traffic of inflammatory cells in the lungs is different from other circulations. There is considerable evidence to show that there is a physiologically prolonged transit time of neutrophils in the lung in comparison with red cells [10, 11]. This is not observed in any other microvascular bed. In addition the mechanism of pathologically increased white cell retention is also different to that of the systemic circulation. The mechanism by which this occurs in the lung is related to the deformability of the cells rather than the presence of adhesion molecules of the immunoglobulin superfamily such as 1C AMI and 1C AM 2; the ligands for the CD11/CD18 adhesion molecules on the neutrophil. For the second part of the response the increased tissue blood flow observed in the peripheral circulation to a phlogistic agent is not seen. Indeed increased pulmonary vascular resistance and pulmonary artery hypertension are a more common consequence of an inflammatory response. Based on these differences it is not unreasonable to suggest that we need to investigate other markers of a pulmonary microvascular inflammatory injury. A logical choice of marker would be something related to breathing and gas transfer.

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Oxygenation as an index of inflammatory lung injury The most obvious pulmonary effect that follows heart surgery is a gas exchange abnormality. In the clinical setting abnormalities of lung function are usually defined in terms of the gas exchanging properties of the lung and respiratory mechanics such as altered compliance. These factors are used in the majority of scoring systems to define severity of lung injury [12]. In the immediate post-operative period many patients will have increased hypoxaemia with stiffer lungs and an abnormal chest radiograph. The first question to address is whether there is any evidence to suggest that these increases or changes in gas exchange and lung mechanics are related to alterations in the permeability or leakiness of the microvasculature to solutes and water. Is this a classic inflammatory response? Solute flux and water accumulation has been measured in humans using three different methods[13]. Macromolecule accumulation into the interstitial can be monitored using radio labeled proteins accumulation. The permeability of the blood gas interface to small solutes can also been measured using the clearance from the lung into the blood of a small radio labeled solute administered by aerosol. Lastly the classic indication dilution technique has been used to calculate permeability-surface area product and an index of lung interstitial water. Using these techniques in patients with a severe lung injury, categorized as the adult respiratory distress syndrome, solute flux measurements were found to be unrelated to gas exchange [14-16]as was an increase in interstitial water [15]. This inconsistency is further highlighted in studies following open-heart surgery. In one study oxygenation and lung mechanics were unrelated to protein accumulation after open-heart surgery [17]. In others the time course of observed changes were different with a statistically significant abnormality in solute flux measured for 48 hours and that for gas exchange at one week[18]. This uncertainty regarding the use of gas exchange and lung function as a marker of an inflammatory response is compounded by two further considerations. First we know that modifications to the plasmatic markers of inflammation (as observed with altered surface coating described above) is not reflected by improved physiology. Second, there are many examples of known pathologies associated with impaired oxygenation after heart surgery, which are not parts of the classic inflammatory response. These include the reduced functional residual capacity associated with general anaesthesia alone, altered shape and motion of the diaphragm and chest wall, atelectasis due to lobar collapse, pleural effusions or following surfactant loss. Many of these factors can, in part be prevented by alterations in the technique of anaesthesia [19]. A possible confounding variable in the system is that patients inhale a mixture that is enriched with added oxygen. This must contribute to the overall "oxidant stress" in the lung microvasculature however quantifying this contribution in humans is difficult. We recognize that rodents are sensitive to the effects of breathing a hyperoxic mixture. Indeed breathing an atmosphere of 100% oxygen is lethal to rats and mice. The precise mechanism of this effect is unclear as • this lethality is not observed in other species such as in birds which show no pathologic pulmonary damage, although hydrogen peroxide accumulates in their airways after exposed to 100% oxygen for prolonged periods. [20] • pulmonary microvascular injury appears to be an all-or-none effect [21] • lethality appears to be related to pleural rather than lung water accumulation [21]. In turn this pleural accumulation in rodents is a consequence of lymphatic drainage rather than parenchymal tissue injury.

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2. Expired breath markers of pulmonary microvascular disease Let us now turn to the possibility of using other markers of pulmonary microvascular injury associated with heart surgery. As with other systems two broad groups are available depending on their presence in the condensate (hydrogen peroxide, isoprostanes) or gas phase (nitric oxide, ethane or pentane). Condensate markers Hydrogen peroxide has been suggested as a marker of oxidant injury to the lung [22]. The authors of this original study showed that condensate hydrogen peroxide was 5 fold higher (1.68±0.35 jamol L"1) in patients with severe lung injury categorized as ARDS than in 16 patients receiving mechanical ventilation without ARDS. The authors also demonstrated that there was no relationship of peroxide concentration to inspired oxygen but there was to plasma lysozyme implying a neutrophil related source for the peroxide. A second report confirmed higher amounts of peroxide with ARDS and also showed no effect of anaesthesia alone on this variable [23]. It is therefore disappointing that the only study of the effects of a period of cardiopulmonary bypass on this end-point showed a 50% reduction in measured peroxide compared to prebypass [24]. This was again not affected by the inspired oxygen tension. Isomers of prostaglandin p20, especially 8-isoprostane has been shown to increase after ischaemia-reperfusion injury to the myocardium in humans [25, 26]. More recently raise concentrations have been measured in expired breath in a variety of chronic inflammatory lung lesions such as asthma and cystic fibrosis [27, 28]. These data suggest a possible role for this measurement in investigation of the lung impairment associated with heart surgery. Gaseous Breath Markers Alkanes such as ethane and pentane have been suggested as markers of oxidant stress and inflammatory injury to the lung. One study demonstrated highly significant, but transient rises in expired ethane during the early reperfusion periods (release of aortic cross clamp and termination of cardiopulmonary bypass) during heart surgery in humans [29]. The relationship of this observation to a pulmonary inflammatory process is unclear as the same study showed that a significant rise was also associate with the use of diathermy or electrocautery. Earlier studies had also shown expired pentane concentration was increased following a 30-minute period of breathing 100% oxygen [18]. The majority of the interest in expired markers an inflammatory response has concentrated on nitric oxide. As discussed in other parts of this monograph the interpretation of a single measurement is confounded by other influences. The first is related to the site of production discussed elsewhere. Second if local production is increased then there may be a concomitant increase in nitrate production. Increased production may be monitored in expired breath or may be shown as an increase in nitrite production. However if the increase in production is related to increase in oxidant stress then there may be a diversion of nitric oxide into peroxinitrite formation. This may be reflected as a decreased nitrite and expired nitric oxide signal despite a total increase in nitric oxide formation. A further explanation may be related to the conditions of the study and the methods of measurement. These factors together may explain the divergent data published thus far. Some studies showed an increase in nitrate formation [30], others have shown decreased [31, 32] or no change in expired nitric oxide concentration [33, 34]. One study also failed to show a change in nitrate production or nitric oxide excretion despite evidence of increased

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neutrophil activation [33]. A further study demonstrated a dichotomy between nitric oxide excretion and nitrate concentrations only when a period of hypoxaemia preceded the period of cardiopulmonary bypass, which produced no effect on its own [35]. We have tried to overcome some of these influences and limitations by measuring the response to an intravenous bolus of glyceryl trinitrate (GTN or nitroglycerine)[36]; a substrate for the microvascular release of nitric oxide. End-tidal nitric oxide is monitored continuously, using a chemiluminescence detector during controlled ventilation with fixed parameters of tidal volume and frequency. The transient increase in expired NO is related to the dose of GTN administered. This observation is not altered by a short period of cardiopulmonary bypass but is significantly depressed with longer periods of ischaemia, as found with lung transplantation [34]. 3. Comment and Conclusions • • • •

If an inflammatory pulmonary lesion occurs with CPB it is transient Endothelial dysfunction does occur but is unlikely due to neutrophil derived oxidant stress. Current data on expired markers is confused due to interaction between oxidants and NO and the NO "sump" in tissues. Unraveling some of these issues will require multiple markers to be measured simultaneously

References 1. Royston D. The evolution of coagulation and inflammation. In: BD S, editor. The Relationship between Coagulation, Inflammation and the Endothelium-A pyramid towards outcome.A Society of Cardiovascular Anesthesiologists Monograph. Philadelphia: Lippincott Williams & Wilkins; 2000. p, 1-30. 2. Fedullo PF, Auger WR, Charmick RN, Moser KM, Jamieson SW. Chronic thromboembolic pulmonary hypertension. Clin Chest Med 1995;16(2):353-74. 3. Welbourn CR, Goldman G, Paterson IS, Valeri CR, Shepro D, Hechtman HB.Neutrophil elastase and oxygen radicals: synergism in lung injury after hindlimb ischemia. Am J Physiol 1991;260(6 Pt 2):H 1852-6. 4. Gu YJ, van Oeveren W, Akkerman C, Boonstra PW, Huyzen RJ, Wildevuur CR. Heparin-coated circuits reduce the inflammatory response to cardiopulmonary bypass. Ann Thorac Surg 1993;55(4):917-22. 5. Svennevig JL, Geiran OR, Karlsen H, Pedersen T, Mollnes TE, Kongsgard U, Froysaker T. Complement activation during extracorporeal circulation. In vitro comparison of Duraflo II heparin-coated and uncoated oxygenator circuits. J Thorac Cardiovasc Surg 1993;106(3):466-72. 6. Gravlee GP. Heparin-coated cardiopulmonary bypass circuits. J Cardiothorac Vase Anesth 1994;8(2):213-22. 7. Westendorp RG, Langermans JA, Huizinga TW, Elouali AH, Verweij CL, Boomsma DI, Vandenbroucke JP, Vandenbrouke JP. Genetic influence on cytokine production and fatal meningococcal disease. Lancet 1997;349(9046): 170-3. 8. Hoare G, Kovesi T, Bundy R, Royston D, Yacoub M, Marczin N. Cardiopulmonary bypass (CPB) and Lipopolysaccharide (LPS) induced inflammatory activity in the plasma of patients undergoing cardiac surgery. Am J Resp Crit Care Med 2001;163(5):A276. 9. Bundy R, Kovesi T, Royston D, Yacoub M, Marczin N. Cardiopulmonary bypass (CPB) and endotoxin (LPS) induced cytotoxic activity in the plasma of patients undergoing coronary bypass surgery. Am J Resp Crit Care Med 2001;163(5):A213. 10. MacNee W, Selby C. New perspectives on basic mechanisms in lung disease. 2. Neutrophil traffic in the lungs: role of haemodynamics, cell adhesion, and deformability. Thorax 1993;48(l):79-88 11. Selby C, MacNee W. Factors affecting neutrophil transit during acute pulmonary inflammation: minireview. Exp Lung Res 1993;19(4):407-28. 12. Moss M, Goodman P, Heinig M, Barkin S, Ackerson L, Parsons P. Establishing the relative accuracy of three new definitions of the adult respiratory distress syndrome. Crit.Care Med 1995;23(10):1629 - 1637. 13. Jones JG, Royston D, Minty BD. Changes in alveolar-capillary barrier function in animals and humans. Am Rev Respir Dis 1983;127(5 Pt 2):S51-9. 14. Rinaldo JE, Pennock B. Effects of ibuprofen on endotoxin-induced alveolitis: biphasic dose response and dissociation between inflammation and hypoxemia. Am J Med Sci 1986;291(l):29-38.

