Clinics E-Book November 2011 by eBook Clinics

CLINICS Editor Mauricio Rocha-e-Silva Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Area Editors Ana Maria de Ulhoa Escobar Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil

Geraldo Busatto Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Paulo Hoff Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Anuar Ibrahim Mitre Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Gustavo Franco Carvalhal Faculdade de Medicina da Pontifı´cia Universidade Cato´lica do Rio Grande do Sul Porto Alegre, Rio Grande do Sul, Brazil

Renato Delascio Lopes Universidade Federal de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Ashok Agarwal The Cleveland Clinic Foundation Cleveland, Ohio, USA Berenice Bilharinho Mendonça Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Bruno Zilberstein Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Carlos Serrano Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Carmen Silvia Valente Barbas Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Claudia Regina Furquim de Andrade Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Edmund Chada Baracat Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Eliete Bouskela Universidade do Estado do Rio de Janeiro Rio de Janeiro, RJ, Brazil Emilia Inoue Sato Universidade Federal de Saõ Paulo Saõ Paulo, SP, Brazil Fulvio Alexandre Scorza Universidade Federal de Saõ Paulo Saõ Paulo, SP, Brazil

Heitor Franco de Andrade Jr. Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Ivete Bedin Prado Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Joaquim Prado Moraes-Filho Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Ludhmila Abrahao Hajjar Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Luı´z Eugeˆnio Garcez-Leme Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Lydia Masako Ferreira Universidade Federal de Saõ Paulo Saõ Paulo, SP, Brazil Maria Cecı´lia Solimene Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Newton Kara-Junior Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Olavo Pires de Camargo Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil

Rubens Belfort Jr. Universidade Federal de Saõ Paulo Saõ Paulo, SP, Brazil Ruy Jorge Cruz Junior University of Pittsburgh Pittsburgh, PA, USA Sergio Paulo Bydlowski Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Silvia Vanessa Lourenço Faculdade de Odontologia da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Simone Appenzeller Universidade Estadual de Campinas Campinas, SP, Brazil Sophie Françoise Mauricette Derchain Faculdade de Cieˆncias Me´dicas, Universidade Estadual de Campinas Campinas, SP, Brazil Suely Kazue Nagahashi Marie Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Thelma Suely Okay Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Vale´ria Aoki Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil

Editorial Board Abhijit Chandra King George’s Medical College Lucknow, India Adamastor Humberto Pereira Universidade Federal do Rio Grande do Sul Porto Alegre, RS, Brazil Adauto Castelo Universidade Federal de Saõ Paulo Saõ Paulo, SP, Brazil Ademar Lopes Fundaçaõ Antoˆnio Prudente, Hospital do Caˆncer Saõ Paulo, SP, Brazil Alberto Azoubel Antunes Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Alexandre Roberto Precioso Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Andrea Schmitt University of Goettingen Goettingen, Germany Arnaldo Valdir Zumiotti Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil

Saõ Paulo, SP, Brazil Cesar Gomes Victora Faculdade de Medicina da Universidade Federal de Pelotas Pelotas, RS, Brasil Daniel Romero Munõz Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Edmund Neugebauer Witten/Herdecke University Witten, North Rhine - Westphalia, Germany Egberto Gaspar de Moura Jr. Universidade do Estado do Rio de Janeiro Rio de Janeiro, RJ, Brazil Ernest Eugene Moore University of Colorado Denver Denver, CO, USA Euclides Ayres Castilho Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Fa´bio Biscegli Jatene Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil

Artur Brum-Fernandes Universite´ de Sherbrooke Que´bec, Canada´

Francisco Laurindo Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Carmita Helena Najjar Abdo Faculdade de Medicina da Universidade de Sa˜o Paulo

Hiroyuki Hirasawa Chiba University School of Medicine Chiba, Japan

Irismar Reis de Oliveira Faculdade de Medicina da Universidade Federal da Bahia Salvador, BA, Brasil Irshad Chaudry University of Alabama Birmingham, AL, USA Ivan Cecconello Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Ke-Seng Zhao Southern Medical University Guangzhou, China Laura Cunha Rodrigues London School of Hygiene and Tropical Medicine - University of London London, UK Marcelo Zugaib Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Marco Martins Amatuzzi Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Maria Aparecida Shikanai Yasuda Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Mauro Perretti William Harvey Research Institute London, UK Michael Gregory Sarr

Mayo Clinic Rochester, MN, USA Milton de Arruda Martins Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Mitchell C. Posner The University of Chicago Medical Center Chicago, IL, USA Moyses Szklo Johns Hopkins Bloomberg School of Public Health Baltimore, USA Navantino Alves Faculdade de Cieˆncias Me´dicas de Minas Gerais Belo Horizonte, MG, Brazil Noedir Antonio Groppo Stolf Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Pedro Puech-Leaõ Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Peter Libby Brigham and Women’s Hospital Boston, Boston, MA, USA Philip Cohen University of Houston Health Center Houston, Texas, USA Rafael Andrade-Alegre Santo Toma´s Hospital Republic of Panama´, Panama´

Ricardo Antonio Refinetti Faculdade de Medicina da Universidade Federal do Rio de Janeiro Rio de Janeiro, RJ, Brazil Roberto Chiesa San Raffaele Hospital Milan, Italy Ronald A. Asherson

Netcare Rosebank Hospital Rosebank, Johannesburg, South A´frica Samir Rasslan Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Tarcisio Eloy Pessoa de Barros Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil

Valentim Gentil Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Wagner Farid Gattaz Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil

Board of Governors Antonio Alci Barone Arnaldo Valdir Zumiotti Berenice Bilharinho de Mendonça Clarice Tanaka Claudia Regina Furquim de Andrade Dalton de Alencar Fischer Chamone Daniel Romero Munõz Edmund Chada Baracat Eduardo Massad Eloisa Silva Dutra de Oliveira Bonfa´ Euclides Ayres de Castilho Euripedes Constantino Miguel Evandro Ararigboía Rivitti Fa´bio Biscegli Jatene Flair Jose´ Carrilho Francisco Vargas Suso Gerson Chadi Giovanni Guido Cerri Irineu Tadeu Velasco Ivan Cecconello

Jorge Elias Kalil Jose´ Antonio Franchini Ramires Jose´ Eduardo Krieger Jose´ Eluf Jose´ Ota´vio Costa Auler Jose´ Ricardo de Carvalho Mesquita Ayres Luiz Augusto Carneiro D’Albuquerque Luiz Francisco Poli de Figueiredo Magda Maria Sales Carneiro-Sampaio Manoel Jacobsen Teixeira Marcelo Zugaib Marcos Boulos Marcus Castro Ferreira Maria Aparecida Shikanai Yasuda Maria Irma Seixas Duarte Miguel Lorenzo Barbero Marcial Miguel Srougi Milberto Scaff Mı´lton de Arruda Martins Noedir Antonio Groppo Stolf

Editorial Director Kavita Kirankumar Patel-Rolim Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Olavo Pires de Camargo Paulo Andrade Lotufo Paulo Hila´rio Nascimento Saldiva Paulo Marcelo Gehm Hoff Pedro Puech-Lea˜o Ricardo Ferreira Bento Roberto Zatz Roger Chammas Samir Rasslan Sandra Josefina Ferraz Ellero Grisi Selma Lancman Tarcı´sio Eloy Pessoa de Barros Uenis Tannuri Umbertina Conti Reed Valentim Gentil Venaˆncio Avancini Ferreira Alves Vicente Odone Wagner Farid Gattaz Wilson Jacob

Editorial Assistant Nair Gomes Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Daniela Aquemi Higa Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil Ariane Maris Gomes Faculdade de Medicina da Universidade de Saõ Paulo Saõ Paulo, SP, Brazil

Editorial Office: Rua Dr. Ovı´dio Pires de Campos, 225 - 6 ˚ Andar Email: clinics@hcnet.usp.br Website: www.scielo.br/clinics

CEP 05403-010 Sa˜o Paulo/SP Tel.: +55-11-2661-6235 Submission: http://www.editorialmanager.com/clinics

Indexations: LILACS; MEDLINE; PubMed; PubMed Central; SciELO; Science Citation Index Expanded (ISI Web of Knowledge); Scopus; Ulrich’s Periodical Directory; Qualis/Capes - Classified as an International Circulation Journal in Medicine. Clinics. Saõ Paulo: Scientific Journal of Hospital das Clıńicas da Faculdade de Medicina da Universidade de Saõ Paulo, 2005Monthly Periodical: January to December ISSN 1678-3379 Formerly Revista do Hospital das Clıńicas da FMUSP, 1946–2004. 1. Medicine-scientific production. 2. Medical Sciences I. Hospital das Clıńicas da Faculdade de Medicina da Universidade de Saõ Paulo. CDD 610

PUBLICATION INFORMATION AND EDITORIAL POLICIES CLINICS publishes peer-reviewed articles of interest to clinicians and researchers in the medical sciences. CLINICS is registered with PubMed Central and SciELO. This complies with the policies of funding agencies, such as, the Wellcome Trust, the Research Councils UK - RCUK, the National Institutes of Health(NIH), and the DFG, German Research Foundation, which request or require deposition of the published articles that they fund into such publicly available databases. CLINICS supports the position of the International Committee of Medical Journal Editors (http://www.icmje.org/) on trial registration. All trials initiated after January 1, 2012 must be registered prospectively (before patient recruitment begins) in a publicly accessible registry. Trials initiated before January 1, 2012 must be registered before submission to our journals. See the ICMJE FAQ on trial registration for further details. Visit http://www. who.int/ictrp/network/list_registers/en/index.html for the WHO’s list of approved registries. CLINICS suggests: http://www. clinicaltrials.gov/, a user friendly site.

Publication Fees CLINICS uses a business model in which expenses are recovered in part by charging a publication fee to the authors or research sponsors for each article they publish. Our 2011 prices are: Original Articles: R$ 1.700,00 (US$ 1,000.00); Review Articles: R$ 1.700,00 (US$ 1,000.00); Case Reports, Technical Notes, and Rapid Communications: R$ 1.000,00 (US$ 600.00); Invited Reviews, Editorials and Letters to the Editors: No charge. Manuscripts involving human subjects or the use of laboratory animals must clearly state adherence to appropriate guidelines and approval of protocols by their institutional review boards. Photographs that may identify patients or other human participants of studies shall be acceptable only when a legally valid permission (form provided when necessary) is signed by the said patient or other human participant or by his/her legally constituted representative. Manuscripts should be submitted online, in English, digitalized in a word.doc-compatible software program. Authors are strongly advised to submit the manuscript in its final form to a spell check for English (USA). Submissions with excessive spelling or syntax mistakes, as well as articles in which the meaning is not sufficiently clear shall be returned to authors for correction. Authors are also very strongly advised to limit abbreviations to the minimum possible. Abbreviations tend to make text unreadable. All abbreviations must be defined when first used. Only terms or expressions used at least 5 times throughput the text should be abbreviated. Never use abbreviations spelt as common English words such as, for instance FUN, PIN, SCORE, SUN, etc. Please make sure to submit your manuscript in the exact format described below. Failure to do so will cause the submission to be returned to you during the preliminary examination by the Editorial Office.

Manuscripts are invited in the following categories: ORIGINAL STUDY: Complete original studies should be submitted in this category. Three sections are offered: Basic, Clinical, and Surgical Research. Original Studies must follow the following format:

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Title (up to 250 characters); Running title (up to 40 characters, letters and spaces); Full address of corresponding author only; Authors’ names (without titles or graduations). Authors should have participated sufficiently in the work to take public responsibility for appropriate portions of the content. Such participation must be declared in this section of the manuscript.

Manuscript:

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Abstract: limited to 250 words and structured into: Objectives, Method, Results, Conclusions. Citations or abbreviations (except internationally recognized such as weights, measures, physical or chemical) are not allowed. Authors are strongly recommended not to display numerical statistical information, but merely to state what is significantly different (or not) between described parameters. Keywords: 3–6 items from the Medical Subject Headings (Mesh) should be used. Introduction: should set the purpose of the study, give a brief summary (not a review) of previous relevant works, and state what new advance has been made in the investigation. It should not include data or conclusions from the work being reported. A final sentence summarizing the novelty to be presented is permissible. Materials and Methods: should briefly give clear and sufficient information to permit the study to be repeated by others. Standard techniques need only be referenced. Previously published methods may be briefly described following the reference. Ethics: when reporting experiments on human subjects, indicate whether the procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional or regional) and with the Helsinki Declaration of 1975, as revised in 1983. When reporting experiments on animals, indicate whether the institution’s or a national research council’s guide for, or any national law on the care and use of laboratory animals was followed. Results: should be a concise account of the new information discovered, with the least personal judgment. Do not repeat in text all the data in the tables and illustrations, but briefly describe what such material contains. Discussion: should be limited to the significance of the new information and relate the new findings to existing knowledge. Only unavoidable citations should be included. Citation to review articles are not encouraged in this section. Acknowledgements: should be short and concise, and restricted to those absolutely necessary. References in text: CLINICS adopts the Vancouver format. Cite references in text with Arabic numerals, in order of appearance, within parentheses (1), after previous word, with spacing as in this example: ‘‘Diabetes (2), hypertension (3,4) and alcoholism (5–9)

are complex medical problems (10).’’ Under exceptional circumstances authors’ names may appear in text: Single author: ‘‘Einstein (11) proposed a new theory …’’ Two authors: ‘‘Watson & Crick (12) reported on the structure of …’’ Three or more authors: ‘‘Smith et al (13) described …’’ Reference List: Only citations that appear in the text should be referenced. Unpublished papers, unless accepted for publication, should not be cited. Work accepted for publication should be referred to as ‘‘in press’’ and a letter of acceptance of the journal must be provided. Unpublished data should only be cited in the text as ‘‘unpublished observations’’, and a letter of permission from the author must be provided. Authors are responsible for the accuracy and completeness of their references and for correct text citation. CLINICS adopts the Vancouver format; references must be restricted to directly relevant published works, papers, or abstracts that have been accepted for publication. Usually the total number of references should not exceed 35. For up to six authors, list all authors; for more than 6 authors, list first six, followed by ‘‘et al’’. Tables and Figures: The maximum number of tables plus figures is six. Tables: Do not be incorporate Tables into the Manuscript. Upload each table individually into the system. Tables should be constructed using the Table feature in your word processor or using a spreadsheet program such as Excel. Tables should be numbered in order of appearance in the text with Arabic numerals. Each table should have a title and, if necessary, an explanatory legend. All tables must be referred to and succinctly described in the text. Under no circumstances should a table repeat data presented in an illustration. Statistical measures of variation (i.e., standard deviation, standard error) should be identified, and decimal places in tabular data should be restricted to those with mathematical and statistical significance. Figures: Do not incorporate Figures into the Manuscript. Photographs, illustrations, charts, drawings, line graphs, etc are all defined as Figures. We do not publish Pictures Graphics, Photos, etc. Number your figures consecutively in Arabic numerals in order of appearance. Upload each Figure individually into the system. Legend(s) should be descriptive and allow examination of the figure without reference to text. Legends should be incorporated into the Main Document, after the Tables (if any) or after the references. Photographic illustrations should must be of professional quality and uploaded as *.tiff files, Typewritten or hand lettering is unacceptable, as are figures generated by dot matrix printers. Generally, figures will be reduced to fit one column of text. Actual magnification of all photomicrographs should be provided, preferably by placing a length scale on the print. Line graphs and charts must never be sent as jpeg illustrations. It is usually best to prepare these as ExcelH files. When ready copy the line graph or chart to a word.doc sheet. Comments: Authors should use this space to describe the novelty contained in their original study. Only the

Editor of CLINICS has access to this section of the submission. FAST TRACK ARTICLES: Fast track articles should follow the same format described above for original studies. Fast track articles must be complete original studies with justifiable urgency for publication. The Editorial Office undertakes to produce a first action response in the shortest possible time and to publish accepted Fast Track articles in the next available issue. Only one article may be submitted as Fast Track in any calendar year by any author or co-author. In Comments, authors must explain the reasons for the claim to Fast Track. Rejection by journals with a higher Impact Factor that ours is an acceptable reason for requesting FAST TRACK, provided that the reviewers’ reports from the previous submission be included in the present submission. Information contained in the comments is limited to the Editor and shall remain confidential. No publication fee discount is allowed for accepted Fast Track articles. REVIEW ARTICLES: Such articles should cover themes relevant to medical practice or mammalian function. Spontaneously submitted reviews are welcome, but potential authors should bear in mind that they are expected to have expertise in the reviewed field. The sections should be arranged as follows:

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CASE REPORTS: Case Reports will be published only if justified by the extreme rarity of the case and/or novelty of adopted procedures. Case reports should be arranged as follows:

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Peer Review: Manuscripts are reviewed by at least two expert consultants. Accepted manuscripts are edited to comply with the Journalâ&#x20AC;&#x2122;s format, to remove redundancies, and to improve clarity and understanding without altering meaning. The edited text will be presented to authors for approval. Submission: A copyright transfer form, signed by all authors, must be submitted by fax (55-11-2661-7524) or by mail as soon as the manuscript is submitted. Any financial or other relationships that could lead to a conflict of interest must be disclosed in the copyright transfer form. If the Editor considers this conflict of interest relevant to the paper, a footnote will be added to show the equity interest in or affiliation with the identified commercial firm(s). As soon as authors are satisfied that the manuscript complies with the Journal format, our site should be accessed through www. clinics.org.br. The system will guide authors through the manuscript submission process and prompt authors to input information into specific fields as they are submitting their manuscript. The Editorial Office will be automatically notified of the submission and will send an email confirming it as soon as the submission letter reaches the office. Progress of the manuscript through the Editorial Office procedures will be available to authors at all times.

ISSN-1807-5932

CLINICS CONTENTS Clinics 2011 66(11):1849–1999

CLINICAL SCIENCES

PROP1 and CTNNB1 expression in adamantinomatous craniopharyngiomas with or without bcatenin mutations Carolina M.G. Cani, Hamilton Matushita, Luciani R. S. Carvalho, Ibere C. Soares, Luciana P. Brito, Madson Q. Almeida, Berenice B. Mendonc¸a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1849

Autologous stem cell transplantation improves quality of life in economically challenged, Brazilian multiple myeloma patients Leina Y. Etto, Vaˆnia Maris Morelli, Vanderleia C. Silva, Vania T. M. Hungria, Rozana M. Ciconelli, Manuella S. S. Almeida, Jose´ Salvador R. de Oliveira, Jose´ Carlos Barros, Brian G. Durie, Gisele W. B. Colleoni . . . . . . . . . . . . . 1855

Association between participation in community groups and being more physically active among older adults from Floriano´polis, Brazil Giovana Zarpellon Mazo, Taˆnia Bertoldo Benedetti, Cinara Sacomori . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1861

Gender differences, polypharmacy, and potential pharmacological interactions in the elderly Carina Duarte Venturini, Paula Engroff, Luı´sa Scheer Ely, Luı´sa Faria de Arau´jo Zago, Guilherme Schroeter, Irenio Gomes, Geraldo Attilio De Carli, Fernanda Bueno Morrone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1867

Non-small cell lung cancer in never smokers: a clinical entity to be identified Ilka Lopes Santoro, Roberta Pulcheri Ramos, Juliana Franceschini, Sergio Jamnik, Ana Luisa Godoy Fernandes . . . . 1873

M2-Polarized tumor-associated macrophages are associated with poor prognoses resulting from accelerated lymphangiogenesis in lung adenocarcinoma Bicheng Zhang, Guoqing Yao, Yafei Zhang, Juan Gao, Bo Yang, Zhiguo Rao, Jianfei Gao . . . . . . . . . . . . . . . . . 1879

A negative expiratory pressure test during wakefulness for evaluating the risk of obstructive sleep apnea in patients referred for sleep studies Salvatore Romano, Adriana Salvaggio, Anna Lo Bue, Oreste Marrone, Giuseppe Insalaco . . . . . . . . . . . . . . . . . . 1887

The characteristics of stress cardiomyopathy in an ethnically heterogeneous population Francisco O. Nascimento, Orlando Santana, Margarita Perez-Caminero, Alexandre M. Benjo . . . . . . . . . . . . . . . . 1895

Preoperative nodal staging of non-small cell lung cancer using

99m

Tc-sestamibi spect/ct imaging

Juliana Muniz Miziara, Euclides Timo´teo da Rocha, Jose´ Elias Abra˜o Miziara, Gustavo Fabene Garcia, Maria Izilda Previato Simo˜es, Marco Antoˆnio Lopes, Lı´gia Maria Kerr, Carlos Alberto Buchpiguel . . . . . . . . . . . . . . . . . . . . . 1901

Assessment of symptoms of urinary incontinence in women with polycystic ovary syndrome Thais Montezuma, Fla´via lgna´cio Antoˆnio, Ana Carolina Japur de Sa´ Rosa e Silva, Marcos Felipe Silva de Sa´, Rui Alberto Ferriani, Cristine Homsi Jorge Ferreira . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1911

The validity and reliability of the Portuguese versions of three tools used to diagnose delirium in critically ill patients Dimitri Gusmao-Flores, Jorge Ibrain Figueira Salluh, Felipe Dal-Pizzol, Cristiane Ritter, Cristiane Damiani Tomasi, Marco Antoˆnio Sales Dantas de Lima, Lauro Reis Santana, Rita Ma´rcia Pacheco Lins, Patrı´cia Pimenta Lemos, ´ vila Chalhub, Melissa Tassano Pitrowsky, Acioly L.T. Lacerda, Gisele Vasconcelos Serpa, Jenisson Oliveira, Ricardo A Karestan C. Koenen, Lucas C. Quarantini . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1917

Do Omega-3 fatty acids prevent atrial fibrillation after open heart surgery? A meta-analysis of randomized controlled trials Luciana Armaganijan, Renato D. Lopes, Jeff S. Healey, Jonathan P. Piccini, Girish M. Nair, Carlos A. Morillo . . . . . 1923

The ADA*2 allele of the adenosine deaminase gene (20q13.11) and recurrent spontaneous abortions: an age-dependent association Daniela Prudente Teixeira Nunes, Lı´gia Cosentino Junqueira Franco Spegiorin, Cinara Ca´ssia Branda˜o de Mattos, Antonio Helio Oliani, Denise Cristina Mo´s Vaz-Oliani, Luiz Carlos de Mattos . . . . . . . . . . . . . . . . . . . . . . . . . . . 1929

Revisting stapled and handsewn loop ileostomy closures: a large retrospective series Emre Balik, Tunc Eren, Dursun Bugra, Yilmaz Buyukuncu, Ali Akyuz, Sumer Yamaner . . . . . . . . . . . . . . . . . . . . 1935

Mechanical evaluation of the resistance and elastance of post-burn scars after topical treatment with tretinoin Maria Fernanda Dematte, Rolf Gemperli, Alessandra Grassi Salles, Marisa Dolhnikoff, Tatiana Lanc¸as, Paulo Hila´rio Nascimento Saldiva, Marcus Castro Ferreira . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1949

Restless legs syndrome in subjects with a knee prosthesis: evidence that symptoms are generated in the periphery Jose´ Carlos Pereira Jr, Joa˜o Luiz Pereira da Silva Neto, Ma´rcia Pradella-Hallinan. . . . . . . . . . . . . . . . . . . . . . . . . 1955

BASIC RESEARCHES

Vimentin and laminin are altered on cheek pouch microvessels of streptozotocin-induced diabetic hamsters Jemima Fuentes R. Silva, Fatima Z. G. A. Cyrino, Marisa M. D. Breitenbach, Eliete Bouskela, Jorge Jose´ Carvalho . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1961

Impact of Plasma-Lyte pH 7.4 on acid-base status and hemodynamics in a model of controlled hemorrhagic shock Danilo Teixeira Noritomi, Adriano Jose´ Pereira, Diogo Diniz Gomes Bugano, Paulo Sergio Rehder, Elie´zer Silva . . . . 1969

REVIEW

An overview of recently published medical papers in Brazilian scientific journals Mauricio Rocha e Silva, Ariane Gomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1975

CASE REPORTS

Immunoglobulin G4-related systemic sclerosing disease in a patient with sclerosing cholangitis, inflammatory pseudotumors of the lung and multiple radiological patterns: a case report Olı´via Meira Dias, Alexandre de Melo Kawassaki, Hironori Haga, Alberto Cukier, Carlos Roberto Ribeiro Carvalho . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1983

The best approach for diagnosing primary sclerosing cholangitis Wellington Andraus, Luciana Haddad, Lucas Souto Nacif, Felipe D. Silva, Roberto Blasbalg, Luiz Augusto Carneiro D’Albuquerque. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1987

Primary Sjo¨gren’s syndrome in children: Is a family approach indicated? Barbara S. Longhi, Simone Appenzeller, Maraisa Centeville, Reinaldo J. Gusma˜o, Roberto Marini . . . . . . . . . . . . 1991

Unusual presentation of multiple aneurysms of the ascending aorta Sergio Francisco Santos Jr, Marcelo Luiz Peixoto Sobral, Anderson da Silva Terrazas, Gilmar Geraldo Santos, Noedir A. G Stolf . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1995

ERRATA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1999

CLINICS 2011;66(11):1849-1854

DOI:10.1590/S1807-59322011001100001

CLINICAL SCIENCE

PROP1 and CTNNB1 expression in adamantinomatous craniopharyngiomas with or without b-catenin mutations Carolina M.G. Cani,I Hamilton Matushita,II Luciani R. S. Carvalho,I Ibere C. Soares,III Luciana P. Brito,I Madson Q. Almeida,I Berenice B. MendonçaI I Faculdade de Medicina da Universidade de Saõ Paulo, Disciplina de Endocrinologia, Unidade de Endocrinologia do Desenvolvimento, Laborato´rio de Hormoˆnios e Gene´tica Molecular LIM/42, Saõ Paulo, SP/Brazil. II Faculdade de Medicina da Universidade de Saõ Paulo, Departamento de Neurologia, Saõ Paulo, SP/Brazil. III Faculdade de Medicina da Universidade de Saõ Paulo, Divisaõ de Anatomia Patolo´gica, Laborato´rio de Patologia Hepa´tica/LIM14, Saõ Paulo, SP/Brazil.

INTRODUCTION: Activating mutations in exon 3 of the b-catenin gene are involved in the pathogenesis of adamantinomatous craniopharyngiomas. Recently, the interaction between b-catenin and PROP1 has been shown to be responsible for pituitary cell lineage determination. We hypothesized that dysregulated PROP1 expression could also be involved in the pathogenesis of craniopharyngiomas. OBJECTIVES: To determine whether dysregulated gene expression was responsible for tumor pathogenesis in adamantinomatous craniopharyngiomas, the b-catenin gene was screened for mutations, and the expression of the b-catenin gene and PROP1 was evaluated. METHODS: The b-catenin gene was amplified and sequenced from 14 samples of adamantinomatous craniopharyngiomas. PROP1 and b-catenin gene expression was assessed by real-time RT-PCR from 12 samples, and b-catenin immunohistochemistry was performed on 11 samples. RESULTS: Mutations in the b-catenin gene were identified in 64% of the adamantinomatous craniopharyngiomas samples. Evidence of b-catenin gene overexpression was found in 71% of the tumors with b-catenin mutations and in 40% of the tumors without mutations, and b-catenin immunohistochemistry revealed a nuclear staining pattern for each of the analyzed samples. PROP1 expression was undetectable in all of the tumor samples. CONCLUSION: We found evidence of b-catenin gene overexpression in the majority of adamantinomatous craniopharyngiomas, and we also detected a nuclear b-catenin staining pattern regardless of the presence of a bcatenin gene mutation. These results suggest that WNT signaling activation plays an important role in the pathogenesis of adamantinomatous craniopharyngiomas. Additionally, this study was the first to evaluate PROP1 expression in adamantinomatous craniopharyngiomas, and the absence of PROP1 expression indicates that this gene is not involved in the pathogenesis of this tumor, at least in this cohort. KEYWORDS: Sellar tumor; Real-time PCR; WNT pathway; Gene expression; Pituitary. Cani CMG, Matushita H, Carvalho LRS, Soares IC, Brito LP, Almeida MQ, et al. PROP1 and CTNNB1 expression in adamantinomatous craniopharyngiomas with or without b-catenin mutations. Clinics. 2011;66(11):1849-1854. Received for publication on June 6, 2011; First review completed on June 14, 2011; Accepted for publication on July 7, 2011 E-mail: carolinacani@gmail.com Tel.: 55 11 2661-7512

Craniopharyngiomas are histologically benign and can be categorized into two primary subtypes with different histopathological features: adamantinomatous and papillary. The former is thought to arise from squamous cell rests of the craniopharyngeal duct, which is a canal that connects the stomodeal ectoderm with Rathke’s pouch,2,3 and the latter subtype results from metaplasia of the adenohypophyseal cells in the pituitary gland.4 The adamantinomatous craniopharyngioma is the most common histological type and occurs at all ages, but it predominantly affects individuals in the first two decades of life. Conversely, papillary craniopharyngiomas have been almost exclusively described in adults.5

INTRODUCTION Craniopharyngiomas are rare intra/suprasellar neoplasms that account for 10% of all pediatric intracranial tumors. During childhood, they are the most common lesions involving the hypothalamo-pituitary regions.1

Copyright ß 2011 CLINICS – This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. No potential conflict of interest was reported.

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cysts and tumors. The phenotype of these mice suggests that dysregulation of PROP1 expression in humans may contribute to the pathogenesis of pituitary tumors.26 Morbidity and mortality frequently result from the aggressive nature of craniopharyngiomas as well as their treatment, which lead to significant medical and social problems. Therefore, it is critically important to better elucidate the pathogenesis of this tumor. Thus, the aim of this study was to analyze PROP1 and CTNNB1 expression in adamantinomatous craniopharyngiomas and correlate these findings with the presence of somatic mutations in CTNNB1.

The pathogenesis of craniopharyngiomas is poorly understood. To date, mutations in the b-catenin gene have been identified only in the adamantinomatous subtype and at a variable frequency.6-9 The b-catenin protein is encoded by the CTNNB1 gene. The association of b-catenin with cadherin adhesion molecules at the plasma membrane provides a link between cadherins and the actin cytoskeleton, which is essential for strong cell-cell adhesion.10 Furthermore, b-catenin is a key mediator of the canonical WNT signaling pathway, which is involved in the control of stem cell pluripotency, cell proliferation, differentiation and migration.11 b-catenin is regulated by a multiprotein complex that contains the tumor suppressor protein adenomatous polyposis coli (APC), the scaffold proteins Axin and Axin2 and the phosphokinases glycogen synthase kinase-3b (GSK-3b) and casein kinase 1. When WNT signaling is inactive, b-catenin is phosphorylated within the multiprotein complex; this phosphorylation results in its ubiquitination and degradation by the proteasome.12 However, WNT activation inhibits GSK-3b, and b-catenin is then able to avoid degradation and accumulate in the cytoplasm. It is then translocated into the nucleus where it binds to the transcription factor T cell factor (TCF)/ lymphoid enhancer factor (LEF) and thereby regulates gene expression.13 The GSK-3b-binding domain of b-catenin corresponds to its degradation targeting box and is encoded by exon 3 of CTNNB1. Activating mutations within this region, which have been described for the adamantinomatous subtype but not the papillary subtype,6-9 promote bcatenin accumulation through the inhibition of its degradation and lead to WNT signaling activation.14 Recently, the interaction between b-catenin and PROP1 was described as a new mechanism for the b-catenindependent regulation of pituitary cell lineage determination in response to WNT signaling. According to this model, the PROP1/b-catenin protein complex would act as a binary switch to simultaneously repress the transcription factor HESX1 and activate the expression of the transcription factor PIT1.15 Interestingly, both factors are involved in pituitary cell proliferation and differentiation. PROP1 is a paired-like homeodomain transcription factor that is essential for pituitary development and function due to its regulation of PIT1 expression,16 which is essential for the terminal differentiation and expansion of somatotropes, lactotropes and thyrotropes.17 PROP1 is encoded by PROP1, a homeobox gene with 3 exons. The homeodomain is characterized by three helices, the third of which is essential for target gene DNA recognition.18 PROP1 can bind to DNA via the carboxy-terminus to activate target genes or via the amino-terminus and the homeodomain to repress target genes. These results suggest that PROP1 can act as both a transcriptional activator and repressor.16,19 Patients with loss-of-function mutations in PROP1 present with combined pituitary hormonal deficiencies that are generally associated with pituitary enlargement.20-25 Magnetic resonance imaging of the sellar regions of these patients can demonstrate a T1 hyperintense signal that resembles craniopharyngioma-like tumors. In aGSU-Prop1 transgenic mice, persistent Prop1 expression may result in increased cellular proliferation, which would enhance the probability of a tumor-initiating mutation. In fact, these mice exhibit defects consistent with the dysregulation of pituitary cell proliferation, such as adenomatous hyperplasia with the formation of Rathke’s cleft

MATERIALS AND METHODS Subjects The study was approved by the ethics committee of the Hospital das Clı´nicas in Sa˜o Paulo, Brazil, and informed written consent was obtained from all of the patients’ parents. We analyzed 14 adamantinomatous craniopharyngiomas that were obtained during the surgical resections of children and adolescents (five males and nine females). The age of these patients ranged from 1.58 to 21.58 years (mean 10 years). The phenotype of these patients was very similar. Most patients presented with headaches, visual deficiencies, hypopituitarism and large tumors, and most patients required two or more surgical procedures for the relief of their neurological symptoms. All samples were used for cDNA sequencing. However, two of the 14 tumor samples were excluded from the expression analysis due to their poor RNA quality. b-catenin immunohistochemistry was performed on ten samples.

RNA extraction and reverse transcription Total RNA was extracted from fresh-frozen tissues, which had been preserved in RNAlaterH (Ambion, Austin, TX USA) at -20 ˚C using the TRIzolH reagent (Invitrogen, Carlsbad, CA, USA). The elapsed time between tissue isolation and RNA extraction was approximately one to two weeks. The integrity of the extracted RNA was evaluated on a 1% agarose gel, and the concentrations were determined using a spectrophotometer (Eppendorf Biophotometer, Hamburg, Germany). The cDNA samples were obtained from a reverse transcription reaction performed on the same day as RNA extraction and were stored at -20 ˚C. The cDNA was synthesized from 1.25 mg of total RNA in a final volume of 50 mL, and a final concentration of 25 ng/mL was obtained using the high-capacity cDNA archive kit (Applied Biosystems, Foster City, CA, USA). The reverse transcription reaction was performed at 25 ˚C for 10 minutes and was followed by a cycle at 37 ˚C for 120 minutes. Next, the remaining RNA was stored at -80 ˚C. The molecular analyses were most often conducted within two weeks of cDNA synthesis.

PCR Exon 3 of CTNNB1 (ENSG00000168036) was amplified using the primers sense 59-GATTTGATGGAGTTGGACATGG-39 and antisense 59-TGTTCTTGAGTGAAGGACTGAG39. The PCR reaction setup was as follows. cDNA (25 ng) from each tumor sample was added to a 25-mL reaction mixture containing 5x PCR buffer (50 mM KCL, 1.5 mM MgCl2 and 10 mM Tris-HCL pH 9.0), 200 mmol of dNTPs, 20 pmol of each primer and 1.5 U of GoTaqH DNA

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Polymerase (Promega, Madison, WI, USA). The PCR reaction mixture was then denatured for 5 minutes at 94 ˚C, cycled 35 times (94 ˚C for 45 sec, 65 ˚C for 45 sec and 72 ˚C for 60 sec) and incubated at 72 ˚C for 10 minutes for a final extension. The PCR reaction yielded a fragment of 218 bp. Each of the amplified fragments was examined using 1.5% agarose gel electrophoresis, stained by ethidium bromide and visualized under UV light. The PCR products were pretreated with an enzymatic combination of exonuclease I and shrimp alkaline phosphatase (United Stated Biochemical Corp., Cleveland, OH, USA) and were directly sequenced using the BigDye terminator cycle sequencing ready reaction kit (PE Applied Biosystems, Foster City, CA) and an ABI PRISM 3100 automatic sequencer (Perkin-Elmer Corp., Waltham, MA, USA).

solution diluted in methanol (v/v) and were then washed with phosphate buffer (10 mM phosphate buffered saline (PBS) pH 7.4). Proteins were blocked using CAS-Block (Zymed). For the detection of b-catenin, monoclonal antibody (clone 14, BD) was used at a 15800 dilution, and the stained cells were incubated at 37 ˚C for 30 minutes and then at 4 ˚C overnight. The final visualization was carried out by chromogenic detection using a solution of PBS containing 0.1% diaminobenzidine, 0.06% H2O2 and 1% dimethyl sulfoxide. The sections were immersed in this solution for 5 minutes at 37 ˚C. Counterstaining was performed using Harris hematoxylin. A known positive control was included, and sections that were incubated with PBS instead of the primary antibody served as negative controls.

Statistical analysis Quantitative real-time RT-PCR

All statistical analyses were performed with SPSS version 16.0 (SPSS, Chicago, IL). Categorical variables were compared using Fisher’s exact test, and p values ,0.05 were considered significant.

The PROP1, PIT1 and CTNNB1 mRNA levels were measured by quantitative real-time RT-PCR from 12 adamantinomatous craniopharyngiomas using the ABI Prism 7000 sequence detection system (Applied Biosystems, Foster City, CA, USA). TaqManH gene expression assays were used to quantify gene expression (PROP1 - Hs00196604_m1; PIT1 Hs00230821_m1; CTNNB1 - Hs00355045_m1). The amplification reactions were performed in triplicate using 12.5 mL of TaqManH universal PCR master mix (Applied Biosystems), 2 mL of cDNA (25 ng/mL) and 1.25 mL of TaqManH gene expression assay solution in a final volume of 25 mL. The geNorm software was used to identify the most adequate internal control gene for the normalization of target gene expression.27 The three most stably expressed housekeeping genes in adamantinomatous craniopharyngiomas were PGK1 (phosphoglyceratekinase 1 - 4326318E), PPIA (cyclophilin A - 4326316E) and HPRT1 (hypoxanthine phosphoribosyltransferase 1 - 4326321E). The geometric mean of the expression of these three genes was used as the internal control gene value. A pooled sample of normal pituitary tissue from 39 adult patients (Human Pituitary Gland Poly A+ RNA, Clontech, Palo Alto, CA, USA) was used as a reference for relative quantification using the 2-DDCT method.28 A no template control (NTC) sample was used in each amplification plate to exclude the possibility of contamination. PIT1 expression was also evaluated to exclude the presence of normal pituitary gene expression in the craniopharyngioma samples. PIT1 is a transcription factor that is a member of the POU domain family, which is essential for the proliferation and differentiation of the somatotrope, lactotrope and thyrotrope pituitary lineages.29 Samples were considered as overexpressed when they demonstrated a two-fold or greater expression change over the normal pooled pituitary samples. Undetectable expression was defined as a CT value greater than 34 due to high variability and poor precision of the relative quantification from the tumor samples above this CT value.

RESULTS Heterozygous missense mutations in exon 3 of CTNNB1 were found in 9 of the 14 adamantinomatous craniopharyngiomas within codons 32, 33, 37 and 41, which affect the target region of GSK-3b (Table 1). All but two of these mutations have been previously described in craniopharyngiomas.6-9 One of these novel mutations was the result of an amino acid substitution of aspartic acid, a hydrophilic amino acid, for valine, a hydrophobic amino acid, at codon 32. The other novel mutation was a serine to proline replacement at residue 37; both of these amino acids are classified as polar and uncharged. CTNNB1 overexpression was found in five of the seven samples with b-catenin mutations (tumor samples 3, 5, 9, 11, and 14), and this expression was found to be between 2.5and 6.2-fold greater in these samples than in those from the normal pituitary. Of the five samples without b-catenin mutations, two were found to have upregulated CTNNB1 expression (fold change of 4.6 and 3.6 for samples 4 and 13, respectively) (Figure 1). However, this difference did not reach statistical significance (p = 0.558). Table 1 - Somatic mutations in exon 3 of CTNNB1 in patients with adamantinomatous craniopharyngiomas. Patients 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Immunohistochemistry Immunohistochemical analyses were performed on 3-mm paraffin sections that were deparaffinized in xylene and rehydrated through a series of ethanol treatments. Antigen retrieval was accomplished by incubating the sections in 10 mM sodium citrate pH 6.0 and heating them with a steamer. To block endogenous peroxidase activity, sections were immersed for 30 minutes in a 6% hydrogen peroxide

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Age (years)

Sex

Coding DNA sequence change

Amino acid change

9.66 4 1.58 10.58 9.9 21 2.41 1.83 13.58 17.58 14.5 11.66 9.25 4

M M F F M M F M F F F F F F

c.122C.T Not found c.110C.T Not found c.109T.C Not found c.94G.A c.122C.T c.98C.T c.94G.A c.98C.G Not found Not found c.95A.T

p.Thr41Ile Not found p.Ser37Phe Not found p.Ser37Pro1 Not found p.Asp32Phe p.Thr41Ile p.Ser33Phe p.Asp32Phe p.Ser33Cys Not found Not found p.Asp32Val1

Mutations not previously described in adamantinomatous craniopharyngiomas.

PROP1 and CTNNB1 expression in craniopharyngiomas Cani CMG et al.

CLINICS 2011;66(11):1849-1854

Figure 1 - CTNNB1 gene expression in adamantinomatous craniopharyngiomas with or without somatic CTNNB1 mutations.

Mutations in exon 3 of CTNNB, which encodes the GSK-3b binding domain of b-catenin, have been previously shown to occur in 16% to 100% of adamantinomatous craniopharyngiomas.6-9 In our cohort, CTNNB1 mutations were identified in 64% of the adamantinomatous craniopharyngiomas. These mutations were found at codon 32 (n = 3), which flanks the phosphorylation site of GSK-3b, as well as codons 33 (n = 1), 37 (n = 3) and 41 (n = 2), which affect the serine/threonine residues of b-catenin that are the target of GSK-3b.14 Recently, mutations in these codons were shown to be responsible for increased b-catenin target gene activation, and this increase was represented by elevated mRNA levels of Axin2 and BMP4 in craniopharyngioma cells with nuclear b-catenin accumulation.30 Axin2 is a well-recognized inhibitor and target gene of b-catenin31, whereas BMP4 plays a crucial role in tooth development and enhances cell proliferation in tumors with b-catenin accumulation.32,33

The PROP1 mRNA levels were undetectable in 10 of 12 samples. Two of these samples showed both PROP1 and PIT1 expression, which indicated contamination from normal pituitary tissue. Therefore, these samples were discarded from the analysis. Immunohistochemical staining for b-catenin expression showed a nuclear staining pattern (Figure 2) in all the analyzed samples, and this same result was also observed for those samples without CTNNB1 mutations (Table 2).

DISCUSSION Craniopharyngiomas are the most common lesions of the hypothalamo-pituitary region in the child population.1 Despite their benign histological appearance, the aggressive behavior and the invasiveness of these tumors into pituitary and parasellar structures, such as the optic nerve and the hypothalamus, can result in significant neurological sequelae and consequently may impair the quality of life.

Table 2 - Immunohistochemical staining for b-catenin expression, the mutational analysis and the expression of CTNNB1 in adamantinomatous craniopharyngiomas. Patients 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Nuclear staining

CTNNB1 mutation

CTNNB1 expression (fold number)

++ Not performed +++ + Not performed Not performed + ++ + + ++ + ++ Not performed

Yes No Yes No Yes No Yes Yes Yes Yes Yes No No Yes

0.97 1.6 6.2 4.5 4.4 1.7 3.3 2.7 2.5 1.9 3.8 1.9 3.5 3.6

Nuclear staining was classified as follows: +, staining in a maximum of 10% of the cells; ++, staining in 10% to 50% of the cells; +++, staining in more than 50% of the cells. Expression is represented by the fold change compared to the normal pituitary pool.

Figure 2 - b-catenin immunohistochemical staining showing the nuclear staining pattern.

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PROP1 and CTNNB1 expression in craniopharyngiomas Cani CMG et al. 4. Asa SL, Kovacs K, Bilbao JM. The pars tuberalis of the human pituitary. A histologic, immunohistochemical, ultrastructural and immunoelectron microscopic analysis. Virchows Arch A Pathol Anat Histopathol. 1983;399:49-59, doi: 10.1007/BF00666218. 5. Adamson TE, Wiestler OD, Kleihues P, Yasargil MG. Correlation of clinical and pathological features in surgically treated craniopharyngiomas. J Neurosurg. 1990;73:12-7. 6. Sekine S, Shibata T, Kokubu A, Morishita Y, Noguchi M, Nakanishi Y, et al. Craniopharyngiomas of adamantinomatous type harbor betacatenin gene mutations. Am J Pathol. 2002;161:1997-2001. 7. Kato K, Nakatani Y, Kanno H, Inayama Y, Ijiri R, Nagahara N, et al. Possible linkage between specific histological structures and aberrant reactivation of the Wnt pathway in adamantinomatous craniopharyngioma. J Pathol. 2004;203:814-21. 8. Buslei R, Nolde M, Hofmann B, Meissner S, Eyupoglu IY, Siebzehnrubl F, et al. Common mutations of beta-catenin in adamantinomatous craniopharyngiomas but not in other tumours originating from the sellar region. Acta Neuropathol. 2005;109:589-97, doi: 10.1007/s00401-005-1004-x. 9. Oikonomou E, Barreto DC, Soares B, De Marco L, Buchfelder M, Adams EF. Beta-catenin mutations in craniopharyngiomas and pituitary adenomas. J Neurooncol. 2005;73:205-9, doi: 10.1007/s11060-004-5232-z. 10. Takeichi M. Cadherin cell adhesion receptors as a morphogenetic regulator. Science. 1991;251:1451-5, doi: 10.1126/science.2006419. 11. Gooding JM, Yap KL, Ikura M. The cadherin-catenin complex as a focal point of cell adhesion and signalling: new insights from three-dimensional structures. Bioessays. 2004;26:497-511, doi: 10.1002/bies.20033. 12. Kikuchi A, Yamamoto H, Kishida S. Multiplicity of the interactions of Wnt proteins and their receptors. Cell Signal. 2007;19:659-71, doi: 10. 1016/j.cellsig.2006.11.001. 13. Hurlstone A, Clevers H. T-cell factors: turn-ons and turn-offs. EMBO J. 2002;21:2303-11, doi: 10.1093/emboj/21.10.2303. 14. Polakis P. Wnt signaling and cancer. Genes Dev. 2000;14:1837-51. 15. Olson LE, Tollkuhn J, Scafoglio C, Krones A, Zhang J, Ohgi KA, et al. Homeodomain-mediated beta-catenin-dependent switching events dictate cell-lineage determination. Cell. 2006;125:593-605, doi: 10.1016/j.cell. 2006.02.046. 16. Sornson MW, Wu W, Dasen JS, Flynn SE, Norman DJ, Oâ&#x20AC;&#x2122;Connell SM, et al. Pituitary lineage determination by the Prophet of Pit-1 homeodomain factor defective in Ames dwarfism. Nature. 1996 Nov 28;384:327-33, doi: 10.1038/384327a0. 17. Zhu X, Gleiberman AS, Rosenfeld MG. Molecular physiology of pituitary development: signaling and transcriptional networks. Physiol Rev. 2007;87:933-63, doi: 10.1152/physrev.00006.2006. 18. Nakayama M, Kato T, Susa T, Sano A, Kitahara K, Kato Y. Dimeric PROP1 binding to diverse palindromic TAAT sequences promotes its transcriptional activity. Mol Cell Endocrinol. 2009;307:36-42, doi: 10.1016/j.mce. 2009.03.010. 19. Showalter AD, Smith TP, Bennett GL, Sloop KW, Whitsett JA, Rhodes SJ. Differential conservation of transcriptional domains of mammalian Prophet of Pit-1 proteins revealed by structural studies of the bovine gene and comparative functional analysis of the protein. Gene. 2002;291:211-21, doi: 10.1016/S0378-1119(02)00599-1. 20. Mendonca BB, Osorio MG, Latronico AC, Estefan V, Lo LS, Arnhold IJ. Longitudinal hormonal and pituitary imaging changes in two females with combined pituitary hormone deficiency due to deletion of A301,G302 in the PROP1 gene. J Clin Endocrinol Metab. 1999;84:942-5, doi: 10.1210/jc.84.3.942. 21. Voutetakis A, Argyropoulou M, Sertedaki A, Livadas S, Xekouki P, Maniati-Christidi M, et al. Pituitary magnetic resonance imaging in 15 patients with Prop1 gene mutations: pituitary enlargement may originate from the intermediate lobe. J Clin Endocrinol Metab. 2004;89:2200-6, doi: 10.1210/jc.2003-031765. 22. Teinturier C, Vallette S, Adamsbaum C, Bendaoud M, Brue T, Bougneres PF. Pseudotumor of the pituitary due to PROP-1 deletion. J Pediatr Endocrinol Metab. 2002;15:95-101, doi: 10.1515/JPEM.2002.15.1.95. 23. Turton JP, Mehta A, Raza J, Woods KS, Tiulpakov A, Cassar J, et al. Mutations within the transcription factor PROP1 are rare in a cohort of patients with sporadic combined pituitary hormone deficiency (CPHD). Clin Endocrinol (Oxf). 2005;63:10-8, doi: 10.1111/j.1365-2265.2005.02291.x. 24. Abrao MG, Leite MV, Carvalho LR, Billerbeck AE, Nishi MY, Barbosa AS, et al. Combined pituitary hormone deficiency (CPHD) due to a complete PROP1 deletion. Clin Endocrinol (Oxf). 2006;65:294-300, doi: 10.1111/j.1365-2265.2006.02592.x. 25. Nascif SO, Vieira TC, Ramos-Dias JC, Lengyel AM, Abucham J. Waxing and waning of a pituitary mass in a young woman with combined pituitary hormone deficiency (CPHD) due to a PROP-1 mutation. Pituitary. 2006;9:47-52, doi: 10.1007/s11102-006-6215-1. 26. Cushman LJ, Watkins-Chow DE, Brinkmeier ML, Raetzman LT, Radak AL, Lloyd RV, et al. Persistent Prop1 expression delays gonadotrope differentiation and enhances pituitary tumor susceptibility. Hum Mol Genet. 2001;10:1141-53, doi: 10.1093/hmg/10.11.1141. 27. Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, et al. Accurate normalization of real-time quantitative RT-PCR data by

Interestingly, 71% of the tumors (five out of seven) with b-catenin somatic mutations were shown to overexpress CTNNB1, and this result was also observed in 40% of the tumor samples without b-catenin mutations (two out of five). Each of the ten samples (seven with CTNNB1 mutations) submitted for immunohistochemical analysis demonstrated nuclear b-catenin staining (Table 2), whereas two out of the three non-mutated samples showed both CTNNB1 overexpression and nuclear bcatenin staining. These results suggested that CTNNB1 overexpression, which leads to WNT signaling activation, plays a key role in the tumorigenesis of adamantinomatous craniopharyngiomas regardless of the presence of CTNNB1 mutations. The pathogenesis of adamantinomatous craniopharyngiomas that do not have mutations in or display the overexpression of CTNNB1 remains to be determined. To investigate other pathways that are likely involved in this process, we performed a PROP1 expression analysis. This gene was chosen based on the fact that b-catenin, which is the only protein shown thus far to be involved in this pathogenesis, interacts with PROP1 to control pituitary cell lineage determination15, as well as the fact that transgenic mice engineered to exhibit persistent expression of Prop1 demonstrate both defects in the control of cellular proliferation and an increased susceptibility to pituitary tumors.26 Moreover, patients with loss-of-function PROP1 mutations can present with pituitary enlargement.20-25 Thus, we investigated whether PROP1 dysregulation could contribute to the tumorigenesis of adamantinomatous craniopharyngiomas. However, PROP1 expression was undetectable in all the samples, which indicated that expression of the PROP1 gene most likely does not affect adamantinomatous craniopharyngioma pathogenesis. In conclusion, CTNNB1 overexpression was found in the majority of the adamantinomatous craniopharyngiomas studied. Our findings also suggest that WNT signaling activation via b-catenin upregulation plays an important role in the pathogenesis of adamantinomatous craniopharyngiomas regardless of the presence of CTNNB1 mutations. Additionally, PROP1 dysregulation was not likely involved in the pathogenesis of adamantinomatous craniopharyngiomas in this cohort of patients. However, this study was the first to analyze PROP1 expression in this type of tumor, and further studies are necessary to confirm the lack of a role for PROP1 in this tumorigenesis.

AUTHOR CONTRIBUTIONS Cani CMG is the first author who wrote the paper and was responsible for the collection of the craniopharyngioma samples, application of the methodology, and analysis of the results. Matushita H was the neurosurgeon responsible for collecting the adamantinomatous craniopharyngioma samples during the therapeutic surgery. Carvalho LRS, Brito LP, and Almeida MQ developed the methodology. Soares IC performed the immunohistochemistry. MendoncÂ¸a BB supervised the study and instructed the group.

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geometric averaging of multiple internal control genes. Genome Biol. 2002;3:RESEARCH0034, doi: 10.1186/gb-2002-3-7-research0034. 28. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001;25:402-8, doi: 10.1006/meth.2001.1262. 29. Li S, Crenshaw EB3rd, Rawson EJ, Simmons DM, Swanson LW, Rosenfeld MG. Dwarf locus mutants lacking three pituitary cell types result from mutations in the POU-domain gene pit-1. Nature. 1990;347:528-33, doi: 10.1038/347528a0. 30. Holsken A, Kreutzer J, Hofmann BM, Hans V, Oppel F, Buchfelder M, et al. Target gene activation of the Wnt signaling pathway in nuclear

beta-catenin accumulating cells of adamantinomatous craniopharyngiomas. Brain Pathol. 2008;19:357-64, doi: 10.1111/j.1750-3639.2008.00180.x. 31. Jho EH, Zhang T, Domon C, Joo CK, Freund JN, Costantini F. Wnt/betacatenin/Tcf signaling induces the transcription of Axin2, a negative regulator of the signaling pathway. Mol Cell Biol. 2002;22:1172-83, doi: 10.1128/MCB.22.4.1172-1183.2002. 32. Ohazama A, Tucker A, Sharpe PT. Organized tooth-specific cellular differentiation stimulated by BMP4. J Dent Res. 2005;84:603-6, doi: 10. 1177/154405910508400704. 33. Kim JS, Crooks H, Dracheva T, Nishanian TG, Singh B, Jen J, et al. Oncogenic beta-catenin is required for bone morphogenetic protein 4 expression in human cancer cells. Cancer Res. 2002;62:2744-8.

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CLINICAL SCIENCE

Autologous stem cell transplantation improves quality of life in economically challenged, Brazilian multiple myeloma patients Leina Y. Etto,I Vaˆnia Maris Morelli,I Vanderleia C. Silva,I Vania T. M. Hungria,II Rozana M. Ciconelli,III Manuella S. S. Almeida,I Jose´ Salvador R. de Oliveira,I Jose´ Carlos Barros,II Brian G. Durie,IV Gisele W. B. ColleoniI I

Universidade Federal de Saõ Paulo (UNIFESP), Disciplina de Hematologia e Hemoterapia, Saõ Paulo/SP, Brazil. II Faculdade de Cieˆncias Me´dicas da Santa Casa de Miserico´rdia de Saõ Paulo, Disciplina de Hematologia e Oncologia, Saõ Paulo, SP/Brazil. III Universidade Federal de Saõ Paulo (UNIFESP), Disciplina de Reumatologia, Departamento de Medicina, Saõ Paulo/SP, Brazil. IV Cedars-Sinai Outpatient Cancer Center, Los Angeles, CA, USA.

OBJECTIVES: 1) To characterize the impact of multiple myeloma on the quality of life of patients treated in two public institutions in Sa˜o Paulo State, Brazil, using a generic Short Form 36 Health Survey and a questionnaire specific for oncologic patients (QLQ-C30) upon diagnosis, after the clinical treatment, and at day +100 after autologous stem cell transplantation; 2) to evaluate whether autologous stem cell transplantation can improve the quality of life of our economically challenged population aside from providing a clinical benefit and disease control. METHODS: We evaluated 49 patients with multiple myeloma (a total of 70 interviews) using the two questionnaires. The scores upon diagnosis, post-treatment/pre-autologous stem cell transplantation, and at D+100 were compared using ANOVA (a comparison of the three groups), post hoc tests (two-by-two comparisons of the three groups), and paired t-tests (the same case at two different times). RESULTS : Of the included patients, 87.8% had a family budget under US $600 (economic class C, D, or E) per month. The generic Short Form 36 Health Survey questionnaire demonstrated that physical function, role-physical, and bodily pain indices were statistically different across all three groups, favoring the D+100 autologous stem cell transplantation group (ANOVA). The questionnaire specific for oncologic patients, the QLQ-C30 questionnaire, confirmed what had been demonstrated by the Short Form 36 Health Survey with respect to physical function and bodily pain, with improvements in role functioning, fatigue, and lack of appetite and constipation, favoring the D+100 autologous stem cell transplant group (ANOVA). The post hoc tests and paired t-tests confirmed a better outcome after autologous stem cell transplantation. CONCLUSION: The questionnaire specific for cancer patients seems to be more informative than the generic Short Form 36 Health Survey questionnaire and reflects the real benefit of autologous stem cell transplantation in the quality of life of multiple myeloma patients in two public Brazilian institutions that provide assistance for economically challenged patients. KEYWORDS: Myeloma; Quality of life; SF-36; QLQ-C30; Transplantation. Etto LY, Morelli VM, Silva VC, Hungria VTM, Ciconelli RM, Almeida MSS, et al. Autologous stem cell transplantation improves quality of life in economically challenged, Brazilian multiple myeloma patients. Clinics. 2011;66(11):1855-1859. Received for publication on April 27, 2011; First review completed on July 7, 2011; Accepted for publication on July 7, 2011 E-mail: gcolleoni@unifesp.br / vmorelli@unifesp.br Tel.: 55 11 5579-1550

personal care.1 MM treatment includes therapy for the underlying disease and supportive therapy to enhance QoL. The aim of MM treatment is to control the disease, particularly its bone destruction. Healthcare professionals involved in MM treatment must minimize any posttreatment complications and ensure the best possible longterm QoL for each patient. International studies assessing the QoL of patients with MM were validated in developed countries. In Brazilian public hospitals, MM patients have important related social, economic, and emotional difficulties, including the following: a low family budget, unemployment, difficulties in receiving government financial support after diagnosis, a long distance between the home

INTRODUCTION Multiple myeloma (MM) has a significant impact on a patient’s quality of life (QoL) because the patient becomes dependent on others, even for routine activity execution and

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cognitive, emotional and social); three symptom scales (fatigue, pain, and nausea and vomiting); and global health and quality-oflife scales (dyspnea, insomnia, lack of appetite, constipation, diarrhea, and financial difficulty).3 These scales have final scores between 0 and 100, where high scores indicate better outcomes for the functional scales and global health and worse outcomes for the symptom and quality-of-life scales. Authorizations for the use of the SF-36 and QLQ-C30 questionnaires (Portuguese [Brazilian] version) were given by Dr. Marcos Bosi Ferraz and Dr. Rozana M. Ciconelli and the EORTC Quality of Life Group, respectively, in May 2006. These questionnaires were administered to patients upon diagnosis, after the clinical treatment (post-treatment/pre-ASCT), and at day +100 after ASCT (D+100 ASCT). Of the 49 patients, 29 were evaluated upon diagnosis, 27 were evaluated post-treatment/pre-ASCT (9 patients were also evaluated at diagnosis), and 14 were evaluated at D+100 ASCT (12 patients were also evaluated post-treatment/pre-ASCT). Thus, the 49 MM patients generated 70 total completed interviews, as depicted in the flow diagram of the study design (Figure 1). All of the interviews were conducted by one of the principal researchers (V.C.S.), and the average time required to complete the questionnaires was approximately 30 minutes (while the patient was waiting for the physician consultation). Statistical analysis. The sample classification (according to sex, isotype, disease stage, and the International Staging System [ISS]) was expressed as absolute numbers and as relative percentages. Chi-squared tests were used to compare the categorical variables among the groups. The ages were expressed as means ¡ standard deviations. For the SF-36 and QLQ-30 questionnaire score analyses, we followed all of the directions provided by each responsible group.2,4,5 The interviews that were generated by the quality-of-life questionnaires upon diagnosis, post-treatment/pre-ASCT and at D+100 ASCT were compared using ANOVA (for comparisons of the three groups), post hoc tests (for two-by-two comparisons of the three groups) and paired t-tests (the same case at two different times). The level of significance for all of the statistical tests was 5%.

and the hospital, transportation difficulties for disable patient, the absence of a caregiver, and poor family support related to social problems. Because of these difficulties, we believe that it is important to evaluate whether high-dose chemotherapy followed by autologous hematopoietic stem cell transplantation (ASCT), could improve the QoL of our patients (other than clinical benefits and disease control). Therefore, the aim of this study was to characterize the impact of MM in the QoL of patients treated in two public institutions in Sa˜o Paulo State, Brazil, using a generic Short Form 36 Health Survey and a questionnaire specific for oncologic patients (QLQ-C30) upon diagnosis, after the clinical treatment, and at day +100 after ASCT. We also evaluated whether ASCT improves the QoL of our economically challenged population.

METHODS Study design. Between March 2006 to August 2007, we evaluated 49 MM patients who were treated at Disciplina de Hematologia e Hemoterapia, Universidade Federal de Saõ Paulo Saõ Paulo/UNIFESP (a total of 26 patients) and Santa Casa de Miserico´rdia de Saõ Paulo (a total of 23 patients). We did not include patients with solitary plasmacytoma or monoclonal gammopathy of undetermined significance or patients without a physical/emotional condition for a 30minute interview. Patients who were more than 70 years old, had chronic renal insufficiency requiring dialysis, congestive cardiac insufficiency, or cardiac amyloidosis did not undergo high-dose chemotherapy followed by ASCT. Written informed consent was obtained from all of the patients, and the study was approved by the ethics committees of these two institutions (CEP no. 0249/06 and CEP no. 374/06, respectively). Economic class assessment. A questionnaire that defined the economic classes of each of the 49 MM patients was applied using Brazil’s Economic Classification (www.ibope.com.br). This classification was created in 2000 and considers how many bathrooms, color TVs, radios, cars, vacuum cleaners, washing machines, DVD players, refrigerators, freezers and housemaids are in the patient’s household. It also considers the highest education level of any household member (not necessarily the MM patient). The final score classifies the patient within one of seven economic classes (A1/2, B1/2, C, D, or E). Based on the dollar value in 2000, an A1-class patient has a family budget of US $5,000/month, and an Eclass patient has a family budget of US $130/month. Quality-of-life questionnaires. Two quality-of-life instruments were used in this study: the Short Form 36 Health Survey (SF36)2 and the European Organization for Research and Treatment of Cancer (EORTC) Quality of Life QuestionnaireC30 (QLQ-C30).3 The SF-36 is an internationally validated instrument that can be self-administered or administered over the phone by trained health professionals. It includes a multiitem scale that assesses the following eight health domains: physical function, role-physical, bodily pain, general health, vitality, social function, role-emotional, and mental health. The physical and mental components are summaries of the eight domains in the SF-36 questionnaire. These domains have final scores between 0 and 100, where zero corresponds to the worst health state, and 100 corresponds to the best health state.2 This questionnaire has been adapted and validated in Portuguese since 1999.2 The EORTC QLQ-C30 questionnaire is an internationally validated instrument in Portuguese (Brazilian) and incorporates nine multi-item scales: five functional scales (physical, role,

RESULTS There were no statistically significant differences among the three evaluated groups with respect to sex, age, isotype, clinical stage, and ISS (Table 1). With respect to economic class, the following classification was obtained: 4.0% in B1, 8.2% in B2, 63.3% in C, 22.5% in D, and 2.0% in E. According to our classification, 87.8% of the included patients had a familial budget of less than US $600 per month (i.e., economic classes C, D, or E). The generic SF-36 questionnaire demonstrated that physical function, role-physical, and bodily pain were statistically different across all three groups and favored the D+100 ASCT group (ANOVA; Table 2). The physical component portion of the SF-36 had borderline statistical significance and favored the D+100 ASCT, which corresponds to the physical function, role-physical, and bodily pain domains. The QLQ-C30 questionnaire confirmed the SF-36 results with respect to physical function and bodily pain and improved role functioning, fatigue, the lack of appetite and constipation and usually favored the D+100 ASCT group (ANOVA; Table 3). The significant results obtained using the post hoc tests (two-by-two comparisons of the three groups,) are shown in Table 4. The outcomes were better after treatment (including ASCT). Paired t-tests (comparing

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Quality of life in Brazilian MM patients Etto LY et al.

Figure 1 - A flow diagram of the study design. ASCT - autologous stem cell transplantation.

ASCT. The SF-36 is an internationally validated instrument that can be self-administered or given over the phone. This questionnaire has been adapted and validated in Portuguese since 1999.2 We found only one previous report that described the use of the SF-36 questionnaire in MM patients to compare QoL pre- and post-kyphoplasty.6 Recently, the EORTC validated a disease-specific questionnaire module (QLQ-MY20) for patients with MM. The study was performed through the EORTC Quality of Life Group using clinical trials in seven countries. The social support scale was removed in the final analysis because it did not discriminate between the groups in this population.7 The differences observed when comparing the MM patients upon diagnosis and at D+100 ASCT were already present in one recent QoL study from the University of Arkansas for Medical Sciences, Little Rock, that used other indices.8 However, in Brazilian public hospitals, the economic situation of MM patients is completely different from that of developed countries, where all of the questionnaires were designed. The majority of patients in this study are in

the same case at two different times) demonstrated improvements in fatigue (p = 0.036) and global health (p = 0.045) after clinical treatment compared to the time of diagnosis (as determined by the QLQ-C30). This finding confirms the post hoc results. Paired t-tests demonstrated improvements in physical function (p = 0.022) and roleemotional (p = 0.043) when the patients at the end of their clinical treatments were compared to the patients at D+100 ASCT using the SF-36 questionnaire. The QLQ-C30 detected significant improvements in the global health (p = 0.034) and social characteristics (p = 0.044) when the same patients in the two aforementioned situations were compared.

DISCUSSION In this study, we characterized the impact of MM on the QoL of patients treated in two public institutions in SaË&#x153;o Paulo State, Brazil, using a generic (SF-36) questionnaire and a questionnaire specific for oncologic patients (QLQ-C30) upon diagnosis, after clinical treatment and at day +100 after

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Table 1 - The clinical and laboratory characteristics of the 49 MM patients (upon diagnosis, post-treatment/pre-ASCT, and D+100 ASCT). Total of 70 interviews. Parameters Sex Female Male Age (years) Isotype IgG IgA IgM Light chain Non-secretor Stage IA IIA IIIA IIIB ISS 1 2 3

Upon diagnosis

Post-treatment/ Pre-ASCT

D+100 ASCT

Total

15 (21.4%) 14 (20%) 55.4¡13.3

13 (18.6%) 14 (20%) 53.9¡9.8

7 (10%) 7 (10%) 54.1¡7.8

35 (50%) 35 (50%)

20 (28.6%) 5 (7.1%) 0 (0%) 3 (4.3%) 1 (1.4%)

21 (30%) 2 (2.9%) 1 (1.4%) 1 (1.4%) 2 (2.9%)

9 (12.9%) 1 (1.4%) 1 (1.4%) 2 (2.9%) 1 (1.4%)

50 (71.5%) 8 (11.4%) 2 (2.8%) 6 (8.6%) 4 (5.7%)

0 (0%) 0 (0%) 13 (18.6%) 1 (1.4%)

3 2 50 15

p-value 0.965

0.856 0.717

0.583 2 (2.9%) 1 (1.4%) 18 (25.7%) 8 (11.5%)

1 1 19 6

(1.4%) (1.4%) (27.1%) (8.6%)

10 (14.3%) 9 (12.9%) 10 (14.3%)

11 (15.7%) 8 (11.4%) 8 (11.4%)

(4.3%) (2.8%) (71.4%) (21.5%) 0.593

5 (7.1%) 7 (10%) 2 (2.9%)

26 (37.1%) 24 (34.3%) 20 (28.6%)

ASCT - autologous stem cell transplantation. The ages are expressed as means ¡ standard deviations. The p-values were calculated using a chi-squared test (for the categorical variables) or ANOVA (for age).

One criticism of this study design is that the patient groups were not the same at the three time-points that were investigated. This could limit the power of the observation; however, the paired t-test results from both questionnaires showed improvements in the patients’ QoL. In conclusion, this study supports the use of the EORTC QLQ-C30 as part of routine clinical care in MM patients in developing countries. Our results also suggest that the questionnaire for cancer patients, QLQ-C30, seems to be more informative than the generic questionnaire, SF-36.

economic class C, which means that 87.8% of the included patients have a family budget of less than US $600/month (i.e., economic class C, D, or E). Therefore, it is important to apply such questionnaires in underdeveloped countries to ensure that ASCT is a good option for their incurable disease in this population. The QLQ-MY20 was not validated in Portuguese (Brazilian) when the questionnaires were applied and, therefore, was not used in our study. Although the questionnaires are designed to be selfcompleted, in this study the questionnaires were given during interviews performed by a principal researcher. The questionnaires were conducted in an interview because most of the patients were considered to be functionally illiterate and/or could not complete the forms alone. Others could read but were unable to understand what needed to be answered. We believe that administering the questionnaire by the same person provides more uniform results.

Table 3 - Analysis of variance (ANOVA) comparing the three groups according to the QLQ-C30 questionnaire.

Scale

Global health 64 (¡24) Functional scale Physical 43 (¡34) Role 39 (¡40) Emotional 64 (¡27) Cognitive 65 (¡30) Social 62 (¡31) Symptom scale Fatigue 53 (¡31) Nausea 14 (¡25) Pain 58 (¡34) Quality of life scale Dyspnea 15 (¡27) Insomnia 43 (¡31) Lack of 34 (¡47) appetite Constipation 36 (¡43) Diarrhea 2 (¡12) Financial 53 (¡41) difficulties

Table 2 - Analysis of variance (ANOVA) comparing the three groups according to the SF-36 questionnaire.

Domains Physical function Role-physical Bodily pain General health Vitality Social function Role-emotional Mental health Physical component Mental component

PostUpon treatment/ diagnosis, Pre-ASCT, D+100 ASCT, Mean (¡SD) Mean (¡SD) Mean (¡SD) p-value 33 (¡33) 31(¡36) 36 (¡26) 60 (¡22) 58 (¡23) 45 (¡38) 49 (¡39) 64 (¡23) 33 (¡11)

44 (¡27) 47(¡28) 53 (¡23) 66 (¡14) 63 (¡17) 54 (¡31) 58 (¡24) 73 (¡19) 37 (¡8)

60 (¡25) 56 (¡29) 55 (¡28) 60 (¡14) 68 (¡24) 68 (¡25) 69 (¡33) 74 (¡21) 40 (¡7)

0.026 0.041 0.025 0.388 0.364 0.094 0.158 0.194 0.053

47 (¡13)

49 (¡8)

51 (¡11)

0.362

Posttreatment/ Upon diagnosis, Pre-ASCT, D+100 ASCT, Mean (¡SD) Mean (¡SD) Mean (¡SD) p-value 70 (¡22)

71 (¡16)

0.455

59 72 70 76 71

71 83 77 69 82

(¡20) (¡29) (¡34) (¡24) (¡32)

0.01 0.001 0.391 0.339 0.186

27(¡24) 5 (¡12) 31(¡27)

21(¡24) 7 (¡22) 34 (¡27)

0.001 0.289 0.002

5 (¡18) 26 (¡32) 11(¡24)

7 (¡19) 21 (¡38) 14 (¡31)

0.239 0.061 0.046

9 (¡20) 0 30 (¡40)

7(¡14) 9 (¡20) 31(¡38)

0.002 0.059 0.071

(¡24) (¡34) (¡28) (¡23) (¡35)

The p-values were calculated using an ANOVA. QoL: Quality-of-life. ASCT- autologous stem cell transplant.

The p-values were calculated using an ANOVA. ASCT - autologous stem cell transplantation.

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Quality of life in Brazilian MM patients Etto LY et al. manuscript, analysis and interpretation of the data, and approval of the final version of the manuscript. Silva VC: collected the data. Hungria VTM and Durie B were responsible for the critical revision of the manuscript regarding important intellectual content. Ciconelli R was responsible for the design of the study, analysis and interpretation of the data. Almeida MSS, Oliveira JSR, Barros JC collected the data. Colleoni GWB conceived and designed the study, drafted the article, analyzed and interpreted the data, critically revised the article for important intellectual content, and gave final approval of the version of the article that was submitted.

Table 4 - Post hoc tests comparing the three groups (two by two). POST HOC Test Physical function (SF-36) Physical (QLQ-C30) Role (QLQ-C30) Role (QLQ-C30) Fatigue (QLQ-C30) Fatigue (QLQ-C30) Pain (QLQ-C30) Pain (QLQ-C30) Lack of appetite (QLQ-C30) Constipation (QLQ-C30) Constipation (QLQ-C30)

Group Upon diagnosis D+100 ASCT { Upon diagnosis D+100 ASCT { Upon diagnosis Post-treatment/Pre-ASCT Upon diagnosis D+100 ASCT { Upon diagnosis Post-treatment/Pre-ASCT Upon diagnosis D+100 ASCT { Upon diagnosis Post-treatment/Pre-ASCT Upon diagnosis D+100 ASCT { Upon diagnosis Post-treatment/Pre-ASCT Upon diagnosis Post-treatment/Pre-ASCT Upon diagnosis D+100 ASCT {

p-value 0.02 0.011 0.003 {

REFERENCES

0.001

1. Desikan R, Jagannath S, Richardson P, Munshi N, Barlogie B. MM and other plasma cell dyscrasias, in Pazdur R, Coia LR, Hoskins WJ, Wagman LD (ed): Cancer management: a multidisciplinary approach. PRR Inc, 6th edition. Melville, NY, 2002;pp667-84. 2. Pereira GI, Costa CD, Geocze L, Borim AA, Ciconelli RM, CamachoLobato L., et al. Traduçaõ para a lıńgua portuguesa e validaçaõ do questiona´rio gene´rico de avaliaçaõ de qualidade de vida SF-36 (Brasil SF36). Rev Bras Reumatol. 1999;39:143-50. 3. Aaronson NK, Ahmedzai S, Bergman B. The European Organization for Research and Treatment of Cancer QLQ-C30: a quality-of-life instrument for use in international clinical trials in oncology. J Natl Can Inst. 1993;85:365-76, doi: 10.1093/jnci/85.5.365. 4. Kemmler G, Holzner B, Kopp M, Du¨nser M, Margreiter R, Greil R, et al. Comparison of two quality-of-life instruments for cancer patients: the functional assessment of cancer therapy-general and the european organization for research and treatment of cancer quality of life questionnaire-C 30. J Clin Oncol. 1999;17:2932-40. 5. Gulbrandsen N, Hjermstad MJ, Wisløff F, Nordic Myeloma Study Group. Interpretation of quality of life scores in multiple myeloma by comparison with a reference population and assessment of the clinical importance of score differences. Eur J Haematol. 2004;72:172-80, doi: 10. 1046/j.0902-4441.2003.00195.x. 6. Dudeney S, Lieberman IH, Reinhardt MK, et al. Kyphoplasty in the treatment of osteolytic vertebral compression fractures as a result of multiple myeloma. J Clin Oncol. 2002;20:2382-7, doi: 10.1200/JCO.2002.09.097. 7. Cocks K, Cohen D, Wisløff F, Sezer O, Lee S, Hippe E, et al. An international field study of the realiabiity and validity of a diseasespecific questionnaire modulo (the QLQ- My20) in assessing the quality of life of patients with multiple myeloma. Eur J Cancer. 2007;43:1670-8, doi: 10.1016/j.ejca.2007.04.022. 8. Sherman AC, Simonton S, Latif U, Plante TG, Anaissie EJ. Changes in quality-of-life and psychosocial adjustment among multiple myeloma patients treated with high-dose melphalan and autologous stem cell transplantation. Biol Blood Marrow Transplant.2009;15:12-20, doi: 10. 1016/j.bbmt.2008.09.023.

0.003 {

0.002 0.003 {

0.042 0.05 {

0.005 {

0.016

ASCT - autologous stem cell transplantation. { The group with the superior outcome.

Additionally, the QLQ-C30 reflects the benefits of ASCT for the QoL of MM patients from two public Brazilian institutions that provide assistance for economically challenged patients.

ACKNOWLEDGEMENTS V.C.S. was supported by CAPES, Brazil.

AUTHOR CONTRIBUTIONS Etto LY was responsible for the draft of the manuscript, analysis and interpretation of the data. Morelli VM was responsible for the draft of the

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DOI:10.1590/S1807-59322011001100003

CLINICAL SCIENCE

Association between participation in community groups and being more physically active among older adults from Floriano´polis, Brazil Giovana Zarpellon Mazo, Taˆnia Bertoldo Benedetti, Cinara Sacomori Universidade do Estado de Santa Catarina (UDESC), Centro de Cieˆncias da Sau´de e do Esporte (CEFID), Floriano´polis/SC, Brazil.