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15. Rinaldo JE, Borovetz HS, Mancini MC, Hardesty RL, Griffith BP. Assessment of lung injury in the adult respiratory distress syndrome using multiple indicator dilution curves. Am Rev Respir Dis 1986;133(6):1006-10. 16. Royston D, Braude S, Nolop KB. Failure of aerosolised 99mTc DTPA clearance to predict outcome in patients with adult respiratory distress syndrome. Thorax 1987;42(7):494-9. 17. Macnaughton PD, Braude S, Hunter DN, Denison DM, Evans TW. Changes in lung function and pulmonary capillary permeability after cardiopulmonary bypass. Crit Care Med 1992;20(9): 1289-94 18. Morita S, Snider MT, Inada Y. Increased N-pentane excretion in humans: a consequence of pulmonary oxygen exposure. Anesthesiology 1986;64(6):730-3. 19. Royston D. Surgery with cardiopulmonary bypass and pulmonary inflammatory responses. Perfusion 1996;ll(3):213-9. 20. Somayajulu RS, Mukherjee SP, Lynn WS, Bennett PB. Pulmonary oxygen toxicity in chickens and rabbits. Undersea Biomed Res 1978;5(l):l-8. 21. Royston BD, Webster NR, Nunn JF. Time course of changes in lung permeability and edema in the rat exposed to 100% oxygen. J Appl Physiol 1990;69(4): 1532-7. 22. Baldwin SR, Simon RH, Grum CM, Ketai LH, Boxer LA, Devall LJ. Oxidant activity in expired breath of patients with adult respiratory distress syndrome. Lancet 1986; 1(8471):! 1-4. 23. Wilson WC, Swetland JF, Benumof JL, Laborde P, Taylor R. General anesthesia and exhaled breath hydrogen peroxide. Anesthesiology 1992;76(5):703-10. 24. Mumby S, Block R, Petros AJ, Gutteridge JM. Hydrogen peroxide and catalase are inversely related in adult patients undergoing cardiopulmonary bypass: implications for antioxidant protection. Redox Rep 1999;4(l-2):49-52. 25. Delanty N, Reilly MP, Pratico D, Lawson JA, McCarthy JF, Wood AE, Ohnishi ST, Fitzgerald DJ. FitzGerald GA. 8-epi PGF2 alpha generation during coronary reperfusion. A potential quantitative marker of oxidant stress in vivo. Circulation 1997;95(11):2492-9. 26. Reilly MP, Delanty N, Roy L, Rokach J, Callaghan PO, Crean P, Lawson JA, FitzGerald GA. Increased formation of the isoprostanes IPF2alpha-I and 8-epi-prostaglandin F2alpha in acute coronary angioplasty: evidence for oxidant stress during coronary reperfusion in humans. Circulation 1997;96(10):3314-20. 27. Montuschi P, Corradi M, Ciabattoni G, Nightingale J, Kharitonov SA, Barnes PJ. Increased 8isoprostane, a marker of oxidative stress, in exhaled condensate of asthma patients. Am J Respir Crit Care Med 1999; 160(1):216-20. 28. Montuschi P, Kharitonov SA, Ciabattoni G, Corradi M, van Rensen L, Geddes DM, Hodson ME, Barnes PJ. Exhaled 8-isoprostane as a new non-invasive biomarker of oxidative stress in cystic fibrosis Thorax 2000;55(3):205-9. 29. Andreoni KA, Kazui M, Cameron DE, Nyhan D, Sehnert SS, Rohde CA, Bulkley GB, Risby TH. Ethane: a marker of lipid peroxidation during cardiopulmonary bypass in humans. Free Radic Biol Med 1999;26(3-4):439-45. 30. Ruvolo G, Greco E, Speziale G, Tritapepe L, Marino B, Mollace V, Nistico G. Nitric oxide formation during cardiopulmonary bypass. Ann Thorac Surg 1994;57(4): 1055-7. 31. Beghetti M, Silkoff PE, Caramori M, Holtby HM, Slutsky AS, Adatia I. Decreased exhaled nitric oxide may be a marker of cardiopulmonary bypass-induced injury. Ann Thorac Surg 1998;66(2):532-4. 32. Ishibe Y, Liu R, Hirosawa J, Kawamura K, Yamasaki K, Saito N. Exhaled nitric oxide level decreases after cardiopulmonary bypass in adult patients. Crit Care Med 2000;28(12):3823-7. 33. Brett SJ, Quinlan GJ, Mitchell J, Pepper JR, Evans TW. Production of nitric oxide during surgery involving cardiopulmonary bypass. Crit Care Med 1998;26(2):272-8. 34. Marczin N, Riedel B, Gal J, Polak J, Yacoub M. Exhaled nitric oxide during lung transplantation. Lancet 1997;350(9092):1681-2. 35. Pearl JM, Nelson DP, Wellmann SA, Raake JL, Wagner CJ, McNamara JL, Duffy JY. Acute hypoxia and reoxygenation impairs exhaled nitric oxide release and pulmonary mechanics. J Thorac Cardiovasc Surg 2000; 119(5):931-8. 36. Marczin N, Riedel B, Royston D, Yacoub M. Intravenous nitrate vasodilators and exhaled nitric oxide. Lancet 1997;349(9067):1742.

Disease Markers in Exhaled Breath N. Marczin and M.H. Yacoub (Eds.) fOS Press, 2002

Oxidative Stress During Cardiac Surgery in Diabetic Patients Tamas K6VESI, Ruth BUNDY, Ginette HOARE, David ROYSTON and Nandor MARCZIN Department of Anaesthesia and Cardiothoracic Surgery, Royal Brompton & Harefield NHS Trust, Harefield Hospital, Harefield, UK Abstract. Accelerated atherosclerosis and specifically, coronary artery disease are major determinants of morbidity and mortality in patients with diabetes mellitus (DM). These patients are at a significantly higher risk when undergoing surgery for coronary revascularisation and frequently develop myocardial and pulmonary injury after cardiopulmonary bypass (CPB). The mechanisms of this injury remain unclear, however, the role of increased oxidant stress and enhanced neutrophil leukocyteendothelial cell interactions leading to disturbed vascular regulation is being increasingly recognised. On the basis of recent observations suggesting that reactive oxygen intermediates (ROI) are generated in higher amounts by diabetic patients, we hypothesised that CPB augments ischaemia-reperfusion-induced oxidant stress in patients with diabetes which could interfere with the endogenous L-arginine-NOcGMP pathways and metabolism and efficiency of exogenous NO donors such as glyceryl trinitrate (GTN). As a first step we have investigated these events by measuring exhaled NO. In addition we have initiated studies to explore the oxidative stress related influence of high glucose concentrations on human endothelial cell survival and inflammation in culture. This chapter presents our preliminary experiments.

1. Introduction Diabetes Mellitus (DM) appears to be a major source of morbidity in developed countries. As the effect of long-term exposure to high glucose concentrations destructive changes develop in many organs of diabetic patients, of them in terms of morbidity and mortality probably the most important is the vascular system. The likelihood of coronary artery disease (CAD) is 3-to 5-fold higher in diabetic patients despite controlling other risk factors such as obesity, hypercholesterinaemia, hypertension and smoking. Since the availability of insulin, up to 3/4 of all deaths among diabetics can be directly attributed to CAD [1,2]. It is not surprising that year-by-year an increasing number of diabetic patients require coronary artery surgery carrying higher risks of perioperative complications. A retrospective study showed increased risk of lung, renal and neurological complications in the postoperative period in diabetic patients compared to a non-diabetic population [3,4], In the majority of the cases coronary artery bypass grafting surgery is performed utilizing cardiopulmonary bypass (CPB), which is associated with two major types of complications. CPB leads to an ischaemia-reperfusion related lung injury with associated dysfunction of the pulmonary endothelium. In addition to this, during CPB the blood is exposed to large areas of synthetic material and the activation of the cellular components induces a systemic inflammatory response.

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2. Oxidative stress during cardiac surgery In previous studies it has been demonstrated that both the CPB-induced oxidative stress and inflammatory responses were higher in patients with DM [5]. Due to chronic hyperglycaemia there is a higher degree of oxidation of the cellular components resulting in an increased formation of lipid hydroperoxides, thiobarbituric acid substances and FIisoprostanes which generates higher amounts of reactive oxygen intermediates [1]. On the other hand, in diabetic patients the antioxidant capacity of the plasma also appears to be reduced. Several factors might contribute to the endothelial dysfunction and the impaired nitric oxide (NO) production-metabolism in diabetics. These include inappropriate utilization of arginine for NO synthesis, abnormal nitric oxide synthase (NOS) activity due to inadequate co-factors, higher NO quenching by advanced glycosylation end-products or the increased synthesis of ROI destroy NO bioactivity [6]. On the basis of these suggestions we wanted to test the hypothesis that the diabetes related oxidative stress might interfere with the NO pathways and results in NO consumption, which could be detected during cardiac surgery at the bedside by monitoring exhaled NO levels. Thus, we investigated the characteristics of oxidative stress and pulmonary endothelial dysfunction with measuring endogenous (epithelial) and nitroglycerine (glycerine trinitrate; GTN)-induced exhaled NO in diabetics vs. controls undergoing coronary artery surgery with CPB. As demonstrated earlier in both animal and human studies, the endogenous NO pathways in the pulmonary microvasculature could be augmented by intravenous administration of exogenous NO-donors resulting in increased NO concentrations in the expired breath [7,8]. It has also been demonstrated that oxidative stress during lung transplantation results in consumption of NO and reduction in exhaled NO. It has been postulated that GTN-induced exhaled NO might be a useful tool in monitoring metabolic function of pulmonary microvasculature [9]. 3. Exhaled NO in diabetic patients

Fig. 1.

Exhaled NO measurements were performed in intubated and mechanically ventilated patients using standardized ventilatory settings before, 1 and 3 hours after CPB [8]. Baseline NO measurements were followed by intravenous administration of 1, 2 and 3 n-g/kg GTN with exhaled NO response recorded. Performing the exhaled NO measurements before and after CPB in both control and diabetic subjects, we found that basal exhaled NO levels

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were not different between these two groups and that exhaled NO remained unchanged folowing CPB (Fig. 1). Intravenous bolus administration of 1, 2 and 3 ug/kg GTN resulted in a rapid, transient and dose-dependent increase in exhaled levels of NO. In non-diabetic patients these GTN-induced exhaled NO responses were significantly smaller at 1 hour and 3 hours after CPB when compared to levels measured before CPB. In the diabetic group the exhaled NO response to GTN appeared to be less and exhibited a further decrease in the post-bypass period (Fig. 2). These preliminary data suggest that ischaemia-reperfusion injury during routine open-heart surgery does not influence basal exhaled NO reflective of epithelial NO mechanisms, but is associated with some microvascular dysfunction as judged from consumption of GTNderived NO. While we found no evidence for altered epithelial NO mechanisms in diabetic patients, the GTN-induced exhaled NO seems to be attenuated potentially suggesting an ongoing oxidative stress in the pulmonary microvasculature. 4. Influence of high glucose on endothelial cell function in culture There are several data published about the influence of high glucose concentrations on endothelial cell function in culture. High glucose has been shown to activate NFKB related gene induction resulting in inflammation process and apoptotic cell injury. However, most of these experiments were performed on either animal cells or on human umbilical vein endothelial cells (HUVECs) with contradicting results. Our aim was to extend these studies to adult human vascular endothelial cells. Therefore, in our experiments we used human aortic (HAEC) and pulmonary endothelial cells (PAEC) to study cell proliferation, viability and NF-KB mediated gene induction in cells exposed to different glucose concentrations in the range of 5.5 — 30 mM, which is relevant to clinical situations in diabetic patients.