OBJECTIVE: In Brazil, older adults frequently participate in community groups. However, the influence of this participation on physical activity levels has not been fully investigated. It is known that both regular physical activity and social support are beneficial for health. The aim of this study is to evaluate the association between participation in community groups and physical activity among older adults from Floriano´polis, Brazil. METHODS: The sample consisted of 1062 adults with a mean age of 71.9 (¡7.6) years. Among these individuals, 293 subjects participated in community groups and 769 did not. A questionnaire to collect sociodemographic data and the long version of the International Physical Activity Questionnaire were used for the assessment. RESULTS: The prevalence of active older adults was 66.6% among participants in community groups and 58.4% among non-participants. Participation in these groups was significantly associated with being more physically active in the transportation and domestic domains, but with being less physically active in the leisure-time domain. Some changes in these associations were observed when the sample was stratified by age, gender, body mass index, and health status. With respect to total physical activity, participation in community groups was associated with being more physically active in only two strata (subjects younger than 70 years and women). CONCLUSION: The results of this study indicate that older adults who participate in community groups are characterized by a greater probability of being more physically active. However, longitudinal studies are needed to determine whether participation in community groups facilitates the adoption of physically active behavior. KEYWORDS: Physical Activity; Community Groups; Older Adults; Health; Social Support. Mazo GZ, Benedetti TB, Sacomori C. Association between participation in community groups and being more physically active among older adults from Floriano´polis, Brazil. Clinics. 2011;66(11):1861-1866. Received for publication on October 3, 2011; First review completed on October 7, 2011; Accepted for publication on October 7, 2011 E-mail: csacomori@yahoo.com.br Tel.: 55 48 3321-8683

status,8,10 and a lower body mass index (BMI).8,9,11 Furthermore, continuous engagement in PA programs has been associated with male gender, nonsmoking, and fast walking speeds.12 In addition to regular PA, social support has also been reported to be beneficial for the health of older adults.2,13,14 One systematic review suggested that participation in social groups generally prevents and alleviates social isolation and loneliness and improves overall well-being.15 In Brazil, there are social programs that contribute to the autonomy and socialization of older adults, such as community centers for the elderly.16 However, population-based studies provide little information about the PA patterns of older adults who participate in community groups. It has been recognized that this participation can foster friendships14 and favor the adoption of a more active lifestyle because leisure, cultural, intellectual, physical, manual, and artistic activities as well as group interactions take place in these groups.17 Older adults who participate in such groups have been suggested to have higher self-esteem, which

INTRODUCTION Physical inactivity among older adults is considered a public health problem because of its high prevalence, with most older adults not achieving recommended physical activity (PA) levels.1-3 Physical inactivity is known to reduce healthy life expectancy.2,3 Therefore, regular engagement of older adults in PA groups may delay age-related decline, prevent the deleterious effects of different chronic diseases, and promote social ties.3-5 In this respect, adequate PA levels are generally associated with younger age, health,6-9 male gender, reduced psychological distress, rural residency,6 higher income, educational level, and socioeconomic

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improves family integration and contributes to the recovery of personal and social values.18 The objective of this study was to compare PA levels between participants in community groups and non-participants and evaluate the association between participation in these groups and being more physically active among older adults from Floriano´polis, Brazil. We hypothesized that being socially active through participating in community groups would be associated with being physically active. It was also expected that gender, age, health status, educational level, and BMI would influence this association.

Instruments Sociodemographic data were collected using a diagnostic questionnaire. The dependent variable, PA level, was evaluated using the long version of the International Physical Activity Questionnaire (IPAQ), which was administered by interview and represented an average week. The total PA level and the different domains of the IPAQ (leisure time, domestic, transportation, and work) were evaluated. Inactive behavior was defined as the sum of time spent sitting on a normal weekday and the time spent sitting on a normal weekend day. The subjects were classified as either active or inactive according to these criteria.

MATERIALS AND METHODS

Statistical analysis

Population and sample

The data were analyzed using the SPSS 15.0 software program (SPSS, Chicago, IL, USA). Descriptive statistics were calculated (absolute and relative frequency, confidence interval, mean, and standard deviation). The MannWhitney test was used to compare groups and the chi-square test to evaluate the association between variables. A 95% confidence interval was adopted. Univariate Poisson regression analysis was used to estimate the prevalence ratio between participation in community groups and being more physically active, with ‘‘participation in community groups’’ (0 = no, 1 = yes) as an independent variable and physical activity in each of the analyzed PA domains as dependent variables. The PA level quantified in all domains was classified as 0 (not very active) or 1 (very active) using a cut-off point of 150 min/week of at least moderate PA, corresponding to activities that require moderate physical effort and cause you to breathe somewhat harder than normal.23 The work-related domain was not analyzed because most of the subjects were retired, and PA at work was rarely reported. Next, the sample was divided into the following categories to determine whether the association between the two main variables of this study would persist: gender (male and female); age (0 = ,70 years and 1 = $70 years); BMI (0 = underweight/normal and 1 = overweight/ obese), and self-reported health status (0 = poor/very poor and 1 = excellent/good).

A cross-sectional, population-based study was conducted involving the elderly population (age $60 years) of the municipality of Floriano´polis, Santa Catarina, Brazil. In 2000, this municipality had a population of approximately 343,047 inhabitants. Of these, 28,816 were older adults (11,979 men and 16,837 women), corresponding to 8.4% of the entire population. Approximately 98% of the population lived in urban residences.19 At the end of this study, there were 33 community groups for the elderly in the 12 districts of Floriano´polis. These groups were mainly attended by women (2,261 women and 246 men) aged 65 or older. Adults $60 years who agreed to participate were included in the study. Subjects with cognitive deficits that prevented participation in the interview, physical immobility, or who were under bed restriction caused by a debilitating disease were excluded. Two databases derived from previous studies20,21 were used for the selection of the sample. These databases were reorganized to analyze the PA of the older adults participating in community groups. Probabilistic, stratified random sampling was used. The sample was stratified by both gender and age21 and by age alone,20 and the sample size was calculated using a formula for adjusting the sample.22 The primary sampling units were census sectors defined by the Brazilian Institute of Geography and Statistics21 and municipal districts.20 The secondary sampling units were the community groups existing in Floriano´polis in 2002.20 The sample of 1,062 older adults was divided into two groups (Figure 1): subjects who regularly participated in community groups, hereafter referred to as participants (n = 293, 27.6%), and subjects who did not, hereafter referred to as non-participants (n = 769, 72.4%). The study was conducted in accordance with the principles of the Declaration of Helsinki and was approved by the Ethics Committees of Universidade Federal de Santa Catarina (protocol 051/2001) and Universidade do Estado de Santa Catarina (protocol 95/2007).

RESULTS Characterization of the participants Table 1 shows the characteristics of the older adults. The mean age was similar in community group participants (72.5¡6.4 years) and non-participants (71.7¡8 years). Most participants were women whose maximum educational level was elementary school and who lived without a partner. In contrast, the proportion of men with an elementary school education level who lived with a partner was higher among non-participants. The BMI distribution was similar in the two groups, and the prevalence of a poor health status was higher among non-participants. An association was observed between participation in community groups and gender, age, educational level, marital status, health status, and the PA domains (total, leisure time, transportation, and domestic). With respect to the total PA level, 66.6% of the older adults participating in community groups and 58.4% of nonparticipants achieved the recommended level of at least 150 min/week. In the other domains, most subjects were classified as not very active (performing less than 150 min PA/week). Older adults who participated in community groups spent significantly more time engaging transportation

Figure 1 - Population and sample.

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Table 1 - Sociodemographic characteristics, health status, body mass index, and physical activity level of older adults participating in community groups and those who do not.

Characteristics Gender* Male Female Age (years)* 60-69 $70 Educational level** Up to elementary school High school or higher Marital status* With a partner Without a partner Health status* Poor/very poor Excellent/good BMI* Underweight/normal Overweight/obese Total PA* Not very active Very active Leisure-time PA* Not very active Very active Domestic PA* Not very active Very active Transportation PA* Not very active Very active Work PA* Not very active Very active

Participants (n = 293) n (%)

Non-participants (n = 769) n (%)

Total n (%)

27 (6.2) 266 (42.6)

410 (93.8) 359 (57.4)

437 (100) 625 (100)

103 (22.5) 190 (31.5)

355 (77.5) 414 (68.5)

458 (100) 604 (100)

251 (35.1) 29 (12.8)

464 (64.9) 197 (87.2)

715 (100) 226 (100)

103 (17.5) 190 (40.2)

486 (82.5) 283 (59.8)

589 (100) 473 (100)

50 (17.6) 243 (31.2)

234 (82.4) 535 (68.8)

284 (100) 778 (100)

120 (27.8) 173 (27.4)

311 (72.2) 449 (72.6)

431 (100) 631 (100)

104 (24.8) 189 (29.4)

315 (75.2) 454 (70.6)

418 (100) 644 (100)

199 (25.7) 94 (32.7)

576 (74.3) 193 (67.3)

775 (100) 287 (100)

183 (22.8) 110 (42.5)

620 (77.2) 149 (57.5)

803 (100) 259 (100)

261 (29.9) 32 (17)

613 (70.1) 156 (83)

874 (100) 188 (100)

278 (27.8) 15 (23.4)

720 (72.2) 49 (76.6)

998 (100) 64 (100)

p-value

170.39

,.001

10.48

.001

23.92

,.001

67.55

,.001

19.11

,.001

0.0

.992

5.92

,.015

5.24

.022

37.97

,.001

12.77

,.001

0.58

.443

BMI: body mass index; PA: physical activity. Total (n = 1062). ** Total (n = 941) due to missing data. *

participation in community groups and the total PA level was observed in the sample as a whole. In contrast, there was a significant association between these two variables in the group of subjects ,70 years of age and among women, with adults ,70 years of age and women who participated in community groups being characterized by a higher probability of being more physically active than those not participating in these groups.

PA and sitting on average, whereas non-participants spent more time engaging in leisure-time PA on average (Table 2).

Association between participation in community groups and physical activity Table 3 shows the results of univariate Poisson regression analysis for the sample as a whole and according to age, health status, gender, and BMI. No association between

Table 2 - Time spent on physical activities and time spent sitting during a normal week for older adults participating in community groups and those who do not. Participants (n = 293)

Physical activity

Total* Leisure time* Domestic* Transportation* Work* Time spent sitting**

Non-participants (n = 769)

Interquartile range

p- value***

225 0 0 80 0 570

390 78 178 180 0 273

220 0 0 30 0 540

410 150 120 120 0 246

.072 .022# .168 ,.001# .743 .003#

Minutes per week. Time spent sitting on a weekday + time spent sitting on a weekend day (min/day). Mann-Whitney test. # Significant difference (p,0.05). Md = Median. **

***

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Table 3 - Association between participation in community groups and physical activity levels in the sample as a whole and according to group. Total sample

Physical Activity Total: Participants Non-participants

Leisure time: Participants Non-participants

Domestic: Participants Non-participants

Transportation: Participants Non-participants

Age groups

Health status groups

Gender groups

Men (n = 437)

Women (n = 625)

BMI groups Underweight/ Overweight/ normal obesity (n = 431) (n = 631)

(n = 1062)

#70 years (n = 458)

.70 years (n = 604)

Poor (n = 284)

Good (n = 778)

1 1.03 (0.99-1.07) p = .096

1 1.07 (1.01-1.13) p = .023*

1 1.01 (0.95-1.06) p = .814

1 1.01 (0.92-1.11) p = .861

1 1.03 (0.99-1.08) p = .113

1 1 1.05 0.99 (0.88-1.11) (1.00-1.10) p = .05* p = .900

1 1.03 (0.96-1.09) p = .421

1 1.04 (0.99-1.09) p = .126

1 0.94 (0.90-0.98) p = .006*

1 0.99 (0.92-1.06) p = .703

1 0.90 (0.85-0.96) p = .001*

1 0.92 (0.84-1.01) p = .070

1 0.94 (0.89-0.99) p = .013*

1 1 0.92 1.01 (0.88-1.16) (0.88-0.97) p = .003* p = .881

1 0.92 (0.86-0.98) p = .008*

1 0.96 (0.90-1.01) p = .139

1 1.06 (1.01-1.11) p = .016*

1 1.10 (1.02-1.18) p = .009*

1 1.03 (0.97-1.09) p = .317

1 1.11 (0.99-1.23) p = .064

1 1.05 (0.99-1.10) p = .079

1 1 0.97 1.09 (0.85-1.10) (1.03-1.15) p = .662 p = .002*

1 1.05 (0.98-1.13) p = .153

1 1.06 (0.99-1.12) p = .065

1 1.08 (1.03-1.13) p = .002*

1 1.08 (1.01-1.16) p = .032*

1 1.07 (1.01-1.14) p = .032*

1 1.07 (0.96-1.19) p = .223

1 1.07 (1.02-1.13) p = .007*

1 1 0.96 1.09 (0.85-1.08) (1.03-1.15) p = .475 p = .002*

1 1.11 (1.03-1.19) p = .005*

1 1.06 (0.99-1.12) p = .065

Results are reported as crude prevalence ratios (95% confidence interval). * Significant at p,.05.

better daily PA scores in older Israeli adults.13 In this respect, community groups probably assume the role of a social network. Shye et al.24 investigated the effect of social network size on mortality risk and observed a protective effect of a larger social network. Physical activity has also been shown to protect older Japanese adults against overall mortality and mortality due to cardiovascular diseases.25 These results indicate that both social networks and PA are important factors for longevity. With respect to the total PA level, an association between participation in community groups and being more physically active was only observed for adults ,70 years of age and women. An association between PA and younger age has been previously reported.1,6-8 However, studies have shown that men are more physically active in old age compared to women.1,26,27 In the leisure-time domain, older adults participating in community groups were less likely to be physically active. It is possible that the community group represents the leisure activity of these subjects. Thus, there is a need to encourage PA in these pre-established groups through integrative activities, such as dancing and competitions. Older adults participating in community groups were more likely to be physically active regarding domestic tasks than non-participants. This association remained significant only in subjects ,70 years of age and in women and can be explained by the traditional gender role that is still predominant in this population.28 Furthermore, the present results suggest that participation in community groups is an essentially female practice, as few men participate in these groups. Another Brazilian study also showed a predominance of women in these groups (86.3%), including widows (49.7%), women in the age range of 65 to 74 years (65.5%), and those with an elementary school education (45.7%).29 It was not the objective of the present study to determine why

With respect to the leisure-time PA domain, older adults participating in community groups presented a lower probability of being physically active during leisure time. When the analysis was performed according to group, this association remained significant only in the group of subjects $70 years of age, subjects with a good health status, women, and subjects with a normal weight. Analysis of the domestic PA domain showed that older adults participating in community groups were more likely to be physically active than non-participants. This association remained significant only in the group of subjects ,70 years of age and in women. Finally, an association was also observed between participation in community groups and being more physically active in the transportation domain, which was no longer significant in the group of subjects with a poor health status and in men.

DISCUSSION The prevalence of physically active older adults observed in the two groups (participants in community groups: 66.6%; non-participants: 58.4%) was similar to that reported in a previous study conducted on older Brazilian adults.8 In addition, the prevalence of older adults who were active during their leisure time was similar to that reported for older Canadian adults (30%)11 and lower than that observed for users of health care plans in Brazil (46% for men and 43% for women).1 However, different criteria and instruments were used in these studies. Analysis of the sample as a whole showed that older adults participating in community groups presented a higher probability of being more physically active in the domestic and transportation domains. With respect to social context, a more diversified social network and relationships with friends and neighbors have consistently been associated

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women participate more frequently in community groups, but the female bias among older individuals may be one reason. For example, female widowhood represents a possible explanation for this pattern because men in Brazil tend to remarry and are more reluctant to engage in cultural, educational, or playful activities.30,31 Finally, an association was also observed between participation in community groups and being more physically active in the transportation domain. This association was no longer significant in the group of subjects with a poor health status and in men. In general, health problems are not an obstacle to PA.6,11,32 In contrast to the present results, a Swedish study reported no marked variation in the proportion of subjects with low PA level as a function of health status.10 In a study performed by Borges et al.,29 older adults participating in community groups were found to present a good or very good health status, whereas Lima-Costa et al.33 observed in a population-based sample study that most of older adults referred a regular health status and those with lower household income presented worse health conditions. With respect to the lack of a significant association being found among men, one hypothesis is that they are more dependent on motor vehicles for transportation, as men more frequently have a driver’s license and continue driving in old age compared to women.34,35 This study demonstrated an association between participation in community groups and PA level (total, leisure time, transportation, and domestic tasks). However, the cross-sectional design of the study did not permit a determination of whether participation in community groups facilitates engagement in physical activity or whether being physically active favors participation in these groups. Longitudinal studies are needed to establish whether participation in community groups has health benefits by facilitating PA habits. Furthermore, it would be interesting to investigate this association in different countries and among various cultural backgrounds. The project was conducted at Centro de Cieˆncias da Sau´de e do Esporte (CEFID), Universidade do Estado de Santa Catarina (UDESC) and Centro de Desportos (CDS), Universidade Federal de Santa Catarina (UFSC), Floriano´polis, Santa Catarina, Brazil.

6. 7. 8.

9. 10.

11. 12.

13. 14.

15.

16.

17. 18. 19. 20. 21. 22. 23.

AUTHOR CONTRIBUTIONS Mazo GZ and Sacomori C were responsible for the project development, collection and management of data, and manuscript writing. Benedetti TB was responsible for the project development, collection and management of data, and manuscript editing.

24.

25.

REFERENCES 1. Ministry of Health, Brazil. Surveillance of Risk and Protection Factors for Chronic Diseases by Telephone Survey - VIGITEL. Rio de Janeiro, Ministry of Health, Secretary Office of Health Surveillance, Secretary Office of Strategic Management; 2009. 2. Chodzko-Zajko WJ, Proctor DN, Fiatarone Singh MA, Minson CT, Nigg CR, Salem GJ, et al. American College of Sports Medicine position stand. Exercise and physical activity for older adults. Med Sci Sports Exerc. 2009;41:1510-30. 3. Hui EK, Rubenstein LZ. Promoting Physical Activity and Exercise in Older Adults. J Am Med Dir Assoc. 2006;7:310–4, doi: 10.1016/j.jamda. 2006.03.006. 4. Shephard RJ. Aging, Physical Activity, and Health. Sa˜o Paulo: Phorte; 2003. 5. Bird SP, Tarpenning KM, Marino FE. Designing resistance training programmes to enhance muscular fitness: a review of the acute

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programme variables. Sports Med. 2005;35:841-51, doi: 10.2165/ 00007256-200535100-00002. Lim K, Taylor L. Factors associated with physical activity among older people: a population-based study. Prev Med. 2005;40:33–40, doi: 10. 1016/j.ypmed.2004.04.046. Schutzer KA, Graves BS. Barriers and motivations to exercise in older adults. Prev Med. 2004;39:1056–61, doi: 10.1016/j.ypmed.2004.04.003. Hallal PC, Victora CG, Wells JCK, Lima RC. Physical Inactivity: Prevalence and Associated Variables in Brazilian Adults. Med Sci Sports Exercise. 2003;35:1894-900, doi: 10.1249/01.MSS.0000093615. 33774.0E. Aslan D, Ozcebe H, Temel F, Takmaz S, Topatan S, Sahin A, et al. What influences physical activity among elders? A Turkish experience from Ankara, Turkey. Arch Gerontol Geriatr. 2008;46:79–88. Lindstrom M, Hanson BS, Ostergren P. Socioeconomic differences in leisure-time physical activity: the role of social participation and social capital in shaping health related behavior. Soc Sci Med. 2001;52:441–51, doi: 10.1016/S0277-9536(00)00153-2. Ashe M, Miller WC, Eng JJ, Noreau L. Older Adults, Chronic Disease and Leisure-Time Physical Activity. Gerontology. 2009;55:64–72, doi: 10. 1159/000141518. Shimada H, Lord SR, Yoshida H, Kim H, Suzuki T. Predictors of Cessation of Regular Leisure Time Physical Activity in CommunityDwelling Elderly People. Gerontology. 2007;53:293–7, doi: 10.1159/ 000103214. Litwin H. Social network type and health status in a national sample of elderly Israelis. Soc Sci Med. 1998;46:599-609, doi: 10.1016/S02779536(97)00207-4. Pitkala KH, Blomquist L, Routasalo P, Saarenheimo M, Karvinen E, Oikarinen U, Mantyranta T. Leading groups of older people: a description and evaluation of the education of professionals. Educ Gerontol. 2004; 30:821–33, doi: 10.1080/03601270490507268. Cattan M, White M, Bond J, Learmouth A. Preventing social isolation and loneliness among older people: a systematic review of health promotion interventions. Ageing Soc. 2005;25:41-67, doi: 10.1017/ S0144686X04002594. PNAS - National Social Policy 2004. Ministe´rio do Desenvolvimento Social e Combate a Fome, Secretaria Nacional e Assisteˆncia Social. Available online at http://www.mds.gov.br/arquivos/pnas_final.pdf [Accessed May 11, 2010]. Mazo GZ, Mota J, Gonçalves LHT, Matos MG, Carvalho J. Physical activity and quality of life of older women from Floriano´polis, Brazil. Rev Port Cien Desp. 2008;8:414–23. Miguel CS, Fortes VLF. Older women of a third-age group: the interfaces of relationship with families. Rev Bras de Cieˆn do Envelh Hum. 2005;2:74-85. Brazilian Institute of Geography and Statistics. Summary of Social Indicators. Rio de Janeiro: Instituto Brasileiro de Geografia e Estatı´stica; 2000. Mazo GZ. Physical activity and quality of life of older women [Thesis]. Porto, Portugal: Faculdade de Cieˆncias do Desporto e de Educaçaõ Fı´sica; 2003. Benedetti TB. Physical activity: a health promotion perspective for older adults in the municipality of Floriano´polis. [Thesis]. Floriano´polis (SC): Universidade Federal de Santa Catarina; 2004. Barbetta PA. Statistics applied to Social Sciences. 5. Ed Floriano´polis: Editora da UFSC; 2003, p.60. Marshal A, Bauman A. The International Physical Activity Questionnaire: Summary Report of the Reliability & Validity Studies. Produzido pelo Comiteˆ Executivo do IPAQ. DRAFT IPAQ – Summary, March; 2001. Shye D, Mullooly JP, Freeborn DK, Pope CR. Gender differences in the relationship between social network support and mortality: a longitudinal study of an elderly cohort. Soc Sci Med. 1995;41:935-47, doi: 10. 1016/0277-9536(94)00404-H. Ueshima K, Ishikawa-Takata K, Yorifuji T, Suzuki E, Kashima S, Takao S, et al. Physical Activity and Mortality Risk in the Japanese Elderly: A Cohort Study. Am J Prev Med. 2010;38:410–8, doi: 10.1016/j.amepre. 2009.12.033. Lord S, Chastin SFM, McInnes L, Little L, Briggs P, Rochester L. Exploring patterns of daily physical and sedentary behaviour in community-dwelling older adults. Age Ageing. 2011;40:205-10, doi: 10. 1093/ageing/afq166. Harris TJ, Owen CG, Victor CR, Adams R, Cook DG. What factors are associated with physical activity in older people, assessed objectively by accelerometry? Br J Sports Med. 2009;43:442-50, doi: 10.1136/bjsm.2008. 048033. Brannon L. Gender: Psychological Perspectives. 2. ed. Boston: Allyn and Bacon; 1999. Borges PLC, Bretas RP, Azevedo SF, Barbosa JMM. Profile of older adults participating in community groups in Belo Horizonte, Minas Gerais, Brazil. Cad Saude Publica. 2008;24:2798-808, doi: 10.1590/S0102311X2008001200008.

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30. Barreto KML, Carvalho EMF, Falca˜o IV, Lessa FJD, Leite VMM. Epidemiological and sociodemographic profile of older women from the Open University of the Third-Age in the State of Pernambuco. Rev Bras Saude Matern Infant. 2003;3:339-54, doi: 10.1590/S151938292003000300013. 31. Ramos LR. Determinant factors of healthy aging in older adults living in an urban center: the Epidoso Project, Sa˜o Paulo. Cad Saude Publica. 2003;19:793-7, doi: 10.1590/S0102-311X2003000300011. 32. Annear MJ, Cushman G, Gidlow B. Leisure time physical activity differences among older adults from diverse socioeconomic neighborhoods. Health Place. 2009;15:482–90, doi: 10.1016/j.healthplace.2008.09.005.

33. Lima-Costa MF, Barreto S, Giatti L. Does socioeconomic status equally affect older and younger adults in Brazil? A study using data from the National Household Sampling Study– PNAD/98. Cien Saude Colet. 2002;7:813-24. 34. Ragland DR, Satariano WA, MacLeod KE. Reasons given by older people for limitation or avoidance of driving. Gerontologist. 2004;44: 237-44, doi: 10.1093/geront/44.2.237. 35. Dickerson AE, Molnar LJ, Eby DW, Adler G, Bee´dard M, Bergweger M, et al. Transportation and aging: a research agenda for advancing safe mobility. Gerontologist. 2007;47:578-90, doi: 10.1093/geront/47.5. 578.

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DOI:10.1590/S1807-59322011001100004

CLINICAL SCIENCE

Gender differences, polypharmacy, and potential pharmacological interactions in the elderly Carina Duarte Venturini,I Paula Engroff,I Luı´sa Scheer Ely,I Luı´sa Faria de Arau´jo Zago,II Guilherme Schroeter,II Irenio Gomes,I Geraldo Attilio De Carli,I Fernanda Bueno MorroneII,III I

Pontifıćia Universidade Cato´lica do Rio Grande do Sul (PUCRS), Instituto de Geriatria e Gerontologia, Porto Alegre/RS, Brazil. II Pontifıćia Universidade Cato´lica do Rio Grande do Sul (PUCRS), Faculdade de Farmaćia, Porto Alegre/RS, Brazil. III Pontifıćia Universidade Cato´lica do Rio Grande do Sul (PUCRS), Programa de Po´s-Graduaçaõ em Biologia Celular e Molecular, Porto Alegre/RS, Brazil.

OBJECTIVE: This study aims to analyze pharmacological interactions among drugs taken by elderly patients and their age and gender differences in a population from Porto Alegre, Brazil. METHODS: We retrospectively analyzed the database provided by the Institute of Geriatric and Gerontology, Porto Alegre, Brazil. The database was composed of 438 elderly and includes information about the patients’ disease, therapy regimens, utilized drugs. All drugs reported by the elderly patients were classified using the Anatomical Therapeutic and Chemical Classification System. The drug-drug interactions and their severity were assessed using the MicromedexH Healthcare Series. RESULTS: Of the 438 elderly patients in the data base, 376 (85.8%) used pharmacotherapy, 274 were female, and 90.4% of females used drugs. The average number of drugs used by each individual younger than 80 years was 3.2¡2.6. Women younger than 80 years old used more drugs than men in the same age group whereas men older than 80 years increased their use of drugs in relation to other age groups. Therefore, 32.6% of men and 49.2% of women described at least one interaction, and 8.1% of men and 10.6% of women described four or more potential drug-drug interactions. Two-thirds of drug-drug interactions were moderate in both genders, and most of them involved angiotensin-converting enzyme inhibitor, non-steroidal anti-inflammatory, loop and thiazide diuretics, and b-blockers. CONCLUSION: Elderly patients should be closely monitored, based on drug class, gender, age group and nutritional status. KEYWORDS: Older Adults; Polytherapy; Pharmacology; Drug-Drug; Interaction; Hazards. Venturini CD, Engroff P, Ely LS, Zago LFA, Schroeter G, Gomes I, De Carli GA, et al. Gender differences, polypharmacy, and potential pharmacological interactions in the elderly. Clinics. 2011;66(11):1867-1872. Received for publication on April 22, 2011; First review completed on May 31, 2011; Accepted for publication on July 11, 2011 E-mail: fernanda.morrone@pucrs.br Tel.: 55 51 3320-3512

require multiple drugs. It is well documented that polypharmacy has a greater potential to lead to drug interactions and adverse events.4 Pharmacological interactions contribute to the decreased in general health in the elderly, leading to disability, reduced quality of life, raising the number of hospital admissions, a longer duration of hospital stays, a greater need for ambulatory services, and increased healthcare costs.5 Physiological alterations in the body with age make the elderly more susceptible to interactions. These processes include a decrease of renal function and hepatic metabolism, gastro-intestinal tract alterations, and nutritional status deficiency.6 Therefore, body fat gains and muscle mass losses are normally seen in the elderly population, and these factors are also determinants of the intensity of drug interactions.7 Considering the prevalent use of polypharmacy and the health hazards due to drug-drug interaction in the elderly, this study aims to analyze potential pharmacological interactions among drugs taken by elderly patients and

INTRODUCTION Aging is the main risk factor for the development of chronic diseases, and this phenomenon is increasing worldwide.1 Although chronologic age is most often used to define the population $65 years old in developed countries and .60 years old in developing countries, the elderly are considered a heterogeneous group, with individuals aging at varying rates.2 The elderly are the predominant users of pharmaceuticals in the population.3 Thus, aging of the population could be a reason for an increased use of pharmaceutical products. These patients often have multiple diseases, and so they

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with the drug administration. For this reason, all type of interactions we found in this study must be considered ‘‘potential interactions.’’ The Institutional Ethics Committee of PUCRS approved this study (number 0502935), and all the study participants signed consent forms. The data were analyzed and tabulated using the computer program SPSS v17.0, and the results were presented as a percentage of the data set. Ninety-five percent confidence intervals (95% CI) were utilized to show differences in the elderly characteristics. Statistical analysis was performed considering age groups ,80 and $80 years old using a Student’s t test to compare the average between the two groups and a Pearson’s Chi-Square test to identify the severity of the interactions. Results were expressed as mean¡sd, and a p-value ,0.05 was considered significant.

their age and gender differences in a population from Porto Alegre, Brazil.

METHODS This observational, cross-sectional study is part of an epidemiological project (Multidimensional Study of Elderly in Porto Alegre) that represents a partnership between the Porto Alegre City Hall and the Instituto de Geriatria e Gerontologia (IGG) - Pontifı´cia Universidade Cato´lica do Rio Grande do Sul (PUCRS). The elderly interviewed were randomly recruited, between January 2006 and May 2007, by the University social service professionals, from different socioeconomic groups as previously described by Faggiani et al.8 To participate in this research study, the following inclusion criteria were used: individuals were able-bodied (to allow for travel to the interview site) and were older than sixty years old. The elderly who refused to participate in the study were excluded. The sample size for this study was in accordance to the number of individuals estimated for each neighborhood of Porto Alegre and was updated by the Brazilian Institute of Geography and Statistics according to an estimation of the population variation through 2005.9 The database generated from Multidimensional Study of Elderly in Porto Alegre was used in the present study for retrospective analysis. The database was constructed from a previously validated pharmacotherapeutic questionnaire that elderly filled out based on their memory and/or using materials supplied to them, such as prescriptions and/or a prescription label. The data were collected, and results were kept in and the individual patient’s file, together with the completed questionnaires. For the present study, we retrospectively analyzed the database provided by the IGG. To evaluate potential drugdrug interactions (DDI), we considered the most frequently interacting drug pairs in a computerized database10 of the pharmacotherapy used in elderly individuals from Porto Alegre-RS, Brazil. The database was composed of 438 elderly and includes information about the elderly general disease and therapy regimens, the prescribed and utilized drugs, the general pharmacological classes, of those drugs, the number of drugs used, and the frequency of selfmedication (all drug administration, except those prescribed by a physician). The Anatomical Therapeutic and Chemical Classification System (ATC)11 was used to classify the drugs that the elderly reported using, and the analysis of the potential DDI was performed according to Reis and Cassiani,12 using the computer software MicromedexH Healthcare Series (Thomson MicromedexTM, Greenwood Village, Co, USA).13 A severity rating scale employing the categories of ‘‘low’’ interaction (risk of adverse outcomes appears small), ‘‘moderate’’ interaction (to avoid administration unless it is determined that the benefit of co-administration outweighs the risk to the elderly), and ‘‘severe’’ interaction (to avoid administration of combination) was used to describe the potential DDI. Furthermore, it has been provided an association of the clinical consequences or adverse reactions to drugs and the characterization of the interaction mechanisms. Polypharmacy was defined as the use of more than three drugs that have the potential to cause drug interactions and side effects.4 The questionnaires contained no information about the timing of or beverage and/or food consumption

RESULTS In this study, a population of elderly individuals provided information on their use of all categories of drugs, and this information was analyzed to determine potential DDI within this population. Preliminarily, we evaluated three age groups (60 to 69, 70 to 79, and $80 years old). According to those results, we performed another statistical analysis considering only two age groups: ,80 and $80 years old. In both analyses, we gathered information about whether individuals used drugs (yes/no), the number of drugs used per day (1 to 3, 4 to 6, and $7), the number of interactions (0, 1, 2, 3, and $4), and the severity of the interactions (low, moderate, and severe) (Table 1).

Use of pharmacotherapy Of the 438 elderly individuals interviewed, 376 (85.8%) used pharmacotherapy, and only 62 (14.2%) people older than 60 years old did not use any drugs. Table 1 shows that the majority of the population (90.4%) that used drugs was female (N = 274). In this case, differences among age groups were not significant. Women ,80 years old used more drugs than men in the same age group (p,0.001); however, in the population $80 years old, the differences between genders were not significant. Men $80 years old tended to increase their use of drugs in relation to other age groups of the same gender (p = 0.057). Regarding the number of drugs used by the elderly, the majority of this population used from one to three drugs (3.20¡2.61), and the average number of drugs used by women 79 years old or younger (3.58¡2.74) was higher than the average used by men (2.20¡2.02) in the same age group (p,0.001). However, among men, the number of drugs used increased with age (4.53¡3.23) (p = 0.010) (Table 1). The majority of women (36.9%) used five or more drugs active principles, whereas the majority of men (41.2%) used between one and two drugs active principles. Among men, the number of drugs’ active principles used increased with age (p = 0.012).

Potential drug-drug interactions We found that the number of potential DDI increased in parallel with the number of drugs used by the elderly. In all age groups analyzed, 32.6% of men and 49.2% of women described at least one interaction. Moreover, 8.1% of men and 10.6% of women reported four or more potential DDI. Among women, the DDI were more

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Table 1 - Drug use and intensity of interaction in the elderly, according to gender and age group (N = 438). VARIABLE

DRUG USE [N(%)] Men Women p** Total NUMBER OF MEDICATIONS (m¡sd) Men Women p* Total NUMBER OF DRUG ACTIVE PRINCIPLES (m¡sd) Men Women p* Total NUMBER OF INTERACTIONS (m¡sd) Men Women p* Total INTENSITY OF INTERACTION [N(%)] Men Low Moderate Severe Women Low Moderate Severe p** Total Low Moderate Severe

AGE GROUP

TOTAL ,80

$80

p-value

102 (75.6) 274 (90.4) ,0,001 376 (85.8)

86 (72.9) 239 (90.9) ,0,001 325 (85.3)

16 (94.1) 35 (87.5) 0,657 51 (89.5)

0.057** 0.499**

2.50¡2.33 3.51¡2.67 ,0.001 3.20¡2.61

2.20¡2.02 3.58¡2.74 ,0.001 3.15¡2.61

4.53¡3.26 3.03¡2.12 0.094 3.47¡2.58

0.010* 0.219*

2.65¡2.59 3.88¡2.99 ,0.001 3.50¡2.92

2.33¡2.25 3.97¡3.07 ,0.001 3.46¡2.93

4.88¡3.64 3.35¡2.36 0.124 3.81¡2.86

0.012* 0.225*

0.87¡1.89 1.33¡2.36 0.048 1.19¡2.23

0.71¡1.50 1.42¡2.49 0.001 1.20¡2.25

2.00¡3.46 0.73¡0.93 0.153 1.11¡2.09

0.149* 0.001*

4 (9.1) 31 (70.5) 9 (20.5)

3 (8.6) 25 (71.4) 7 (20.0)

1 (11.1) 6 (66.7) 2 (22.2)

0.955**

13 (8.7) 104 (69.8) 32 (21.5) 0.988

9 (7.0) 92 (71.3) 28 (21.7) 0.936

4 (20.0) 12 (60.0) 4 (20.0) 0.842

0.157**

17 (8.8) 135 (69.9) 41 (21.2)

12 (7.3) 117 (71.3) 35 (21.3)

5 (17.2) 18 (62.1) 6 (20.7)

0.216**

0.399**

0.390*

0.403*

0.760*

(m¡sd): mean ¡ standard deviation. p-values were based on Student’s T-test; ** p-values were based on Pearson’s Chi-Square test. *

frequent in individuals younger than 80 years old (p = 0.001), whereas men experienced more potential DDI as age increased; most DDI in men occurred after age 80. Regarding the intensity of drug interactions, two-thirds of them were of moderate severity in both genders, and there were no statistically significant differences among all age groups analyzed. Among those individuals who used drugs, there were 591 interactions reported. The 30 most frequent types of DDI are shown in Table 2 (intensity and possible effects), most of them involving angiotensin-converting enzyme inhibitors (e.g., captopril, enalapril), non-steroidal anti-inflammatory (NSAIDs) agents (e.g., aspirin, diclofenac, ibuprofen), loop and thiazide diuretics (e.g., furosemide, hydrochlorothiazide), and b-adrenergic blockers (e.g., propranolol). According to our data, cardiovascular system drugs were the most prescribed drug class (62.3%). The second most frequently used drugs were analgesic and anti-inflammatory agents (34.6%), followed by central nervous system drugs (20.8%). Unexpectedly, vitamin and mineral supplements are used by 15.1% of the patients studied.

and the potential DDI as well the intensity of DDI in both genders and in several age groups of elderly patients. Other studies reporting drug utilization by the elderly from different countries have already been performed and have shown a high incidence of polytherapy.14,15 Although there are several published studies describing polypharmacy and pharmacological interactions, such data on Brazilian patients over 80 years old are lacking. Furthermore, only a few studies have compared differences between genders in this age population. We found that most of the elderly who use drugs are female. Although it is known that women both consult a physician more frequently and participate more often in research studies, data from the Brazilian Institute of Geography and Statistics show that female residents in Porto Alegre outnumber male residents.9,16 Previous studies conducted in Porto Alegre, Brazil, using the elderly population have shown that the elderly use around 3.2 drugs per day and that females uses more drugs.8,16 As reported by Flores and Mengue,16 the average number of medications can be higher because people do not commonly consider some agents to be drugs, such as therapies for obesity, allergy, pain, diarrhea, disorders of the kidney or bladder or digestive tract, as well nutritional (vitamins and mineral) supplements.

DISCUSSION In this study, we examined an elderly population from Porto Alegre, RS, Brazil, and analyzed drug consumption

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Table 2 - The 30 most frequent Drug-Drug Interactions (DDI) in the elderly.

Interaction

Intensity*

N (%)

Possible Effects

Verapamil - Sinvastatin Digoxin - Spironolactone Captopril - Aspirin Enalapril - Thiazide Diuretics Captopril - Thiazide Diuretics Propranolol - Hydrochlorothiazide Aspirin - Enalapril Aspirin - Furosemide Enalapril - Metformin Aspirin - Verapamil Aspirin - Aluminum, Calcium or Magnesium Containing Products Diclofenac - Hydrochlorothiazide Furosemide - Digoxin Captopril - Furosemide Captopril â&#x20AC;&#x201C; Ibuprofen Ibuprofen - Hydrochlorothiazide Enalapril - Furosemide Aspirin - Ibuprofen Diclofenac - Captopril Glibenclamide - Hydrochlorothiazide Levothiroxine - Sinvastatine Aspirin - Spironolactone Aspirin - Diclofenac Propranolol - Nifedipine Fluoxetine - Aspirin Metformin - Propranolol Aspirin - Glibenclamide Propranolol - Furosemide Alendronate - Calcium (Ca Co3) Ibuprofen - Propranolol

Severe Severe Moderate Moderate Moderate Moderate Moderate Moderate Moderate Moderate Moderate

10 (1.7) 5 (0.9) 33 (5.7) 30 (5.2) 25 (4.3) 25 (4.3) 23 (3.9) 19 (3.3) 14 (2.4) 14 (2.4) 12 (2.1)

Increased risk of myopathy or rhabdomyolysis Digoxin toxicity (nausea, vomiting, cardiac arrhythmias) Decreased captopril effectiveness Postural hypotension (first dose) Postural hypotension (first dose) Hyperglycemia, hypertriglyceridemia Decreased effectiveness of enalapril Blunting of the diuretic effect of furosemide Hyperkalemic lactic acidosis Increased risk of bleeding Decreased salicylate effectiveness

Moderate Moderate Moderate Moderate Moderate Moderate Moderate Moderate Moderate Moderate Moderate Moderate Moderate Moderate Moderate Moderate Moderate Low Low

9 (1.6) 9 (1.6) 9 (1.6) 8 (1.4) 8 (1.4) 8 (1.4) 8 (1.4) 6 (1.0) 6 (1.0) 6 (1.0) 6 (1.0) 6 (1.0) 6 (1.0) 6 (1.0) 5 (0.9) 5 (0.9) 5 (0.9) 13 (2.3) 7 (1.2)

Decreased diuretic and antihypertensive efficacy Digoxin toxicity (nausea, vomiting, cardiac arrhythmias) Postural hypotension (first dose) Decreased antihypertensive and natriuretic effects Decreased diuretic and antihypertensive efficacy Postural hypotension (first dose) Decreased antiplatelet effect of aspirin Decreased antihypertensive and natriuretic effects Decreased glyburide effectiveness Decreased levothyroxine efficacy Decreased spironolactone effectiveness Reduced diclofenac efficacy Hypotension and/or bradycardia. Increased risk of bleeding. Hypoglycemia, hyperglycemia, or hypertension Increased risk for hypoglycemia Hypotension, bradycardia Reduced alendronate absorption Decreased antihypertensive effect

Intensity Low: risk of adverse outcomes appears small; Intensity Moderate: to avoid administration unless it is determined that the benefit of coadministration outweighs the risk to the elderly; Intensity Severe: to avoid administration of combination.

old,7 and this fate can explain why men start to visit doctors later in their life.23 Similarly to other studies, our results show that the majority of the elderly patients in this study used cardiovascular system drugs, central nervous system drugs, and anti-inflammatory agents.8,16 The majority of interactions was of moderate intensity and also involved cardiovascular system drugs and NSAIDs. In fact, there are DDI studies in the literature, but there is little agreement among them with respect to the severity and clinical importance of interactions.24 According to Schroeter et al.,25 the majority of the elderly use the following combinations of cardiovascular drugs: a diuretic and a b-adrenergic blocker, or a diuretic, a badrenergic blocker, and an angiotensin-converting enzyme inhibitor (ACE). Even though diuretics are widely recommended by the World Health Organization (WHO)26 to optimize therapeutic effects and diminish adverse events, loop and thiazide diuretics largely contribute to moderateseverity interactions, mainly when they are used with captopril, propranolol and aspirin. These associations might result in pharmacodynamic interactions, leading to a loss of drug efficacy. For example, the use of NSAIDs in patients receiving antihypertensive therapy with b-adrenoceptor antagonists (b-blockers), thiazides, or ACE inhibitors can result in a loss of antihypertensive action.27 In the elderly, pharmacodynamic interactions are of particular relevance because they can reduce homeostatic mechanisms, and the elderly are therefore particularly

Surprisingly, after their eighties, men seem to take more drugs and drug active principles. Similarly, in this study, we found that women used more drugs than men only until the age of 79. Women are more concerned with their health and consult health services more often and earlier than men, and women are more accustomed to the use of drugs. In addition, more health programs are developed for women, such as colon and breast cancer prevention programs.17 Furthermore, women more easily adopt the sick role; they tend to recognize and experience more health problems, more underlying gender-related psycho-social and behavioral influences, and to perceive more symptoms than men.18,19 These factors could explain the greater use of medications by women and therefore the higher number of DDI in among women younger than 79 years old. These attitudes also provides to this group better medical monitoring and treatment of disease and early diagnosis. For some time, geriatricians have preferred to minimize the number of drugs they prescribe to elderly patients to prevent side effects and interactions.20 This medical management could explain the decreased use of drugs among the over-80 population, especially because recent studies have shown that inappropriate prescriptions is a great risk factor for adverse drug events and interactions in the elderly population.21,22 Otherwise, men generally visit doctors later than women, after their disease process has already begun or when the symptoms have already presented.19 It is important to highlight the fact that, in the south of Brazil, the life expectancy is around 81 years

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kinds of medications to be drugs.16 Female patients have a greater risk of developing adverse drug reactions than males due to multiple pharmacokinetic parameters, such as a lower rate of drug absorption, reduced hepatic clearance, a greater percentage of body fat with age that can increase distribution volumes for lipophilic drugs, and higher oral bioavailability.7 In the same way, gastric acid secretion, gastrointestinal and renal blood flow, and immunological differences, are several factors that might contribute to sexrelated differences in pharmacokinetics.38 Greater body mass in men results in alterations of the distribution volume and total clearance of most medications. Besides, gender differences in oxidative metabolism, conjugation, and renal filtration; secretion, and reabsorption lead to faster drug clearance in men compared with women.38 There are also pharmacodynamic differences between men and women, particularly for cardiac and psychotropic medications. For instance, various antipsychotics appear more effective in women than men for the same dosage and plasma concentration. Furthermore, women are at increased risk for half-life prolongation with certain anti-arrhythmic drugs compared with men, even at equivalent serum concentrations.7 In order to prevent DDI or diminish its consequences, several measures should be considered for elderly patients, including periodic review of their medicines and their adverse events, preference for monotherapy as soon as possible instead of associations containing fixed dosages, choice of drugs with proven efficacy, and suspension of drug use as soon as possible. Therefore, the comprehensiveness of the prescriptions and the pharmacologic orientations beyond simply the regimen should be evaluated.39 For reducing drug-drug and food-drug interactions, it might also be important to change the administration schedule for elderly patients at risk for nutritional deficiency.6 This practice aims to diminish potential adverse drug reactions and prevent aggravation of the clinical features that occurs due to the great number of drugs utilized by each elderly individual. The present study demonstrates that potential drug interactions in the elderly correlate with the concomitant use of multiple drugs. Furthermore, elderly patients should be closely monitored for adverse drug reactions and potential pharmacological interactions, particularly with drugs that target the cardiovascular system, given our finding that they were the most commonly used class of drug and the most frequently involved in drug interactions.

sensitive to combined postural hypotensive or sedative effects of drugs. In the case of aspirin, an NSAID, many interactions can occur when it is combined with other drugs. In most of them, aspirin leads to a decrease in the effectiveness of other drugs, which might result in an increase in the dosage prescribed. Otherwise, aspirin might increase the effect (or side effect) of other drugs. In both cases, co-administration with aspirin might increase the number of adverse events, which can be confused with the severity of comorbidities. The consequences of such interactions include a longer duration of hospital stays with administration of more drugs to patients, resulting in a higher probability of DDI.28 Another combination frequently used is digoxin and furosemide, as reported by Moura et al.29 While in our results this pair of drugs does not represent the most frequent interaction, it is responsible for moderate-intensity interactions that might precipitate or contribute to the development of arrhythmias, especially in patients with preexisting cardiac abnormalities. These effects can be prevented by dietary sodium restriction or by addition of potassium-sparing diuretics.30 We also observed in our results a few severe interactions that mainly involving the following drug combinations: verapamil and sinvastatin that are both CYP3A4 inhibitors, and their co-prescription might result in an increase in the bioavailability of the statin, with a greater risk of myopathy or rhabdomyolysis, as was well-documented by Molden et al.31 and Jacobson;32 digoxin and spironolactone due to this last drug reduces renal clearance and increases serum concentration of digoxin, thus its dose should be reduced in cases of combination with spironolactone.33 Although we could not evaluate real side effects, the computer program used in this study provided information about the associated clinical consequences or adverse reactions to drugs and characterized the interactions mechanisms. The same methodology has been previously used by others.12,34,35 In the last several decades, polypharmacy among the elderly has increased due to several reasons such as the increase of life expectancy, the prevalence of non-degenerative chronic diseases with complex pharmacotherapeutic regimens,14 the introduction of new drugs to the pharmaceutical market, self-medication including herbal drugs, misuse of medication, poor quality of doctors’ choices of prescriptions, doctors’ over-prescription of drugs, additional medicines prescribed to treat side effects, and poor doctor-patient relationships.3,15 Physiological changes related to age can lead to pharmacokinetic and pharmacodynamic changes in elderly patients, and these changes seem to be more serious among women.7 Furthermore, women take more medicines and self-report worse health than men. Older women who have more income use more prescribed drugs.36 As Wortman et al.37 reported, in the city of Porto Alegre, Brazil, the use of benzodiazepines is higher among older and widowed or divorced women. It is important to emphasize that female life expectancy is about 7.62 years higher than that of males in Brazil.23 In this study, we found that until 79 years of age, women had more interactions than men in the same age group, probably because women also use higher numbers of drugs until this age. Thereafter, it is possible that women selfmedicate more often and that they do not consider several

ACKOWLEDGMENTS This study was supported by the following agencies: Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq) grant # 475137/20094 and Porto Alegre City Hall, Brazil. The authors thank American Journal Experts for English correction.

AUTHOR CONTRIBUTIONS Venturini CD, Engroff P, Ely LS were responsible for the study’s methodology, data interpretation and final writing of the paper. Zago LFA, Schroeter G and Gomes I were responsible for the study’s methodology and data interpretation. De Carli GA and Morrone FB were responsible for the preparation and design of the study, final writing, and correction of the paper.

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20. Phillips SL, Carr-Lopez SM. Impact of a pharmacist on medication discontinuation in a hospital-based geriatric clinic. Am J Hosp Pharm. 1990;47:1075-9. 21. Green JL, Hawley JN, Rask KJ. Is the number of prescribing physicians an independent risk factor for adverse drug events in an elderly outpatient population? Am J Geriatr Pharmacother. 2007;5:31-9, doi: 10. 1016/j.amjopharm.2007.03.004. 22. Tulner LR, Kuper IM, Frankfort SV, van Campen JP, Koks CH, Brandjes DP, et al. Discrepancies in reported drug use in geriatric outpatients: relevance to adverse events and drug-drug interactions. Am J Geriatr Pharmacother. 2009;7:93-104, doi: 10.1016/j.amjopharm.2009.04.006. 23. Instituto Brasileiro de Geografia e Estatı´stica. Breves notas sobre a mortalidade no Brasil no perıódo 1991/2007. Rio de Janeiro: 2008. 24. Vitry AI. Comparative assessment of four drug interaction compendia. Br J Clin Pharmacol. 2007;63:709-14, doi: 10.1111/j.1365-2125.2006.02809. x. 25. Schroeter G, Chaves LL, Engroff P, Faggiani FT, De Carli GA, Morrone FB. Estudo de Utilizaçaõ de Anti-Ulcerosos na Populaçaõ Idosa de Porto Alegre, RS, Brasil. Rev HCPA. 2008;28:89-95. 26. World Health Organization. International Society of Hypertension (ISH). Guidelines for the management of hypertension. J Hypert. 1999;17:15183. 27. Seymour RM, Routledg PA. Important Drug-drug interactions in the elderly. Drugs Aging. 1998;12:485-94, doi: 10.2165/00002512-19981206000006. 28. Terleira A, Portoles A, Rojas A, Vargas E. Effect of drug-test interactions on length of hospital stay. Pharmacoepidemiol Drug Saf. 2007;16:39-45, doi: 10.1002/pds.1330. 29. Moura C, Acurcio F, Belo N. Drug-drug interactions associated with length of stay and cost of hospitalization. J Pharm Pharm Sci. 2009;2:26672. 30. Gomez R, Venturini CD. Interaçaõ entre alimentos e medicamentos. Porto Alegre: Suliani Letra e Vida. 2009.p.64 31. Molden E, Skovlund E, Braathen P. Risk management of simvastatin or atorvastatin interactions with CYP3A4 inhibitors. Drug Saf. 2008;31:58796, doi: 10.2165/00002018-200831070-00004. 32. Jacobson TA. Comparative pharmacokinetic interaction profiles of pravastatin, simvastatin, and atorvastatin when coadministered with cytochrome P450 inhibitors. Am J Cardiol. 2004;94:1140-6, doi: 10.1016/j. amjcard.2004.07.080. 33. Fenster PE, Hager WD, Goodman MM. Digoxin-quinidine-spironolactone interaction. Clin Pharmacol Ther. 1984;36:70-3, doi: 10.1038/clpt. 1984.141. 34. Nobili A, et al. Potentially severe drug interactions in elderly outpatients: results of an observational study of an administrative prescription database. J Clin Pharm Ther. 2009;34:377-86, doi: 10.1111/j.1365-2710. 2009.01021.x. 35. Lin CF, Wang CY, Bai CH. Polypharmacy, aging and potential drugdrug interactions in outpatients in Taiwan: a retrospective computerized screening study. Drugs Aging. 2011;28:219-25, doi: 10.2165/11586870000000000-00000. 36. Chrischilles EA, Foley DJ, Wallace RB, Lemke JH, Semla TP, Hanlon JT, et al. Use of medications by persons 65 and over: data from the established populations for epidemiologic studies of the elderly. J Gerontol. 1992;47:137-44. 37. Wortmann AC, Gru¨dtner MC, Fialho AF, Jardim Neto JC, Schaefer LG, Sehn F, et al. Consumo de benzodiazepıńicos em Porto Alegre. Rev Assoc Med Bras. 1994;40:265-70. 38. Schwartz JB. The influence of sex on pharmacokinetics. Clin Pharmacokinet. 2003;42:107-21, doi: 10.2165/00003088-200342020-00001. 39. Rozenfeld S, Pepe VLE. Guia Terapeûtico Ambulatorial. Porto Alegre: Artes Me´dicas; 1992.

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DOI:10.1590/S1807-59322011001100005

CLINICAL SCIENCE

Non-small cell lung cancer in never smokers: a clinical entity to be identified Ilka Lopes Santoro, Roberta Pulcheri Ramos, Juliana Franceschini, Sergio Jamnik, Ana Luisa Godoy Fernandes Federal University of Sa˜o Paulo, Respiratory Division, Sa˜o Paulo/SP, Brazil.

OBJECTIVES: It has been recognized that patients with non-small cell lung cancer who are lifelong never-smokers constitute a distinct clinical entity. The aim of this study was to assess clinical risk factors for survival among neversmokers with non-small cell lung cancer. METHODS: All consecutive non-small cell lung cancer patients diagnosed (n = 285) between May 2005 and May 2009 were included. The clinical characteristics of never-smokers and ever-smokers (former and current) were compared using chi-squared or Student’s t tests. Survival curves were calculated using the Kaplan-Meier method, and log-rank tests were used for survival comparisons. A Cox proportional hazards regression analysis was evaluated by adjusting for age (continuous variable), gender (female vs. male), smoking status (never- vs. ever-smoker), the Karnofsky Performance Status Scale (continuous variable), histological type (adenocarcinoma vs. non-adenocarcinoma), AJCC staging (early vs. advanced staging), and treatment (chemotherapy and/or radiotherapy vs. the best treatment support). RESULTS: Of the 285 non-small cell lung cancer patients, 56 patients were never-smokers. Univariate analyses indicated that the never-smoker patients were more likely to be female (68% vs. 32%) and have adenocarcinoma (70% vs. 51%). Overall median survival was 15.7 months (95% CI: 13.2 to 18.2). The never-smoker patients had a better survival rate than their counterpart, the ever-smokers. Never-smoker status, higher Karnofsky Performance Status, early staging, and treatment were independent and favorable prognostic factors for survival after adjusting for age, gender, and adenocarcinoma in multivariate analysis. CONCLUSIONS: Epidemiological differences exist between never- and ever-smokers with lung cancer. Overall survival among never-smokers was found to be higher and independent of gender and histological type. KEYWORDS: Lung neoplasm; Non-small cell lung cancer; Adenocarcinoma; Never-smoker; Smoking. Santoro IL, Ramos RP, Franceschini J, Jamnik S, Fernandes ALG. Non-small cell lung cancer in never smokers: a clinical entity to be identified. Clinics. 2011;66(11):1873-1877. Received for publication on March 4, 2011; First review completed on March 30, 2011; Accepted for publication on July 12, 2011 E-mail: ilkasantoro@pneumo.epm.br Tel.: 55 11 5549-1830

The proportion of never-smokers with lung cancer is expected to increase in parallel with successful smoking prevention and smoking cessation programs. Although the incidence of lung cancer may be increasing among never-smoker patients, it is unclear whether this increase represents are real increase in the lung cancer incidence among never-smokers or arises from the increasing prevalence of never-smokers in the general population.6 It is noteworthy that differences in epidemiological characteristics and histological subtypes between smokers and never-smokers have been demonstrated, especially among Asian patients.7,8 This suggests that the pathogenesis of non-small cell lung cancer (NSCLC) in never-smokers might be different than in smokers. It was recently demonstrated that a subgroup of patients with NSCLC exhibits a specific activating mutation in the epidermal growth factor receptor gene associated with better responses to target drugs, such as tyrosine kinase inhibitor drugs, and overall survival.9 There is limited literature regarding never-smoker lung cancer patients in the Western hemisphere, especially in

INTRODUCTION Lung cancer remains the leading cause of cancer mortality, accounting for more deaths than breast, colon, and prostate cancer combined. Smoking was established as a risk factor for lung cancer in the early 1950s.1-3 Moreover, smoking is associated with both lung cancer carcinogenesis and the prognosis of lung cancer patients.4 Due to the overwhelming etiological role of tobacco smoking, lung cancer is mainly considered a smoking-related disease; consequently, the never-smoker population is usually under-represented in lung cancer studies.5 Only recently has attention turned toward the small number of never-smokers with this disease.

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Survival curves and the five-year survival rates were calculated according to the Kaplan-Meier method, while a log-rank test was used to assess the differences in survival between the groups. A multivariate analysis (Cox proportional hazard regression) was evaluated by adjusting for known prognostic factors and potential confounders. The number of independent variables (factors) was limited by the occurrence of the event (death). The factors considered for inclusion were gender (female vs. male), smoking status (never- vs. ever-smoker), histological type (adenocarcinoma vs. non-adenocarcinoma), AJCC stage (early vs. advanced staging), and treatment (chemotherapy and/or radiotherapy vs. the best treatment support). The age at diagnosis and the Karnofsky Performance Status Scale were adjusted as continuous variables in the Cox regression model. Statistical analyses were performed using IBMâ&#x20AC;&#x2122;s Statistical Package for the Social Sciences, version 19.0. All hypothesis tests were two-tailed, and the level of significance was set at 5%.

Brazil. The aim of this study was to assess the epidemiological characteristics of never-smoker patients with lung cancer, focusing on clinical risk factors and survival.

METHODS The study described here was a prospective cohort study conducted between May 2005 and May 2009 using an electronic database. All consecutive patients with pathologically proven NSCLC who presented at our outpatient lung cancer clinic were eligible. The Institutional Review Board of our center approved this study. Patient consent was obtained for entry into the database. Data were collected at diagnosis as part of routine clinical practices using a structured data collection format. The selected epidemiological characteristics included age, gender, and ethnicity. NSCLC was further divided into three major histological subtypes: squamous cell carcinoma (SCC), adenocarcinoma and other types of carcinoma (large-cell carcinoma, poorly or undifferentiated carcinoma and not otherwise specified NSCLC). The vast majority of the lung specimens that were used for diagnosis were bronchial biopsies, and immunohistochemistry was employed to better identify the histological types when required. The American Joint Committee on Cancer (AJCC) staging system (sixth edition) was applied, and for analysis purposes, disease stage was categorized into two groups: early disease (Stages Ia to IIIa) and advanced disease (Stages IIIb and IV); moreover, among Stage IV patients, the database included whether metastases were intra- or extrathoracic. Weight loss, comorbidity, the Karnofsky Performance Status Scale, smoking status and treatments were documented. Weight loss was categorized into two groups: 10% or more and less than 10%. Comorbidities included one or more of the following conditions: hypertension, ischemic heart disease, diabetes mellitus, chronic obstructive pulmonary disease, asthma, and pulmonary tuberculosis sequelae. Smoking status was classified into two levels: ever-smoker and never-smoker. Patients with any history of smoking (current and former smokers) were classified as eversmokers, and the intensity of tobacco exposure was measured in pack-years. Never-smokers were defined as those who had never smoked in the past. Data related to passive exposure to environmental tobacco smoke and cooking fumes were not consistently available. Consequently, for the purpose of analysis, the never-smoker category did not account for passive exposure. Treatment status was stratified into two categories: treated (surgery, chemotherapy, or/and irradiation) and best treatment support. The date of the last follow-up or of death was collected for each patient. Overall survival was measured from the date of histological diagnosis to the date of death or the date that the patient was last known to be alive for censored observation.

RESULTS Two hundred eighty-five NSCLC patients diagnosed between May 2005 and May 2009 were included in our analysis. The majority were ever-smokers (76%). The median tobacco exposure was 41 pack-years (range: 1 to 210 pack-years). Among the never-smokers (56 patients), there were significantly more women (68%) and adenocarcinomas (70%). There were no significant differences between never- and ever-smokers with regard to age, the Karnofsky Performance Status Scale, weight loss, ethnicity, comorbidities, AJCC stage, and patient treatment. The majority of the patients presented with advanced disease, although no significant difference in the proportion of advanced disease or extrathoracic disease was observed between the groups (Table 1). Supplementary analyses were completed for the neversmoker group to examine the behavior of the variables analyzed above according to gender. The patients of both genders were similar with respect to age, the Karnofsky Performance Status Scale, the proportion of histological types, weight loss, comorbidities, extrathoracic metastasis, and patient treatment. However, female never-smokers exhibited a greater proportion of Stages IIIb and IV than their male counterparts (Table 2). The overall five-year survival rates of never-smokers and ever-smokers were significantly different (p = 0.049) (Figure 1). The median overall survival was 15.7 months (95% CI: 13.2 to 18.2). The median survival time was 14.9 months (95% CI: 12.9 to 16.9 months) for ever-smokers and 22.1 months (95% CI: 9.5 to 34.6 months) for never-smokers. To complement the survival study, a multivariate analysis was conducted using the Cox proportional hazard model. Being a never-smoker (vs. an ever-smoker), a higher Karnofsky Performance Status, early staging and treatment (vs. the best treatment support) were independent and favorable prognostic factors for overall survival after adjusting for age, gender, and adenocarcinoma (vs. nonadenocarcinoma) (Table 3). Moreover, a subgroup analysis of never-smokers revealed that the survival of the never-smoker-population did not show any influence of female gender (vs. male) or adenocarcinoma (vs. non-adenocarcinoma) on the Cox regression [-2log likelihood = 105,803; chi-squared = 2,027; p = 0.363).

Statistical analyses Differences between the never- and ever-smokers were compared using the chi-squared test for categorical variables and Studentâ&#x20AC;&#x2122;s t-test for continuous data, as were the differences between the two genders within the group of never-smokers.

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Table 1 - Characteristics of NSCLC patients grouped according to their smoking status at diagnosis. Variables Age mean (SD) Karnofsky Performance Status mean (SD) Female, n (%) Ethnicity Caucasian African descent Asian Histological type, n (%) Squamous cell Adenocarcinoma Other Weight loss $10%, n (%) Comorbidity,n (%) Staging, n (%) IIIb to IV (advanced disease) Extrathoracic metastasis, n (%) Treatment, n (%) Surgery Chemotherapy Radiotherapy Combined therapies Best support treatment

Never-smoker n = 56

Ever-smoker n = 229

61.6 (13.9) 81.6 (13.6) 38 (68)

63.4 (11.2) 80.3 (13.8) 73 (32)

42 (75) 11 (20) 3 (5)

161 (70) 60 (26) 8 (4) 0.03

11 39 06 17 14

(20) (70) (10) (30) (25)

87 116 26 99 44

(38) (51) (11) (43) (19)

0.08 0.34

46 17 40 07 36 07 13 16

(82) (30) (71) (12) (64) (12) (23) (28)

169 56 161 34 139 32 45 68

(74) (24) (70) (15) (61) (14) (20) (30)

0.19 1 0.57 1 0.87 1 0.651 0.621 0.771 0.601 0.871

p-value 0.31 ¥ 0.51 ¥ ,0.001 1 0.52

n = number of patients; SD = standard deviation. 1 Chi-squared. ¥ t-test.

In our study the presence of comorbidities did not differ between ever or never-smokers, although it has been described with tobacco-related disease. Conversely, in other studies the presence of comorbidities justified the worst survival among lung cancer patients.12 For a more comprehensive analysis, we performed a Cox proportional hazards regression adjusted for known prognostic factors. This multivariate analysis also confirmed that never-smokers exhibited a decreased risk of dying. As

DISCUSSION Our main finding was that never-smokers had better fiveyear survival rates than ever-smokers. This confirms the results of the other studies, which have shown survival benefits associated with a never-smoking status in NSCLC patients. Never-smoking status has been reported as an independent predictor of improved survival at five years (16% for current smokers, 23% for never-smokers),10 and a poorer survival outcome in patients with a history of smoking was described in a retrospective analysis.4 However, there is controversy regarding this finding in the current literature. Another study did not find differences in survival between NSCLC patients stratified according to their smoking status.11

Table 2 - Subject characteristics of never-smoker patients by gender. Variables Age mean (SD) Karnofsky performance status mean (SD) Histological type n (%) Squamous cell Adenocarcinoma Other $10% Weight loss n (%) Comorbidity n (%) Staging n (%) IIIb to IV Extra thoracic metastasis n (%) Treatment n (%)

Female n = 38

Male n = 18

62.8 (12.6) 59.3 (16.4) 81.3 (13.6) 82.2 (13.9)

p-value 0.39 ¥ 0.82 ¥ 0.59 1

08 (21) 27 (71) 03 (8) 13 (34) 09 (24)

03 12 03 04 05

(17) (68) (17) (22) (28)

0.361 0.74 1

34 (90) 10 (26) 27 (71)

12 (67) 07 (39) 13 (72)

0.04 1 0.20 1 0.93 1

n = number of patients; SD = standard deviation. 1 Chi-squared. ¥ t-test.

Figure 1 - Sixty-month survival Kaplan-Meier curve for NSCLC patients grouped according to smoking status.

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Table 3 - Multivariate analysis of factors related to overall survival in 285 NSCLC patients, using Cox regression (-2 log likelihood = 1,265.5; chi-squared = 60.0; p,0.001). Variable #

Age Female Never-smoker KPS # Adenocarcinoma Early disease detection Treatment

Coefficient

Adjusted HR

95% CI

p-value

-0.02 -0.25 -0.54 -0.03 0.08 0.82 -0.57

0.01 0.19 0.27 0.01 0.18 0.23 0.21

0.98 0.77 0.58 0.97 1.09 0.44 0.57

0.97-1.00 0.53-1.13 0.34-0.99 0.96-0.99 0.76-1.56 0.28-0.69 0.38-0.85

0.050 0.180 0.047 ,0.001 0.650 ,0.001 0.006

KPS = Karnofsky Performance Status Scale; SE = standard error; HR = hazard ratio; CI = confidence interval for HR. # = continuous variable.

lung adenocarcinoma have been described: the KRAS and the epidermal growth factor receptor (EGFR)20 pathways. KRAS mutations are generally linked to tobacco consumption, and the EGFR pathway is generally associated with nonsmokers.26-28 Recent studies have indicated that patients with mutations in the EGFR gene respond better to treatment with EGFR tyrosine kinase inhibitors.29,30 Although KRAS mutations are historically considered to represent a tumorigenic pathway in smokers, their prevalence has been reported to be similar in smokers and nonsmokers; however, differences in mutation type have been reported.31 Though this is still controversial, molecular differences between groups may be responsible for distinct clinical manifestations and responses to treatment. As biomarkers may be used for risk stratification and treatment selection, new pathogenic pathways are being studied.32,33 In contrast to studies in Asian populations, we did not find differences in the age of diagnosis among neversmokers compared with ever-smokers.34 Our findings are consistent with the results of studies performed in Europe and the United States, where this disease occurs mainly in older adults. These differences may be explained by the hypothesis that indoor air pollutants, such as cooking fumes, play a role in lung carcinogenesis in developing countries, although there is some controversy surrounding this issue.35 Exposure to cooking fumes is common in Brazil; however, we lacked information regarding our participantsâ&#x20AC;&#x2122; exposure levels to such fumes. As lung cancer is considered a disease of smokers,36,37 never-smoker patients may experience either late presentation or late diagnosis on the part of physicians. The majority of our patients presented with the disease at later stages; however, when the group of never-smokers was analyzed for gender associations, we found that women were more likely than men to have lung cancer diagnosed at a more advanced stage. It is noteworthy that we did not find differences in extrathoracic disease between neversmokers and ever-smokers. Consequently, the clinical threshold for investigating symptomatic never-smokers must be lower. The limitations of our study are related to the lack of information about passive smoking and cooking fume exposure as well as molecular analyses of the tumors. However, the epidemiological behavior of our neversmoker sample confirmed that even a racially varied population, as found in Brazil, follows the same model as that of never-smokers in other parts of the world. In conclusion, the vast majority of never-smoker lung cancer patients were female and exhibited adenocarcinoma as the predominant histological type. Additionally, the female

expected, early disease diagnosis, patient treatment and higher Karnofsky Performance Status scores were also favorable, independent predictors of survival. Interestingly, neither female gender nor having adenocarcinoma reached the specified significance level in the multivariate survival analyses, although both variables accounted for a significantly higher proportion of never-smoker patients in the univariate analysis. Never-smokers constituted 24% of the NSCLC patients in our population. The literature supports the assertion that several characteristics are more commonly seen in NSCLC patients who are never-smokers. Our study found that women were more likely than men to have non-smokingassociated lung cancer (68%). The risk of developing lung cancer among women who smoke has been described as higher than that of men who are exposed to the same smoking rate.13-18 These results are still controversial, and the possibility that women have an increased susceptibility to the effects of smoking is not yet clearly defined. However, the predominance of females among never-smokers with tumors, even without exposure to cigarette smoke carcinogens, has been previously described, suggesting that aspects related to hormonal factors may interfere with tumor carcinogenesis.6,8,15 The role of estrogen in the carcinogenesis of other types of tumors in women is well established. Growing evidence indicates the effects of estrogen on lung cancer cells. The presence of estrogen receptors (ERs) in pulmonary tumor cells suggests that this hormone plays a role in the carcinogenesis of lung cancer.19-21 There are two main types of ERs in humans: ER-alpha and ER-beta. ER-beta receptors are the major mediators of estrogen activity in lung cells. They are active receptors in lung tissue and can contribute to the growth of neoplastic cells. Although the prevalence of ERs in tumor cells is similar in men and women, gender differences in survival exist.22,23 The mechanism underlying these sex-based differences is unclear, but genetic and metabolic factors, hormonal influences, and the presence of specific isoforms of ER-beta may be involved. Another interesting finding is the predominance of adenocarcinoma among never-smoking patients, which is consistent with the results of previous studies.7,24,25 Although this histological variant is most commonly found in women, never-smoking men also showed a higher proportion of adenocarcinoma in our study, suggesting that a factor unrelated to sex is responsible for the predominance of adenocarcinomas in the never-smoking population. Several genetic alterations have been described that may contribute to the development of adenocarcinoma in nonsmokers. Two main pathways for the development of

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never-smoker patients showed a higher proportion of advanced disease, although the proportion with extrathoracic metastasis was similar to that of male never-smokers. Among NSCLC patients, after adjusting for age, female gender and adenocarcinoma, being a never-smoker with early treatment of the disease and having a higher Karnofsky Performance Status Scale were associated with a better prognosis. Lung cancer in never-smokers has a different clinical profile, with a distinctly lower mortality rate compared to lung cancer among smokers, which reflects a singular clinical behavior and natural history. Financial disclosure: Prof. Ilka Lopes Santoro and Dr. Se´rgio Jamnik are coinvestigators in a Phase III research project supported by AstraZeˆnecaH, BoehringerH, AbbottH and BristolH. They are also involved in a Phase II project supported by Merck Sharp DomeH. The other authors do not have any financial disclosures to declare.

16. 17. 18. 19. 20.

21. 22.

23.

AUTHOR CONTRIBUTIONS Santoro IL and Fernandes ALG conceived and designed the study, were responsible for the analysis and interpretation of the data, critical revision, and final approval. Ramos RP, Franceschini J, and Jamnik S were responsible for the collection, analysis and interpretation of data, and draft of the article.

24.

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CLINICS 2011;66(11):1879-1886

DOI:10.1590/S1807-59322011001100006

CLINICAL SCIENCE

M2-Polarized tumor-associated macrophages are associated with poor prognoses resulting from accelerated lymphangiogenesis in lung adenocarcinoma Bicheng Zhang,I Guoqing Yao,I Yafei Zhang,II,I Juan Gao,III Bo Yang,I Zhiguo Rao,I Jianfei GaoI I Wuhan General Hospital of Guangzhou Command, People’s Liberation Army, Department of Oncology, Wuhan, Hubei Province, China. II Southwest Hospital, Third Military Medical University, Department of Gastroenterology, Chongqing, China. III Wuhan General Hospital of Guangzhou Command Department of Gastroenterology, People’s Liberation Army, Wuhan, Hubei Province, China.

OBJECTIVES: Tumor-associated macrophages have been implicated in promoting tumor growth, progression and metastasis. However, the activated phenotype (M1 or M2) of tumor-associated macrophages remains unknown in solid tumors. Therefore, this study examined the density and prognostic significance of M2-polarized tumorassociated macrophages in lung adenocarcinoma. METHODS: Tumor specimens from 65 lung adenocarcinoma patients were assessed by ELISA for Th1/Th2 cytokine concentrations. The activated phenotype (M1 or M2) of tumor-associated macrophages was determined utilizing immunofluorescence staining. Additionally, to evaluate lymphangiogenesis, peritumoral lymphatic microvessel density was measured using D2-40. The correlation between tumor-associated macrophage subtype and overall patient survival was analyzed using the Kaplan-Meier method and compared using the log-rank test. RESULTS: A shift toward Th2 cytokine expression was detected within lung adenocarcinoma microenvironments. Approximately 79.71¡16.27% of tumor-associated macrophages were M2 polarized; the remaining 20.35¡5.31% were M1 polarized. The infiltration of M2-polarized macrophages was significantly associated with P-TNM staging and lymph node metastasis. The peritumoral lymphatic microvessel density was significantly higher in the high M2polarized tumor-associated macrophage group than in the low M2-polarized tumor-associated macrophage group. A significant difference in overall patient survival was detected not only between patients with tumors with high and low macrophage counts but also between patients with tumors with high and low counts of M2-polarized macrophages. CONCLUSION: Tumor-associated macrophages in lung adenocarcinoma have an M2-polarized subtype and are associated with poor prognoses, perhaps resulting from accelerated lymphangiogenesis and lymph node metastasis. KEYWORDS: M2-polarized macrophages; Tumor-associated macrophages; Lymphangiogenesis; Lung adenocarcinoma; Prognosis. Zhang B, Yao G, Zhang Y, Gao J, Yang B, Rao Z, et al. M2-Polarized tumor-associated macrophages are associated with poor prognoses resulting from accelerated lymphangiogenesis in lung adenocarcinoma. Clinics. 2011;66(11):1879-1886. Received for publication May 29, 2011; First review completed on July 4, 2011; Accepted for publication on July 12, 2011 E-mail: jianfeigao1957@hotmail.com Tel.: 86 27 6887-8461

their protumoral effects on lung adenocarcinoma, the role of TAMs in promoting tumors is supported by clinical studies that identified a correlation between high macrophage numbers in tumor tissues and poor patient prognoses.3-5 However, the mechanisms underlying TAM functional changes within tumor microenvironments remain unknown; one potential cause is the altered polarization of TAMs with associated changes in their activated phenotypes.6 There are at least two different subpopulations of activated macrophages within tumor microenvironments. The first type, known as classically activated macrophages (M1 macrophages), are activated by lipopolysaccharides (LPS) or by double signals from interferon (IFN)-c and tumor necrosis factor-a (TNF-a). M1-polarized macrophages exhibit an

INTRODUCTION Recently, compelling evidence has emerged that macrophages infiltrating the tumor microenvironment, also known as tumor-associated macrophages (TAMs), promote processes such as angiogenesis, lymphangiogenesis, tumor growth and progression in solid tumors.1,2 In addition to

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IL-12high, IL-23high, IL-10low phenotype and produce TNF-a and nitric oxide (NO); they are potent effector cells that kill microorganisms and tumor cells.7,8 The second type of macrophages is known as alternatively activated macrophages (M2 macrophages). Exposure to IL-4, IL-13, vitamin D3, glucocorticoids or transforming growth factor-b (TGF-b) decreases macrophage antigen-presenting capability and upregulates the expression of macrophage mannose receptors (MMR, also known as CD206), scavenger receptors (SR-A, also known as CD204), CD163, dectin-1 and DC-SIGN.9 M2polarized macrophages exhibit an IL-12low, IL-23low, IL-10high phenotype and play an important role in stroma formation, tissue repair, tumor growth, angiogenesis and immunosuppression.10,11 Although several studies throughout the past decade have suggested that TAMs exhibit a polarized M2 phenotype,12-14 limited reports describing the polarization of activated TAMs (such as M1 and M2) within tumor microenvironments have only recently been published.15-18 Lymphangiogenesis is considered to be an initial and necessary event for lymphatic and regional lymph node metastasis.19 A number of studies have demonstrated lymphatic microvessel density (LMVD) to be an independent prognostic factor in solid tumors.20-23 Additionally, several studies have shown that TAMs contribute to lymphangiogenesis and lymphatic vessel invasion in malignant tumors.24,25 However, as TAMs are heterogeneous in different tumor microenvironments and have multiple activated phenotypes, further investigations are necessary to clarify whether all TAMs or only M2-polarized TAMs correlate with lymphangiogenesis and poor prognosis in solid tumors. Here, we investigated the density of infiltrating M2polarized TAMs and the association between M2-polarized TAMs, lymphangiogenesis, and prognosis. We found that TAMs in lung adenocarcinoma exhibit a M2-polarized subtype and are associated with poor prognosis, perhaps as a result of lymphatic metastasis.

tuberculomas, six cases of inflammatory pseudotumors, five cases of hamartomas and three cases of lung cysts, were also included in this study. The clinicopathologic parameters of the lung adenocarcinoma patients are shown in Table 1.

ELISA Fresh human lung adenocarcinoma tissues and benign lung lesions were obtained from surgical specimens for the detection of Th1/Th2 cytokines by ELISA. Briefly, the specimens were rinsed three times in phosphate-buffered saline (PBS). Next, 1 g of each tissue sample was homogenized in 1 ml of PBS. Tissue homogenates were then centrifuged at 12,000 g for 1 min at 25 ˚C. After centrifugation, the supernatants were collected for further analysis. Cytokine production was quantified by cytokine-specific ELISA. Human IFN-c, IL-12, IL-4, and IL-10 (Biosource, Camarillo, CA, USA) ELISA kits were utilized; all procedures were performed according to the manufacturer’s instructions. Sample absorbances were determined using an ELISA plate reader (Dynatech, Chantilly, VA, USA).