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There was no evidence of any morphological changes in HAEC characteristic for apoptosis and cytotoxicity as a result of high glucose exposure. Elevated glucose (15 mM) had no effect on HAEC proliferation, only high glucose concentrations (30 mM) had moderate influence on serum-induced replication of human aortic endothelial cells in culture (Fig. 3). Control experiments using similar concentrations of mannitol suggested that this inhibition was specific for glucose and was unlikely to be related to osmotic effects. Similarly to data obtained in the presence of serum, high glucose concentrations remained ineffective in causing endothelial injury in the absence of serum. Finally, we have studied the influence of glucose on endothelial survival in cytokine environment, in a model of cytokine-induced apoptosis in cycloheximide sensitised endothelial cells. Endothelial cells were grown in normal, medium and high glucose and were exposed to different concentrations of bacterial lipopolisaccharyde (LPS) and cytokines such as interleukin-1 (IL-1). As shown in Figure 4, IL-1 reduced endothelial cell survival in a concentration dependent manner, however this was the same in the presence of high glucose. Thus high glucose failed to potentiate LPS and cytokine-induced apoptosis. In studying cytokine induced NF-KB activation and upregulation of endothelial adhesion molecule (ICAM) expression, we compared the influence of high glucose on ICAM expression in the absence or presence of cytokine. High glucose concentrations failed to effectively activate the NFKB pathway and induce ICAM upregulation. NF-KB activation and ICAM induction was however evident in response to IL-1. Interestingly, glucose appeared to attenuate IL-1 induced ICAM induction (Fig. 5). Summary During routine cardiac surgery utilizing CPB ischaemia-reperfusion injury is associated with some microvascular dysfunction as judged from consumption of GTNderived NO. When compared to control patients, basal exhaled NO either before or after CPB remains similar in diabetic patients suggesting that the basal NO production of the airway epithelium remains unaltered. However, in diabetic patients the GTN-induced exhaled NO appears less even before CPB, which might suggest attenuated GTN metabolism and/or consumption of released NO perhaps due to ongoing oxidative stress in

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the microvasculature. These issues will be clarified in future experiments utilising plasma and exhaled breath condensate markers of oxidative stress in the control and diabetic patients. Our cell culture experiments investigating the influence of high glucose concentrations on human aortic endothelial cells suggest only a moderate effect on cell replication and apoptosis. In addition, glucose did not potentiate cytokine-induced cytotoxicity and not only failed to activate the NF-KB pathway and adhesion molecule upregulation but appeared to attenuate cytokine-mediated 1CAM induction. These data might imply species and cell type specific responses to pathological glucose concentrations and suggest that the adult endothelial tested are equipped with effective protective mechanism against high glucose induced cellular injury. The mechanisms of glucoseinduced attenuation of cytokine-induced NF-KB responses require much further study but if proved correct, might have significant clinical implications regarding wound healing problems in diabetic patients and their susceptibility to perioperative indfections. References [1]

Keaney JFJ, Loscalzo J: Diabetes, oxidative stress, and platelet activation [editorial; comment]. Circulation 1999 ;99:189-191

[2]

Bierman EL: George Lyman Duff Memorial Lecture. Atherogenesis in diabetes. Arterioscler Thromb 1992;12:647-656

[3]

Peters A, Kerner W: Perioperative management of the diabetic patient. Exp Clin Endocrinol Diabetes 1995;I03:213-218

[4]

Pallas F, Larson DF: Cerebral blood flow in the diabetic patient. Perfusion 1996;11:363-370

[5]

Chello M, Mastroroberto P, Cirillo F, Bevacqua E, Carrano A, Perticone F, Marchese AR: Neutrophil-endothelial cells modulation in diabetic patients undergoing coronary artery bypass grafting. Eur J Cardiothorac Surg 1998; 14:373-379

[6]

Pieper GM: Review of alterations in endothelial nitric oxide production in diabetes: protective role of arginine on endothelial dysfunction. Hypertension 1998;31:1047-1060

[7]

Husain M, Adrie C, Ichinose F, Kavosi M, Zapol WM: Exhaled nitric oxide as a marker for organic nitrate tolerance. Circulation 1994;89:2498-2502

[8]

Marczin N, Riedel B, Royston D, Yacoub M: Intravenous nitrate vasodilators and exhaled nitric oxide. Lancet 1997;349:1742-1742

[9]

Marczin N, Riedel B, Gal J, Polak J, Yacoub M: Exhaled nitric oxide during lung transplantation. Lancet 1997;350:1681-1682

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Part VI.

New Technological Developments

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Disease Markers in Exhaled Breath N. Marczin and M.H. Yacoub (Eds.) IOS Press, 2002

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Exhaled Breath Condensate Gunther BECKER FILTLung and Chest Diagnostics Ltd., Berlin, Germany, www.fih.de Abstract. The study was aimed at recovering mediators of inflammation from the airways by collecting expired air samples using a new technical approach for mediator sampling in diseased states and in the normal bronchial system. Predicted value of a certain marker (LTB4) was checked in 106 volunteers without any history of airway diseases. Significant levels of Leukotriene B4 and hydrogen peroxide were found in healthy volunteers too. The LTB4 content of exhaled breath condensate was doubled after bronchial challenge with allergen in asthmatics. In healthy people with irritative inhalative exposition LTB4 was 50 % higher in mean than in non-exposed healthy controls. The exhaled breath condensate seems to be a sufficient tool for noninvasive sampling of specimen from the lower airways. Nevertheless, there is a strong need for further standardisation of the method. Exhaled breath condensate is still lacking on predicted values in healthy and disease state. We need more common knowledge about influences of pattern of breathing and environmental factors.

1. Introduction Breath condensate is the fall-out from exhaled air during cooling down to about zero degrees. This condensate contains a couple of non-volatile substances like cell markers, markers of inflammation and degradation substances from protein and fatty-acid metabolism [1; 2, 4, 9]. The following paper describes the possibility of sampling breath condensate and its use as a diagnostic tool in airway diseases. 2. Methods Exhaled breath condensate (EBC) was sampled using ECoScreen™ (EJAEGER Inc.). The principle of non-rebreathing sampling system was patented by FILT in May 1994 (PCT/DE95/00633, EP 0759169). Exhaled air is thereby directed by a non-rebreathing valve through a disposable cooling tube with a temperature of approximately minus 30 °C. After subjects had been breathing normally for a period of 15 minutes, approximately 3-5 cc of primarily breath condensate was taken. During the entire experiment, forced ventilation was not permitted. The recommended sampling time was over 10 minutes [5, 6]. Cooling capacity was increased by special sized flow-directed laminar tubes. The condensate was stored at minus 80 °C until it was analysed. Analysis of exhaled breath condensate was performed by laboratory tests, specially fitted for this cell-free solution with low protein content. Commercially available tests were adapted for breath condensate by special standard solutions from pooled condensate with added standard substances. Leukotriene B4 (LTB4) was measured by enzyme linked immunoassay (EIA) of CoulterImmunotec.

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H2O2 was measured by chemiluminescence method using lucigenine for signal amplification. NO was measured in exhaled air by a mass spectrometer (EcoPhysics™ Konstanz). In three different studies, significance of exhaled breath parameters were checked for usefulness and reproducibility in healthy volunteers and patients with asthmatic diseases and COPD: 1. LTB4 in EBC measurements were conducted in healthy volunteers with no previous history of lung and airway diseases to determine the effects of exposure to irritative inhalatives. 100 office workers and 100 hairdressers of both genders participated as volunteers. Within the study, during a random week, measurements were taken on day 1 and 5. 2. Bronchial challenge test (BCT) was performed in 30 allergic asthmatics of both genders with the specific allergen. The antigen was determined by RAST and skin Prick test. Patients had to inhale a 1 to 1.000 dilution of the allergen from an airbag of 10 1 volume administered by PARI-Boy jet nebulizer###. Allergen inhalation was stopped after reaching a significant decrease of FEV, or after the airbag was empty. The target parameter in EBC was LTB4. 3. H2O2, LTB4 in EBC and NO in exhaled air were measured in study with both healthy volunteers (n=19) and patients with COPD (n=25). FEV, was checked for control purpose. The three studies were performed independent of each other. Patient examinations were performed according to ethical rules and regulations. Each patient had to sign an informed consent form. 3. Results EBC sampling was possible all patients and volunteers. The subjects experienced no inconveniences while using the ECoScreen™. Detectable amounts of the analytes were found in all samples. Concentrations of markers measured in EBC, in relation to lung function, are detailed in the Table 1.below. Lung function of healthy volunteers were almost normal. Table 1: LTB4 in healthy volunteers at the work-place (office vs. barbershop) on day 1 and 5. Group Day N Unit Mean SD Median t-Test 1 vs. 5 t-Test O. vs. B.

Office 1 95 pg/m! 99,4 91,04 75,4 P=0,002

Office 5 91 pg/ml 114,9 121,62 76,4 P=0,04 P=0,0035

% Diff. 15,57

Barbershop I 80 pg/ml 157,7 124,55 136,1

Barbershop 5 77 pg/ml 166,5 139,70

% Diff. 5,61

152,2 0,18

There were no differences in lung function tests (FVC, FEV,, R) between the groups. Lung function in all volunteers were within normal ranges. Trends in lung function according to day, time and age were not detectable.

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Table 2: LTB4 and FEVl (Mean±SEM) in EBC before and after BCT with specific allergen Unit

Before BCT

LTB4

Pg/ml

77±16,0

145±32,0

164±39,8

24 h after BCT 197±48,3

FEVl

A%

-

-14,7±16

+3,0±27,6

+ 1,4±9,9

Immediately after BCT

4-6h after BCT

Table 3: FEVt, hydrogen peroxide (H2O2), LTB4 and NO (Mean±SD) in EBC in healthy controls and patients with COPD without exacerbation. Unit N FEVl

1

LTB4

pg/ml

NO

jiMol ppb

HA

Healthy control

COPD

Significance

19 3,77±0,7 110±88 0,501±1,2 3,77±2,5

25 1,92±1,2 12351450 1,722+1,78 6,37±5,1

P< 0,05 P< 0,05 P< 0,05 n.s.

4. Discussion Significant levels of the markers were found in all volunteers. There were significant differences between healthy volunteers working in an office in comparison to hairdressers exposed to irritative inhalative agents in a barbershop. It is worth mentioning that LTB4 seems to be one part of the physiological airway regulation in healthy people. It was possible to show significant changes of exhaled LTB4 during irritative airway exposition. Changes in LTB4 were not correlated to differences in traditional lung function tests, FVC, FEV, and resistance, e.g. [7]. A significant increase of exhaled LTB4 was shown after bronchial challenge tests with allergen. This increase was stable for at least 24 hours. A socalled late response with sustained or repeated decrease of airway function 4 to 6 hours after challenge was not detected in any of the patients. In contrast a long-lasting activation of LTB4 release was visible. This could be a hint for an activation of neutrophils also found in slight allergic response. The preliminary study data provides some new information about allergic airway response. It seems to be possible to detect cellular activation in the airways in an early phase with or without non-significant reduction of lung function [3], Measurement of ECP or EPX was only successful in 9 out of 30 patients in EBC. There were no significant differences between pre and post BCT data. The number of cases were too small to give sufficient data about reproducibility and trends of eosinophilic markers ECP and EPX in EBC [3]. It was possible to measure NO, LTB4 and hydrogen peroxide simultaneously in the third preliminary study [8]. Patients with moderate and severe airflow limitations during spontaneous breathing were able to use the breath condensate sampler for a duration of 10 to 15 minutes.

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Significant concentrations of H2O2, LTB4 in EBC and NO in exhaled air were found. In smokers with COPD significant higher levels of the parameters were detected compared to healthy controls. The differences in H2O2 and LTB4 were significant between both study groups (p< 0,05). Whereas, NO concentrations in exhaled air showed a tendency to higher values in COPD, but not were not statistically significant. The sampling of EBC was performed on an outpatient basis by a pneumologist. The whole sampling process was integrated into be integrated into a routine diagnostic procedure. It was possible to show significant levels LTB4 in EBC in healthy volunteers and in inhalative exposed workers. In the case of specific allergen challenge in asthma, EBC parameters were able to give more sensitive information than traditional lung function tests. At least the safety of BCT could be improved by early detection of airway response or response of cells in the airways. Also in airflow limitations breath condensate was easy to obtain during spontaneous breathing. Sampling of exhaled breath condensate is a useful non-invasive procedure to obtain samples from the deeper airways. 5. Implications Exhaled breath condensate could be the tool for development of non-invasive methods of airway diagnostic in inflammatory airway diseases. There is a current lack in the immediate analysis of markers because of unsolved problems in analysis of EBC. The introduction of biosensors for immediate analysis will give the possibility to use EBC just like traditional lung function tests. References [1] A. Antczak, D. Nowak, B. Shariati, M. Krol, G. Piasekka, Z. Kurmanowska: Increased hydrogen peroxide and thiobarbituric acid -reactive products in expired breath condensate of asthmatic patients. Eur Respir J 10(1997)1235-1241. [2] P.J. Barnes, S.A. Kharitonov: Non-invasive determination of inflammatory airway diseases. Jaeger-info breath-condensate, Suppl. 1, march (2001) 3-7. [3] G. Becher, E. Beck, G. Neubauer, E. Stresemann, K. Norpoth, W. Schiitte, St. Hummel, J. Lichey: Leukotrienes in Breathing Condensate Released During Bronchial Challenge Test. Amer J Respir Crit Care Med 153 /4 (1996) A214. [4] G. Becher, K. Winsel, E. Beck, E. Stresemann: Leukotriene B4 in breathing condensate of patients with bronchopulmonary diseases and in normals. J Appl Cardiopulm Pathophysio! 5 (1995) 215-219. [5] G. Becher, E. Beck, M. Rothe, G. Neubauer, E. Stresemann: Vorrichtung zur Sammlung von nichtgasformigen Bestandteilen der Ausatemluft durch Ausfrieren (Atemkondensat). mt Medizintechnik. 117(1997) H3; 89-95. [6] G. Becher, E. Beck, M. Rothe, E. Stresemann: Das Atemkondensat als Methode zur nichtinvasiven Erfassung von Entztindungsmediatoren aus den unteren Atemwegen. Pneumologie 51 Sonderheft 2(1997) 456-459. [7] G. Becher, M. Rothe, R. Stresemann, E. Beck, F. Falck: Bestimmung von Entzundungsparametem im Atemkondensat zur Friiherkennung von Inhalationsschaden. Schr.-reihe der BAfAAM FB 788. Dortmund-Berlin 1998. [8] E. Beck, G. Becher, M. Rothe, M. Heinze, B. Neubauer, A. Bodner: Vergleich der Konzentration von NO und H2O2 im Exhalat von Patienten mit COPD sowie gesunden Nichtrauchem. 40. Kongr. Dt. Gesell. Pneumol., Bad-Reichenhall, 17.-20. Marz 1999 (oral presentation). [9] L. Scheideler, H-G. Manke,U. Schwulera, O. Inacker, H. Hammerle: Detection of non-volatile macromolecules in breath. Am Rev Respir Dis 148 (1993) 778-784.