Immunofluorescence staining For the simultaneous visualization of CD68 and MMR or CD68 and inducible nitric oxide synthase (iNOS) on the same tissue section, double-label immunofluorescence staining was performed. The tumor tissues were acetone-fixed at 4 ˚C and then cut into 10-mm frozen sections. After being rinsed three times in PBS, the slides were treated with 3% H2O2 to inactivate endogenous peroxidases and then with normal goat serum to block nonspecific binding sites. Next, slides were immunostained using a monoclonal mouse antihuman CD68 antibody (15100; Zhongshan Company, Beijing, China). Slides were also stained with rabbit anti-human MMR antibodies (15200; BioLegand, San Diego, CA, USA) or rabbit anti-human iNOS antibodies (15200; Santa Cruz Biotechnology, Santa Cruz, CA, USA). All slides were incubated overnight at 4 ˚C. Next, the slides were rinsed three times in PBS and incubated with TRITC-conjugated goat anti-mouse IgG for 30 min and then with FITCconjugated goat anti-rabbit IgG for 30 min. Nuclei were counterstained with 49-6-diamidino-2-phenylindole (DAPI) for 5 min. All slides were examined under a laser confocal microscope (Leica TCS SP5, Leica, German) fitted with TRITC (red fluorescence) and FITC (green fluorescence) filters. The excitation wavelength for FITC was 488 nm; the excitation wavelength for TRITC was 568 nm. Red CD68-positive fluorescence was located in the cytoplasm, and green MMR-positive fluorescence was located at the cell membrane and in the cytoplasm. However, green iNOS-positive fluorescence was only observed in the cytoplasm. If CD68 and MMR or CD68 and iNOS were coexpressed, the fluorescence was yellow. All nuclei were blue.

MATERIALS AND METHODS Ethics statement This study was approved by the Ethics Committee of Wuhan General Hospital, Guangzhou Command of the People’s Liberation Army. Written informed consent was obtained from all patients.

Patients and tissue samples A total of 65 patients with lung adenocarcinoma who underwent either lobectomy or pneumonectomy at Wuhan General Hospital were included in the study. The study group was composed of 38 men and 27 women with a mean age of 51.5 years (age range: 32-76 years). The patients underwent tumor resection from 2003 to 2006. None of the patients had received any preoperative chemotherapy or radiotherapy. The lesions of each patient were classified into stages according to the UICC 2010 pTNM classification scheme (7th edition); stage I, II, III, and IV lesions were present in 12, 27, 24, and 2 patients, respectively. Histologically, 16 tumors were graded as well-differentiated adenocarcinoma, 20 as moderately differentiated, and 29 as poorly differentiated. Lymph node metastasis occurred in 35 patients; no metastatic lymph nodes were observed in the remaining 30 patients. Follow-up visits were conducted and outcomes were recorded for all patients. A total of 20 patients with benign lung lesions, including six cases of

Immunohistochemistry To evaluate LMVD, D2-40 was used as the immunohistochemical marker for lymphatic endothelial cells (LECs).26 Resected tissue specimens were fixed in formalin, embedded in paraffin, and cut into 5-mm serial sections. Next, these sections were deparaffinized in xylene and rehydrated with solutions of graded alcohol and deionized water. The slides were immunostained with the mouse monoclonal antibody D2-40 (15200; Signet Laboratories, Dedham, MA, USA) overnight at 4 ˚C. Next, the slides were

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Table 1 - The relationships between TAM, M1- or M2-polarized TAM counts and clinicopathologic features ( x¡ s). Clinicopathologic features Gender Male Female Age (yrs.) $55 ,55 Differentiation Well-to-moderate Poor Lymph node metastasis Positive Negative pTNM staging I+II III+IV *

Cases

TAMs

p-value

M1-polarized TAMs

p-value

M2-polarized TAMs

p-value

38 27

104.19¡15.93 103.07¡15.74

0.712

21.15¡6.11 20.61¡5.73

0.948

83.04¡12.70 82.15¡11.26

0.903

34 31

105.45¡18.96 101.88¡14.11

0.895

21.29¡5.10 20.58¡6.26

0.705

84.84¡12.04 82.00¡10.61

0.681

36 29

103.17¡15.15 103.98¡11.67

0.984

20.63¡6.96 20.80¡6.16

0.157

82.23¡10.43 82.87¡10.17

0.645

35 30

120.44¡35.83 94.26¡14.52

0.042*

24.09¡4.56 18.85¡7.27

0.882

95.99¡15.60 70.26¡12.16

0.003*

39 26

96.38¡21.55 115.49¡30.94

0.037*

20.31¡4.71 23.45¡5.35

0.431

76.81¡10.32 92.05¡18.14

0.029*

Statistically significant.

were compared using a x2 test. The relationship between TAM counts and LMVD was assessed using the Spearman correlation test. On the basis of TAM or M2-polarized TAM count, patients were classified into two groups, and the overall survival rate was then compared between the high and low groups, respectively. Overall survival time was calculated from the date of surgery until death or, if the patient was still alive, until the last follow-up visit. Death from any cause was considered in the calculations of overall survival. Two overall survival rates were calculated by the Kaplan-Meier method and compared using the log-rank test. Each prognostic factor was evaluated with regard to survival in a multivariate analysis using a Cox proportional hazards regression model. A p-value of ,0.05 was considered statistically significant. All statistical analyses were performed with SPSS 13.0 (SPSS Inc., Chicago, IL, USA).

exposed to a biotinylated secondary antibody for 20 min and then treated with streptavidin peroxidase. For color development, the slides were stained with 3, 39diaminobenzidine (DAB). Hematoxylin and eosin (H&E) were used as a counterstain. A reddish-brown precipitate in the cytoplasm of LECs indicated a positive reaction. After the immunostained sections were scanned at low magnification (6100), the regions with the greatest numbers of distinctly stained lymphatic foci (hot spots) were determined simultaneously by two different observers. Then, two observers blinded to the tumor status or the stains used independently counted the slides for LMVD staining under 400x magnification (0.03 mm2 field) in three regions. An intratumoral compartment was identified as the area encompassing the cancerous tissue in the H&E sections. A peritumoral compartment was defined as a 1-mm-wide band surrounding the intratumoral compartment that included the edge of the tumor and just outside the tumor. Single immunoreactive endothelial cells and endothelial cell clusters separate from other microvessels were counted as vessels according to previously used procedures.24

RESULTS Th2 shift in lung adenocarcinoma To detect Th1/Th2 cytokines in the microenvironment of lung adenocarcinoma, we obtained fresh lung tumor tissues and benign lung lesions from surgical resections and assessed the expression of the Th1 cytokines IFN-c and IL12 and the Th2 cytokines IL-4 and IL-10 in tissue homogenates using a cytokine-specific ELISA. As shown in Figure 1, IFN-c and IL-12 expression was lower in fresh

Statistical analysis The numbers of M1- or M2-polarized TAMs and intratumoral LMVD and peritumoral LMVD are expressed as the mean¡SD. Statistical differences between the means were analyzed using the independent samples t-test. Rates

Figure 1 - Th1/Th2 cytokine expression in fresh lung adenocarcinoma tissue and benign lung lesion homogenates (pg/ml). Fresh lung adenocarcinoma homogenates contained significantly more IL-4 and IL-10 than benign lung lesion homogenates. * p,0.01.

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lung adenocarcinoma homogenates than in benign lung lesion homogenates. However, fresh lung adenocarcinoma samples contained significantly more Th2 cytokines than benign lung lesion samples. IL-10 was the predominant Th2 cytokine present in the lung adenocarcinoma samples.

The correlation of M2-polarized TAMs with peritumoral LMVD D2-40 positive lymph vessels were observed in each of the 65 lung adenocarcinoma examined. Lymphatic vessels with the characteristic irregular morphology, empty lumina without red blood cells, and thin endothelia were distinct and stained strongly with D2-40 when present. Vessels containing red blood cells did not stain positive with D2-40. In lung adenocarcinoma samples, D2-40-positive vessels were detected more frequently in the peritumoral stroma (Figure 3A) than in the intratumoral compartment (Figure 3B). The D2-40-positive peritumoral LMVD count was 11.56ยก10.73, which was higher than the D2-40-positive intratumoral LMVD count (2.96ยก1.15) (Figure 3C, p,0.01). Because peritumoral LMVD is a significant prognostic factor for non-small-cell lung cancer (NSCLC),27 we explored the correlation between M2-polarized TAMs and peritumoral LMVD. We divided all of the cases into two groups according to their CD68 grade (cutoff value = 102); the low-TAM group included 25 cases, and the high-TAM group was comprised of 40 cases. There was a significant difference in peritumoral LMVD between the two groups (p = 0.047). Next, we divided all of the cases into two groups according to their levels of CD68/MMR expression (cutoff values = 82): the low M2polarized TAM group (16 cases) and the high M2-polarized TAM group (49 cases). The peritumoral LMVD was significantly higher in the high M2-polarized TAM group than in the low M2-polarized TAM group (p = 0.009). Lastly, we divided all of the cases into two groups according to their levels of CD68/iNOS expression: the low M1-polarized TAM group (31 cases) and the high M1-polarized TAM group (34 cases). There was no significant difference in peritumoral LMVD between the two groups.

M2-polarized macrophages in lung adenocarcinoma To identify and quantify the infiltrated TAMs associated with the M1 or M2 phenotype, we utilized anti-iNOS, antiMMR, and anti-CD68 antibodies for immunofluorescence staining. CD68-positive macrophages were detected in varying concentrations in all 65 lung adenocarcinoma cases and in all 20 benign lung lesion samples. In the lung adenocarcinoma samples, CD68-positive TAMs were predominately located in the peritumoral stroma and tumor tissues, e.g., in the lumen of the lung adenocarcinoma; in particular, TAMs were observed along the invasive tumor front. MMR-positive signals predominately colocalized in macrophages, but iNOS expression was detected in both lung tumor cells and TAMs. M1-polarized TAMs were defined as CD68+iNOS+, whereas M2-polarized TAMs were distinguished by the expression of CD68 and MMR.15 M1and M2-polarized TAMs were detected in each of the 65 lung adenocarcinoma tissue samples. Overall, the percentage of CD68+MMR+ TAMs was 79.71ยก16.27% (Figure 2A), and the percentage of CD68+iNOS+ TAMs was 20.35ยก5.31% (Figure 2B). CD68 and iNOS coexpression was observed in 100% of the benign lung lesion samples; the percentage of CD68+iNOS+ TAMs was 89.5%.

The correlation of M2-polarized TAMs with clinicopathologic features Table 1 also depicts the correlations between TAMs, M1or M2-polarized TAMs and clinicopathologic features. TAM number and M2-polarized TAM number were significantly associated with p-TNM staging (p = 0.037, 0.029) and lymph node metastasis (p = 0.042, 0.003); however, these factors were not associated with gender, age or tumor differentiation. Interestingly, the number of M2-polarized TAMs was more strongly correlated with lymph node metastasis than the number of infiltrating CD68-positive TAMs. The number of M1-polarized TAMs failed to correlate with any of the clinicopathologic features included in this study.

The prognostic significance of M2-polarized TAMs To assess the prognostic significance of M2-polarized TAM infiltration, patients were first classified into two groups based on their TAM count; patients in each group were then further divided into two subgroups based on their M2-polarized TAM count. . We used median values of 102 and 82 as the cutoff values for the TAM and M2polarized TAM counts, respectively. The median survival times were 36 and 11 months for patients with TAM counts of #102 and .102, respectively. The five-year survival rates in the low TAM group and the high TAM group were 28.0%

Figure 2 - M2- and M1-polarized TAMs in lung adenocarcinoma. (A) The coexpression of CD68 and MMR in TAMs in human lung adenocarcinoma sections. (B) The coexpression of CD68 and iNOS in TAMs in human lung adenocarcinoma sections.

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Figure 3 - Peritumoral (A) and intratumoral LMVD (B) in lung adenocarcinoma samples. D2-40-positive staining was detected in lymph vessels by immunohistochemical staining (6400, SP method). The red arrow indicates the LMVD detected in lung tissues. (C) The LMVD of lung adenocarcinoma tissues and adjacent peritumoral tissues. * p,0.01.

rejection reactions.28 Human NSCLC cells express a type-2 cytokine pattern both in situ and in vitro, which may play an active immunoregulatory role in the lung cancer microenvironment.29 In patients with several malignant tumors, including lung tumors, a Th2 cytokine shift has been positively correlated with the degree of malignancy.28,30 In our study, IL-4 and IL-10 were detected in high amounts in lung adenocarcinoma tissue homogenates. Macrophages are plastic cells; for example, they can switch between an activated M1 state and an activated M2 state depending upon specific signals within their microenvironment. Because IL-4 is an activator of M2-polarized macrophages and IL-10 is known to function to promote the development of M2 macrophages from monocytes,31 the cytokine signals required for M2 activation were abundantly present within the microenvironment of the examined lung adenocarcinoma. Recently, Ohtaki et al. found that IL-10 was significantly correlated with the number of CD204-positive macrophages (M2 macrophages) within the stroma of lung adenocarcinoma.32 M2-polarized TAMs were detected predominantly in lung adenocarcinoma and were significantly associated with p-TNM staging and lymph node metastasis. In a series of reviews, Martinez et al. proposed that TAMs might polarize toward an M2 phenotype.14 Hagemann et al. demonstrated that when co-cultured, ovarian cancer cells switched co-cultured macrophages to an M2 phenotype similar to that of ovarian TAMs.15 Initially, macrophages in solid ovarian tumors were identified by CD68 expression; in serial sections, these macrophages also expressed SR-A. Using flow cytometric analysis, these authors observed that a large proportion of CD68(+) macrophages in samples of ovarian cancer ascites were also positive for MMR

and 7.5%, respectively. Figure 4A depicts the difference in the overall survival rates between the high-TAM-count group and the low-TAM-count group (p,0.001). The median survival times were 52 and 12 months for patients with M2-polarized TAM counts of #82 and .82, respectively. The five-year survival rates in the low M2-polarized TAM group and the high M2-polarized TAM group were 37.5% and 8.2%, respectively; the overall survival rate was significantly lower in the high M2-polarized TAM group than in the low M2-polarized TAM group (p,0.001) (Figure 4B).

Multivariate analysis TAM count, M1-polarized TAM count, M2-polarized TAM count, intratumoral LMVD, peritumoral LMVD, and other factors, including gender, age, tumor differentiation, lymph node metastasis, and P-TNM staging, were analyzed using Cox proportional hazards regression models in all of the lung adenocarcinoma patients. TAM count, M2-polarized TAM count, peritumoral LMVD, lymph node metastasis, and P-TNM staging were independent prognostic factors for overall survival (Table 2).

DISCUSSION Our data demonstrate that a shift toward the production of Th2 cytokines, a factor that induces alternative macrophage activation, occurs within the lung adenocarcinoma tumor microenvironment. Under normal conditions, Th1/Th2 cytokines in the body are in a dynamic balance. When a Th2 shift occurs, the immune system can become suppressed, making the host more susceptible to microbial infections, tumorigenesis and progression, allergic reactions, and graft

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Table 2 - Hazard ratios (HR), 95% confidence interval (CI) and p-value for the 65 patients with lung adenocarcinoma.

Factor

p-value

95% CI

Gender Age Differentiation Lymph node metastasis P-TNM staging TAM count M1-polarized TAMs count M2-polarized TAMs count Intratumoral LMVD Peritumoral LMVD

0.303 0.393 0.670 0.011* 0.039* 0.015* 0.860 0.031* 0.327 0.038*

0.574 1.317 1.175 2.778 3.021 3.602 0.881 4.280 1.189 1.073

0.200-1.649 0.700-2.480 0.558-2.476 1.260-6.123 1.055-8.649 1.279-10.142 0.214-3.628 1.146-15.984 0.841-1.679 1.004-1.147

Statistically significant.

more strongly correlated with lymph node metastasis than were CD68-positive TAMs. These data suggest the important role of M2-polarized TAMs in tumor progression and metastasis. Our study also showed that M2-polarized TAMs were associated with poor prognosis in lung adenocarcinoma, perhaps resulting from accelerated lymphangiogenesis and lymph node metastasis. Because previous studies concerning the relationship between TAM infiltration and clinical outcome in various cancers did not describe TAM heterogeneity and presented contradicting conclusions, the significance of macrophage infiltration in patient survival has been controversial. Recently, several studies have demonstrated that M2-polarized TAMs in gliomas,17 lung adenocarcinoma,32 intrahepatic cholangiocarcinomas,34 and angioimmunoblastic T-cell lymphoma adenocarcinoma35 are associated with tumor progression and prognosis. In the present study, we have also found M2polarized TAMs to be associated with poor prognosis in lung adenocarcinoma patients. The patients with high M2polarized TAM counts exhibited poor survival. Our multivariate Cox proportional hazards analysis indicated that M2-polarized TAM density was a useful prognostic marker for overall survival in lung adenocarcinoma cases. Two research papers previously reported that in patients with extended survival, macrophages infiltrating the tumor islets in NSCLC were predominantly of the M1 phenotype.36,37 These results appear to differ from our findings. However, in our research, the types of lung cancer involved were restricted to lung adenocarcinoma, not NSCLC, which may be primarily responsible for the conflicting findings. Metastatic tumors that are spread through the blood or lymphatic vessels occur in most forms of human cancer, with peritumoral lymphangiogenesis and regional lymph node metastasis often being the most important prognostic factors for carcinoma patients.38 In addition to tumor cells, TAMs and other inflammatory cells in the tumor stroma can express VEGF-C and VEGF-D and promote peritumoral lymphangiogenesis and lymph node metastasis.24,25 Previously, we reported that alternatively activated TAMs can induce peritumoral lymphangiogenesis in a murine Lewis lung adenocarcinoma model.39 In the present study, for the first time, a positive correlation between M2-polarized TAM count and peritumoral LMVD, but not intratumoral LMVD, was found in patients with lung adenocarcinoma. Interestingly, the

Figure 4 - Kaplan-Meier curves for overall survival by TAM count (A) and M2-polarized TAM count (B) in patients with lung adenocarcinoma. A shows the difference in the overall survival rate between the high and the low TAM count groups (p,0.001). B shows the difference in the overall survival rate between the high and the low M2-polarized TAM groups (p,0.001).

(62.73ยก16.27%) and SR-A (66.28ยก4.31%). Further, coculturing of human primary ovarian cancer cells with human macrophages increased macrophage MMR and SRA surface expression. In murine colon adenocarcinoma-38 and GL261 murine glioma, over 90% of tumor-infiltrating myelomonocytoid cells were of the CD11b+ F4/80+ monocyte/macrophage lineage. These cells also had a myeloidderived suppressor cell phenotype, as they suppressed the proliferation of activated splenic CD8+ T cells and exhibited a CD11b+ CD11c+ Gr-1low IL-4Ra+ phenotype. In addition, tumor-infiltrating myelomonocytoid cells expressed higher levels of MMR and lower levels of CXCL10, which are alternative and classical macrophage activation markers, respectively.33 In the present study, M1 and M2 macrophages coexisted in the tumor microenvironment, which showed the heterogeneity of TAMs in lung adenocarcinoma. However, a greater number of M2-polarized TAMs were detected than M1-polarized TAMs. Further, M2-polarized TAMs were

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M2-Polarized TAMs in lung adenocarcinoma Zhang B et al. 13. Sica A, Schioppa T, Mantovani A, Allavena P. Tumour-associated macrophages are a distinct M2 polarised population promoting tumour progression: potential targets of anti-cancer therapy. Eur J Cancer. 2006;42:717-27, doi: 10.1016/j.ejca.2006.01.003. 14. Martinez FO, Sica A, Mantovani A, Locati M. Macrophage activation and polarization. Front Biosci. 2008;13:453-61, doi: 10.2741/2692. 15. Hagemann T, Wilson J, Burke F, Kulbe H, Li NF, Plu¨ddemann A, et al. Ovarian cancer cells polarize macrophages toward a tumor-associated phenotype. J Immunol. 2006;176:5023-32. 16. Kawamura K, Komohara Y, Takaishi K, Katabuchi H, Takeya M. Detection of M2 macrophages and colony-stimulating factor 1 expression in serous and mucinous ovarian epithelial tumors. Pathol Int. 2009;59:300-5, doi: 10.1111/j.1440-1827.2009.02369.x. 17. Komohara Y, Ohnishi K, Kuratsu J, Takeya M. Possible involvement of the M2 anti-inflammatory macrophage phenotype in growth of human gliomas. J Pathol. 2008;216:15-24, doi: 10.1002/path.2370. 18. Niino D, Komohara Y, Murayama T, Aoki R, Kimura Y, Hashikawa K, et al. Ratio of M2 macrophage expression is closely associated with poor prognosis for Angioimmunoblastic T-cell lymphoma (AITL). Pathol Int. 2010;60:278-83, doi: 10.1111/j.1440-1827.2010.02514.x. 19. Alitalo K, Tammela T, Petrova TV. Lymphangiogenesis in development and human disease. Nature. 2005;438:946-53, doi: 10.1038/nature 04480. 20. Birner P, Schindl M, Obermair A, Breitenecker G, Kowalski H, Oberhuber G. Lymphatic microvessel density as a novel prognostic factor in early-stage invasive cervical cancer. Int J Cancer. 2001;95:29-33, doi: 10.1002/1097-0215(20010120)95:1,29::AID-IJC1005.3.0.CO;2-W. 21. Kim HS, Sung W, Lee S, Chang SG, Park YK. Lymphatic vessel densities of lymph node-negative prostate adenocarcinoma in Korea. 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VEGF-C expressing tumor-associated macrophages in lymph node positive breast cancer: impact on lymphangiogenesis and survival. Surgery. 2006;139:839-46, doi: 10.1016/j.surg.2005.12.008. 26. Kahn HJ, Marks A. A new monoclonal antibody, D2-40, for detection of lymphatic invasion in primary tumors. Lab Invest. 2002;82:1255-7. 27. Anagnostou VK, Tiniakos DG, Fotinou M, Achimastos A, Syrigos KN. Multiplexed analysis of angiogenesis and lymphangiogenesis factors predicts outcome for non-small cell lung cancer patients. Virchows Arch. 2011;458:331-40, doi: 10.1007/s00428-010-1015-4. 28. Kidd P. Th1/Th2 balance: the hypothesis, its limitations, and implications for health and disease. Altern Med Rev. 2003;8:223-246. 29. Huang M, Wang J, Lee P, Sharma S, Mao JT, Meissner H, et al. Human non-small cell lung cancer cells express a type 2 cytokine pattern. Cancer Res. 1995;55:3847-53. 30. Ito N, Nakamura H, Tanaka Y, Ohgi S. Lung carcinoma: analysis of T helper type 1 and 2 cells and T cytotoxic type 1 and 2 cells by intracellular cytokine detection with flow cytometry. Cancer. 1999;85:2359-67, doi: 10. 1002/(SICI)1097-0142(19990601)85:11,2359::AID-CNCR10.3.0.CO;2-A. 31. Mantovani A, Sica A, Sozzani S, Allavena P, Vecchi A, Locati M. The chemokine system in diverse forms of macrophage activation and polarization. Trends Immunol. 2004;25:677-86, doi: 10.1016/j.it.2004.09. 015. 32. Ohtaki Y, Ishii G, Nagai K, Ashimine S, Kuwata T, Hishida T, et al. Stromal macrophage expressing CD204 is associated with tumor aggressiveness in lung adenocarcinoma. J Thorac Oncol. 2010;5:150715, doi: 10.1097/JTO.0b013e3181eba692. 33. Umemura N, Saio M, Suwa T, Kitoh Y, Bai J, Nonaka K, et al. Tumorinfiltrating myeloid-derived suppressor cells are pleiotropic-inflamed monocytes/macrophages that bear M1- and M2-type characteristics. J Leukoc Biol. 2008;83:1136-44, doi: 10.1189/jlb.0907611. 34. Hasita H, Komohara Y, Okabe H, Ashimine S, Kuwata T, Hishida T, et al. Significance of alternatively activated macrophages in patients with intrahepatic cholangiocarcinoma. Cancer Sci. 2010;101:1913-9, doi: 10. 1111/j.1349-7006.2010.01614.x. 35. Niino D, Komohara Y, Murayama T, Aoki R, Kimura Y, Hashikawa K, et al. Ratio of M2 macrophage expression is closely associated with poor prognosis for Angioimmunoblastic T-cell lymphoma (AITL). Pathol Int. 2010;60:278-83, doi: 10.1111/j.1440-1827.2010.02514.x. 36. Ohri CM, Shikotra A, Green RH, Waller DA, Bradding P. Macrophages within NSCLC tumour islets are predominantly of a cytotoxic M1

LMVD in cases with high numbers of infiltrating M2-polarized TAMs was significantly higher than in cases with low numbers of M2-polarized TAMs. These data suggest that in lung adenocarcinoma patients, M2-polarized TAM infiltration is correlated with poor survival, likely as a result of accelerated lymphangiogenesis and lymph node metastasis. Our data indicate that M2-polarized TAMs in lung adenocarcinoma are a predictor of poor prognosis as a result of their promotion of lymphangiogenesis and lymphatic metastasis. Because M2-polarized TAMs appear to play an important role in peritumoral lymphangiogenesis and decreased survival in lung adenocarcinoma patients, they represent a potential target for therapeutic intervention. It is possible that the use of exogenous agents to switch the phenotype of the TAMs from M2 to M1 during tumor progression could decrease lymphatic metastasis and prolong lung adenocarcinoma patient survival.

ACKNOWLEDGEMENTS We thank Manli Qi (Department of Pathology, Wuhan General Hospital of Guangzhou Command, People’s Liberation Army, Wuhan, China) for her excellent technical assistance. This study was supported by the Natural Science Foundation of Hubei Province, China (Number 2010CDB09204).

AUTHOR CONTRIBUTIONS Zhang B drafted the first manuscript and performed the experiments. Zhang Y performed the experiments, analyzed the data, prepared the figures, and performed the statistical analysis. Yao G, Gao J, Yang B, Rao Z and Gao J were responsible for the experiments. Zhang B and Yao G contributed equally to this work.

REFERENCES 1. Porta C, Subhra Kumar B, Larghi P, Rubino L, Mancino A, Sica A. Tumor promotion by tumor-associated macrophages. Adv Exp Med Biol. 2007;604:67-86, doi: 10.1007/978-0-387-69116-9_5. 2. Mantovani A. La mala educacio´n of tumor-associated macrophages: diverse pathways and new players. Cancer Cell. 2010;17:111-2, doi: 10. 1016/j.ccr.2010.01.019. 3. Ryder M, Ghossein RA, Ricarte-Filho JC, Knauf JA, Fagin JA. Increased density of tumor-associated macrophages is associated with decreased survival in advanced thyroid cancer. Endocr Relat Cancer. 2008;15:106974, doi: 10.1677/ERC-08-0036. 4. Lee CH, Espinosa I, Vrijaldenhoven S, Subramanian S, Montgomery KD, Zhu S, et al. Prognostic significance of macrophage infiltration in leiomyosarcomas. Clin Cancer Res. 2008;14:1423-30, doi: 10.1158/10780432.CCR-07-1712. 5. Shieh YS, Hung YJ, Hsieh CB, Chen JS, Chou KC, Liu SY. Tumorassociated macrophage correlated with angiogenesis and progression of mucoepidermoid carcinoma of salivary glands. Ann Surg Oncol. 2009;16:751-60, doi: 10.1245/s10434-008-0259-6. 6. Lewis CE, Pollard JW. Distinct role of macrophages in different tumor microenvironments. Cancer Res. 2006;66:605-12, doi: 10.1158/0008-5472. CAN-05-4005. 7. Mosser DM. The many faces of macrophage activation. J Leukoc Biol. 2003;73:209-12, doi: 10.1189/jlb.0602325 8. Edwards JP, Zhang X, Frauwirth KA, Mosser DM. Biochemical and functional characterization of three activated macrophage populations. J Leukoc Biol. 2006;80:1298-307, doi: 10.1189/jlb.0406249. 9. Stein M, Keshav S, Harris N, Gordon S. Interleukin 4 potently enhances murine macrophage mannose receptor activity: a marker of alternative immunologic macrophage activation. J Exp Med. 1992;176:287-92, doi: 10.1084/jem.176.1.287. 10. Gordon S. Alternative activation of macrophages. Nat Rev Immunol. 2003;3:23-35, doi: 10.1038/nri978. 11. Gratchev A, Kzhyshkowska J, Ko¨the K, Muller-Molinet I, Kannookadan S, Utikal J, et al. Mphi1 and Mphi2 can be re-polarized by Th2 or Th1 cytokines, respectively, and respond to exogenous danger signals. Immunobiology. 2006;211:473-86, doi: 10.1016/j.imbio.2006.05.017. 12. Mantovani A, Allavena P, Sica A. Tumour-associated macrophages as a prototypic type II polarised phagocyte population: role in tumour progression. Eur J Cancer. 2004;40:1660-7, doi: 10.1016/j.ejca.2004.03.016.

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38. Pepper MS. Lymphangiogenesis and tumor metastasis: myth or reality? Clin Cancer Res. 2001;7:462-8. 39. Zhang B, Wang J, Gao J, Guo Y, Chen X, Wang B, et al. Alternatively activated RAW264.7 macrophages enhance tumor lymphangiogenesis in mouse lung adenocarcinoma. J Cell Biochem. 2009;107:134-43, doi: 10.1002/jcb.22110.

phenotype associated with extended survival. Eur Respir J. 2009;33:11826, doi: 10.1183/09031936.00065708. 37. Ma J, Liu L, Che G, Yu N, Dai F, You Z. The M1 form of tumor-associated macrophages in non-small cell lung cancer is positively associated with survival time. BMC Cancer. 2010;10:112, doi: 10.1186/1471-2407-10-112.

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DOI:10.1590/S1807-59322011001100007

CLINICAL SCIENCE

OBJECTIVE: Obstructive sleep apnea is characterized by increased upper airway collapsibility during sleep. The present study investigated the use of the negative expiratory pressure test as a method to rule out obstructive sleep apnea. METHODS: Flow limitation was evaluated in 155 subjects. All subjects underwent a diurnal negative expiratory pressure test and a nocturnal sleep study. The severity of sleep apnea was determined based on the apneahypopnea index. Flow limitation was assessed by computing the exhaled volume at 0.2, 0.5, and 1.0 s (V0.2, V0.5, and V1.0, respectively) during the application of a negative expiratory pressure and expressed as a percentage of the previous exhaled volume. Receiver-operating characteristic curves were constructed to identify the optimal threshold volume at 0.2, 0.5, and 1.0 s for obstructive sleep apnea detection. RESULTS: Mean expiratory volumes at 0.2 and 0.5 s were statistically higher (p,0.01) in healthy subjects than in all obstructive sleep apneic groups. Increasing disease severity was associated with lower expiratory volumes. The V0.2 (%) predictive parameters for the detection of sleep apnea were sensitivity (81.1%), specificity (93.1%), PPV (98.1%), and NPV (52.9%). Sensitivity and NPV were 96.9% and 93.2%, respectively, for moderate-to-severe obstructive sleep apnea, and both were 100% for severe obstructive sleep apnea. CONCLUSION: Flow limitation measurement by V0.2 (%) during wakefulness may be a very reliable method to identify obstructive sleep apnea when the test is positive and could reliably exclude moderate and severe obstructive sleep apnea when the test is negative. The negative expiratory pressure test appears to be a useful screening test for suspected obstructive sleep apnea. KEYWORDS: Sleep; Upper Airways; Respiratory Measurement; Screening Test. Romano S, Salvaggio A, Lo Bue A, Marrone O, Insalaco G. A negative expiratory pressure test during wakefulness for evaluating the risk of obstructive sleep apnea in patients referred for sleep studies. Clinics. 2011;66(11):1887-1894. Received for publication on May 9, 2011; First review completed on May 30, 2011; Accepted for publication on July 13, 2011 E-mail: insalaco@ibim.cnr.it Tel.: (+39) 0916809110

Studies have estimated that OSA, which is defined based on an apnea-hypopnea index of 5 and excessive daytime somnolence, may affect at least 2–4% of the middle-aged general population.9 Interestingly, prevalence estimates are much higher when individuals are divided based on demographic variables, such as age, sex, and body mass index (BMI).10 In certain subgroups of the population, such as obese subjects10-11 and professional drivers,12 the prevalence of OSA may be higher than in the general population. OSA diagnostic procedures are expensive and time consuming, and studies have estimated that nearly 80% of men and 93% of women with moderate-to-severe sleep apnea are undiagnosed.13 Although effective screening procedures could help in evaluating OSA risk, current screening criteria remain unsatisfactory. The identification of simple, noninvasive, predictive, and reliable tools to detect OSA would be useful in several fields.

INTRODUCTION Obstructive sleep apnea (OSA) is a risk factor for serious health problems, such as cardiovascular disease1 and postoperative complications,2 and could have consequences for the safety of subjects because of decreased physical abilities.3-5 The under-recognition of OSA may result in important health and social costs6 and reduce the quality of life of affected individuals.7 Obstructive sleep apnea is a disorder characterized by repetitive episodes of upper airway closure during sleep, and increased upper airway collapsibility is one of its main determinants.8

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Negative expiratory pressure (NEP) is a noninvasive tool that was initially designed to evaluate flow limitation in patients with obstructive lung disease; however, Koulouris and coworkers have suggested that the flow limitation induced by the NEP application may also reflect upper airway collapsibility.14 NEP has been applied in subjects with obesity and/or OSA15-22 to evaluate whether NEP responses could predict OSA, and the results have been variable. Some studies15-20 have found that NEP flowvolume curves and quantitative indices during wakefulness are useful in the detection of upper airway collapsibility, whereas other studies21-22 have showed that the NEP technique appears to have limited usefulness as a clinical tool for the routine screening of OSA patients during wakefulness. The difference between these results could be related to the various flow-limitation measurements that were applied. Effective screening procedures could help to distinguish subjects with different probabilities of being affected by OSA. We have previously demonstrated the effectiveness of the NEP test in identifying increased upper airway collapsibility during tidal expiration in severe OSA patients.23 The aim of the present study was to evaluate whether the flow limitation induced by NEP during wakefulness could be a valuable method to detect subjects with mild or moderate OSA.

NEP test NEP tests were performed in awake subjects at -10 cm H2O during the morning before the portable home monitor testing session. The NEP tests were performed while patients were in the sitting position with the neck in a neutral position and quietly breathing. All subjects were wearing a nose clip. The NEP test was performed as previously described.23 A Super Air Amplifier (Exair model 120021 Cincinnati, Ohio, USA) was able to generate a range of negative pressures as compressed air was delivered through the device. Air was supplied by a tank of compressed air and controlled using an electrically operated solenoid valve (Norgren Ltd model 95004, Warwickshire, UK). The solenoid was automatically activated during early tidal expiration and kept open for 2 s by software control. The Super Air Amplifier device was placed at the distal end of a pneumotachograph (Hans Rudolph model 3830; Kansas City, MO, USA), and mouth pressure was measured using a pressure transducer (PCLA0050, Sensortechnics GmbH, Puchheim, D). The signals recorded by the pneumotachograph and the mouth pressure transducer were filtered and digitized at 200 Hz. The volume was obtained by numerically integrating the airflow signal. Airflow and mouth pressure were displayed in real time on the computer screen and recorded for subsequent analysis. Signal analysis and solenoid valve control were performed using software developed in our laboratory using LabVIEW 8.2 (National Instruments; Austin, TX, USA). The NEP of –10 cm H2O was set by occluding the pneumotachograph with a stopper and adjusting the flow of the compressed air to the air amplifier. NEP application during tidal expiration could produce a sudden and variable flow reduction that was dependent on upper airway narrowing. Flow limitation was evaluated by measuring the expired volume at 0.2, 0.5, and 1.0 s (V0.2, V0.5, and V1.0, respectively) immediately after NEP application (Figure 1). We waited for at least four regular breaths (as observed by visual inspection of the flow signal on the computer screen) between consecutive NEP applications. All subjects underwent seven NEP applications, and a minimum of four valid measures of expiratory volumes at 0.2, 0.5, and 1.0 s were averaged for each subject. These values were expressed as the percent of the mean expiratory volumes of the three breaths preceding each NEP application. Measured volumes at 0.2, 0.5, and 1.0 s were only accepted under regular breathing conditions (i.e., the differences between inspiratory and expiratory volumes for each of the three breaths before each NEP application were less than 10%).

MATERIALS AND METHODS The present study included 155 subjects (130 males and 25 females) who were referred to our sleep laboratory (we evaluated spirometry to exclude subjects with bronchial obstruction from the study). Patients with major craniofacial or upper airway abnormalities and/or acute or known chronic pulmonary or neuromuscular disease were excluded from the study. The mean age of the subjects was 51¡11 years, and the mean BMI was 32.6¡7.1 kg/m2 (the BMI was $30 for 58.7% of the subjects). Pulmonary function tests were performed during the day using a MEDGRAPHICS E´lite plethysmograph (MEDGRAPHICS Corporation; St. Paul, Minnesota, USA). Each patient gave informed consent, and the study protocol was approved by the local ethical committee. All subjects underwent a diurnal NEP test and nocturnal monitoring using a portable cardio-respiratory system.24 Nocturnal monitoring was performed using a portable computerized system (Somte` Compumedics Inc.; Abbotsford, VIC, Australia). The recorded signals were airflow, snoring, thoracic effort, abdominal effort, limb movement, body position, electrocardiogram, arterial oxygen saturation, pulse rate, and pulse waveform. All were recorded for at least 6 h. Polygraphic recordings were analyzed manually by scorers who were blinded to the results of the NEP test. Apneas were defined based on the lack of airflow or a .90% reduction in the airflow signal for at least 10 s. Hypopneas were defined based on a discernible drop in flow by $30% of the baseline value for a period lasting at least 10 s, followed by a $4% drop in SaO2.25 The apnea-hypopnea index (AHI) was calculated as the total number of apneas and hypopneas per hour of estimated total sleep time. Subjects with an AHI $5 were classified as having OSA, and subjects with an AHI ,5 were classified as healthy. OSA severity was determined based on the AHI as mild (5# AHI ,15), moderate (15# AHI #30), or severe (AHI .30).

Statistical analysis Data are reported as means¡SD. A p-value of ,0.05 was considered significant. Differences between healthy and OSA subjects with different severity were evaluated using the Tukey-Kramer test. Logistic regression for the analysis of continuous factors with categorical responses was applied. Receiver-operating characteristic (ROC) curves were constructed to determine the optimal V0.2 (%), V0.5 (%), and V1.0 (%) threshold values for OSA detection. We also calculated sensitivity, specificity, and positive (PPV) and negative predictive values (NPV) at various levels of the measured volumes. A ROC curve was also constructed

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Figure 1 - Negative expiratory pressure (NEP) application during tidal expiration. Airflow and mouth pressure (Pm) during tidal respiration and NEP application. The hatched areas under the flow curve measure the expired volume at 0.2 s (V0.2), 0.5 s (V0.5), and 1.0 s (V1.0) after NEP application. Insp: inspiration; Exp: expiration.

AHI and V0.2 (%), V0.5 (%), and V1.0 (%) are shown in Figure 2. Figure 3 shows the mean expiratory volumes at V0.2 (%), V0.5 (%), and V1.0 (%) for the healthy subjects and the subjects in the three different OSA severity groups. Exhaled V0.2 (%) and V0.5 (%) volumes were significantly lower (p,0.01) in all OSA severity groups, than in healthy subjects. The exhaled V1.0 (%) volume, however, was only significant for the healthy subjects compared with the moderate and severe OSA subjects. The expiratory volumes were associated with disease severity (i.e., the expiratory volume decreased as disease severity increased), and the highest volumes were found in the healthy subjects. A ROC curve analysis was applied to evaluate the predictive effectiveness of V0.2 (%), V0.5 (%), and V1.0 (%) for OSA subjects with AHI $5, AHI $15 and AHI .30. The predictive parameters for V0.2 (%), V0.5 (%), and V1.0 (%) are reported in Table 2. Interestingly, V1.0 (%) was the least

for BMI. Statistical analysis was performed using the commercial software JMP 8.0.1 (SAS Institute Inc., Cary, NC, USA).

RESULTS All subjects had normal forced expiratory flow volume loops (FVC 99¡16%, FEV1 99¡16%, and FEF25-75 88¡27% of the predicted values, respectively). The entire spectrum of OSA severity was represented in the study population. Nocturnal monitoring showed a mean AHI of 32¡28 events/h in the study population. Table 1 shows the anthropometric and respiratory characteristics of the healthy and OSA subjects with different degrees of severity. The mean NEP test results for V0.2 (%), V0.5 (%), and V1.0 (%) in the study population were 19.4¡12.2%, 56.4¡29.9% and 100.4¡46.0%, respectively. The relationships between

Table 1 - Anthropometric and respiratory data of the subjects arranged by AHI. Variables

AHI ,5/h (n = 29)

5# AHI ,15/h (n = 28)

15# AHI #30/h (n = 34)

AHI .30/h (n = 64)

Age (y) BMI (kg/m2) Neck circumference (cm) AHI (events/h) FVC (% predicted) FEV1 (% predicted) FEF25-75 (% predicted)

41¡12 27.5¡3.5 39.5¡2.8 2.0¡1.4 100¡14 105¡15 96¡23

56¡9* 29.9¡5.7 40.2¡3.0 9.1¡2.8* 100¡14 100¡15 86¡27

53¡10* 34.5¡7.71 42.6¡3.61 21.4¡3.8* 99¡15 99¡15 88¡30

53¡11* 35.1¡7.1* 43.7¡4.1* 61.1¡18.4* 96¡18 96¡8 86¡28

BMI = body mass index; AHI = apnea-hypopnea index; FVC = forced vital capacity; FEV1 = forced expiratory volume in one second; FEF25-75 = forced expiratory flow 25-75%. Values are presented as the means¡SD. * p,0.0001, 1 p,0.005; healthy vs. OSA severity groups.

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(p,0.007) than V0.2 (%) in discriminating OSA subjects. The areas under the ROC curves for BMI and V0.2 (%) were 0.79 (95% CI: 0.72 to 0.85) and 0.91 (95% CI: 0.85 to 0.95), respectively. Figure 4 illustrates the relationship between AHI and V0.2 (%) graphically. The optimal cutoff point to discriminate subjects with an AHI .30 was 23%, and only five subjects with values of V0.2 lower than 23% (positive for OSA) were healthy. No subject with a V0.2 value higher than 23% (negative for OSA) had severe OSA. Only three subjects with a V0.2 value higher than 23% had moderate OSA, and 15 subjects had mild OSA (1.9 and 9.6%, respectively).

DISCUSSION The present study verified the efficacy of the flow limitation induced by NEP and evaluated by V0.2 (%), V0.5 (%) and V1.0 (%) as a screening tool to detect OSA risk. Our results indicated that the V0.2 (%) parameter is more sensitive in detecting patients with moderate and severe OSA. V0.2 (%) identified subjects with moderate and severe OSA with very high sensitivity and identified healthy subjects with very high specificity. When the NEP test was negative for OSA, there was a high probability that the subject did not have moderate or severe OSA. When the test was positive, most OSA subjects (AHI $5) were identified. For a positive test, the predictive value to identify OSA was very high, but the predictive value for distinguishing severity was lower. Conversely, a negative test was predictive of subjects who did not have moderate and severe OSA. Thus, the measurement of flow limitation by V0.2 (%) induced by NEP is a valuable predictor for OSA and can reliably distinguish between OSA and healthy subjects. Obesity and increased upper airway collapsibility are considered two of the most important determinants of OSA. Previous studies regarding obese male subjects have shown that at least 40% are affected by OSA.26-27 Because obesity is considered to be a major cause of OSA, we compared the ROC curve for V0.2 (%) induced by NEP to the ROC curve for BMI to identify OSA subjects. The V0.2 (%) curve had better discriminatory power than BMI, which agreed with the supposition that collapsibility is the main mechanism of upper airway obstruction during sleep. It is important to heighten public awareness regarding the potential hazards and health risks that surround untreated sleep apnea.1-7,28-34 In addition, certain groups of individuals, such as commercial drivers, need to realize that OSA is dangerous to them and to others. Physicians and the general population are widely aware of the potentially deleterious effects of OSA on an individual patientâ&#x20AC;&#x2122;s health and wellbeing, and there is even increasing awareness among public administrators of the traffic hazards and accidents at work that are linked to untreated OSA. Many patients who present for surgical procedures may have undiagnosed OSA, despite an improved awareness and increased frequency of diagnosis.29-31 Untreated OSA patients are known to have higher incidences of difficult intubation, postoperative complications, increased intensive care unit admissions, and longer hospital stays.32 Several case reports have documented an increase in the incidence of postoperative complications and deaths among patients suspected of having OSA.33-34 In previous studies, 24% of patients with OSA had significant postoperative complications, compared with 9% of the patients in the control

Figure 2 - The relationships between AHI and V0.2 (%), V0.5 (%), and V1.0 (%).

effective volume parameter in distinguishing between healthy and OSA subjects. V0.2 (%) identified subjects with AHI $15 and .30 (i.e., moderate-to-severe OSA) with very high sensitivity and identified subjects with an AHI ,5 (i.e., healthy subjects) with very high specificity. A negative test result indicated 100% probability of having an AHI not exceeding 30, 93.2% probability of having an AHI not exceeding 15, but an equal probability of being healthy or having mild OSA. A positive test result indicated 98% probability of having an AHI $5, 84.8% probability of having an AHI $15 and 56.1% probability of having an AHI .30. Thus, the measurement of flow limitation using V0.2 (%), which was determined using NEP, was an effective predictor of OSA. The ROC curve analysis applied to the BMI measurements showed that BMI was significantly less reliable

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Figure 3 - Expiratory volumes of each subject at 0.2, 0.5, and 1.0 s expressed as the % of the mean expiratory volume of the three breaths preceding NEP application for healthy subjects and subjects from the 3 OSA severity groups. The means and SD are shown. NS: not significant, *p,0.0001, 1p,0.003, #p,0.01; healthy vs. OSA severity groups.

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group.29,30 Identifying patients at risk for OSA is the first step in preventing postoperative complications due to OSA. Polysomnography is the gold standard for diagnosis of OSA, but increasing awareness of the importance of identifying patients with OSA has increased the waiting lists for diagnosis and proper treatment. Nevertheless, it would take several years to satisfy the current requirement for polysomnography in the general population with existing resources.35 The use of portable monitoring devices for the diagnosis of OSA would probably not be sufficient to overcome the growing demand for diagnostic testing.36 Although sleep recordings are mandatory for OSA diagnosis, it would be useful to identify simple, reliable and inexpensive screening methods for physicians to detect subjects who are more likely to be at risk for upper airway obstruction. A highly sensitive and positive predictive screening tool with an acceptable specificity and negative predictive ability is a basic requirement when screening patients for a disease that has an important impact on health. OSA screening tests have demonstrated considerable variability in predictive ability, which depends on the screening tool, the patient population, and the OSA severity. An OSA screening questionnaire that was tested in patients from sleep centers resulted in 70% to 93% sensitivity.37-41 Ross et al.42 published a meta-analysis of screening tests for OSA in 2000 and found that a lack of discriminatory analysis makes it difficult to recommend an evidence-based choice of tests for preoperative screening. In addition, the Berlin questionnaire41 has shown varied results that depend on the patient population studied. For example, the sensitivity was 86% in primary care patients40 and 57-68% in sleep laboratory patients.43

Previous studies have used different methods to evaluate the flow limitation induced by NEP during wakefulness and assess upper airway collapsibility in OSA and healthy subjects.14-22 Some studies have applied methods that were the same as those adopted for the evaluation of flow limitation in patients with bronchial obstruction,14 whereas others have introduced new methods to evaluate upper airway obstruction.15-22 The various methods have demonstrated that NEP can play a role in the identification of upper airway flow limitation without reaching entirely satisfactory discriminatory levels. Ferretti et al.21 evaluated flow limitation by measuring the expiratory volume at 0.5 and 1.0 s after NEP application and concluded that the NEP test appears to have a limited usefulness as a clinical tool for routine screening. In addition, Van Meerhaeghe et al.17 expressed the expiratory flow limitation as the percentage of the expired tidal volume over which the NEP-induced flow did not exceed the spontaneous flow, and they concluded that NEP may be useful in predicting OSA with AHI $15 in a clinic-based population (the sensitivity was 81.9% and the specificity was 69.1%). Tamisier et al.20 used the ratio between the areas under the curves of the NEP flow-volume loop and the spontaneous flow-volume loop and reported that the quantitative index was higher in healthy subjects than in patients with any type of breathing-related sleep disorder (the predictive positive value was 96.6% and the predictive negative value was 76.9%). Subjects with breathing-related sleep disorders and healthy subjects exhibited significant differences, there was a trend toward a lower quantitative index in patients with severe OSA who exhibited greater expression of a collapsible upper airway. Subjects with mild OSA, however, were not included in this study. In our study, we measured the expiratory volume

Table 2 - Receiver-operating characteristic curve analysis for NEP volume at V0.2, V0.5, and V1.0.

AHI $5/h Optimal cutoff point Sensitivity, % Specificity, % PPV, % NPV, % Area under the ROC curve AHI $15/h Optimal cutoff point Sensitivity, % Specificity, % PPV, % NPV, % Area under the ROC curve AHI .30/h Optimal cutoff point Sensitivity, % Specificity, % PPV, % NPV, % Area under the ROC curve

V0.2

V0.5

22.2 81.1 (73.2-87.5) 93.1 (77.2-99.2) 98.1 (95.5-100) 52.9 (39.2-66.6) 0.91 (0.85-0.95)

71.0 84.3 (76.7-90.1) 89.7 (72.6-97.8) 97.3 (94.3-100) 56.5 (42.2-70.8) 0.90 (0.84-0.94)

84.1 82.8 95.5 54.5 0.88

127.4 (76.6-90.0) (64.2-84.2) (91.7-99.3) (39.8-69.2) (0.81-0.92)

22.9 96.9 (91.3-99.4) 70.7 (57.3-81.9) 84.8 (90.7-98.9) 93.2 (85.8-100) 0.90 (0.84-0.94)

93.9 74.1 86.0 87.8 0.90

69.8 (87.1-97.7) (61.0-84.7) (79.4-82.5) (78.6-97.0) (0.85-0.95)

82.6 79.0 86.2 72.6 0.90

108.7 (73.7-89.6) (66.1-88.6) (79.2-93.2) (61.5-83.7) (0.84-0.94)

23.0 100 (94.4-100) 45.6 (35.2-56.4) 56.1 (47.0-65.2) 100 (100-100) 0.78 (0.71-0.84)

69.1 95.3 (86.9-99.0) 51.1 (40.4-61.7) 57.5 (48.1-66.9) 94.0 (87.4-100) 0.79 (0.72-0.85)

70.3 71.4 70.3 71.7 0.78

93.8 (57.6-81.1) (61.0-80.4) (59.1-81.5) (62.5-80.9) (0.70-0.84)

Data are presented as the means (95% confidence interval). AHI = Apnea-hypopnea index; PPV = positive predictive value; NPV = negative predictive value; ROC = receiver operating characteristic; V0.2 = volume for 200 ms after NEP application; V0.5 = volume for 500 ms after NEP application; V1.0 = volume for 1 s after NEP application.

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NEP test to evaluate OSA risk Romano S et al.

Figure 4 - The relationship between AHI and V0.2 (%) with the optimal cutoff point (dashed vertical line) to discriminate subjects with AHI.30.

(expressed as the percent of the mean expiratory volume of the three breaths preceding NEP application) at the very beginning of expiration (V0.2), at 0.5 s (V0.5), and at 1.0 s (V1.0) in a large sample of subjects and showed that V0.2 and V0.5 were predictive of OSA (V1.0 was less useful). The NEP test may provide an objective assessment to identify patients at high risk for OSA and could be widely applied to reduce socioeconomic impact of this disease. This investigation was limited by the various anthropometric characteristics of the different groups. In addition, we only enrolled subjects who had attended a sleep laboratory, and these subjects may not represent the general population. Importantly, the present study included subjects who represented the entire spectrum of disease severity, which prevented the exaggeration of diagnostic accuracy that could occur when a diagnostic test is evaluated in a sample with a limited severity spectrum.44 In conclusion, the evaluation of flow limitation during wakefulness using the NEP technique was predictive of OSA. The NEP test was able to reliably discriminate among subjects who had an AHI $5 and subjects who had an AHI ,15. The NEP test could be adopted as a screening test for the evaluation of suspected OSA patients because it appears to be a very reliable diurnal test that can objectively predict OSA. The NEP test may be even more useful when combined with anamnestic data, such as the symptoms of snoring and hypersomnolence. Further studies should be performed to evaluate whether OSA can be screened using NEP in the general population. The measurement of flow limitation based on V0.2 (%) during wakefulness may be a very reliable method to identify healthy subjects when the test is negative and subjects with OSA when the test is positive. Further data are required to determine the operating characteristics of the technique for OSA screening in patients who are awake.

ACKNOWLEDGEMENTS The authors are indebted to Dr. Pietro Abate and Mr. Giovanni Sciortino for their technical support. This study was supported by the Italian National Research Council grant number ME.P01.006.

AUTHOR CONTRIBUTIONS Romano S and Insalaco G were responsible for the design and coordination of the study, statistical analysis, preparation and approval of the final version of the manuscript. Salvaggio A was responsible for the design of the study, recruitment and clinical care of subjects, compilation of database, revision and approval of the final version of the manuscript. Lo Bue A was responsible for the recruitment of subjects, compilation of database, reading and approval of the final version of manuscript. Marrone O was responsible for the recruitment, revision and approval of the final version of the manuscript.

REFERENCES 1. Somers VK, White DP, Amin R, Abraham WT, Costa F, Culebras A, et al. Sleep apnea and cardiovascular disease. Circulation. 2008;118: 1080-111. 2. Chung SA, Yuan H, Chung F. A systemic review of obstructive sleep apnea and its implications for anesthesiologists. Anesth Analg. 2008; 107:1543-63. 3. Sassani A, Findley LJ, Kryger M, Goldlust E, George C, Davidson TM. Reducing motor-vehicle collisions, costs, and fatalities by treating obstructive sleep apnea syndrome. Sleep. 2004;27:453-8. 4. Lindberg E, Carter N, Gislason T, Janson C. Role of snoring and daytime sleepiness in occupational accidents. Am J Respir Crit Care Med. 2001;164:2031-5. 5. Kim HC, Young T, Matthews CG, Weber SM, Woodward AR, Palta M. Sleep-disordered breathing and neuropsychological deficits. A population-based study. Am J Respir Crit Care Med. 1997;156:1813-9. 6. Smith R, Ronald J, Delaive K, Walld R, Manfreda J, Kryger MH. What are obstructive sleep apnea patients being treated for prior to this diagnosis? Chest. 2002;121:164-72. 7. Flemons WW, Tsai W. Quality of life consequences of sleep-disordered breathing. J Allergy Clin Immunol. 1997;99:S750-6. 8. Fogel RB, Malhotra A, White DP. Sleep. 2: pathophysiology of obstructive sleep apnoea/hypopnoea syndrome. Thorax. 2004;59:159-63.

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9. Young T, Palta M, Dempsey J, Skatrud J, Weber S, Badr S. The occurrence of sleep-disordered breathing among middle-aged adults. N Engl J Med. 1993;323:1230–5. 10. Young T, Peppard PE, Gottlieb DJ. Epidemiology of obstructive sleep apnea: a population health perspective. American Journal of Respiratory and Critical Care Medicine. 2002;165:1217–39. 11. Stradling JR, Crosby JH. Predictors and prevalence of obstructive sleep apnoea and snoring in 1001 middle aged men. Thorax 1991;46: 85–90. 12. Pack AI, Dinges DF, Maislin G. A study of the prevalence of sleep apnea among commercial truck drivers. Washington: FMCSA;2002. Publication No. DOT-RT-02–030. 13. Young T, Evans L, Finn L, Palta M. Estimation of the clinically diagnosed proportion of sleep apnea syndrome in middle-aged men and women. Sleep. 1997;20:705-6. 14. Koulouris NG, Valta P, Lavoie A, Corbeil C, Chasse´ M, Braidy J, et al. A simple method to detect expiratory flow limitation during spontaneous breathing. Eur Respir J. 1995;8:306-13. 15. Liistro G, Veriter C, Dury M, Aubert G, Stanescu D. Expiratory flowlimitation in awake sleep-disordered breathing subjects. Eur Respir J. 1999;14:185-90. 16. Verin E, Tardif C, Portier F, Similowski T, Pasquis P, Muir JF. Evidence for expiratory flow limitation of extrathoracic origin in patients with obstructive sleep apnoea. Thorax. 2002;57:423-8. 17. Van Meerhaeghe A, Delpire P, Stenuit P, Kerkhofs M. Operating characteristics of the negative expiratory pressure technique in predicting obstructive sleep apnoea syndrome in snoring patients. Thorax. 2004;59:883-8. 18. Baydur A, Wilkinson L, Mehdian R, Bains B, Milic-Emili J. Extrathoracic expiratory flow limitation in obesity and obstructive and restrictive disorders. Chest. 2004;125:98-105. 19. Insalaco G, Romano S, Marrone O, Salvaggio A, Bonsignore G. A new method of negative expiratory pressure test analysis detecting upper airway flow limitation to reveal obstructive sleep apnea. Chest. 2005; 128:2159-65. 20. Tamisier R, Wuyam B, Nicolle I, Pe´pin JL, Orliaguet O, Perrin CP, et al. Awake flow limitation with negative expiratory pressure in sleep disordered breathing. Sleep Med. 2005;6:205-13. 21. Ferretti A, Giampiccolo P, Redolfi S, Mondini S, Cirignotta F, Cavalli A, et al. Upper airway dynamics during negative expiratory pressure in apneic and non-apneic awake snorers. Respir Res. 2006;7:54-63. 22. Guillot M, Costes F, Sforza E, Maudoux D, Bertoletti L, Barthe´le´my JC, et al. Is tidal expiratory flow limitation predictive of sleep-related disorders in the elderly? Eur Respir J. 2010;36:842-8. 23. Romano S, Salvaggio A, Pastrello Hirata R, Lo Bue A, Picciolo S, et al. Upper airway collapsibility evaluated by a negative expiratory pressure test in severe obstructive sleep apnea. Clinics. 2011;66:567-72. 24. Collop NA, Anderson WM, Boehlecke B, Claman D, Goldberg R, Gottlieb DJ, et al. Portable Monitoring Task Force of the American Academy of Sleep Medicine: Clinical guidelines for the use of unattended portable monitors in the diagnosis of obstructive sleep apnea in adult patients. J Clin Sleep Med. 2007;3:737-47. 25. Iber C, Ancoli-Israel S, Chesson A, Quan S. For the American Academy of Sleep Medicine. 1st ed. Westchester: IL: American Academy of Sleep Medicine; 2007. The AASM manual for the scoring of sleep and associated events: rules, terminology and technical specifications.

26. Vgontzas AN, Tan TL, Bixler EO, Martin LF, Shubert D, Kales A. Sleep apnea and sleep disruption in obese patients. Arch Intern Med. 1994; 154:1705-11. 27. Resta O, Foschino-Barbaro MP, Legari G, Talamo S, Bonfitto P, Palumbo A, et al. Sleep-related breathing disorders, loud snoring and excessive daytime sleepiness in obese subjects. Int J Obes Relat Metab Disord. 2001;25:669-75. 28. Neves C, Tufik S, Chediek F, Poyares D, Cintra F, Roizenblatt M, et al. Effects of sildenafil on autonomic nervous function during sleep in obstructive sleep apnea. Clinics. 2010;65:393-400. 29. Boushra NN. Anaesthetic management of patients with sleep apnoea syndrome. Can J Anaesth. 1996;43:599-616. 30. Gupta RM, Parvizi J, Hanssen AD, Gay PC. Postoperative complications in patients with obstructive sleep apnea syndrome undergoing hip or knee replacement: A case-control study. Mayo Clin Proc. 2001;76:897-905. 31. Reeder MK, Muir AD, Foex P, Goldman MD, Loh L, Smart D. Postoperative myocardial ischaemia: Temporal association with nocturnal hypoxaemia. Br J Anaesth. 1991;67:626-31. 32. Gupta RM, Parvizi J, Hanssen AD, Gay PC. Postoperative complications in patients with obstructive sleep apnea syndrome undergoing hip or knee replacement: A case-control study. Mayo Clin Proc. 2001;76:897905. 33. Lofsky A. Sleep apnea and narcotic postoperative pain medication: A morbidity and mortality risk. Anesth Patient Safety Found Newsletter. 2002;17:24-25. 34. Hwang D, Shakir N, Limann B, Sison C, Kalra S, Shulman L, et al. Association of sleep-disordered breathing with postoperative complications. Chest. 2008;133:1128-34. 35. Flemons WW, Douglas NJ, Kuna ST, Rodenstein DO, Wheatley J. Access to diagnosis and treatment of patients with suspected sleep apnea. Am J Respir Crit Care Med. 2004;169:668-72. 36. Littner MR. Portable monitoring in the diagnosis of the obstructive sleep apnea syndrome. Semin Respir Crit Care Med. 2005;26:56-67. 37. Kapuniai LE, Andrew DJ, Crowell DH, Pearce JW. Identifying sleep apnea from self-reports. Sleep. 1988;11:430-6. 38. Douglass AB, Bornstein R, Nino-Murcia G, Keenan S, Miles L, Zarcone VP Jr, et al. The Sleep Disorders Questionnaire: I. Creation and multivariate structure of SDQ. Sleep. 1994;17:160-7. 39. Roth T, Zammit G, Kushida C, Doghramji K, Mathias SD, Wong JM, et al. A new questionnaire to detect sleep disorders. Sleep Med 2002;3: 99-108. 40. Ramachandran SK, Josephs LA. A Meta-analysis of Clinical Screening Tests for obstructive sleep apnea. Anesthesiology. 2009;110:928-39. 41. Netzer NC, Stoohs RA, Netzer CM, Clark K, Strohl KP. Using the Berlin questionnaire to identify patients at risk for the sleep apnea syndrome. Ann Intern Med. 1999;131:485-491. 42. Ross SD, Sheinhait IA, Harrison KJ, Kvasz M, Connelly JE, Shea SA, et al. Systematic review and meta-analysis of the literature regarding the diagnosis of sleep apnea. Sleep. 2000;23:519-32. 43. Ahmadi N, Chung SA, Gibbs A, Shapiro CM. The Berlin questionnaire for sleep apnea in a sleep clinic population: Relationship to polysomnographic measurement of respiratory disturbance. Sleep Breath. 2008;12:39-45. 44. Knottnerus JA, Leffers P. The influence of referral patterns on the characteristics of diagnosis tests. J Clin Epidemiol. 1992;45:1143-54.

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DOI:10.1590/S1807-59322011001100008

CLINICAL SCIENCE

The characteristics of stress cardiomyopathy in an ethnically heterogeneous population Francisco O. Nascimento,I Orlando Santana,I Margarita Perez-Caminero,II Alexandre M. BenjoII,III I Columbia University, Division of Cardiology at Mount Sinai Heart Institute, Miami Beach, Florida, USA. II Mount Sinai Medical Center, Department of Medicine, Miami Beach, Florida, USA. III Universidade Federal Fluminense, Pathology Department, Niteroi/RJ, Brazil.

OBJECTIVES: Stress cardiomyopathy is a cardiac syndrome that is characterized by transient left ventricular systolic dysfunction in the absence of obstructive coronary artery disease. Its epidemiology has been described in homogeneous Asian, Caucasian and Black populations, but its characteristics in heterogeneous populations are poorly understood. Our aim was to assess the characteristics of stress cardiomyopathy in a heterogeneous population that included a large percentage of Hispanics. METHODS: We reviewed 59 consecutive cases of stress cardiomyopathy that were confirmed by coronary angiography and were in agreement with the Mayo Clinic diagnostic criteria. RESULTS: The mean age of the patients was 74 years (range, 39-91 years), and 37 patients were female (62.7%). Twenty-nine patients (49.2%) were Latino/Hispanic, 26 (44%) were Caucasian, 3 (5%) were Asian, and 1 patient (1.7%) was Black. The most common chief symptom was dyspnea, followed by chest pain and an absence of symptoms in 54.2, 28.8, and 18.6% of the patients, respectively. The primary EKG abnormalities consisted of a T wave inversion, an ST segment elevation, and ST segment depression in 69.5%, 25.4%, and 15.3% of the patients, respectively. The stressor event was identified in 90% of the cases. In 32 cases (54%), the stressor event was physical stress or a medical illness, and in 21 cases (35.6%), the stressor event was emotional stress. The in-hospital mortality rate was 8.5%. CONCLUSIONS: In our heterogeneous study population, stress cardiomyopathy presented with a 3:2 female-to-male ratio, and dyspnea was the most common chief complaint. Stress cardiomyopathy exhibited a T wave inversion as the primary EKG abnormality. These findings differ from previous cases that have been reported, and further studies are needed. KEYWORDS: Stress; Cardiomyopathy; Takotsubo; Hispanics; Epidemiology. Nascimento FO, Santana O, Perez-Caminero M, Benjo AM. The characteristics of stress cardiomyopathy in an ethnically heterogeneous population. Clinics. 2011;66(11):1895-1899. Received for publication on July 8, 2011; First review completed on July 13, 2011; Accepted for publication on July 15, 2011 E-mail: osantana@msmc.com Tel.: 305 674-2168

Usually, the cardiac biomarkers are elevated, and multiple wall motion abnormalities with non-obstructive coronary artery disease (as determined by angiography) are hallmarks of this syndrome.1-5 Following its original description, several SC case series have been published from Eastern countries, suggesting that the syndrome affects mostly Asian patients.1-5 More recently, other authors have reported SC in Caucasian6-12 and Black13,14 populations in Europe and North America. No series that included Hispanics has been reported. Miami Beach, Florida, USA, is an ethnically heterogeneous region, and the reported population is composed primarily of Hispanics (53% in 2006) and Caucasians.15 Our aim was to study the demographic data, clinical characteristics, and in-hospital prognoses of patients with SC in the Miami Beach population.

INTRODUCTION Stress cardiomyopathy (SC) or Takotsubo cardiomyopathy is a cardiac syndrome that is characterized by transient, left ventricular systolic dysfunction in the apical portion and hyperkinesia in the basal segments with an absence of obstructive coronary artery disease.1 SC was first described in 1990 by Sato et al.,2 who introduced the term ‘‘takotsubo’’, the Japanese word for ‘‘octopus trap’’, which has a similar shape as the ballooned apex in cardiac systole. Clinically, SC typically affects women who are in their seventh decade of life and who have had physical or emotional stress; the most common symptom is chest pain. The electrocardiographic changes suggest an acute coronary syndrome (more specifically, an ST segment elevation myocardial infarction).

METHODS After obtaining approval from the Institutional Review Board, we retrospectively reviewed 76 consecutive cases of presumed acute coronary syndrome who underwent coronary

No potential conflict of interest was reported.

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angiograms and were found to lack obstructive coronary disease at our institution from June 2005 through July 2010.

Caucasian, 3 patients (5%) were Asian, and 1 patient (1.7%) was Black (Figure 1A and 1B).

Inclusion criteria

Symptoms

The selected patients satisfied the Mayo Clinic criteria13 for the diagnosis of SC as follows: (1) transient akinesis or dyskinesis in the left ventricular apical and mid-ventricular segments with regional wall motion abnormalities extending beyond a single epicardial vascular distribution; (2) the absence of obstructive coronary disease or angiographic evidence of an acute plaque rupture; (3) new electrocardiographic abnormalities (either ST segment elevation or T wave inversion); and (4) the absence of recent significant head trauma, intracranial bleeding, pheochromocytoma, obstructive epicardial coronary artery disease, myocarditis or hypertrophic cardiomyopathy.

The most prevalent chief symptom was dyspnea, which was followed in prevalence by chest pain and finally the absence of symptoms in 54.2, 28.8, and 18.6% of the patients (Figure 1C). Of the patients who were classified as asymptomatic, 2 (3.4%) presented with cardiac arrest. In 1 patient (1.7%), it was impossible to determine the chief complaint, and both symptoms were considered.

Electrocardiography Fifty-four patients (91.5%) exhibited electrocardiographic abnormalities, and some cases had overlapping findings. The remaining 5 cases (8.5%) had pacemaker rhythms that precluded further analysis. The primary abnormalities were T wave inversion (TWI), ST segment elevation, and ST segment depression in 69.5, 25.4, and 15.3% of the cases, respectively (Figure 1D).

Exclusion criteria We excluded patients with borderline obstructive coronary artery disease (as determined by angiographic evidence of 5070% stenosis in at least one epicardial vessel that was associated with the affected wall motion abnormality), patients with unresolved wall motion abnormalities, and patients without a follow-up echocardiogram to reassess left ventricular function.

Cardiac biomarkers and echocardiographic findings

The demographic data, medical history, cardiac biomarkers, 12-lead electrocardiogram, echocardiogram, coronary angiogram and ventriculogram findings, time of the event, and discharge summaries were reviewed.

Troponin I levels were elevated in 91.5% of the cases, with a mean of 7.6¡18 mg/dL. The mean left ventricular ejection fraction at the time of presentation was 30¡9%, and this subsequently improved to 56¡9% during a mean follow-up evaluation of 10 days. Reverse Takotsubo, a variant of stress cardiomyopathy that consists of basal hypokinesis with normal apical contractions, occurred in two patients (3.4%).

Ethnicity definitions

Trigger event

The patients were categorized as Caucasian, Hispanic, Black or Asian. The patients were considered of Hispanic origin if they were born in any Central or South American country or any Caribbean country with Hispanic colonization. The white patients were considered to be Caucasian if they did not meet criteria for Hispanic ethnicity. The Black patients were not considered Hispanic regardless of their country of birth. Any immediate descendants from two Asian parents were not considered to be Hispanic, regardless of the country of birth.

The stressor event was identified in 90% of the cases. In 32 (54%) of the cases, the stressor event was physical stress or a medical illness, and in 21 (35.6%) of the cases, the stressor event was emotional stress.

Data revision

Recurrence and mortality rates The syndrome recurred in two patients (3.4%) during the time frame of our study, and one patient experienced two recurrences. The in-hospital mortality rate was 8.5% (five cases), and all of these deaths were non-cardiac-related. The demographic data, clinical presentations, diagnostic data and in-hospital mortality rates are presented in Table 1.

Statistical analysis The continuous variables with normal distributions were analyzed using Student’s t-test and are presented as mean +/1 standard deviation. The categorical data are presented as percentages and frequencies and were analyzed using the X2 test. A univariate analysis was performed to compare the Hispanics and non-Hispanics, and the variables with p,0.2 were analyzed using multivariate analyses. A p-value of ,0.05 was considered to be significant. The statistical analyses were performed using SPSS version 17 (SPSS Institute, Chicago, IL).

Ethnic differences We compared the characteristics of the Hispanics and nonHispanics using Student’s t-test and the X2 test for the continuous and categorical variables, respectively (Table 2). For further evaluation, we performed a univariate analysis that included age, gender, symptoms upon arrival (e.g., chest pain, shortness of breath and arrest), EKG presentation (ST elevation or depressed or inverted T waves), troponin I peak, the trigger (psychological or physical) and in-hospital death. ST elevation and in-hospital death emerged as potentially significant variables (p = 0.019 and p = 0.154, respectively); however, only the presentation of ST elevation in Hispanics reached statistical significance in the multivariate analysis (OR 4.06, 95% CI = 1.22-13.59, p = 0.022).

RESULTS We initially evaluated 76 cases, of which 17 were excluded due to the presence of at least one exclusion criterion. Therefore, we analyzed 59 consecutive cases of SC.

Demographics

DISCUSSION

The mean age of the 59 patients was 74 years (range, 39-91 years), and 37 patients were female (62.7%). Twenty-nine patients (49.2%) were Hispanic, 26 patients (44%) were

The demographic and clinical characteristics of SC have been documented extensively by several studies of Asian,1-5

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Figure 1 - The gender (A) and ethnic (B) distributions, chief complaints (C) and electrocardiographic findings (D) of the patients presenting with stress cardiomyopathy.

Caucasian6-12 and Black13-14 patients within mostly homogeneous populations. Stress cardiomyopathy accounts for 1.7-2.2% of all patients who are admitted with a presumed diagnosis of an acute coronary syndrome.6,16 SC is more commonly described in women (90%) who are older than 70 years of age and who have experienced physical and/or emotional stress, including an acute medical illness. Clinically, the most common presenting symptoms are chest pain (63-90%) and shortness of breath (16-24%). SC is associated with electrocardiographic changes that resemble an acute coronary syndrome, specifically, ST-segment elevation myocardial infarction (67-75%), with troponin being elevated in approximately 70% of the cases.1-14,16,17 Our data demonstrate some findings that are similar to previous case studies. However, the Miami Beach population exhibited some minor differences. Although the mean age was 74 years old, three of the patients were premenopausal women. There was a female predominance of 62.7% in our population, which is lower than what is usually reported. Our gender distribution (an approximately 3:2 female:male ratio) differs markedly from the 9:1 ratio that is described in the majority of reports. It is unclear why our study contained such a relatively high prevalence

of men, given a similar gender distribution in the community. In addition, as expected, the ethnicities of the patients in our study population were also markedly different from prior studies. According to the 2006 US Census Bureau15, the city of Miami Beach, FL, USA, is composed of 53.4% of individuals of Hispanic or Latino origin, 41.2% of Caucasian (excluding Caucasians of Hispanic origin), 4% Black, and 1.4% of Asian individuals. These demographic characteristics were concordant with our studied population. The leading symptom in our study population was dyspnea, which was seen in 54% of the cases, and is in contrast to chest pain, which has been classically described in several reports.1,5,7,13,16,17 Of note, there was a high incidence (over 18%) of asymptomatic patients who had abnormal objective data (EKG, troponin I, or echocardiography). TWI was the most common EKG abnormality. This finding differs from other studies that identified ST segment elevation as the primary finding. Another difference in our cohort was the high prevalence of elevated troponin I (in over 90% of the patients), with a high mean peak level (7.6 mg/dL). The initial and follow-up left ventricular ejection fractions were similar to previous reports. Two of

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Table 1 - The demographic and clinical characteristics of 59 patients in the Stress Cardiomyopathy Registry. Total Gender Female Male Age (mean¡SD) Race Hispanic Non-Hispanic Caucasian Asian Black Symptoms Chest pain Dyspnea No symptoms EKG findings Abnormal ST elevation ST depression T wave inversion Troponin I Elevated Not elevated Peak (mean¡SD) Ejection fraction Initial (mean¡SD) Final (mean¡SD) Trigger event Emotional Physical Unidentifiable Reverse Takotsubo Recurrence Death Any cause

n = 59

37 22 74¡13

62.7 37.3

29 30 26 3 1

49.2 50.6 44 5 1.7

17 32 11

28.8 54.2 18.6

54 15 9 41

91.5 25.4 15.3 69.5

54 5 7.6¡18

91.5 8.5

Table 2 - The demographic and clinical data of the Hispanic and non-Hispanic groups.

Age Female Chest Pain Dyspnea Arrest ST Segment Elevation ST Segment Depression T wave Inversion Peak Troponin Psychological Stress Physical Stress Death

35.6 54 10 3.4 3.4

8.5

Non-Hispanic (n = 30)

p-value

74.2+/-15.4 17 (59%) 8 (28%) 17 (59%) 1 (3%) 13 (45%) 5 (17%) 19 (66%) 9.97+/-25.1 10 (34%) 15 (52%) 4 (14%)

73.9+/-12.2 20 (67%) 9 (30%) 14 (47%) 1 (3%) 5 (17%) 4 (13%) 22 (73%) 4.9+/-8.8 11 (37%) 17 (57%) 1 (3%)

0.40 0.52 0.84 0.36 0.98 0.02 0.68 0.51 0.31 0.86 0.70 0.15

valuable diagnostic and follow-up information. The treatment is supportive, approximately 20% of patients require vasopressors.1,27 The prognosis for SC is favorable, with the complete recovery of left ventricular function typically occurring within 1-3 weeks, and the in-hospital mortality rate ranges from 1-8%.1,6,27 In our patient cohort, five patients (8.5%) died during hospitalization, which is a slightly higher rate than has been described previously. However, none of these five deaths were cardiac-related, which suggests that stress cardiomyopathy itself may represent a marker of a generally impaired health status and may not be the cause of death. The four-year recurrence rate of SC is reported to be 311%.1,4,6,7 During the time frame of our study, two (3.4%) of the patients had a recurrence of their stress cardiomyopathy. Of note, one patient had two recurrences of SC within four years and fully recovered from all three episodes.

30¡9.3 56¡9 21 32 6 2 2

Hispanic (n = 29)

our patients (3.4%) had the ‘‘reverse Takotsubo’’ variety of SC, and this rate is comparable to previous reports.6 In our population, the Hispanics and non-Hispanics had nearly identical presentations that differed from previously described cohorts. We speculate that environmental factors might explain these differences, as it is known that diet and medication can have protective effects in cardiovascular pathologies.18,19 We subjectively perceive that our population has a high level of activity and sun exposure that would produce a satisfactory level of vitamin D, but we recognize that the lack of documentation in this regard is a study limitation. Further studies should be performed to clarify whether these factors are involved in our findings, as vitamin D deficiency is known to play a role in cardiomyopathy.20-22 Further studies will also clarify whether exercise has a protective effect against SC as previously described for other specific cardiac situations.23-25 Based on these hypotheses, future studies will compare the physical activities and vitamin D levels of SC patients at various Columbia Hospitals. The cause of SC remains unknown. Microvascular dysfunction due to excessive catecholamines and myocyte stunning due to overt sympathetic stimulation are among the most commonly accepted theories; however, metabolic abnormalities involving myocardial calcium overload and impaired glucose metabolism may also play roles.26,27 SC is essentially a diagnosis of exclusion and is usually given after a coronary angiogram has excluded the possibility of an acute myocardial infarction. Echocardiography provides

Study Limitations This study was a retrospective observational analysis of a single-center experience. The evaluation of the patients was limited to the in-hospital setting.

CONCLUSIONS In our heterogeneous population, stress cardiomyopathy presented with a 3:2 female:male ratio, with dyspnea as the primary chief complaint and TWI as the primary EKG abnormality. These findings differ from previous reports. In a subgroup analysis, an initial EKG presentation ST wave elevation was more common among Hispanics, although it was not the most common electrocardiographic abnormality. Future studies that focus on Hispanic populations and environmental factors are needed.

AUTHOR CONTRIBUTIONS Nascimento FO and Perez-Caminero M were responsible for the data collection and statistical analysis. Santana O was responsible for the supervision of the study and writing of the manuscript. Benjo AM was responsible for the data collection, statistical analysis, supervision of the study and writing of the manuscript.