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Smell as a Diagnostic Tool in the 21st

Century: The Portable Electronic Nose Timothy E. BURCH and Steven A. SUNSHINE Cyrano Sciences, Inc., 73 N. VinedoAve., Pasadena, CA91107 USA Abstract. The use of electronic sensor arrays in medical science has been demonstrated recently. Differentiation and identification of upper respiratory bacteria is possible in the laboratory using the electronic nose. From the in vitro results, it may be possible to use this device for point of care diagnosis of bacterial infection, pulmonary disease and other significant diseases within the primary care physician's office. Early results obtained with a Cyrano prototype medical electronic nose demonstrate the device can distinguish patients with differing severity of intrapulmonary bacterial infection. Further, the results may be correlated with clinical diagnostic indicators of infection. Validation studies are in progress.

1. Introduction The sense of smell has been a part of medicine for thousands of years, from early Chinese and Greek practitioners, to modern day aromatherapy. Reviews of "medical olfaction" include compilations of anecdotal descriptions of odors [1,2]. However, smell has not been utilized for routine diagnostic purposes, perhaps due to the subjective nature of odor recognition as well as the possibly complex biological origins of chemical signatures that constitute a medical odor. Advances in odor sensing technology, signal processing and diagnostic algorithms have created chemical sensing and identification devices called "electronic noses". The electronic nose has been used for several years in a wide variety of industrial and commercial applications. In the laboratory, these devices have been shown to differentiate bacteria cultures [3,4]. In veterinary science, an electronic nose device was used to identify dietary ketosis in dairy cattle from breath measurement. In medicine, applications have been reported that range from the detection of pneumonia to differentiation of serum from cerebrospinal fluid [5,6]. Recently, the potential use of the electronic nose as a screening tool was demonstrated for bacterial vaginosis and urinary tract infection [7,8]-

2. The Portable Electronic Nose The Cyranose™ 320 (Figure 1) is a commercial handheld chemical vapor detector containing a sensor array of 32 chemically sensitive resistors, a sampling system, a data acquisition system, and a processor. The sampling system delivers ambient air and the sample vapor to the sensors in sequence. The processor and embedded pattern recognition software collect and analyze the differential responses of the sensors to the sample vapor.

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The pattern of response from the sensor array is "learned" by the device, which enables subsequent identification of unknown vapors. Each sensor in the Cyrano array is a thin-film of an insulator (polymer) containing conductive particles (carbon black) that make the composite a resistor (Figure 1). The polymer-composite resistors absorb chemical vapors from the air, and in so doing, their electrical resistance increases. The response of each individual sensor within the array depends, in part, on the thermodynamic partitioning of chemicals between the sample vapor and the insulator and the diffusion coefficient of the vapor in the film.

Figure 1. The commercial Cyranose™ 320 handheld chemical vapor detector and the Cyrano sensor array.

3. Case Studies using the Cyrano Electronic Nose Cyrano Sciences is developing prototype medical electronic noses for detection and diagnosis of a variety of disease states. One area of focus is the diagnosis of bacterial infection, in particular, upper respiratory infection (URI). The gold standard for URI diagnosis is microbiological culture. In practice however, cultures are not always prescribed for reasons that include the time required for laboratory results, typically 48 hours. As a screening device in the primary care physician's office, a medical electronic nose could aid in detection and diagnosis of bacterial URI. Effective screening could reduce the number of negative samples sent for microbiological culture and the number of prescriptions for broad-spectrum antibiotics, thereby reducing drug resistance of bacteria. As a first proof of technical feasibility, pure cultures of the most common URI pathogens (Streptococcus pneumoniae, Streptococcus pyogenes, Staphylococcus aureus, Branhemalla catarrhalis, Haemophilus influenzae) were presented to the sensor array along with uninoculated medium controls. Representative results are shown in Figure 2. The in vitro results show distinct clustering by species or genus and demonstrate that the Cyrano sensor array can detect unique chemistries in the air above growing cultures. The patterns of response to these chemical differences are stored as digital "smellprints". Training the device on pure cultures allows for identification of unknown cultures that are presented for analysis. Over a two-week period, the prototype device correctly identified the five bacteria with a 93% to 100% correct success rate after a single training session on the first day.

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Figure 2. Canonical discriminant scores for the principle bacteria responsible for upper respiratory infection (URJ) in 24 hr cultures. Identification of bacteria is possible due to the distinct clustering of bacteria by species.

The Cyrano prototype medical electronic nose is being evaluated for early diagnosis of pneumonia infection in patients on mechanical ventilators at the University of Pennsylvania Hospital. Ventilator associated pneumonia (VAP) occurs in 10-25% of patients intubated for longer than 48 hours, and has an associated mortality rate of 25-70% [9]. Early detection of VAP would allow for timely and effective treatment and a significant improvement in patient outcome. In the clinical study, air is sampled directly from the ventilator circuit of patients and the response from the sensor array is compared to clinical indicators of infection. The combined pulmonary infection score (CPIS) incorporates patient temperature, leukocyte count, radiography and several other measures to estimate the risk for pneumonia. Preliminary data for 15 patients (Figure 3) shows a close correspondence between the CPIS score and the response of the Cyrano sensor array. [One patient with medium CPIS score in Fig. 3 was subsequently diagnosed with ARDS; the patient died the folio-wing day]. Additional testing is underway to verify these early results and to develop a predictive model for the degree and timing of infection.

4. Summary Laboratory results and preliminary clinical studies using a prototype medical electronic nose indicate there may be some conditions where electronic olfaction can be used for detection and diagnosis of illness. These early results require validation through additional clinical study. The link between detection and identification of a chemical vapor and a defined illness will require confirmation by chemical analyses and correlation with appropriate clinical diagnostic measures of disease. Once confirmed, "smart" devices using sensor array technology may become a routine part of disease diagnosis and patient care.

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Figure 3. Correlation of the predicted and actual pneumonia score for ventilator patients determined using the Cyrano medical prototype. The diagonal line has unit slope. Patients with medium to high score (> 40) would be treated for infection while patients with low score (0-20) would not.

Acknowledgements We thank Olivia Deffenderfer for assistance with data analysis and our clinical collaborators Dr. William Hanson, Dr. Erica Thaler and Meta Phillips, RN. In vitro experimentation was supported in part by WelchAllyn, Inc.

References [1] G. Hayden, Olfactory Diagnosis in Medicine, Postgrad. Med. 67 (1980) 110-118. [2] M. Smith, The Use of Smell in Differential Diagnosis, Lancet 1 (1982) 1452. [3] J.W. Gardner, H.W. Shin, E.L. Hines, An Electronic Nose System to Diagnose Illness, Sensors and Actuators B 70 (2000) 19-24. [4] A.K. Pavlou and A.P.F. Turner, Sniffing out the Truth: Clinical Diagnosis Using the Electronic Nose, Clin. Chem. Lab. Med. 38 (2000) 99-112. [5] C.W. Hanson, H.A. Steinberger, The Use of a Novel 'Electronic Nose' to Diagnose the Presence of Intrapulmonary Infection, Anesthesiology 87 (1997) A269. [6] E.R. Thaler, F. Bruney, D. Kennedy, C.W. Hanson, Use of an Electronic Nose to Distinguish Cerebrospinal Fluid from Serum, Archives ofOtolaryngol. Head Neck Surg. 126 (2000) 71-74. [7] S. Chandiok, et al, Screening for Bacteria Vaginosis: A Novel Application of Artificial Nose Technology, J. Clin. Pathol. 50 (1997) 790-791. [8] S. Aathithan et al, Diagnosis of Bacteriuria by Detection of Volatile Organic Compounds in Urine Using an Automated Headspace Analyzer with Multiple Conducting Polymer Sensors. J. Clin. Microbiol. 39 (2001)2590-2593. [9] C.W. Hanson, Pneumonia. In: M.J. Murray et al (eds.), Critical Care Medicine: Perioperative Management, 2nd Ed. Lippincott, Williams & Wilkins, Philadelphia, 2002.

Disease Markers in Exhaled Breath N. Marczin and M.H. Yacoub (Eds.) fOS Press, 2002

39!

Biosensors and Express Biochemical Diagnostics of Some Diseases Nickolaj F. STARODUB, Andrew V. REBRIEV, Valentyna M. STARODUB Dept. Biochemistry of Sensory and Regulatory Systems of Institute of Biochemistry of Ukrainian National Academy of Sciences, 9 Leontovicha Str., 01030 Kiev-30, Ukraine E-mail: nstarodub@hotmail. com; web site: http://www. biochem. kiev. ua Abstract. This paper presents experimental data about the development of optical and electrochemical immune and enzyme biosensors based on the ion-sensitive field effect transistors and photoluminescence of porous silicon. These are intended for the determination of the level of myoglobin, glucose and urea in blood for the diagnostics of myocardial infarction, diabetes and kidney diseases. It was demonstrated that these biosensors are sensitive as well as selective towards analytes and can provide rapid, simple and inexpensive analysis. The electrochemical biosensors are prepared with the use of a biocompatible photopolymer, which provides long lasting preservation of enzyme activity and moreover, its use allows simple manufacturing of a biological membrane that can be fulfilled simultaneously with production of the transducer. The optical immune sensors are based on very cheap and replaceable sensitive elements. Both types of biosensors have potential for use in medical practice.

1 Introduction Environmental monitoring, control of biotechnological processes and biochemical medical diagnostics demand more and more selective, sensitive, rapid and inexpensive methods of analysis. During the last decades a number of biological sensors have appeared [1,2]. Theoretically these instrumental electronic devices are able to fulfil all these demands. Unfortunately, biosensors have not found yet a wide practical application in spite of an impressive number of their existing prototypes. In our opinion one of main reasons for this is a problem connected with difficulties in manufacturing the biosensors. It is necessary to choose the optimal type of the transducer and to optimise the manufacturing process. Success in this area will likely depend on the combination of the biologically sensitive material and its long-term durability in real conditions or on the use of very cheap and replaceable sensitive elements. This paper presents results on both of these possibilities.