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Angina Pectoris–Myocardial Infarction Investigations in Japan. J Am Coll Cardiol. 2001;38:11–8, doi: 10.1016/S0735-1097(01)01316-X. Satoh H, Tateishi H, Uchida T. Takotsubo-type cardiomyopathy due to multivessel spasm. In: Kodama K, Hon M, editors. Clinical Aspect of Myocardial Injury: From Ischemia to Heart Failure (in Japanese). Tokyo: Kagakuhyouronsya Co. 1990:56-64. Kawai S, Suzuki H, Yamaguchi H, Tanaka K, Sawada H, Aizawa T, et al. Ampulla cardiomyopathy (‘Takotusbo’ cardiomyopathy)—reversible left ventricular dysfunction: with ST segment elevation. Jpn Circ J. 2000;64:156–9, doi: 10.1253/jcj.64.156. Kurisu S, Sato H, Kawagoe T, Ishihara M, Shimatani Y, Nishioka K, et al. Tako-tsubo-like left ventricular dysfunction with ST-segment elevation: a novel cardiac syndrome mimicking acute myocardial infarction. Am Heart J. 2002;143:448–55, doi: 10.1067/mhj.2002.120403. Akashi YJ, Nakazawa K, Sakakibara M, Miyake F, Koike H, Sasaka K. The clinical features of takotsubo cardiomyopathy. QJM. 2003;96:563–73, doi: 10.1093/qjmed/hcg096. Eshtehardi P, Koestner SC, Adorjan P, Windecker S, Meier B, Hess OM, et al. Transient apical ballooning syndrome – clinical characteristics, ballooning pattern, and long-term follow-up in a Swiss population. Int J Cardiol. 2009;135:370-75, doi: 10.1016/j.ijcard.2008.03.088. Sharkey SW, Windenburg DC, Lesser JR, Maron MS, Hauser RG, Lesser JN, et al. Natural history and expansive clinical profile of stress (TakoTsubo) cardiomyopathy. J Am Coll Cardiol. 2010;55:333-41, doi: 10.1016/ j.jacc.2009.08.057. Parodi G, Del Pace S, Carrabba N, Salvadori C, Memisha G, Simonetti I, et al. Incidence, clinical findings, and outcome of women with left ventricular apical ballooning syndrome. Am J Cardiol. 2007;99:182–5, doi: 10.1016/j.amjcard.2006.07.080. Previtali M, Repetto A, Panigada S, Camporotondo R, Tavazzi L. Left ventricular apical ballooning syndrome: prevalence, clinical characteristics and pathogenetic mechanisms in a European population. Int J Cardiol. 2009;134:91-6, doi: 10.1016/j.ijcard.2008.01.037. Vidi V, Rajesh V, Singh PP, Mukherjee JT, Lago RM, Venesy DM, et al. Clinical characteristics of tako-tsubo cardiomyopathy. Am J Cardiol. 2009;104:578-82, doi: 10.1016/j.amjcard.2009.04.028. Athanasiadis A, Vogelsberg H, Hauer B, Meinhardt G, Hill S, Sechtem U. Transient left ventricular dysfunction with apical ballooning (tako-tsubo cardiomyopathy) in Germany. Clin Res Cardiol. 2006;95:321-8, doi: 10. 1007/s00392-006-0380-0. Auer J, Porodko M, Berent R, Punzengruber C, Weber T, Lamm G, et al. Transient left ventricular apical ballooning mimicking acute coronary syndrome in four patients from central Europe. Croat Med J. 2005;46:9429. Pezzo SP, Hartlage G, Edwards CM. Takotsubo cardiomyopathy presenting with dyspnea. J Hosp Med. 2009;4:200-2, doi: 10.1002/jhm. 402.

14. Patel HM, Kantharia BK, Morris DL, Yazdanfar S. Takotsubo syndrome in African American women with atypical presentation: a single-center experience. Clin Cardiol 2007;30:14-8. 15. http://quickfacts.census.gov/qfd/states/12/1245025.html 16. Gianni M, Dentali F, Grandi AM, Sumner G, Hiralal R, Lonn E. Apical ballooning syndrome or takotsubo cardiomyopathy: a systematic review. Eur Heart J. 2006;27:1523–9, doi: 10.1093/eurheartj/ehl032. 17. Pilgrim TM, Wyss TR. Takotsubo cardiomyopathy or transient ballooning syndrome: A systematic review. Int J Cardiol 2008;124:283-92. 18. Andrade AC, Cesena FH, Consolim-Colombo FM, Coimbra SR, Benjo´ AM, Krieger EM, et al. Short-term red wine consumption promotes differential effects on plasma levels of high-density lipoprotein cholesterol, sympathetic activity, and endothelial function in hypercholesterolemic, hypertensive, and healthy subjects. Clinics. 2009;64:435-42, doi: 10.1590/S1807-59322009000500011. 19. Carnieto A Jr, Dourado PM, Luz PL, Chagas AC. Selective cyclooxygenase-2 inhibition protects against myocardial damage in experimental acute ischemia. Clinics. 2009;64:245-52, doi: 10.1590/S1807-59322009 000300016. 20. Zittermann A, Schleithoff SS, Koerfer R. Vitamin D insufficiency in congestive heart failure: why and what to do about it? Heart Fail Rev. 2006;11:25-33, doi: 10.1007/s10741-006-9190-8. 21. Wang TJ, Pencina MJ, Booth SL, Jacques PF, et al. Vitamin D deficiency and risk of cardiovascular disease. Circulation. 2008;117:503-11, doi: 10. 1161/CIRCULATIONAHA.107.706127. 22. Mitka M. Vitamin D deficits may affect heart health. JAMA. 2008; 299:753-4. 23. Negrao CE, Middlekauff HR. Adaptations in autonomic function during exercise training in heart failure. Heart Fail Rev. 2008;13:51-60. 24. Farah C, Meyer G, Andre´ L, Boissie`re J, Gayrard S, Cazorla O, et al. Moderate exercise prevents impaired Ca2+ handling in heart of COexposed rat: implication for sensitivity to ischemia-reperfusion. Am J Physiol Heart Circ Physiol. 2010;299:H2076-81, doi: 10.1152/ ajpheart.00835.2010. 25. Shen YJ, Pan SS, Zhuang T, Wang FJ. Exercise preconditioning initiates late cardioprotection against isoproterenol-induced myocardial injury in rats independent of protein kinase C. J Physiol Sci. 2011;61:13-21, doi: 10. 1007/s12576-010-0116-9. 26. Sharkey SW, Lesser JR, Zenovich AG, Maron MS, Lindberg J, Longe TF, et al. Acute and reversible cardiomyopathy provoked by stress in women from the United States. Circulation. 2005;111:472–9, doi: 10.1161/01.CIR. 0000153801.51470.EB. 27. Kurowski V, Kaiser A, von Hof K, Killermann DP, Mayer B, Hartmann F, et al. Apical and midventricular transient left ventricular dysfunction syndrome (tako-tsubo cardiomyopathy): frequency, mechanisms, and prognosis. Chest. 2007;132:809–16, doi: 10.1378/chest.07-0608.

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DOI:10.1590/S1807-59322011001100009

CLINICAL SCIENCE

Preoperative nodal staging of non-small cell lung cancer using 99mTc-sestamibi spect/ct imaging Juliana Muniz Miziara,I Euclides Timo´teo da Rocha,I Jose´ Elias Abra˜o Miziara,I Gustavo Fabene Garcia,I Maria Izilda Previato Simo˜es,I Marco Antoˆnio Lopes,I Lı´gia Maria Kerr,I Carlos Alberto BuchpiguelII I

Hospital de Caˆncer de Barretos, Barretos/SP, Brazil. II Faculdade de Medicina da Universidade da Saõ Paulo, Hospital das Clıńicas da Faculdade de Medicina da Universidade de Saõ Paulo, Radiologia, Saõ Paulo/SP, Brazil.

OBJECTIVES: The proper nodal staging of non-small cell lung cancer is important for choosing the best treatment modality. Although computed tomography remains the first-line imaging test for the primary staging of lung cancer, its limitations for mediastinum nodal staging are well known. The aim of this study is to evaluate the accuracy of hybrid single-photon emission computed tomography and computed tomography using 99mTc-sestamibi in the nodal staging of patients with non-small cell lung cancer and to identify potential candidates for surgical treatment. METHODS: Prospective data were collected for 41 patients from December 2006 to February 2009. The patients underwent chest computed tomography and single-photon emission computed tomography/computed tomography examinations with 99mTc-sestamibi within a 30-day time period before surgery. Single-photon emission computed tomography/computed tomography was considered positive when there was focal uptake of sestamibi in the mediastinum, and computed tomography scan when there was lymph nodes larger than 10 mm in short axis. The results of single-photon emission computed tomography and computed tomography were correlated with pathology findings after surgery. RESULTS: Single-photon emission computed tomography/computed tomography correctly identified six out of 19 cases involving hilar lymph nodes and one out of seven cases involving nodal metastases in the mediastinum. The sensitivity, specificity, positive predictive value, and negative predictive value for 99mTc-sestamibi single-photon emission computed tomography/computed tomography in the hilum assessment were 31.6%, 95.5%, 85.7%, and 61.8%, respectively. The same values for the mediastinum were 14.3%, 97.1%, 50%, and 84.6%, respectively. For the hilar and mediastinal lymph nodes, chest tomography showed sensitivity values of 47.4% and 57.1%, specificity values of 95.5% and 91.2%, positive predictive values of 90% and 57.1% and negative predictive values of 67.7% and 91.2%, respectively. CONCLUSION: Single-photon emission computed tomography/computed tomography with 99mTc-sestamibi showed very low sensitivity and accuracy for the nodal staging of patients with non-small cell lung cancer, despite its high level of specificity. In addition, the performance of single-photon emission computed tomography/computed tomography added no relevant information compared to computed tomography that would justify its use in the routine preoperative staging of non-small cell lung carcinoma. KEYWORDS: Lung cancer; Lymph nodes; MIBI; Single-photon emission computed tomography; Functional imaging. Miziara JM, Rocha ET, Miziara JEA, Garcia GF, Simo˜es MIP, Lopes MA, et al. Preoperative nodal staging of non-small cell lung cancer using sestamibi spect/ct imaging. Clinics. 2011;66(11):1901-1909.

99m

Tc-

Received for publication on May 28, 2011; First review completed on June 16, 2011; Accepted for publication on July 18, 2011 E-mail: julimiziara@ig.com.br Tel.: 55 17 3321-6600

mediastinal lymph nodes is a major determinant of both the prognosis and the therapeutic approach. Proper staging is important for selecting patients who may benefit from surgical resection and for defining the treatment modalities of patients who will undergo radiotherapy. The histopathologic evaluation of lymph nodes is considered the gold standard in assessing the presence or absence of metastases in the mediastinum. There are several invasive methods that can be used for this purpose: mediastinoscopy, anterior mediastinotomy, transthoracic needle aspiration, endobronchial or esophageal ultrasound with needle aspiration, and thorachoscopy.1,2 Mediastinoscopy is the most

INTRODUCTION The survival of lung cancer patients is related to the extent of their disease at the time of diagnosis. In the absence of distant metastases, the spread of tumors to the

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CT scanners. Sequentially, data from both CT and SPECT are acquired. The two images are merged, creating SPECT images that are superimposed on corresponding anatomical planes. This image fusion may help to differentiate between tumors and other areas of physiological activity.14,15 The aim of this study is to evaluate the accuracy of SPECT/CT using the radiotracer 99mTc-sestamibi in the mediastinal lymph node staging of patients with non-small cell lung cancer and candidates to surgical treatment.

common invasive test. It has a mean sensitivity of up to 80%, with a range of 44% to 97%. With this method, only high and low paratracheal, pre-tracheal, and subcarinal lymph nodes are accessible; there is also a low but real risk of morbidity and mortality.1 In an attempt to reduce the frequency of invasive methods or to guide the most appropriate procedures for lymph node biopsies, noninvasive imaging tests are used when applicable. Computed tomography (CT) is the imaging method of choice in the evaluation and staging of primary cancers. The diagnostic CT criteria for the involvement of lymph nodes are based on their sizes, especially when their minor axes are longer than ten millimeters. However, small lymph nodes that are considered normal according to such criteria may contain tumor cells, while inflammatory and infectious diseases may be responsible for enlarged lymph nodes, limiting the overall effectiveness of this diagnostic test. In a meta-analysis by Toloza that evaluated 20 studies, chest CT examinations showed a sensitivity of 57%, specificity of 82%, and positive and negative predictive values of 56% and 83%, respectively.3 Tomographic imaging in nuclear medicine is based on the metabolic activity of tissues and may be useful for identifying pathological changes before they are detected by radiological examinations such as CTs. PET (positron emission tomography) scans with 18F-FDG (18F-fluorodeoxyglucose) have superior sensitivity and specificity compared to chest CTs and are considered the most accurate imaging method for staging patients with lung cancers.3-5 However, there are limitations related to positive predictive value of this method because there may be FDG uptake in inflammatory cells.6 The sensitivity can also be decreased when lymph node metastasis is microscopic or below the spatial resolution threshold of current, state-of-the-art scanners.7 In Brazil, the availability of PET is restricted to a few institutions because of equipment costs and availability of comercial doses of FDG regarding the number of cyclotrons installed in Brazil. Alternatively, single-photon emission computed tomography (SPECT) is widely available, has lower costs than PET, and does not require the presence of a cyclotron adjacent to the hospital. Sestamibi (hexakis-2-methoxyisobutyl-isonitrile) labeled with technetium (99mTc-sestamibi) is a lipophilic cation that is routinely used in myocardial perfusion imaging and has been used as a tumor-seeking agent.8 Encouraging results have been obtained with SPECT scanning using sestamibi to detect primary lung malignancies9-11 and to perform mediastinal staging with a higher diagnostic accuracy higher than chest CTs.12,13 Despite the positive results obtained with sestamibi, particularly regarding its specificity, there are difficulties in the analysis of mediastinal images. The main difficulty is related to the limited spatial resolution of SPECT. Furthermore, the vascular structures in the mediastinum and heart, where sestamibi is taken up, interfere with the correct interpretation of the images and could cause false positive results and decrease the diagnostic capability of the method. The association of functional images and anatomical information from CTs may be useful in interpreting SPECT by providing more accurate data regarding the location and extent of tumor lesions. Hybrid devices usually have dual detectors, with scintillation cameras and low-dose

MATERIALS AND METHODS Patients A cross-sectional study with prospective data collection was conducted from December 2006 to February 2009 at the Hospital de CaË&#x2020;ncer de Barretos-SP. The study was approved by the Institutional Ethics Committee. The inclusion criteria were as follows: 1) patients of either sex, 2) patients who were at least 18 years old, 3) patients with histological diagnoses of non-small cell lung cancer (e.g., squamous cell carcinoma, adenocarcinoma or large cell carcinoma) or pulmonary lesions that were strongly suspicious for neoplasia, 4) patients with clinical stages I, II, or III, as classified by the sixth edition of TNM16 with performance status that allowed surgery enrollment (ECOG PS zero or 1), and 5) patients who agreed to participate in the study and signed the informed consent form. Patients were excluded if their diagnosis of non-small cell lung cancer was not confirmed after surgical resection. Patients with bulky lymph node metastases that were considered unresectable and pregnant patients were also excluded. The clinical evaluation included: physical examination, hematologic and biochemic screening, cardiologic evaluation, bronchoscopy when the pulmonary lesions were considered accessible for this method, bone scan, chest and upper abdomen CT, brain MRI or CT. Forty one patients were enrolled in the study. All of the patients were submitted to surgical procedures for diagnosis and treatment which were performed within 30 days after chest CT and SPECT/CT. The type of resection performed on each patient was defined by the thoracic surgery team in accordance with the extent of the primary tumor. Mediastinal systematic lymph node dissection was performed for adequate pathological staging according to the tumor location. For tumors of the right lung, a mediastinal dissection included the ipsilateral hilar region as well as the upper and lower paratracheal, subcarinal and paraesophageal lymph nodes. For left lung tumors, the left hilar region, lower paratracheal area, aortopulmonary window, and para-aortic, subcarinal, and paraesophageal lymph nodes were sampled. Lymph nodes were identified according to the Mountain nodal station classification system16 and were sent for histopathological analysis, which was performed by an experienced pathologist with expertise in lung cancer. The largest diameter of the metastatic foci in the lymph nodes was divided into two groups: those ,10 mm and those $10 mm. 99m

Tc-sestamibi SPECT/CT

The GE Medical Systems, Millennium VG, Hawkeye hybrid equipment was used. Every patient received an injection of 1,110 MBq (30 mCi) of 99mTc-sestamibi, which was prepared following the manufacturerâ&#x20AC;&#x2122;s instructions (Bristol-Myers Squibb Medical Imaging, Massachusetts,

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(UICC), which is the classification proposed by the American Thoracic Society and modified by MountainDresler (MD-ATS).17

USA). Images were acquired 10 minutes after the administration of sestamibi with patients placed in supine positions with their arms elevated. SPECT: Emission images were acquired using a dual-head, large field-of-view scintillation camera equipped with a lowenergy, high-resolution collimator (VPC-45). The images were acquired every 20 seconds, at a 3 ˚ angle, in a circular orbit of 180˚ per detector array, and using a 1286128 matrix. The equipment was calibrated for a photopeak of 140 keV with a symmetric 20% window. CT images were acquired sequentially in a non-dedicated 3rd-generation scanner installed in the SPECT camera gantry, with a 10 mm slice thickness (maximum of 40 slices), a maximum current of 2.5 mA and a 140 kV potential. The raw data from SPECT and CT were transferred from the acquisition equipment to an Entegra workstation (General Electric Healthcare), and the tools provided by the manufacturer were used to process the data. The following iterative processing protocol in standard 2-D was used. The image reconstruction was made after automatic pre-processing, which included the reconstruction of the SPECT plane imaging, the reformatting of the anatomical and functional sections according to the type of organ and the creation of maximum-intensity projection imaging. The Butterworth filter was used with a cutoff frequency of 0.28 Nyquist and an order of 10. After this initial step, the images were reoriented to obtain transaxial, coronal, and sagittal views. All images were independently interpreted by two nuclear physicians who were blinded to the chest CT and pathology findings. Disagreements in the analysis were resolved by consensus. The following aspects were analyzed regarding the primary pulmonary lesion: 1) presence or absence of sestamibi uptake; 2) qualitative visual assessment of sestamibi uptake, which was classified as mild, moderate or intense. As a parameter, the uptake in primary tumor was compared with uptake in physiological thoracic structures as follows: more than soft tissues (mild) and less than hepatic uptake; similar to hepatic uptake (moderate); similar to cardiac uptake (intense). The intensity of uptake was correlated with histology and tumor size. 3) a semiquantitative analysis obtained by outlining the regions of interest (ROI) over the tumor (T) and in the contralateral normal lung (L). ROI was defined manually on coronal images that showed the tumors’ highest degree of uptake. The maximum ROI values were measured, and the TL/T index was calculated. This analysis was correlated with the histology, tumor size and tumor size was analyzed as a continuous variable and also divided into three categories: 1) less than or equal to 3.0 cm, 2) greater than 3.0 cm and less than or equal to 7.0 cm and 3) greater than 7.0 cm. With regard to the lymph nodes, the following aspects were analyzed: 1) the presence or absence of focal uptake in the mediastinum or hilum; 2) a qualitative visual assessment of the sestamibi uptake, which was classified as mild, moderate or intense; 3) a semi-quantitative analysis that was performed in the same manner as the analysis conducted for the primary lesion, comparing radiotracer uptake in the lymph nodes and the lung; and 4) the identification of uptake lymph node chains in accordance with the international system adopted by the American Joint Committee on Cancer (AJCC) and the International Union Against Cancer

Chest CT All CT scans were performed on a Hispeed CT (GE, Milwaukee) after intravenous administration of an iodinated contrast agent at a dose of 1.0 ml/kg. Helical CT scans were performed with a slice thickness of 7 mm, pitch of 1.5 and reconstruction thickness of 5 mm. The scans had a window for the lung parenchyma and a window for the mediastinum and were acquired from the lower cervical region to the upper lumbar region, including the adrenal glands. The images were read independently by two radiologists who were blinded to the SPECT and pathological findings. Disagreements in the assessments were resolved by consensus. In the axial plane, those lymph nodes larger than 10 mm on their smallest axis were considered suspicious for metastasis. The lymph node stations were identified according to the international system adopted by the AJCC and the UICC.17

Statistical analysis To calculate the diagnostic efficacy as determined by the sensitivity, specificity, positive predictive value and negative predictive value, the SPECT/CT results were compared to pathology analyses, which were defined as the reference test. The same assessment was conducted to evaluate the accuracy of chest CTs. A descriptive analysis was performed by measuring the central tendency, dispersion, and relative frequencies of patient and tumor characteristics. The inter-observer agreement was calculated by the Kappa index. In the analysis of the correlation between numerical variables, a Spearman’s correlation coefficient test was used. The chi-square test was used to assess the relationships between categorical variables. Kruskal-Wallis and Mann-Whitney nonparametric tests were used to evaluate the relationships between continuous and categorical variables. The level of significance was set at 0.05. All statistical analyses were performed with the SPSS (version 18.0) software package.

RESULTS From December 2006 to February 2009, 46 patients with potentially resectable lung lesions were included in our study. Among these patients, five were excluded for the following reasons. In one patient, the pathological results revealed a carcinoid tumor. For another patient, a chest CT was not performed at our institution. In three other cases, SPECT evaluations were not performed. The characteristics of the 41 patients that were included in this study are summarized in Table 1. Two lesions, a squamous cell carcinoma, and an adenocarcinoma, were diagnosed in one patient. Therefore, we analyzed a total of 42 primary lung lesions in 41 patients.

Primary tumor Primary tumor: SPECT/CT. We found evidence of radiotracer uptake in 39 out of 42 pulmonary lesions (92.8%). Thirty-one lesions were classified as mild in intensity, and eight lesions were classified as moderate in intensity. In three patients (7.2%), the primary tumor did not

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With regard to the pathological staging of the lymph nodes, 21 patients (51.3%) had no regional metastases and were therefore classified by the TNM system as pN0. Thirteen (31.7%) had metastases only in the hilar lymph nodes and were staged as pN1, and seven patients (17%) were classified as pN2 because they had metastases in the mediastinal lymph nodes.

Table 1 - Patient characteristics (n = 41). Age (years), range (mean) Gender (male/female) Histopathology diagnosis Thoracotomy Fine-needle aspiration Bronchoscopy Histology Adenocarcinoma Squamous cell carcinoma Broncholoalveolar Adenosquamous Non-small cell carcinoma Primary tumor size (cm), range (mean) Tumor classification (TNM) T1 T2 T3 T4 Lesion size Range (mean) without sestamibi uptake (cm) Range (mean) with sestamibi uptake (cm) Number of resected hilar lymph nodes Number of resected mediastinal lymph Hilar metastasis Mediastinal metastasis Size of metastatic foci Hilar lymph nodes ,10 mm $10 mm Mediastinal lymph nodes ,10 mm $10 mm

45-78 (62.6) 28 (68.3%)/13 (31.7%) 24 (58.5%) 11 (26.8%) 6 (14.6%)

Lymph nodes: SPECT/CT and CT

22 (52.2%) 13 (31%) 5 (12%) 1 (2.4%) 1 (2.4%) 1.5-11.5 (4.7)

SPECT/CT with sestamibi correctly identified six out of 19 cases with involvement of the hilar lymph nodes (Figure 1). In four of these cases, the metastatic foci were larger than or equal to 10 mm. In two cases, the foci were smaller than 10 mm. In ten out of 13 cases in which SPECT was falsely negative, the metastases were smaller than 10 mm (i.e., below the spatial resolution threshold of SPECT). In 22 cases, there was no involvement of the hilar lymph nodes by pathology examination, and sestamibi correctly staged 21 of these cases. Therefore, there was one false positive result where the analysis of this lymph node showed a reactive process, with the presence of sinus histiocytosis and follicular hyperplasia. Regarding mediastinum staging by SPECT/CT, the method correctly evaluated only one out of seven patients with nodal metastases, as confirmed by pathology examinations. The positive node in this case was located on the aorto-pulmonary window, and the metastasis was larger than 10 mm (Figure 2). In the six false negative cases, the sizes of the lymph node metastases were smaller than 10 mm in three cases and larger than 10 mm in three other cases. SPECT/CT made a correct diagnosis in 33 out of 34 patients without lymph node involvement, as confirmed by pathology examinations. Therefore, there was one false positive result, which occurred in the same patient in which there was a false positive finding in the hilum. The degree of uptake was considered to be mild in all cases. The mediastinal lymph node uptake index was 0.14 and ranged from 0.2 to 0.47 for the hilar lymph nodes. CT provided the correct diagnosis in nine out of 19 cases with metastasis of the hilum. Of the 22 patients without lymph node involvement, CT was negative in 21 patients. Regarding the mediastinum, CT correctly assessed four out of seven cases with nodal metastases and 31 out of 34 patients without lymph node involvement. The sensitivity, specificity, positive predictive value, and negative predictive value of SPECT/CT and CT in the analyses of the hilar and mediastinal lymph nodes are described in Table 2. Table 3 describes the locations and sizes of the primary tumors, histology, chains with positive nodes, sizes of lymph node metastases, and diagnoses of the two imaging methods (SPECT/CT and CT) for the seven patients with positive lymph nodes in the mediastinum. The reproducibility analysis of the imaging method in relation to the pathology results revealed regular kappa values for CT in the analysis of the mediastinum (0.5) and the hilum (0.4) and a weak agreement for SPECT/CT for the mediastinum (Kappa 0.1) and the hilum (Kappa 0.3). Between the two observers who evaluated the SPECT/CT results, there was complete diagnostic agreement in all 41 cases for the evaluation of the mediastinum (Kappa 1) and disagreement in two cases for the hilum analysis (kappa 0.8). For the CT images, the diagnosis differed in only one

9 (21.4%) 23 (54.8%) 9 (21.4%) 1 (2.4%) 1.5-2.5 (1.9) 2.0-11.5 (5.0) 2-28 (10.3) 4-44 (22.3) 19 (46.3%) 7 (17.7%)

12 (63.1%) 7 (36.9%) 3 (42.9%) 4 (57.1%)

concentrate sestamibi. The mean size of these lesions was 1.9 cm. For those tumors that concentrated sestamibi, the mean size was 5.0 cm. Although the mean size of those lesions that showed sestamibi uptake was significantly higher than the mean size of those lesions that did not (p = 0.009), no statistically significant differences between the intensity of uptake (mild or moderate) and the tumor size (p = 0.8) or between the degree of uptake and the tumor histological type (p = 0.2) were found. Regarding the quantitative analysis, the rate of uptake in primary tumors ranged from 0.17 to 0.76 (average of 0.50). There was no correlation between the rate of uptake and the tumor size (correlation coefficient = 0.081, p = 0.6). Furthermore, there was no statistically significant difference between the rate of uptake and primary tumor lesions that were smaller than or equal to 3.0 cm, greater than 3.0 cm and less than or equal to 7.0 cm or greater than 7.0 cm (p = 0.8). The histological type did not influence the rate of uptake (p = 0.55).

Lymph Nodes The mean number of resected hilar and mediastinal lymph nodes and the number of patients with hilar and mediastinal metastases by pathology examination, which was considered to be the reference method, are described in Table 1. Of the seven patients with metastases in the mediastinal lymph nodes, six also had metastases in the hilum. There was only one case of metastasis in the mediastinum with no involvement of the hilar lymph nodes (skip metastasis). The metastatic foci were smaller than 10 mm in 12 out of 19 patients (63.1%) with metastases of the hilar lymph nodes and in three out of the seven patients (42.9%) with mediastinal lymph node metastases.

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Figure 1 - SPECT/CT showing left hilar sestamibi uptake in a patient with an adenocarcinoma of the left lung.

are the main radiological criteria used to classify lymph nodes as suspicious for metastatic disease; they are usually considered positive when the short axis diameter is larger than 10 mm. However, lymph nodes of a normal size may contain tumor cells and thus lead to false negative results and low sensitivity. With chest CTs, our study found a sensitivity of 57% in the evaluation of the mediastinum, which was similar to the sensitivity described in other studies.3,18 For the hilar lymph nodes, the sensitivity of CT

case in the assessment of the hilum (Kappa 0.9) and in two cases in the assessment of the mediastinum (Kappa 0.8).

DISCUSSION Chest CT is the most commonly performed imaging test for the primary staging of lung cancers. However, it has low sensitivity and may compromise the selection of treatment options for patients. Currently, the sizes of the lymph nodes

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Figure 2 - SPECT/CT showing aorto-pulmonary window (station five) uptake in a patient with an adenocarcinoma of the left lung.

was 47.4%, which was lower than the mediastinal CT sensitivity. In a prospective study that assessed the role of chest CT in the preoperative staging of 49 patients with non-small cell lung cancer, the hilar CT sensitivity was only 11%, which was lower than the sensitivity of 67% found for mediastinal lymph nodes.19 Perhaps this difference is due to the greater difficulty in evaluating the hilar region using this method

than in evaluating other regions. In our study, CT specificity was higher than 90% for both the pulmonary hilum and the mediastinum. Such a high specificity might be related to the study population, which included only patients who were being considered for surgical treatment and who therefore had no bulky adenopathies. Toloza et al. found a mean specificity of 82% (ranging from 57% to 93%) in their metaanalysis. Moreover, the accuracy of chest CTs in the staging

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in staging lung cancers, especially considering its higher availability and lower costs compared to PET.9-13 SPECT/CT showed mild or moderate sestamibi uptake in 39 out of 42 pulmonary lesions (92.85%). Only three lesions did not concentrate sestamibi, and these lesions were the smallest tumors in the sample. However, the mean size of the lesions that showed no uptake was 1.9 cm, which is considered to be within the diagnostic power of modern SPECT scanners. In 31 cases, the degree of uptake was mild according to the criteria adopted in the study (i.e., lower than the degree of uptake in the liver and higher than the degree of uptake in soft tissues). In eight cases, the degree of uptake was similar to the degree of liver uptake and was therefore considered moderate. Although the degree of uptake was considered mild for most lesions, it was much higher than the degree of uptake seen in soft tissues. It is possible that the comparison criteria that were selected in an attempt to make the interpretation more objective were not adequate because we observed that the degree of liver uptake was very similar to the degree of heart uptake, which can be considered intense in these two organs. In contrast to the findings reported with FDG-PET in lung cancer, our study did not show any direct correlation between the degree of sestamibi uptake in primary lesions and lymph nodes and the size and histology classification of tumors. That means, larger lesions or more agressive histology types of tumors were not associated with higher sestamibi uptake. One potential explanation is a difference in the transmembrane electrical potential, while another is the degree of neovascularity. Although aggressive histologies should show higher levels of sestamibi uptake, this phenomenon was not observed in our study sample. The size of the tumor is important because larger tumors are more easily detected by SPECT imaging. However, larger tumors have larger necrotic areas. In addition, necrotic, fibrotic and/or inflammatory tissues were not quantified by the pathology examinations in our study, and no corrective measurements were taken according to the histology findings. In a study evaluating 25 patients with indeterminate pulmonary nodules, Minai et al. found a sensitivity of 85.7% and a specificity of 100% for sestamibi SPECT. Two of the three false negative cases occurred with the smallest nodules in the study, which had sizes ranging from 1.2 to

Table 2 - SPECT/CT and CT results for the analyses of the hilar and mediastinal lymph nodes. Test SPECT/CT (hilum) CT (hilum) SPECT/CT (mediastinum) CT (mediastinum)

Sensitivity

Specificity

PPV

NPV

31.6% 47.4% 14.3%

95.5% 95.5% 97.1%

85.7% 90% 50%

61.8% 67.7% 84.6%

57.1%

91.2%

57.1%

91.2%

Note: PPV: positive predictive value; NPV: negative predictive value.

of the mediastinum was not superior to that found in earlier meta-analyses, suggesting that despite the superior resolution of most modern scanners, CTs did not lead to better accuracy for nodal staging.3 However, in a recent retrospective study that evaluated the accuracy of multi-slice chest CTs in mediastinum nodal staging in 86 patients with non-small cell lung cancer, the sensitivity, specificity, positive and negative predictive values, and accuracy were 100%, 98.5%, 94.4%, 100%, and 98.8%, respectively. For its diagnostic criteria, this study used not only lymph node size but also the locations of the lymph nodes in relation to the position of the primary tumor and the lymph node structure. For example, the presence of central calcification and fat density were used as common signs of negative or normal nodes. Peripheral calcifications and necrosis were also considered as positive findings. When only the sizes of the lymph nodes (short axis larger than 10 mm) were considered, the effectiveness of the test was decreased, with sensitivity, specificity, positive and negative predictive values and accuracy values of 64%, 61%, 87%, 40%, and 62%, respectively. These findings suggest that when factors other than size are also considered, the diagnostic efficacy of multi-slice CTs can be enhanced.20 Although PET results have been shown to be superior to chest CTs in the evaluation of regional lymph nodes, and although the association between PET and CT (PET/CT) seems to be more effective in staging the mediastinum than simply relying on PET imaging,4-6 PET/CT was not available in our institution. The rationale for performing this study was based on the scarce and controversial evidence regarding the value of SPECT/CT with sestamibi

Table 3 - Patients with mediastinal lymph node metastasis (n = 7). Primary tumor localization

Size of primary tumor (cm)

Histology

SRL

2.7

Adenocarcinoma

3 8

ILL ILL

4.5 11.5

Adenocarcinoma Adenocarcinoma

ILL

7.0

Adenocarcinoma

IRL

2.4

Adenocarcinoma

SLL

11.0

Adenocarcinoma

SLL

7.5

SCC

Patient

Positive lymph node stations 2 3 4R 7 5 8 7 8 4R 7 8 4L 5 6 5

Note: SRL superior right lobe; ILL inferior left lobe; IRL inferior right lobe; SLL superior left lobe.

1907

Metastasis size ,10 ,10 $10 ,10 $10 ,10 $10 $10 ,10 ,10 $10 ,10 ,10 ,10 ,10

mm mm mm mm mm mm mm mm mm mm mm mm mm mm mm

SPECT

+ -

+1.7 cm +1.2 cm + 1.1 cm +1.2 cm

Lung cancer nodal staging with SPECT/CT Miziara JM et al.

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1.3 cm.9 Santini et al. also studied the role of sestamibi in the diagnosis of pulmonary lesions. There was sestamibi uptake in 91.6% of the malignant lesions, while only 8.3% of the identified tumors showed no sestamibi uptake.11 These results are consistent with the results of our study. SPECT/CT with sestamibi correctly identified six of 19 cases with involvement of the hilar lymph nodes, with a sensitivity of 31.6%. Only one out of seven patients with mediastinal nodal metastases, identified by pathology examinations, were correctly identified, leading to a sensitivity of only 14.3%. SPECT/CT sensitivity values for both the hilum and the mediastinum were inferior to the sensitivity of chest CT; these values were also lower than the sensitivity values described in previous studies. One hypothesis is that the sizes of the metastases in the lymph nodes were smaller than the sizes reported in previous studies. In the 13 cases in which SPECT have a false negative result for the hilum, the majority of metastases were smaller than 10 mm (n = 10; 76.9%). In the mediastinum, 50% of the lymph nodes were smaller than 10 mm. Regarding mediastinal nodal staging by sestamibi SPECT, Chiti et al. evaluated 36 patients with pulmonary lesions and showed a higher accuracy for SPECT than for chest CT. SPECT correctly staged 10 out of 11 patients with mediastinal metastases and 21 out of 25 without mediastinal metastases, with sensitivity and specificity values of 91% and 84%, respectively. However, node enlargement was seen by CT in the majority of the patients with confirmed nodal involvement (80%), in contrast to our findings.12 Nosotti et al. studied 87 patients with non-small cell lung cancer and found a sensitivity of 54.5% and a specificity of 100% for sestamibi SPECT in the analysis of nodal staging. The lymph nodes were evaluated by mediastinoscopy or thoracotomy; however, the numbers and sizes of the lymph nodes that were resected were not described in detail.13 In a prospective study evaluating the value of PET/CT in the nodal staging of 51 patients with lung cancers who were scheduled for surgery, the sensitivity was as low as 40%, mainly because of the small sizes of the lymph nodes (,10 mm).7 The sensitivity of PET/CT in the preoperative staging of regional thoracic lymph nodes was 54.2% in a study involving 159 patients with resectable lung cancers. The specificity was 91.9%. The mean size of the lymph nodes that were falsely negative on PET/CT was 7.8 mm, with a minimum size of 3 mm and a maximum size of 15 mm. The method correctly identified 85.3% of the lymph nodes that were larger than 10 mm and 32.4% of the patients with lymph node metastases that were smaller than 10 mm. The authors concluded that the spatial resolution of PET/CT appears to be inadequate to detect lymph node metastases that are smaller than 10 mm.21 This finding is also more relevant for SPECT that has lower spatial resolution than PET. Although the hybrid system used in the present study had an attached CT with a much lower resolution than a multidetector CT, the method was effective in excluding the areas of physiological uptake and possibly decreasing the rate of false positive results. There was only one false positive result, which was represented by an inflammatory reaction in the affected lymph node and found through a pathology examination. Previous studies have shown that the fusion of SPECT and CT can help to differentiate tumor activity and

physiological activity.15 The main advantage of SPECT/CT in our study was the ability to distinguish areas of tumor uptake from those of cardiac and/or vascular uptake, helping to reduce the frequency of false positive results while simultaneously providing higher specificity (.95%). Despite the limited resolution of SPECT, which made it difficult to interpret the results due to low sensitivity and many false negative results, the strong agreement between observers in the analysis of the mediastinum and the hilum should be noted. This agreement suggests that image interpretation was consistent between experts and that the poor results were likely due to the limitations of the method. In conclusion, SPECT/CT using 99mTc-sestamibi seems to play no role in the nodal staging of patients with non-small cell lung carcinoma because the technique is associated with a much lower sensitivity than CT or pathology analyses. Moreover, no incremental diagnostic value of SPECT could be observed in the present study, especially when compared to the staging information provided by chest CT. However, more studies are needed that use devices equipped with multidetector CT scanners, which could provide more accurate registration and simultaneously offer more adequate attenuation corrections for emission scans.

AUTHOR CONTRIBUTIONS Miziara JM was responsible for the design and coordination of the project, and writing of the manuscript. Buchpiguel CA was responsible for guiding the project’s implementation and writing of the manuscript. Rocha ET was responsible for guiding the project’s implementation and performing nuclear medicine image analyses. Miziara JEA was responsible for the design of the project and surgical procedures. Garcia GF and Pinheiro MAL were responsible for the analysis of the radiological images. Simo˜es MIP was responsible for the nuclear medicine image analyses. Kerr LM was responsible for all the histopathology analyses.

REFERENCES 1. Detterbeck FC, Jantz MA, Wallace M, Vansteenkiste J, Silvestri GA. Invasive mediastinal staging of lung cancer: ACCP evidence-based clinical practice guidelines (2nd edition). Chest. 2007;132(3 Suppl):202S20S, doi: 10.1378/chest.07-1362. 2. Guimara˜es MD, Chojniak R, Gross JL, Bitencourt AG. Predictive success factors for CT-guided fine needle aspiration biopsy of pulmonary lesions. Clinics (Sao Paulo). 2009;64:1139-44. 3. Toloza EM, Harpole L, McCrory DC. Noninvasive staging of non-small cell lung cancer: a review of the current evidence. Chest. 2003;123(1 Suppl):137S-46S, doi: 10.1378/chest.123.1_suppl.137S. 4. Konishi J, Yamazaki K, Tsukamoto E, Tamaki N, Onodera Y, Otake T, et al. Mediastinal lymph node staging by FDG-PET in patients with nonsmall cell lung cancer: analysis of false-positive FDG-PET findings. Respiration. 2003;70:500-6, doi: 10.1159/000074207. 5. Vansteenkiste JF, Stroobants SG, De Leyn PR, Dupont PJ, Verschakelen JA, Nackaerts KL, et al. Mediastinal lymph node staging with FDG-PET scan in patients with potentially operable non-small cell lung cancer: a prospective analysis of 50 cases. Leuven Lung Cancer Group. Chest. 1997;112:1480-6. 6. Vansteenkiste JF, Stroobants SS. PET scan in lung cancer: current recommendations and innovation. J Thorac Oncol. 2006;1:71-3, doi: 10. 1097/01243894-200601000-00014. 7. Perigaud C, Bridji B, Roussel JC, Sagan C, Mugniot A, Duveau D, et al. Prospective preoperative mediastinal lymph node staging by integrated positron emission tomography-computerised tomography in patients with non-small-cell lung cancer. Eur J Cardiothorac Surg. 2009;36:731-6, doi: 10.1016/j.ejcts.2009.05.044. 8. Piwnica-Worms D, Holman BL. Noncardiac applications of hexakis(alkylisonitrile) technetium-99m complexes. J Nucl Med. 1990;31:1166-7. 9. Minai OA, Raja S, Mehta AC, Sullivan EJ, Khan SU, Dasgupta A, et al. Role of Tc-99m MIBI in the evaluation of single pulmonary nodules: a preliminary report. Thorax. 2000;55:60-2, doi: 10.1136/thorax.55.1.60. 10. Sergiacomi G, Schillaci O, Leporace M, Laviani F, Carlani M, Manni C, et al. Integrated multislice CT and Tc-99m Sestamibi SPECT-CT evaluation of solitary pulmonary nodules. Radiol Med. 2006;111:213-24, doi: 10.1007/s11547-006-0022-7.

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Lung cancer nodal staging with SPECT/CT Miziara JM et al. 17. Mountain CF, Dresler CM. Regional lymph node classification for lung cancer staging. Chest. 1997;111:1718-23, doi: 10.1378/chest.111.6.1718. 18. Webb WR, Gatsonis C, Zerhouni EA, Heelan RT, Glazer GM, Francis IR, et al. CT and MR imaging in staging non-small cell bronchogenic carcinoma: report of the Radiologic Diagnostic Oncology Group. Radiology. 1991;178:705-13. 19. Sioris T, Jarvenpaa R, Kuukasjarvi P, Helin H, Saarelainen S, Tarkka M. Comparison of computed tomography and systematic lymph node dissection in determining TNM and stage in non-small cell lung cancer. Eur J Cardiothorac Surg. 2003;23:403-8, doi: 10.1016/s1010-7940(02) 00806-0. 20. Volterrani L, Mazzei MA, Banchi B, Voltolini L, La Sala F, Carbone SF, et al. MSCT multi-criteria: A novel approach in assessment of mediastinal lymph node metastases in non-small cell lung cancer. Eur J Radiol. 11. 21. Bille A, Pelosi E, Skanjeti A, Arena V, Errico L, Borasio P, et al. Preoperative intrathoracic lymph node staging in patients with nonsmall-cell lung cancer: accuracy of integrated positron emission tomography and computed tomography. Eur J Cardiothorac Surg. 2009;36:440-5, doi: 10.1016/j.ejcts.2009.04.003.

11. Santini M, Fiorello A, Mansi L, Rambaldi PF, Vicidomini G, Busiello L, et al. The role of technetium-99m hexakis-2-methoxyisobutyl isonitrile in the detection of neoplastic lung lesions. Eur J Cardiothorac Surg. 2009; 35:325-31, doi: 10.1016/j.ejcts.2008.09.033. 12. Chiti A, Maffioli LS, Infante M, Grasselli G, Incarbone M, Gasparini MD, et al. Assessment of mediastinal involvement in lung cancer with technetium-99m-sestamibi SPECT. J Nucl Med. 1996;37:938-42. 13. Nosotti M, Santambrogio L, Gasparini M, Baisi A, Bellaviti N, Rosso L. Role of (99m)tc-hexakis-2-methoxy-isobutylisonitrile in the diagnosis and staging of lung cancer. Chest. 2002;122:1361-4, doi: 10.1378/chest. 122.4.1361. 14. Chowdhury FU, Scarsbrook AF. The role of hybrid SPECT-CT in oncology: current and emerging clinical applications. Clin Radiol. 2008;63:241-51, doi: 10.1016/j.crad.2007.11.008. 15. Katyal S, Kramer EL, Noz ME, McCauley D, Chachoua A, Steinfeld A. Fusion of immunoscintigraphy single-photon emission computed tomography (SPECT) with CT of the chest in patients with non-small cell lung cancer. Cancer Res. 1995;55(23 Suppl):5759s-63s. 16. Mountain CF. Revisions in the International System for Staging Lung Cancer. Chest. 1997;111:1710-7, doi: 10.1378/chest.111.6.1710.

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DOI:10.1590/S1807-59322011001100010

CLINICAL SCIENCE

Assessment of symptoms of urinary incontinence in women with polycystic ovary syndrome Thais Montezuma,I Fla´via Igna´cio Antoˆnio,II Ana Carolina Japur de Sa´ Rosa e Silva,III Marcos Felipe Silva de Sa´,III Rui Alberto Ferriani,III Cristine Homsi Jorge FerreiraII I

Universidade de Saõ Paulo, Faculdade de Medicina de Ribeiraõ Preto, Physiotherapy Course, Ribeiraõ Preto/SP, Brazil. II Universidade de Saõ Paulo, Faculdade de Medicina de Ribeiraõ Preto, Department of Biomechanics, Medicine and Rehabilitation of the Locomotor Apparatus, Ribeiraõ Preto/SP, Brazil. III Universidade de Saõ Paulo, Faculdade de Medicina de Ribeiraõ Preto, Department of Obstetrics and Gynecology, Ribeiraõ Preto/SP, Brazil.

OBJECTIVES: The pelvic floor muscles are sensitive to androgens, and due to hyperandrogenism, women with polycystic ovary syndrome can have increased mass in these muscles compared to controls. The aim of this study is to compare reports of urine leakage and quality of life between women with and without polycystic ovary syndrome. METHODS: One hundred thirteen 18- to 40-year-old nulliparous women with polycystic ovary syndrome or without the disease (controls) were recruited at the University Hospital of School Medicine of Sa˜o Paulo University at Ribeira˜o Preto City, Brazil. The subjects were not taking any hormonal medication, had not undergone previous pelvic surgery and did not exercise their pelvic floor muscles. The women were divided into the following four groups: I- polycystic ovary syndrome with normal body mass index (n = 18), II- polycystic ovary syndrome with body mass index $25 (n = 32), III- controls with normal body mass index (n = 29), and IV- controls with Body Mass Index $25 (n = 34). Quality of life was evaluated using the SF-36 questionnaire, and the subjects with urinary complaints also completed the International Consultation on Incontinence Questionnaire Short Form to evaluate the severity of their urinary incontinence. RESULTS: The replies to the International Consultation on Incontinence Questionnaire Short Form revealed a significant difference in urinary function between groups, with 24% of the subjects in group IV reporting urinary incontinence. The mean scores for the SF-36 questionnaire revealed that group II had the lowest quality of life. CONCLUSIONS: The control obese group (IV) reported a higher prevalence of urinary incontinence. There was no difference in the reported frequency of urine loss between the polycystic ovary syndrome and control groups with normal body mass index or between the polycystic ovary syndrome and control groups with body mass index $25. KEYWORDS: PCOS; Androgens; Pelvic floor muscle; Urinary incontinence; Quality of life. Montezuma T, Antoˆnio FI, Rosa eSilva ACJS, Sa´ MFS, Ferriani RA, Ferreira CHJ. Assessment of symptoms of urinary incontinence in women with polycystic ovary syndrome. Clinics. 2011;66(11):1911-1915. Received for publication on June 21, 2011; First review completed on July 12, 2011; Accepted for publication on July 18, 2011 E-mail: cristine@fmrp.usp.br Tel.: 55 16 3602-3058

is plausible that women with PCOS may have an increased muscle mass relative to controls. Anthropometric characteristics, including excess fat mass, percent body fat, and body fat androgen distribution, have been well documented in PCOS.3 However, only a limited amount of information is available regarding the possible connection between PCOS and increased muscle mass in general.3 The association between obesity and PCOS was first described by Stein and Leventhal.4 Obesity among women with PCOS has implications for general health and is also a risk factor for pelvic floor dysfunction.5,6 Obesity can increase intra-abdominal pressure and thus expose the pelvic support structures and organs to a chronic state of stress, with subsequent pelvic floor muscle fatigue and/or chronic stretch of the pudendal nerve.7 According to some studies, these structures are important for the maintenance of urinary continence; therefore, obesity represents a risk factor for urinary incontinence (UI).8-10

INTRODUCTION Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders in women of reproductive age, affecting an average of seven percent of all women of reproductive age. PCOS is associated with clinical and biochemical hyperandrogenism, menstrual irregularities, and acne.1,2 It is commonly known that the use androgens and anabolic steroids increases muscle mass and strength. Because of the characteristics of obesity and hyperandrogenism in PCOS, it

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Women with PCOS had clinical or biochemical hyperandrogenism, and the control women had normal ovulatory cycles (which ranged from 26 to 33 days).

Recent studies have shown that androgens may potentially play an important role in changes in the pelvic floor and lower urinary tract, as the muscles in these structures— particularly the levator ani and the urethral sphincter—are sensitive to androgens.11,12 Urinary incontinence is a common health problem among women and is highly prevalent in older women; however, according to some studies, urinary incontinence is unexpectedly prevalent among young females as well.13-15 Ha¨gglund et al13 investigated the prevalence of urinary incontinence among females with a special focus on younger women, and their principal finding was that urinary incontinence was reported by 23% of nulliparous women, with one-eighth of women below 30 years of age reporting problems with urinary incontinence. Polycystic ovary syndrome (PCOS) typically manifests during the reproductive period, and no information is available on the prevalence of pelvic floor dysfunction (and urinary incontinence in particular) among women with PCOS. Because there is a high incidence of obesity among women with PCOS, two counteracting variables might affect the function of the pelvic floor muscles and cause problems related to this function, including UI. These variables are obesity itself as a source of overload on the muscles of the pelvic floor and hyperandrogenism as a factor that is likely to improve the function of the pelvic floor muscles. In light of the paucity of previous studies regarding this topic, the objective of the present investigation was to assess UI rates, symptoms of urine loss and quality of life in obese and non-obese women with PCOS and to compare these findingm to obese and non-obese women without PCOS.

Evaluation Each subject completed the International Consultation on Incontinence Questionnaire (ICIQ-SF) to assess her UI symptoms; the questionnaire was validated and translated into Portuguese. Women who reported an involuntary loss of urine one or more times per week over the preceding three months were classified as incontinent.17 Each participant also completed a questionnaire to assess her health-related quality of life (SF-36), and the incontinent women additionally completed the International Consultation on Incontinence Questionnaire-Short Form (ICIQ-SF), which is a specific questionnaire that assesses the severity of UI symptoms. The ICIQ-SF consists of six questions on the subject’s reports of urinary incontinence during the previous four weeks. Based on the responses to Q3, Q4, and Q5, a score is calculated from the sum of the score for each answer; this score ranges from zero (when there is no report of urine leakage, reflecting no impact on the quality of life) to 21 (highest severity of UI and the maximum impact on the quality of life). Based on the mean scores for Q3, Q4, and Q5, Klovning et al.18 classified the index of severity of UI and related QOL into the following five levels: slight (1-5), moderate (6-12), severe (13 -18), and highly severe (19-21). The SF-36 is a widely used and validated instrument that includes the following eight domains: functional capacity, limitation of physical aspects, pain, general health, vitality, social aspects, emotional aspects, and mental health. These eight domains range from 0 (zero) to 100 (one-hundred), where 0 = worst and 100 = best score for each domain.19

MATERIALS AND METHODS

Statistical Analysis

We conducted an observational cross-sectional controlled study at the University Hospital, Faculty of Medicine of Ribeira˜o Preto, University of Sa˜o Paulo (HC-FMRPUSP). The project was approved by the Research Ethics Committee of HC-FMRP (protocol no. HCRP 11165/2007), and each subject provided written informed consent prior to her participation. The study included a sample of 113 women with PCOS and controls who were seen at this institution; they were divided into the following four groups: group I consisted of 18 women with normal body mass index (BMI) (between 18.5 and 24.9 kg/m2) and PCOS; group II consisted of 32 overweight or obese women (BMI 25 to 39.9 kg/m2) with PCOS; group III consisted of 29 women without PCOS and with normal BMI; and group IV consisted of 34 women without PCOS and with BMI $25 kg/m2.

We analyzed the data using the PROC MEANS and PROC FREQ features of the SAS 9.0 software program. A quantile regression model was used to analyze the quantitative variables20 to compare the median K values between the various groups of interest. The results were obtained using the STATA software program. The qualitative variables were analyzed with the Fisher’s exact test using the SAS 9.0, PROC FREQ software program.

RESULTS The mean age (and range) was 24.78 years (18-32) for group I, 28.75 years (18-38) for group II, 31.69 (23-39) years for group III, and 30.61 (21-38) for group IV. Groups II and IV were similar with respect to age (p = 0.32), whereas groups I and III were not (p,0.01). The groups were similar with respect to skin color (p = 0.63). Table 1 shows the prevalence of UI in the four groups. Group IV had the highest prevalence (24%) of urinary loss and was followed by groups III, II, and then I. The Fisher’s exact test revealed a difference between all four of the groups (p = 0.04). However, when the groups with similar BMI were examined (Table 2), we found no significant difference between groups I and III (p = 0.52) or between groups II and IV (p = 0.08). An analysis of the ICIQ-SF results revealed no statistically significant differences between the groups with regard to the severity of the urinary complaints.

Subjects The diagnosis of PCOS was based on the criteria of the 2003 Rotterdam Consensus,16 as used by the Department of Endocrine Gynecology, HC-FMRP-USP. The following inclusion criteria were applied: nulliparous women age 18 to 40 years, no previous pelvic surgery or pregnancy, not engaged in training of pelvic floor muscles, and not using any hormonal medicine that could influence the muscle. We also excluded women with an androgen-secreting neoplasm, pituitary adenoma, adrenal hyperplasia, acromegaly or Cushing’s syndrome.

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Table 1 - Prevalence of incontinence among the women in the four groups, and the association of the groups with reports of urinary incontinence. Groups

Women with urinary loss (N)

Percentage of women with urinary loss

I- PCOS with normal BMI II- PCOS with BMI $25 kg/m2 III- Control with normal BMI BMI IV- Control with BMI $25 kg/m2

18 32 29 34

0 2 2 8

0% 6% 7% 24%

Table 3 summarizes the average values that were obtained for each domain of the SF-36 in each of the various groups. Group II had the lowest averages with regard to functional capacity, limitation of physical aspects, pain, general health, vitality, social aspects, and mental health. A comparison of groups II and IV using the quantile regression model revealed significant differences in the functional capacity (p,0.02), general health (p,0.01) and social aspects (p,0.01) domains.

p-value

0.04

reported UI more frequently, and this effect was especially true in the group without PCOS. In this respect, these findings agree with previous reports that indicate that being overweight (and obesity in particular) has a negative effect on the structures of the pelvic floor by weakening the muscles and fascia that are important for maintaining continence and by possibly causing neurologic damage and increased intra-abdominal pressure.7 Thus, obesity is a risk factor for the development of UI.8-10 The women in the control group with BMI $25 reported the highest prevalence of UI (24% of the women in this group) and presented the highest frequency of urine loss, the highest impact of urine loss on daily life and the highest number of activities that were affected by this loss. An analysis of the results of the quality of life questionnaire (SF-36) revealed that the PCOS group with BMI $25 reported the lowest mean values with regard to functional capacity, limitation of physical aspects, pain, general health status, vitality, social aspects, and mental health. These results may be explained by previous studies that demonstrated that PCOS involves some risk factors for the development of cardiovascular disease, such as insulin resistance, dyslipidemia, diabetes mellitus, systemic arterial hyeon, endothelial dysfunction, central obesity, chronic proinflammatory markers, systemic arterial hypertension, and poor physical fitness.5,25,26 A non-dipping blood pressure (BP) pattern can be considered to be an independent predictor of future cardiovascular events. A recent crosssectional study by Kargili and collaborators27 was performed to identify any relationship between changes in BP patterns during sleep and PCOS. Their study revealed that a non-dipping BP pattern is highly prevalent among PCOS patients. This condition contributes to the risk of developing cardiovascular disease in women with PCOS and to a subsequent decrease in the quality of life. These factors, together with the other risk factor, obesity, tend to impair the quality of life of women with PCOS and with a BMI $25.5,28 According to a review by Spritzer,29 women with hirsutism—which is highly prevalent among women with PCOS—experience marked psychosocial discomfort that

DISCUSSION In the present study, we investigated the hypothesis that hyperandrogenism in women with PCOS might act as a protective factor against UI. This hypothesis was based on studies reporting that androgens can play an important role in changes in the pelvic floor and in the lower urinary tract, as the muscles of these structures—in particular, the levator ani and the urethral sphincter—express high number of androgen receptors and are therefore sensitive to androgens.11,12,21,22 In addition, androgen receptors are present in the smooth muscle of various female urogenital tissues, thereby suggesting the importance of androgens in the lower urinary tract.23 In a 2011 study, Mammadov et al24 verified the effect of testosterone treatment on the urodynamic findings and histopathomorphology of the pelvic floor muscle in female rats with experimental stress-induced urinary incontinence. These authors observed that after testosterone administration, there were increases both in pressure loss in the urodynamics findings and in the muscle’s cross-sectional area. Although the aforementioned hypothesis is plausible, the present study is the first to compare the frequency of reports of urine loss between women with and without PCOS. To help compensate for the effects of BMI, we investigated women with and without PCOS who were divided into groups of BMI that were ,25 or $25. The two PCOS groups reported UI less frequently than did the control groups. The first analysis revealed a difference between the four groups; however, this difference disappeared when the groups were analyzed according to BMI. The difference between the four groups was due to the fact that the women with BMI $25

Table 2 - Comparison of the groups with the same BMI using the Fisher’s exact test. Total n

Urinary complaints

Groups No

I- PCOS with normal BMI III- Control with normal BMI II- PCOS with BMI $25 kg/m2 IV- Control with BMI $25 kg/m2

p-value

Yes

18 0% 100% 27 93% 30 94% 26 76%

0 93%% 94 76

0 0% 2 7% 2 6% 8 24%

0 7 6 24

1913

18 29 32 34

0.52 0.08

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The Center for Quantitative Methods (CEMEQ-FMRP-USP) for valuable advice regarding the statistical analysis.

Table 3 - Average scores for each SF-36 domain within the different groups.

I Functional capacity Limitation of physical aspects Pain General health Vitality Social aspects Emotional aspects Mental health

AUTHOR CONTRIBUTIONS

Group

Domain

III

Montezuma T was responsible for the experiments. Antonio FI wrote the manuscript and performed the experiments. Rosa e Silva ACJS, Silva de Sa´ MF, Ferriani RA contributed to the final version of the manuscript. Ferreira CHJ contributed to the final version of the manuscript, conceived, and designed the experiments.

95.56 91.67

80.65 84.68

94.79 91.67

90.76 85.61

73.94 72.50 64.72 80.56 74.04 66.22

66.35 68.77b 55.65 70.97b 73.12 60.77

75.88 80.46 62.71 84.90 72.22 65.67

78.27 82.79b 66.67 83.33b 80.81 70.18

REFERENCES 1. Thomson RL, Buckley JD, Moran LJ, Noakes PM, Clifton PM, Norman RJ, et al. Comparison of aerobic exercise capacity and muscle strength in overweight women with and without polycystic ovary syndrome. An International Journal of Obstetrics and Gynecology. 2009;116:1242-50, doi: 10.1111/j.1471-0528.2009.02177.x. 2. Mason H, Colao A, Blume-Peytavi U, Rice S, Qureshi A, Pellatt L, et al. Polycystic ovary syndrome (PCOS) trilogy: a translational and clinical review. Clinical Endocrinology. 2008;69:831-44, doi: 10.1111/j.1365-2265. 2008.03329.x. 3. Douchi T, Yamamoto S, Oki T, Maruta K, Kuwahata R, Nagata Y. Serum androgen levels and muscle mass in women with polycystic ovary syndrome.Obstetrics Gynecology. 1999;94:337-40. 4. Stein IF and Leventhal ML. Ammenorrhoea associated with bilateral polycystic ovaries. American Journal Obstetrics Gynecology. 1935; 29:181-91. 5. Kousta E, Tolis G, Franks S. Polycystic Ovary Syndrome. Revise diagnostic criteria and long-term health consequences. Hormones 2005;4:133-47. 6. Whitcomb EL, Lukacz ES, Lawrence JM, Nager CW, Luber KM. Prevalence and degree of bother from pelvic floor disorders in obese women. International Urogynecology Journal and Pelvic Floor Dysfunction. 2009;20:289-94, doi: 10.1007/s00192-008-0765-x. 7. Noblett KL, Jensen JK, Ostergard DR. The relationship of body mass index to intra-abdominal pressure as measured by multichannel cystometry. International Urogynecology Journal and Pelvic Floor Dysfunction. 1997;8:323-6, doi: 10.1007/BF02765589. 8. Han MO, Lee NY, Park HS. Abdominal Obesity is Associated with Stress Urinary Incontinence in Korean Women. International Urogynecology Journal. 2005;17:35-9, doi: 10.1007/s00192-005-1356-8. 9. Subak LL, Whitcomb E, Shen H, Saxton J, Vittinghoff E, Brown JS. Weight Loss: A Novel and Effective Treatment for Urinary Incontinence. Journal of Urology. 2005;174:190-5, doi: 10.1097/01.ju.0000162056.30326.83. 10. Cummings JM, Rodining CB. Urinary Stress Incontinence Among Obese Womem: Review of Pathophysiology Therapy. International Urogynecology Journal. 2000;11:41-44, doi: 10.1007/s001920050008. 11. Nnodim JO. Quantitative study of the effects of denervation and castration on the levator ani muscle of the rat. The Anatomical Record. 1999;255:324-33, doi: 10.1002/(SICI)1097-0185(19990701)255:3,324::AIDAR8.3.0.CO;2-1. 12. Nnodim JO. Testosterone mediates satellite cell activation in denervated rat levator ani muscle. The Anatomical Record. 2001;263:19-24, doi: 10. 1002/ar.1072. 13. Ha¨gglund D, Olsson H, Leppert J. Urinary incontinence: an unexpected large problem among young females. Results fromapopulation-based study. Family Practice. 1999;16:506-9, doi: 10.1093/fampra/16.5.506. 14. Simpson L. Stress incontinence in younger women: prevention and treatment. Nursing Standard. 2000;14:49-54; quiz 56,58. 15. Brown SJ, Donath S, MacArthur C, McDonald EA, Krastev AH. Urinary incontinence in nulliparous women before and during pregnancy: prevalence, incidence, and associated risk factors. International Urogynecology Journal Pelvic Floor Dysfunction. 2010;21:193-202, doi: 10.1007/s00192-009-1011-x. 16. The Rotterdam ESHRE/ASRM – Sponsored PCOS consensus workshop group. Revised 2003 consensus on diagnostic criteria and long-term health risks related to polycystic ovary syndrome (PCOS). Hum Reprod. 2004;19:41-7, doi: 10.1093/humrep/deh098. 17. Tamanini JT, Dambros M, D’ancona CA, Palma PC, Netto NR. Validation of the ‘‘International Consultation on Incontinence Questionnaire — Short Form’’ (ICIQ-SF) for Portuguese. Revista Sau´de Pu´blica. 2004;38:438-44. 18. Klovning A, Avery K, Sandvik H, Hunskaar S. Comparison of two questionnaires for assessing the severity of urinary incontinence: The ICIQ-UI SF versus the incontinence severity index. Neurourology and Urodynamics. 2009;28:411-5, doi: 10.1002/nau.20674. 19. McHorney CA, Ware JE, Raczek AE. The MOS 36-item short-form health survey (SF-36): II. Psychometric and clinical tests of validity in measuring physical and mental health constructs. Medical Care. 1993;31:247-63. 20. Koenker R, Bassett G. Regression quantiles. Econometrica. 1978; 46:33-50, doi: 10.2307/1913643. 21. Copas P, Bukovsky A, Asbury B, Elder RF, Caudle MR. Estrogen, progesterone, and androgen receptor expression in levator ani muscle

p-value = 0.02. p-value,0.01. Quantile regression analysis comparing the PCOS group with BMI $25 to its control with the same BMI. b

leads to conflicts that compromise their quality of life; we observed this situation in the present study as well. The general quality of life that was evaluated in these women might not depend on UI, as several aspects were evaluated, and several studies that did not assess UI also indicated a poorer general quality of life among obese women with PCOS. The hypothesis that PCOS may serve a protective role against UI is not supported by the present findings. However, some limitations of the present study should be discussed, and the most important of these is the fact that a convenience sample was used that only included nulliparous women. On one hand, this choice controlled the parity variable, which is known to be related to UI; on the other hand, the prevalence of UI among nulliparous women is much lower than among parous women. It should also be noted that, even though a validated instrument was used to assess the report of UI and although all interviews were conducted by one examiner, several studies have indicated that UI tends to be underreported.30,31 This effect may explain the seemingly small number of affected patients in this study, and it represents a limitation. Moreover, the stringent inclusion criteria may have also contributed to the small sample size. It is also important to note that a physical examination was not performed. This omission represents another limitation of this study because a symptom-based diagnosis can underestimate the true presence of urinary incontinence when diagnosed by physical examination. Despite these limitations, this first report should serve as the basis for future studies with representative stratified samples of nulliparous and multiparous women in which reports of UI can be correlated to androgen levels and PFM to clarify the impact of PCOS on these variables. A higher percentage of obese women in the control (nonPCOS) group reported UI. There was no difference in the frequency of reports of urine loss between the PCOS group with normal BMI and its control group with normal BMI, nor between the PCOS group with BMI$25 and its control group with BMI$25. The quality of life of women with PCOS and BMI$25 is lower than that of women without PCOS.

ACKNOWLEDGEMENTS We gratefully acknowledge the Foundation for the Support of Research of Sa˜o Paulo State (FAPESP) and Professor Edson Zangiacomi Martinez from

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22. 23.

24.

25.

Urinary incontinence in Polycystic Ovary Syndrome Montezuma T et al.

and fascia. Journal of Women’s Health and Gender-Based Medicine. 2001;10:785-95, doi: 10.1089/15246090152636541. Celayir S, Ilce Z, Dervisoglu S. The sex hormone receptors in the bladder in childhood-I: preliminary repost in male subjects. European Journal of Pediatric Surgery. 2002;12:312-7, doi: 10.1055/s-2002-35951. Berman JR, Almeida FG, Jolin J, Raz S, Chaudhuri G, Gonzalez-Cadavid NF. Correlation of androgen receptors, aromatase, and 5-alpha reductase in the human vagina with menopausal status. Fertility and Sterility. 2003;79:925-31, doi: 10.1016/S0015-0282(02)04923-3. Mammadov R, Simsir A, Tuglu I, Evren V, Gurer E, Ozyurt C. The effect of testosterone treatment on urodynamic findings and histopathomorphology of pelvic floor muscles in female rats with experimentally induced stress urinary incontinence.International Urology and Nephrology. Int Urol Nephrol. 2011 Mar 29. [Epub ahead of print]. de Azevedo GD, Costa EC, Micussi MT, de Sa´ JC. [Lifestyle modifications in the polycystic ovary syndrome: role of physical exercise and importance of multidisciplinary approach]. [Article in Portuguese]. Rev Bras Ginecol Obstet. 2008;30:261-7.

26. Wild S, Pierpoint T, McKeigue P, Jacobs H. Cardiovascular disease in women with polycystic ovary syndrome at long-term follow-up: a retrospective cohort study. Clinical Endocrinology. 2000;52:595-600, doi: 10.1046/j.1365-2265.2000.01000.x. 27. Kargili A, Karakurt F, Kasapoglu B, Derbent A, Koca C, Selcoki Y. Association of polycystic ovary syndrome and a non-dipping blood pressure pattern in young women. Clinics. 2010;65:475-9, doi: 10.1590/ S1807-59322010000500004. 28. Gigante DP, Barros FC, Post CL, Olinto MTA. Prevaleˆncia de obesidade em adultos e seus fatores de risco. Rev Saude Publ. 1997;31:236-46, doi: 10.1590/S0034-89101997000300004. 29. Spritzer PM. Revisitando o Hirsutismo. Arquivos Brasileiros de Endocrinologia e Metabologia. 2002;46:127-36. 30. Rodrigues RAP, Mendes MMR. Incontineˆncia urina´ria em idosos: proposta para a conduta da enfermeira. Rev Latino-Am Enfermagem. 1994;2(2):5-20. 31. Butler RN, Maby JI, Montella JM, Yong GGPH. Urinary incontinence: keys to diagnosis of the older woman. Geriatrics. 1999;54(10):29-30.

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DOI:10.1590/S1807-59322011001100011

CLINICAL SCIENCE

The validity and reliability of the Portuguese versions of three tools used to diagnose delirium in critically ill patients Dimitri Gusmao-Flores,I,VII Jorge Ibrain Figueira Salluh,II,III Felipe Dal-Pizzol,IV Cristiane Ritter,IV Cristiane Damiani Tomasi,IV Marco Antoˆnio Sales Dantas de Lima,V Lauro Reis Santana,VI Rita Ma´rcia Pacheco Lins,VI Patrıćia Pimenta Lemos,VI Gisele Vasconcelos Serpa,VI Jenisson Oliveira,VI Ricardo A´vila Chalhub,VII Melissa Tassano Pitrowsky,II Acioly L.T. Lacerda,VIII Karestan C. Koenen,IX Lucas C. QuarantiniVI,VII I Universidade Federal da Bahia, University Hospital Prof. Edgar Santos, Intensive Care Unit, Salvador, BA/Brazil. II D’Or Institute of Research and Education, Rio de Janeiro, RJ/Brazil. III Instituto Nacional do Caˆncer (INCA), Intensive Care Unit, Rio de Janeiro, RJ/Brazil. IV Universidade do Extremo Sul Catarinense, Laborato´rio de Fisiopatologia Experimental and Instituto Nacional de Cieˆncia e Tecnologia Translacional em Medicina (INCT-TM), Programa de Po´s-Graduaçaõ em Cieˆncias da Sau´de, Unidade Acadeˆmica de Cieˆncias da Sau´de, Criciu´ma/SC, Brazil. V Instituto Nacional do Caˆncer (INCA), Neurosurgery Section, Rio de Janeiro, RJ/Brazil. VI University Hospital Prof. Edgar Santos, Universidade Federal da Bahia, Department of Psychiatry, ´ rgaõs e Sistemas, Instituto de Salvador, Bahia/Brazil. VII Universidade Federal da Bahia, Programa de Po´s-graduaçaõ em Processos Interativos dos O Cieˆncias da Sau´de (ICS), Salvador, BA/Brazil. VIII Universidade Federal de Saõ Paulo, Laborato´rio Interdisciplinar de Neuroscieˆncias Clıńicas – LiNC, Department of Psychiatry, Saõ Paulo/SP, Brazil. IX Harvard School of Public Health, Departments of Society, Human Development, and Health & Epidemiology.

OBJECTIVES: The objectives of this study are to compare the sensitivity and specificity of three diagnostic tools for delirium (the Intensive Care Delirium Screening Checklist, the Confusion Assessment Method for Intensive Care Units and the Confusion Assessment Method for Intensive Care Units Flowsheet) in a mixed population of critically ill patients, and to validate the Brazilian Portuguese Confusion Assessment Method for Intensive Care Units. METHODS: The study was conducted in four intensive care units in Brazil. Patients were screened for delirium by a psychiatrist or neurologist using the Diagnostic and Statistical Manual of Mental Disorders. Patients were subsequently screened by an intensivist using Portuguese translations of the three tools. RESULTS: One hundred and nineteen patients were evaluated and 38.6% were diagnosed with delirium by the reference rater. The Confusion Assessment Method for Intensive Care Units had a sensitivity of 72.5% and a specificity of 96.2%; the Confusion Assessment Method for Intensive Care Units Flowsheet had a sensitivity of 72.5% and a specificity of 96.2%; the Intensive Care Delirium Screening Checklist had a sensitivity of 96.0% and a specificity of 72.4%. There was strong agreement between the Confusion Assessment Method for Intensive Care Units and the Confusion Assessment Method for Intensive Care Units Flowsheet (kappa coefficient = 0.96). CONCLUSION: All three instruments are effective diagnostic tools in critically ill intensive care unit patients. In addition, the Brazilian Portuguese version of the Confusion Assessment Method for Intensive Care Units is a valid and reliable instrument for the assessment of delirium among critically ill patients. KEYWORDS: CAM-ICU; ICDSC; CAM-ICU Flowsheet; Critical care; Delirium. Gusmao-Flores D, Salluh JIF, Dal-Pizzol F, Ritter C, Tomasi CD, Lima MASD, et al. The validity and reliability of the Portuguese versions of three tools used to diagnose delirium in critically ill patients. Clinics. 2011;66(11):1917-1922. Received for publication on May 19, 2011; First review completed on June 14, 2011; Accepted for publication on July 18, 2011 E-mail: dimitrigusmao@gmail.com Tel.: 00 55 71 3245-2515

intensive care units (ICU) and is associated with increased mortality, longer hospital stays, and long-term cognitive impairment.1-4 Despite its high prevalence and its negative impact on outcomes, the epidemiology and clinical management of delirium have long been compromised by the lack of uniform terminology and validated instruments for detecting and monitoring at-risk patients. Recently, an international effort culminated in a uniform definition and terminology for delirium.5 The need for a specialized professional to evaluate patients according to the Diagnostic and Statistical Manual of

INTRODUCTION Delirium is an acute and fluctuating disturbance of the consciousness that occurs in up to 80% of patients in

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The ICDSC and the CAM-ICU were previously translated into Portuguese using the recommendations of the International Society of Pharmacoeconomics and Outcomes Research (ISPOR)14 by Salluh and Dal-Pizzol (www.icudelirium.com). Thirty minutes after the intensivist’s initial evaluation based on the CAM-ICU, a psychiatrist or neurologist applied the DSM-IV criteria as a reference standard. Subsequently, the patient was evaluated by the same intensivist using the CAM-ICU Flowsheet15 and the ICDSC. The items evaluated by the ICDSC included the following: changes in the level of consciousness, inattention, disorientation, hallucinations, delusions, psychosis, psychomotor agitation or retardation, speech or inappropriate mood, sleep/wake cycle disturbance, and symptom fluctuations.16 Patients were considered to have delirium if the ICDSC was equal to or greater than 4. Scores between 1 and 3 indicated subsyndromal delirium. The CAMICU Flowsheet was developed from the CAM-ICU and involves switching the original numbering of features 3 and 4, as most ICU patients with delirium are given positive scores in the order of the Flowsheet; switching the numbering allows the CAM-ICU Flowsheet to be completed with only three features and the fourth feature is only necessary in a minority of patients. The intensivists who performed the CAM-ICU and ICDSC were kept unaware of the clinical diagnoses made by the psychiatrist or neurologist. Patients diagnosed with delirium on any scale were classified into one of three groups: hypoactive, hyperactive, or mixed.17 Delirium subtypes were classified into motoric subtype groupings according to the Richmond Agitation Sedation Scale (RASS). Patients were considered to have hypoactive delirium if their RASS ratings were between 23 and 0, and were considered to have hyperactive delirium if their RASS ratings were between 1 and 4; mixed-type delirium was defined as alternating between these two ranges. Demographic and clinical characteristics were collected in addition to the APACHE II score. Patients were followed for up to 28 days, and the following outcomes were recorded: the ICU length of stay, the duration of mechanical ventilation, and 28-day mortality.

Mental Disorders (DSM-IV) and the dependence on clinical evaluations rather than validated diagnostic tools frequently leads to the underdiagnosis of delirium.6,7 In 2001, an adapted Confusion Assessment Method (CAM) was validated in a cohort of critically ill patients.8,9 Since then, the CAM-ICU and other tools, such as the Intensive Care Delirium Screening Checklist (ICDSC), have been tested in various ICU populations.10 Compared to the delirium checklist (ICDSC), the CAM-ICU demonstrated good agreement with delirium detection for critical care surgical subjects.11 In a large prospective evaluation, Pun et al. showed that the CAM-ICU demonstrated good compliance and excellent interrater reliability when implemented on a large scale by nursing staff.12 Recently, the CAM-ICU Flowsheet derived from the CAM-ICU was developed to reduce the time required for patient assessment.13 Although a Brazilian national survey of ICU physicians showed that the Brazilian Portuguese version of the CAMICU is the most widely used diagnostic tool for delirium diagnosis in critically ill patients, no validation of this tool had been performed prior to the present study.7 The main objectives of the present study were to compare the sensitivity and specificity of three diagnostic tools for delirium (the ICDSC, the CAM-ICU and the CAM-ICU Flowsheet) in critically ill patients and to validate the CAMICU in Brazilian Portuguese.

MATERIALS AND METHODS The study was conducted in four medical-surgical intensive care units (two general ICUs in university hospitals, one medical-surgical ICU in a tertiary hospital and one medicalsurgical ICU in a comprehensive cancer center) in three cities in diverse regions of Brazil (Salvador/Bahia in the Northeast, Rio de Janeiro/RJ in the Southeast, and Criciu´ma/Santa Catarina in the South). Each institution recruited a different number of patients. Two units in Salvador (one general ICU in a university hospital and one in a tertiary hospital) enrolled a total of 30 patients, one center in Rio de Janeiro (medical-surgical ICU) recruited 25 patients, and one center in Criciu´ma (general ICU) recruited 64 patients. Data collection was conducted between July and November 2010. The local ethics committees approved the study. Non-consecutive patients over 18 years of age and hospitalized in the ICU for more than 48 hours were included. This convenience sample was obtained with two evaluations every week according to the availability of participating neurologists and psychiatrists to perform evaluations using DSM-IV criteria. All patients had to be arousable (with a score of greater than or equal to -3 according to the Richmond Agitation Sedation Scale) for the evaluation. To prevent the effects of withdrawal, patients were excluded if they had a history of alcohol or narcotic abuse. Those who were unable to communicate (i.e., because of hearing and/or visual impairment) or who did not understand Portuguese were also excluded. Only one intensivist in each unit was responsible for the application of delirium scales. All intensivists who applied the CAM-ICU and CAM-ICU Flowsheet were trained and had expertise in the use of the tools. With the exception of one center that used two psychiatrists to simultaneously rate the patients, the DSM-IV evaluation was conducted by only one neurologist or psychiatrist.

Statistical Analysis Standard descriptive statistics were applied. Using 2x2 tables, the diagnostic values of the CAM-ICU, the CAM-ICU Flowsheet and the ICDSC were described with regard to sensitivity (true positive/[true-positive + false-negative]), specificity (true-negative/[false-positive + true-negative]), positive predictive value (true-positive/[true-positive + falsepositive]), and negative predictive value (true-negative/[falsenegative + true-negative]). The kappa test was used to verify the reproducibility between instruments, and the chi-square test was used to detect differences in the diagnoses based on the instruments. A receiver operating characteristic (ROC) curve was used to evaluate the performance of the ICDSC in classifying delirium. Statistical analyses were performed with the statistical software package STATA (version 10.0) using a significance level of 5%.