2. Electrochemical enzyme biosensors for glucose and urea determination Among biosensors [3-7] that have been developed in our department we choose to discuss in this section only those of them, which are based on the ion-sensitive field effect transistors (ISFETs). Analysis of glucose, urea and myoglobin (Mb) in blood is the most often performed in clinical practice and therefore, we will present approaches to create biosensors for the detection of these substances. These biosensors should be organised in a

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special array, which will provide multi-parametrical complex biochemical analysis of blood. 2.1. Experimental procedure The semiconductor chips were produced in the Institute of Biocybernetics and Biomedical Engineering PAN (Warsaw). Two ISFETs were placed on the platform and worked independently. An enzyme was attached to the gate surface of one of the ISFETs from its solution also containing a photopolymer (working mixture). Another ISFET served as a reference structure and was covered with bovine serum albumin. The polymer mixture includes 78% of l-vinyl-2-pyrrolidinone, 10% of oligocarbonate methacrylate, 10% of oligourethan methacrylate and 2% of photoinitiator. At first water enzyme solution was mixed with l-vinyl-2-pyrrolidinone. Then water was removed by applying vacuum, and the remaining components were added. 0.25 ul of the working mixture was placed on the gate surface of the ISFET and the chip was subsequently placed in the vacuum2 and illuminated with ultraviolet lamp (Xmax=30°-400 nm and intensity 10.54 Watt/m ) for 10 min. As a rule the working mixture was prepared in advance. The sensor response was registered in buffer solution containing the substrate (for calibration) and in buffer containing the sample to be analysed (at the analysis of the test samples). 2.2. Results It was shown that under optimal conditions of the immobilisation of enzymes in the matrix of photopolymerisable composition based on 1 -vinyl-2-pyrrolidinone the residual enzyme activity was about 48 % for p_glucose oxidase (GOD) and 22 % for urease from Jeack beans. To investigate the efficiency of the sensor performance in real conditions human blood serum was used. The measurements with the developed biosensors were compared to standard colourimetric methods. The obtained data reveal high correlation between these two approaches. The difference in the values of the methods was not higher than 10%. The intensity of the urease sensor response to urea decreased slowly in course of 40 days, but the magnitude of this decrease was less than 20%. There also exist a possibility to prolong the lifetime of the sensor. Urease is a very non-stable enzyme since it contents SHgroup in its active centre that determines loss of the enzyme activity during the lifetime of the sensor. In the literature [3, 8] it is pointed out that the maximum lifetime of such a sensor is 60 days. In our case, this may be longer up to 6 months. Moreover, the biosensors based on the biological membranes prepared from fresh mixtures and mixtures stored for 46 days in refrigerator exibit the same level of responses. It thus allows using preprepared working mixture at a later time. In special experiments it was shown that urease activity in polymerised membrane didn't decrease during a 2-month storage in refrigerator. Only after 6 months we observed approximately 30% reduction of the activity. In case of the storage of GOD in polymerised membrane we observed a 17% decrease in activity over 3 months but this mixture could still be used after 6 months. Furthermore, other methods can be used to stabilize enzymes in pre-prepared mixtures. One of them is addition of appropriate substrate. For example, in the presence of thiourea in the mixture (0.5%), an increase of the urease activity of 11.3% was observed. These data allow us to consider this mixture as a stable biological reagent at the biosensor manufacturing in a photolytographic way. In our opinion the developed approach of the preparation of a biological membrane will promote wide practical application of biosensors.

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3. Optical immune sensors for Mb determination The determination of Mb is very important because Mb serves as one of biochemical markers of myocardial infarction. We created three variants of immune sensors for the control of this substance in blood serum. One of them is based on the ISFETs, the second and the third use effects of the surface plasmon resonance (SPR) and photoluminescence (PhL) of porous silicon (PS), respectively [9-14]. In this paper we will present results only on the two last types of immune sensors, which serve as an example of a biosensor with replaceable sensitive elements. 3.1. Experimental design Specific antibodies (Ab) in the concentration of 100 M,g/ml were immobilized on the glass surface treated sequentially with gold and dodecanthiol or on the surface of PS (4x4 mm). The immobilization was accomplished by physical sorption for 1 h at room temperature. The surface was then washed with tris-HCl buffer (pH 7.3) containing 140 mmol/1 sodium chloride and 0.01% of Twin-20. The level of PhL and reflective angle were measured at the immersion of the PS sample in control or test solution or at the introduction of these solutions in the measuring cell of the SPR, respectively. The visible PhL was excited by He-Cd laser at 440 nm and maximum of its irradiation was observed at 650 nm. The experimental details are presented in [9 and 13]. 3.2. Results It was shown that the sensitivity of the SPR based immune sensor to Mb was at the level of 100 ng/ml. It corresponds to the maximum level of this substance, which may be found in blood of healthy people. In patients with myocardial infarction Mb concentration may raise up to 1 ng/ml and higher. The linear plot of the sensor output lies in the range of 0.1-1.0 [ig/ml and therefore covers the range, which is of interest to medical practice. In the reverse situation when the concentration of specific Ab to Mb was determined we obtained sensitivity on the level of 1 ng/ml and linear plot in the range from 1.0-10 ng/ml to 10 (ig/ml. The observed difference in the sensitivity at the determination of Mb and its specific Ab is apparently connected with the differences of molecular weights and accordingly with their molecular dimensions. The last stipulate formation of a larger layer on the surface and as a result of this the reflection angle changes more considerably. The overall time of the analysis was not more than 30 min including time for immobilization of the sensitive layer. After destruction of the immune complex with the solution of pH 2.2 it is possible to fulfil up to 7-9 cycles of analysis. The sensitivity of the immune sensor based on the PS PhL to Mb is 10 ng/ml. This coincides with the sensitivity of the ELISA-method, This biosensor is capable to detect Mb concentrations in the range from 10 ng/ml to 10 ng/ml. Overall time of one measurement is about 15-30 min, which is significantly shorter than the duration of the ELISA method. In case of the immune sensor overall time of the experiment can be even shorter if measurements are accomplished in kinetic regime. From the experiments, in which the operational stability of immune sensor was studied it can be concluded that it can be used only for one cycle of measurements. The main reason of this is low stability of the PS surface itself. Nevertheless this lack doesn't prevent the practical use of this biosensor since silicon is very cheap and the procedure of PS preparation is very simple. To provide good biosensor calibration PS wafers should be manufactured by a common technology and than scribed in small pieces, which then will serve as a transducer for a number of immune sensors both for control and test sample measurements.

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Both of the described optical immune sensors exhibit somewhat less sensitivity than the sensor based on the ISFETs, which may reveal Mb in the concentration of 1 ng/ml [14] with approximately the same overall time of analysis. Unfortunately this immune sensor is more complicated since it demands specific conjugates of Ab with enzymes, special conditions and algorithm for the determination of enzyme activity of the formed immune complex. Moreover, this immune sensor is capable of providing only one cycle of measurements and after this it is necessary to change transducer and to repeat its cleaning and immobilization of a biological material on the ISFET gate surface. Both procedures, and in particular the manufacturing of the transducer, remain relatively complicated tasks. 4. Conclusion The developed biosensors can provide sensitive, simple and fast analysis of different analytes. The proposed principles of preparation of biosensors will allow a novel way of their industrial production whereby transducers are combined with biological layer during the manufacturing process. This will promote wide practical application of these instrumental devices. Reference

[I] A.P.F. Turner et al., Biosensors: Fundamentals and Applications, Oxford, Oxford Univ. Press, (1989)3-12. [2] N.F. Starodub, Immune biosensors - a new direction in biochemical diagnostics. Biopolymers and cell 5 (1989) 5-15. [3] N.F. Starodub, Electrochemical biosensors and biochemical diagnostics. ln:Proc. of Int. Center of Biocybemetics ofAcad. ofSci. of Socialist. Countries, Jablonna, Poland 3 (1990) 173-202. [4] N.F. Starodub at al., Optoimmunosensors for analysis of specific and non-specific classes of immunoglobulins. Sensors & Actuators B 7 (1992) 371-375. [5] N.F. Starodub et al., Fiber optic immunosensors for detection of some drugs. Sensors and Actuators B 13-14 (1993) 728-731. [6] N.F. Starodub et al., Fiber optic immunosensors based on enhanced chemiluminescence and their application to determine different antigens. Sensors & Actuators B 18-19 (1994) 161-165. [7] N.F. Starodub et al., Multi-enzymatic electrochemical sensor: field measurements and their optimisation. Anal. Chim. Acta 385 (1999) 461-466. [8] O.A. Boubriak et al., Determination of urea in blood serum by a urease biosensor based on an ion-sensitive field-effect transistor. Sensors and Actuators B 26-27 (1995) 429-431. [9] N.F. Starodub et al., Development of the myoglobin immune sensor based on the surface plasmon resonance. Ukr. Biochem. J. 41 (1999) 33-37. [10] N.F. Starodub et al., Extinguishing visible photoluminescence of porous silicon stimulated by antigen-antibody immunocomplex formation. In: SPIE Proc. Optical Organic and Semiconductor Inorganic Materials, August 26-29, 1996, Riga, Latvia. 2968 (1997) 73-76. [II] V.M. Starodub et al., Antigen-antibody interaction: new aspects of the signal generation at the specific complex formation. The FASEB Journal. 11 (1997) Al 163. [12] V.M. Starodub et al., Control of a myoglobin level in solution by the bioaffinic sensors based on the photoluminescence of porous silicon. In: Proc. XII European Conference on Solid-Slate Transducers and the IX UK Conference on Sensors and their Applications, Sept. 13-16, 1998, Southampton, UK. Edited by White, N.M. Bristol: Inst. of Physics. 1 (1998) 817-820. [13] V.M. Starodub et al., Control of myoglobin level in a solution by an immune sensor based on the photoluminescence of porous silicon. Sensors & Actuators B 58 (1999) 409-414. [14] V.M. Starodub, N.F. Starodub, Electrochemical immune sensor based on the ion-selective field effect transistor for the determination of the level of myoglobin. In: Proc. of the 13' European Conference on Solid-State Transducers, September 12-15, 1999, the Hague, the Netherlands: (1999)185-188.

Disease Markers in Exhaled Breath N. Marczin and M.H. Yacoub (Eds.) IOS Press, 2002

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Infrared Laser Spectrometer for Real-Time Analysis of Ethane in Exhaled Human Breath Manfred MURTZ, Hannes DAHNKE, Sandra STRY, and Peter HERING Heinrich Heine University Hospital, Institute for Laser Medicine 40225 Diisseldorf, Germany Abstract. We demonstrate real-time analysis of ethane fractions in exhaled human breath using laser absorption spectroscopy. Our measurements are carried out by means of mid-infrared cavity leak-out spectroscopy, a ring-down technique utilizing a cw laser. This method proves to be an unique tool with very high sensitivity and specificity for rapid and precise breath testing. The detection limit achieved is 300 volume parts per trillion ethane in human breath (integration time: 5s). In contrast to conventional gas chromatographic analysis our method enables ethane monitoring without preconcentration of the breath sample.

1. Introduction The analysis of trace compounds in exhaled breath on the ppb (parts per billion) level can be carried out by a number of different methods. For some compounds, very sensitive analysers based upon a specific chemical reaction are available. A prominent example is the chemilummescence analyzer for nitric oxide (NO) which enables online analysis of a single exhalation. The analysis of hydrocarbons, such as ethane, in exhaled breath is more complicated. Generally, this is performed via gas chromatography plus mass spectrometry (GC/MS). Due to the insufficient sensitivity of the GC/MS technique, the breath sample must be preconcentrated and passing through a trap-and-purge process before analysis. This multi-step procedure is not only time-consuming but also prone to errors. Especially the GC/MS analysis of exhaled ethane is a very difficult and laborious task [1], Ethane is considered to be the most important volatile marker for lipid peroxidation and there is increasing interest in the online analysis of breath ethane. For these reasons we are currently investigating laser-based breath analysis techniques. Infrared laser spectroscopy shows great advantages for sensitive and specific trace gas analysis since most relevant trace compounds exhibit a characteristic fingerprint spectrum in the mid-infrared. We have recently developed an ultra-sensitive infrared spectroscopy method called cavity leak-out (CALO) spectroscopy which takes advantage of modern continuous-wave infrared laser sources [2]. In this article we briefly describe the benefits of this infrared laser spectrometer for breath analysis. The set-up of our CALO spectrometer is presented and its application to real-time monitoring of exhaled ethane is demonstrated.