RESULTS The characteristics of the 119 patients who met the inclusion criteria are presented in Table 1.

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The ICDSC classified delirium adequately when compared with the DSM-IV (area under the ROC curve = 0.91). The ROC curve is displayed in Figure 1. A diagnostic cutoff value of 5 or more for the ICDSC total score provided 67.5% sensitivity and 96.2% specificity for diagnosing delirium. With this cutoff, delirium was correctly classified in 86.5% of cases.

Table 1 - The primary patient characteristics. Gender Male Female Age (mean ¡ SD) Apache II (mean ¡ SD) Ventilation Spontaneous Mechanical Non-invasive Type of patient Medical Surgical Main reason for ICU admission Cardiovascular Sepsis Respiratory failure Neurologic Trauma Abdominal surgery Renal failure* Other Delirium diagnosed according to DSM-IV 28-day mortality

70 (58.3%) 49 (42.7%) 57¡16 15¡6 50 (41.6%) 58 (49.1%) 11 (9.3%)

DISCUSSION The aim of this study was to validate the Brazilian Portuguese version of the CAM-ICU according to DSM-IV criteria. The scale showed an overall sensitivity of 72.5% (95% CI: 55.9% – 84.9%) and specificity of 96.2% (95% CI: 88.5% – 99%). Moreover, the scale demonstrated high positive (90.6%; 95% CI: 73.8% – 97.5%) and negative (87.4%; 95% CI: 78.1% – 93.2%) predictive values, which suggests that very few cases of delirium remain unidentified when the scale is used systematically. Thus, our data demonstrate that the CAM-ICU is valid in Brazilian Portuguese. In addition, there is high accuracy for delirium diagnosis among the three tools (CAM-ICU, CAM-ICU Flowsheet, and ICDSC), and the CAM-ICU and CAM-ICU Flowsheet can be used interchangeably. The development and validation of diagnostic tools is important to a thorough understanding of clinical disorders. Unfortunately, studies have demonstrated that a clinical impression is insufficient for delirium diagnosis.18 Recently, a Dutch group observed a low sensitivity for delirium diagnosis with only clinical observation (45%),19 making it necessary to develop and validate diagnostic tools. In 1990, using DSM-III-R criteria, Inouye20 created and validated the Confusion Assessment Method (CAM), an algorithmic technique that uses only four of the DSM-III-R criteria for delirium. In the intensive care environment, the CAM has been adapted as the CAM-ICU because many patients are unable to speak after being intubated and ventilated. The first validation study of the CAM-ICU included only 38 patients.21 Two nurses and two intensivists compared the CAM-ICU method with the standard DSM-IV. In addition to a high specificity and sensitivity, an excellent interrater correlation was observed. The same investigators published a second study that included 111 patients who were on mechanical ventilation; in addition to confirming a high interrater correlation (kappa coefficient: 0.99, 95% CI: 0.92 – 0.99), they reported a sensitivity and specificity of approximately 100%.22 Our study differs in some respects from the studies by Ely et al. described above.21,22 First, we did not observe as high a sensitivity for the CAM-ICU, which varied in Ely et al. from 93% to 100%. In our study, the sensitivity of the CAM-ICU was 72.5%. Although there is not a clear explanation, the difference is not likely related to the implementation of the CAM-ICU in Portuguese, as similar

66 (55.4%) 53 (44.6%) 27 (22.8%) 11 (9.3%) 17 (14%) 08 (6.7%) 03 (2.5%) 20 (16.9%) 03 (2.5%) 29 (24.5%) 46 (38.6%) 20 (17%)

*chronic renal failure (requiring dialysis). DSM-IV: Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition.

As demonstrated by their severity-of-illness scores (APACHE II scores of 15¡6), the sample of patients was determined to be critically ill. The patients represented a mixed ICU population. Using standard evaluation technique (DSM-IV), delirium was observed in 38.6% (46/119) of the patients. The types of delirium observed in these patients included hypoactive (69.5%), hyperactive (19.5%), and mixed (11%) delirium. Most patients (76.4%) were easily arousable at the time of their evaluation (RASS-0 or RASS-1). The CAM-ICU identified 26.8% of the delirious patients and showed an overall sensitivity of 72.5% and specificity of 96.2%. The CAM-ICU Flowsheet showed similar accuracy. The ICDSC identified 25.2% of the patients as delirious and had a sensitivity of 96% and a specificity of 72.4% (Table 2). The kappa coefficient was used to detect the correlation between the diagnostic tools. We observed a concordance of 98.32% with a kappa of 0.96 between the CAM-ICU Flowsheet and the CAM-ICU (Table 3). The McNemar test (p = 1.00) suggested that there were no significant differences between the two instruments. To assess the correlation between the ICDSC and the CAM-ICU, it was necessary to exclude patients with a diagnosis of subsyndromal delirium that was diagnosed based only on the ICDSC (27 patients). We found a kappa of 0.59 (Table 4). As expected, similar findings were observed when comparing the CAM-ICU Flowsheet and the ICDSC.

Table 2 - The sensitivity and specificity of the CAM-ICU, CAM-ICU Flowsheet and ICDSC in 119 critically ill patients.

CAM-ICU CAM-ICU Flowsheet ICDSC

Sensitivity (95% CI)

Specificity (95% CI)

PPV (95% CI)

NPV (95% CI)

72.5 (55.9 – 84.9) 72.5 (55.9 – 84.9) 96.0 (81.5 – 99.8)

96.2 (88.5 – 99.0) 96.2 (88.5 – 99.0) 72.4 (58.6 – 83.0)

90.6 (73.8–97.5) 90.6 (73.8–97.5) 65.0 (49.7–78.2)

87.4 (78.1–93.2) 87.4 (78.1–93.2) 97.7 (86.2–99.9)

PPV: positive predictive value; NPV: negative predictive value.

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The ICDSC checklist is an eight-item screening tool (one point for each item) that is based on DSM criteria and applied to data that can be collected through medical records or on information obtained from a multidisciplinary team. Bergeron et al. observed a high sensitivity (99%) when a cutoff of 4 was used, but a moderate specificity was observed (64%).16 Similarly, our study found a high sensitivity (96%) and a moderate specificity (72.4%). Other studies have reported a low sensitivity for the ICDSC (43%) compared with the standard method of diagnosis (DSMIV).31 In the mixed population of patients in this earlier study, the CAM-ICU showed a higher sensitivity (64%) but a lower specificity. More recently, the German version of the ICDSC was compared with the CAM-ICU in a population of surgical patients with a close correlation (kappa coefficient: 0.8; 95% CI: 0.78 â&#x20AC;&#x201C; 0.84; p,0.001).13 In our study, we observed a low correlation between the CAM-ICU and the ICDSC (kappa coefficient: 0.59). A change in the cutoff would likely change the correlation between these diagnostic tools. A cutoff of 5 correctly identified 86.5% of cases, whereas a cutoff of 4 correctly identified 80.6% of cases. For this analysis, it was necessary to exclude cases that were considered to be subsyndromal delirium (a cutoff of 3). Evaluating all 119 cases, we observed a high degree of accuracy with this tool and the DSM-IV, with an area under the ROC curve of 0.91. Because the CAM-ICU and CAMICU Flowsheet responses are dichotomous (yes or no), it was not possible to draw an ROC curve. We found the CAM-ICU and the CAM-ICU Flowsheet to be similar and to be highly accurate for delirium diagnosis, which suggests that these are appropriate tools for developing a diagnostic profile. However, because of its high specificity and only moderate sensitivity, the ICDSC may be more useful in stratifying patients with delirium. Recently, Tomasi et al. suggested that the CAM-ICU is a better predictor of outcomes than the ICDSC, which is probably because of the high rate of false positives with the ICDSC.32 Our study has some notable limitations. First, as the study was performed in different regions, the tools evaluated (CAM-ICU, CAM-ICU Flowsheet, and ICDSC) were applied by different intensivists in different ICUs. Thus, we could not perform an interrater correlation with the tools that were applied. However, we believe that the evidence is strong enough to demonstrate a close correlation between the raters because the tool is simple and easily applied. We measured the performance of the scales against the DSM-IV, which is considered to be the standard technique for clinical assessment. Therefore, the application of the CAM-ICU and the CAM-ICU Flowsheet by the same investigator does not imply an evaluation bias. Our study also has several strengths. Not only was our study the first to validate the CAM-ICU and the CAM-ICU Flowsheet for Brazilian Portuguese, but it was performed as a multicenter evaluation in three different and representative regions of Brazil. The study evaluated a mixed population of critically ill patients, including ventilated and non-ventilated patients. These methodological characteristics increase the external validity of the results. The present data demonstrate that the CAM-ICU and the ICDSC are valid tools that can be used in Brazilian Portuguese with a high degree of accuracy. The CAM-ICU Flowsheet has an excellent agreement with the CAM-ICU (kappa coefficient = 0.96) and can be employed as a fast,

Table 3 - A comparison of the CAM-ICU and the CAM-ICU Flowsheet. CAM-ICU

CAM-ICU Flowsheet

Positive

Negative

Total

Positive

Negative Total

1 32

86 87

87 119

Kappa coefficient: 0.96.

results have been observed in other languages, including Spanish.23 One possible explanation is a change in the sensitivity of the CAM-ICU when it is used in a cohort of mechanically ventilated and sedated patients. We observed that most patients had a RASS score of zero (60.5%), which may not only represent the lower degree of severity in our cohort but may also represent a current trend toward less sedation in ICU patients.24 When comparing diagnostic instruments for delirium, Luetz et al. demonstrated that the sensitivity of the CAM-ICU is higher in patients with a RASS score of higher than 0.25 However, a common feature of every published study is the high specificity of the CAM-ICU. The CAM-ICU has been translated and validated in many languages 26,27 and has become the most frequently employed tool for diagnosing delirium in ICU patients.7,28 A distinct advantage of this tool is that it does not require the patient to speak, which can be useful in patients who are on mechanical ventilation. In our study, we observed no difference in the accuracy of the CAM-ICU between the patients who were ventilated and those who were not. We also observed different scales in patients undergoing noninvasive ventilation (NIV). Eleven patients were observed with NIV (three of whom presented with delirium), and there was a 100% correlation among the CAM-ICU Flowsheet, the CAM-ICU and the ICDSC. The CAM-ICU Flowsheet was developed to decrease the time required for the evaluation of patients with suspected delirium. However, to the best of our knowledge, only a single study has evaluated its performance.29 In our study, we observed an excellent correlation between the CAM-ICU and the CAM-ICU Flowsheet, with a kappa of 0.96. Guenther et al. evaluated the CAM-ICU Flowsheet in German (with a duration of application that did not exceed 2 minutes) and noted a sensitivity of 88% to 92% and a specificity of 100% with close interobserver correlation.30 In our study, the sensitivity was 72.5%, and the specificity was 96.2%. In some cases, less than one minute was necessary for completion of the instrument. Table 4 - A Comparison of the CAM-ICU/CAM-ICU Flowsheet and the ICDSC. ICDSC

CAM-ICU Flowsheet CAM-ICU

Positive

Negative

Total

Positive

Negative Total

19 49

43 43

62 92

Kappa coefficient: 0.59.

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Three tools used to diagnose delirium Gusmao-Flores D et al.

Figure 1 - Receiver operating characteristic curve based on the total score (0-8) obtained on the ICDSC.

practical and reliable tool. Finally, the ICDSC has a high sensitivity for diagnosing delirium but a moderate specificity and a poor correlation with the CAM-ICU and CAMICU Flowsheet.

ACKNOWLEDGMENTS

This project was partially supported by the National Council of Technological and Scientific Development (CNPq): [474869/2010-5] Edital Universal MCT/CNPq 14/2010.

AUTHOR CONTRIBUTIONS 8.

Gusma˜o-Flores D and Salluh JIF designed the study and wrote the protocol, applied the scales and performed the data collection, undertook the statistical analysis, and contributed to the writing of the first draft of the manuscript. Pitrowsky MT applied the scales, performed the data collection, and contributed to the writing of the first draft of the manuscript. dal-Pizzol F, Ritter C, Tomasi CD designed the study and wrote the protocol, applied the scales and performed the data collection. Lima MASD, Santana LR, Lins RMP, Lemos PP, Serpa G and Oliveira J applied the scales, performed the data collection and contributed to the writing of the first draft of the manuscript. Quarantini LC designed the study and wrote the protocol, contributed to writing the first draft of the manuscript, applied the scales and performed the data collection. Chalhub RA applied the scales and performed the data collection. Lacerda ALT and Koenen KC contributed to the writing of the first draft of the manuscript. All authors contributed to and have approved the final manuscript.

10. 11.

12.

REFERENCES

13.

1. Lin SM, Liu CY, Wang CH, Lin HC, Huang CD, Huang PY, et al. The impact of delirium on the survival of mechanically ventilated patients. Crit Care Med. 2004;32:2254-59, doi: 10.1097/01.CCM.0000110878.49476.42. 2. Lima DP, Ochiai ME, Lima AB, Curiati JA, Farfel JM, Filho WJ. Delirium in hospitalized elderly patients and post-discharge mortality. Clinics. 2010;65:251-5, doi: 10.1590/S1807-59322010000300003. 3. Salluh JI, Soares M, Teles JM, Ceraso D, Raimondi N, Nava VS, et al. The DECCA (Delirium Epidemiology in Critical Care) Study Group. Delirium epidemiology in critical care (DECCA): an international study. Crit Care. 2010;14:R210. 4. Girard TD, Jackson JC, Pandharipande PP, Pun BT, Thompson JL, Shintani AK, et al. Delirium as a predictor of long-term cognitive

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impairment in survivors of critical illness. Crit Care Med. 2010;38:151320, doi: 10.1097/CCM.0b013e3181e47be1. Morandi A, Pandharipande P, Trabucchi M, Rozzini R, Mistraletti G, Trompeo AC, et al. Understanding international differences in terminology for delirium and other types of acute brain dysfunction in critically ill patients. Intensive Care Med. 2008;34:1907-15, doi: 10.1007/s00134008-1177-6. McNicoll L, Pisani MA, Zhang Y, Ely EW, Siegel MD, Inouye SK. Delirium in the intensive care unit: occurrence and clinical course in older patients. J Am Geriatr Soc. 2003;51:591-8, doi: 10.1034/j.1600-0579. 2003.00201.x. Salluh JI, Dal-Pizzol F, Mello PV, Friedman G, Silva E, Teles JM, et al. Brazilian Research in Intensive Care Network. Delirium recognition and sedation practices in critically ill patients: a survey on the attitudes of 1015 Brazilian critical care physicians. J Crit Care. 2009;24:556-62, doi: 10. 1016/j.jcrc.2009.04.004. Ely EW, Margolin R, Francis J, May L, Truman B, Dittus R, et al. Evaluation of delirium in critically ill patients: validation of the confusion assessment method for the intensive care unit (CAM-ICU). Crit Care Med. 2001;29:1370-9, doi: 10.1097/00003246-200107000-00012. Ely EW, Inouye SK, Bernard GR, Gordon S, Francis J, May L, et al. Delirium in mechanically ventilated patients: validity and reliability of the confusion method assessment for the intensive care unit (CAM-ICU). JAMA. 2001;286:2703-10, doi: 10.1001/jama.286.21.2703. van Eijk MM, van Marum RJ, Klijn IA, de Wit N, Kesecioglu J, Slooter AJ. Comparison of delirium assessment tools in a mixed intensive care unit. Crit Care Med. 2009;37:1881-5, doi: 10.1097/CCM.0b013e3181a00118. Plaschke K, von Haken R, Scholz M, Engelhardt R, Brobeil A, Martin E, et al. Comparison of the confusion assessment method for the intensive care unit (CAM-ICU) with the Intensive Care Delirium Screening Checklist (ICDSC) for delirium in critical care patients gives high agreement rate(s). Intensive Care Med. 2008;34:431-6, doi: 10.1007/ s00134-007-0920-8. Pun BT, Gordon SM, Peterson JF, Shintani AK, Jackson JC, Foss J, et al. Large-scale implementation of sedation and delirium monitoring in the intensive care unit: a report from two medical centers. Crit Care Med. 2005;33:199-205, doi: 10.1097/01.CCM.0000166867.78320.AC. Guenther U, Popp J, Koecher L, Muders T, Wrigge H, Ely EW, et al. Validity and reliability of the CAM-ICU Flowsheet to diagnose delirium in surgical ICU patients. J Crit Care. 2010;25:144-51, doi: 10.1016/j.jcrc. 2009.08.005. Wild D, Grove A, Martin M, Eremenco S, McElroy S, Verjee-Lorenz A, et al. Principles of good practice for the translation and cultural adaptation process for patient reported outcomes (PRO) measures: report of the ISPOR taskforce for translation and cultural adaptation. Value Health. 2005;8:94–104, doi: 10.1111/j.1524-4733.2005.04054.x. Amirfarzan H, Pun BT, Ely EW. CAM-ICU Flowchart CAM-ICU. www.icudelirium.org. Bergeron N, Dubois MJ, Dumont M, Dial S, Skrobik Y. Intensive Care Delirium Screening Checklist: evaluation of a new screening tool. Intensive Care Med. 2001;27:859–64, doi: 10.1007/s001340100909.

Three tools used to diagnose delirium Gusmao-Flores D et al.

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17. Peterson JF, Pun BT, Dittus RS, Thomason JW, Jackson JC, Shintani AK, et al. Delirium and its motoric subtypes: a study of 614 critically ill patients. J Am Geriatr Soc. 2006;54:479-84, doi: 10.1111/j.1532-5415.2005. 00621.x. 18. Spronk PE, Riekerk B, Hofhuis J, Rommes JH. Occurrence of delirium is severely underestimated in the ICU during daily care. Intensive Care Med. 2009;35:1276-80, doi: 10.1007/s00134-009-1466-8. 19. van Eck van der Sluijs JF, Oldenbeuving AW, Roks G, Tilanus JJ. Delirium on intensive care frequently missed: clinical impression alone is not enough. Ned Tijdschr Geneeskd. 2010;154:A1290. 20. Inouye S, van Dyck C, Alessi CA, Balkin S, Siegal A, Horwitz R. Clarifying confusion: Th Confussion Assesment Method. Ann Inter Medicine. 1990;113:941-8. 21. Ely EW, Margolin R, Francis J, May L, Truman B, Dittus R, et al. Evaluation of delirium in critically ill patients: validation of the Confusion Assessment Method for the Intensive Care Unit (CAM-ICU). Crit Care Med. 2001;29:1370-9, doi: 10.1097/00003246-200107000-00012. 22. Ely EW, Inouye SK, Bernard GR, Gordon S, Francis J, May L, et al. Delirium in mechanically ventilated patients: validity and reliability of the confusion assessment method for the intensive care unit (CAM-ICU). JAMA. 2001;286:2703-10, doi: 10.1001/jama.286.21.2703. 23. Tobar E, Romero C, Galleguillos T, Fuentes P, Cornejo R, Lira MT, et al. Confusion Assessment Method for diagnosing delirium in ICU patients (CAM-ICU): cultural adaptation and validation of the Spanish version. Med Intensiva. 2010;34:4-13, doi: 10.1016/j.medin.2009.04.003. 24. Strøm T, Martinussen T, Toft P. A protocol of no sedation for critically ill patients receiving mechanical ventilation: a randomised trial. Lancet. 2010;375:475-80, doi: 10.1016/S0140-6736(09)62072-9. 25. Luetz A, Heymann A, Radtke FM, Chenitir C, Neuhaus U, Nachtigall I, et al. Different assessment tools for intensive care unit delirium: which

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score to use? Crit Care Med. 2010;38:409-18, doi: 10.1097/CCM. 0b013e3181cabb42. Toro AC, Escobar LM, Franco JG, Dı´az-Go´mez JL, Mun˜oz JF, Molina F, et al. Spanish version of the CAM-ICU (Confusion Assessment Method for the Intensive Care Unit). Pilot study of validation. Med Intensiva. 2010;34:14-21. Chuang WL, Lin CH, Hsu WC, Ting YJ, Lin KC, Ma SC. Evaluation of the reliability and validity of the Chinese version of the confusion assessment method for the intensive care unit. Hu Li Za Zhi. 2007;54:45-52. Patel RP, Gambrell M, Speroff T, Scott TA, Pun BT, Okahashi J, et al. Delirium and sedation in the intensive care unit: survey of behaviors and attitudes of 1384 healthcare professionals. Crit Care Med. 2009;37:825-32, doi: 10.1097/CCM.0b013e31819b8608. Vreeswijk R, Toornvliet A, Honing MLH, Bakker K, de Man T, Daas G, et al. Validation of the Dutch version of the Confusion Assessment Method (CAM-ICU) for delirium screening in the Intensive Care Unit. Neth J Crit Care. 2009;13:73-8. Guenther U, Popp J, Koecher L, Muders T, Wrigge H, Ely EW, et al. Validity and reliability of the CAM-ICU Flowsheet to diagnose delirium in surgical ICU patients. J Crit Care. 2010;25:144-51, doi: 10.1016/j.jcrc.2009.08.005. van Eijk MM, van Marum RJ, Klijn IA, de Wit N, Kesecioglu J, Slooter AJ. Comparison of delirium assessment tools in a mixed intensive care unit. Crit Care Med. 2009;37:1881-5, doi: 10.1097/CCM.0b013e3181a 00118. Tomasi CD, Grandi C, Salluh J, Soares M, Giombelli VR, Cascaes S, et al. Comparison of CAM-ICU and ICDSC for the detection of delirium in critically ill patients focusing on relevant clinical outcomes. J Crit Care. 2011;Jul 5. [Epub ahead of print].

CLINICS 2011;66(11):1923-1928

DOI:10.1590/S1807-59322011001100012

CLINICAL SCIENCE

Do Omega-3 fatty acids prevent atrial fibrillation after open heart surgery? A meta-analysis of randomized controlled trials Luciana Armaganijan,I,II Renato D. Lopes,III Jeff S. Healey,II Jonathan P. Piccini,III Girish M. Nair,II Carlos A. MorilloII I Electrophysiology and Cardiac Arrhythmias, Dante Pazzanese Institute of Cardiology, Sa˜o Paulo/SP, Brazil. II Department of Medicine, Cardiology Division, Arrhythmia Service, McMaster University, Population Health Research Institute, Hamilton, ON, Canada. III Department of Medicine, Division of Cardiology, Duke Clinical Research Institute, Duke University, Durham, NC, USA.

OBJECTIVES: N-3 polyunsaturated fatty acids have been proposed as a novel treatment for preventing postoperative atrial fibrillation due to their potential anti-inflammatory and anti-arrhythmic effects. However, randomized studies have yielded conflicting results. The objective of this study is to review randomized trials of N-3 polyunsaturated fatty acid use for postoperative atrial fibrillation. METHODS: Using the CENTRAL, PUBMED, EMBASE, and LILACS databases, a literature search was conducted to identify all of the studies in human subjects that reported the effects of N-3 polyunsaturated fatty acids on the prevention of postoperative atrial fibrillation in cardiac surgery patients. The final search was performed on January 30, 2011. There was no language restriction, and the search strategy only involved terms for N-3 polyunsaturated fatty acids (or fish oil), atrial fibrillation, and cardiac surgery. To be included, the studies had to be randomized (open or blinded), and the enrolled patients had to be $18 years of age. RESULTS: Four randomized studies (three double-blind, one open-label) that enrolled 538 patients were identified. The patients were predominantly male, the mean age was 62.3 years, and most of the patients exhibited a normal left atrial size and ejection fraction. N-3 polyunsaturated fatty acid use was not associated with a reduction in postoperative atrial fibrillation. Similar results were observed when the open-label study was excluded. CONCLUSIONS: There is insufficient evidence to suggest that treatment with N-3 polyunsaturated fatty acids reduces postoperative atrial fibrillation. Therefore, their routine use in patients undergoing cardiac surgery is not recommended. KEYWORDS: Omega-3 fatty acids; Atrial fibrillation; Meta-analysis; Open Heart surgery; Prevention. Armaganijan L, Lopes RD, Healey JS, Piccini JP, Nair GM, Morillo CA. Do Omega-3 fatty acids prevent atrial fibrillation after open heart surgery? A meta-analysis of randomized controlled trials. Clinics. 2011;66(11):1923-1928. Received for publication on May 24, 2011; First review completed on June 16, 2011; Accepted for publication on July 19, 2011 E-mail: renato.lopes@duke.edu Tel.: 919 668-8241

Furthermore, antiarrhythmic drugs have not been effective in preventing postoperative AF.2 Recent clinical and experimental studies have shown that N-3 polyunsaturated fatty acids (PUFAs) are effective in preventing AF,7,8 and PUFAs have been used to prevent AF in patients undergoing cardiac surgery. However, the studies supporting this treatment have reported conflicting results on its efficacy in preventing postoperative AF.9-13 The objective of this analysis is to estimate the effect of PUFA on preventing postoperative AF through a metaanalysis of randomized controlled clinical trials.

INTRODUCTION Atrial fibrillation (AF) is the most common arrhythmia following cardiac surgery. Its incidence ranges from 20 to 70%, depending on the patient age and the complexity of the surgical procedure.1-3 Although well tolerated in most cases, postoperative AF is associated with prolonged hospitalization, increased costs, morbidity, and mortality.4-6 The etiology of postoperative AF is multifactorial, and although several risk factors are associated with this condition, the underlying mechanisms are controversial.

METHODS Study Search

Using the Cochrane Central Register of Controlled Trials (CENTRAL, The Cochrane Library, Issue 3, 2008), PUBMED, EMBASE, and LILACS databases, a literature search was conducted to identify all of the studies that reported the

No potential conflict of interest was reported.

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AF as any episode lasting $30 seconds in the monitor recording (primary outcome). Clinically recognized AF (as documented in the patientâ&#x20AC;&#x2122;s clinical records by the treating clinician) and the AF burden (defined as the percentage of time that a given patient had AF) were the secondary outcomes.

effects of PUFAs on the prevention of postoperative AF in human cardiac surgery patients. The final literature search was performed on January 30th, 2011. To maximize the sensitivity, no language restriction was used, and the search strategy involved only terms for PUFA (or fish oil), AF, and cardiac surgery. All of the included studies were randomized, both open and blinded studies were considered, and the enrolled patients had to be $18 years of age. In addition, the reference lists from the included studies were scrutinized to identify additional citations and specialists in the field, and the authors of the included publications were contacted and asked for unpublished data to avoid publication bias.

Statistical Analysis The individual unit of analysis was the patient (not patient-years). Heterogeneity was evaluated using the Cochran Q and chi-squared tests. For the purpose of this analysis, we interpreted a chi-squared of $25 as indicative of significant heterogeneity. All of the analyses were performed according to the intention-to-treat principle. The overall treatment effects were reported via odds ratios with 95% confidence intervals. Random effects modeling with the Mantel-Haenszel test was performed to generate a pooled estimate of the effect of PUFA treatment on postoperative AF. To exclude ascertainment bias and any potential confounding due to concomitant treatments in open-label designs, we performed a sensitivity analysis that excluded the open-label trial. The statistical testing was twotailed, and statistical significance was defined as p,0.05. All of the statistical analyses were conducted with the R Development Core Team software.

Data Abstraction The outcome of interest was the occurrence of AF. Two reviewers (LA and JSH) independently screened, extracted data from, and performed quality assessment of the cohort trials identified by the literature search. Any disagreements between the examiners were settled by consensus. A funnel plot of the results was generated to detect any publication bias. Figure 1 demonstrates the study selection process according to the PRISMA flow diagram.

Definition of Atrial Fibrillation The definition of AF varied between the studies. Calo et al.9 defined postoperative AF as any electrocardiographically confirmed episode of AF of more than 5 minutes in duration or requiring intervention for angina or compromised hemodynamics during the hospitalization period. In the Heidt study,11 AF was detected by monitoring or 12-lead electrocardiography during the intensive care unit period and was defined as any confirmed episode of AF lasting $15 minutes. In the Heidarsdottir study,12 all of the patients underwent continuous electrocardiographic monitoring during hospitalization, and postoperative AF was defined as any episode lasting more than 5 minutes. The patients exited the study upon discharge from the hospital or two weeks after the surgery. Finally, Saravanan et al.13 defined

RESULTS Search Results Four clinical studies (538 patients) reporting the effects of PUFAs on the prevention of postoperative AF were identified and included in the final analyses of this study.9,11-13 Of the four studies, two12,13 reported no effects on postoperative AF, and two9,11 reported a significant reduction in the incidence of AF shortly after cardiac surgery.

Study Characteristics and Study Quality The characteristics of the studies are summarized in Table 1. Most of the studies used encapsulated fish oil, but one11 examined the effects of intravenously administered n3 fatty acids. Two studies12,13 had a placebo control, one11 administered saturated fatty acids as a control, and the other one9 enrolled patients who had not received PUFA as the control group. The Calo,9 Heidt,11 and Saravanan13 studies included patients who were .18 years of age, while Heidarsdottir12 included all consecutive patients who were .40 years old and scheduled for elective or urgent (not emergent) open heart surgery. The Calo9 and Heidt11 studies excluded patients with unstable hemodynamic conditions before surgery, and Saravanan13 included patients who underwent elective isolated coronary artery bypass graft surgery. Calo,9 Heidt,11 and Saravanan13 all excluded concomitant valvular surgery.

Baseline Patient Characteristics The preoperative baseline characteristics are shown in Table 2. In general, the patients in both control and study groups tended to be male with a mean age of 62.3 years. Most of the patients in all of the studies had a normal left atrial size and ejection fraction. Systemic hypertension was more frequently observed in the Calo9 and Heidarsdottir12 populations. The concomitant use of beta-blockers was more common in the Heidarsdottir12 and Saravanan13 studies.

Figure 1 - The PRISMA flow diagram.

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Omega-3 Fatty Acids and Atrial Fibrillation Armaganijan L et al.

Table 1 - Studies characteristics. Results (POAF) Study

Study design

Control

Inclusion criteria

Calo

160

No PUFA

Age .18, NSR, stable hemodynamic conditions before surgery

Heidt

102

Open-label, prospective, randomized, controlled trial with parallel groups; not blinded Prospective, randomized double-blinded trial

Exclusion criteria

Heidarsdottir

168

Prospective, randomized, double-blinded, placebocontrolled trial

Placebo

Saravanan

103

Prospective, randomized, double-blinded, placebocontrolled trial

Placebo

Concomitant valvular surgery, a prior history of any SVT, the current use of an AAD other than BB, CCB, or digitalis Concomitant valvular surgery, a Age .18, NSR, stable prior history of any SVT, the hemodynamic conditions current use of an AAD other before surgery, no angina than BB or CCB at rest All of the consecutive Age ,40, prior history of any patients scheduled for SVT, the current use of elective or open heart amiodarone or sotalol, an surgery emergent operation A prior history of any atrial Age .18, scheduled to undergo elective isolated arrhythmia, the current use of a class-1 or class-3 AAD, fish oil CABG use within 3 mos prior to surgery

IV saturated free fatty acids

PUFA

Control

15.2%

33.3%

17.3%

30.6%

54.2%

54.1%

56%

43%

AAD, antiarrhythmic drugs; BB, beta-blockers; CABG, coronary artery bypass grafting; CCB, calcium channel blockers; IV, intravenous; NSR, normal sinus rhythm; POAF, postoperative atrial fibrillation; PUFA, polyunsaturated fatty acid; SVT, supraventricular tachycardia.

In contrast, Saravanan et al.13 found no difference in the length of the hospital stay between the groups. No drugrelated complications were reported by Saravanan et al.13

Atrial Fibrillation and Clinical Outcomes Figure 2 shows a forest plot comparing the total mortality of the patients that received PUFAs to those that did not receive PUFAs. The use of PUFAs was not associated with a reduction in the occurrence of postoperative AF in the patients undergoing cardiac surgery compared to the untreated patients (odds ratio, 0.79; 95% confidence interval, 0.56 - 1.13; p = 0.195). Similar results were observed when the open-label study was excluded from the analyses (odds ratio, 0.99; 95% confidence interval, 0.65 - 1.49; p = 0.963). Heidarsdottir et al.12 reported no differences in the postoperative outcomes (such as blood transfusion, the reoperation frequency, and major bleeding) between the groups. Two patients in the PUFA group died. One patient died due to a massive myocardial infarction and heart failure three days after surgery, and another patient died from an ascending aortic rupture two days after surgery. One patient in the PUFA arm and three patients in the placebo group suffered cerebrovascular accidents. Gastrointestinal discomfort was more common in the PUFA group and resulted in the discontinuation of two patients. In the Calo9 study, no significant adverse reactions were observed in the PUFA patients, except for a single case of an allergic reaction, and there were no differences in the incidence of non-fatal complications or death between the groups. In both this study and in the study by Heidt et al.,11 PUFA treatment was associated with a shorter hospital stay.

DISCUSSION This analysis showed no benefit of PUFAs for reducing AF following cardiac surgery. Although several strategies involving PUFAs have been developed for preventing postoperative AF, none have proven its efficacy or justify its routine use in this setting. Clinical and experimental studies have shown that the administration of PUFAs reduces major cardiac events, including sudden cardiac death, ventricular fibrillation, and AF.14-16 In a cohort of 4815 patients with 12 years of followup, Mozaffarian et al.17,18 demonstrated that fish intake is associated with a lower incidence of AF. Several studies have demonstrated the protective effect of PUFAs against ventricular arrhythmias. Hyperpolarization of the resting membrane potential and an increase in the phase-4 refractory period have been postulated to be the main antiarrhythmic effects of PUFAs.7,19 Moreover, PUFAs have been shown to reduce the asynchronous contractile activity of myocytes,20 increase electrical stability by prolonging the inactivation of fast outward sodium channels, and modulate calcium release as a result of their direct interaction with the sarcoplasmic reticulum.

Table 2 - Baseline characteristics. Age

Calo Heidt Heidarsdottir Saravanan

Male (%)

Control

PUFA

Control

PUFA

64.9¡9.1 NR 67 (43, 82) 68 (64, 73)

66.2¡8.0 NR 67 (45, 82) 64 (58, 71)

84 64 76.9 82

86 73 81.9 77

LA (mm) Control

PUFA

EF (%) Control

39.7¡5.2 39.7¡5.1 55.3¡11.4 40.5¡5.1 40.0¡5.1 52.3¡15.6 NR NR 60 (15, 77.5) 6% had 4% had 8% had LA$2.3 LA$2.3 cm/ EF#55% 2 2 cm/m m

HTN (%)

Beta-blocker (%)

PUFA

Control

PUFA

Control

PUFA

56.3¡12.1 52.0¡15.0 60 (15, 70) 10% had EF#55%

81.5 NR 64.7 29

78.5 NR 61.4 35

56.8 NR 74.1 82

58.2 NR 78.3 88

The values are presented as the mean ¡ standard deviation or as the median (25th, 75th), unless otherwise indicated. EF, ejection fraction; HTN, hypertension; LA, left atrium; NR, not reported; PUFA, polyunsaturated fatty acid.

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Calo et al.9 first demonstrated that PUFA administration during hospitalization significantly reduced the occurrence of postoperative AF in patients undergoing coronary artery bypass graft surgery (18.1% absolute risk reduction [RR]; 54.4% relative RR) and was associated with a shorter hospital stay (7.3¡2.1 days vs. 8.2¡2.6 days for PUFA patients and control patients, respectively; p = 0.017). In the multivariable analysis, age and the use of PUFAs were significant independent predictors of postoperative AF. However, this study was not blinded or placebo-controlled, and it excluded patients undergoing valvular surgery and those with a history of AF, which are factors that increase the risk for postoperative AF. Another limitation of this study was that the patients in the PUFA group were hospitalized for fewer days and may have had unidentified asymptomatic AF after discharge. The results of this study are congruent with those found by Heidt.11 Only patients undergoing coronary artery bypass graft procedures were included in that study. The patients treated with PUFAs developed significantly less AF than control patients (17.3% vs. 30.6%, respectively; p,0.05). More recently, Mariscalco et al. demonstrated that preoperative PUFA therapy is associated with a decreased incidence of early AF after cardiac surgery but does not prevent late AF. PUFA administration was independently associated with a 46% relative reduction in the risk of early AF development (OR, 0.54; 95% CI, 0.31–0.92). This study included coronary artery bypass surgery procedures and valve repair, combined surgeries, and other types of cardiac surgery. Independent predictors of early AF were age, the left atrial area, reoperation, and the use of an intra-aortic balloon pump. However, this study was limited by its lack of randomization; therefore, a causal relationship between PUFAs and postoperative AF cannot be accurately assessed. Contrary to previous studies, two recent, randomized, double-blind, and placebo-controlled studies failed to demonstrate any beneficial effects of PUFA administration.12,13 In addition, the recently published Efficacy and Safety of Prescription Omega-3 Acid Ethyl Esters (P-OM3) for the Prevention of Recurrent Symptomatic Atrial Fibrillation trial showed similar results. This was a prospective, randomized, and double-blinded trial of 663 AF patients (average age, 61 years; 56% male) enrolled at 96 sites in which the use of a fish oil-derived product (4 grams/day) was compared to the use of a placebo.31 The two main findings of this study were that the paroxysmal AF patients who received omega-3 did not differ significantly from the patients of the placebo group in terms of the time to the first recurrence of symptoms, and there was no significant difference in persistent AF between the treated and placebo groups. Differences in the patient profiles, the type of surgery, the definition of arrhythmia, and the method of arrhythmia surveillance could explain the contradictory results of these studies. In particular, the method used to evaluate the occurrence of AF and its burden had the most significant effect. Calo et al.9 and Heidarsdottir et al.12 defined AF as any episode lasting more than 5 minutes, Heidt et al.11 used a cutoff of 15 minutes and Saravanan et al.13 used a cutoff of 30 seconds. This difference likely explains the higher overall incidence of AF in the latter study. The method of rhythm surveillance (continuous monitoring vs. electrocardiogram monitoring only if symptoms are present) might also affect the detection of AF. Unsurprisingly, the occurrence of AF was higher in the

Figure 2 - A meta-analysis of the randomized studies examining the incidence of postoperative atrial fibrillation.

However, other studies have demonstrated that PUFAs shorten the effective refractory period and action potential,21-23 which favors the initiation and maintenance of AF. In fact, a few experimental studies have demonstrated a pro-arrhythmic effect of PUFAs.24,25 In terms of adverse side effects, the studies reported different outcomes and results, but in general, PUFAs do not seem to be directly associated with any bleeding or ischemic complications. Clinical studies have reported several benefits of PUFA treatment for chronic and acute inflammation.26-28 EPA and DHA can inhibit the conversion of arachidonic acid to prostaglandin E2 and leukotriene B4, and they also decrease the synthesis of tumor necrosis factor alpha and interleukin1 beta.29 The trauma caused by surgery initiates both local and systemic inflammatory responses, reflected by increased C-reactive protein levels. Moreover, it is possible that local atrial inflammation may produce atrial conduction abnormalities and consequent reentrant arrhythmias. In a study conducted by Halonen et al.,30 the administration of intravenous hydrocortisone reduced the incidence of AF after cardiac surgery, suggesting that an exaggerated inflammatory response could be an etiological factor for the development of postoperative AF. A study by Mariscalco et al.10 showed higher white blood cell counts after surgery in patients affected by early AF and lower levels of postoperative C-reactive protein levels in the PUFA-treated group, suggesting that PUFAs have antiinflammatory properties. This was contradicted by a study by Saravanan,13 which showed that C-reactive protein levels were not affected by PUFAs. This discrepancy might be due to the low doses of PUFAs used in this study, which may have been below the threshold required to exert any antiinflammatory effects. Interestingly, patients in the Calo et al. study population were more likely to have a history of myocardial infarction and/or off-pump surgery. This might also explain the better response to PUFA therapy in this study compared to other studies.

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Table 3 - The PUFA type and dose, the definition of AF, and the rhythm surveillance method. Study Calo

Heidt

Heidarsdottir

Saravanan

Type/dose of PUFA

Definition of AF

Rhythm surveillance

Daily doses of 850–882 mg EPA and DHA in an average ratio of 1:2 EPA:DHA, initiated at least 5 days before surgery and continued until hospital discharge Infusion pump (100 mg soya oil/kg body weight/day) started at least 12 hours before surgery and continued until transfer to the ward Two capsules twice daily for a daily dose of 1240 mg EPA, initiated 5–7 days before surgery and continued until hospital discharge or a maximum of 2 weeks after surgery Daily doses of 2 mg of a commercially available n-3 PUFA preparation, providing 85–88% EPA+DHA as ethyl esters in a ratio of 15251 and initiated at least 5 days before CABG and continued until the day of discharge

Any episode lasting more than 5 min or requiring intervention Any episode lasting more than 15 min

Continuous rhythm monitoring for the first 4–5 days, followed by daily ECG until hospital discharge Monitoring or 12-lead ECG during the ICU period

Any episode lasting more than 5 min

Continuous ECG monitoring during hospitalization

Any episode lasting more than 30 sec

Heart rhythm monitoring (Holter) from the immediate post-op period to 5 days after surgery; if the patient was hospitalized for longer than 5 days, ECG was performed daily

CABG, coronary artery bypass grafting; DHA, docosahexaenoic acid; ECG, electrocardiograph; EPA, eicosapentaenoic acid; ICU, intensive care unit; PUFA, polyunsaturated fatty acid.

PUFA treatment and its effects on the incidence of postoperative AF. The definition of AF also varied among the studies, which could have affected our findings. Finally, the overall incidence of postoperative AF depended on the type and duration of the monitoring techniques, which varied among the studies. Despite the heterogeneity of the studies included in our analysis, we demonstrated that there is currently insufficient evidence to suggest that PUFA treatment reduces AF following cardiac surgery. In addition, little information on the adverse events associated with PUFA treatment is available. A large, multicenter, randomized trial with welldefined clinical outcomes and a consistent method of detection of postoperative AF is still needed to assess the potential effect of PUFAs on postoperative AF and its potential adverse outcomes.

studies that used continuous electrocardiographic monitoring, which was observed in the Saravanan sample. Another important issue that must be considered is the concomitant use of beta-blockers and statins. In Calo et al.,9 for example, the percentage of patients taking both drugs was much lower than in Saravanan et al. (57.5% on betablockers and 56.9% on statins compared to 85% and 98%, respectively). This may have offset the beneficial effects of PUFAs in the Saravanan population (Table 3).

CLINICAL IMPLICATIONS Atrial fibrillation is the most common arrhythmia following cardiac surgery, and inflammation plays a role in its genesis. PUFAs have some anti-inflammatory properties and antiarrhythmic effects. However, this meta-analysis showed that PUFA treatment is not associated with a reduction in the incidence of AF following cardiac surgery. Thus, the routine use of PUFAs in patients undergoing cardiac surgery for the prevention of postoperative AF is not recommended.

AUTHOR CONTRIBUTIONS Armaganijan L conceived the study aims and design, contributed to the systematic review and data extraction, performed the analysis and interpreted the results, drafted the manuscript. Lopes RD, Healey JS conceived the study aims and design, contributed to the systematic review and data extraction, performed the analysis and interpreted the results. Nair GM, Morillo CA, Piccini JP contributed to the data extraction, interpretation of results, and to the revision of the manuscript.

LIMITATIONS Our study has some limitations. The major limitation is the clinical and statistical heterogeneity of the included studies. To overcome this degree of heterogeneity, it would be necessary to perform a large and adequately powered randomized study, which would be difficult to be conducted and expensive. Therefore, the lack of statistical power and the heterogeneity among the studies could, in part, have explained our findings. We only included randomized studies because they are the gold standard for assessing treatment effects. However, the population involved in randomized clinical trials is usually strictly selected and might not reflect the true profiles of the patients treated in clinical practice. Thus, caution should be taken when extrapolating our results to this setting. Furthermore, we could not systematically assess the adverse events associated with PUFA treatment because these outcomes were not systematically and consistently reported in the included studies. Various fatty acids and dosages were used in the studies, and we were unable to investigate the influence of the duration and dosage of preoperative

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7. Kang JX, Leaf A. Antiarrhythmic effects of polyunsaturated fatty acids. Recent studies. Circulation. 1996;94:1774-80. 8. Kang JX, Leaf A. Protective effects of free polyunsaturated fatty acids on arrhythmias induced by lysophosphatidylcholine or palmitoylcarnitine in neonatal rat cardiac myocytes. Eur J Pharmacol. 1996;297:97-106, doi: 10.1016/0014-2999(95)00701-6. 9. Calo L, Bianconi L, Colivicchi F, Lamberti F, Loricchio ML, de Ruvo E, et al. N-3 fatty acids for the prevention of atrial fibrillation after coronary artery bypass surgery: A randomized, controlled trial. J Am Coll Cardiol. 2005;45:1723-8, doi: 10.1016/j.jacc.2005.02.079. 10. Mariscalco G, Sarzi Braga S, Banach M, Borsani P, Bruno VD, Napoleone M, et al. Preoperative n-3 polyunsatured fatty acids are associated with a decrease in the incidence of early atrial fibrillation following cardiac surgery. Angiology. 2010;61:643-50, doi: 10.1177/0003319710370962. 11. Heidt MC, Vician M, Stracke SK, Stadlbauer T, Grebe MT, Boening A, et al. Beneficial effects of intravenously administered n-3 fatty acids for the prevention of atrial fibrillation after coronary artery bypass surgery: A prospective randomized study. Thorac Cardiovasc Surg. 2009;57:27680, doi: 10.1055/s-0029-1185301. 12. Heidarsdottir R, Arnar DO, Skuladottir GV, Torfason B, Edvardsson V, Gottskalksson G, et al. Does treatment with n-3 polyunsaturated fatty acids prevent atrial fibrillation after open heart surgery? Europace. 2010;12:356-63, doi: 10.1093/europace/eup429. 13. Saravanan P, Bridgewater B, West AL, Oâ&#x20AC;&#x2122;Neill SC, Calder PC, Davidson NC. Omega-3 fatty acid supplementation does not reduce risk of atrial fibrillation after coronary artery bypass surgery: A randomized, doubleblind, placebo-controlled clinical trial. Circ Arrhythm Electrophysiol. 2010;3:46-53, doi: 10.1161/CIRCEP.109.899633. 14. de Lorgeril M, Renaud S, Mamelle N, Salen P, Martin JL, Monjaud I, et al. Mediterranean alpha-linolenic acid-rich diet in secondary prevention of coronary heart disease. Lancet. 1994;343:1454-9, doi: 10.1016/S0140-6736 (94)92580-1. 15. Burr ML, Fehily AM, Gilbert JF, Rogers S, Holliday RM, Sweetnam PM, et al. Effects of changes in fat, fish, and fibre intakes on death and myocardial reinfarction: Diet and reinfarction trial (dart). Lancet. 1989;2:757-61, doi: 10.1016/S0140-6736(89)90828-3. 16. Siscovick DS, Raghunathan TE, King I, Weinmann S, Wicklund KG, Albright J, et al. Dietary intake and cell membrane levels of long-chain n3 polyunsaturated fatty acids and the risk of primary cardiac arrest. JAMA. 1995;274:1363-7, doi: 10.1001/jama.274.17.1363. 17. Mozaffarian D, Psaty BM, Rimm EB, Lemaitre RN, Burke GL, Lyles MF, et al. Fish intake and risk of incident atrial fibrillation. Circulation. 2004;110:368-73, doi: 10.1161/01.CIR.0000138154.00779.A5. 18. McLennan PL, Bridle TM, Abeywardena MY, Charnock JS. Dietary lipid modulation of ventricular fibrillation threshold in the marmoset monkey. Am Heart J. 1992;123:1555-61, doi: 10.1016/0002-8703(92)90809-A. 19. Fynn SP, Todd DM, Hobbs WJ, Armstrong KL, Garratt CJ. Role of dispersion of atrial refractoriness in the recurrence of clinical atrial

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CLINICS 2011;66(11):1929-1933

DOI:10.1590/S1807-59322011001100013

CLINICAL SCIENCE

The ADA*2 allele of the adenosine deaminase gene (20q13.11) and recurrent spontaneous abortions: an age-dependent association Daniela Prudente Teixeira Nunes,I Lı´gia Cosentino Junqueira Franco Spegiorin,II,III Cinara Ca´ssia Brandaõ de Mattos,I Antonio Helio Oliani,II,III Denise Cristina Mo´s Vaz-Oliani,II,III Luiz Carlos de MattosI I Faculdade de Medicina de Saõ Jose´ do Rio Preto (FAMERP), Immunogenetics Laboratory, Molecular Biology Department - Saõ Jose´ do Rio Preto, SP, Brazil. II Faculdade de Medicina de Saõ Jose´ do Rio Preto (FAMERP), Gynecology and Obstetrics Department, Saõ Jose´ do Rio Preto, SP, Brazil. III Hospital de Base - Fundaçaõ Faculdade Regional de Medicina de Saõ Jose´ do Rio Preto (HB-FUNFARME), Gynecology and Obstetrics Outpatient Clinic, Saõ Jose´ do Rio Preto, SP, Brazil.

OBJECTIVE: Adenosine deaminase acts on adenosine and deoxyadenosine metabolism and modulates the immune response. The adenosine deaminase G22A polymorphism (20q.11.33) influences the level of adenosine deaminase enzyme expression, which seems to play a key role in maintaining pregnancy. The adenosine deaminase 2 phenotype has been associated with a protective effect against recurrent spontaneous abortions in European Caucasian women. The aim of this study was to investigate whether the G22A polymorphism of the adenosine deaminase gene is associated with recurrent spontaneous abortions in Brazilian women. METHODS: A total of 311 women were recruited to form two groups: G1, with a history of recurrent spontaneous abortions (N = 129), and G2, without a history of abortions (N = 182). Genomic DNA was extracted from peripheral blood with a commercial kit and PCR-RFLP analysis was used to identify the G22A genetic polymorphism. Fisher’s exact test and odds ratio values were used to compare the proportions of adenosine deaminase genotypes and alleles between women with and without a history of recurrent spontaneous abortion (p,0.05). The differences between mean values for categorical data were calculated using unpaired t tests. The Hardy-Weinberg equilibrium was assessed with a chi-square test. RESULTS: Statistically significant differences were identified for the frequencies of adenosine deaminase genotypes and alleles between the G1 and G2 groups when adjusted for maternal age. CONCLUSIONS: The results suggest that the adenosine deaminase *2 allele is associated with a low risk for recurrent spontaneous abortions, but this association is dependent on older age. KEYWORDS: Adenosine deaminase; Recurrent spontaneous abortions; ADA gene. Nunes DPT, Spegiorin LCJF, Branda˜o de Mattos CC, Oliani AH, Vaz-Oliani DCM, de Mattos LC. The ADA*2 allele of the adenosine deaminase gene (20q13.11) and recurrent spontaneous abortions: an age-dependent association. Clinics. 2011;66(11):1929-1933. Received for publication on July 15, 2011; First review completed on July 15, 2011; Accepted for publication on July 22, 2011 E-mail: luiz.carlos@famerp.br / imunogenetica.famerp@gmail.com Tel.: 55 17 3201-5857

The enzyme adenosine deaminase (ADA; EC 3.5.4.4) catalyzes the deamination of adenosine and deoxyadenosine, thereby affecting the methylation process, cell growth and differentiation, apoptosis, DNA replication and immune functions.4,5 The genetic deficiency of ADA in humans results in severe combined immunodeficiency syndrome (SCID) of both the humoral and cellular immune responses.6 The polymorphism of the ADA gene (20q13.11) resulting from the substitution of G by A at nucleotide 22 of exon 1 replaces the Asp amino acid (ADA*1 allele) with Asn (ADA*2 allele) amino acid in position 8 of the enzyme. Consequently, individuals with the ADA*2 allele express low levels of ADA compared to homozygous ADA*1/*1 individuals.7 ADA activity is critical in maintaining a normal pregnancy.8,9 In a previous report, Nicotra et al.10 observed that the frequency of the ADA*2 allele is lower in European

INTRODUCTION Recurrent spontaneous abortion (RSA) is defined by two or more consecutive miscarriages before 20 weeks of gestation.1,2 The etiology of RSA is varied, but immunological, anatomical, endocrine, and genetic aspects are considered the most important factors. These factors may act on fetus selection in such a way as to impede the implantation of the zygote or to prevent fertilization.3

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Promega), 2.1 mL of DMSO (Nuclear), 1.0 mL of 2-mercaptoethanol (200 mM, Vetec), 1.0 mL of sense primer (5 pM, IDT; 59-GCCCGGCCCGTTAAGAAGAGC-39), 1.0 mL of antisense primer (5 pM, IDT; 59- GGTCAAGTCAGGGGCAGAAGCAGA-39), 4.0 mL of dNTPs (1.25 mM, Invitrogen), 0.2 mL of GoTaq Hot Start DNA polymerase (5 U, Promega), and 2.0 mL of genomic DNA. As an internal contamination control, a tube was prepared under identical conditions but without the genomic DNA (blank). The amplification conditions were as follows: 94 ˚C for 15 minutes, 36 cycles of 94 ˚C for 40 seconds, 66 ˚C for 80 seconds, 72 ˚C for 80 seconds, and 1 cycle of 72 ˚C for 8 min, with the product remaining at 4 ˚C ad infinitum. The amplified 397 bp fragment was analyzed by 2% agarose gel electrophoresis (Invitrogen) and ethidium bromide staining (Invitrogen). The PCR product (7.0 mL) was incubated at 65 ˚C with 0.7 mL of Taq I Fast Digest (1 U, Fermentas) and 1.34 mL of enzyme buffer (10x, Fermentas) for 20 minutes. After an electrophoresis run of 30 minutes at 100 volts in 2% agarose gel (Invitrogen), the fragments were viewed using ethidium bromide staining. The PCR product corresponding to the ADA*1 allele (G22) was then cleaved into two fragments: a 245 bp fragment and a 152 bp fragment. The ADA*2 allele (22A) was identified by the absence of the Taq I restriction site.

women who suffer RSA than those who do not, suggesting a protective effect of this allele against RSA and higher fertility rates among women with ADA*2 allele. Recently, these same authors observed a synergic effect of ACP1 (low molecular weight protein tyrosine phosphatase) and ADA polymorphisms with respect to RSA.11 These authors concluded that women with high ADA activity and low ACP1 activity have a higher susceptibility to RSA. A European ethnic background is present in the Brazilian population,12 but data are scarce on the importance of the ADA G22A genetic polymorphism in RSA in Brazilian women. The aim of this study was to investigate whether the ADA G22A polymorphism is associated with RSA in Brazilian women.

PATIENTS AND METHODS Ethical considerations This study was approved by the Research Ethics Committee of the Medical School in Sa˜o Jose´ do Rio Preto - FAMERP (# 308/2008). The objectives of the investigation and all procedures performed in the study were explained to selected patients, and those who agreed to participate in the study gave their written consent.

Patient selection

Statistical analysis

Two groups of pregnant women were selected from the Gynecology and Obstetrics Clinic, Hospital de Base from the Regional Medical School Foundation in Sa˜o Jose´ do Rio Preto, Sa˜o Paulo State, Brazil. The first group (G1, N = 129) included only women who had suffered at least two consecutive spontaneous abortions with the same partner according to their medical records and to reports from the patients themselves. The second group (G2, N = 182) included only women with at least two successive spontaneously conceived pregnancies and no history of spontaneous abortion. Patients who were younger than 18 years old were excluded from the study.

The GraphPad Instat computer program version 3.06 was used for all statistical calculations. Fisher’s exact test was used to detect differences in the distribution of ADA genotypes and alleles with respect to RSA; a 5% alpha error was considered acceptable. The differences between mean values for categorical data were calculated by the unpaired t test. Odds ratios and 95% confidence intervals were also calculated. The chi-square test was applied to compare the overall frequencies of ADA genotypes and verify whether the distribution of the ADA genotypes was in HardyWeinberg equilibrium using the Online Encyclopedia for Genetic Epidemiology studies (OEGE) (http://www.oege. org/software/hwe-mr-calc.shtml).

Data collection An epidemiological questionnaire was completed by all participating patients, and these data were later confirmed with their medical records. Comorbidities such as a history of diabetes, hypertension, polycystic ovarian syndrome, uterine malformation, antiphospholipid antibodies, and endometriosis were also collected from the medical records.

RESULTS This study evaluated women with (G1) and without (G2) a history of RSA. There were statistically significant differences with respect to the mean age (G1: 31.9¡5.7 vs. G2: 29.2¡5.8; p = 0.0001) and average number of pregnancies (G1: 4.6¡1.5 vs. G2: 3.5¡0.9; p = 0.0001). The mean number of spontaneous abortions in G1 was 2.7 (¡0.8), ranging from 2 to 6, and the average numbers of live births in G1 and G2 were 0.5 (¡0.3) and 2.6 (¡1.0), respectively (p = 0.0001). The identification of ADA genotypes was accomplished based on the electrophoretic profile of the 397 bp fragment of exon 1 of the ADA gene after digestion with the Taq I enzyme (Figure 1). The ADA*1/*1 and ADA*1/*2 genotypes were found in G1 and G2, but just one patient with the ADA*2/*2 genotype was observed in G2 (Table 1). The distributions of the ADA genotypes were found to be in Hardy-Weinberg equilibrium in G1 (x2 = 0.26, DF 1) and G2 (x2 = 0.57, DF 1). The frequencies of the ADA*1/*1, ADA*1/*2 and ADA*2/*2 genotypes were similar and showed no statistically significant differences (p = 0.6771, x2 = 0.78; DF 2). The differences remained insignificant even when the

Collection of blood samples and genomic DNA extraction Five milliliters of peripheral blood was collected from each participant in tubes containing EDTA anticoagulant. Genomic DNA was extracted from 200 mL of whole blood using a commercial kit (PureLinkTM Genomic DNA Mini Kit, Invitrogen). The manufacturer’s instructions were strictly followed.

Identification of the ADA*1 and ADA*2 alleles The identification of the ADA*1 and ADA*2 alleles was achieved using PCR-RFLP analysis following the protocol of Safranow et al.13 A gene amplification reaction (25 mL final volume) was performed for each sample of genomic DNA under the following conditions: 7.2 mL of MilliQ water, 5.0 mL of PCR Buffer Green (5x, Promega), 1.5 mL MgCl2 (25 mM,

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The ADA gens and recurrent spontaneous abortions Nunes DPT et al.

postulated that the ADA G22A genetic polymorphism might be associated with RSA in Brazilian women. The series of this study is representative of the population of the northwestern region of Sa˜o Paulo State, which is chiefly composed of Italian, Spanish, Portuguese, and African descendants.14 To avoid possible bias, a control group was formed of women who had at least two successful pregnancies and no history of miscarriages. The women who had experienced RSA who were enrolled in this study (G1) had a higher mean age compared to those who had not (G2). This difference may result from the fact that women without risk factors for spontaneous abortions achieve reproductive success and their desired number of children at an earlier age, whereas women with reproductive problems are encouraged to persist longer in their attempts to conceive and reproduce.15 Moreover, older age is a risk factor for RSA.1,3 This observation is supported by data from a recent demographic survey carried out in Brazil, which showed that maternal age of higher than 35 years contributes to an increased prevalence of RSA.16 In the present study, women who had experienced RSA became pregnant a significantly higher number of times than those who had not. Spontaneous abortions were confirmed based on clinically recognized pregnancy losses before 20 weeks of gestation; this condition has great predictive value for establishing the occurrence of unsuccessful pregnancies.1,3 Women who experience RSA persist in attempts to become pregnant even if the pregnancy does not often reach full term. Moreover, there is evidence that some early fetal losses are not clinically recognized in women with a predisposition to RSA because these pregnancies do not last long enough to be characterized as such. These fetal losses are not included in the statistics, and the rate of spontaneous abortions is therefore underestimated.17 Several women in G1 gave birth to live babies, but the average number of births was lower than those in G2. This finding is consistent with the finding that women at low risk for RSA are more likely to produce children. Moreover, in women at risk for RSA, the chance of spontaneous abortions increases with each fetal loss.18,19 It seems that reproductive success is achieved at a younger age, especially when mothers have no risk factors for RSA, contributing to a greater number of live births in this group.16,19 The frequencies of ADA genotypes and the ADA*1 and ADA*2 alleles were similar between G1 and G2 and were not associated with the occurrence of RSA. Hence, from a preliminary overview, the results presented in this study are different from those observed for Italian women in whom

Figure 1 - The electrophoretic profile of the fragment of 397 base pairs (bp) of exon 1 of the ADA gene after digestion with the Taq I enzyme showing the ADA*1/*1 (1), ADA*1/*2 (2) and ADA*2/*2 (3) genotypes and the marker (L) of 100 base pairs.

comparison between G1 and G2 was restricted to the ADA*1/*2 and ADA*2/*2 genotypes (p = 0.8342, OR = 1.177, 95% CI = 0.536-2.586). Although the frequencies of the ADA*1 and ADA*2 alleles were different in G1 and G2, the difference was not statistically significant (p = 0.7050, OR: 1.237, 95% CI: 0.578-2.646). A comparative analysis of the frequencies of the ADA*1 and ADA*2 alleles when controlling for age revealed statistically significant differences between the G1 and G2 groups (Table 2). The data for G1, grouped according to the ADA genotypes (ADA*1/*1 and ADA*1/*2), are shown in Table 3. There were no statistically significant differences in the mean age, average number of pregnancies, number of live births or comorbidities between women with the ADA*1/*1 genotype and those with the ADA*1/*2 genotype. Controlling for comorbidities, these analyses were not statistically significant (diabetes: OR: 0.385, CI 95%: 0.0453.297, p = 0.3601; hypertension: OR: 1.817, CI 95%: 0.23514.017, p = 1.000; polycystic ovarian syndrome: OR: 4.120, CI 95%: 0.243-69.710, p = 0.3984; uterine malformation: OR: 1.411, CI 95%: 0.080-24.737, p = 1.000; antiphospholipid antibodies: OR: 1.046, CI 95%: 0.132-8.241, p = 1.000; endometriosis: OR: 0.569, CI 95%: 0.159-2.031, p = 0.4198).

DISCUSSION The aim of this study was to investigate the association between the G22A polymorphism of the ADA gene in Brazilian women with a history of RSA. Because much of the Brazilian population has a clear European ancestry,12 we

Table 1 - The frequencies of ADA genotypes and alleles in women with (G1) and without (G2) a history of recurrent spontaneous abortions. G1 (N = 129) ADA genotypes ADA*1/*1 ADA*1/*2 ADA*2/*2 Alleles ADA*1 ADA*2

G2 (N = 182)

OR*

CI 95%

p-value**

118 11 0

91.5 8.5 0.0

164 17 1

90.2 9.3 0.5

1.177 0.904 0.426

0.536 – 2.586 0.408 – 2.003 0.017 – 10.552

0.8435 0.8436 1.0000

247 11

95.7 4.3

345 19

94.8 5.2

1.237

0.578 – 2.646

0.7050

*Unadjusted odds ratio. **Calculated by Fisher’s exact test.

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Table 2 - A comparison of the frequencies of ADA alleles and genotypes in women with (G1) and without (G2) RSA aged 35 years or younger and those older than 35 years of age. Genotypes 35 or younger ADA*1/*1 ADA*1/*2 Alleles ADA*1 ADA*2 Older than 35 ADA*1/*1 ADA*1/*2 ADA*2/*2 Alleles ADA*1 ADA*2 *

G1 (N = 129)

G2 (N = 182)

N = 85

N = 155

OR*

CI 95%

p-value**

76 9

89.4 10.6

146 9

94.2 5.8

0.5205

0.198 – 1.366

0.2042

161 9

94.7 5.3

301 9

97.1 2.9

0.5349

0.208 – 1.374

0.2126

(N = 44)

(N = 27)

42 2 0

95.5 4.5 0.0

18 8 1

66.7 29.6 3.7

10.500 0.1131 0.1985

2.059 – 53.539 0.021 – 0.584 0.007 – 5.055

0.0019 0.0050 0.3803

86 2

97.7 2.3

44 10

81.5 18.5

9.773

2.051 – 46.573

0.0012

Adjusted odds ratio. Calculated by Fisher’s exact test.

carriers of at least one ADA*2 allele have higher levels of circulating and intracellular adenosine.24 Because adenosine acts as a hormone that regulates blood flow, neurotransmission and platelet aggregation and is a potent vasodilator,25,26 its presence in the uterus and the placenta could contribute to a reduced rate of early loss of zygotes or fetuses, thus protecting ADA*2-carrying women against RSA.10,11 This protection may be especially relevant for women older than 35 years of age. Additionally, the increased adenosine resulting from the ADA*2 allele could contribute to vascular integrity, thus increasing uterine and placental blood flow.9 Therefore, evaluations of ADA levels at different times during the gestational period among women carrying distinct ADA genotypes could help to clarify the potential effects of the ADA*2 allele during successful pregnancies and shed additional light on the biological and clinical impact of this enzyme on RSA and in assisted reproduction. Different studies have observed low frequencies of the ADA*2 allele in couples with sterility problems, women suffering from RSA, those with great variability in gestation time and those who have low birth weight newborns.10,11 Because this enzyme has an important role in modulating the immune response, a reduction in its expression seems to affect the fertility rate of women older than 35 years of age. To assess the differential effects of the ADA*1 and ADA*2 alleles on miscarriages, the comorbidities of pregnant women in G1 with the ADA*1/*1 and ADA*1/*2 genotypes were compared. There were no statistically significant differences in mean age, the average numbers of pregnancies and spontaneous abortions, the average number of live births, diabetes (including pregnancy-related and type II diabetes), hypertension, polycystic ovarian syndrome, uterine malformations, antiphospholipid antibodies or endometriosis. Therefore, the ADA*1 and ADA*2 alleles, when controlled for comorbidities, are not associated with the presence or absence of RSA. These comorbidities are associated with miscarriages, but their influence may be independent of the ADA alleles and genotypes. The small number of ADA*1/*2 women in G1, which reduces the power of the test, does not allow an analysis of whether either allele provides a biological advantage with

the ADA*2 allele was associated with a lower risk of RSA.10,11 Different factors may contribute to the differences observed. It is possible that the ADA*2 allele has a low frequency in the Brazilian female population as a result of ethnic mixing and that its protective effect is overshadowed by other determinants of RSA. In fact, population analysis revealed that the frequency of the ADA*2 allele is 0.12 in Caucasians and varies between 0.03 and 0.04 in African descendants.7,20 The frequencies of the ADA*2 allele found in both groups of this study were lower than those reported for Caucasians but very close to those observed in African descendants. Because much of the population of the northwestern region of Sa˜o Paulo State has African ancestry,14 this characteristic could contribute to the reduced frequency of the ADA*2 allele in the casuistic analyzed in this study. The frequency of the ADA G22A genetic polymorphism in different Brazilian regions has been little explored. A recent study found that the frequency of the ADA*2 allele in healthy men and women in the State of Rio Grande do Sul is 11.7%.21 This percentage is approximately twice that found in the present study, but the ethnic background of the population of southern Brazil is different from that of the northwestern region of Sa˜o Paulo State.14,22,23 It is possible that the frequency of the ADA*2 allele among Brazilians is influenced by ethnic and racial admixture. Investigations focusing the ADA G22A polymorphism in other regions of Brazil could confirm this proposition. The ADA genotypes and alleles were compared between the G1 and G2 groups when controlling for maternal age because this variable was found to be associated with the risk for RSA. The results did not reveal statistically significant differences in the frequency of the ADA*2 allele among those aged 35 years or younger, whereas an association between this allele and low risk for RSA was observed for women older than 35 years of age. The role of the ADA*2 allele in reducing the risk of RSA is not completely understood. The presence of an ADA*2 allele reduces ADA enzyme expression to between 15 and 20% of that found in the homozygous ADA*1/*1 genotype, which enables an increase in adenosine levels.7 Consequently,

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The ADA gens and recurrent spontaneous abortions Nunes DPT et al.

Table 3 - The demographic characteristics, reproductive histories and comorbidities of women who had suffered two or more recurrent spontaneous abortions according to the ADA*1/*1 and ADA*1/*2 genotypes (N = 129). Characteristics Mean age (SD) Pregnancies (SD) Live births (SD) Miscarriages (SD) Comorbidities Diabetes Hypertension Polycystic ovarian syndrome Uterine malformation Antiphospholipid antibodies Endometriosis

ADA*1/*1 (N = 118)

ADA*1/*2 (N = 11)

p-value*

32.1 (¡5.7) 4.7 (¡1.5) 0.7 (¡0.5) 2.7 (¡0.8)

30.0 (¡5.1) 3.6 (¡1.1) 0.5 (¡0.3) 2.5 (¡0.6)

0.2301 0.2417 0.1953 0.6765 p-value ** 0.3030 1.0000 0.3607 1.0000 1.0000 0.1602

N 3 16 18 6 9 14

% 1.9 13.3 15.2 2.8 8.5 9.5

N 1 1 0 0 1 3

% 9.1 9.1 0.0 0.0 9.1 27.3

*Calculated by an unpaired t test. **Calculated by Fisher’s exact test.

10. Nicotra M, Bottini N, Grasso M, Gimelfarb A, Lucarini N, Cosmi E, et al. Adenosine deaminase and human reproduction: a comparative study of fertile women and women with recurrent spontaneous abortions. Am J Reprod Immunol. 1998;39:266-70, doi: 10.1111/j.1600-0897.1998. tb00363.x. 11. Nicotra M, Bottini N, La Torre M, Amante A, Bottini E, Gloria-Bottini F. Repeated spontaneous abortion. Cooperative effects of ADA and ACP1 genetic polymorphisms. Am J Reprod Immunol. 2007;58:1–10, doi: 10. 1111/j.1600-0897.2007.00483.x. 12. Bertonha J F. O Brasil, os imigrantes italianos e a polı´tica externa fascista, 1922-1943. Rev Bras Polı´ Int. 1997;40:106-30, doi: 10.1590/S003473291997000200005. 13. Safranow K, Rzeuski R, Binczak-Kuleta A, Czyzycka E, Skowronek J, Jakubowska K, et al. ADA*2 Allele of the Adenosine Deaminase Gene May Protect against Coronary Artery Disease. Cardiology. 2007;108:27581, doi: 10.1159/000099096. 14. Conjuntura Econoˆmica de Saõ Jose´ do Rio Preto. Secretaria Municipal de Planejamento e Gestaõ Estrate´gica; 2010. Available from http://www.rio preto.sp.gov.br/PortalGOV/do/subportais_Show?c = 146 (Accessed in 2011 (Mar 2). 15. Bottini N, Magrini A, MacMurray J, Cosmi E, Nicotra M, Gloria-Bottini F, et al. Smoking, haptoglobin and fertility in humans. Tob Induc Dis. 2002;1:3-6, doi: 10.1186/1617-9625-1-1-3. 16. Cecatti JG, Guerra GVQL, Sousa MH, Menezes GMS. Aborto no Brasil: um enfoque demogra´fico. Rev Bras Ginecol Obstet. 2010;32:105-11, doi: 10.1590/S0100-72032010000300002. 17. Choudhury SR, Knapp LA. Human reproductive failure I: Immunological factors. Hum Reprod Update. 2000;7:113-34, doi: 10. 1093/humupd/7.2.113. 18. Knudsen UB, Hansen V, Juul S, Secher NJ. Prognosis of a new pregnancy following previous spontaneous abortions. Eur J Obstet Gynecol Reprod Biol. 1991;39:31-6, doi: 10.1016/0028-2243(91)90138-B. 19. Andersen AMN, Wohlfahrt J, Cristens P, Olsen J, Melbye M. Maternal age and fetal loss: population based register linkage study. BMJ. 2000;320:1708-12, doi: 10.1136/bmj.320.7251.1708. 20. Weissmann J, Vollmer M, Pribilla O. Survey of the distribution of adenosine deaminase and superoxide dismutase markers in different populations. Hum Hered. 1982;32:344-56, doi: 10.1159/000153321. 21. Dutra GP, Ottoni GL, Lara DR, Bogo MR. Lower frequency of the low activity adenosine deaminase allelic variant (ADA1*2) in schizophrenic patients. Rev Bras Psiquiatr. 2010;32:275-8, doi: 10.1590/S151644462010005000003. 22. Correa SMS. Linha e´tnica entre ‘‘alema˜es’’ e ‘‘brasileiros’’ em a´rea de colonizaçaõ no Rio Grande do Sul. XXIV Simpo´sio Nacional de Histo´ria. 2007. 23. IBGE (Brasil); Histo´rico de Porto Alegre, Rio Grande do Sul. http:// www.ibge.gov.br/cidadesat/historicos_cidades/historico_conteudo.php? codmun = 431490 (acessado em 20.07.2010). 24. Napolioni V, Lucarini N. Gender-specific association of ADA genetic polymorphism with human longevity. Biogerontology. 2010;11:457-62, doi: 10.1007/s10522-010-9266-7. 25. Arch JRS, Newsholme EA. The control of the metabolism and the hormonal role of adenosine. Essays Biochem. 1978;14:182-3. 26. Wen X, Perrett D, Jones N, Tozer AJ, Docherty SM, Iles RK. High follicular fluid adenosine levels may be pivotal in the metabolism and recycling of adenosine nucleotides in the human follicle. Metabolism. 2010;59:1145-55, doi: 10.1016/j.metabol.2009.09.037.

respect to early fetal loss as established by other determinants of RSA. In summary, the data of this study show that the risk for RSA increases with maternal age and that the ADA*2 allele of the ADA gene is associated with low risk of RSA among older women.

ACKNOWLEDGEMENTS AND FUNDING This work was supported by the Higher Education Professional Training Board (CAPES) – Ministry of Education, Brazilian Government (to CCBM and DPTN), and by a research grant from the Medical School in Sa˜o Jose´ do Rio Preto - BAP-FAMERP (to AHO and LCM).

AUTHOR CONTRIBUTIONS Nunes DPT and Spegiorin LCJF contribute equally like first author. Nunes DPT and Branda˜o de Mattos CC performed the genomic analysis. Spegiorin LCJF, Oliani AH and Vaz-Oliani DCM are MD and selected the casuistic and provided samples. de Mattos LC conceived the study and prepared the manuscript.

REFERENCES 1. Rai R, Regan L. Recurrent Miscarriage. Lancet. 2006;9535:601-11, doi: 10. 1016/S0140-6736(06)69204-0. 2. Zegers-Hochschild F, Adamson GD, de Mouzon J, Ishihara O, Mansour R, Nygren K, et al. International Committee for Monitoring Assisted Reproductive Technology (ICMART) and the World Health Organization (WHO) revised glossary of ART terminology, 2009. Fertility and Sterility. 2009;92:1520-4, doi: 10.1016/j.fertnstert.2009.09.009. 3. Ford HB, Schust DJ. Recurrent Pregnancy Loss: Etiology, diagnosis and therapy. Rev Obstet Gynecol. 2009;2:76-83. 4. Siaw MFE, Mitchello BS, Koller CA, Coleman MS, Hutton JJ. ATP depletion as a consequence of adenosine deaminase inhibition in man. Proc Natl Acad Sci U S A. 1980;77:6157-61, doi: 10.1073/pnas.77.10.6157. 5. Dong RP, Kameoka J, Hegen M, Tanaka T, Xu Y, Schlossman SF, et al. Characterization of adenosine deaminase binding to human CD26 on T cells and its biologic role in immune response. J Immunol. 1996;156:1349-55. 6. Hirschhorn R, Candotti F. Immunodeficiency due to defects of purine metabolism. In: Ochs HD, Smith CIE, Puck JM, editors. Primary Immunodeficiency diseases: A molecular and genetic approach. 2nd edition. New York: Oxford University Press; 2007:p169-96. 7. Hirschhorn R, Yang DR, Israni A. An Asp8Asn substitution results in the adenosine deaminase (ADA) genetic polymorphism (ADA 2 allozyme): occurrence on different chromosomal backgrounds and apparent intragenic crossover. Ann Hum Genet. 1994;58:1-9, doi: 10.1111/j.14691809.1994.tb00720.x. 8. Yoneyama Y, Sawa R, Suzuki S, Ishino H, Miura A, Kuwabara Y, et al. Regulation of plasma adenosine deaminase levels in normal pregnancy. Gynecol Obstet Invest. 2002;53:71-4, doi: 10.1159/000052995. 9. Lee SJ, Hwang HS, Kim BNR, Kim MA, Lee JW, Park YW, et al. Changes in serum adenosine deaminase activity during normal pregnancy. J Korean Med Sci. 2007;22:718-21, doi: 10.3346/jkms.2007.22.4.718.

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DOI:10.1590/S1807-59322011001100014

CLINICAL SCIENCE

Revisiting stapled and handsewn loop ileostomy closures: a large retrospective series Emre Balik,I Tunc Eren,II Dursun Bugra,I Yilmaz Buyukuncu,I Ali Akyuz,I Sumer YamanerI I Istanbul University, Istanbul Faculty of Medicine, General Surgery, Department of General Surgery Millet Caddesi, Sehremini Capa, Turkey. Medeniyet University Goztepe Education & Research Hospital, Department of General Surgery, Istanbul, Turkey.