2. Trace gas analysis using infrared CALO spectroscopy: experimental details The basic arrangement for laser absorption spectroscopy consists of a wavelength tunable laser, an absorption cell containing the gas sample of interest, and a photodetector which

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monitors the transmitted laser power. Probing of the laser power transmitted at different wavelengths yields a characteristic spectral pattern of absorption lines that mirrors the kind and density of molecules inside the cell; this absorption spectrum provides an unique

Ring-down cell

Pnotodetector

Fig. 1: Schematic of the CALO spectrometer. The ring-down cell (length: L=50 cm) contains the breath sample to be analyzed. The effective optical pathlength is 3.6 km. cw laser = continuous-wave laser. 0,«0 0.36

Laser Rower

0.30

035

Leek-Out Sgnal

OX 0.15 0.10 0.05 0.00

2

4

6

8

10

Time[(js]

12

14

16

18

20

Fig. 2: Typical leak-out signal as monitored by the photodetector after the laser is turned off. The absorption is determined from the 1/e decay time T according to : a= c"' (t*1 - TO~'), where TO is the decay time when no absorber is present.

fingerprint and can be used to identify the molecules and to quantify their fraction in the breath sample. For most molecules the fingerprint spectral region is between 3 and 10 \im (mid-infrared). In many cases, the infrared spectra even allow to identify different isotopomers of a volatile compound. For example, with a high-resolution laser spectrometer one can distinguish between two stable isotopomers of nitric oxide, i.e. I4NO and 15 NO. This enables the use of tracer techniques, e.g., for pharmacological studies. Due to the extremely low concentration of trace gases the absorption of laser light inside the cell is generally very small, typically in the order of 10~9/cm for ppb fractions. In order to achieve spectra from such low-concentrated trace gases we have recently developed a novel spectroscopic technique which provides very high sensitivity. This technique is described in detail in [2,3]. The major trick is to enclose the gas sample into an optical cavity formed by two highly reflective mirrors (reflectivity: R =99.985%) which acts as a "light trap" where the laser beam is reflected back and forth several times (Fig. 1). In this way, the laser light travels more than 3 km in the 50 cm long absorption cell. For determination of the absorption we measure the decay time of the trapped light (Fig. 2). This exponential power decay is monitored via the light leaking out of the cavity after the laser power is turned off for a short time. This time-domain technique enables very precise and sensitive absorption measurements. For the generation of mid-infrared laser radiation we use two different sources: a carbon monoxide laser (COL) and alternatively a difference frequency generation (DFG) system. The COL operates on about 300 laser lines in the wavelength region between 2.6 and 4.0 urn. By mixing the laser light with microwave radiation in an electro-optic modulator, tunable laser sidebands are generated (power: ca. 100 u,W), tunable in the spectral range of 8 to 18

M. Miirtz et al. / Infrared Laser Spectrometer for Real-Time Analysis of Ethane

397

GHz above and below each laser line. For details please refer to [4]. The COL is a bulky laboratory set-up and not portable. To overcome this, we built a DFG system that can be made portable in the near future. Here, tunable infrared radiation is generated in a non-linear crystal which is optically pumped by two compact near-infrared lasers. The infrared output powers amounts to about 20 - 30 uW in the 3 um region; the modehop-free tuning range is about 50 GHz. 3.0n 2.5I 2.0"9 O

j«d % 1.00.50.0 2960

2980 3000 \Afewerturrter [cm1]

3020

Fig. 3: Fingerprint absorption spectrum of ethane near 3 um. The arrow indicates the specific ethane peak which has been selected for the analysis.

3. Monitoring of ethane in exhaled human breath by means of CALO spectroscopy Ethane has a unique spectral fingerprint in the 3u,m region (Fig. 3). For the measurement of the ethane concentration in a breath sample it is sufficient to just monitor one of the isolated lines (marked by an arrow in Fig. 3). It is important to note that the line we selected is almost free of interferences with lines from other compounds in breath. Using CALO spectroscopy we achieve a detection limit of about 300 ppt ethane (integration time: 5 s) (5). To our knowledge this result is unique and this is currently the only technique for real-time ethane measurements in exhaled air without accumulation and preconcentration of the breath sample. Though the spectrometer clearly has the potential for online measurements we are currently carrying out offline measurements of breath samples collected in bags, since the spectrometer is not yet portable. The patients are asked to blow their complete lung capacity after a deep inhalation into a Tedlar bag under spirometric control. The bag is then sealed and send to our laboratory. The period (for up to two days) between breath sample acquisition and analysis has no effect on the measurements. During analysis a gas flow from the bag through the cell is maintained by a rotary pump. This gas flow is controlled by a flow controller to be 100 ccm per minute. We have placed a cooling trap (temperature: 120 K) between the sample bag and the absorption cell. This removes breath components like water, isoprene and other volatile compounds present in exhaled human breath whose spectra may interfere with the ethane spectrum near 3 urn. To ensure high discrimination against interfering compounds which do pass the cooling trap, like methane and ethylene, we reduced the pressure inside the absorption cell to 100 mbar. This reduces cross interference since the separation of the spectral lines improves due to the reduction of the pressure broadening. Fig. 4 shows the absorption versus wavelength for a typical breath sample near the selected ethane line. The spectrum shows a clear peak indicating 50 ppb ethane in this example and a second peak which indicates methane, about 4 ppm in this case. In order to demonstrate the application of our CALO spectrometer for precise time- resolved breath testing, we monitored the ethane fraction exhaled by a smoker after smoking a cigarette.

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We took breath samples about every 30 minutes in a period of 4 hours after smoking. During this period the proband volunteered not to smoke. The first breath sample was not taken before 30 minutes after the cigarette was finished. This ensured that no cigarette smoke was present in the lungs. Fig. 5 shows the results. We observed a strong increase and subsequent decay of the ethane fraction after smoking. The measurements of the data points displayed include several absorption measurements on top of and next to the ethane line; this took about a total of 1 minute for each data point. The data presented here have been corrected for the corresponding atmospheric background ethane fraction; thus what Bhane(SOppb)

30-

'.2520-

15-

Methane (4 pprrj

10-

53000.15

300020

300025

3000.30

Wsvenunntoer [cm'1]

Fig. 4: CALO spectrum of exhaled breath near 3.3 urn

0

0

60

120 180 Timefmin]

240

Fig. 5: Ethane fraction in breath after smoking

we observe here is of endogenous origin. The uncertainty of the analysis is about 1 to 2 ppb, including the statistical error of the consecutive absorption measurements and the error introduced by the background correction. It is well known that the ethane fraction is enhanced by smoking due to the free-radical induced damage in the respiratory system [6]. Our analysis confirms this fact very clearly. Height and decay time strongly depend on the condition of the individual, on the smoking frequency and even on the cigarette brand. However, one should consider that part of the ethane observed is not endogenously generated but originates from the cigarette smoke, which has been absorbed in the respiratory system and is then slowly released over time. These measurements certainly should not be considered as systematic study. But the results demonstrate that our spectroscopic method is well suited for such investigations. 4. Conclusion Modem methods of infrared laser spectroscopy enable sensitive, isotope-selective, and precise real-time monitoring of various trace gases in human breath (NO, CO, C^, etc.). We have found that the analysis of ethane fractions in human breath can be performed with high sensitivity, specificity and speed. We achieved a detection limit of 300 ppt (integration time: 5 s) which is sufficient for online measurements without preconcentration. The presented approach has the potential to become a versatile tool for non-invasive monitoring of in-vivo lipid peroxidation and for other breath tests based on exhaled trace gases. To our knowledge there is no analysis method with a higher sensitivity for ethane and a sufficiently low cross sensitivity to other gases, such as methane, ethylene, etc. The device presented is still in an experimental stage. A portable device is currently in progress. References [1] T.H. Risby, S.S. Sehnert, Clinical application of breath biomarkers of oxidative stress status, Free Radic Biol. Med. 27 (1999) 1182-1194

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[2] M. Miirtz, B. Freeh, W. Urban, High-resolution cavity leak-out absorption spectroscopy in the 10 u.m region, Appl. Phys. B 68 (1999) 243-249 [3] D. Kleine, H. Dahnke, W. Urban, P. Hering, M. Murtz, Real-time detection of 13CH4 in ambient air by use of mid-infrared cavity leak-out spectroscopy, Opt. Lett. 25 (2000) 1606-1608 [4] M. MUrtz, B. Freeh, P. Palm, R, Lotze, W. Urban, Tunable carbon monoxide overtone laser sideband system for precision spectroscopy from 2.6 to 4.1 urn, Opt. Lett. 23 (1998) 58-60 [5] H. Dahnke, D. Kleine, P. Hering, M. Miirtz, Real-time monitoring of ethane in human breath using midinfrared cavity leak-out spectroscopy, Appl, Phys. B 72 (2001) 971-975 [6] M.P. Habib, N.C. Clements, H.S. Garewal, Cigarette smoking and ethane exhalation in humans, Am. J. Respir. Crit. Care Med. 151 (1995)2368-2871

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Disease Markers in Exhaled Breath N. Marczin and M.H. Yacoub(Eds.) IOS Press. 2002

Methods of tuneable diode laser absorption spectroscopy applied to the analysis of exhaled breath Gianfranco GIUBILEO ENEA, Via E.Fermi 45, 00044 Frascati (Roma), Italy Tel: +39.06.94005 768, e-mail: giubileo@frascati. enea. it Abstract The analysis of high resolution molecular absorbing spectra in the infrared region can be based on particular features of tuneable diode lasers. This possibility can be usefully applied in the detection of trace gases in the exhaled breath. In this lecture the high precision tuning of a diode laser and the highresolution molecular spectroscopy principles will be recalled and the basic experimental set-up necessary to perform a tuneable diode laser absorption spectroscopy (TOLAS) will be discussed. Many species may be detected by this sensitive technique, eventually offering the possibility of a non-invasive diagnosis of a number of diseases. Values of the minimum detectable amount for different species will be given. Attention will be devoted to the breath test analysis for the diagnosis of infection by the bacterium Helicobacter Pylori, which is believed to cause peptic ulcer while infecting the gastric mucous layer. The TOLAS apparatus realised in the Molecular Spectroscopy Laboratory of ENEA in Frascati will be illustrated. The operating conditions, the calibrating method and the analytical procedure will be discussed.

1. Introduction The infrared spectroscopy is based on selective molecular absorption of IR radiation passing through a gas mixture, the absorption spectrum consisting in a well defined set of lines in the IR region, each set being characteristic for each molecular species and for each isotope likewise a fingerprint. The appropriate radiation source for this measure is the tuneable diode lasers (TDL), which emits a narrow line of IR radiation continuously tuneable over small spectral intervals. A fine-tuning of TDL radiation frequency makes it possible to measure the absorption line of a single molecular transition with high resolution, allowing the fine spectroscopic characterisation of a chemical species. Consequently high selective detection of chemical species and accurate isotopic analysis in gaseous mixtures are possible. This means that high-resolution IR molecular spectroscopy may be put at work in the non-invasive medic diagnosis field to detect gaseous molecular species in the human breath. In the following we shall review the principles of molecular spectroscopy based on tuneable diode laser (TOLAS), the scheme of an experimental apparatus for TOLAS, and the application of TOLAS to the diagnosis of Helicobacter pylori infection. 2. Tuneable diode laser based absorption spectroscopy A typical IR emitting TDL device is a homogeneous structure consisting of a ternary lead salt crystal supporting a p-n junction. In this device the laser inversion is realised

G. Giubileo f Methods of Tuneable Diode Laser Absorption Spectroscopy

40 ]

Laser source

Digital oscilloscope

PC

Fig. 1 - Schematic general layout of a TOLAS system for trace gas analysis.

through a forward biasing of the junction by a dc voltage supply. These lasers furnish coherent radiation in the IR region. Typically we find laser structure thickness of 1mm with lum active zone thickness, giving ImW total output power with 1A bias current. The energy gap between the two laser levels is depending on the temperature and the refractive index is depending on the injection current. The operating temperature of the TDL we use is around the liquid nitrogen temperature. Once the laser has been placed in a cryostat supplied by liquid nitrogen and the baseline temperature has been fixed to select the proper laser mode, the emission radiation frequency may be tuned continuously within the defined limits of a small spectral interval by modulating the injection current within the corresponding limits. The spectrum of TDL radiation consists in a very narrow line, the spectral line width being order of magnitude less than the absorption linewidth of any molecular spectrum. The emitted single mode radiation frequency can be moved in a given spectral region by tuning the emission frequency making it possible to scan a definite absorption line of a given gaseous substance (see fig. 2). A calibration of the line intensity makes it possible to measure the gas concentration inside the examined sample of breath. The general scheme of an experimental apparatus for TOLAS is reported in fig. 1. Major components of the optical system are the laser, the current controller, the temperature controller, the sample cell, the radiation detector and the data storage and analysis system. The laser is located inside the cryostat to maintain the basic operating temperature of the semiconductor. The radiation emitted by the laser is forced to pass through a monochromator to select the proper laser mode and eliminate eventual undesired modes. Subsequently it is allowed to enter the sample cell. The residual laser radiation emerging from the cell will shot the radiation detector connected to the acquisition system. The cell contains the sample during the measure while contains the reference mixture or vacuum during the calibration of the system. A vacuum pump connected to the cell makes it possible a fast evacuation of the cell itself. Many chemical species whose IR spectrum details are known may be detected by this sensitive technique, eventually offering the possibility of a non-invasive diagnosis of as number of diseases [1,2]. The TOLAS has already been successfully used to perform high-resolution detection of gas traces in atmosphere at very low concentration [3]. The Table 1 (modified from Ref. 3) reports a partial list of gaseous species detectable by