Istanbul

OBJECTIVE: To compare the surgical outcomes of stapled and handsewn closures in loop ileostomies. METHODS: The data of 225 patients requiring loop ileostomies from 2002 to 2007 were retrospectively evaluated. The patients underwent partial small-bowel resections and either handsewn or stapled anastomoses for the ileostomy closures. They were followed up postoperatively with routine surgical examinations. RESULTS: The study group consisted of 124 men and 101 women with a mean age of 49.12 years. The ileostomy closure was performed with handsewn in 129 patients and with stapled in 96 patients. The mean time to the first postoperative flatus was 2.426 days in the handsewn group and 2.052 days in the stapled group (p,0.05). The mean time to the first postoperative defecation was 3.202 days in the handsewn group and 2.667 days in the stapled group (p,0.05). The mean duration of patient hospital stay was 8.581 days for the handsewn group and 6.063 days for the stapled group (p,0.05). CONCLUSIONS: Patients who underwent ileostomy closure with stapled recovered faster in the postoperative period and required shorter hospital stays than those whose closures were performed with handsewn. In our opinion, stapled should be considered the gold standard for loop ileostomy closures. KEYWORDS: Loop ileostomy; Closure technique; Handsewn anastomosis; Stapled anastomosis; Surgical outcomes. Balik E, Eren T, Bugra D, Buyukuncu Y, Akyuz A, Yamaner S. Revisiting stapled and handsewn loop ileostomy closures: a large retrospective series. Clinics. 2011;66(11):1935-1941. Received for publication on June 26, 2011; First review completed on July 21, 2011; Accepted for publication on August 2, 2011 E-mail: ebalik@istanbul.edu.tr Tel.: 90 532 204 40 00

confirmed, any systemic factors are corrected, and any fistulae are controlled or corrected, these ileostomies are typically closed through the stoma site without a formal laparotomy.5 Both loop ileostomy construction and subsequent closure are generally believed to be fairly straightforward, safe procedures with relatively low associated morbidity and mortality.7 Many opinions exist about the optimal method for performing these closures. Proponents of each method claim several advantages, including a diminished risk of anastomotic complications and favorable surgical outcomes.5 Routine stoma closure can be performed either with a handsewn, end-to-end anastomosis or through various techniques using staples.8–10 Proponents of stapled (functional) anastomoses often claim that these have a larger diameter than sutured anastomoses and thus will likely have a lower risk of small bowel obstruction or anastomotic leakage. Stapling proponents also claim that these anastomoses are typically faster to construct and result in decreased operative times and potential cost reduction.5 Several studies have examined the differing surgical techniques for closing loop ileostomies to identify the method that minimizes perioperative morbidity, as measured in terms of bowel obstruction, wound infection, anastomotic leakage, operative time, and postoperative outcomes, such as the time to first flatus, time to first defecation, time to initiation of oral intake, and duration of

INTRODUCTION Stoma is a Greek word for mouth or opening.1 An intestinal stoma is an opening of the intestinal tract into the abdominal wall. Ileostomies were first described by the German surgeon Baum in 1879 and later by the Bohemian surgeon Maydl in 1883.2 In 1952, Brooke published his experiences with ileostomy construction and introduced a new method for suturing the mucosa to the skin.3 Unlike the first colostomies, the first ileostomies were end stomas. Turnbull and Weakley were the first surgeons to describe the loop ileostomy (in 1971).4 The diverting loop ileostomy is a commonly used stoma that is often employed to diminish the consequences of an anastomotic leak in low-colorectal anastomoses, ileal pouchanal anastomoses, and in situations in which reversible patient factors increase the risk of an anastomotic dehiscence.5 A defunctioning loop ileostomy is traditionally closed 6 to 12 weeks after the initial surgery.6 Once anastomotic healing is

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hospital stay.5 Unfortunately, most of these studies have either not found significant differences or have reported conflicting results.7 As a result, no significant patient safety differences have been found between stapled and sutured anastomoses, and there is no consensus on the best method for loop closure.9,11 Thus, we performed a review of 225 patients to help answer this question.

following the initiation of the liquid diet, the patients received semi-solid and then full-solid diets. The intravenous fluid support was gradually decreased and was ceased on the day that the full-solid diet was started. When sufficient flatus and defecation were confirmed, patients with adequate oral intake and analgesia were discharged from the hospital, and the duration of their stay was recorded. All complications diagnosed within the first 30 days after surgery were considered to be postoperative, including those specifically related to the operative procedure and general complications. The complications were further classified as ‘‘surgical’’ or ‘‘medical’’ according to the specific treatment required. A statistical analysis of the two ileostomy closure techniques compared the patients in the HA group with those in the SA group. In addition, the effects of the primary surgeries on the outcomes of the ileostomy closures were examined by dividing the patients into those who underwent a total proctocolectomy-ileal-pouch-anal anastomosis (the TPC group) and those who received a low-anterior resection (the LAR group). Student’s t-tests were used to evaluate the independent variables of postoperative surgical outcomes (the time to the first postoperative flatus, first defecation, and the initiation of oral intake, and the duration of hospital stay). Fisher’s exact test and the chi-squared test were used to evaluate postoperative complications. The SPSS program was used to perform the statistical analyses. Differences were considered statistically significant when p,0.05.

METHODS This study retrospectively evaluated the data of all patients at a single-center institution (Istanbul University, Istanbul Faculty of Medicine, Department of General Surgery) who required an ileostomy closure due to a protective loop ileostomy for coloanal or ileoanal anastomosis between 2002 and 2007. All patients who underwent ileostomy closure during this period were included in the study, without exclusion criteria. Approval for this study was obtained from the Ethics Committee of Istanbul University, Istanbul Faculty of Medicine. Signed informed consent was obtained from all patients who were included in the study. Clinical and epidemiological data on file and information on the primary and ileostomy closure operations were analyzed and correlated with complications in the first 30 postoperative days, the need for additional surgery, overall morbidity and mortality, and the surgical outcomes (i.e., the patients’ early postoperative follow-up parameters, such as the time to the postoperative first flatus, the first defecation, and the initiation of oral intake, and the duration of the hospital stay). All patients’ oral intake was discontinued the night before the ileostomy closure. No special bowel preparation was performed preoperatively. Prophylactic intravenous antibiotics (second-generation cephalosporin) were administered during anesthesia induction. The same highly experienced surgeons (a senior surgeon assisted by a resident) performed the procedures in all cases. All patients received general anesthesia. The ileostomy closure was initially attempted through a peristomal incision without midline laparotomy. To accomplish the ileostomy closures, all patients operated on between 2002 and 2005 underwent a partial small bowel resection and handsewn end-toend anastomosis (the HA group), while every patient operated on between 2005 and 2007 underwent a partial resection and stapled functional anastomosis (the SA group). All handsewn anastomoses were performed using a single extramucosal seromuscular layer of 3/0 polyglactin sutures (VicrylH, Ethicon, Somerville, NJ, USA), and all the stapled anastomoses used one of two GIA 80/3.8 mm-type linear cutters (AutosutureH, Covidien, Norwalk, CT, USA). The GIA 80/3.8 mm was preferred to the GIA 60/3.8 mm because it created a larger anastomotic lumen. The mesenteric layers were not closed after the anastomoses in any of the patients. All patients were postoperatively followed with daily routine surgical examinations that included auscultation for bowel sounds, abdominal examinations and wound care. Administration of narcotic analgesics was ceased on the first postoperative day, and analgesia was maintained with intravenous administration of nonsteroidal anti-inflammatory drugs for the next three postoperative days. The patients were offered a clear liquid diet at the time of their first flatus and were questioned to determine the time of their first postoperative defecation. On consecutive days

RESULTS Two hundred twenty-five patients who underwent diverting loop ileostomy closures were retrospectively evaluated. Our study group consisted of 124 men (55.1%) and 101 women (44.9%), with a mean age of 49.12 years (range, 17 – 85). The mean time between the initial surgery and stoma closure was 10 weeks (range, 8 – 16). The distribution of the patients according to their diagnoses and surgical interventions is summarized in Table 1. All patients with an initial diagnosis of rectal cancer underwent a low-anterior resection (LAR), and all patients with diagnoses of familial adenomatous polyposis (FAP) or ulcerative colitis (UC) underwent a total proctocolectomyileal-pouch-anal anastomosis (TPC). The secondary procedure (the ileostomy closure) was performed using handsewn end-to-end anastomosis (HA) in 129 patients (57.33%) and a stapled functional anastomosis (SA) in 96 patients (42.67%). The overall mean time to the first postoperative flatus was 2.205 days (range, 1 – 6). The mean time was 2.426 days (range, 1 – 6) in the HA group and 2.052 days (range, 1 – 6) in the SA group (p = 0.000 for the independent samples student’s t-test). The overall mean time to the first postoperative defecation was 2.878 days (range, 1 – 8). The mean time was 3.202 days (range, 1 – 8) in the HA group and 2.667 days (range, 1 – 6) in the SA group (p = 0.006 for the independent samples student’s t-test). The overall mean time to the initiation of oral intake was 3.822 days (range, 1 – 25). The mean time was 4.047 days (range, 1 – 25) in the HA group and 3.521 days (range, 2 – 13) in the SA group (p = 0.078 for the independent samples student’s t-test).

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Loop ileostomy closures Balik E et al.

Table 1 - The distribution of the patients according to their diagnoses and surgical interventions. Primary diagnosis

Number of patients %

Secondary operation (ileostomy closure)

Primary operation

FAPa

UCb

RCc

Total proctocolectomy (TPC)

Low-anterior resection (LAR)

23 10.22

61 27.11

141 62.67

84 37.33

141 62.67

129 57.33

96 42.67

Familial adenomatous polyposis. Ulcerative colitis. c Rectal cancer. (HA, partial small bowel resection and handsewn end-to-end anastomosis; SA, partial resection and stapled functional anastomosis). b

The overall mean duration of the hospital stay was 7.205 days (range, 2 – 60). The mean duration was 8.581 days (range, 3 – 60) in the HA group and 6.063 days (2 – 23) in the SA group (p = 0.002 for the independent samples student’s t -test). The above results are summarized in Table 2. The mean time to the first postoperative flatus was 2.46 days (range, 1 – 6) in the TPC group and 2.15 days (range, 1 – 6) the LAR group (p = 0.053 for the independent samples student’s t-test). The mean time to the first postoperative defecation was 3.08 days (range, 1 – 8) in the TPC group and 2.95 days (range, 1 – 8) in the LAR group (p = 0.518 for the independent samples student’s t-test). The mean time to the initiation of oral intake was 4.19 days (range, 1 – 10) in the TPC group and 3.60 days (range, 1 – 25) in the LAR group (p = 0.054 for the independent samples student’s t-test). The mean duration of the hospital stay was 8.35 days (range, 3 – 27) in the TPC group and 7.01 days (range, 2 – 60) in the LAR group (p = 0.103 for the independent samples student’s t-test). These results are summarized in Table 3. The postoperative medical complications consisted of acute renal failure and early postoperative fever, and the postoperative surgical complications consisted of anastomotic leakage, small bowel obstruction and wound infection. The distribution of postoperative medical and surgical complications according to the surgical technique used for the ileostomy closure is shown in Table 4. The medical complications were treated with simple, conservative therapies, which, in all cases, resulted in rapid improvement of the clinical condition and discharge without the need for any additional medical care. The cases of small bowel obstruction and wound infection were successfully treated with conservative therapies and did not require additional surgical interventions. Additional surgery due to anastomotic leakage was needed in seven patients (3.11%). A new ileostomy was created in four (1.78%) of these patients, and three (1.33%) patients underwent partial small bowel resection and endto-end anastomosis. All patients with anastomotic leakage

were eventually discharged from the hospital without additional medical or surgical complications in the postoperative follow-up periods for the additional surgeries. The overall complication rate was 19.4% in the HA group and 18.8% in the SA group (p.0.05, using the chi-squared test). There were no statistically significant differences between the two groups in the rates of the individual complications (i.e., wound infection and anastomotic leakage) (p.0.05, using the chi-squared and Fisher exact tests) (Table 4). Additionally, the overall complication rate for the TPC group was 21.4%, whereas the overall complication rate for the LAR group was 17.7%, revealing no statistically significant difference between these two groups in terms of the primary operation’s effects on the secondary operation (p.0.05, using the chi-squared test).

DISCUSSION Loop ileostomies are frequently used after ileoanal or coloanal anastomoses in colorectal surgery to prevent probable complications associated with the anastomosis itself. They are most frequently performed for colorectal cancer and inflammatory bowel disorders (IBD). Recently, the use of neoadjuvant chemoradiation therapy has resulted in an increase in sphincter-saving operations, leading to higher rates of low-colorectal and even coloanal anastomosis procedures.12–14 This decrease in the rate of abdominoperineal resection in favor of sphincter-saving operations may have led to an increase in the rate of diverting loop ileostomies. In terms of surgical complications, the postoperative morbidity and mortality rates for these lowcolorectal, coloanal, and ileoanal anastomoses, when performed alone, are so remarkably high that fecal diversion has become a routine recommendation. Since the first report of the procedure by Turnbull and Weakley15 in 1966, loop ileostomies have increased in popularity because of their technical simplicity, lack of odor, liquid discharge, and decreased rates of parastomal

Table 2 - A comparison of the early postoperative outcomes of stapled (SA) versus handsewn (HA) loop ileostomy closures. Early postoperative outcomes (days) First flatus First defecation Initiation of oral intake Duration of hospital stay

Overall study group

HA group

SA group

p-value

2.205 (1 – 6) 2.878 (1 – 8) 3.822 (1 – 25) 7.205 (2 – 60)

2.426 (1 – 6) 3.202 (1 – 8) 4.047 (1 – 25) 8.581 (3 – 60)

2.052 (1 – 6) 2.667 (1 – 6) 3.521 (2 – 13) 6.063 (2 – 23)

0.000 0.006 0.078 0.002

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considerations (stapled vs. handsewn suture techniques).7,8,22,27–33 In this study, our primary aim was to compare the two principal anastomotic techniques with respect to their surgical outcomes and complication rates. Hull et al.7 compared handsewn and stapled loop ileostomy closures and found no significant differences in the time to the first defecation, solid diet, or discharge. The complications were similar for the two groups. Similar results were reported by Pittman et al.34, who found no significant difference in the anastomotic leak rate, length of surgery, or length of hospitalization in patients with sutured versus stapled anastomoses. In a systematic review of randomized controlled trials, Lustosa et al.35 found more frequent stenosis and shorter procedure times when performing colorectal anastomoses using stapling, but there was insufficient evidence to demonstrate the superiority of stapling over handsewing. In this study, we compared the postoperative surgical outcomes of patients who underwent ileostomy closures using the two different surgical techniques. When the HA and SA groups were compared, the statistical analysis revealed a significantly shorter time to the first flatus, time to first defecation and duration of hospital stay in the SA group (p,0.05). No statistically significant difference in the time to initiation of oral intake could be demonstrated between the two groups (p.0.05); however, this outcome was not of primary importance because of its dependence on the surgeon’s decisions and methods during routine postoperative follow-up. These results show a faster recovery for the patients who underwent ileostomy closure with the SA technique compared to those who underwent the HA technique (Table 2). One of the major advantages observed with the SA technique was the shorter hospital stay. The mean durations of hospital stay were 6.063 and 8.581 days in the in the SA and HA groups, respectively (p,0.05). At first glance, these times may seem excessive following such a surgical intervention. This explanation is related to our institution’s nature; being a university hospital, it is obliged to accept all patients, most of whom are from the rural areas of our country. The patients often have to travel long distances, and so the risk of any kind of postoperative complication must be ruled out before they can be sent home. Although the mean times to the initiation of oral intake were 3.521 and 4.047 days in the SA and HA groups, respectively, the patients were followed in our clinic until they received fullsolid diets without any complications before they were discharged from the hospital. Despite this relatively long clinical follow-up, the results of our study still reflect a

Table 3 - A comparison and statistical analysis of the association between the presence of a remnant colon and the early postoperative outcomes, using independent samples student’s t-tests. Early postoperative outcomes (days) First flatus First defecation Initiation of oral intake Duration of hospital stay

Overall study group

TPC Group

LAR Group

p-value

2.21 2.88

2.46 3.08

2.15 2.95

0.053 0.518

3.82

4.19

3.60

0.054

7.21

8.35

7.01

0.103

hernia and prolapse.12,16–21 In addition to these advantages, surgeons have also preferred protective loop ileostomies over protective colostomies because of the expected decrease in morbidity and mortality associated with the stoma closure.12,16–20 Nevertheless, ileostomy closure is not by any means a morbidity-free procedure. The reported overall complication rates for ileostomy closure range from 10% to 17%, and can reach 30% when performed to divert ileoanal pouches.17,21,22 The most frequent complication after ileostomy closure is reported to be small bowel obstruction.17,23 This complication has been particularly associated with extensive pelvic dissection during the primary surgical procedure, ileal vessel distention, and inflammatory disease affecting the remaining small bowel in patients primarily treated for IBD with proctocolectomy and ileal pouch.17,21 The relatively high rates of this complication associated with IBD have also motivated studies examining the real benefits of protective fecal diversion in this specific situation.17,19,23,24 The temporary loop ileostomy is generally thought to be simple to construct and easy to close, with low perioperative morbidity and mortality. The two principal anastomotic techniques are end-to-end handsewn (HA) and functional stapled (SA) anastomoses. Numerous studies have compared the integrity of handsewn versus stapled bowel anastomoses, and it is generally thought that their complication rates are similar.25,26 Several factors are associated with an increased risk of postoperative complications after ileostomy closure, such as the interval between primary surgery and closure, the use of bowel preparation, antibiotic prophylaxis, and technical

Table 4 - The postoperative medical and surgical complications according to the surgical technique used for the ileostomy closure. Postoperative Complications Following Ileostomy Closure Medical Acute renal failure Surgical technique

Number of patients 1 % 0.44 p-value .0.05a a

SA 0 0

Surgical Fever

Anastomotic leakage

Small bowel obstruction

Wound infection

4 1.78 .0.05a

1 0.44

5 2.22 .0.05a

2 0.89

8 3.56 .0.05b

6 2.67

7 3.11 .0.05b

9 4.00

Fisher’s exact test. Chi-squared test.

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Figure 1 - A statistical analysis of the overall postoperative complications according to the surgical anastomotic technique used for the ileostomy closure.

Figure 2 - A statistical analysis of the overall postoperative complications according to the initial diagnoses and the primary surgical intervention.

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in the postoperative period and required shorter hospital stays than those who underwent the HA technique. There were no statistically significant differences between the two anastomotic techniques in postoperative complication rates. The nature of the primary surgical intervention did not significantly alter the postoperative surgical outcomes or the complication rates of the ileostomy closure. In our opinion, the SA technique should be considered the gold standard for loop ileostomy closures.

shorter hospital stay for the SA group compared with the HA group. There have been studies comparing the operative times and costs of the two techniques in addition to their surgical outcomes. Horisberger et al.36 found that the average operative time was 17.8 minutes shorter for the SA technique than for the HA technique, and that the costs associated with operative time were significantly higher for the HA group compared to the SA group. Although the material costs for the anastomosis were significantly higher for the SA group, there were no significant differences between the HA and SA groups in the overall costs (including the surgical costs and hospital stay).36 Previous studies have shown that, in general, a reduction of the operative time by 15 minutes through the use of a stapled anastomosis reduced the overall cost per case.7 We focused on postoperative surgical outcomes and surgical complications in our study, and we did not evaluate the operative time and costs, which is a weakness of our study. The effects of the primary surgical intervention on the secondary procedure (ileostomy closure) were also investigated in our study, with the aim of determining whether the presence of remnant colonic segments affected the surgical outcomes of the ileostomy closures. We compared the postoperative surgical outcomes of patients who had previously undergone TPC and LAR. No statistically significant differences between the TPC group and the LAR group were found in the postoperative times to first flatus, first defecation, and initiation of oral intake or in the duration of the hospital stay (p.0.05) (Table 3). These results indicate that the primary surgical technique and the presence of a remnant colon did not significantly affect the outcomes of ileostomy closures. The complications in the post-closure interval that required surgery were small bowel obstruction and anastomotic leakage. There have been reports of obstruction and leakage frequencies after ileostomy closure of 0% to 18% and 0% to 3%, respectively.12,21,37–40 Others studies have reported obstruction rates of 0% to 9% for the enterotomy suture technique, and 0% to 0.5% for the stapled anastomosis technique.12,21,37 Advocates have suggested that the stapled anastomosis provides a larger lumen; this consideration is particularly important in the presence of a malfunctioning distal limb, which may have a smaller diameter.37,38,40 The distribution of postoperative medical and surgical complications according to the surgical techniques used for ileostomy closure is summarized in Table 4. No statistically significant differences were found between the HA and the SA groups in the overall complication rate (p.0.05, using the chi-squared test). There were also no statistically significant differences between the two groups in the rates of the individual complications (i.e., wound infection and anastomotic leakage) (p.0.05) (Figure 1). The overall complication rates for the TPC and LAR groups were also compared, and no statistically significant differences were found between these two groups (p.0.05, using the chi-squared test) (Figure 2). These results show that the nature of the primary surgical technique and the presence of a remnant colon did not significantly increase the complications rate for the secondary procedure. The two principal anastomotic techniques for ileostomy closures are HA and SA. Patients who underwent ileostomy closures with the SA technique recovered significantly faster

ACKNOWLEDGEMENTS The authors would like to thank Sedat ZIYADE, MD for his contributions to the statistical work-up of this study. Istanbul Vakıf Gureba Training & Research Hospital, Department of Thoracic Surgery.

AUTHOR CONTRIBUTIONS Balik E supervised and executed the study, performed most of the operations of the principal (or CPC, or RAF), and secondary operations (ileostomy closure), and operated the follow-up of patients. Eren T performed the study throughout his residence, participated in almost all operations, performed the surgery follow-up of patients in the department, collected the data, created Excel charts, performed the statistical evaluations and wrote the manuscript. Bugra D was the leader in establishing the study protocol and coordination of the surgical team during the implementation of this Protocol, held two primary operations (or CPC, or RAF), and secondary operations (ileostomy closure), and participated in the surgery follow-ups of patients in the department, read and revised the article. Buyukuncu Y performed primary operation (or CPC, or RAF), and secondary operations (ileostomy closure), and participated in the surgery follow-ups of patients in the department, revised the writing of the article. Akyuz A, chief surgeon of the surgical team, held two primary operations (or CPC, or RAF), and secondary operations (ileostomy closure), and participated in the surgery follow-ups of patients in the department, he oversaw all steps of the study and performed the last revision of the article before submission. Yamaner S had the inspiration for this work, raised the idea of such study, conducted operations both primary (or CPC, or RAF), and secondary operations (ileostomy closure), and participated in the surgery follow-ups of patients in the department, supervised every stage of the study and revised the article in order to eliminate possible errors.

REFERENCES 1. Stredman’s Medical Dictionary. 27th ed. Baltimore: Lippincott Williams & Wilkins; 2000. 2. Kaidar-Person O, Person B, Wexner SD. Complications of construction and closure of temporary loop ileostomy. J Am Coll Surg. 2005;201:759– 73, doi: 10.1016/j.jamcollsurg.2005.06.002. 3. Brooke BN. The management of an ileostomy including its complications. Dis Colon Rectum. 1993;36:512–6, doi: 10.1007/BF02050020. 4. Turnbull RB Jr, Weakley FL. Atlas of intestinal stomas. St Louis: Mosby; 1967:32–9. 5. Leung TT, Maclean AR, Buie WD, Dixon E. Comparison of stapled versus handsewn loop ileostomy closure: A meta-analysis. J Gastrointest Surg. 2008;12:939–44, doi: 10.1007/s11605-007-0435-1. 6. Chand M, Nash GF, Talbot RW. Timely closure of loop ileostomy following anterior resection for rectal cancer. Eur J Cancer Care. 2008;17:611–5. 7. Hull TL, Kobe I, Fazio VW. Comparison of handsewn with stapled loop ileostomy closures. Dis Colon Rectum. 1995;39:1086–9, doi: 10.1007/ BF02081405. 8. Hasegawa H, Radley S, Morton DG, Keighley MR. Stapled versus sutured closure of loop ileostomy: a randomized controlled trial. Ann Surg. 2000;231:202–4, doi: 10.1097/00000658-200002000-00008. 9. Mansfield SD, Jensen C, Phair AS, Kelly OT, Kelly SB. Complications of loop ileostomy closure: a retrospective cohort analysis of 123 patients. World J Surg. 2008;32:2101–6, doi: 10.1007/s00268-008-9669-7. 10. Shelygin YA, Chernyshov SV, Rybakov EG. Stapled ileostomy closure results in reduction of postoperative morbidity. Tech Coloproctol. 2010;14:19–23, doi: 10.1007/s10151-009-0550-y.

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11. Allal AS, Kahne T, Reverdin AK, Lippert H, Schlegel W, Reymond MA. Radioresistance-related proteins in rectal cancer. Proteomics. 2004;4:2261–9, doi: 10.1002/pmic.200300854. 12. Fasth S, Hulten L. Loop ileostomy: a superior diverting stoma in colorectal surgery. World J Surg. 1984;8:401–7, doi: 10.1007/BF01655089. 13. Habr-Gama A, Perez RO, Kiss DR, Rawet V, Scanavini A, Santinho PM, et al. Preoperative chemoradiation therapy for low rectal cancer. Impact on downstaging and sphincter-saving operations. Hepatogastroenterology. 2004;51:1703–7. 14. Habr-Gama A, Perez RO, Nadalin W, Nahas SC, Ribeiro U Jr, Silva E Sousa AH Jr, et al. Long-term results of preoperative chemoradiation for distal rectal cancer correlation between final stage and survival. J Gastrointest Surg. 2005;9:90–9, doi: 10.1016/j.gassur.2004.10.010. 15. Turnbull RB Jr, Weakley FL. Ileostomy technics and indications for surgery. Rev Surg. 1966;23:310–4. 16. Cheape JD, Hooks VH 3rd. Loop ileostomy: a reliable method of diversion. South Med J. 1994;87:370–4, doi: 10.1097/00007611-19940300000014. 17. Edwards DP, Leppington-Clarke A, Sexton R, Heald RJ, Moran BJ. Stoma-related complications are more frequent after transverse colostomy than loop ileostomy: a prospective randomized clinical trial. Br J Surg. 2001;88:360–3, doi: 10.1046/j.1365-2168.2001.01727.x. 18. GooszeN AW, Geelkerken RH, Hermans J, Lagaay MB, Gooszen HG. Temporary decompression after colorectal surgery: randomized comparison of loop ileostomy and loop colostomy. Br J Surg. 1998;85:76–9, doi: 10.1046/j.1365-2168.1998.00526.x. 19. Law WL, Chu KW, Choi HK. Randomized clinical trial comparing loop ileostomy and loop transverse colostomy for faecal diversion following total mesorectal excision. Br J Surg. 2002;89:704–8, doi: 10.1046/j.13652168.2002.02082.x. 20. Sakai Y, Nelson H, Larson D, Maidl L, Young-Fadok T, Ilstrup D. Temporary transverse colostomy vs loop ileostomy in diversion: a casematched study. Arch Surg. 2001;136:338–42, doi: 10.1001/archsurg.136.3. 338. 21. Van de Pavoordt HD, Fazio VW, Jagelman DG, Lavery IC, Weakley FL. The outcome of loop ileostomy closure in 293 cases. Int J Colorectal Dis. 1987;2:214–7, doi: 10.1007/BF01649508. 22. Shellito PC. Complications of abdominal stoma surgery. Dis Colon Rectum. 1998;41:1562–72, doi: 10.1007/BF02237308. 23. Petit T, Maurel J, Lebreton G, Javois C, Gignoux M, Segol P. Results and indications of lateral ileostomy functionally terminated in colorectal surgery. Ann Chir. 1999;53:949–53. 24. Rullier E, LE Toux N, Laurent C, Garrelon JL, Parneix M, Saric J. Loop ileostomy versus loop colostomy for defunctioning low anastomoses during rectal cancer surgery. World J Surg. 2001;25:274–7, doi: 10.1007/ s002680020091.

25. Macrae HM, Mcleod RS. Handsewn vs. stapled anastomoses in colon and rectal surgery: a meta-analysis. Dis Colon Rectum. 1998;41:180–9, doi: 10.1007/BF02238246. 26. Yamamoto T, Bain IM, Mylonakis E, Allan RN, Keighley MRB. Stapled functional end-to-end anastomosis versus sutured end-toend anastomosis after ileocolonic resection in Crohn Disease. Scand J Gastroenterol. 1999;7:708–13. 27. Berry DP, Scholefield JH. Closure of loop ileostomy. Br J Surg. 1997;84:524, doi: 10.1002/bjs.1800840424. 28. Carlsen E, Bergan AB. Loop ileostomy: technical aspects and complications. Eur J Surg. 1999;165:140–3, doi: 10.1080/110241599750007324. 29. Altomare DF, Pannarale OC, Lupo L, Palasciano N, Memeo V, Rubino M. Protective colostomy closure: the hazards of a ‘‘minor’’ operation. Int J Colorectal Dis. 1990;5:73–8, doi: 10.1007/BF00298472. 30. Mann LJ, Stewart PJ, Goodwin RJ, Chapuis PH, Bokey EL. Complications following closure of loop ileostomy. ANZ J Surg. 1991;61:493–6, doi: 10. 1111/j.1445-2197.1991.tb00275.x. 31. Phang PT, Hain JM, Perez-Ramirez JJ, Madoff RD, Gemlo BT. Techniques and complications of ileostomy takedown. Am J Surg. 1999;177:463–6, doi: 10.1016/S0002-9610(99)00091-4. 32. Kestenberg A, Becker JM. A new technique of loop ileostomy closure after endorectal ileoanal anastomosis. Surgery. 1985;98:109–11. 33. Amin SN, Memon MA, Armitage NC, Scholefield JH. Defunctioning loop ileostomy and stapled side-to-side closure has low morbidity. Ann R Coll Surg Engl. 2001;83:246–9. 34. Pittman DM, Smith LE. Complications of colostomy closure. Dis colon rectum. 1985;28:836–43, doi: 10.1007/BF02555488. 35. Lustosa SA, Matos D, Atallah AN, Castro AA. Stapled versus handsewn methods for colorectal anastomosis surgery: a systematic review of randomized controlled trials. Sao Paulo Med J. 2002;120:132–6, doi: 10. 1590/S1516-31802002000500002. 36. Horisberger K, Beldi G, Candinas D. Loop Ileostomy Closure: Comparison of Cost Efectiveness between Suture and Stapler. World J Surg. 2010;34:2867-71, doi: 10.1007/s00268-010-0787-7. 37. Wexner SD, Taranow DA, Johansen OB, Itzkowitz F, Daniel N, Nogueras JJ, et al. Loop ileostomy is a safe option for fecal diversion. Dis Colon Rectum. 1993;36:349–54, doi: 10.1007/BF02053937. 38. Feinberg SM, Mcleod RS, Cohen Z. Complications of loop ileostomy. Am J Surg. 1987;153:102–7, doi: 10.1016/0002-9610(87)90209-1. 39. Senapati A, Nicholls RJ, Ritchie JK, Tibbs CJ, Hawley PR. Temporary loop ileostomy for restorative proctocolectomy. Br J Surg. 1993;80:628–30, doi: 10.1002/bjs.1800800529. 40. Khoo RE, Cohen MM, Chapman GM, Jenken DA, Langevin JM. Loop ileostomy for temporary fecal diversion. Am J Surg. 1994;167:519–22, doi: 10.1016/0002-9610(94)90249-6.

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DOI:10.1590/S1807-59322011001100015

CLINICAL SCIENCE

Development and analysis of a low-cost screening tool to identify and classify hearing loss in children: a proposal for developing countries Alessandra Giannella Samelli, Camila Maia Rabelo, Ana Paula Chaparin Vespasiano Universidade de Saõ Paulo, Centro de Doceˆncia e Pesquisa em Fonoaudiologia, Fisioterapia e Terapia Ocupacional da Universidade de Saõ Paulo, Saõ Paulo/Brazil.

OBJECTIVES: A lack of attention has been given to hearing health in primary care in developing countries. A strategy involving low-cost screening tools may fill the current gap in hearing health care provided to children. Therefore, it is necessary to establish and adopt lower-cost procedures that are accessible to underserved areas that lack other physical or human resources that would enable the identification of groups at risk for hearing loss. The aim of this study was to develop and analyze the efficacy of a low-cost screening tool to identify and classify hearing loss in children. METHODS: A total of 214 2-to-10 year-old children participated in this study. The study was conducted by providing a questionnaire to the parents and comparing the answers with the results of a complete audiological assessment. Receiver operating characteristic (ROC) curves were constructed, and discriminant analysis techniques were used to classify each child based on the total score. RESULTS: We found conductive hearing loss in 39.3% of children, sensorineural hearing loss in 7.4% and normal hearing in 53.3%. The discriminant analysis technique provided the following classification rule for the total score on the questionnaire: 0 to 4 points – normal hearing; 5 to 7 points – conductive hearing loss; over 7 points – sensorineural hearing loss. CONCLUSION: Our results suggest that the questionnaire could be used as a screening tool to classify children with normal hearing or hearing loss and according to the type of hearing loss based on the total questionnaire score. KEYWORDS: Questionnaire; Hearing loss; Children; Low-cost screening; Hearing. Samelli AG, Rabelo CM, Vespasiano APC. Development and analysis of a low-cost screening tool to identify and classify hearing loss in children: a proposal for developing countries. Clinics. 2011;66(11):1943-1948. Received for publication on July 3, 2011; First review completed on August 3, 2011; Accepted for publication on August 8, 2011 E-mail: alesamelli@usp.br Tel.: 55 11 3091-7455

Chronic otitis media is considered the main cause of mild-to-moderate hearing loss.5-7 The prevalence ranges from 1% to 46% around the world and is higher in developing countries. This condition is considered by the World Health Organization to be a public health problem.5,8 Children with slight/mild hearing loss (,40 dBHL in one or both ears) are an underreported and understudied group. The majority of studies have focused on children with higher levels of hearing loss,9 although studies have shown that even slight/mild hearing loss can result in negative consequences for the biopsychosocial development of children.9,10 In Brazil, primary care does not address hearing health or even recognize this demand, which is partially due to the limited number of health professionals in this area.1 Moreover, the territorial extension, the cost of equipment and a lack of human resources make it difficult to implement such programs. The Family Health Strategy Program has been growing in Brazil, but it is still lacking with regard to actions to address hearing health in primary

INTRODUCTION The prevalence of permanent mild or more severe hearing loss is estimated at one in every ten people.1 In Brazil, many localities already require newborn hearing screening by law, but only in a few cities. However, because newborn hearing screening is not universally implemented in many areas,2 permanent congenital and early-onset hearing loss in children may be detected late in developing countries, with the mean age varying from approximately 2 to 7 years.3 Otitis media is one of the most common disorders in childhood, and approximately 80% of school-age children suffer from temporary hearing loss every year.4

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as a preliminary screening tool to decrease the number of audiological assessments that are necessary in Brazil. Parents answered the questionnaire (Appendix 1), and each answer represented a risk factor for hearing loss. For each subject, every factor that was present was scored with a 1. The score for each question and the total score were calculated. Prior to administering the questionnaires and conducting the other procedures, we studied the questionnaire testretest reproducibility. For this stage, 20 parents of children belonging to the study area and not participating in the other procedures (corresponding to approximately 10% of the total sample) answered the questionnaire twice at an interval of three months. The questionnaire was applied by one researcher the first time and by another the second time. The aim of this step was to verify the clarity and replicability of the answers. The intraclass correlation coefficient showed moderate concordance (0.7) between the values of the total score in the two applications of the questionnaire, suggesting that the understanding and clarity of the questions was suitable.

care. Development of a strategy that involves low-cost screening may help fill the current gap in the care of children with hearing problems in developing countries. Therefore, it is necessary to establish and adopt lower-cost procedures, such as the use of screening and/or questionnaires, that are accessible to underserved areas of the country that lack the physical or human resources necessary to identify groups of children who are at risk for hearing loss as early as possible.11 Although several studies on this topic have been conducted, many have neglected to consider slight or mild and/or unilateral hearing loss. Furthermore, most of the previous studies have been conducted using low-cost instruments and have failed to classify hearing loss by type. Therefore, the aim of the present study was to develop and analyze the efficacy of a low-cost screening tool to identify and classify hearing loss in children through comparison with the results of a complete audiological assessment.

MATERIALS AND METHODS This study was carried out in the Hearing Health Primary Care Laboratory with the approval of the Sa˜o Paulo University School of Medicine Ethics Committee (Protocol # 778/08). All participants provided written informed consent to participate in the study.

Procedures This study was double-blind, as different evaluators performed the questionnaire and the audiological assessment; the parents only knew the results at the end of the investigation. An audiological assessment was conducted on all participants. Following otoscopic inspection, tympanometry was carried out using a GSI 38 Auto Tymp (Grason-Stadler). An audiometric evaluation for pure-tone thresholds was carried out with an Itera II Clinical Audiometer (Madsen) using standard audiometric techniques in a sound-attenuated testing room (from 250 to 8000 Hz). These results were compared with the questionnaire scores, establishing the sensitivity and specificity of this tool. The results of the audiological assessment were considered normal when hearing the thresholds were normal, i.e., #15 dBHL, and when tympanometry presented a type-A curve and acoustic reflexes were detected in both ears. Hearing loss was indicated when the pure tone thresholds were elevated17,18 (.15 dB HL), when the tympanogram was type B, C, As, or Ad and/or when there was an absence of acoustic reflexes with one or both ears.

Study population We carried out a hearing health survey among children who were 2 to 10 years of age in the District of Butanta˜, which is located in the western region of Sa˜o Paulo, Brazil. The study area comprised Sa˜o Remo community, a slum area with approximately 10,000 inhabitants attended by a public primary health care unit staffed by health community agents included in the Family Health Program (more than 2,000 indexed families). Children 2 to 10 years old constitute approximately 18% of this population, but there is no established hearing health care program despite a federal law guaranteeing people’s access to all levels of hearing care. Universal newborn hearing screening and hearing screening in schools are not current practices in this region or across the country, and therefore, late identification of hearing loss in children is common. Children who met the inclusion criteria and were between the ages of 2 and 10 years old (the age criterion applied was selected based on the United Nations’ definition of the beginning of adolescence, i.e., only children up to 10 years of age were included) were invited to participate in this study. The children were evaluated in their family homes, schools and kindergartens in the region, as well as at the health unit. A total of 214 2-10 year-old children of both genders took part in this study.

Statistical analyses The statistical analyses were performed using SPSS 15.0 for Windows. The Kolmogorov-Smirnov test was applied to determine the normality of the variable distribution. To assess the existence of an association between hearing status and gender and between hearing status and age group, the chi-square test (and Fisher’s exact test, when appropriate) was used. The distribution of the total score on the questionnaire was compared between children with normal hearing and those with hearing loss and between children with normal hearing, children with conductive hearing loss and children with sensorineural hearing loss; the results were evaluated using the Kruskal-Wallis test. When necessary, the Bonferroni procedure was used to identify differences between the distributions. An ROC curve was constructed to establish a cutoff value of the total score that would classify a child as either having normal hearing or affected by hearing loss based on the questionnaire. The discriminant analysis technique was used to classify a child

Questionnaire To investigate the problem (late identification of hearing loss in children), we developed a questionnaire to identify children at risk for hearing loss (Appendix 1) with questions regarding health history (pre-, peri-, and post-natal), development, communication skills, and hearing complaints based on previous investigations.3,12-16 This questionnaire was validated in the present investigation and will be applied by health community agents of the family health program in the future to expand access to hearing health primary care services. This questionnaire will also be used

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in one of three categories of hearing (normal hearing, conductive hearing loss, and sensorineural hearing loss) based on the total score. The level of significance was set at 0.05.

Table 2 - Statistics describing the total scores for the questionnaire based on the type of hearing loss or normal audiological assessment.

RESULTS

Children

Characterization of the sample Girls composed 40.7% of the sample, and boys represented 59.3%. There was a significantly greater number of boys participating (p,0.01). Of these 214 children, 114 (53.3%) had normal hearing and 100 (46.7%) had hearing loss (Table 1). When the type of hearing loss was considered, 84 (39.3%) had conductive hearing loss (slight-mild to moderate), and 16 (7.4%) had sensorineural hearing loss (severe to profound). Questionnaire scores (by age and type of hearing loss) Children in the hearing loss category presented higher scores than children in the normal category (Table 1). Furthermore, the total score values were higher in the sensorineural hearing loss group than in the conductive hearing loss group (p,0.001) and the normal group (p,0.001) (Table 2).

2-10 years

Based on the rule described above, without regard to questions 3 and 4 on the questionnaire, which refer to prior Table 1 - Descriptive statistics for the total score in the questionnaire in both categories of audiological assessment (normal and hearing loss).

2-10 years

Normal Hearing loss Total

114 100 214

3.8 6.1 4.9

1.9 3.0 2.7

114 84 16 214

3.8 5.2 10.6 4.9

1.9 2.3 1.6 2.7

In the studied sample, we observed a large number cases of slight or mild-to-moderate conductive hearing loss (39.3%) and severe-to-profound sensorineural hearing loss (7.4%). This prevalence of hearing loss is consistent with other studies that have found high rates of hearing loss in ‘‘disadvantaged’’ groups in developing countries,1,3,5,19 such as the population studied here. Furthermore, Olusanya3 pointed out that several studies have shown a significant prevalence of hearing disorders in children in developing countries, but the authors did not consider the differences in the test protocols employed. The prevalence of hearing loss in developing countries varies from approximately 4% to 25%, with the middle ear disorders ranging from 7% to 36%, which is consistent with the findings of our study. The largest number of slight-to-mild conductive hearing loss observed in our study is consistent with a previous study,20 which found a lower prevalence of hearing loss in a population of children aged 6 to 12 years. It is known that the prevalence of otitis media is highest in children between 2 and 5 years of age,21,22 which coincides with the age range investigated here. Above the age of 5, the prevalence of otitis media decreases, and this may explain the low prevalence reported in this investigation.20 As seen from our results, there is great variability regarding the prevalence of hearing loss reported in various developing countries. This variability is probably due to the different screening procedures and criteria for hearing loss, as well as socioeconomic, health, and other intrinsic and extrinsic factors of each population, which often are not directly related to hearing loss.20 It is noteworthy that the prevalence of hearing loss in this study cannot be extrapolated to the general population because the research was developed within a specific district of Sa˜o Paulo, Brazil. Furthermore, not all of those

- Total score from 0 to 4: normal hearing; - Total score from 5 to 7: conductive hearing loss; - Total score from 8 to 14: sensorineural hearing loss.

Standard deviation

Normal Conductive Sensorineural Total

Characterization of the sample

A rule was also established to classify each subject as having normal hearing, conductive hearing loss or sensorineural hearing loss, and this rule was also based on the cutoff values of the total score. Table 2 shows that the total score distribution was not the same in the three groups (normal, conductive hearing loss and sensorineural hearing loss). Thus, the technique of discriminant analysis yielded the following classification rule:

Mean

Standard Deviation

This study developed a low-cost screening tool to identify hearing loss in children in developing countries that may be used by community agents of the Family Health Program in the future. Furthermore, we analyzed the efficacy of this tool when compared to the results of an audiological assessment. Our findings suggest that this screening tool could be used to classify children as having normal hearing or hearing loss and to identify the type of hearing alteration (conductive or sensorineural hearing loss) based on the scores of the questionnaire.

- An individual could be classified as having ‘‘hearing loss’’ when the total score was greater than or equal to 6; - An individual could be classified as having ‘‘normal hearing’’ when the total score was less than 6.

Mean

DISCUSSION

To determine a cutoff value of the total score for ranking the results of the audiological assessment in normal children or children with hearing loss, ROC analysis was performed (Figure 1). The nearest point on the upper left corner of the curve represents the maximum sensitivity and specificity (0.44 sensitivity and 0.87 specificity). The area under the curve was 0.72, indicating good discriminatory power of the total score. Thus, the point that simultaneously provides the highest sensitivity and specificity corresponds to 5.5. Therefore, the classification rule was established as follows:

Categories

hearing assessment, the questionnaire correctly classified 85% of children with normal hearing, 68% of those with conductive hearing loss and all of the subjects with sensorineural hearing loss.

Cutoff scores of the questionnaire

Children

Type

p-value ,0.001

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Figure 1 - ROC Curve.

invited to participate in the study attended the audiological assessment, which may constitute a limitation of this study; it can be hypothesized that the parents of children suspected of suffering from hearing loss were more willing to participate in the study, and thus the true prevalence of hearing loss could be somewhat lower. However, identification of hearing loss prevalence in the general population was not a goal of the present study.

Slight and mild hearing losses are the most difficult to detect and are often overlooked in studies of hearing health. Taha et al.20 emphasized the use of questionnaires as a method of screening to address parentsâ&#x20AC;&#x2122; concerns regarding hearing loss, although previous studies have not reached a consensus on this subject because of variations in the sensitivity of the proposed instruments. Furthermore, the authors stressed the importance of further investigations on the cost-effectiveness of using these screening tools in some communities. They reported that instruments based on information collected from parents can improve parentsâ&#x20AC;&#x2122; awareness about hearing loss and may increase parentsâ&#x20AC;&#x2122; motivation to access the appropriate hearing health care resources. Our analysis of the total scores for the questionnaire also allowed for a subdivision of the subjects based on the results of their audiological hearing loss assessment: a total score of 0 to 4 points indicated a higher chance of having normal hearing, 5-7 points a higher chance of having conductive hearing loss, and more than 7 points a greater likelihood of sensorineural hearing loss. According to this classification, the questionnaire correctly classified 85% of children with normal hearing, 68% of those with conductive hearing loss and all children with sensorineural hearing loss. It is important to emphasize that this kind of classification based on a low-cost tool that can distinguish children with normal hearing from those with hearing loss and can differentiate between different types of hearing loss (conductive or sensorineural hearing loss) represents a novel contribution that may improve health care in developing countries by reducing the number of referrals to specialized services. Such specialized services are often inadequate, especially in regions far from major urban centers and at the periphery of large Brazilian cities.2 It is important to note that all children with sensorineural hearing loss were classified correctly, indicating that, at least for this type, this low-cost instrument is effective. With regard to conductive hearing loss, the losses are usually intermittent and are often slight or mild and/or unilateral, making it harder for parents to recognize the factors presented in the questionnaire. This

Scores on the questionnaire (by age and type of hearing loss) The difference between the scores of children with normal hearing or hearing loss was evident among the children in this study (2-10 years of age). We also detected differences between the scores for children with normal hearing, those with conductive hearing loss and those with sensorineural hearing loss. Thus, we verified that children with sensorineural hearing loss presented significantly higher scores than children with conductive hearing loss, who in turn presented significantly higher scores than children with normal hearing.

Cutoff scores of the questionnaire Based on the ROC curve, we established a cutoff value that simultaneously provided the highest sensitivity and specificity (5.5 points) for the total score of the questionnaire. This analysis indicated that children who obtained a total score greater than or equal to 6 were more likely to have hearing loss detected at the audiological assessment. Meanwhile, children with a score lower than 6 are more likely to have normal hearing based on the audiological evaluation. For this cutoff value (5.5 points), the sensitivity of the questionnaire was 44% and the specificity was 87%, which are similar to values obtained in other studies12,16 that used questionnaires as a form of screening. However, our results differ from the findings of one investigation,15 which obtained the highest values of sensitivity. This was because the authors considered only hearing loss greater than 40 dBHL, whereas in the present study, we also considered slight and mild hearing losses, expanding the range of hearing loss involved.

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may explain the low level of agreement between the questionnaire data and the audiological assessment in these cases.3,15,16,19,21,22 However, the instrument showed a high sensitivity concerning the identification of children with normal hearing. The lower sensitivity of the method of assessing hearing loss by questionnaire in general for slight or mild hearing loss as well as conductive hearing loss has been previously reported.25 The authors indicated that children with slight or mild hearing loss were not significantly different with respect to the measures of speech, language, reading, academic performance, behavior, or quality of life when compared to children with normal hearing, and this could explain why parents did not suspect hearing loss in these cases. This may also account for the higher sensitivity of the questionnaire for sensorineural hearing loss in our study, as these children presented severe-to-profound hearing loss and therefore may also present the largest deficits in development; this could facilitate the parents’ ability to recognize the signs of hearing loss. With respect to the reproducibility of the results from the questionnaire, we administered the questionnaire to a group of test subjects twice at an interval of three months. It is possible that the interval between the two applications should be shorter. Over a period of three months, it is possible that health problems could arise that could affect answers given on the questionnaire. This would reduce the level of agreement between the answers given at the two time points. For future studies aiming to evaluate reproducibility of this type of questionnaire, we suggest an interval of 15 days between applications26. Although the instrument requires some adjustments to increase its sensitivity to conductive hearing loss, we believe it can be introduced into the practice of the community agents of the Family Health Program and the nurses and pediatricians in primary health care as a means of initial screening. This would allow the detection of possible hearing loss and also serve to attract the attention of parents and health professionals to the issue of hearing care, as it is known that hearing loss, even slight or mild, can have important negative consequences for children and society.19,20,25,27 Actions that improve primary health care are important to enhance this level of attention and prioritize the resources according to the needs of each country, as has been stated by the World Health Assembly (1995) and other authors.3,5,19,20,29 It should be emphasized that hearing loss presents an additional and unique challenge because of its often invisible nature. Therefore, the efforts of countries to create effective prevention programs are essential,19,28 and these programs are complemented by early hearing detection and intervention for children (infants, preschool, and school-aged children).29 Our results suggest that the questionnaire we developed could be used as a screening tool to classify children as having normal hearing or hearing loss as well as to distinguish the type of hearing loss (conductive or sensorineural).

AUTHOR CONTRIBUTIONS Samelli AG conceived and designed the study, was responsible for the study material and recruitment of subjects, collection and assembly of data, data analysis and interpretation, manuscript writing and final approval of the manuscript. Rabelo CM was responsible for the collection and assembly of data, data analysis and interpretation, manuscript writing, and final approval of the manuscript. Vespasiano APC was responsible for the collection of data, and final approval of the manuscript.

REFERENCES 1. Swanepoel DW, Clark JL, Koekemoer D, Hall III JW, Krumm M, et al. Telehealth in audiology: The need and potential to reach underserved communities. Int J Audiol. 2010;49:195-203, doi: 10.3109/ 14992020903470783. 2. Bevilacqua MC, Alvarenga KF, Costa OA, Moret ALM. The universal newborn hearing screening in Brazil: From identification to intervention. Int J Pediatr Otorhinolaryngol. 2010;74:510–5, doi: 10.1016/j.ijporl.2010.02.009. 3. Olusanya B. Early Detection of Hearing Impairment in a Developing Country: What Options? Audiology. 2001;40:141-7, doi: 10.3109/ 00206090109073109. 4. Klausen O, Moller P, Holmefjord A, Reisaerter S, Asbjornsen A. Lasting effects of otitis media with effusion on language skills and listening performance. Acta Otolaryngol Suppl. 2010;543:73-6. 5. Smith AW. WHO activities for prevention of deafness and hearing impairment in children. Scand Audiol Suppl. 2001;30:93-100, doi: 10. 1080/010503901750166808. 6. Shrestha R, Baral K, Weir N. Community ear care delivery by community ear assistants and volunteers: a pilot program. J Laringol Otol. 2001;115: 869-73. 7. Silveira-Netto LF, Costa SS, Sleifer P, Braga MEL. The impact of chronic suppurative otitis media on children’s and teenagers’ hearing. Int J Pediatr Otorhinolaryngol. 2009;73:1751-6, doi: 10.1016/j.ijporl.2009. 09.033. 8. Maharjan M, Bhandari S, Singh I, Mishra SC. Prevalence of otitis media in school going children in Eastern Nepal. Kathmandu University Medical Journal. 2006;4:479-82. 9. Keogh T, Kei J, Driscoll C, Khan A. Children with Minimal Conductive Hearing Impairment: Speech Comprehension in Noise. Audiol Neurotol. 2010;15:27-35, doi: 10.1159/000218360. 10. Bess FH, Dodd-Murphy J, Parker RA. Children with minimal sensorineural hearing loss: prevalence, educational performance, and functional status. Ear Hear. 1998;19:339-54, doi: 10.1097/00003446-199810000-00001. 11. Ito K, Naito R, Murofushi T, Iguchi R. Questionnaire and interview in screening for hearing impairment in adults. Acta Otolaryngol. 2007;127:24-28, doi: 10.1080/03655230701595279. 12. Hammond PD, Gold MS, Wigg NR, Volkmer RE. Preschool hearing screening: Evaluation of a parental questionnaire. J Pediatr Child Health. 1997;33:528-30, doi: 10.1111/j.1440-1754.1997.tb01664.x. 13. Stewart MG, Ohlms LA, Friedman EM, Sulek M, Duncan NO, et al. Is parental perception an accurate predictor of childhood hearing loss? A Prospective study. Otolaryngol Head Neck Surg. 1999;120:340-4, doi: 10.1016/S0194-5998(99)70272-X. 14. Joint Committee on Infant Hearing. Year 2000 position statement: principles and guidelines for early hearing detection and intervention programs. Am J Audiol. 2000;9:9-29, doi: 10.1044/1059-0889(2000/005). 15. Newton VE, Macharia I, Mugwe P, Ototo B, Kan SW. Evaluation of the use of a questionnaire to detect hearing loss in Kenyan pre-school children. Int J Ped Otorhinolaryngol. 2001;57:229–34, doi: 10.1016/S01655876(00)00453-5. 16. Bu X, Li X, Driscoll C. The Chinese Hearing Questionnaire for School Children. J Am Acad Audiol. 2005;16:687-97, doi: 10.3766/jaaa.16.9.6. 17. Gil D, Iorio MC. Formal auditory training in adult hearing aid users. Clinics. 2010;65:165-74, doi: 10.1590/S1807-59322010000200008. 18. Oiticica J, Bittar RS. Metabolic disorders prevalence in sudden deafness. Clinics. 2010;65:1149-553, doi: 10.1590/S1807-59322010001100017. 19. Czechowicz JA, Messner AH, Alarcon-Matutti E, Alarcon J, QuinonesCalderon G, et al. Hearing impairment and poverty: the epidemiology of ear disease in Peruvian schoolchildren. Otolaryngol Head Neck Surg. 2010;142:272-7, doi: 10.1016/j.otohns.2009.10.040. 20. Taha AA, Pratt SR, Farahat TM, Abdel-Rasoul GM, Albtanony MA, et al. Prevalence and risk factors of hearing impairment among primaryschool children in Shebin El-kom District, Egypt. Am J Audiol. 2010;19:46-60, doi: 10.1044/1059-0889(2010/09-0030). 21. Chen CH, Lin CJ, Hwang YH, Ku CJ. Epidemiology of otitis media in Chinese children. Clin Otolaryngol. 2003;28:442-5, doi: 10.1046/j.13652273.2003.00741.x. 22. American Academy of Family Physicians. American Academy of Otolaryngology-Head and Neck Surgery and American Academy of Pediatrics. Subcommittee on otitis media with effusion. Pediatrics. 2004;113:1412-1429.

ACKNOWLEDGEMENTS This study was financially supported by Fundaçaõ de Amparo a` Pesquisa do Estado de Saõ Paulo (Fapesp), Process: 2008/08496-7, Saõ Paulo, Brazil.

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Cad Sau´de Pu´blica. 2007;23:971-6, doi: 10.1590/S0102-311X20070004000 24. 27. Gierek T, Gwozdz-Jezierska M, Markowski J, Witkowska M. The assessment of hearing organ of school children in Upper Silesia region. Int J Pediatr Otorhinolaryngol. 2009;73:1644-59, doi: 10.1016/j.ijporl.2009.08. 009. 28. Olusanya BO. Hearing impairment prevention in developing countries: making things happen. Int J Pediatr Otorhinolaryngol. 2000;55:167-71, doi: 10.1016/S0165-5876(00)00392-X. 29. Olusanya BO, Newton VE. Global burden of childhood hearing impairment and disease control priorities for developing countries. Lancet. 2007;369:1314-7, doi: 10.1016/S0140-6736(07)60602-3.

23. Olusanya BO, Luxon LM, Wirz SL. Screening for early childhood hearing loss in Nigeria. J Med Screen. 2005;12:115–8, doi: 10.1258/0969141054855274. 24. Lo PS, Tong MC, Wong EM, Van Hasselt CA. Parental Suspicion of hearing loss in children with otitis media with effusion. Eur J Pediatr. 2006;165: 851-7, doi: 10.1007/s00431-006-0181-5. 25. Cone BK, Wake M, Tobin S, Poulakis Z, Rickards FW. Slight-mild sensorineural hearing loss in children: audiometric, clinical, and risk factor profiles. Ear Hear. 2010;31:202-212, doi: 10.1097/AUD. 0b013e3181c62263. 26. Leite LHM, Waissmann W, Veggi AB. Reprodutibilidade de um questiona´rio para avaliaçaõ de conhecimentos, percepço˜es e pra´ticas em segurança sanita´ria alimentar de portadores de HIV/AIDS ambulatoriais.

APPENDIX 1 - Questionnaire to identify the risk for hearing loss (children 2-10 years of age).

1. Identification 2. Birth locality (maternity/city) 3. Did the child undergo any hearing screening at birth? - Yes - No (1 point) 4. Has the child ever undergone a hearing test? - Yes - No (1 point) 5. Were any risk indicators for hearing loss present?[14] – All risks are enumerated. - Yes (1 point) - No 6 In what position was the child breastfed? (to describe) - Lying down (1 point) - Sitting 7. Family history: Is there any history of deafness in your family? Who? - Yes (1 point) - No 8. Has the child had any health problems? (Some diseases are enumerated: meningitis, brain injury, frequent otitis media or ear infection). - Yes (1 point) - No 9. Does the child pay attention to loud noises? - Yes - No (1 point) 10. Does the child pay attention when he/she is called by name? - Yes - No (1 point) 11. Does he/she require a gesture or a voice used at high intensity to understand? - Yes (1 point) - No 12. Does your child hear as well as other children of the same age? - Yes - No (1 point) 13. Does your child speak as well as other children of the same age? - Yes - No (1 point) 14. Does your child understand orders, even if he/she is not looking at the speaker? For example: Bring the spoon to your mother (without pointing to the object)? - Yes - No (1 point) 15. Does your child like music? - Yes - No (1 point) 16. Has anyone commented that your son/daughter does not hear well or that his/her speech is very bad? - Yes (1 point) - No

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DOI:10.1590/S1807-59322011001100016

CLINICAL SCIENCE

Mechanical evaluation of the resistance and elastance of post-burn scars after topical treatment with tretinoin Maria Fernanda Dematte,I Rolf Gemperli,II Alessandra Grassi Salles,III Marisa Dolhnikoff,IV Tatiana Lanças,IV Paulo Hila´rio Nascimento Saldiva,IV Marcus Castro FerreiraII I Hospital das Clıńicas, Faculty of Medicine, University of Saõ Paulo, Saõ Paulo, Brazil. II Division of Plastic Surgery, University of Saõ Paulo School of Medicine, Saõ Paulo, Brazil. III Division of Plastic Surgery and Burns Unit, Hospital das Clıńicas, University of Saõ Paulo School of Medicine, Saõ Paulo, Brazil. IV Department of Pathology, University of Saõ Paulo School of Medicine, Saõ Paulo, Brazil.

OBJECTIVE: After burn injuries, scarred skin lacks elasticity, especially in hypertrophic scars. Topical treatment with tretinoin can improve the appearance and quality of the skin (i.e., texture, distensibility, color, and hydration). The objective of this prospective study was to examine the effects of treatment with 0.05% tretinoin for one year on the biomechanical behavior and histological changes undergone by facial skin with post-burn scarring. Setting: Tertiary, Institutional. METHOD: Fifteen female patients who had suffered partial thickness burns with more than two years of evolution were selected. Skin biopsies were obtained initially and after one year of treatment. The resistance and elastance of these skin biopsies were measured using a mechanical oscillation analysis system. The density of collagen fibers, elastic fibers, and versican were determined using immunohistochemical analysis. RESULTS: Tretinoin treatment significantly lowered skin resistance and elastance, which is a result that indicates higher distensibility of the skin. However, tretinoin treatment did not significantly affect the density of collagen fibers, elastic fibers, or versican. CONCLUSION: Topical tretinoin treatment alters the mechanical behavior of post-burn scarred skin by improving its distensibility and thus leads to improved quality of life for patients. KEYWORDS: Burn scars; Topical treatment; Tretinoin; Physical property of skin. Dematte MF, Gemperli R, Salles AG, Dolhnikoff M, Lanc¸as T, Saldiva PHN, et al. Mechanical evaluation of the resistance and elastance of post-burn scars after topical treatment with tretinoin. Clinics. 2011;66(11):1949-1954. Received for publication on May 10, 2011; First review completed on June 16, 2011; Accepted for publication on August 25, 2011 E-mail: rgemperli@sti.com.br Tel.: 55 11 3167-2090

abnormalities. Within the dermis, changes in the extracellular matrix (ECM) include increased concentrations of collagen fibers, which can be arranged in whorls or nodules, and decreased levels of elastic fibers.4,5 Versican, a proteoglycan with hydrophilic properties, is usually present in large amounts in recent hypertrophic scar tissue. As the scar matures and becomes less rigid, the amount of versican decreases.4 After tissue repair, alterations in ECM composition and organization lead to changes in the mechanical properties of the skin that result in decreased elasticity, especially in hypertrophic scars.4,6-8 The severity of these changes varies, depending on the depth of the original injury and other factors. When spontaneously restored, burns that involved deep dermal injury can take from three to five weeks to epithelize completely. Injuries that take longer to epithelize are more likely to evolve with hypertrophy.9 Due to the limitations of surgical techniques, many burn patients require additional treatment to improve the texture and appearance of their skin.10,11 The use of topical tretinoin

INTRODUCTION The evolution of medical knowledge and techniques, including improved intensive care units and surgical procedures, has increased the survival of major burn patients.1-3 Burn victims live with sequelae that may decrease their self-esteem and, therefore, their quality of life. A specialized multi-disciplinary professional team that can treat the multiple aspects of rehabilitation is critical for the proper re-integration of patients into their familiar professional and social environments. Post-burn scars induce profound changes in skin structure, such as epidermal and dermal

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improves the appearance of keloids and hypertrophic scars and seems to increase skin elasticity in scar sequelae.12-15 The topical use of tretinoin with glycolic acid in both restored skin and in grafted skin has been shown to increase the extent of mouth opening in burn patients with facial scarring.10 Several studies addressing the properties of post-burn scars or their cicatricial sequelae have characterized the tegument qualitatively by examining coloration, smoothness (texture, flexibility), or thickness. Previous authors have highlighted the importance of using objective measurements to analyze skin mechanical properties with such equipment as linear extensometers or cutometers (devices that measure cutaneous elasticity by suction).6,7 Measurements of skin elastance have been used in studies assessing the mechanical properties of the skin in patients with systemic sclerosis.16 Using an instrument called an ‘‘elastometer,’’ the skin elastance of these patients was found to be significantly greater than that of control individuals. In addition, this measurement was correlated with clinical scores that indicated thickening.17 In our hands, these devices (i.e., extensometers, autometers, and elastometers) did not provide homogeneous measurements when we attempted to reproduce these data in pilot studies. An alternative method of measuring the mechanical oscillation of tissue using a computer to record tissue resistance and elastance has been described for analyzing lung tissue in various diseases.18,19 The mechanical oscillation of tissue has been used previously to assess the viscoelastic properties of the superficial aponeurotic muscle system in rhytidoplasties20, but it has not yet been used for skin. To investigate more accurately the reported improvements in the distensibility of post-burn scarred skin after topical treatments with tretinoin, we examined skin tissue using mechanical oscillation analysis to measure biomechanical behavior and immunohistochemistry to study possible mechanisms underlying these improvements.

their faces again. This procedure was to be performed once daily for one year, unless clinical signs of dermatitis (i.e., erythema or redness) appeared. If dermatitis was observed, they were instructed to interrupt the treatment and to call the researchers. To minimize bias in the study, patients were instructed not to use sunscreen but were strongly advised to avoid sun exposure and to use mechanical sun protection, such as hats and umbrellas. To ensure adequate treatment and to check for possible complications, such as signs of dermatitis (e.g., redness or itching), patients were required to visit the hospital weekly during the two first months of treatment and bimonthly over the following ten months. After one year of treatment, a second biopsy of the injured skin was taken, from a point 1 cm below the ear lobe (‘‘treated’’) and at least 1 cm from the first biopsy scar. We chose to perform the two biopsies on the same side of each patient’s face to ensure similar scar quality when comparing treated versus non-treated skin (Figure 1). Skin strips were obtained from the biopsies (10.06 2.062.0 mm each) and were subjected to oscillatory mechanical analysis, and resistance (R) and elastance (E) were measured, as previously described21 (Figure 2). Briefly, the resting length (Lr) of each strip was first measured. Metal clips were glued to either end of the tissue strip with cyanoacrylate. Steel wires (0.5 mm in diameter) were attached to the clips; one end was connected to a force transducer (Model 404A; Cambridge Technologies), and the other end was connected to a servo-controlled lever arm (Model 300B; Cambridge Technologies). The lever arm was connected to a function generator (Model 3030; BK Precision, Chicago, IL), which controlled the frequency, amplitude and waveform of the oscillation. The resting force (T) was set by movement of a thumb-wheel screw system, which effected slow vertical displacements of the force transducer. Length and force signals were converted from analogue to digital and recorded in a compatible computer.

METHODS Fifteen female patients between 18 and 40 years of age (mean age = 26.7) were included in the study. These patients exhibited residual facial scars resulting from superficial partial thickness facial burns that had been caused by ethanol and spontaneously restored with conservative treatment two or more years before. All patients signed an informed consent form approved by the Review Board and Ethics Committee of the institution in accordance with the Declaration of the World Medical Association (number 515/ 5). Exclusion criteria included previous surgeries, corticosteroid treatment, keloid scars, systemic diseases, pregnancy, and smoking. All patients were instructed to use contraceptive methods because of the potential teratogenic effects of tretinoin. All selected patients had whole-face scarring. At the beginning of the study, we performed an initial biopsy of the injured skin of each patient in the pre-auricular (‘‘nontreated’’) region. The patients received a supply of tretinoin hydroalcoholic solution (tretinoin – 0.05%; propylene glycol – 5% and alcohol 50% - 20 ml) to be used every night on the face. Patients were instructed to wash their faces at night with water and neutral soap. After washing, they were to apply five drops of the tretinoin solution over their faces without massaging. In the morning, they were instructed to wash

Figure 1 - Pre-treatment biopsy (superior arrow) and planning for the post-treatment biopsy (inferior arrow).

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Post-burn scars treated with tretinoin Dematte MF et al.

Figure 3 - Mean resistance values comparing treated and nontreated areas after one year of topical treatment with tretinoin (p = 0.003).

Figure 2 - Schematic of the mechanical tissue analysis. Skin strips were suspended in a solution and connected to a force transducer and an oscillatory lever arm for measurements of resistance and elastance.

p = 0.047, respectively). The mean decreases in resistance and elastance after treatment were 31.4% and 14.8%, respectively (Figures 3 and 4). There were no evident histological differences in the distributions of ECM components between treated and nontreated specimens. The quantification of total collagen, collagen III, elastic fibers, and versican content by image analysis did not reveal significant differences between treated and non-treated specimens in any of the layers of the skin dermis. Figure 5 shows representative photomicrographs of the post-burn scarred skin stained for total collagen fibers, collagen III, elastic fibers, and versican. There were also no differences in epidermal thickness, as measured pre- and post-treatment.

The oscillation frequency was 0.1 Hz (six excursions per minute), and the amplitude was 1% Lr. Estimations of R and E were determined by applying the recursive least-squares algorithm to the equation of motion: T = EDl+R (Dl/Dt)+K; where R = resistance, E = elastance, T = force, l = length, Dl/ Dt is the change in l per unit time, and K is a constant reflecting resting force22. Resistance and elastance measurements were recorded for ten minutes. After the mechanical oscillation, the strips were fixed in 10% formalin and embedded in paraffin for histological analysis. Next, 5-mm-thick slices were stained with Sirius red and resorcin-fuchsin to identify total collagen and elastic fibers, respectively. Type III collagen fibers and versican were stained by immunohistochemistry.23 Using image analysis software (Image-ProH Plus 4.1 for WindowsH), we determined the densities of type III collagen fibers, total collagen fibers, elastic fibers, and versican in the superficial, middle, and deep layers of the dermis. Protein density was calculated as the relationship between areas of stained protein and total tissue area and was expressed as a percentage. The Wilcoxon test for paired non-parametric variables was used to compare the treated and non-treated areas from each of the 15 patients. The level of significance was established at p,0.05.

DISCUSSION There is a high demand for cutaneous treatment among burn patients. The skin is a communication organ and is vital for the perception of the limits of an organism and for the body image of an individual. Improvements in skin

RESULTS All patients completed the entire tretinoin treatment. Five patients exhibited temporary clinical signs of dermatitis (e.g., erythema, desquamation, pruritus) with spontaneous remission after a one-week interruption of tretinoin use. Because they continued with tretinoin treatment after this period, these patients were not excluded from the study. At the end of the study, three patients exhibited post-inflammatory hyperpigmentation, one exhibited mild telangiectasis on the malar region, and one had keloid development in the biopsy scar. The hyperpigmentations were later treated with 4% hydroquinone and sunscreen for three months. Significant decreases in the mean values of resistance (R) and elastance (E) were observed in treated scarred skin compared to the non-treated scarred skin (p = 0.003 and

Figure 4 - Mean elastance values comparing treated and nontreated areas after one year of topical treatment with tretinoin (p = 0.047).

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Figure 5 - Photomicrographs of total collagen fibers (A), elastic fibers (B), collagen III (C) and versican (D) stained for histological analysis (400X magnification).

scarring in patients. Additionally, the biopsies were not taken from an extremely visible area. Topical treatments with tretinoin have been shown to increase skin distensibility in post-burn scarring and acne scars with high levels of patient satisfaction.10-13,24,26,27 However, the mechanisms underlying these clinical effects are not completely understood. Retinoids, such as tretinoin, affect cell growth and differentiation and alter cell-cell cohesion. In the dermis, tretinoin modulates ECM synthesis by fibroblasts and increases angiogenesis.10,13,28,29 Scar bleaching and texture improvements achieved by topical treatments can promote secondary improvements in self-esteem and quality of life.10,30 Topical therapy with tretinoin should always include sunscreen preparations to prevent hyperpigmentation, and patients should be encouraged to avoid sun exposure.10 Because of the potential teratogenic effects of tretinoin, patients receiving this drug should use contraceptive methods.28 The most common adverse effect of topical tretinoin is local and temporary cutaneous irritation (i.e., ‘‘retinoid dermatitis’’) characterized by erythema, peeling, dryness, and itching.31 This effect was observed in 20% of the patients in this study. This high incidence is probably related to the high concentration (0.05%) of tretinoin that was used in this study. These cases were treated by temporary interruption of tretinoin use, which resulted in spontaneous remission. Other adverse effects included hyperpigmentation and increased telangiectasias in 5% of patients. We currently prefer to use lower doses of tretinoin (0.025%) to minimize skin irritation. Patients with facial burns with over two years of evolution were selected to avoid the period when the

quality, especially in the face, can contribute to increased self-esteem and quality of life for burn patients.10,24,25 We have been using topical tretinoin to treat burn patients for more than a decade. Satisfaction is high among the patients, especially with respect to the observed improvements in tegument quality. The potential for topical treatments using tretinoin combined with glycolic acid to improve the mechanical properties of scarred skin has been indirectly shown through mouth-opening measurements.10 In this study, clinical improvements in burned skin distensibility after tretinoin topical treatment were demonstrated by a decrease in resistance and elastance, as determined by biomechanical analysis. Tissue resistance represents the energy loss resulting from the opposition to movement, and elastance (i.e., the inverse of compliance) is a measure of the tendency of tissue to recoil toward its original dimensions upon the removal of a distending or compressing force. Therefore, lower elastance (or greater compliance) corresponds to greater distensibility in a given tissue. In this study, the mechanical properties of burned skin with and without tretinoin treatment were assessed by adapting a mechanical tissue oscillation method used for analyzing the mechanical behavior of lung tissue in various diseases.18,19 We are not aware of any other reports in the literature in which this method has been used to evaluate post-burn scarred skin. The main disadvantage of this method was the requirement for skin biopsies, which can lead to scar complications. The potential cosmetic concerns associated with biopsies were largely avoided in this protocol because the biopsies were taken from the center of existing burn scars and thus did not cause additional

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Post-burn scars treated with tretinoin Dematte MF et al. 4. Scott PG, Dodd CM, Tredget EE, Ghahary A, Rahemtulla F. Immunohistochemical localization of the proteoglycans decorin, biglycan and versican and transforming growth factor-b in human post-burn hypertrophic and mature scar. Histopathology. 1995;26:423-31, doi: 10. 1111/j.1365-2559.1995.tb00249.x. 5. Kamath NV, Ormsby A, Bergfeld WF, House NS. A light microscopic and immunohistochemical evaluation of scars. J Cutan Pathol. 2002;29:27-32, doi: 10.1034/j.1600-0560.2002.290105.x. 6. Clark JA, Cheng JCY, Leung PC. Mechanical characterization of human postburn hypertrophic skin during pressure therapy. J Biomechanics. 1987;20:397-406, doi: 10.1016/0021-9290(87)90047-9. 7. Krusche T, Worret W. Mechanical properties of keloids in vivo during treatment with intralesional triamcinolone acetonide. Arch Dermatol Rev. 1995;287:289-93, doi: 10.1007/BF01105081. 8. Clark CP3rd. Alpha hydroxy acids in skin care. Clin Plast Surg. 1996;23:49-56. 9. Pownell PJ, Rohrich RJ. Burn reconstruction. Selected Readings in Plastic Surgery. 1998;8:10-37. 10. Salles AG, Gemperli R, Toledo PN, Ferreira MC. Combined tretinoin and glycolic acid treatment improves mouth opening for postburn patients. Aesth Plast Surg. 2006;30:356-62, doi: 10.1007/s00266-004-0151-0. 11. Isaac C, Carvalho VF, Paggiaro AO, Maio M. Intralesional pentoxifylline as na adjunvant treatment for perioral post-burn hypertrophic scars. Burns, 2010;36:831-5. 12. Hansen DA. Treatment of hypertrophic scars with retinoic acid. South African Medical Journal (SAMJ). 1979;22:1114. 13. Janssen de Lipens AMP. The local treatment of hypertrophic scars and keloid with topical retinoic acid. Br J Dermatol. 1980;103:319-23, doi: 10. 1111/j.1365-2133.1980.tb07251.x. 14. Mizutani H, Yoshida T, Nouchin N, Hamanaka H, Shimizu M. Topical tocoretinate improved hypertrophic scar, skin sclerosis in systemic sclerosis and morphea. J Dermatol. 1999;26:11-7. 15. Panabiere-Castaings MH. Retinoic acid in the treatment of keloids. J Dermatol Surg Oncol. 1988;14:1275-6. 16. Balbir-Gurman A, Denton CP, Nichols B, Knight CJ, Nahir AM, Martin G, et al. Non-invasive measurement of biomechanical skin properties in systemic sclerosis. Ann Rheum Dis. 2002;61:237–41, doi: 10.1136/ard.61. 3.237. 17. Ballou SP, Mackiewicz A, Lysikiewicz A, Neuman MR. Direct quantitation of skin elasticity in systemic sclerosis. J Rheumatol. 1990;17:790-94. 18. Dolhnikoff M, Mauad T, Ludwig MS. Extracellular matrix and oscillatory mechanics of rat lung parenchyma in bleomycin-induced fibrosis. Am J Resp Crit Care Med. 1999;160:1750–7. 19. Lanc¸as T, Kasahara DI, Prado CM, Tibe´rio IF, Martins MA, Dolhnikoff M. Comparison of early and late responses to antigen of sensitized guinea pig parenchymal lung strips. J Appl Physiol. 2006;100:1610-6, doi: 10.1152/japplphysiol.00828.2005. 20. Har-Shai Y, Bodner SR, Egozy-Golan D, Lindenbaum ES, Ben-Izhak O, Mitz V, et al. Viscoelastic properties of the superficial musculoaponeurotic system (SMAS): a microscopic and mechanical study. Aesth Plast Surg. 1997;21:219-24, doi: 10.1007/s002669900113. 21. Dolhnikoff M, Morin J, Ludwig MS. Human lung parenchyma responds to contractile stimulation. Am J Respir Crit Care Med. 1998;158:1607-12. 22. Lauzon AM, Bates HT. Estimation of time-varying respiratory mechanical parameters by recursive least squares. J Appl Physiol. 1991;71:115965. 23. Araujo BB, Dolhnikoff M, Silva LF, Elliot J, Lindeman JHN, Ferreira DS, et al. Extracellular matrix components and regulators in the airway smooth muscle in asthma. Eur Respir J. 2008;32:61-9, doi: 10.1183/ 09031936.00147807. 24. Jenkins SC, Henke J. Retinoic acid modifies scars from self-injury by burning. Am J Psychiatry. 1993;150:1125. 25. Balakrishnan C, Hashim M, Gao D. The effect of partial-thickness facial burns on social functioning. J Burn Care Rehabil. 1999;20:224-5, doi: 10. 1097/00004630-199905000-00012. 26. Harris DWS, Buckey CC, Ostlere LS, Rustin MHA. Topical retinoic acid in the treatment of fine acne scarring. Br J Dermatol. 1991;125:81-82, doi: 10.1111/j.1365-2133.1991.tb06048.x. 27. Schmidt JB, Donath P, Hannes J, Perl S, Neumayer R, Reiner A. Tretinoin-iontophoresis in atrophic acne scars. Int J Dermatol. 1999;38:149-53, doi: 10.1046/j.1365-4362.1999.00586.x. 28. Bhawan J. Short and long-term histologic effects of topical tretinoin on photodamaged skin. Int J Dermatol. 1998;37:286-92, doi: 10.1046/j.13654362.1998.00433.x. 29. Humphries JD, Parry EJ, Watson REB, Garrod DR, Griffiths CEM. Alltrans retinoic acid compromises desmosome expression in human epidermis. Br J Dermatol. 1998;139:577-84, doi: 10.1046/j.1365-2133. 1998.02451.x. 30. Poh-Fitzpatrick MB. Skin care of the healed burned patient. Clin Plast Surg. 1992;19:745-51.

spontaneous involution of the tegument occurs because of cicatricial process maturity.6,8,9,30,32,33 ECM remodeling depends on the equilibrium between fiber synthesis and degradation. Collagen fibers are responsible for the tensile force of connective tissue, and elastic fibers return the deformed collagen network to its previously relaxed condition through traction forces. When elastic fibers are injured, there is a loss of elasticity. Additionally, maturation of post-burn scars depends on the normalization of dermal proteoglycan levels.31,34,35 Collagenase production may be favored in the cicatricial tissue with the use of tretinoin, in keeping with what is seen after the compression of hypertrophic areas.10,36 The histological analyses and the quantitative assessments of collagen, elastic fibers, and versican content performed in this study were not sensitive enough to explain the observed changes in the mechanical behavior of the post-burn skin after treatment. The results and tables with these comparisons are available in a doctoral thesis;37 however, they were not referenced here because they were not statistically significant. It is possible that the structural arrangement, distribution and/or organization of the fibers play a more relevant role in determining the mechanical behavior of the tissue than do the quantitative changes. In addition, it is possible that the mechanical experiments may have disturbed the subsequent histological analyses. In the previous studies performed in the lungs, this phenomenon was not observed, but it may be beneficial to use different skin strips for mechanical and histological analyses in future studies. Long-term maintenance of the beneficial effects of topical tretinoin treatment can be achieved through sustained adherence to the recommended treatment regimen. This was observed in this study, as patients completely adhered to the treatment to improve their appearances.8,10 We conclude that topical treatment with tretinoin changes the mechanical behavior of the skin in patients with facial post-burn scarring, thereby improving distensibility.

ACKNOWLEDGMENTS The authors thank Dr. Luiz Fernando Ferraz da Silva for technical support with image analyses and Claudia Garcia (RC Farma´cia Dermatolo´gica) for manufacturing the topical products.

AUTHOR CONTRIBUTIONS Dematte MF conceived and designed the study. Gemperli R supervised the study and was responsible for the final version of the manuscript. Salles AG conceived and designed the study, coordinated one of the groups in the institution, guided the analyses of clinical results, and helped in the preparation of the manuscript. Dolhnikoff M supervised the laboratorial data and was also responsible for the statistical analyses. Lanc¸as T was responsible for the laboratorial measurements. Ferreira MC supervised the clinical care of the patients.

REFERENCES 1. Santos SR, Campos EV, Sanches C, Gomez DS, Ferreira MC. Fluconazole plasma concentration measurement by liquid chromatography for drug monitoring of burn patients. Clinics. 2010;65:237-43, doi: 10.1590/S180759322010000200017. 2. Braz LG, Braz DG, Cruz DS, Fernandes LA, Mo´dolo NS, Braz JR. Mortality in anesthesia: a systematic review. Clinics. 2009;64:999-1006, doi: 10.1590/S1807-59322009001000011. 3. Coelho da Mota DS, Furtado E, Bottino DA, Bouskela E. Effects of buflomedil and pentoxifylline on hamster skin-flap microcirculation: prediction of flap viability using orthogonal polarization spectral imaging. Clinics. 2009;64:797-802.

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31. Kang S, Fisher GJ, Voorhees JJ. Photoaging: pathogenesis, prevention and treatment. Clin Geriatr Med. 2001;17:643-59, doi: 10.1016/S07490690(05)70091-4. 32. Hopkinson I, Anglin IE,Evans DL, Harding KG. Collagen VII expression in human chronic wounds and scars. J Pathology. 1997;182:192-96, doi: 10.1002/(SICI)1096-9896(199706)182:2,192::AID-PATH857.3.0. CO;2-F. 33. Carvalho DA, Mariani U, Gomez DS, Gemperli R, Ferreira MC. A study of the postburned restored skin. Burns. 1999;25:385–94, doi: 10.1016/ S0305-4179(98)00177-6. 34. Scott PG, Dodd CM, Tredget EE, Ghahary A, Rahemtulla F. Chemical characterization and quantification of proteoglycans in human post-burn hypertrophic and mature scars. Clin Sci. 1996;90:417-25.

35. Garg HG, Siebert JW, Garg A, Neame PJ. Inseparable iduronic acid containing proteoglycan PG (IdoA) preparations of human skin and post-burn scar tissues: evidence for elevated levels of PG (IdoA) in hipertrophic scar by N-terminal sequencing. Carbohydrate Res. 1996;284:223-8, doi: 10.1016/0008-6215(95)00403-3. 36. Silfen R, Amir A, Feinmesser M, Hauben DJ. Subdermabrasion in the treatment of post-burn facial hypertrophic scars. Aesth Plast Surg. 2002;26:139-41, doi: 10.1007/s00266-001-0035-5. 37. Be´lico FDS. Ana´lise mecaˆnica e histolo´gica do tegumento facial com sequëla de queimadura apo´s tratamento to´pico com tretinoıńa.(Tese) Saõ Paulo. Faculdade de Medicina Universidade de Saõ Paulo, 2008. http:// www.fm.usp.br/plastica/resumo-65.html.

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DOI:10.1590/S1807-59322011001100017

CLINICAL SCIENCE

Restless legs syndrome in subjects with a knee prosthesis: evidence that symptoms are generated in the periphery Jose´ Carlos Pereira, Jr.,I Joa˜o Luiz Pereira da Silva Neto,II Ma´rcia Pradella-HallinanIII I

Faculdade de Medicina de Jundiaı´, Pediatria, Jundiaı´, SP/Brazil. II Faculdade de Medicina de Jundiaı´, Orthopedy, Jundiaı´, SP/Brazil. III Universidade Federal de Sa˜o Paulo, Instituto do Sono, Departamento de Psicobiologia, Sa˜o Paulo, SP, Brazil.

OBJECTIVE: There are no data adressing the prevalence of restless legs syndrome in subjects who have knee prosthesis. Therefore, we conducted a cross-sectional survey of subjects who underwent knee prosthesis surgery. METHOD: A total of 107 subjects (30 male, 77 female) were interviewed over the telephone regarding restless legs syndrome symptoms. If the patients exhibited symptoms of the syndrome, we conducted face-to-face interviews. Lastly, a therapeutic test with pramipexole was proposed for each subject. RESULTS: In our cohort, 7 males (23%) and 30 females (39%) had restless legs syndrome. Of these, 6 males and 23 females were submitted to face-to-face-interview. Of the males, 5 (83%) had restless legs after the knee surgeryexclusively in the operated leg- and reported no family restless legs history. One man had a prior case of bilateral restless legs syndrome, a positive family history and claimed exacerbation of symptoms in the operated leg. Among the females, 16 (69%) had restless legs prior to surgery. A total of 10 female patients reported bilateral symptoms, with fewer symptoms in the operated leg, while 6 displayed a worse outcome in the operated leg. The 7 females (31%) without restless legs prior to surgery and without a family history experienced symptoms only in the operated leg. All subjects responded favorably to the pramipexole therapeutic test. CONCLUSION: Our results suggest that secondary unilateral restless legs syndrome may ensue from knee prosthesis surgery and that the symptoms are generated in the peripheral nervous system. KEYWORDS: Restless Legs Syndrome; RLS pathophysiology; Knee prosthesis. Pereira Jr JC, Silva Neto JLP, Pradella-Hallinan M. Restless legs syndrome in subjects with a knee prosthesis: evidence that symptoms are generated in the periphery. Clinics. 2011;66(11):1955-1959. Received for publication on August 3, 2011; First review completed on August 18, 2011; Accepted for publication on September 3, 2011 E-mail: joscarlospereirajr@gmail.com (corresponding author) Tel.: 55 11 45215706

amputated limb, which is also known as ‘‘phantom RLS’’.4,5 Ekbom, who described one of the first phantom RLS cases, suggested that RLS symptoms could be generated in the periphery.4 Furthermore, RLS may be primary or secondary to various other diseases. The currently accepted definition of adult RLS from the ‘‘International Restless Legs Syndrome Study Group (IRLSSG)’’6 is as follows: 1) an urge to move the legs is usually accompanied or caused by uncomfortable and unpleasant sensations in the legs; 2) the urge to move or unpleasant sensations begin or worsen during periods of rest or inactivity; 3) the urge to move or unpleasant sensations are partially or totally relieved by movements such as walking or stretching, as long as the activity continues; and 4) the urge to move or unpleasant sensations are worse in the evening or at night than during the day, or only occur in the evening or night. Some supportive clinical features of RLS include a positive family history, periodic limb movements during sleep, and periodic limb movements during wakefulness. An additional supportive clinical feature is the ‘‘improvement of RLS symptoms with dopaminergic agonist’’ therapy.6

INTRODUCTION Restless legs syndrome (RLS) was described as an independent clinical condition by Karl-Axel Ekbom, a Swedish neurologist who also described its main features, in 1945.1 RLS, or Ekbom’s syndrome, is a common neurological disturbance with sensory and motor components.2 The pathophysiology of RLS is considered unknown; however, a recent theory has suggested that the pathophysiology of RLS is secondary to an imbalance between thyroid hormones system and the dopaminergic system.3 It has also been debated whether RLS symptoms are generated in the central or peripheral nervous system.2 In addition, RLS may also occur in patients that have had their leg amputated. In this case, patients report the symptoms as coming from the

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To the best of our knowledge, there are no data indicating the prevalence of RLS among patients with knee prosthesis surgery (KPS). Therefore we conducted a cross-sectional survey of this patient group. We also sought determine if the KPS was capable of causing RLS in patients who previously did not suffer from this condition.

was severe osteoarthritis; in 7 women from the 23 that attended the FFI, KPS has been recommended mainly due to injuries that severely damaged the knee. Only 3 women from the entire group knew that they suffered from RLS, even though their problems had not been addressed. Several women reported that their RLS symptoms were present since they were children. The majority of the patients from the n2 and n3 groups sometimes complained of their unpleasant sensations to their orthopedists or clinicians; however, the possibility that they had RLS has never been considered. The majority of our patients also complained of pain, of varying intensity, concomitant with the more typical RLS symptoms. Dysesthesias, mainly a burning pain around the knee, in the limb with the prosthesis was also a common complaint. A total of 5 men among 6 (83%) and 7 women among 23 (30%) developed RLS postoperatively to the KPS. More specific results from this survey are presented in summaries 1 and 2.

PATIENTS/METHODS A total of 107 patients underwent KPS, which was performed by one of the members of our surgical team (JLPSN). These patients (n1 = 107), 30 males (28%) and 77 females (72%) were interviewed over the telephone regarding RLS symptoms. Their age varied from 41 years to 81 years (mean 63 years). Subsequently, the patients that were positive for RLS symptoms were invited to a face-to-face interview (FFI). A total of 6 men attended the FFI, which were considered group n2 (n2 = 6). A total of 23 women attended the FFI, which made up group n3 (n3 = 23). All patients from group n1 were interviewed by one of us (JCPJr.) over the telephone regarding RLS symptoms. During this interview, the patient was questioned if they were, or not, sufferers from RLS symptoms. A positive history of RLS was only considered when the 4 defining criteria of the syndrome were reported.6 The n2 and n3 groups, comprised of individuals who suffer from RLS, were submitted to the FFI. At this point of the study, we asked the patient the following questions: 1) if the patient was aware of his/her RLS; 2) if the RLS was present prior to or after the KPS; 3) whether the RLS symptomatology was unilateral or bilateral, and if the symptoms were bilateral, were they more intense in the limb that underwent the KPS, or in the limb without the surgery; 4) if there was a positive family history of RLS; and 5) which was the medical condition that warranted the KPS. All patients from the n2 and n3 groups, except 2 women, underwent a pramipexole therapeutic test (PTT) for at least 3 days with a 0,125 mg dose 2 or 3 hours before bedtime. Women from the n3 group, who did not receive the PTT, were already taking clonazepam, which had been prescribed by their doctors as a therapy for insomnia. It has been established that an alleviation of more than 50% in the severity of RLS symptoms, with PTT or clonazepam, was necessary to consider the patient as an RLS sufferer. If not present a normal cognition to attend coherently the FFI would be a motive to exclude the patient of the study. Other comorbidities, or other sleep disturbances, that the patients might had were not studied in this survey as its main objective was to examine the association between RLS and KPS. Therefore, we also did not consider whether the RLS previously to KPS was primary or secondary to other diseases, iron deficiency, or medications.

Summary 1: Males (n2 = 6) A total of 5 patients with unilateral KPS reported symptoms only in the limb with the prosthesis, presented RLS only after the KPS, and did not have relatives with RLS. One of these 5 patients also had RLS symptomatology beyond the legs, which presented in the contralateral wrist and hand. Complaints in the wrist and hand started at the same time as that of the leg, which was weeks after the KPS. One man had RLS prior to the KPS, but after the surgery the symptoms worsened in the KPS limb compared with the limb without prosthesis. The median IRLSSG severity score rating6 from group n2 patients was 23. All did well in the PTT; however, a single male subject had insomnia. In 5 patients the elapsed time between KPS and the first RLS unilateral symptoms was of ‘‘2 or 3 weeks to 2 or 3 months’’. A 71-years-old gentleman began to feel RLS symptoms in the operated leg only 9 years after the surgery, when to him was prescribed escitalopram due to a mild depression.

Summary 2: Females (n3 = 23) A total of 16 patients from the n3 group were sufferers of RLS prior to KPS and 14 of them knew relatives with similar symptoms; from these 16 patients, 10 (62%) reported that the symptomatology was bilateral, however, the KPS had ameliorated the symptoms in the limb with the prosthesis in relation to the limb without it. Among the 16 patients, 6 (38%) reported bilateral symptomatology that became worse in the limb with the prosthesis compared with the limb without KPS. A total of 7 patients (30%) from the n3 group declared RLS only postoperatively to the KPS and did not have relatives with similar symptoms; their symptoms were felt only in the KPS limb. A total of 3 patients from group n3 with RLS prior to the KPS also had symptoms in both thighs, and 2 patients also had symptoms in the arms. One patient from those without RLS prior to KPS had to remove the prosthesis device due to serious infection in the articulation. She was submitted to various surgeries, and the operated limb, although without the prosthesis, manifests now severe RLS symptoms. The 2 patients that were taking clonazepam reported that they had RLS prior to KPS, and clonazepam had ameliorated their RLS symptoms.

RESULTS General aspects: Of our initial group (n1 = 107), 7 males among 30 (23%), and 30 females among 77 (39%) reported RLS during the telephone interview. All of these patients exhibited the 4 criteria that define RLS in accordance with the IRLLSSG.6 A total of 6 men in this study, that attended the FFI (n2 = 6), were submitted to KPS due to sport or vehicles accidents that damaged the knee articulation. Notably, none of these patients complained of osteoarthritis. Among the 23 women that attended the FFI (n3 = 23), 16 of them, indicated that the main reason they underwent KPS

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Restless Legs in patients with a knee prosthesis Pereira JC Jr. et al.

The median IRLSSG severity rating score6 from the n3 group patients was 24. All did well with PTT; however, 1 patient had severe nausea, 1 patient had a significant headache, and 1 patient had insomnia. To patients that already had primary RLS prior to KPS the elapsed time between the KPS and the worsening or ameliorating the symptoms in the operated leg was of ‘‘3 to 6 months’’; and to patients that initiated their RLS symptoms after the KPS was of ‘‘3 or 4 weeks to 3 or 4 months’’.

this study demonstrates, and may be better explained by discussing the basic mechanisms underlying the generation of peripheral perceptions. The dorsal sensory ganglia axons contain a number of receptors. These include proprioceptors, thermoreceptors, nociceptors, mechanoreceptors, and combinations of these receptors families.7We assume that the hyper-stimulation of various types of tegument receptors can cause a variety of different sensations experienced by RLS patients. This assumption is made considering that sensations felt as coming from the periphery (be they normal or abnormal) have to follow their appropriate physiological mechanisms inherent to the sensation phenomena. There are receptors that adapt slowly (e.g., free nervous endings, Merkel receptors, and hair receptors), and rapidly (e.g., Meissner, Ruffini, and Pacinian corpuscles). Proprioceptors all adapt rapidly.7,8 The rapidly adapting receptors may be more involved than the others in RLS symptoms, in what the symptomatology has of ‘‘intriguing’’. After rapid receptors have detected a specific tissue alteration, they become quiescent and do not signal for a short time. Then, they promptly recover their detection ability.8 That is, they ‘‘glitter’’ continuously, which may result in a perception of vibrations deep within the legs. All the leg’s sensory receptors combined in an enhanced signaling network may generate the unpleasant limb sensations that RLS patients report. We postulate that an overstimulation of peripheral receptors in the legs and/or an insufficient modulation of the inputs on the synapses they have to pass through, as they travel to cortex, lead to (after KPS) RLS symptoms, and we suggest that this might be the case of primary RLS as well. Thus, it could be postulated that RLS should be considered as a functional neuropathy. There are other types of neurons interspersed among the somatosensory neurons along their pathway to the cortex,7 which include many dopaminergic neurons.9 The majority of these interspersed neurons have neuromodulatory functions.7 Because dopamine agonists are effective in ameliorating RLS symptoms, a diminished modulation of these sensory inputs to the sensory cortex by the dopaminergic system might be central to RLS pathophysiology.3 We also postulate that RLS may, more informatively, be considered as a ‘‘functional peripheral neuropathy’’. Peripheral receptors transform different stimuli into neural activity and then send these signals to the somatosensory cortex. These inputs are interpreted by the cortex as sensations, which are in accordance with the area of the neural map that receives the input signal. If these peripheral receptors are inadequately stimulated, then transmissions inputs may be ‘‘read’’ as abnormal sensations.10 These sensory receptors may undergo one, or more than one, of the following ‘‘dysfunctions’’: 1) diminishing of their threshold for the stimuli received from the environment (hyperesthesia), or elevation of the threshold (hypoesthesia) or, 2) to become susceptible to stimuli that are not specific for that kind of receptor (dysesthesia).10 Our study indicates that the RLS symptoms secondary to KPS are generated in the periphery of the somatosensory system. Therefore, not being logical that RLS symptoms to be felt can prescind the somatosensory physiological apparatus, we postulate that in primary RLS, as the same, the symptoms are generated through an ‘‘adulteration’’ of the receptive ability of peripheral receptors. It also might be possible that normal

DISCUSSION In this study, the overall prevalence of RLS among KPS patients was elevated (male 23%, female 39%) when compared with the normal population (10%).6 These numbers are explained partly by our cohort being formed mainly of older subjects. However, as many of our patients developed RLS only after the KPS (37%, overall), we can state that KPS may initiate RLS in subjects who never had the syndrome, which may underlie the elevated prevalence of RLS among KPS patients. From our study, secondary RLS (i.e., postoperatively to KPS) should be considered as a relatively common complication of total knee arthroplasty. In our patients, unilateral RLS in the limb with the prosthesis was common. Similarly, RLS symptoms worsened in the knee with prosthesis for some of these patients that had RLS prior to the KPS. However, also among the many patients that had RLS prior to the KPS, in some of them (n3 group), RLS symptomatology in the limb with the prosthesis was ameliorated compared with the one without prosthesis. During KPS, it is unavoidable that various nerve bundles are injured. Subsequently, the scaring of neural and non-neural tissues occurs differently among different subjects. The various ways with which tissues heal, and recover their new histological stability, are what likely differentiate the outcomes in relation to RLS symptoms after the KPS. One of our patients had to remove the prosthesis device, yet still suffers from RLS (acquired only after the KPS). This implies that it is not the prosthesis itself that causes the syndrome. However, the unilateral nature of this patient’s RLS implies that her symptomatology was secondary to the KPS-induced damage of the peripheral nerves within the affected leg. To gain a better understanding of this, somatosensory system physiology and its ‘‘principle of labeled lines’’ need to be discussed. Nevertheless, our findings might suggest that RLS symptoms are generated in the periphery of the nervous system, which is what Ekbom in 1961 had already assumed.4 In the somatosensory system, various sensory receptors capture different stimuli and convey them to the sensory cortex. Each type of receptor is specialized, whereby it receives the stimulus to which it is predetermined to receive. Immediately after it is stimulated, the receptor sends a signal to the somatosensory cortex via nerve fibers. Accordingly, the area of the cortex that receives this signal determines the mode of the subsequent perception.7,8 This mechanism is called the principle of ‘‘labeled’’ lines. In other words, somatic receptors are the structures designated to receive stimuli, however, if their afferent fibers are stimulated at any point while approaching the cortex, the mode of perception by the cortex is identical to when the somatic receptor is stimulated directly.8 The typical dysesthesias associated with RLS secondary to KPS are generated in the peripheral of sensorial system as

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remitting distal cutaneous sensory neuropathy.14 Considering that spontaneous inflammatory processes in the superficial fibular nerve may occur, we believe that an identical process occurs secondary to KPS. One of our patients, a 43-year-old man, presented not only with RLS in the operated leg but also in the contralateral wrist and hand, which both initiated 2-3 weeks after the KPS. We hypothesize that his wrist and hand symptoms are a form of referred paresthesia, whereby the plethora of inputs coming from the superficial fibular nerve ‘‘contaminate’’ the sensory nerves that come from the contralateral wrist and hand. In the PTT, this patient experienced notable amelioration of RLS symptoms in both regions. We believe that this study contributes to the understanding of the neural basis of RLS pathophysiology. Examining the role of the superficial fibular nerve (and perhaps saphenous nerve) in RLS secondary to KPS may improve our understanding of not only of RLS secondary to peripheral neuropathies but also of primary RLS. Our study suggested a peripheral etiopathogenesis of RLS secondary to KPS, and possibly also primary RLS, whereby the sensory receptors of the leg may have a diminished threshold for the initiation of inputs that will be perceived by the cortex as RLS symptoms. The spinal cord and thalamus, the pathways transmitting sensory stimuli to the cortex, have a number of interspersed neurons that serve to modulate the synapses connecting the sensory receptors with the cortex. It is possible that a decrease in this modulation function also may be part of the RLS pathophysiology. The notable effects of dopamine agonists to relieve RLS symptoms are an indication that these medications may act through the modulatory functions of the dopaminergic system in the spinal cord. The reader is encouraged to read the review (1983) by Lindvall and colleagues.9 One of the effects of dopamine release is a down regulation of the thyroid axis.15,16 This pharmacological fact cannot be considered as a simple side effect of this neurotransmitter’s release. It is plausible that an undermodulation of the thyroid axis by the dopaminergic system may result in RLS symptoms.3 The principle of the labeled-lines that we have remembered in this article may be the reasoning that allows us to consider that there is resemblance between both conditions RLS secondary to KPS and phantom limb RLS.17 Orthopedists should be aware that KPS may cause secondary RLS, so that they can better address this potential condition in their patients. Refinement of the KPS procedure may reduce the risk of injury to the superficial fibular nerve, thereby diminishing the incidence of RLS secondary to KPS. This study has been approved by the ‘‘Committee of Ethics of Research on Humans’’ of our institution Faculdade de Medicina de Jundiaı´, Sa˜o Paulo, Brasil.

stimuli received by normal receptors are not sufficiently modulated when the inputs travel to sensory cortex. In a previous article we hypothesized that primary RLS may be secondary to an insufficient modulation of thyroid hormone by the dopaminergic system.3 Considering the findings of the current study, we believe that injuries to sensitive nerves adjoining the knee articulation during KPS result in secondary RLS. During the KPS, infrapatellar branches of the saphenous nerve are cut. In addition, the superficial fibular nerve that passes by the lateral side of the knee commonly suffers variable damage secondary to severe stretching and circulatory stress due to tourniquet application to the limb.11,12 Inflammatory involvement of the fibular nerve and compression of the nerve due to severe edema also frequently occur.12 Due to its proximity to the knee articulation, it is possible that the saphenous nerve that passes by the medial side of the knee13 also suffers similar damage from the KPS, however, this has not been reported in the literature. These three mentioned nerves are sensitive, so damage to any of them may induce disturbance of sensory stimuli but not necessarily motor activity.13 Injuries to peripheral nerves may evoke variable histopathological responses, such as Wallerian degeneration. Damage to the myelin sheath may induce variable degrees of segmental degeneration, and axon compression may damage the axon’s inner canaliculi.10,11 This neural damage may induce variable degrees of hypoesthesia or even anesthesia, or this damage may induce opposite effects such as hyperesthesias, and paresthesias/dysesthesias. Nerves that have been damaged become more exposed to tissue environment and may be stimulated by nonspecific stimuli (tissue pressure on the damaged nerve) that are converted to action potentials10,11 that are, then, turned into specific sensations by the sensory cortex. In addition, the axon may be damaged and become unable to isolate neural potentials so that they ‘‘jump’’ to another axon, which is known as ephaptic neurotransmission.10 Furthermore, if the nerve is cut, neuromas may ensue, which can be a source of pain. Sometimes these are so small that they cannot be detected even by RMI.11 Whatever the cause of action potential firing, or whether the signal is receptor mediated or not, the somatosensorial cortex will always perceive the input as coming from the sensory receptor located at the distal end of the sensory neuron’s axon (i.e., principle of labeled lines).8 We postulate that abnormal neural transmissions initiated at the region of the nerve around the knee (mainly from the sensitive fibular nerve and perhaps also the saphenous nerve) are the cause of the newly diagnosed RLS cases after KPS in the cohort presently studied. As we observed, some patients in this study that had already suffered from RLS prior to KPS had an amelioration of their RLS symptoms after the KPS, when comparing the limb with the prosthesis to the one without it. In these cases we believe that the damage to nerves in their region around the knee led to neural hypoesthesia, thereby minimizing the RLS symptoms that the patients previously felt from their primary RLS. It is interesting to note that the superficial fibular nerve is highly predisposed to sensorial neuropathies.11 Asbury described (1972) that this nerve is occasionally subjected to inflammation restricted to the perineurium, which causes compression of the nerve bundles. The clinical condition that ensues is one of a patchy, burning, painful, partially

ACKNOWLEDGEMENTS We are indebted to Prof Karl Ekbom Jr. from the Department of Neurology, Karolinska University Hospital/Huddinge, Stockolm, Sweden, for his helpful critique of the manuscript and kind attention.

AUTHOR CONTRIBUTIONS Pereira Jr JC conceived and designed the study, was also responsible for the telephone and face-to-face interviews, the writing of the manuscript, and researches for the pertinent literature. Silva Neto JLP has operated all the patients (total knee arthroplasty), and introduced concepts regarding surgery techniques. Pradella-Hallinan M was responsible for the revision of the manuscript and provided important notes to it.

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Restless Legs in patients with a knee prosthesis Pereira JC Jr. et al. 8. Guytom AC, Hall JE. Textbook of Medical Physiology. 11th edition. Elsevier. Ltd; 2006. 9. Lindvall O, Bjorklund A, Skagerberger G. Dopamine-Containing Neurons in the Spinal Cord: Anatomy and Some Functional Aspects. Annal Neurol. 1983;14:255-60, doi: 10.1002/ana.410140302. 10. Silbernagl S, Lang F. Color Atlas of Pathophysiology. 1st ed. Georg Thieme Verlag. 2000; Stutgard, Germany. 11. Ropper AH, Samuels MA. Adams’ and Victor’s Principles of Neurology, 9th ed. McGraw Hill. 2009. 12. Tooms RE. Artroplastia do tornozelo e joelho, em Crenshaw AH, editor: Cirurgia Ortope´dica de Campbell. Editora Manole Ltda. 1996;pp.411-65. (Translation to Portuguese from Campbell’s Operative Orthopedics, 8th edition). ´ rga˜os 13. Kahle W, Frotscher. Anatomia, texto e atlas: Sistema Nervoso e O do Sentido, Artmed 2005, Brasil. (Translation to Portuguese from Tascheatlas der Anatomie, Nerve System und Sinnersorgane, 9th ed). 14. Asbury AK, Picard EH, Baringer JR. Sensory perineuritis. Arch Neurol. 1972;26:302. 15. Delitala G. Dopamine and TSH secretion in man. The Lancet. 1977;310:760-1, doi: 10.1016/S0140-6736(77)90259-8. 16. Wojcikowski J, Gocen Biowska K, Wladystawa AD. Regulation of liver cythocrome P450 by activation of brain dopaminergic system. Biochem Pharmacol. 2008;76:258-67, doi: 10.1016/j.bcp.2008.04.016. 17. Pereira jr JC, AlvesRC. The labelled-lines principle of the somatosensory physiology might explain the phantom limb phenomenom. Medical Hypotheses, doi: 10.1016/j.mehy. 2011.07.054 (article in press; available online 20 august 2011).

REFERENCES 1. Ekbom KA. Restless legs: a clinical study. Acta Med Scand. 1945;158:1122. 2. Montplaisir J, Allen RP, Walters A, Ferini-Strambi L. Restless Legs Syndrome and Periodic Limb Movements during Sleep; in Kryger MH, Roth T, Dement WC; editors: Principles and Practice of Sleep Medicine, fifth edition. Elsevier Saunders, 2011;pp.1026-37 3. Pereira JR JC, Pradella-Hallinan M, Lins Pessoa JH. Imbalance between thyroid hormones and the dopaminergic system might be central to restless legs syndrome pathophysiology: A hypothesis. Clinics. 2010;65:547-54, doi: 10.1590/S1807-59322010000500013. 4. Ekbom KA. Restless Legs in Amputees. Acta Med Scand. 1961;169:41921, doi: 10.1111/j.0954-6820.1961.tb07851.x. 5. Hanna PA, kumar S Walters AS. Restless legs symptoms in a patient with above knee amputation: A case of phantom restless legs; Clin Neuropharmacology. 2004;27:87-9. 6. Allen R, Picchietti D, Hening W, Trenkwalder C, Walters AS, Montplaisir J. Restless Legs Syndrome: Diagnostic criteria, special considerations, and epidemiology. A report from the restless legs syndrome diagnosis and epidemiology workshop at the National Institutes of Health. Sleep Med. 2003;4:101-19, doi: 10.1016/S1389-9457(03) 00010-8. 7. Hendry S, Hsiao S. Somatosensory System. In: Squire LR, Berg D, Bloom DS, Ghosh A, Spitzer NC, editors. Fundamental Neuroscience, 3rd edition. Elsevier Inc, San Diego. 2008;581-608

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DOI:10.1590/S1807-59322011001100018

BASIC RESEARCH

Vimentin and laminin are altered on cheek pouch microvessels of streptozotocin-induced diabetic hamsters Jemima Fuentes R. Silva,I Fatima Z. G. A. Cyrino,III Marisa M. D. Breitenbach,II Eliete Bouskela,III Jorge Jose´ CarvalhoI I Universidade do Estado do Rio de Janeiro, Laboratory of Cellular Ultrastructure and Tissue Biology, Biomedical Center, Institute of Biology, Rio de Janeiro/RJ, Brazil. II Universidade do Estado do Rio de Janeiro, Department of Physiological Sciences, Biomedical Center, Rio de Janeiro/RJ, Brazil. III Universidade do Estado do Rio de Janeiro, Clinical and Experimental Research Laboratory on Vascular Biology (BioVasc), Biomedical Center, Rio de Janeiro/RJ, Brazil.

OBJECTIVES: Normal endothelial cells respond to shear stress by elongating and aligning in the direction of fluid flow. Hyperglycemia impairs this response and contributes to microvascular complications, which result in deleterious effects to the endothelium. This work aimed to evaluate cheek pouch microvessel morphological characteristics, reactivity, permeability, and expression of cytoskeleton and extracellular matrix components in hamsters after the induction of diabetes with streptozotocin. METHODS: Syrian golden hamsters (90–130 g) were injected with streptozotocin (50 mg/kg, i.p.) or vehicle either 6 (the diabetes mellitus 6 group) or 15 (the diabetes mellitus 15 group) days before the experiment. Vascular dimensions and density per area of vessels were determined by morphometric and stereological measurements. Changes in blood flow were measured in response to acetylcholine, and plasma extravasation was measured by the number of leakage sites. Actin, talin, a-smooth muscle actin, vimentin, type IV collagen, and laminin were detected by immunohistochemistry and assessed through a semiquantitative scoring system. RESULTS: There were no major alterations in the lumen, wall diameters, or densities of the examined vessels. Likewise, vascular reactivity and permeability were not altered by diabetes. The arterioles demonstrated increased immunoreactivity to vimentin and laminin in the diabetes mellitus 6 and diabetes mellitus 15 groups. DISCUSSION: Antibodies against laminin and vimentin inhibit branching morphogenesis in vitro. Therefore, laminin and vimentin participating in the structure of the focal adhesion may play a role in angiogenesis. CONCLUSIONS: Our results indicated the existence of changes related to cell–matrix interactions, which may contribute to the pathological remodeling that was already underway one week after induction of experimental diabetes. KEYWORDS: Diabetes Mellitus; Microcirculation; Cytoskeleton; Extracellular Matrix; Immunohistochemistry. Silva JFR, Cyrino FZGA, Breitenbach MMD, Bouskela E, Carvalho JJ. Vimentin and laminin are altered on cheek pouch microvessels of streptozotocininduced diabetic hamsters. Clinics. 2011;66(11):1961-1968. Received for publication on April 2, 2011; First review completed on May 19, 2011; Accepted for publication on July 11, 2011 E-mail: jjcarv@gmail.com Tel.: 55 21 2587-6135

exhibiting identical properties.1 In reality, however, the structures of microvessel networks are highly heterogeneous. This heterogeneity has important effects on the functional behavior of the microcirculation.2 Consequently, the physiology, biochemistry, and pharmacology vary according to the portion of the network that is studied and the location within different tissues.1 Diabetes mellitus is caused by physical or functional loss of beta-cell mass, which results in hyperglycemia. In turn, hyperglycemia contributes to microvascular complications, which result in deleterious effects on the endothelium. Endothelial and smooth muscle cells are largely responsible for regulating vascular tone and permeability, and they make up the subendothelial matrix. Endothelial dysfunction

INTRODUCTION The microcirculation represents a unique environment that acts as the site of gas and nutrient exchange between blood and tissues.1 Microvessel networks are often idealized as a set of vessels of different categories that are connected in a series with the parallel vessels in each category

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procedures were carried out in accordance with the conventional guidelines for experimentation with animals. Twenty-four male Syrian golden hamsters (Mesocricetus auratus), seven to ten weeks old and weighing 90-130 g, were maintained in separate cages under controlled temperature and humidity and a 12/12 h light/dark cycle. Animals had free access to food and water. Syrian golden hamsters were randomly divided into a control group (CG) and a diabetic group. Experimental type 1 diabetes mellitus was induced by intraperitoneal (i.p.) injections of STZ (50 mg/kg body weight/day, Sigma Chemical Co., St. Louis, MO, USA) dissolved in 50 mmol/ l sodium citrate buffer (pH 4.5) for three consecutive days (a total dose of 150 mg/kg) at intervals of 24 h. Hamsters in the CG (n = 8) received sodium citrate buffer alone. Diabetic animals were further divided according to the duration after STZ administration: 6 days (n = 8; DM6) or 15 days (n = 8; DM15) of diabetes development. Before the STZ injection and at 1 h, 24 h, and 3, 6, and 15 days after the last injection, postprandial blood glucose and body mass were measured to confirm diabetes ($210 mg/dl). Three days after the last STZ injection, the diabetic condition was confirmed. Only animals showing weight loss (due to uncontrolled diabetes) and severe hyperglycemia were considered for the analyses.

is regarded as an important factor in the pathogenesis of microangiopathy.3 Numerous hyperglycemia-related mechanisms have been hypothesized to mediate micro- and macrovascular complications. These mechanisms include the polyol and hexosamine pathways, protein kinase C (PKC) activation, generation of reactive oxygen species, poly(ADP-ribose) polymerase (PARP) activation, and the accumulation of advanced glycoxidation or glycation end-products (AGEs).4 Microvascular deterioration in diabetes has been associated with increased blood flow, venular dilation, basement membrane thickening, increased exudation of plasma proteins, decreased density of microvessels, and decreased oxygen supply to the tissues.5 Several investigators have described an increase in microvascular permeability6 and changes in endotheliumdependent relaxation7 in streptozotocin (STZ)-induced diabetic animals during the prediabetic period (six–eight weeks). These changes could be due to increased intravascular pressure, which occurs as a result of the increased shear stress on the vascular luminal surface,8 and the accumulation of extracellular matrix and cytoskeleton proteins glycated by AGEs.9 The regulation of these processes occurs via the mechanically stimulated release of potent, shear-responsive, endothelial-derived factors, such as nitrovasodilators, prostaglandins, lipoxygenases, hyperpolarizing factors, growth factors.10 In contrast to vessel changes observed during acute vasoregulation, sustained changes in the local hemodynamics promote the adaptive structural remodeling of the arterial wall through endothelium-dependent regulation of gene and protein expression.11 Moreover, morphologic alteration of arterioles, capillaries, and venules has been demonstrated after chronic hyperglycemia in diabetic human subjects, rats, and mice.12 Prolonged hyperglycemic states result in the generation of reactive oxygen species, which leads to morphological changes in response to endothelial dysfunction.13 These morphological changes ultimately require the dynamic cytoskeleton and its interaction with the extracellular matrix (ECM).14 Most previous analyses of microvessels in the hamster cheek pouch model and other animal models have been performed after a period of two weeks from the development of diabetes.5,6 Early alterations (first two weeks) in the microvasculature that may contribute to the onset and progression of type 1 diabetes, however, remain elusive. Thus, an integrated analysis of the structural and physiological parameters of the microcirculation during the first two weeks of diabetes development is necessary. The main purpose of this work was to qualitatively describe the structural alterations that occur in microvessels and in the cytoskeleton and ECM components during the early stage of diabetes mellitus development. In addition, we wanted to study the sensitivity of the microvascular membrane to histamine and the endothelium-dependent relaxation induced by acetylcholine.

Intravital Microscopy of the Hamster Cheek Pouch Hamsters from each group were prepared for intravital microscopy according to the methods described in Svensjo¨ (1999)6 at 6 or 15 days after the STZ treatment. Animals were anesthetized with an injection of sodium pentobarbital (60 mg/ml, i.p.) supplemented with i.p. dose of a-chloralose (100 mg/kg) through a femoral vein catheter, and body temperature was kept at 36.5 ˚C. To facilitate spontaneous breathing, a tracheostomy was performed. The right femoral vein was cannulated for the administration of fluoresceindextran (FITC–dextran, MW 150,000, TdB Consultancy, Uppsala, Sweden), which was used to measure the restrictiveness of the microvascular membrane permeability. Alterations in the postcapillary venular permeability were measured using semiquantitative methods by counting sites with extravasation of fluorescent plasma (leaky sites = leaks) at 0, 2, and 5 min after the topical application of histamine (10-6 mol). To determine the vascular reactivity, the luminal diameter of selected arterioles and venules (40-90 mm) was measured both before and 10 min after the microapplication of acetylcholine (10-4 mol, micropipette 10-12 mm and constant volume of 200 ml/min). Microvessels were analyzed using a millimetric ocular lens of a light microscope and observed with a computer-assisted method on analog– digital-converted, video-recorded images. To avoid bias due to single-operator measurements, two independent blinded operators measured vessel diameters.

Morphometric and Stereologic Analysis To reduce measurement errors, intimal (IL) and media layers (ML) were analyzed by different techniques. To determine the thickness of the IL, the cheek pouch was fixed in 2.5% glutaraldehyde and 2.0% paraformaldehyde in 0.15 M cacodylate buffer (pH 7.2) for 2 h. The IL was then postfixed in 1% osmium tetroxide, stained in 2% uranyl acetate, dehydrated in acetone, and embedded in epoxy resin (Epon-812, SEM, Hatfield, PA, USA). Thin sections (1 mm thick) were stained with 0.25% toluidine blue in 1% acetic acid (Vetec, Brazil) and observed under a light

MATERIALS AND METHODS Animals and Induction of Diabetes Mellitus The study protocol was approved by the Ethics Committee for Experimental Animals, no. CEA 004/2009, of the State University of Rio de Janeiro, Brazil. All

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USA). In all cases, p,0.05 was considered statistically significant.

microscope (Axiophot, Carl Zeiss, Germany). Media layer thickness was determined using light microscopy with immunolabeling for a-smooth muscle actin (a-SMA). For details on immunohistochemistry, please see section ‘‘Immunohistochemistry Assay’’. The digital images of 20 vessels for each hamster (TIFF format, 36-bit color, 1,280 X 1,024 pixels) were acquired using a 100X oil-immersion objective lens and manually quantified with the Image-Pro Plus 4.5 program (Image Pro Plus 4.5, Media Cybernetics, Bethesda USA). This sample size was based on previous studies and was designed to avoid biases related to inter-animal variability.15,16 Vascular dimensions were measured taking into account three parameters: lumen diameter, wall thickness and wall/ lumen ratio. We obtained four measurements of IL and ML thickening (i.e., at 0˚C, 90˚C, 180˚C and 270˚C). The luminal area was measured from the internal circumferential line, and the lumen diameter was calculated from the area. The percentage of thickening was measured by the lumen/wall ratio.17 It is important to highlight that the lumen diameter, wall thickness and lumen/wall ratio measurements in hamsters were obtained from a previous work,5 and the measures from the previous study were used as a pattern for the species. The volume density was analyzed by stereology using a test system composed of 36 test points (PT)15 and estimated by the following equation: Vv [structure] = Pp [structure]/ Pt, where Pp is the number of points that hit the structure, and Pt is the total number of test points inside the test system. The density per area was estimated for vessels (arterioles or venules) with the following equation: QA[structure] = N[structure]/AT (1 mm2), where N is the number of profiles counted in the test area (i.e., the frame AT), considering the forbidden line and its extensions.15

RESULTS Effects of STZ-Induced Diabetes on Body Weight and Morphofunctional Parameters As expected, glycemia was extremely high in the hamsters in the diabetic groups. The hamsters were only persistently hyperglycemic after the third day after STZ administration. Compared with the CG, glycemia was increased two-fold in diabetic hamsters 6 days (DM6) after STZ administration and increased almost three-fold in diabetic hamsters 15 days (DM15) after the induction of diabetes (p,0.001 for the CG vs. DM6 and DM15). The degree of glycemia was also different between DM6 and DM15 (p,0.05, Figure 1A). In addition, diabetic hamsters suffered a significant reduction in body weight. The control group showed a weight gain of approximately 5% from day 0 to day 6, whereas the DM6 hamsters displayed a loss in body weight of around 15% for the same time period (p,0.05 for the CG vs. DM6). DM15 hamsters lost 20% compared with the control (p,0.001 for the control group vs. DM15) (Figure 1B). There were significant differences (p,0.05) in the microvascular permeabilities of both the diabetic subgroups (105¡3 and 112¡8 leakage sites/cm2 for the DM6 and DM15 groups, respectively) compared with the control group (158¡13 leakage sites/cm2) 2 min after the end of the topical application of histamine. The peak vascular response, however, usually occurred at 3 to 5 min after the application and dissipated within 10 or 20 min (data not shown). Responses were not significantly different between any two groups at 5 min (p.0.05, Figure 1C). For acetylcholine, there were no significant differences between the diabetic and control hamsters in any determination for any vessel tested (arterioles or venules) (p.0.05 for comparison between groups, Figure 1D). Table 1 shows that no significant differences between groups were observed for morphometric or stereological parameters. Microvessel density per cheek pouch area (QA) and volume density (Vv) were estimated to determine the amount of cheek pouch vascularization in age-matched control and diabetic hamsters. For both parameters, the number of venules was two times greater than the number of arterioles, but no significant difference between groups was observed in any analysis (p.0.05).

Immunohistochemistry Assay Paraffin sections of cheek pouches were deparaffinized, rehydrated, and incubated (10 min) with 0.3% H2O2 in methanol to block endogenous peroxidase. Nonspecific protein binding was blocked by incubation with 1% bovine serum albumin diluted in phosphate-buffered saline (PBS/ BSA). Antigen retrieval for ECM components was performed with trypsin (3%) diluted in distilled water for 10 min at 37 ˚C and the sections were incubated with the primary antibody diluted 15100 with 1% PBS/BSA overnight (4 ˚C, in a humid atmosphere). Cytoskeleton protein sections were incubated in 10 mM sodium citrate buffer (pH 6.0, Antigen Unmasking Solution, H-3300; Vector Laboratories, CA, USA) at 98 ˚C for 20 min and subsequently incubated with the primary antibody diluted 1525. After washing with PBS, slides were incubated with reagent from the Labeled Streptavidin-Biotin System (LSAB) immunohistochemical kit (LSAB-kit, Dako, Carpinteria, CA). Sections were then counterstained with hematoxylin and mounted using an aqueous mounting medium. Control sections were processed after the omission of the primary antibody. Immunohistochemistry staining was evaluated based on a semiquantitative scoring system: score 0, no label; score 1, poor label; score 2, medium label; score 3, strong label.16

Immunohistochemistry Score Actin expression increased in arterioles of the DM6 hamsters compared with the CG. DM15 hamsters do not alter actin expression compared with the CT but showed decrease compared with the DM6 hamsters (CG, score 1; DM6, score 2; DM15, score 1). Actin immunoreactivity was seen in all endothelial and smooth muscle cells (data not shown). There is no difference in actin or talin immunostaining in venules between the groups. The a-SMA immunostaining in arterioles do not change between the groups. In arterioles, talin immunostaining showed an increase in DM6 hamsters when compared to control hamsters. Talin-positive vessels decreased in DM15 hamsters when compared to DM6 hamsters and there is no different relationship with the control group (CG, score 1; DM6, score 3; DM15, score 1). Immunoreactivity for talin was positive in endothelial cells and smooth muscle cells (data not

Data Analysis All data are presented as the mean ¡ the standard error of the mean (SEM) and tested with one-way ANOVA followed by Tukey’s test, using the GraphPad Prism program (5.0 version, GraphPad Software San Diego,

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Figure 1 - The mean ¡ the standard error of the mean (SEM) for glycemia, body weight, and microvascular responses to histamine and acetylcholine in age-matched controls and hamsters at 6 (DM6) and 15 days (DM15) after the induction of diabetes with streptozotocin (STZ, 50 mg/kg). Statistical significance was determined using one-way ANOVA followed by Tukey’s post-hoc analysis. A. Glycemia (mg/ dl): p,0.001 for the control group vs. both DM6 and DM15, and p,0.05 for the DM6 group compared with the DM15 group. B. Body weight (g): p,0.05 for the control group vs. the DM6 group, p,0.001 for the control group vs. the DM15 group, and p,0.05 for the DM6 group vs. the DM15 group. C. Microvascular responses (number of dextran 150kDa leakage sites per cm2) to histamine (10-6 M). There were no significant differences between the groups (p.0.05). D. Responses (as percent increase in lumen diameter) of arterioles and venules to topical application of acetylcholine. There were no significant differences between the groups (p.0.05).

shown). a-SMA expression in venules was increased in the DM6 hamsters compared with the control and the DM15 hamsters (CG, score 1; DM6, score 3; DM15, score 1). Vimentinpositive arterioles and venules increased in DM6 and DM15 hamsters when compared to control hamsters (CG, score 1; DM6, score 2; DM15, score 2). In the CG, the anti-vimentin antibody only reacted with some endothelial cells, but

immunoreactivity was also apparent in smooth muscle cells in both of the diabetic groups. Furthermore, a greater number of cells stained positively for vimentin in the DM6 and DM15 groups compared with the CG (Figure 2). The expression of laminin was more apparent in hamsters in the first week after STZ induction of diabetes than in the control hamsters. Laminin labeling was maintained for 15

Table 1 - Morphological and stereological measurements of microcirculatory vessels of age-matched control and diabetic hamsters. Measurements/Groups/Vessels

Thickness of the intimal and media layers (mm) Lumen diameter Wall thickness Wall/lumen ratio Thickness of the media layer (mm) Lumen diameter Wall thickness Wall/lumen ratio Density per cheek pouch area (QA) (mm2.102) QA Volume density (Vv) (%) Vv

Control

6 days of diabetes

15 days of diabetes

Arterioles

Venules

Arterioles

Venules

Arterioles

Venules

31¡2.7 5.7¡0.7 0.2 0

40¡3 3.6¡0.6 0.1 0

33¡1.2 5.4¡0.9 0.2 0

42¡2.4 2.7¡0.4 0.1 0

25¡2.5 3.8¡0.4 0.2 0

35¡3.1 2.4¡0.2 0.1 0

38¡3.7 5.3¡0.3 0.1 0

41¡2.4 2.6¡0.2 0.1 0

30¡3.8 5.4¡0.2 0.2 0

32¡2.1 2.5¡0.1 0.1 0

28¡7.4 5¡0.8 0.2 0

39¡5.4 2.4¡0.1 0.1 0

3.2¡0.7

8.8¡1.3

3.2¡0.5

7.2¡1

3.5¡0.7

9.7¡1

5.5¡1.3

11¡1.5

5.3¡1.1

11¡1.6

5.2¡1.4

10¡1.9

Values are the mean¡the standard error of the mean. p-values are not significant for any data measured.

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Figure 2 - Changes of vimentin expression in arterioles (A-C) and venules (D-F) by immunohistochemical staining (brown color) (light microscopy, 1,000X). a. The control group only showed a few vimentin-immunoreactive endothelial cells (arrow). b. The diabetic group (6 days) showed positively-stained endothelial (arrow) and smooth muscle cells. c. The diabetic group (15 days) showed vimentinimmunoreactive endothelial (arrow) and smooth muscle cells. d. The control group with few endothelial cells immunoreactive for vimentin (arrowhead). e. The diabetic group (6 days) with a moderate amount of vimentin expression in the intimal and media layers. f. The diabetic group (15 days) with a moderate amount of vimentin expression in the intimal and media layers. The scale bar = 10 mm.

vasorelaxation at an early stage in STZ-induced diabetes. In our work, the vasorelaxation derived from the endothelium remained unchanged, at least until the 15th day of diabetes development. Our results agreed with those of Pieper (1999),19 who showed that the vasorelaxation derived from the endothelium increased on the first day after the application of STZ in STZ-induced diabetic rats. This event was followed by a reversal phase (one-two weeks), in which vasorelaxation was normal, and a delayed phase (eight weeks), during which the vasorelaxation became engaged. Intravital microscopy, which has been extensively used in microcirculation studies, allows in vivo measurements of vascular permeability to various compounds.6 The present study demonstrated the occurrence of changes in microvascular leakage within 2 min after histamine application in diabetic hamsters. Nevertheless, the peak of the response usually occurred 3 to 5 min after histamine application and dissipated within 10 or 20 min. Histamine

days in the diabetic group for both vessel types (arterioles and venules). Figure 3 shows that the IL was immunoreactive for laminin in the CG; however, higher levels of laminin expression were observed in all layers of the vascular wall in the diabetic groups (CG, score 1; DM6, score 2; DM15, score 2). There was no difference in collagen IV staining between the two diabetic groups and the control hamsters. Table 2 shows the distribution scores for cytoskeleton ECM proteins.

DISCUSSION Hyperglycemia exerts deleterious effects on the endothelium, which is the regulator of the vascular wall.3 Abnormalities in the modulatory roles played by the endothelium and/or smooth muscle may be critical and can act as initiating factors in the development of diabetic vascular disease.18 We observed unaltered endothelium-dependent

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Figure 3 - Changes of laminin expression in arterioles (A-C) and venules (D-F) by immunohistochemical staining (light microscopy, 1000X). a. The intimal layers of the hamsters in the control group were immunoreactive for laminin. b and c. All layers showed positive laminin staining for both diabetic groups. d. The control group showed a poorly labeled intimal layer. e. The diabetic group (6 days) with all layers staining positive. f. The diabetic group (15 days) with all layers staining positive. The scale bar = 10 mm.

changes were observed after 5 min of histamine application between the two diabetes groups and the controls. These data agreed with the results of Llorach and coworkers (1976),20 which did not show alterations in onetwo-week diabetic animal models. Many reports have been published about functional and morphological alterations that occur during the early stage of diabetic disease in multiple animal models. In the present

binding to cell-surface receptors triggers subtle or rapid changes in the contractility of endothelial cells that depends on the cytoskeletal organization and cellular adhesion. Therefore, we can suggest that hyperglycemia influences vascular function at 6 and 15 days of diabetes development and that this change may be associated with subtle changes in the expression and distribution of endothelial cell cytoskeleton components. No significant

Table 2 - Distribution of actin, talin, a-SMA, vimentin, laminin, and type-IV collagen immunohistochemistry scores in age-matched control and 6- and 15-day diabetic hamsters. Control

Antibodies

Actin Talin a-SMA Vimentin Laminin Type-IV collagen

6 days of diabetes

15 days of diabetes

Arteriole

Venule

Arteriole

Venule

Arteriole

Venule

+ + ++ + ++ ++

+ + + + + ++

++ +++ ++ ++ +++ ++

+ + +++ ++ ++ ++

+ + ++ ++ +++ ++

+ + + ++ ++ ++

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Vimentin and laminin in diabetic hamsters Silva JF et al.

We also found that vimentin was concomitantly increased with laminin expression in blood vessels. All three major cytoskeletal networks have been shown to adapt their structures by elongating their cell shapes and reorienting parallel to the flow direction.31 Intermediate filament cables, which are initially not under tension, contribute to cell stiffness during large shear deformations. This strain-hardening behavior in the intermediate cytoskeleton filament likely prevents the excessive deformation of the cell at finite strains that would otherwise rupture the actin cytoskeleton.32 Intermediate filaments may also indirectly interact with focal adhesion sites. Indeed, vimentin intermediate filaments are anchored to a6b4integrins in microvascular endothelial cells by plectin. Because a6b4 binds to laminin, these interactions are consistent with the hypothesis that intermediate filaments are recruited to provide mechanical stability during physiological adaptation of the microfilament network to hemodynamic shear stress.31 Thus, the increased expression of vimentin is associated with a proliferative phenotype, possibly due to hyperglycemia.33 Antibodies against laminin and vimentin inhibit branching morphogenesis in vitro. Therefore, this adhesion structure may play a role in angiogenesis.34 Like the endothelial cell cytoskeleton, smooth muscle cells are important in maintaining endothelial structural integrity and in regulating endothelial repair.33 The expression of a-SMA in venules was enhanced in the DM6 hamsters, and increased expression of a-SMA has been shown to result in increased contractile force generation.35 In addition, increased expression of a-SMA has been shown to be associated with vessel stability.32 The primary function of smooth muscle cells (SMC) is the maintenance of blood flow and pressure through their contractility, and a-SMA is necessary for SMC and pericytes to interact with endothelial cells to form a fully functional bloodâ&#x20AC;&#x201C;tissue barrier.35 Our results suggest that changes related to cell-matrix interactions occur in response to a new metabolic condition (6 days of a diabetic state). These changes may contribute to pathological remodeling of the microvessels and persist for at least 15 days after the induction of diabetes.

study, no significant changes were observed in morphometric or stereological measurements, in wall thickening or microvessel lumen diameters, in the density of the vessels per cheek pouch area (QA) or in volume density (Vv) during the early stage of disease development. Morphological changes require the accumulation of ECM proteins glycated by AGEs9 and are associated with basement membrane thickening.14 In part, this thickening may occur when cells synthesize increased amounts of collagen IV.21 The expression of typeIV collagen, however, did not change in cheek pouch microvessels, which may have resulted from a compensatory mechanism to hyperglycemia in vascular cells that promoted a change in the metabolism of type-IV collagen and increased its turnover. An increase in the turnover of type-IV collagen would avoid accumulation of type-IV collagen and result in a thickening of the basement membrane. There is some clinical and experimental evidence of augmented blood flow at the early stage of diabetes.22 Small arteries have the most important influence on local blood flow and are subjected to chemical and neurohormonal influences as well as the continuous effect of mechanical factors generated by the blood stream.8 The endothelium responds to mechanical stresses through mechanotransduction to regulate the vasomotion and function of the vascular wall. Focal adhesions, which significantly contribute to endothelial cell tethering, mediate the attachment of the actin cytoskeleton to the ECM through a cytoplasmic protein complex (i.e., the focal adhesion plaque).23 Blood vessel endothelial cells produce focal adhesion molecules, including focal adhesion kinase, vinculin, talin, and cytoskeletal b-actin. In arterioles, we found that several proteins involved in focal adhesion, such as actin, talin, vimentin, and laminin, were increased in the DM6 group; however, the immunolabels for actin and talin in arterioles were decreased in the DM15 hamsters. Talin is part of the cytoskeletal protein complex that binds the cytoplasmic tail of integrins to the b-actin cytoskeleton in focal adhesions and is essential for focal adhesion function.24 Talin is essential for integrin activation, and it is the initial weak link between integrins and the actin cytoskeleton.25 Talin,26 actin and tubulin readily undergo nonenzymatic glycosylation.27 Loufrani and Henrion (2008)28 reported that AGEs promote an initial depolymerization of actin filaments (actin F) and a subsequent decrease in actin G expression, which corresponds with a reduction in actin microfilaments and the disruption of cell-cell and cell-matrix junctions. In our study, we used a single antibody to immunolabel both forms of actin; this method could explain the increased expression of actin at 6 days after the onset of diabetes. Talin and actin are key proteins in focal adhesion and represent a mechanical interaction between the cell and the ECM. The force of contraction of the cell is related to focal adhesion.29 Chronic hyperglycemia produces a nonenzymatic glycation of talin and actin, which may contribute to reduced steadystate adhesion strength and mechanosensing responses. In addition, hyperglycemia can also promote further changes in vascular permeability. Diabetic hamsters presented increased labeling for laminin in both vessel types. Because laminin is the primary determinant of basement membrane assembly,30 increased laminin expression may suggest a structural remodeling of vessel basement membranes.

AUTHOR CONTRIBUTIONS Silva JF was responsible for the morphometric and stereologic analysis. Carvalho JJ was responsible for the immunohistochemistry assay. Breitenbach MMD was responsible for the induction of diabetes mellitus and data analysis. Cyrino FZGA was responsible for the intravital microscopy of the hamster cheek pouch. Bouskela E participated in the intravital microscopy technique, interpretation of intravital microscopy, immunohistochemistry, analysis of morphometric and stereological techniques, and was responsible for the draft of the introduction, discussion, and conclusions sections.

REFERENCES 1. Aguiar LG, Villela NR, Bouskela E. [Microcirculation in diabetes: implications for chronic complications and treatment of the disease]. Arq Bras Endocrinol Metabol. 2007;51:204-11. 2. Duling BR, Damon DH. An examination of the measurement of flow heterogeneity in striated muscle. Circ Res. 1987;60:1-13. 3. Schalkwijk CG, Stehouwer CD. Vascular complications in diabetes mellitus: the role of endothelial dysfunction. Clin Sci (Lond). 2005;109:143-59. 4. Meerwaldt R, Links T, Zeebregts C, Tio R, Hillebrands JL, Smit A. The clinical relevance of assessing advanced glycation endproducts accumulation in diabetes. Cardiovasc Diabetol. 2008;7:29.

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5. Joyner WL, Mayhan WG, Johnson RL, Phares CK. Microvascular alterations develop in Syrian hamsters after the induction of diabetes mellitus by streptozotocin. Diabetes. 1981;30:93-100. 6. Svensjo E, Cyrino F, Michoud E, Ruggiero D, Bouskela E, Wiernsperger N. Vascular permeability increase as induced by histamine or bradykinin is enhanced by advanced glycation endproducts (AGEs). J Diabetes Complications. 1999;13:187-90. 7. Sotnikova R, Skalska S, Okruhlicova L, Navarova J, Kyselova Z, Zurova J, et al. Changes in the function and ultrastructure of vessels in the rat model of multiple low dose streptozotocin-induced diabetes. Gen Physiol Biophys. 2006;25:289-302. 8. Davies PF. Hemodynamic shear stress and the endothelium in cardiovascular pathophysiology. Nat Clin Pract Cardiovasc Med. 2009;6:16-26. 9. Mott RE, Helmke BP. Mapping the dynamics of shear stress-induced structural changes in endothelial cells. Am J Physiol Cell Physiol. 2007;293:C1616-26. 10. Moncada S. Adventures in vascular biology: a tale of two mediators. Philos Trans R Soc Lond B Biol Sci. 2006;361:735-59. 11. Zhang H, Sunnarborg SW, McNaughton KK, Johns TG, Lee DC, Faber JE. Heparin-binding epidermal growth factor-like growth factor signaling in flow-induced arterial remodeling. Circ Res. 2008;102:1275-85. 12. Jarvisalo MJ, Raitakari M, Toikka JO, Putto-Laurila A, Rontu R, Laine S, et al. Endothelial dysfunction and increased arterial intima-media thickness in children with type 1 diabetes. Circulation. 2004;109:1750-5. 13. Singh R, Barden A, Mori T, Beilin L. Advanced glycation end-products: a review. Diabetologia. 2001;44:129-46. 14. Liu Y, Li H, Bubolz AH, Zhang DX, Gutterman DD. Endothelial cytoskeletal elements are critical for flow-mediated dilation in human coronary arterioles. Med Biol Eng Comput. 2008;46:469-78. 15. Mandarim-de-Lacerda CA. Stereological tools in biomedical research. An Acad Bras Cienc. 2003;75:469-86. 16. Mendonca Lde S, Fernandes-Santos C, Mandarim-de-Lacerda CA. Cardiac and aortic structural alterations due to surgically-induced menopause associated with renovascular hypertension in rats. Int J Exp Pathol. 2007;88:301-9. 17. Bonnet F, Cao Z, Cooper ME, Cox AJ, Kelly DJ, Gilbert RE. Tranilast attenuates vascular hypertrophy, matrix accumulation and growth factor overexpression in experimental diabetes. Diabetes Metab. 2003;29:386-92. 18. Matsumoto T, Oda SI, Kobayashi T, Kamata K. Flow-induced endothelium-dependent vasoreactivity in rat mesenteric arterial bed. J Smooth Muscle Res. 2004;40:1-14. 19. Pieper GM. Enhanced, unaltered and impaired nitric oxide-mediated endothelium-dependent relaxation in experimental diabetes mellitus: importance of disease duration. Diabetologia. 1999;42:204-13. 20. Llorach MA, Bohm GM, Leme JG. Decreased vascular reactions to permeability factors in experimental diabetes. Br J Exp Pathol. 1976;57:747-54. 21. Iglesias-de la Cruz MC, Ziyadeh FN, Isono M, Kouahou M, Han DC, Kalluri R, et al. Effects of high glucose and TGF-beta1 on the expression

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of collagen IV and vascular endothelial growth factor in mouse podocytes. Kidney Int. 2002;62:901-13. Kobayashi T, Kaneda A, Kamata K. Possible involvement of IGF-1 receptor and IGF-binding protein in insulin-induced enhancement of noradrenaline response in diabetic rat aorta. Br J Pharmacol. 2003;140:285-94. Harrington MA, Daub R, Song A, Stasek J, Garcia JG. Interleukin 1 alpha mediated inhibition of myogenic terminal differentiation: increased sensitivity of Ha-ras transformed cultures. Cell Growth Differ. 1992;3:241-8. Weber E, Rossi A, Solito R, Sacchi G, Agliano M, Gerli R. Focal adhesion molecules expression and fibrillin deposition by lymphatic and blood vessel endothelial cells in culture. Microvasc Res. 2002;64:47-55. Wegener KL, Partridge AW, Han J, Pickford AR, Liddington RC, Ginsberg MH, et al. Structural basis of integrin activation by talin. Cell. 2007;128:171-82. Lee TY, Gotlieb AI. Early stages of endothelial wound repair: conversion of quiescent to migrating endothelial cells involves tyrosine phosphorylation and actin microfilament reorganization. Cell Tissue Res. 1999;297:435-50. Pekiner C, Cullum NA, Hughes JN, Hargreaves AJ, Mahon J, Casson IF, et al. Glycation of brain actin in experimental diabetes. J Neurochem. 1993;61:436-42. Loufrani L, Henrion D. Role of the cytoskeleton in flow (shear stress)induced dilation and remodeling in resistance arteries. Med Biol Eng Comput. 2008;46:451-60. Lee SE, Kamm RD, Mofrad MR. Force-induced activation of talin and its possible role in focal adhesion mechanotransduction. J Biomech. 2007;40:2096-106. Sasaki T, Fassler R, Hohenester E. Laminin: the crux of basement membrane assembly. J Cell Biol. 2004;164:959-63. Helmke BP. Molecular control of cytoskeletal mechanics by hemodynamic forces. Physiology (Bethesda). 2005;20:43-53. Schopferer M, Bar H, Hochstein B, Sharma S, Mucke N, Herrmann H, et al. Desmin and vimentin intermediate filament networks: their viscoelastic properties investigated by mechanical rheometry. J Mol Biol. 2009;388:133-43. Skalli O, Bloom WS, Ropraz P, Azzarone B, Gabbiani G. Cytoskeletal remodeling of rat aortic smooth muscle cells in vitro: relationships to culture conditions and analogies to in vivo situations. J Submicrosc Cytol. 1986;18:481-93. Gonzales M, Weksler B, Tsuruta D, Goldman RD, Yoon KJ, Hopkinson SB, et al. Structure and function of a vimentin-associated matrix adhesion in endothelial cells. Mol Biol Cell. 2001;12:85-100. Tomasek JJ, Haaksma CJ, Schwartz RJ, Vuong DT, Zhang SX, Ash JD, et al. Deletion of smooth muscle alpha-actin alters blood-retina barrier permeability and retinal function. Invest Ophthalmol Vis Sci. 2006;47:2693-700.

CLINICS 2011;66(11):1969-1974

DOI:10.1590/S1807-59322011001100019

BASIC RESEARCH

Impact of Plasma-Lyte pH 7.4 on acid-base status and hemodynamics in a model of controlled hemorrhagic shock Danilo Teixeira Noritomi,I Adriano Jose´ Pereira,I Diogo Diniz Gomes Bugano,II Paulo Sergio Rehder,II Elie´zer SilvaI I

Hospital Israelita Albert Einstein, Intensive Care Unit, Saõ Paulo/SP, Brazil. II Faculdade de Medicina da Universidade de Saõ Paulo, Saõ Paulo/SP, Brazil.

OBJECTIVE: Intravenous infusion of crystalloid solutions is a cornerstone of the treatment of hemorrhagic shock. However, crystalloid solutions can have variable metabolic acid-base effects, perpetuating or even aggravating shock-induced metabolic acidosis. The aim of this study was to compare, in a controlled volume–driven porcine model of hemorrhagic shock, the effects of three different crystalloid solutions on the hemodynamics and acid-base balance. METHODS: Controlled hemorrhagic shock (40% of the total blood volume was removed) was induced in 18 animals, which were then treated with normal saline (0.9% NaCl), Lactated Ringer’s Solution or Plasma-Lyte pH 7.4, in a blinded fashion (n = 6 for each group). Using a predefined protocol, the animals received three times the volume of blood removed. RESULTS: The three different crystalloid infusions were equally capable of reversing the hemorrhage-induced low cardiac output and anuria. The Lactated Ringer’s Solution and Plasma-Lyte pH 7.4 infusions resulted in an increased standard base excess and a decreased serum chloride level, whereas treatment with normal saline resulted in a decreased standard base excess and an increased serum chloride level. The Plasma-Lyte pH 7.4 infusions did not change the level of the unmeasured anions. CONCLUSION: Although the three tested crystalloid solutions were equally able to attenuate the hemodynamic and tissue perfusion disturbances, only the normal saline induced hyperchloremia and metabolic acidosis. KEYWORDS: Acidosis; Crystalloid Solution; Hyperchloremia; Hemorrhagic Shock; Strong Ion Difference. Noritomi DT, Pereira AJ, Bugano DDG, Rehder PS, Silva E. Impact of Plasma-Lyte pH 7.4 on acid-base status and hemodynamics in a model of controlled hemorrhagic shock. Clinics. 2011;66(11):1969-1974. Received for publication on February 24, 2011; First review completed on April 4, 2011; Accepted for publication on July 13, 2011 E-mail: eliezer@einstein.br/danilotn@einstein.br Tel.: 55 11 8111-5529

acidosis.6,7 The use of so-called ‘‘balanced solutions,’’ which are more similar to human plasma in terms of electrolytes and acid-base chemistry, may avoid this problem.4 Lactated Ringer’s (LR) Solution and Plasma-Lyte pH 7.4 are two balanced solutions that are commonly used in clinical practice. To our knowledge, no study has compared the effects of these three solutions on the quantitative acid-base and hemodynamic profiles in a hemorrhagic scenario. Therefore, the aim of this study was to compare, in a controlled, volume-driven porcine model of hemorrhagic shock, the effects of three different crystalloid solutions on the hemodynamics and quantitative acid-base balance.

INTRODUCTION Hemorrhagic shock is a well-known cause of metabolic acidosis, which is associated with a poor prognosis.1 Hypothermia, coagulopathy, and acidosis comprise what has been termed the ‘‘trauma triad of death.’’2 Intravenous infusion of crystalloid solutions is a cornerstone of hemorrhagic shock treatment, and it can both restore the intravascular compartment and maintain cardiac output and tissue perfusion.3 However, crystalloid solutions can have variable metabolic acid-base effects, sometimes perpetuating or aggravating metabolic acidosis.4 Normal saline is a very frequently used solution,5 but its use has been associated with the development of hyperchloremic

MATERIALS AND METHODS Animals

All experiments were carried out in accordance with the National Institutes of Health (1985) and the American Physiological Society (1995) principles on the care, manipulation, and protection of laboratory animals, after the

No potential conflict of interest was reported.

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approval of the Animal Ethics Committee from Hospital Israelita Albert Einstein, Sa˜o Paulo, Brazil. Eighteen healthy, male white pigs (30.4¡4.2 kg) were used in the experiments. The animals were evaluated by a veterinarian before and during all experiments. Access to food was suspended 12 hours prior to surgery, but free access to water was always provided.

Table 1 - Characteristics of the fluids used for resuscitation.

Sodium (mEq/l) Potassium (mEq/l) Calcium (mEq/l) Magnesium (mEq/l) Chloride (mEq/l) Lactate (mEq/l) Gluconate (mEq/l) Acetate (mEq/l) Osmolarity (mOsm/l) pH

Surgical procedures Animals were anesthetized using the following protocol: the pigs were pre-medicated with ketamine (10 mg/kg, i.m.) and midazolam (0.25 mg/kg, i.m.). Propofol (5 mg/kg i.v.) was used to induce anesthesia, which was maintained with a continuous i.v. infusion of fentanyl (2.5 mg/kg/h), pancuronium (0.4 mg/kg/h) and isoflurane (1.5%). After induction, the animals were orotracheally intubated (6.5–7.0 tubes) and mechanically ventilated (Origami Takaoka, K. Takaoka, Brazil) using the volume assistcontrol mode with the following parameters: tidal volume, 10 ml/kg body weight; positive ending expiratory pressure (PEEP), 5 cmH2O; and 25–30% of fractional inspiratory O2 concentration (FiO2). The goal was to maintain a normal partial pressure of oxygen (PaO2, 60–100 mmHg) in an arterial blood sample. The respiratory rate (RR) was adjusted to obtain a normal carbon dioxide partial pressure (PaCO2, 35–45 mmHg) in an arterial blood sample. Thermal blankets were used to keep the core temperature around 38 ˚C. During all anesthetic and surgical procedures, continuous electrocardiography monitoring was performed (Dixtal 2021 monitor, Brazil). Arterial and venous lines were placed via left inguinal (femoral) dissection. The arterial line was connected to a pressure transducer (Edwards Lifesciences, USA) to provide continuous arterial pressure measurements (Dixtal 2021 monitor, Brazil). The right jugular vein was also dissected to allow for pulmonary artery catheterization (7F Swan-Ganz, Edwards Lifesciences, USA) and continuous cardiac output measurement (Vigilance, Edwards Lifesciences, USA). A mini-laparotomy and a cystostomy were performed in all animals to quantify urinary output.

Normal Saline

Lactated Ringer’s Solution

Plasma-Lyte pH 7.4

154 0 0 0 154 0 0 0 308 5.50

130 4 3 0 109 28 0 0 275 6.75

140 5 0 3 98 0 23 27 294 7.40

and the infusion was performed using a pressurized system (300 mmHg). The heart rate, blood pressure, cardiac output and SvO2 were monitored during all phases of the experiment: baseline, after each blood withdrawal, 30 min after finishing the bleeding process, immediately after resuscitation and 1 hour after resuscitation. Blood samples were collected before the first blood withdrawal, 1 hour after the final blood withdrawal and 1 hour after the resuscitation process. After the protocol was complete, the animals were euthanized via anesthetic overdose followed by injection of a 20 ml KCl (19.1%) i.v. bolus.

Laboratory samples Arterial blood samples were collected in heparinized syringes and analyzed using a blood-gas analyzer (ABL 700, Radiometer, Copenhagen, Denmark). The analyzer measured samples at 37 ˚C. We collected the following data from the analyzer output: pH, partial pressure of carbon dioxide, bicarbonate, standard base excess (SBE), and ionized calcium. The plasma sodium, chloride, lactate, and potassium levels were measured using ion-selective electrodes in the blood-gas analyzer. The machine calculated the bicarbonate concentration using the Henderson– Hasselbach equation. The SBE was calculated according to Wooten’s equation,9 the formula least sensitive to PCO2, because our experiment was prone to large variations in the PCO2. Additionally, 5 ml of plasma was collected and stored at -80 ˚C. The magnesium (calorimetric method using Formazan dye and Randox blue xylitol, Vitros System, Johnson & Johnson, USA), albumin (calorimetric method using Randox Bromocresol green, dry chemistry, Vitros System, Johnson & Johnson, USA), and phosphate (calorimetric method and dry chemistry, Vitros System, Johnson & Johnson, USA) levels were measured in these plasma samples.

Experimental protocol During surgical preparation, 20 ml/kg of normal saline was administered to all animals. After performing the sedation, mechanical ventilation, and vascular access, controlled hemorrhagic shock was induced in the animals. Based on the calculated ratio of 76 ml blood/kg of body weight,8 we estimated the total blood volume to guide the exsanguination process. Forty percent of the total blood volume was removed via manual blood withdrawal through the arterial catheter in three equal aliquots, with a 30-minute interval between withdrawals. The animals were then randomly allocated to one of the three resuscitation groups (six animals per group): Normal Saline (0.9% NaCl, Baxter Viaflex, Brazil), LR solution (Lactated Ringer’s Solution, Baxter, Viaflex, Brazil), or Plasma-Lyte pH 7.4 (Baxter Healthcare Corporation, USA) in a blinded fashion. The composition of the solutions is described in Table 1. We started the resuscitation with one of the three crystalloid solutions one hour after the bleeding was completed. The total volume infused was three times the total blood volume removed. The total time to infuse the crystalloid solution was 10–15 min per animal,

Conceptual framework for acid-base analysis To separate the independent components of the acid-base disturbances, we performed a quantitative physicochemical analysis using Stewart’s methodology, as modified by Figge et al. and others.10-12 In this approach, the H+ concentration and, therefore pH, is determined using three independent variables: the strong ion difference (SID); the total concentration of weak acids (Atot), which is mainly determined by the albumin and phosphate levels; and the PCO2. This method initially involves the calculation of the apparent strong ion difference (SIDa):

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Model of controlled hemorrhagic shock Noritomi DT et al.

The hemorrhage induced an increase in the heart rate, a decrease in the cardiac output and a trend towards a decrease in the blood pressure; these changes were of the same magnitude in the three groups. The resuscitation phase took less than 15 min for all animals. The total volume of fluid infused per animal was 2865 (¡488) ml in the Normal Saline group, 2774 (¡381) ml in the LR group and 2681 (¡314) ml in the Plasma-Lyte pH 7.4 group, with no significant difference among the three groups with a single p-value reported. The three crystalloid solutions were equally able to attenuate the hemodynamic alterations (Table 3). The post-hoc analysis comparing the equilibrium and baseline time points showed a complete reversion of the decreased cardiac output (p = 0.25) and a partial reversion of the increased heart rate (p = 0.02). From the beginning of the hemorrhage until the beginning of the fluid resuscitation, all animals were anuric. The urine output after the beginning of fluid resuscitation was not different among the three groups: 218 (¡147) ml/h for the normal saline group, 154 (¡75) ml/h for the LR group, and 205 (¡240) ml/h for the Plasma-Lyte pH 7.4 group (p = 0.753). Oxygen delivery (DO2) and consumption (VO2) and mixed oxygen saturation (SvO2) had similar patterns among the three groups. The decrease in the DO2 and SvO2 were only partially reversed following fluid infusion in all groups (equilibrium vs. baseline; p,0.01 for both variables). The three groups were similar with respect to all laboratory values recorded after hemorrhage, including SIG. The infusion of normal saline induced a negative SBE and a highly significant increase in the plasma chloride concentration, whereas the infusion of Ringer’s lactate and Plasma-Lyte pH 7.4 induced a positive SBE and a negative plasma chloride concentration (Figure 1). The Plasma-Lyte pH 7.4 infusion did not induce the accumulation of unmeasured anions, as demonstrated by the SIG values (Table 4).

SIDa ~ ½Na z ½K z Mg2z z Ca2z { z

½Cl{ {½lactate{ ðAll concentrations in mEq=lÞ Because lactate is an independent determinant of mortality in critically ill patients following ICU admission,13,14 the SIDa was ‘‘partitioned’’ into the inorganic ion difference ([Na+] + [K +] + [Mg2+] + [Ca2+] - [Cl2]) and the plasma lactate level. Alternatively, the SID can be calculated by taking into account the role of weak acids (e.g., CO2, albumin, and phosphate) in the balance of electrical charges in the plasma water. This method results in the effective strong ion difference (SIDe). The formula for SIDe, as determined by Figge et al.,12 is as follows: SIDe ~2:46|10pH{8 |PCO2 z ½Albumin |(0:123|pH { 0:631) z ½Phosphate |(0:309|pH { 0:469)

PCO2 is measured in mmHg, albumin is measured in g/L, and phosphate is measured in mmol/L. The difference between the SIDa and SIDe should equal 0 (electrical charge neutrality), unless there are unmeasured charges to explain this ‘‘ion gap.’’ Such charges are described by the strong ion gap (SIG):11 SIG ~ SIDa { SIDe A positive SIG represents unmeasured anions (e.g., sulfate, keto acids, citrate, pyruvate, acetate, gluconate, etc.), which must be included to account for the measured pH.

DISCUSSION In this study, we demonstrate, using an experimental model of hemorrhagic shock, that Plasma-Lyte pH 7.4 and LR Solution cause an increase in the SBE, while normal saline lowers the SBE. Applying a quantitative acid-base approach, we demonstrated that Plasma-Lyte pH 7.4 did not cause an accumulation of unmeasured anions. Accordingly, the difference in the SBE was mainly attributable to fluidinduced variations in the serum chloride level. The three solutions were equally effective in attenuating the macrohemodynamic alterations. While the lactate levels tended to decrease following the administration of normal saline and Plasma-Lyte pH 7.4, but not LR solution, this difference was not statistically significant. Hyperchloremic acidosis is a well-known phenomenon.15 Furthermore, in clinical practice, the intravenous infusion of chloride-rich solutions, such as normal saline, is commonly associated with the development of hyperchloremic acidosis.16 This association has been described in several scenarios, including in normal volunteers,6 but seems to be increased in patients with inflammation.17 After development, hyperchloremic acidosis takes a few days to resolve.18 However, the consequences of hyperchloremic acidosis are not well established.19 Some studies report no impact on clinical outcome,14 while other studies found an impact.10 Small studies have suggested that large infusions

Statistical analysis The data were assessed for a normal distribution using the Kolmogorov-Smirnov goodness-of-fit model and are presented as the mean and standard deviation. The baseline mean values were compared using a one-way analysis of variance (one-way ANOVA), with a post-hoc Tukey’s test. Hemodynamic, oxygen and laboratory variables were compared by two-way ANOVA, focusing on the group vs. time interaction. The significance level was set at p,0.05 for single comparisons. The commercially available SPSS 10.0 statistical package (Chicago, Illinois, USA) was used to perform the statistical analysis.