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Table 1 - Partial list of substances detectable in breath by TDL spectroscopy and their minimum detectable concentration (mdc).l pptv = 10*'^ volume mixing ratio. Species CO2 mdc | 160

03 100

N2O 5

CO

11

CH4 36

NO 140

SO2 130

NO2 20

NH3 9

HNO3 H202 220 1 130

TOLAS and the respective minimum detectable concentration. In the following chapter an exemplary use of TDL AS in the breath analysis will be outlined. 3. Urea breath test The urea breath test (UBT) allows a non-invasive diagnosis of infection by Helicobacter pylori. H.pylori is a spiral-shaped gram-negative bacterium that may infect the gastric mucous layer in humans and cause the peptic ulcer disease [4]. The bacterial toxins weaken the stomach protective mucus allowing the stomach cells to be susceptible to the damaging effects of acid and pepsin. It has been discovered that the bacterium produces urease, an enzyme that make it possible to dissociate the urea molecule (N2H4CO) into ammonia (NR|OH) and carbon dioxide (CCh), so the presence of the bacterium can be revealed by detecting the enzymatic activity of urease. The urease enzymatic activity can be assessed by measuring the chemical conversion of urea to carbon dioxide in a breath test. For this purpose it is necessary to ingest a small amount of urea labelled by the non-radioactive isotope I3C. If H.pylori is present inside the stomach it will digest the labelled urea producing labelled COi, which is collected by the haematic flow, brought to the lungs and exhaled with the breath, determining a measurable increase of the 13CO2/I2CO2 isotopic ratio in the breath after 15-20 minutes after the ingestion. A healthy people is not able to split the ingested labelled urea, which will be expelled unmodified, and will not produce an increase in the I3C breath content. An increase of more than 5% in the I3C breath content is considered as the definite sign of the infection [5]. The value of the isotopic ratio may be determined detecting a defined absorbing line for each one of the two isotopes and calculating the ratio between the respective intensities. The natural isotopic content of the I3C isotope in the breath is 1.095% of the total CCh, and very low amounts of I3C are involved to increase the isotopic ratio of 5%. The precision reached by TOLAS is sufficient to perform the analysis. The general scheme of a TOLAS apparatus has already been given in fig. 1. In the system realised at ENEA Frascati Laboratories the TDL source has been tuned to 4.33 urn to allow the scanning of both Pas and Ra2 lines selected in the CC>2 spectrum to measure the isotopic ratio (see fig. 2). Reference mixtures have been used to calibrate the system. A more detailed discussion has been reported in [6].

G. Giubileo / Methods of Tuneable Diode Laser Absorption Spectroscopy

403

100

75

8 50

6 25

0

2305.7

2305.8 2305.9 Wavenumber (cm"') Fig. 2 - Small part of CO2 absorption spectum in the 4.3 (jm region. The absorbing lines are identified as P35(UCO2) and R32( I2 CO 2 ).

4. Conclusions. A sensitive analysis of trace gases in the exhaled breath is possible through the high resolution molecular Spectroscopy based on tuneable diode laser (TOLAS). An exemplary application of TDLAS in gastroenterology is the diagnosis of the Helicobacter pylori infection through the urea breath test based on the measurement of i3 CO2/12CO2 isotopic ratio in the exhaled breath. References. [1] G. Baldacchini, F. D'Amato, G. Giubileo, S. Martellucci, Tunable diode laser detection of small traces of gases for medical diagnostic5, Kluwer Academic Publishers Serie E: Biomedical Optical Instrumentation and Laser-Assisted Biotechnology, 325, (1996) 185-195. [2] G. Giubileo, Laser based assessment of lipid peroxidation in humans; 5/Y£3405 (1988) 642-653 [3] P. Werle, SPIE, Substance Detection System 2092 (1993) 4-15 |4]

J. Alper, Science, 260 (1993) 159-160

[5] L. G. Sandstrom, S. H. Lundqvist, A. B. Petterson, M. S. Shumate, IEEE J. Sel. Top. Quantum Electron, 5, n.4, (1999), 1040-1048 [6] G. Giubileo, L. De Dominicis, M. Giorgi, R. Pulvirenti, M. Snels, A TDLAS system for the diagnosis of Helicobacter Pylori infection in humans, Laser Phys. 11 (2001) n.l, 154.

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Part VII. Industrial Forum

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Disease Markers in Exhaled Breath N. Marczin and M.H. Yacoub (Eds.) JOS Press, 2002

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Industrial Forum - Aerocrine AB Tryggve HEMMINGSSON Aerocrine AB, Smidesvagen 12, S-171 41 SOLNA, Sweden Abstract. This presentation describes the recent and future activities of our company, Aerocrine AB of Sweden, where I am currently working as a Project Manager.

1. Aerocrine and its founders Aerocrine was founded in 1997, by researchers from the Karolinska Institute in Stockholm, one of them was Professor Lars Gustafsson. Professor Gustafsson was also the first to find endogenous Nitric Oxide in exhaled air in 1991. This finding was followed by Associate Professor Kjell Alving, working at another institution at the Karolinska Institute who in 1993 found elevated NO levels in asthmatics. This triggered a lot of research at Karolinska, and in other institutions throughout the world. 2. Our application areas for NO measurement The two groups at Karolinska Institute also measured elevated gaseous levels in other hollow organs, like the intestines and urinary tracts, during the following years. These findings also reflect the patent portfolio of Aerocrine today. However, these are three vast application areas, and therefore the company decided to focus on airway inflammation monitoring, primarily asthma, since it is a serious and growing global health problem. Today, there is no alternative simple non-invasive method, which can be used for clinical routine measurement of inflammation, for this widespread chronic disease. 3. Scientific network and cooperation We at Aerocrine are very proud to see within our scientific network • many of the worlds leading scientists in Nitric Oxide research, which give us good access to the scientific community. Within the company, we have in key positions •

a very experienced management and development team, with long experience from the pharmaceutical industry and from medical device companies, which puts us in a unique position, Aerocrine is cooperating with strategic partners, and is working with the business strategy of outsourcing functions like product development, manufacturing, logistics and distribution. 4. In our Scientific Advisory Board which initially was formed by our founders, we are pleased to have:

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•

Professor Peter Barnes at the Imperial College in London, who is well known among all of you, as a world leading asthma scientist • Professor Lou Ignarro, at the University of California in Los Angeles, has collaborated with our founders, and through that connection, he joined the Advisory Board. He became one of the Nobel Price Laureates in 1998 for his work on nitric oxide • Professor Salvador Moncada is also one of the leading scientists in the area, today working at Wolfson Institute in London Until last year, Professor Jeff Drazen, was one of the Advisory Board Members, but as he was appointed Editor in Chief of the New England Journal of Medicine, he had to leave our Advisory Board. We are working with an extensive clinical network, which we have established throughout Europe and the US. 5. NO measurement in clinical praxis At Aerocrine we see the clinical potential of using exhaled nitric oxide measurement in clinical praxis. By monitoring exhaled nitric oxide, the doctor can follow the patient's compliance with anti-inflammatory medication, and follow the therapeutic response to these drugs. We can also see the potential in optimising the dose of anti-inflammatory therapy, and to predict exacerbations in asthma patients. Other areas of interest are - the differentiation between COPD and asthma and to make differential diagnosis of Cystic Fibrosis and Primary Ciliary Dyskinesia. Our target is to establish nitric oxide as the primary marker of inflammation within the following target groups, actively working from the top and down:

6. Achievements During last year, to pick a few samples, Aerocrine: • achieved to establish the infrastructure of the company • got our NIOX® instrument approved for sales, according to the Medical Device Directive in Europe. This means that NIOX® is the only Nitric Oxide measurement instrument on the market, approved for clinical use • performed European launch of NIOX* at the ERS congress last autumn

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During this year, we are setting up our service organization. We have started to get into clinical practice and building our distribution organization, through partners. As of today, we have established a distributors network for our products, in the major countries of Europe, and there are around 50 NIOX® installations throughout Europe and the United States. We have established reference centres all over Europe, and in the US. One of our customers in the United States is the US Army. 6. Development plans Looking at our future plans, except for expanding our sales and marketing business with NIOX®, and our active work on an FDA approval in the US, our activities within product development also include development of an Offline unit for NIOX®, and the development of a Tidal Breathing application for NIOX®. There is also work in progress for bringing exhaled nitric oxide analysis to the General Practitioner's, and to the patient, for home care use. Our patent portfolio covers our priority markets in Europe, the United States and Japan, and we are continuously developing our intellectual property. 7. Owners The company's main owners are: • Healthcap - a Swedish Venture Capital Company, focused on life sciences • Investor - the biggest Industrial Group of Sweden, with a large ownership in for example Ericsson and AstraZeneca • Health & Brands - an International Venture Capital Company • Sjatte AP-fonden - one of the main Swedish Pension Funds • Skandia - a major Scandinavian Insurance Company. We at Aerocrine welcome new contacts for cooperation, as it is important for us to expand our network with new research clinics and organisations. If you are interested in our company, please visit our Web page at www.aerocrine.com for the latest news and contact information.

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Disease Markers in Exhaled Breath N. Marczin and M.H. Yacoub (Eds.) IOS Press. 2002

Biochemical Diagnostics of the Lung with

ECoScreen™ L. NEUMANN, W. STEINHAEUSSER Erich JAEGER GmbH, D-97204 Wuerzburg, Germany Abstract. Exhaled breath condensate (EBC) delivers a variety of new biochemical parameters additional to conventional lung function analysis. The ECoScreen™ is a powerful system for non-invasive collection of EBC with a proven inert sample vial. The method is based on the so-called counter current principle, where the substances are cooled down to a temperature of about -10°C. The system allows stable collection conditions by controlling temperature and volume closely. Obtaining a sample of 2 millilitres takes about 5 to 15 minutes. For EBC collection in breathing circuits, an adapter is available for scientific applications and for the monitoring of respiratory parameters during collection. An optional volume sensor measures tidal volume (VT), peak flow (PEF) and breathing frequency (BF). Meanwhile, the system has proven its reliability in many studies worldwide. With the development of biosensors for disease markers, this method may be beneficial for the diagnosis and treatment of pulmonary diseases even in point-of-care settings.

1. Exhaled breath condensate Normally, breath condensate is collected during tidal breathing whereby the composition of the bronchial and alveolar fluids remain unchanged. According to the current state of research, we assume that biomolecules reach the breath condensate by bronchial and alveolar aerosol forming as well as by evaporation. The cause of aerosol formation is a partial expiratory collapse of some bronchioli and/or alveoli, especially during RV-manoeuvres. During inspiration, aerosol particles are torn off from the alveolar lining fluid due to separation of the collapsed surfaces and subsequently exhaled (see Fig.l) [1].

Fig. 1. Hypothetic mechanism of aerosol generation during inspiratory reopening of collapsed alveoli according to Brand [1].

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The sample primarily contains metabolic products from the airways, and to a lower extent from other organs, which reach the lung via the blood. Besides, exhaled substances, which were incorporated from the environment via air, intestine or skin can be identified.

2. Collection of exhaled breath condensate with the ECoScreen™ -system The ECoScreen™ is a powerful basic system for non-invasive collection of exhaled air substances, i.e. aerosols and vapour. The method is based on the counter current principle, where the substances are cooled down to a temperature of about -10°C. In the loop, impaction and turbulent flow enhances precipitation of aerosols and vapour allowing obtainment of a 2 millilitre sample within 5 to 15 minutes. Due to an additional impaction device and a saliva trap, no saliva contamination occurs during normal tidal breathing. Furthermore, the proven inert sample vial with low surface area in relation to sample volume, avoids molecular adhesion and subsequent relevant loss of low-concentrated sticky molecules. The principle is patented by FILT GmbH (EP 0759169): [4]. Tab. 1. Features of the ECoScreen™ Constructive features of the sampling unit: Stable collection conditions Controlled temperature Volume measured Sampling tube and mouth piece with internal air guidance No saliva contamination Proven inert sample vial with low surface area to avoid adhesion Disposable sampling tube Closed circuit sampling option Optional fractional sampling, volume triggered Double-wall insulated cooling tube incl. A freezing circuit and central bore for precise sampling tube fit

Fig. 2. Counter-current principle for energy saving cooling. Within the tube, the exhaled air is cooled down to a temperature of about -10°C, whereby water vapour, aerosols, hydrogen peroxide, eicosanoids and other nongaseous components precipitate at the inner wall of the sampling tube.

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Fig. 3. (a) Optional adapter for closed respiratory circuits (protected functional sample No. 297 12 836.1 (DE)); (b) optional volume sensor for tidal volume, peak flow or breathing frequency.

3. Options for the ECoScreen™ Several studies [2, 3] showed that the amount of breath condensate collected with the help of the ECoScreen™ is directly proportional to the breathing volume expired through the cooling system. The optional volume sensor (Fig.3b) records ventilatory parameters and displays them numerically on a display. Thus, the collection procedure can be stopped as soon as the desired amount of condensate has been collected. In addition to the total volume, tidal volume (VT), peak flow (PEF) or breathing frequency (BF) can be displayed. With short sampling times expiratory flow and respiratory volume may be checked via a feedback mechanism. For collecting breath condensate within closed respiratory circuits an optional adapter (Fig.3a) assures that the sampling tube can be connected and removed without pressure decrease during positive pressure respiration. Moistening of breath, which results in a considerable dilution of the condensate (no standardization) is still difficult. Consequently, it is appropriate to interrupt humidifying during collection. As far as calculation of the concentrations in the diluted condensate is concerned, experience is so far lacking. 4. Perspectives The new methods deliver a variety of new parameters which broaden the spectrum of conventional lung function analysis. Their true meaning for lung function diagnosis cannot yet be completely assessed. With the development of biosensors for disease markers, this method will be of special interest in the future for physicians in clinics and private practice. Meanwhile, the ECoScreen™ -system has proven to work reliably in many studies worldwide [5]. Studies with exhaled breath condensate to use optical biosensors, as well as electrochemical biosensors, are on the way. References [1] P. Brand, Exhaled Condensate - A source of biochemical information, Jaeger-Info: Special Edition Breath Condensate, Hoechberg, 1(2001) p 12. [2] P. Reinhold et al., Die Gewinnung von Atemkondensat - EinfluG des Atemmusters auf die Probenmenge, Tagung der Sektion Pathophysiologie der Atmung in der DGP, Bad Honnef. 1998. [3] Ch. Gessner, Linearer Zusammenhang des Atemkondensatvolumens und der Ventilation, Tagung der Sektion Pathophysiologie der Atmung in der DGP, Bad Honnef. Germany 1999. [4] G. Becher et a/., Vorrichtung zur Sammlung von nichtgasformigen Bestandteilen der Ausatemluft durch Ausfrieren (Atemkondensat). mt Medizintechnik, 117(1997) H3; 89-95 [5] S. A. Kharitonov, P. J. Barnes, Exhaled Markers of Pulmonary Disease, Am J Respir Crit Care Med 163 (2001)1693-1722.

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Portability and Flexibility in Exhaled Breath Condensate Collection John W. VAUGHAN Respiratory Research, Inc. 1167 Raintree Drive Charlottesville, VA 22901 Abstract. Measurement of inflammatory markers in exhaled breath condensate (EBC) provides a new non-invasive research tool to study lung disease. These assays have potential clinical utility in patient management. With rapid expansion in the classes of biomarkers studied to date in EBC—each perhaps with different optimal conditions for collecting and assaying—there is need for EBC collectors that are flexible—allowing for different condensate temperatures, flows, and attachments. As researchers extend their research out of the laboratory and into schools, clinics and worksites, portability and technical flexibility in EBC collection devices will be increasingly beneficial. The RTube™ and pHTube™ systems were designed to accommodate the evolving needs of researchers in the field and to assist the progress of these assays into the clinical realm.

1. Exhaled Breath Condensate: Not Yet Ready for Standardization Along with the explosion in interest in EBC assays, many technical questions have been raised concerning EBC collection methodologies. EBC contains aerosols and vapor phase components, and both volatile and non-volatile compounds. Each component of and compound within EBC likely has ideal collection and storage conditions that may be unique. The technical controls necessary for collection and assay of each individual compound will be identified through careful evaluation and experimentation that cannot be performed with static, inflexible methodologies. Isolating and controlling suspected sources of EBC variability will allow empiric investigation of the technical factors that must be understood before a potential standard system is developed for any individual biomarker of interest. The RTube™ system, and its companion system the pHTube™, were designed to address the needs of the conscientious researcher to identify and control for variability, allowing simplification and optimization of each investigator's specific protocol. 2. Description and Specifications of the RTube/pHTube System Materials As used in medical syringes, the condensing surface of the RTube and pHTube are manufactured from polypropylene, which is non-reactive with most compounds of interest and has been recommended as the most functional material when assaying for bioactive lipid mediators[l]. It is proven effective for inorganic compounds as well. The exhalation valve doubles as a syringe-style plunger that is used to rapidly collect condensate from the condenser walls. Manufactured from silicon and teflon, it is a flexible but durable appliance that tolerates extremes of temperature well. The small

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J. W. Vaughan / Portability and Flexibility in Exhaled Breath Condensate Collection

Figure 1. The RTube: (a) aluminum cooling sleeve; (b) collection chamber; (c) exhalation valve and syringe-style plunger; (d) optional filter; (e) mouthpiece assembly.

mouthpiece attachment through which the patient breathes along with the vertical orientation of the condensing chamber inherently prevents gross saliva contamination. Attachable to the device is a 0.3 micron particle filter that can be placed proximal to the condenser and used to select for small particles and further minimize saliva contamination, or distal to the condenser to assist in preventing microbial egress into the atmosphere. The effect of the removable filter on inorganic constituents of EBC is insignificant, but the effect on larger molecules has not been evaluated. For cooling, the RTube/pHTube system employs a sleeve that surrounds the condensing chamber. Because of its excellent heat transfer capability, this is manufactured from aluminum (Figure 1). Cooling The unique exhalation valve of the RTube and pHTube causes pronounced turbulence within the condenser, which along with the excellent heat conducting properties of the aluminum sleeve, promotes rapid cooling and condensation of exhaled air. The sleeve can be chilled at practically any temperature prior to use, down to as low as -80°C. Using the system with custom alterations, precise temperatures can be maintained throughout collection. The proper collection temperature may vary for different compounds and may need to be individually investigated. Cooling sleeves used in studies outside of the laboratory or clinical environment can be stored in the patient's home freezer (roughly -4 to -17°C). Duration of Collection Duration of collection on EBC reproducibility is beginning to be examined empirically. It can be anticipated that concentrations of certain volatile compounds with high water-partition coefficients may show collection-time dependency. In general, an average EBC volume of approximately one milliliter is collected in seven minutes. But, because of the syringe-styled RTube condensing chamber, collections of 100-150 microliters can be performed with easy access to sample in only one minute. This feature has been particularly helpful in performing multiple repeated collections on the same patient. Colder condensing temperatures result in modest

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improvements in the efficiency of extraction of water vapor out of exhaled air, with increased final volumes of EEC. Attachments The RTube™ and pHTube™ are designed with standard size fittings to attach directly to varied respiratory monitoring devices. The RTube™ system has been attached inline with ventilators in the intensive care unit and respirators used in the operating room. With few or no adapters, it connects to ETCO2 monitors, nitric oxide analyzers, flow meters, and devices for measuring humidity, temperature and pressure. The inhalation port can be readily attached to dry or humidified gases including supplemental oxygen for research subjects who require it. Furthermore, it can be attached to a nebulizer. Storage After the subject exhales through the RTube, the collected EBC can be frozen and stored in the collection chamber. Alternatively, the syringe can be plunged and the sample immediately removed to a polypropylene microcentrifuge tube or processed. Depending on the temperature stability of the exhaled biomarker of interest, subjects involved in home, school, clinic or workplace studies are able to cap the ends of the collection chamber, write their name and date on the label, and store the sealed chamber in the freezer. The samples can later be shipped on ice or simply brought to the laboratory in a small ice chest. For pH assays, the samples can be stored and transported at ambient temperatures. 3. Conclusion The RTube™ and pHTube™ EBC collection devices were designed by researchers to be maximally flexible, and to function in a variety of investigational settings. These devices are handheld, readily portable, and designed for use even by the unsupervised patient in the home, workplace, laboratory, hospital, or clinic. Temperature at initiation of collection can be controlled, Filters, flow meters, end-tidal gas monitors, temperature probes, hygrometers, and other instruments can readily be fitted into the standard size fittings incorporated within the disposable collection chamber. The RTube can be directly attached to endotracheal and tracheostomy tubes. The inhalation port can be connected to dry or humidified gases, or directly to nebulizers. The system therefore allows for rigorous investigations into the causes of variability that inevitably appear in EBC assays. As each discovered biomarker may reveal unique optimum conditions for collection and assay, the RTube system will allow individualization of collection method. It is useful for identifying anatomic and physiologic determinants of biomarker concentration. Finally, the combination of attributes of the RTube and pHTube make the system particular useful for ready collection of samples from diverse patient groups in most any setting. Reference 1. Mutlu GM, Garey K.W, Robbins RA, Danziger LH, Rubinstein I. Collection and Analysis of Exhaled Breath Condensate in Humans. Am J Respir Crit Care Med 2001; 164(5):731 -737.

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Author Index Adcock, I.M. Adding, L.C. Antczak, A. Aubier, M. Bach, F.H, Balint, B. Baltopoulos, G.J. Barnes, P.J. Becher, G. Bellenis, I. Berberat, P. Berry, M.J. Boczkowski, J. Boughton-Smith, N.K. Bundy, R. Burch, T.E. Chatzimichalis, A. Choi, A.M.K. Chrysofakis, G. Dahnke, H. Davis, I.C. Demoncheaux, E. Dougenis, D. Dweik, R.A. Erzurum, S.C. Fotopoulos, V. Gaston, B. Giubileo, G. Gratziou, C. Guenther, L. Gustafsson, L.E. Hall, J. Hardiman, K.M. Hemmingsson, T. Hering, P. Hickman-Davis, J.M. Higenbottam, T. Hoare, G. Hogman, M. Horvath, I. Hunt, J.F. Ito, K. Jilling, T. Kajland-Wilen, L.

151 35,52 333 79 267 234 30 133,234 383 300 267 242 79 24 375 387 300 73 209 395 344 49 300 11,93,159 3,143,199 300 18 400 191 267 35,52 96 354 407 395 344 49 375 187 234 167 151 223 64

304 Kallio, E.A. 125 Kellermayer, M. Kharitonov, S.A. 57,62,177,218,234 Kokinis, K. 300 300 Koletsis, E.N. Korovesi, I. 319 304 Koskinen, P.K. 319,366 Kotanidou, A. 319 Koutsoukou, A. 375 Kovesi, T. 354 Lazrak, A. 304 Lemstrom, K.B. 105 Lindstrom, A.B. Marczin, N. 88,274,291,375 344,354 Matalon, S. 319 Mavrommati, I. 344 McArdle, P. 96 McCarthy, M. 284 McRae, K. Miekisch, W. 338 395 Mtirtz, M. 30 Myrianthefs, P.M. 410 Neumann, L. 354 Nielsen, V. 338 Noldge-Schomburg, G.F.E. 344 O'Reilly, P. 319 Orfanos, S.E. 73 Otterbein, L.E. 366 Papapetropoulos, A. 105 Pleil, J.D. 261 Polyakova, V. 319 Psevdi, E. 202 Rami, J. Rebriev, A.V. 391 196 Ricciardolo, F.L.M. 113 Risby, T.H. 246,257 Rolla, G. 319,366 Roussos, C. Royston, D. 369,375 73 Sarady, J.K. Schubert, J.K. 338 84 Sethi, J.M. Siafakas, N.M. 209 Scares, M.P. 267

418

Starodub, N.F. Starodub, V.M. Steinhaeusser, W. Sterk, P.J. Stry, S. Sunshine, S.A. Taille, C.

391 391 410 196 395 387 79

Tikkanen, J.M. Tzanakis,N. Vaughan, J.W. Wright, I.G. Xagorari, A. Yacoub, M.

304 209 413 274 366 304