RESULTS Table 2 shows the baseline characteristics for the animals in each experimental group. The groups were not different with respect to any of the studied characteristics, except for a slightly higher SIG in the Plasma-Lyte pH 7.4 group when compared to the LR group. The mean blood volume removed in each group was 955 (¡163) ml in the Normal Saline group, 925 (¡127) ml in the LR group and 896 (¡105) ml in the Plasma-Lyte pH 7.4 group, with no statistical difference among the three groups (p = 0.75).

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Table 2 - Baseline metabolic characteristics of the groups. Characteristics pH PaCO2 (mmHg) Bicarbonate (mEq/l) SBE (mEq/l) SIDai (mEq/l) Sodium (mEq/l) Chloride (mEq/l) Lactate (mEq/l) Albumin (g/dl) Hemoglobin (g/dl) SIG (mEq/l)

Normal Saline (n = 6)

Lactated Ringerโ s Solution (n = 6)

Plasma-Lyte pH 7.4 (n = 6)

p-value *

7.39ยก0.06 44ยก7 26.4ยก2.7 2.8ยก2.1 42.3ยก2.0 136.1ยก2.4 102ยก4 1.7ยก1.3 2.4ยก0.3 10.6ยก0.8 2.3ยก1.9

7.40ยก0.06 42ยก2 25.8ยก2.0 1.0ยก2.1 39.5ยก3.3 135.9ยก2.6 103ยก5 1.5ยก0.7 2.2ยก0.4 9.7ยก0.9 0.8ยก1.5

7.43ยก0.05 39ยก5 25.0ยก1.5 1.2ยก1.3 41.5ยก0.8 137.0ยก2.8 103ยก3 1.2ยก0.5 2.4ยก0.2 11,0ยก1.0 3.6ยก1.3

0.593 0.253 0.564 0.292 0.134 0.744 0.653 0.684 0.581 0.068 0.030

Data are expressed as the mean ยก SD. * p-values were calculated using a one-way ANOVA.

of normal saline may be detrimental to renal function.21 Despite the controversy regarding its impact on clinical outcome, hyperchloremia can have deleterious effects, such as increase in the tumor necrosis factor (TNF), interleukin (IL)-6, and IL-10 levels,22 a decrease in urine output,23 and coagulation disturbances.24,25 However, there is no definitive conclusion as to whether these deleterious effects are directly related to the hyperchloremia or if they are mediated by metabolic acidosis and/or acidemia. The use of balanced solutions has been studied in some experimental and clinical situations. In experimental sepsis, Kellum et al.26 demonstrated that animals resuscitated with Hextend (a colloid based balanced solution) survived 45% longer and had significantly less metabolic acidosis than animals treated with normal saline. Furthermore, the survival time was positively correlated with the changes in pH and negatively correlated with the increase in serum

chloride after initial resuscitation. Similar results were found in a massive hemorrhagic model. Rats resuscitated with normal saline were significantly more acidotic than rats resuscitated with an equal volume of LR and had significantly worse survival (50% vs. 100%, respectively).27 In a clinical study of elderly surgical patients,28 the use of LR solution and Hextend prevented the development of hyperchloremic metabolic acidosis and resulted in improved gastric mucosal perfusion when compared with saline-based solutions. Few studies have specifically examined the use of PlasmaLyte pH 7.4. Results similar to ours have been reported following its use in patients who underwent general surgery or kidney transplantation.21 It should be noted, however, that a quantitative approach was not used in these studies; therefore, the unmeasured anions could not be precisely estimated. Based on our results, Plasma-Lyte pH 7.4 and LR

Table 3 - Effects of crystalloid infusion on the hemodynamic and oxygenation parameters. {{

Parameter

MAP

PWP

SvO2

DO2

VO2

Group

Baseline

Hemorrhage

Equilibrium{

p-value *

Normal Saline Lactated Ringerโ s Solution Plasma-Lyte pH 7.4 Normal Saline Lactated Ringerโ s Solution Plasma-Lyte pH 7.4 Normal Saline Lactated Ringerโ s Solution Plasma-Lyte pH 7.4 Normal Saline Lactated Ringerโ s Solution Plasma-Lyte pH 7.4 Normal Saline Lactated Ringerโ s Solution Plasma-Lyte pH 7.4 Normal Saline Lactated Ringerโ s Solution Plasma-Lyte pH 7.4 Normal Saline Lactated Ringerโ s Solution Plasma-Lyte pH 7.4

99.5ยก31.0 107.5ยก24.2 101.8ยก26.6 114.4ยก29.7 143.0ยก28.8 141.7ยก27.1 5.6ยก2.3 5.2ยก1.4 5.7ยก2.0 10.0ยก3.5 12.8ยก6.6 9.6ยก3.0 70.5ยก7.2 70.3ยก5.5 74.5ยก6.3 541.6ยก164.3 470.2ยก100.8 604.6ยก233.9 142.5 + 82.4 119.3 + 12.3 120.9 + 24.9

78.8ยก27.9 87.7ยก18.1 68.0ยก34.0 207.4ยก31.6 208.8ยก25.1 204.5ยก16.4 2.7ยก0.7 3.2ยก0.6 2.5ยก0.4 6.7ยก2.5 9.5ยก3.7 7.8ยก2.2 37.9ยก6.2 46.3ยก3.4 44.9ยก12.6 253.6ยก39.2 283.1ยก34.3 244.9ยก67.0 142.7 + 26.0 136.3 + 19.4 129.7 + 47.8

80.7ยก29.0 107.5ยก31.8 85.7ยก33.2 159.4ยก29.4 168.2ยก40.8 181.3ยก15.6 4.7ยก1.7 5.1ยก0.7 3.8ยก0.7 7.3ยก2.5 10.4ยก4.3 8.8ยก1.7 53.8ยก7.5 46.2ยก17.0 57.1ยก11.7 360.8ยก136.4 328.6ยก55.4 321.8ยก73.9 145.9 + 41.2 140.8 + 23.9 163.4 + 97.9

0.125* 0.124# ,0.0001* 0.075# ,0.0001* 0.221# 0.620* 0.092# ,0.0001* 0.283# ,0.0001* 0.762# 0.471* 0.827#

Data are expressed as the mean ยก SD. { Equilibrium refers to the post-resuscitation phase, after 60 min of observation. {{ MAP, mean arterial pressure; HR, heart rate; CO, cardiac output; PWP, pulmonary wedge pressure; SvO2, mixed-venous oxygen saturation; DO2, oxygen delivery; VO2, oxygen consumption. * Two-way ANOVA for the factor Time (compared to BL, Hemorrhage, Equilibrium). # Two-way ANOVA for the factor Group (no statistical significance was found for the Time 6 Group interaction).

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Model of controlled hemorrhagic shock Noritomi DT et al.

Figure 1 - Standard base excess (SBE), lactate and chloride plasma concentrations in the three groups.

solution have the same general acid-base effect (SBE). This result is consistent with previous studies in renal transplantation patients, except when larger volumes were infused, such as in the pump prime during cardiopulmonary bypass.18 Importantly, Plasma-Lyte pH 7.4 did not cause an accumulation of unmeasured anions as evidenced by the sustained SIG values. Some concerns exist regarding the use of LR, especially due to its low osmolarity (255–273 mOsm/ L). Some authors suggest that its use should be limited and should be avoided for cases at risk of intracranial hypertension.29 In addition, the induction or maintenance of hyperlactemia by LR solution (as suggested by our results) may be significant when larger volumes are infused. Our study suggests that Plasma-Lyte pH 7.4 is a safe alternative for the treatment of hemorrhagic shock. Perhaps the most important point of our study is the use of the metabolic acidosis magnitude as a clinical monitoring tool. According to our results, resuscitation with normal saline can disrupt the relationship between base deficit and hypovolemia (an increased base deficit during resuscitation). The misdiagnosis of worsening acidosis as tissue hypoperfusion can be a factor leading to the overhydration of many patients after major surgical procedures and is correlated with the length of the intensive care unit stay.16 Fluid overload has also been associated with a prolonged time on mechanical ventilation30 and a lower survival rate31 in critically ill patients. Avoidance of normal saline, whenever base deficit needs to be monitored, can prevent some of these problems. A similar consideration can be made with respect to LR solution and the serum lactate levels; however, based on our results and the results of others, this point seems to be less important. Our study has some limitations. First, our model caused only low-level metabolic acidosis in the majority of the animals. We would probably need to prolong the hypotensive period to achieve a more significant acidosis, but this extension could lead to death before resuscitation. Because the shock-induced acidosis was not fundamental to our study, we do not believe its absence is a significant limitation to the interpretation of the results. Second, we could not a priori define the sample size needed to detect potential differences because there have been no similar

Table 4 - Effects of crystalloid infusion on the acid-base parameters. Parameter pH

Group

Normal Saline Lactated Ringer’s Solution Plasma-Lyte pH 7.4 PaCO2 (mmHg) Normal Saline Lactated Ringer’s Solution Plasma-Lyte pH 7.4 Bicarbonate Normal Saline (mEq/L) Lactated Ringer’s Solution Plasma-Lyte pH 7.4 SBE (mEq/L) Normal Saline Lactated Ringer’s Solution Plasma-Lyte pH 7.4 SIDai (mEq/L) Normal Saline Lactated Ringer’s Solution Plasma-Lyte pH 7.4 Sodium (mEq/ Normal Saline L) Lactated Ringer’s Solution Plasma-Lyte pH 7.4 Chloride (mEq/ Normal Saline L) Lactated Ringer’s Solution Plasma-Lyte pH 7.4 Lactate (mEq/L) Normal Saline Lactated Ringer’s Solution Plasma-Lyte pH 7.4 SIG (mEq/L) Normal Saline Lactated Ringer’s Solution Plasma-Lyte pH 7.4

Hemorrhage Equilibrium{ p-value* 7.31¡0.07 7.32¡0.03

7.31¡0.09 7.31¡0.13

0.506

7.36¡0.10 55¡12 50¡3

7.43¡0.08 51¡14 57¡18

0.425

44¡10 26.8¡3.2 25.0¡2.0

40¡8 24.6¡3.0 26.8¡2.3

0.045

23.7¡1.1 2.0¡3.1 0.3¡2.0

26.0¡1.1 0.96¡2.97 2.11¡2.57

0.042

-0.8¡1.2 41.9¡3.4 40.7¡3.3

1.67¡1.05 38.6¡3.3 41.7¡2.2

0.152

42.8¡2.2 135.5¡1.8 135.5¡2.7

43.6¡2.6 136.8¡1.9 134.4¡3.5

0.388

136.1¡3.4 102¡3 103¡5

134.7¡3.5 107¡3 101¡ 3

0.018

102¡2 3.0¡1.3 2.5¡1.2

98¡2 1.5¡0.5 2.7¡1.8

0.213

2.8¡0.6 -0.1¡2.7 1.0¡1.7

1.9¡0.4 2.7¡1.8 2.0¡2.4

0.511

4.7¡1.9

7.4¡2.3

Data are expressed as the mean ¡ SD. { Equilibrium refers to the post-resuscitation period, after 60 min of observation. * Two-way ANOVA for the time factor (Hemorrhage/Equilibrium) interaction. + p,0.05 for the group factor (Normal Saline, Lactated Ringer’s Solution and Plasma-Lyte pH 7.4).

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Model of controlled hemorrhagic shock Noritomi DT et al.

CLINICS 2011;66(11):1969-1974

10. Stewart PA. Modern quantitative acid-base chemistry. Can J Physiol Pharmacol. 1983;61:1444-61, doi: 10.1139/y83-207. 11. Kellum JA, Kramer DJ, Pinsky MR. Strong ion gap: a methodology for exploring unexplained anions. J Crit Care. 1995;10:51-5, doi: 10.1016/ 0883-9441(95)90016-0. 12. Figge J, Rossing TH, Fencl V. The role of serum proteins in acid-base equilibria. J Lab Clin Med. 1991;117:453-67. 13. Smith I, Kumar P, Molloy S, Rhodes A, Newman PJ, Grounds RM, et al. Base excess and lactate as prognostic indicators for patients admitted to intensive care. Intensive Care Med. 2001;27:74-83, doi: 10.1007/ s001340051352. 14. Gunnerson KJ, Saul M, He S, Kellum JA. Lactate versus non-lactate metabolic acidosis: a retrospective outcome evaluation of critically ill patients. Crit Care. 2006;10:R22, doi: 10.1186/cc3987. 15. Kellum JA. Saline-induced hyperchloremic metabolic acidosis. Crit Care Med. 2002;30:259-61, doi: 10.1097/00003246-200201000-00046. 16. Waters JH, Miller LR, Clack S, Kim JV. Cause of metabolic acidosis in prolonged surgery. Crit Care Med. 1999;27:2142-6, doi: 10.1097/ 00003246-199910000-00011. 17. Kellum JA, Bellomo R, Kramer DJ, Pinsky MR. Etiology of metabolic acidosis during saline resuscitation in endotoxemia. Shock. 1998;9:364-8, doi: 10.1097/00024382-199805000-00009. 18. Liskaser FJ, Bellomo R, Hayhoe M, Story D, Poustie S, Smith B. Role of pump prime in the etiology and pathogenesis of cardiopulmonary bypass-associated acidosis. Anesthesiology. 2000;93:1170-3, doi: 10.1097/ 00000542-200011000-00006. 19. Handy JM, Soni N. Physiological effects of hyperchloraemia and acidosis. Br J Anaesth. 2008;101:141-50, doi: 10.1093/bja/aen148. 20. Noritomi DT, Soriano FG, Kellum JA, Cappi SB, Biselli PJ, Libo´rio AB, et al. Metabolic acidosis in patients with severe sepsis and septic shock: a longitudinal quantitative study. Crit Care Med. 2009;37:2733-9, doi: 10. 1097/CCM.0b013e3181a59165. 21. Hadimioglu N, Saadawy I, Saglam T, Ertug Z, Dinckan A. The effect of different crystalloid solutions on acid-base balance and early kidney function after kidney transplantation. Anesth Analg. 2008;107:264-9, doi: 10.1213/ane.0b013e3181732d64. 22. Kellum JA, Song M, Almasri E. Hyperchloremic acidosis increases circulating inflammatory molecules in experimental sepsis. Chest. 2006;130:962-7, doi: 10.1378/chest.130.4.962. 23. Wilcox CS. Regulation of renal blood flow by plasma chloride. J Clin Invest. 1983;71:726-35, doi: 10.1172/JCI110820. 24. Martin G, Bennett-Guerrero E, Wakeling H, Mythen MG, el-Moalem H, Robertson K, et al. A prospective, randomized comparison of thromboelastographic coagulation profile in patients receiving lactated Ringer’s solution, 6% hetastarch in a balanced-saline vehicle, or 6% hetastarch in saline during major surgery. J Cardiothorac Vasc Anesth. 2002;16:441-6, doi: 10.1053/jcan.2002.125146. 25. Ferrara A, MacArthur JD, Wright HK, Modlin IM, McMillen MA. Hypothermia and acidosis worsen coagulopathy in the patient requiring massive transfusion. Am J Surg. 1990;160:515-8, doi: 10.1016/S00029610(05)81018-9. 26. Kellum JA. Fluid resuscitation and hyperchloremic acidosis in experimental sepsis: improved short-term survival and acid-base balance with Hextend compared with saline. Crit Care Med. 2002;30:300-5, doi: 10. 1097/00003246-200202000-00006. 27. Healey MA, Davis RE, Liu FC, Loomis WH, Hoyt DB. Lactated ringer’s is superior to normal saline in a model of massive hemorrhage and resuscitation. J Trauma. 1998;45:894-9, doi: 10.1097/00005373-19981100000010. 28. Wilkes NJ, Woolf R, Mutch M, Mallett SV, Peachey T, Stephens R, et al. The effects of balanced versus saline-based hetastarch and crystalloid solutions on acid-base and electrolyte status and gastric mucosal perfusion in elderly surgical patients. Anesth Analg. 2001;93:811-6, doi: 10.1097/00000539-200110000-00003. 29. Shackford SR, Zhuang J, Schmoker J. Intravenous fluid tonicity: effect on intracranial pressure, cerebral blood flow, and cerebral oxygen delivery in focal brain injury. J Neurosurg. 1992;76:91-8, doi: 10.3171/jns.1992.76. 1.0091. 30. National Heart, Lung, and Blood Institute Acute Respiratory Distress Syndrome (ARDS) Clinical Trials Network, Wiedemann HP, Wheeler AP, Bernard GR, Thompson BT, et al. Comparison of two fluid-management strategies in acute lung injury. N Engl J Med. 2006;354:2564-75, doi: 10. 1056/NEJMoa062200. 31. Vincent JL, Sakr Y, Sprung CL, Ranieri VM, Reinhart K, Gerlach H, et al. Sepsis in European intensive care units: results of the SOAP study. Crit Care Med. 2006;34:344-53, doi: 10.1097/01.CCM.0000194725.48928.3A.

studies that we could use to estimate the magnitudes of the effects. Therefore, we cannot exclude the possibility that Plasma-Lyte pH 7.4 causes a greater increase in the SBE than LR solution. However, if this difference does exist, it is probably of a clinically irrelevant magnitude. In summary, the three crystalloid solutions were able to reverse the hemorrhagic shock–induced hemodynamic disturbances. While normal saline was associated with hyperchloremic acidosis, LR and Plasma-Lyte pH 7.4 did not result in hyperchloremic acidosis and led to a more acceptable metabolic profile. Based on our results, PlasmaLyte pH 7.4 should be considered a reasonable alternative in the treatment of hemorrhagic shock. However, future clinical studies should address this specific point. This is the first study that simultaneously compared the effects of three different crystalloid solutions on acid-base and hemodynamic profiles following hemorrhagic shock. Although the three crystalloid solutions tested were equally able to attenuate the hemodynamic and tissue perfusion changes following induction of hemorrhagic shock, only normal saline induced hyperchloremia and metabolic acidosis.

AUTHOR CONTRIBUTIONS Noritomi DT, Pereira AJ, and Silva E conceived and designed the study, conducted the experiment, performed the analysis of the results, reviewed the drafts of the manuscript, and approved the final version of the manuscript. Bugano DDG and Rehder PS conducted the experiment, reviewed the drafts of the manuscript, and approved the final version of the manuscript.

REFERENCES 1. Kaplan LJ, Kellum JA. Initial pH, base deficit, lactate, anion gap, strong ion difference, and strong ion gap predict outcome from major vascular injury. Crit Care Med. 2004;32:1120-4, doi: 10.1097/01.CCM.0000125517. 28517.74. 2. Mikhail J. The trauma triad of death: hypothermia, acidosis, and coagulopathy. AACN Clin Issues. 1999;10:85-94, doi: 10.1097/00044067199902000-00008. 3. Moore FA, McKinley BA, Moore EE, Nathens AB, West M, Shapiro MB, et al. Inflammation and the Host Response to Injury, a large-scale collaborative project: patient-oriented research core—standard operating procedures for clinical care. III. Guidelines for shock resuscitation. J Trauma. 2006;61:82-9. 4. Morgan TJ. The meaning of acid-base abnormalities in the intensive care unit: part III — effects of fluid administration. Crit Care. 2005;9:204-11, doi: 10.1186/cc2946. 5. Ho AM, Karmakar MK, Contardi LH, Ng SS, Hewson JR. Excessive use of normal saline in managing traumatized patients in shock: a preventable contributor to acidosis. J Trauma. 2001;51:173-7, doi: 10. 1097/00005373-200107000-00033. 6. Reid F, Lobo DN, Williams RN, Rowlands BJ, Allison SP. (Ab)normal saline and physiological Hartmann’s solution: a randomized doubleblind crossover study. Clin Sci (Lond). 2003;104:17-24, doi: 10.1042/ CS20020202. 7. eingraber S, Rehm M, Sehmisch C, Finsterer U. Rapid saline infusion produces hyperchloremic acidosis in patients undergoing gynecologic surgery. Anesthesiology. 1999;90:1265-70, doi: 10.1097/00000542199905000-00007. 8. Dingley J, Foe¨x BA, Swart M, Findlay G, DeSouza PR, Wardrop C, et al. Blood volume determination by the carbon monoxide method using a new delivery system: accuracy in critically ill humans and precision in an animal model. Crit Care Med. 1999;27:2435-41, doi: 10.1097/00003246199911000-00019. 9. Wooten EW. Calculation of physiological acid-base parameters in multicompartment systems with application to human blood. J Appl Physiol. 2003;95:2333-44.

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DOI:10.1590/S1807-59322011001100020

REVIEW

An overview of recently published medical papers in Brazilian scientific journals Mauricio Rocha e Silva, Ariane Gomes Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo.

A brief review intended as information to the readership of Clinics on papers recently published under various medical headings in Brazilian scientific journals recently indexed or about to be indexed in ISI-THOMSON Journal Citation Reports. Journals covered in this review are Acta Ortope´dica Brasileira, Arquivos Brasileiros de Cardiologia, Jornal Brasileiro de Pneumologia, Revista Brasileira de Cirurgia Cardiovascular and Revista da Associac¸a˜o Me´dica Brasileira. KEYWORDS: Medical Headings; Brazilian Scientific Output; New Journals. Rocha-e-Silva M, Gomes A. An overview of recently published medical papers in Brazilian scientific journals. Clinics. 2011;66(11):1975-1982. Received for publication on October 3, 2011; First review completed on October 7, 2011; Accepted for publication on October 7, 2011 E-mail: mauricio.silva@pobox.com Tel.: 55 11 2661 6235

material and found that cytogenetic alterations are an important cause of pregnancy loss and their detection helps the genetic counseling to the couple. Trisomy 16 is the most often found change. Santos et al.4 evaluated Cesarean uterine scars by the grey-level histogram in women with a previous Cesarean section, performed either during labor or before labor (elective cesarean) and found a significant difference in results, namely a larger tissue change in the elective Cesarean. The perinatal period was the object of five studies. Carvalho et al.5 determined the prevalence and factors associated with episiotomy in a reference center of Pernambuco. Prevalence was 29% and associated factors were maternal diseases and absence of previous vaginal delivery. Ferreira et al.6 assessed the impact of breast-feeding on the occurrence of anthropometric deviations among preschool children of the semiarid region of Alagoas (Brazil) and possible associated factors. They find that breast-feeding for a minimum period of thirty days had a protective effect against overweight in preschool children of the semiarid region of Alagoas. Nardozza et al.7 assessed and compared accuracy of birth weight prediction using a combination of two-dimensional (abdominal circumference - AC and femur length - FL) and three-dimensional parameters (fetal arm -VolArm and thigh -VolTh volumes). And developed a formula using VolArm, VolTh, AC and FL which was more accurate for prediction of birth weight than formulae using only two-dimensional parameters. Assunc¸a˜o et al.8 evaluated the perinatal outcome of twin pregnancies delivered in a tertiary teaching hospital according to chorionicity and conclude that the rate of preterm deliveries and low birth weight is higher in monochorionic pregnancies when compared to dichorionic twins. But survival rates were similar in both groups. Nomura et al.9 studied fetal vitality assessed in pregnancies after gastroplasty with Roux-en-Y gastric bypass to check for maternal complications and perinatal results and conclude that fetal vitality was not compromised in pregnancies after the procedure. The main

INTRODUCTION This paper represents an informative insight report into general medical research articles recently published in Brazilian journals. Selected journals were recently introduced or are about to be introduced to the Journal Citation Reports of ISI THOMSON. We understand that this contributes information to the readers of Clinics. A total of 80 articles were collected from recent years in four Brazilian journals. Articles relating strictly to the cardiopulmonary system were excluded for presentation in a later insight review. Table 1 displays the various medical headings into which this review is divided.

REVIEW The following is a brief review of the eighty highlighted articles. Medical headings are sequenced according to frequency of citations in each section.

GYNECOLOGY Sixteen papers cover the general field of gynecology. Gomes and da Silva1 described prevalence of stress urinary incontinence in women over 20 years of age, who participate in the Family Health Program in the city of Dourados, State of Mato Grosso do Sul, Brazil and conclude that prevalence is similar to that of other studies. Tanuri et al.2 compared results of retropubic and transobturator sling for surgical treatment of female stress urinary incontinence which were effective at one-year follow-up. Rolnik et al.3 described chromosomal abnormalities in spontaneous abortion

1975

Recently published medical papers in Brazilian scientific journals Rocha e Silva M and Gomes A

ORTHOPEDICS

Table 1 - General distribution of articles under medical headings*. SUBJECT

REFERENCES

SUBJECT

REFERENCES

Gynecology Orthopedics Education Metabolism

16 13 8 7

4 4 4 4

Oncology Tobacco & Smoking

7 7

Infectology Psychiatry Trauma Urology & Nephrology Ethics Endocrinology Gastroenterology

Ophthalmology Geriatrics

2 4

Pediatrics

CLINICS 2011;66(11):1975-1982

Thirteen papers cover orthopedic pathology. Belangero et al.17 evaluated the performance of HIMEX, an extensible nail with hooks, in osteogenesis imperfecta deformities and claim that it significantly reduced the number of fractures, presenting lower incidence of migration and higher survival rates than those described in literature. Canto et al.18 determined the influence of vertebral posterior elements decortication in bone graft integration, and find that it accelerated the histologic process of integration. Cruz et al.19 analyzed the biomechanical characteristics of balance in elderly people, based on pressure center oscillation in five foot positions, with open and closed eyes and find that visual feedback contributed to posture control. Ferreira et al.20 implemented a ‘‘Back School’’ program for low-back chronic pain. And claim that it improved the quality of life and functional capacity of the participants. Morelli et al.21 compares results of the treatment of patients with proximal humerus fractures using two different fixation methods: the t plate vs. the locking screw plate and found no early or late differences between groups. The treatment and the angular deviations tolerated in diaphyseal forearm fractures in children evoke divergent opinions in literature. In view of this controversy, Nicolini et al.22 evaluate the preferred treatment methods for this injury, during the 39th Brazilian Congress on Orthopedics and Traumatology and found that among orthopedic surgeons less than 30 years old, the choice for less invasive treatments and greater acceptance of angular values prevailed Pires-Neto et al.23 determined whether a biopsy of the synovia of the carpal tunnel is able to identify systemic diseases that were not diagnosed by clinical examination and laboratory tests and conclude it did not contribute to early diagnosis, and added extra costs to the procedure. Silva and Fucs24 retrospectively evaluated the treatment of the spastic pes planus valgus with the technique proposed by Pisani; they also examined 12 radiographic parameters and concluded that the technique is still a good option when compared with osteotomies and arthrodesis for the treatment of flexible deformities. Souza et al.25 conducted an epidemiological study of facial fractures in children in an emergency room and alert that a policy of prevention is necessary, with special attention to traffic accidents and falls, which were the etiologic agents that caused most of the fractures. The effect of the neuregulins 1-alpha and 1-beta on the regeneration the sciatic nerves of male adult C57BL/6J mice, using the tubulization technique was examined by Souza et al.26, who found that the addition of neuregulins provided a significant increase in the number of myelinized fibers. Umeda et al.27 examined the clinical results of triple arthrodesis in Cerebral Palsy patients and determined whether there is any correspondence between the results and the AOFAS scale, and changes in radiographic angles between the preand postoperative periods. They conclude that the procedure corrects or improves these deformities but that the AOFAS scale had low correlation with the result. Two articles cover Geriatric orthopedics. Gai et al.28 checked factors related to falls of a group of independent and autonomous elderly women and found that body balance condition was the major factor related to fall occurrences. Gawryszewski29 analyzed characteristics of fall related injuries, with emphasis on falls on the same level, of those with 60 years or more of age resident in the state of Sa˜o

3 2

Total (93) exceeds number of references because some references fall under more than one Medical heading.

complication was anemia, requiring specific nutritional counseling and a broad evaluation for micronutrient deficiencies at early pregnancy. Endometriosis is covered by two studies. Clinical characteristics of endometriosis and Th1/Th2 immune response patterns were analyzed by Podgaec et al.10 and found that when specific clinical data are associated with a higher production of certain cytokines, there is a Th1 response pattern that may be related to deep infiltrating endometriosis. Bellelis et al.11 found that endometriosis is most often diagnosed in the fourth decade of life. Menopause was studied by Del Giorno et al.12 and by Wolff et al.13 The former evaluated effects of treatment with Trifolium pratense on climacteric symptoms and sexual satisfaction in postmenopausal women and found no results on menopausal symptoms or sexual satisfaction. The latter evaluated the endometrial morphology and histology of non-bleeding postmenopausal women. They claim that diagnostic hysteroscopy associated with aspiration biopsy (Pipelle) performed in the day care facility can reveal endometrial alterations that cannot be diagnosed by transvaginal ultrasound. Three articles cover gynecological oncology. Bagnoli et al.14 verified the expression levels and correlation of aromatase, matrix metalloproteinase 2 (MMP-2), matrix metalloproteinase 9 (MMP-9) and CD44 in ductal carcinoma in situ (DCIS) and infiltrating ductal carcinoma (IDC) when both are found in the same breast and conclude that aromatase through local estrogen synthesis in breast tissue plays an important role in breast carcinogenesis, mainly influencing MMP-2 and MMP-9 which are important participants in tumor cell invasion and dependence of their connection to CD44 for action. Bacchi et al.15 looked at the 21-gene expression assay as a support to the decision regarding use of chemotherapy in early breast cancer. They conclude that considering the current price for the 21-gene expression assay in Brazil, our economic analysis suggests that such testing is an overall cost-saving, from the perspective of third party payers. Carvalho et al.16 evaluated the distribution of molecular subtypes of breast tumors diagnosed in young Brazilian women as well as the frequency of loss of heterozygocity (LOH) in BRCA1 among different molecular subtypes of early-onset breast cancer. They claim that basal-like tumor was the second most frequent molecular type was only observed in women diagnosed under the age of 35 years.

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Recently published medical papers in Brazilian scientific journals Rocha e Silva M and Gomes A

Paulo and conclude that urgent programs to implement fall prevention are required.

writing centered on the reader allowed us to make recommendations to render the consent form easier to read.

EDUCATION

METABOLISM

Education contributed eight highlighted articles. Two of these regard scientific initiation. Fede et al.30 evaluated the number of studies presented during oral sessions, selected for awards and published from 2002 to 2007 at the Undergraduate Medical Congress of the ABC Foundation School of Medicine. They understand that institutional scientific production is relevant and should continue to be stimulated. Fernandes et al.31 evaluated the experience gained through LCCT implementation and understand that it has met its goals, since most current and former members recognized its role in their medical training. Colicchio and Passos32 studied traffic behavior of medical students comparing current patterns with those observed in a study previously carried out in the same institution. Risky traffic behavior was found to be very frequent among these participants directly involved in accidents with victims. Because this is a segment of the population, that is aware and has direct contact with the consequences of traffic accidents, authors would hope to find a lower incidence of such high risk behavior. Information provided by this investigation should lead to a reflection from the academic community intending to introduce educational programs to effectively change student behavior. On a similar note, Petroianu et al.33 assessed the prevalence of alcohol, tobacco and psychotropic drug consumption by students of the Medical School of the Federal University of Minas Gerais, Brazil, and found that alcohol was the drug most used and was related to other drug addictions. Drugs were most frequently used by single, male students, who live alone and do not support themselves. On a different note, Lourençaõ et al.34 highlight the relationship between health and quality of life among the resident medical staff and find that an adequate training program is needed not only to increase professional qualification and personal quality of life, but also to provide safety during patient treatment. Silva et al.35 evaluated the competence of senior medical students in diagnosing tuberculosis based on their reading of chest X-rays and found that in this sample of medical students, who had received formal training in radiology early in their medical school course, the competence in interpreting the chest X-rays of TB patients was good. Since being introduced as an elective discipline (non-obligatory) in the second semester of 2002, acupuncture has been taught in the University of Saõ Paulo School of Medicine. Amadera et al.36 conducted a study to verify interest and acceptance among under graduate medical students and to discern a positive influence contributing to improving their skills. A genuine interest to learn acupuncture was noted with almost 30% of all students volunteering for a brief training in the field while some of them choose additional training of two years due to the importance of acupuncture as part of professional skills. Results indicate that acupuncture should be included as an elective discipline for medical students. On a different note, Araujo et al.37 endeavored to determine the literacy level of Hospital das Clıńicas da Faculdade de Medicina da Universidade de Saõ Paulo outpatients, for the purpose of identifying recommendations to adapt the writing of the informed consent form to the outpatients literacy level. The results and the orientations for the text

Nine highlighted articles cover metabolism, with a special emphasis on dyslipidemia. Pereira et al.38 assessed diagnostic validity of four reference tables for waist circumference in female teenagers in order to detect lipid alterations, hyperinsulinism, elevated homeostasis model assessment, hyperleptinemia and excess of body adiposity. The claim that the cutoffs for waist circumference used by McCarthy et al. were the most appropriate for populational assessments. Freedman’s et al. proposal is appropriate for clinical use since it presents higher specificity. In addition, it can substitute high costs exams, out of the professionals’ reach such as insulin and leptin. Poeta et al.39 evaluated healthrelated quality of life of obese children and claim that such children had a poorer quality of life related to health when compared to normal children. Pannunzio et al.40 find that some stimulated whole saliva parameters (phosphate, free sialic acid, proteins and peroxiclase activity) are influenced by an increase of Body Mass Index creating conditions favorable for dental caries. Mendonc¸a et al.41 endeavored to establish prevalence of overweight and obesity in children and adolescents from the city of Maceio´, Alagoas, Brazil, and investigate the association of risk of overweight and obesity with gender, age and type of school. The prevalence of overweight and obesity were, respectively 9.3% and 4.5%. Overweight and obesity were significantly more frequent among private school students. Feliciano-Alfonso et al.42 estimated prevalence and distribution of modifiable cardiovascular risk factors and Metabolic Syndrome (MS) in young individuals admitted to the National University of Colombia in Bogota and found it to justify promotion of therapeutic lifestyle changes among this age group in Colombia. Costa et al.43 correlated anthropometric data and respiratory muscle strength of normal-weight and obese women. They found that bioelectrical impedance and obesity showed a direct correlation with RMS. WC and WHR had no influence on RMS of obese women; however, a relevance to risk factors for associated diseases was observed. These results appear to be due to an adjustment to excess body weight over the years. Trevisan et al.44 analyzed the influence of soy protein ingestion and exercises with weights, on resting energy expenditure of women in post-menopause. They found them to be determinants for increase in resting energy expenditure of women in post-menopause, and can be potentiated by ingestion of soy protein.

ONCOLOGY Seven articles are highlighted with respect to oncological findings. Cancer mortality rates began to decline in developed countries in the 1990s, but their behavior in developing countries is less well-known, which led Fonseca et al.45 to re-examine the Brazilian situation finding that, as seen in developed countries, general cancer mortality rates tended to decline in Brazilian state capitals over the period 1980-2004, a tendency largely due to a decline in stomach cancer death rates for both genders. Younes et al.46 evaluated the accuracy of preoperative clinical staging with computed tomography in predicting the correct pathological stage. And conclude that clinical staging presents limited efficacy for the correct staging of non-small cell lung cancer

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patients in a sample of the Brazilian population. Zuliani et al.47 compared survival and toxicity of three different treatments for stage IIIB cervix cancer: low-dose-rate, highdose-rate, brachytherapy and association of high-dose-rate and chemotherapy. They find that patients receiving highdose-rate had better survival. The high-dose-rate cum chemotherapy association showed no benefit when compared to high-dose-rate only. Toxicity rates showed no difference between the three groups. Leme et al.48 endeavored to establish the clinical and demographic profile and identify risk factors among patients with head and neck cancer and relate them to the polymorphism of GSTT1 and GSTM1. An association was established between GSTM1 null genotypes and head and neck cancer. Bagnoli et al.14 verified the expression levels and correlation of aromatase, matrix metalloproteinase 2 (MMP-2), matrix metalloproteinase 9 (MMP-9) and CD44 in ductal carcinoma in situ (DCIS) and infiltrating ductal carcinoma (IDC) when both are found in the same breast and conclude that aromatase through local estrogen synthesis in breast tissue plays an important role in breast carcinogenesis, mainly influencing MMP-2 and MMP9 which are important participants in tumor cell invasion and dependence of their connection to CD44 for action. Matos et al.49 present a series of 93 consecutive patients who had elective surgery for known gallbladder polyps, treated from January 2003 to December 2007 and claim that cholecystectomy is the appropriate surgical treatment for gallbladder polyps, when removal is warranted. Saad et al.50 have previously suggested that pretreatment levels of CA 19-9 correlate with overall survival among patients with advanced pancreatic cancer treated with gemcitabine; they sought to confirm the prognostic role of the pretreatment level of CA 19-9 and found further evidence in favor of the concept. Fanger et al.51 describe high prevalence rates of depression and suicidal behavior among cancer patients and suggest that simple screening instruments and specific questions during interviews are needed to detect these clinical conditions.

treatment, and correlate them with sociodemographic characteristics, clinical data, stage of readiness to change, and severity of nicotine dependence. The findings of this study highlight the role of health professionals in the approach to smoking cessation and suggest the importance of interventions that are more targeted, in view of the differences between men and women. Castro et al.57 compared smokers and never smokers in terms of quality of life; BMI; hospitalizations; functionality; family history of mental disorder; tobacco-related diseases; depression; and psychoactive substance use. This study suggests that, for smoking cessation programs, subgroups of smokers with specific characteristics (early age at smoking onset, tobaccorelated diseases, depressive disorders and Use of psychoactive substances) should be identified. To determine the prevalence of respiratory symptoms and smoking, as well as pulmonary function parameters among charcoal production workers in three cities in southern Brazil, Souza et al.58 conducted a study which found that respiratory symptoms and airflow reduction were more common in the smoking workers.

GASTROENTEROLOGY We highlight six articles on the gastrointestinal system. Nomura et al.9 studied fetal vitality assessed in pregnancies after gastroplasty with Roux-en-Y gastric bypass to check for maternal complications and perinatal results and conclude that fetal vitality was not compromised in pregnancies after the procedure. The main complication was anemia, requiring specific nutritional counseling and a broad evaluation for micronutrient deficiencies at early pregnancy. Portela et al.59 investigated whether liver transplants were cost effective at a University Hospital in the state of CearaÂ´, Brazil. Results were similar to those found by other Brazilian transplant centers showing that it is an economically viable procedure based upon the financial transfer from the Brazilian National Health Service (SUS). Macedo et al.60 determined the occurrence of hepatopulmonary syndrome in cirrhosis patients who are candidates for liver transplantation and found a high prevalence of the syndrome, which however did not correlate with any of the variables analyzed. Matos et al.49 present a series of 93 consecutive patients who had elective surgery for known gallbladder polyps, treated from January 2003 to December 2007 and claim that cholecystectomy is the appropriate surgical treatment for gallbladder polyps, when removal is warranted. The etiology of acute pancreatitis cannot be found by clinical, biological and imaging investigations in 30% of these cases. This led Ardengh et al.61 to evaluate results from endoscopic ultrasonography for diagnosis of gallbladder microlithiasis in patients with unexplained (idiopathic) acute pancreatitis and found it to be a very reliable procedure to diagnose gallbladder microlithiasis and should be used for the management of patients with unexplained acute pancreatitis. Saad et al.50 have previously suggested that pretreatment levels of CA 19-9 correlate with overall survival among patients with advanced pancreatic cancer treated with gemcitabine; they sought to confirm the prognostic role of the pretreatment level of CA 19-9 and found further evidence in favor of the concept. Coelho et al.62 compared data of patients submitted to appendectomy for acute appendicitis at a public and at a private hospital. They found important differences in the

TOBACCO and SMOKING Seven papers on problems related to smoking are highlighted. Neto et al.52 studied factors associated with smoking experimentation and initiation among adolescent students (11-14 years of age). And found that alcohol consumption and the influence of peers and relatives who smoke showed a strong association with smoking experimentation. Paternal smoking and media influence were found to increase the chances of adolescent smoking. Smoking prevalence was the object of Malta et al.53 who evaluated the prevalence of smoking in the adult population of Brazil. Estimates indicate a reduction in the prevalence of smoking, which was, however, still greater among males than among females. Souza et al.54 developed a new scale aimed at evaluating smoking motivation by incorporating questions and domains from the 68-item Wisconsin Inventory of Smoking Dependence Motives into the Modified Reasons for Smoking Scale. They claim that the new scale provides an acceptable framework of motivational factors for smoking, with satisfactory psychometric properties and reliability. At the geriatric end of the scale, Carvalho et al.55 evaluated the prevalence of smoking among elders and find that such patients in Brasilia exhibit a high prevalence of smoking and a low motivation for smoking cessation. On a similar note, Russo and Azevedo56 list reasons smokers give for seeking smoking cessation

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diagnosis and outcomes between the two types of institutions.

TRAUMA Trauma is highlighted in four original articles. Parreira et al.69 assessed characteristics of trauma patients who sustained falls from their own height, focusing on presence of severe injuries, diagnosis and treatment and conclude that the importance of the trauma mechanism in victims of falls from own height should be emphasized due to a considerable possibility of occult severe injuries, mainly in the cephalic segment. On the same note, Parreira et al.70 assessed characteristics of trauma in the elderly by comparison to a group of younger trauma patients. And found that the elderly group was characterized by a higher frequency of falls from their own height concomitant diseases and severe injuries in the head, mainly subdural hematomas, cerebral contusions and subarachnoid hemorrhage. Nunes and Nascimento71 identified clusters of municipalities with high rates of hospitalization due to motorcycle accidents in the ‘‘Vale do Paraı´ba’’ region of the state of Sa˜o Paulo, thereby locating the residence of those hospitalized for motorcycle accidents, highlighting municipalities where preventive measures should be enforced. Colicchio and Passos32 studied traffic behavior of medical students comparing current patterns with those observed in a study previously carried out in the same institution. Risky traffic behavior was found to be very frequent among these participants directly involved in accidents with victims. Because this is a segment of the population, that is aware and has direct contact with the consequences of traffic accidents, authors would hope to find a lower incidence of such high risk behavior. Information provided by this investigation should lead to a reflection from the academic community intending to introduce educational programs to effectively change student behavior.

GERIATRICS Four papers on geriatrics are reviewed. Salgado et al.63 investigated the relationship between serum albumin levels, drugs used, length of hospitalization and number of diseases within the prognosis of hospitalized elderly and describe a mathematical equation obtained by this study which shows that albumin and days of hospitalization were inversely correlated with death and directly correlated with the number of drugs bound to albumin. A previously discussed study by Carvalho et al.55 covers the prevalence of smoking among elderly patients Also previous discussed are two studies, one by Gai et al.28 on factors related to falls of a group of independent and autonomous elderly women, one by Gawryszewski29 on characteristics of fall related injuries.

INFECTOLOGY Infectology is the highlighted subject of four studies, with prominence to human immunodeficiency virus infection, which has increased considerably among middle-aged women as found by Valadares et al.64 On a different note, and because sexually transmitted diseases are common reasons for seeking medical assistance, Passos et al.65 analyzed the temporal distribution of first appointments in a sexually transmitted disease clinic from January 1993 to December 2005 to verify whether there is a seasonal increase in sexually transmitted disease after Carnival. No such influence was detected. An analysis of the professional profile of physicians who prescribe antiretroviral drugs to HIV infected persons in the State of Sa˜o Paulo led Scheffer et al.66 to conclude that the absolute majority of HIV patients receive their prescriptions from duly trained and experienced physicians. Nevertheless, the large number of nonqualified physicians together with the reduced number of physicians in HIV high incidence regions make up the major challenge for comprehensive and adequate care of HIV patients. A study by Teixeira and Taquette67 identified factors associated with unprotected sexual activity in females under the age of 15 years and found that multiple types of violence suffered by teenagers, including structural, intrafamily, and sexual violence, increase their vulnerability to early, unprotected sexual activity and to STIs and unexpected pregnancy.

UROLOGY and NEPHROLOGY Urology and nephrology contribute four studies to this series. In two previously discussed articles Gomes and da Silva1 described prevalence of stress urinary incontinence in women over 20 years of age, who participate in the Family Health Program in the city of Dourados, State of Mato Grosso do Sul, Brazil and conclude that prevalence is similar to that of other studies. Tanuri et al.2 compared results of retropubic and transobturator sling for surgical treatment of female stress urinary incontinence which were effective at one-year follow-up. Oliveira et al.72 evaluated risk factors related to occurrence of female urinary incontinence and found that independent risk factors include age, vaginal delivery, forceps delivery, and weight of largest infant and as a protective factor, cesarean section delivery. Carvalho et al.73 assessed frequency and type of cardiovascular and renal/collecting system abnormalities seen in a sample of patients with Turner Syndrome and found that abnormalities found in this study was similar to those of previous studies, but most were found in routine exams after Turner Syndrome diagnosis. Thus, early detection of associated anomalies depends on early detection of TS.

PSYCHIATRY Four articles fall under the heading of Psychiatry. As previously noted, Teixeira and Taquette67 identified factors associated with unprotected sexual activity in females under the age of 15 and their consequences. On a different note, Junges and Bagatini68 present data on how chronically ill persons reframe life through their discursive practices and show that self-satisfaction relies on finding the thread to bring together the elements of existence based on a new model permitting a new identity. As shown above, Fanger et al.51 determine prevalence rates of depression and suicidal behavior among cancer inpatients and factors associated with these conditions. Also discussed is a paper by Ferreira et al.6 who assessed the impact of breast-feeding on the occurrence of anthropometric deviations among preschool children of the semiarid region of Alagoas (Brazil).

ETHICS Ethics is the subject of three articles. Fortes74 writes about the meaning assigned to ethical thought that should guide Brazilian bioethicists regarding the equity of a Health System and found a pluralism of values that jeopardizes determination of moral equity in the health system. Extensive discussion among all interested parties is necessary to reach

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a possible consensus. Duque et al.75 present their practical experience in obtaining informed consent for a study performed at the ‘‘Instituto Nacional de Caˆncer’’ involving research on stored biologic samples from patients operated for colon cancer from 2000 to 2004. And conclude that obtaining consent by postal and phone communication for retrospective genetic research with stored tissue samples is feasible. Most responded to contact and consented to participate, but there were costs and risks that cannot be neglected. As noted above, Teixeira and Taquette 67 identified factors associated with unprotected sexual activity in females under the age of 15 and refer to the multiple types of violence suffered by teenagers, including structural, intrafamily, and sexual violence, increase their vulnerability to early, unprotected sexual activity and to STIs and unexpected pregnancy. The synergistic effects of poverty, low educational achievement, and low self-esteem reduces the odds that adolescents will build the tools required for self-protection and exposes them to further victimization outside the family environment.

the majority of pediatricians in the city of Sa˜o Paulo were not familiar with the Guidelines for Prevention of Atherosclerosis in Childhood and Adolescence. Individual strategies were rarely found. Medical education and adequate disclosure of the Guidelines are necessary for active control of populational risk factors.

REFERENCES 1. Gomes GV, da Silva GD. Stress urinary incontinence in women belonging to the Family Health Program of Dourados/MS. Rev Assoc Med Bras. 2010;56(6):649-54, doi: 10.1590/S0104-42302010000600011. 2. Tanuri ALS, Feldner PC, Jarmy-Di Bella ZIK, Castro RA, Sartori MGF, Girao M. Retropubic and transobturator sling in treatment of stress urinary incontinence. Rev Assoc Med Bras. 2010;56(3):348-54, doi: 10. 1590/S0104-42302010000300022. 3. Rolnik DL, de Carvalho MHB, Catelani A, Pinto A, Lira JBG, Kusagari NK, et al. Cytogenetic analysis of material from spontaneous abortion. Rev Assoc Med Bras. 2010;56(6):681-3, doi: 10.1590/S0104-423020100006 00017. 4. Santos OD, Nardozza LMM, Araujo E, Rolo LC, Camano L, Moron AF. Cesarean uterine scar evaluation by the grey-level histogram. Rev Assoc Med Bras. 2010;56(1):99-102, doi: 10.1590/S0104-42302010000100023. 5. de Carvalho CCM, Souza ASR, Moraes OB. Prevalence and factors associated with practice of episiotomy at a maternity school in Recife, Pernambuco, Brazil. Rev Assoc Med Bras. 2010;56(3):333-9, doi: 10.1590/ S0104-42302010000300020. 6. Ferreira HD, Vieira EDF, Cabral CR, de Queiroz MDR. Breastfeeding for at least thirty days is a protective factor against overweight in preschool children from the semiarid region of Alagoas. Rev Assoc Med Bras. 2010;56(1):74-80, doi: 10.1590/S0104-42302010000100020. 7. Nardozza LMM, Araujo E, Vieira MF, Rolo LC, Moron AF. Estimate of birth weight using two- and three-dimensional ultrasonography. Rev Assoc Med Bras. 2010;56(2):204-8, doi: 10.1590/S0104-42302010000200020. 8. de Assuncao RA, Liao AW, Brizot MD, Krebs VLJ, Zugaib M. Perinatal outcome of twin pregnancies delivered in a teaching hospital. Rev Assoc Med Bras. 2010;56(4):447-51, doi: 10.1590/S0104-42302010000400018. 9. Nomura RMY, Dias MCG, Igai AMK, Liao AW, Miyadahira S, Zugaib M. Assessment of fetal vitality and perinatal results in pregnancies after gastroplasty with roux-en-y gastric bypass. Rev Assoc Med Bras. 2010;56(6):670-4, doi: 10.1590/S0104-42302010000600015. 10. Podgaec S, Dias JA, Chapron C, de Oliveira RM, Baracat EC, Abrao MS. TH1 and TH2 immune responses related to pelvic endometriosis. Rev Assoc Med Bras. 2010;56(1):92-8, doi: 10.1590/S0104-42302010000100022. 11. Bellelis P, Dias JA, Podgaec S, Gonzales M, Baracat EC, Abrao MS. Epidemiological and clinical aspects of pelvic endometriosis - a case series. Rev Assoc Med Bras. 2010;56(4):467-71, doi: 10.1590/S0104-42302 010000400022. 12. Del Giorno C, da Fonseca AM, Bagnoli VR, de Assis JS, Soares JM, Baracat EC. Effects of trifolium pratense on the climacteric and sexual symptoms in postmenopause. Rev Assoc Med Bras. 2010;56(5):558-62, doi: 10.1590/S0104-42302010000500017. 13. Wolff LPG, do Monte AA, Atti ACD, Monteiro IMU. Assessment of endometrial morphology and histology in postmenopausal women. Rev Assoc Med Bras. 2010;56(6):711-4, doi: 10.1590/S0104-42302010000600023. 14. Bagnoli F, de Oliveira VM, da Silva M, Taromaru GCM, Rinaldi JF, Aoki T. The interaction between aromatase, metalloproteinase 2,9 and CD44 in breast cancer. Rev Assoc Med Bras. 2010;56(4):472-7, doi: 10.1590/S010442302010000400023. 15. Bacchi CE, Prisco F, Carvalho FM, Ojopi EB, Saad ED. Potential economic impact of the 21-gene expression assay on the treatment of breast cancer in Brazil. Rev Assoc Med Bras. 2010;56(2):186-91, doi: 10.1590/S010442302010000200017. 16. de Carvalho LV, Pereira EM, Frappart L, Boniol M, Bernardo WM, Tarricone V, et al. Molecular characterization of breast cancer in young Brazilian women. Rev Assoc Med Bras. 2010;56(3):278-87. 17. Belangero WD, Livani B, Belangero VMS. Survival rates of the HIMEX extensible nail in the treatment of children with osteogenesis imperfecta. Acta ortop bras. 2010;18(6):343-8, doi: 10.1590/S1413-78522010000600008. 18. Canto FRT, Garcia SB, Issa JPM, Marin A, Del-Bel E, Defino HLA. Influence of vertebral decortication on tissue neoformation at bone graft interface. Acta ortop bras. 2010;18(4):187-90, doi: 10.1590/S1413-785220 10000400002. 19. Cruz A, Oliveira EM, Melo SIL. Biomechanical analysis of equilibrium in the elderly. Acta ortop bras. 2010;18(2):96-9, doi: 10.1590/S1413-785220 10000200007. 20. Ferreira MS, Navega MT. Effects of a guidance program to adults with low back pain. Acta ortop bras. 2010;18(3):127-31. 21. Morelli RSeS, Travizanuto REdS. Proximal humerus fractures: comparative study of two different fixation methods. Acta ortop bras. 2010;18(2):79-84, doi: 10.1590/S1413-78522010000200004.

ENDOCRINOLOGY Diabetes is the subject of two articles. Rodrigues et al.76 evaluated the prevalence of chronic vascular complications and associated factors in patients with type 1 diabetes mellitus and found a high prevalence of microvascular complications. The relation between diabetes and physical activity was studied by Pitanga et al.77 in terms of how much total physical activity (intensity and duration) is required and its different domains (work, commuting, household, and leisure time) as predictors of absence of diabetes in the black ethnicity population. They conclude that physical activity accumulated in different domains should be suggested in amounts suitable for a black ethnicity population in order to contribute to diabetes prevention.

OPHTHALMOLOGY Two articles on ophthalmology are reviewed. Rosa et al.78 describe the clinical characteristics of a sample of patients with cat eye syndrome who were seen at their service. The phenotype observed in cat eye syndrome is highly variable and may be superimposed on the phenotype of the ocularauricular-vertebral spectrum. Although these patients usually have good prognosis, including from a neurological point of view, they believe that all patients with the syndrome should be assessed very early on for the presence of cardiac, biliary and anorectal malformations, which may avoid possible complications in the future, including patient deaths. Toledo et al.79 examined the early detection of visual impairment in schoolchildren and investigated its association with academic performance. Results suggest that there is an association between low visual accuracy and poor academic performance in the sample assessed.

PEDIATRICS Two pediatric papers are highlighted. The previously reported study by Nardozza et al.7 assessed and compared accuracy of birth weight prediction using a combination of two-dimensional (abdominal circumference - AC and femur length - FL) and three-dimensional parameters (fetal arm VolArm and thigh -VolTh volumes). They developed a formula using VolArm, VolTh, AC and FL which was more accurate for prediction of birth weight than formulae using only two-dimensional parameters. Grosso et al.80 report that

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22. Nicolini AP, Jannarelli B, Gonçalves MHL, Blumetti FC, Dobashi ET, Ishida A. Treatment of forearm fractures in children and adolescents. Acta ortop bras. 2010;18(1):35-8, doi: 10.1590/S1413-78522010000100007. 23. Pires-Neto PJ, Pires RES, Conde´ LG, Rezende FM, Souza AFJd. Pathological study of the synovial tissue of patients who underwent open carpal tunnel release. Acta ortop bras. 2010;18(4):200-3, doi: 10. 1590/S1413-78522010000400005. ˆ d, Fucs PMMdB. Surgical treatment of planovalgus foot in 24. Silva LAA cerebral palsy by Pisani’s arthroereisis. Acta ortop bras. 2010;18(3):162-5. 25. Souza DFMd, Santili C, Freitas RR, Akkari M, Silveira FMJPS. Epidemiology of children’s facial fractures in the emergency room of a tropical metropolis. Acta ortop bras. 2010;18(6):335-8, doi: 10.1590/ S1413-78522010000600006. 26. Souza FId, Zumiotti AV, Silva CF. Neuregulins 1-alpha and 1-beta on the regeneration the peripheral nerves. Acta ortop bras. 2010;(5):250-4, doi: 10.1590/S1413-78522010000500003. 27. Umeda K, Fucs PMdMB, Yamada HH, Assumpçaõ RMCd, Svartman C. Triple arthrodesis in cerebral palsy. Acta ortop bras. 2010;18(5):261-70, doi: 10.1590/S1413-78522010000500005. 28. Gai J, Gomes L, Nobrega OD, Rodrigues MP. Factors related to falls of elderly women residents in a community. Rev Assoc Med Bras. 2010;56(3):327-32, doi: 10.1590/S0104-42302010000300019. 29. Gawryszewski VP. The importance of falls on the same level among the elderly in Saõ Paulo state. Rev Assoc Med Bras. 2010;56(2):162-7, doi: 10. 1590/S0104-42302010000200013. 30. Fede AB, Miranda MD, Lera AT, Ueda A, Antonangelo DV, Schaffhausser HD, et al. Experience with the ABC Foundation School of Medicine undergraduate meeting. Rev Assoc Med Bras. 2010;56(3):313-7, doi: 10.1590/S0104-42302010000300016. 31. Fernandes FG, Hortencio LDS, Unterpertinger FD, Waisberg DR, PegoFernandes PM, Jatene FB. Cardiothoracic Surgery League from University of Saõ Paulo Medical School: twelve years in medical education experience. Rev Bras Cir Cardiovasc. 2010;25(4):552-8, doi: 10.1590/S0102-76382010000400020. 32. Colicchio D, Passos ADC. Traffic behavior of medical students. Rev Assoc Med Bras. 2010;56(5):535-40, doi: 10.1590/S0104-42302010000500013. 33. Petroianu A, dos Reis DCF, Cunha BDS, de Souza DM. Prevalence of alcohol, tobacco and psychotropic drug consumption by medical students of the ‘‘Universidade Federal de Minas Gerais’’. Rev Assoc Med Bras. 2010;56(5):568-71, doi: 10.1590/S0104-42302010000500019. 34. Lourencao LG, Moscardini AC, Soler Z. Health and quality of life of medical residents. Rev Assoc Med Bras. 2010;56(1):81-91, doi: 10.1590/ S0104-42302010000100021. 35. da Silva VMC, Luiz RR, Barreto MM, Rodrigues RS, Marchiori E. Competence of senior medical students in diagnosing tuberculosis based on chest X-rays. J Bras Pneumol. 2010;36(2):190-6, doi: 10.1590/S180637132010000200006. 36. Amadera JED, Pai HJ, Hsing WT, Teixeira MZ, Martins MD, Lin CA. The teaching of acupuncture in the University of Saõ Paulo School of Medicine, Brazil. Rev Assoc Med Bras. 2010;56(4):458-61, doi: 10.1590/ S0104-42302010000400020. 37. de Araujo DVP, Zoboli E, Massad E. How to make consent forms easier to read? Rev Assoc Med Bras. 2010;56(2):151-6, doi: 10.1590/S010442302010000200011. 38. Pereira PB, de Arruda IKG, Cavalcanti A, Diniz AD. Lipid Profile of Schoolchildren from Recife, PE. Arq Bras Cardiol. 2010;95(5):606-13, doi: 10.1590/S0066-782X2010005000136. 39. Poeta LS, Duarte MDD, Giuliano IDB. Health-related quality of life of obese children. Rev Assoc Med Bras. 2010;56(2):168-72, doi: 10.1590/ S0104-42302010000200014. 40. Pannunzio E, Amancio OMS, Vitalle MSD, de Souza DN, Mendes FM, Nicolau J. Analysis of the stimulated whole saliva in overweight and obese school children. Rev Assoc Med Bras. 2010;56(1):32-6, doi: 10.1590/ S0104-42302010000100012. 41. Mendonca MRT, da Silva MAM, Rivera IR, Moura AA. Prevalence of overweight and obesity in children and adolescents from the city of Maceio´ (AL). Rev Assoc Med Bras. 2010;56(2):192-6, doi: 10.1590/S010442302010000200018. 42. Feliciano-Alfonso JE, Mendivil CO, Ariza IDS, Perez CE. Cardiovascular risk factors and metabolic syndrome in a population of young students from the National University of Colombia. Rev Assoc Med Bras. 2010;56(3):293-8, doi: 10.1590/S0104-42302010000300012. 43. Costa TR, Lima TP, Gontijo PL, de Carvalho HA, Cardoso FPD, Faria OP, et al. Correlation of respiratory muscle strength with anthropometric variables of normal-weight and obese women. Rev Assoc Med Bras. 2010;56(4):403-8, doi: 10.1590/S0104-42302010000400011. 44. Trevisan MC, de Souza JMP, Marucci MDN. Influence of soy protein and exercises with weights on the resting energy expenditure of women in post-menopause. Rev Assoc Med Bras. 2010;56(5):572-8, doi: 10.1590/ S0104-42302010000500020. 45. Fonseca LAM, Eluf-Neto J, Wunsch V. Trends of cancer mortality in Brazilian state capitals, 1980-2004. Rev Assoc Med Bras. 2010;56(3):30912, doi: 10.1590/S0104-42302010000300015.

46. Younes RN, Schutz FAB, Gross JL. Preoperative and pathological staging of NSCLC: retrospective analysis of 291 cases. Rev Assoc Med Bras. 2010;56(2):237-41, doi: 10.1590/S0104-42302010000200026. 47. Zuliani AC, Cunha MD, Esteves SCB, Teixeira JC. Brachytherapy for stage IIIB squamous cell carcinoma of the uterine cervix: survival and toxicity. Rev Assoc Med Bras. 2010;56(1):37-40, doi: 10.1590/S0104-4230 2010000100013. 48. Leme CVD, Raposo LS, Ruiz MT, Biselli JM, Galbiatti ALS, Maniglia JV, et al. GSTM1 and GSTT1 genes analysis in head and neck cancer patients. Rev Assoc Med Bras. 2010;56(3):299-303, doi: 10.1590/S0104-423020 10000300013. 49. de Matos ASB, Baptista HN, Pinheiro C, Martinho F. Management of Gallbladder Polyps. Rev Assoc Med Bras. 2010;56(3):318-21, doi: 10. 1590/S0104-42302010000300017. 50. Saad ED, Reis PT, Borghesi G, Machado MC, Simon SD, Tabacof J, et al. Further evidence of the prognostic role of pretreatment levels of CA 19-9 in advanced pancreatic cancer. Rev Assoc Med Bras. 2010;56(1):22-6, doi: 10.1590/S0104-42302010000100010. 51. Fanger PC, de Azevedo RCS, Mauro MLF, Lima DD, Gaspar KC, da Silva VF, et al. Depression and suicidal behavior of cancer inpatients: prevalence and associated factors. Rev Assoc Med Bras. 2010;56(2):1738, doi: 10.1590/S0104-42302010000200015. 52. Neto ADM, Andrade TM, Napoli C, Abdon L, Garcia MR, Bastos FI. Determinants of smoking experimentation and initiation among adolescent students in the city of Salvador, Brazil. J Bras Pneumol. 2010;36(6):67482, doi: 10.1590/S1806-37132010000600003. 53. Malta DC, Moura EC, Silva SA, de Oliveira PPV, Silva V. Prevalence of smoking among adults residing in the Federal District of Brasilia and in the state capitals of Brazil, 2008. J Bras Pneumol. 2010;36(1):75-83, doi: 10. 1590/S1806-37132010000100013. 54. de Souza EST, Crippa JAD, Pasian SR, Martinez JAB. University of Sa˜o Paulo Reasons for Smoking Scale: a new tool for the evaluation of smoking motivation. J Bras Pneumol. 2010;36(6):768-78. 55. de Carvalho AA, Gomes L, Loureiro AML. Smoking in elderly patients admitted to long-term care facilities. J Bras Pneumol. 2010;36(3):339-46, doi: 10.1590/S1806-37132010000300012. 56. Russo AC, de Azevedo RCS. Factors that motivate smokers to seek outpatient smoking cessation treatment at a university general hospital. J Bras Pneumol. 2010;36(5):603-11, doi: 10.1590/S1806-37132010000500012. 57. de Castro MRP, Matsuo T, Nunes SOV. Clinical characteristics and quality of life of smokers at a referral center for smoking cessation. J Bras Pneumol. 2010;36(1):67-74, doi: 10.1590/S1806-37132010000100012. 58. de Souza RM, de Andrade FM, Moura ABD, Teixeira PJZ. Respiratory symptoms in charcoal production workers in the cities of Lindolfo Collor, Ivoti and Presidente Lucena, Brazil. J Bras Pneumol. 2010;36(2):210-7. 59. Portela MP, Neri EDR, Fonteles MMF, Garcia JHP, Fernandes MEP. Cost of liver transplantation at a university hospital of Brazil. Rev Assoc Med Bras. 2010;56(3):322-6, doi: 10.1590/S0104-42302010000300018. 60. Macedo LG, Lopes EPD, de Albuquerque M, Markman-Filho B, Veras F, de Araujo A, et al. Occurrence of hepatopulmonary syndrome in patients with cirrhosis who are candidates for liver transplantation. J Bras Pneumol. 2010;36(4):432-40, doi: 10.1590/S1806-37132010000400007. 61. Ardengh JC, Malheiros CA, Rahal F, Pereira V, Ganc AJ. Microlithiasis of the gallbladder: role of endoscopic ultrasonography in patients with idiopathic acute pancreatitis. Rev Assoc Med Bras. 2010;56(1):27-31, doi: 10.1590/S0104-42302010000100011. 62. Coelho JCU, Fernandes FM, Cortiano LGG, Leme GMD, Sadowski JA, Artner CL. Appendectomy. comparative study between a public and a private hospital. Rev Assoc Med Bras. 2010;56(5):522-7, doi: 10.1590/ S0104-42302010000500011. 63. Salgado FXC, Vianna LG, Giavoni A, de Melo GF, Karnikowski MGD. Albumin and drug therapy in the prognosis of hospitalized elderly. Rev Assoc Med Bras. 2010;56(2):145-50, doi: 10.1590/S0104-42302010000200010. 64. Valadares ALR, Pinto-Neto AM, Abdo C, de Melo VH. HIV in middleaged women: associated factors. Rev Assoc Med Bras. 2010;56(1):112-5, doi: 10.1590/S0104-42302010000100025. 65. Passos MRL, Arze WNC, Mauricio C, Barreto NA, Varella RD, Cavalcanti SMB, et al. IS There an increase in STDs during carnival? time series of diagnoses in a STD clinic. Rev Assoc Med Bras. 2010;56(4):420-7, doi: 10.1590/S0104-42302010000400014. 66. Scheffer MC, Escuder MM, Grangeiro A, de Castilho EA. Professional background and experience of antiretroviral prescribing physicians in the state of Sa˜o Paulo. Rev Assoc Med Bras. 2010;56(6):691-6, doi: 10. 1590/S0104-42302010000600020. 67. Teixeira SAM, Taquette SR. Violence and unsafe sexual practices in adolescents under 15 years of age. Rev Assoc Med Bras. 2010;56(4):440-6, doi: 10.1590/S0104-42302010000400017. 68. Junges JR, Bagatini T. Making sense of the narratives of chronically ill persons. Rev Assoc Med Bras. 2010;56(2):179-85, doi: 10.1590/S010442302010000200016. 69. Parreira JG, Vianna AMF, Cardoso GS, Karakhanian WZ, Calil D, Perlingeiro JAG, et al. Severe injuries from falls on the same level. Rev Assoc Med Bras. 2010;56(6):660-4, doi: 10.1590/S0104-42302010000600 013.

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Recently published medical papers in Brazilian scientific journals Rocha e Silva M and Gomes A

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70. Parreira JG, Solda SC, Perlingeiro JAG, Padovese CC, Karakhanian WZ, Assef JC. Comparative analysis of trauma characteristics between elderly and younger trauma patients. Rev Assoc Med Bras. 2010;56(5):541-6, doi: 10.1590/S0104-42302010000500014. 71. Nunes MN, Nascimento LFC. Hospitalization due to motorcycle accidents in the paraiba valley using spatial analysis. Rev Assoc Med Bras. 2010;56(6):684-7, doi: 10.1590/S0104-42302010000600018. 72. Oliveira E, Zuliani LMM, Ishicava J, Silva SV, Albuquerque SSR, de Souza AMB, et al. Evaluation of factors related to the occurrence of female urinary incontinence. Rev Assoc Med Bras. 2010;56(6):688-90, doi: 10.1590/S0104-42302010000600019. 73. Carvalho AB, Guerra G, Baptista MTM, de Faria APM, Marini S, Guerra ATM. Cardiovascular and renal anomalies in turner syndrome. Rev Assoc Med Bras. 2010;56(6):655-9, doi: 10.1590/S0104-42302010000600012. 74. Fortes PAD. Equity in the health system according to brazilian bioethicists. Rev Assoc Med Bras. 2010;56(1):47-50, doi: 10.1590/S010442302010000100015. 75. Duque CG, Ramalho DMD, Casali-da-Rocha JC. Consent procedures and research on stored biological samples. Rev Assoc Med Bras. 2010;56(5):5637, doi: 10.1590/S0104-42302010000500018.

76. Rodrigues TC, Pecis M, Canani LH, Schreiner L, Kramer CK, Biavatti K, et al. Characterization of patients with type 1 diabetes mellitus in southern Brazil: Chronic complications and associated factors. Rev Assoc Med Bras. 2010;56(1):67-73, doi: 10.1590/S0104-4230201000010 0019. 77. Pitanga FJG, Lessa I, Barbosa PJB, Barbosa SJO, Costa MC, Lopes AD. Physical activity in the prevention of diabetes in black ethnicity: How much is required? Rev Assoc Med Bras. 2010;56(6):697-704, doi: 10.1590/ S0104-42302010000600021. 78. Rosa RFM, Mombach R, Zen PRG, Graziadio C, Paskulin GA. Clinical characteristics of a sample of patients with cat eye syndrome. Rev Assoc Med Bras. 2010;56(4):462-5, doi: 10.1590/S0104-42302010000400021. 79. Toledo CC, Paiva APG, Camilo GB, Maior MRS, Leite ICG, Guerra MR. Early detection of visual impairment and its relation to academic performance. Rev Assoc Med Bras. 2010;56(4):415-9, doi: 10.1590/S010442302010000400013. 80. Grosso AF, Santos RD, da Luz PL. Lack of knowledge of guidelines for prevention of atherosclerosis in childhood and adolescence by pediatricians in the city of SaË&#x153;o Paulo. Rev Assoc Med Bras. 2010;56(2):157-61, doi: 10.1590/S0104-42302010000200012.

1982

CLINICS 2011;66(11):1983-1986

DOI:10.1590/S1807-59322011001100021

CASE REPORT

Hospital das Clıńicas da Faculdade de Medicina da Universidade de Saõ Paulo, Instituto do Coraçaõ (Incor), Pulmonology Division, Saõ Paulo/SP, Brazil. Hokkaido University Hospital, Department of Surgical Pathology, Hokkaido, Hokkaido/Japan.

Email: meiradias@yahoo.com.br Tel.: 55 11 2661-7577

consolidations with a ground-glass halo (Figure 1 and 2). The symptoms and signs improved after treatment with prednisone but reoccurred after tapering off of the corticosteroid treatment. Several tests were performed to look for evidence of autoimmune diseases, but these tests yielded negative results. The association of relatively rare conditions, such as an indolent sclerosing cholangitis, presumed autoimmune pancreatitis with endocrine insufficiency, and inflammatory pseudotumors of the lung, led us to the diagnosis of IRSSD. The serum IgG4 concentration was 936 UI (reference value: ,140 UI). An anatomopathological revision of the previous pulmonary nodule biopsy revealed a dense fibrosis with plasmacytic infiltration and focal lymphoid aggregation in the nodular lesion. Small numbers of eosinophils were detected (,5 cells/high-power field [HPF] in most areas). Some vessels were partially or completely obstructed by inflammatory cells. Most of the infiltrating plasma cells were positive for IgG4, and the IgG4/IgG ratio was greater than 90% (Figure 3). Light chain restriction (normal range of kappa:lambda) and Epstein-Bar virus encoded RNA, which was evaluated using in situ hybridization, were not detected. There were no signs of malignancy. The patient received prednisone (20 mg/daily) with recrudescence of the tomographic findings. We prescribed a low dose of prednisone (10 mg/daily) to avoid recrudescence of the pulmonary nodules.

INTRODUCTION IgG4-related systemic sclerosing disease (IRSSD) is a new clinical entity that is characterized by elevated levels of serum IgG4; plasma cell infiltrates in tissues, including the pancreas, lung, liver, kidney and breast; and a good response to corticosteroid therapy.1-4 We report a case of a patient with sclerosing cholangitis and pulmonary inflammatory pseudotumors. The diagnosis was made twelve years after the onset of the initial symptoms. The aim of this report is to describe a case with different radiological patterns.

CASE REPORT A 70-year-old male patient was referred to our institution in November 2009 for the evaluation of recurrent pulmonary infiltrates. His clinical problems started in 1998. The patient was diagnosed with sclerosing cholangitis after a cholecystectomy for gallstones. Dilatation of the bile ducts using endoscopic retrograde cholangiopancreatography was performed. Symptomatic control was achieved using ursodeoxycholic acid. No inflammatory bowel disease was detected. In addition, the patient has suffered from diabetes since 2000 and became insulindependent just after diagnosis. In 2004, a lower lobe pulmonary nodule was detected in a routine abdominal computed tomography (CT). This same exam revealed no signs of liver disease but demonstrated atrophy of the pancreas and irregularities in the pancreatic ducts, suggesting chronic pancreatitis. The pulmonary nodule was resected and diagnosed as an inflammatory pseudotumor. Four years later, the patient developed recurrent episodes of dyspnea and fatigue. Chest CTs over the years showed different image patterns and indicated predominantly bilateral peripheral nodules or subpleural

DISCUSSION High serum IgG4 that is associated with sclerosing pancreatitis was first described in 2001 by Hamano and colleagues and suggests a distinct disease entity due to its responsiveness to corticosteroids.5 This report and other reports have indicated the involvement of multiple organs containing inflammatory pseudotumors with plasma cells expressing IgG4, such as the pancreas, bile ducts, gallbladder, breast, salivary glands, retroperitoneum, kidney, lung, and prostate.1-4 Recently, a new clinical entity, IRSSD, has been proposed as an etiology for hyper-IgG4 gammaglobulinemia and IgG4 plasma cell infiltrates in multiple organs and is responsive to glucocorticoids.1-3,6

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IgG4-related systemic sclerosing disease Dias OM et al.

CLINICS 2011;66(11):1983-1986

Figure 1 - Chest computer tomography (CT) scans showing different image patterns along the years. A: December, 2004: the initial clinical presentation as a subpleural solitary pulmonary nodule in the right inferior lobe with adjacent subpleural opacities. The nodule was resected and diagnosed as an inflammatory pseudotumor. B: Three years later, CT scans with multiple spiculated pulmonary nodules surronded with ground glass halo and architectural distortion of the adjacent parenchyma. C and D: June, 2008: Pulmonary nodule with ground glass halo in the middle lobe along with consolidation in the left inferior lobe resembling organizing pneumonia. There was a complete response after corticosteroid treatment.

Zen et al. examined clinical and histological features of inflammatory pseudotumors and described lesions that are characterized by dense lymphoplasmacytic infiltrates intermixed with fibrosis, eosinophilic infiltration, irregular narrowing of the bronchioles that are entrapped in nodules, obliterative phlebitis or arteritis, and an interstitial pneumonia pattern at the boundaries of nodules. Immunostaining

revealed many IgG4+ plasma cells diffusely distributed within the nodules, and the ratio of IgG4+ plasma cells to other plasma cells was extraordinarily high.7-8 Convincing clinical criteria have not been clearly defined. IgG4 serum levels are frequently elevated in the majority of patients (above 140 mg/dl). However, 25% of patients display normal IgG4 levels.2 Immunostaining has revealed

Figure 2 - Chest CT scans obtained in 2009. On the left column, solitary pulmonary nodule in the upper right lobe along with parenchimal bands in the inferior right lobe. Five months later, the right upper nodule disappeared, but other nodules surrounded with ground glass halo appeared in the inferior lobes bilaterally.

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CLINICS 2011;66(11):1983-1986

IgG4-related systemic sclerosing disease Dias OM et al.

Figure 3 - Lung biopsy of the pulmonary nodule. A: Nodular fibrous lesion with immunopositivity for IgG4 in the infiltrating plasma cells (Verhoeff-Masson trichrome stain, original magnification x 2); B: Plasmacytic infiltration of the fibrous lesion (H&E stain, magnification 6 100); C: Occlusive vascular lesion with plasmacytic infiltration (H&E stain, magnification 6 100); D: Immunopositivity for IgG4 in the infiltrating plasma cells (Verhoeff-Masson trichrome stain, original magnification 6 100).

or biliary disease. The drug regimens used for the treatment of lung disease were empirical. In conclusion, when faced with inflammatory pseudotumors (histologically corresponding to plasma cell granuloma) or migratory pulmonary infiltrates that respond to corticosteroid therapy, the diagnosis of IRSSD should be considered in the differential diagnosis. Accurate diagnoses may lead to effective treatments and prevent unnecessary lung resections.

IgG4+ plasma cells. IgG4+ cells that number between .60 and 100 cells/high-power field (HPF) and a ratio IgG4+/ IgG+ cells between .40% and 50% are highly suggestive of IgG4-related disease.9 Although its prevalence is still unknown, previous studies have reported an increased incidence in male patients in their sixties who usually present with dry cough or dyspnea or are asymptomatic.1 Asian patients seem to have a genetic predisposition for IRSSD.10 However, cases in non-Asian patients, including the current case subject, have also been reported. Due to the heterogeneity of radiological presentations, IRSSD must be included as a differential diagnosis of other interstitial diseases. IRSSD is categorized into four major subtypes: the solid nodular type; the round-shaped groundglass opacity type; the alveolar interstitial type, which includes honeycombing, bronchiectasis, and diffuse ground-glass opacities; and the bronchovascular type, which is characterized by thickening of the bronchovascular bundles and interlobular septa.11 To our knowledge, we report herein the case with the longest radiological followup (seven years) and the first case with multiple documented radiological patterns in the same patient. In addition, we noticed a pattern of consolidation as a possible pulmonary involvement of IRSSD. Corticosteroids appear to be effective in the majority of the reported cases. The most commonly used dosage is prednisolone at 1 mg/kg/day with gradual tapering every 1-2 weeks for a total treatment period of 11 weeks. Clinical reports have also indicated spontaneous resolution without treatment.10 However, relapses during corticosteroid tapering are also possible, as in this current case. Therefore, low dose prednisone may be necessary as a maintenance treatment. Alternative drug regimens for corticosteroidresistant cases have not been studied. Treatment data were extrapolated from clinical studies of patients with pancreatic

AUTHOR CONTRIBUTIONS Dias OM was responsible for the patientâ&#x20AC;&#x2122;s diagnosis, wrote the first draft of the manuscript, conceived the review of the literature on the subject. Kawassaki AM was involved in the review and critical revision of manuscript before submission. Cukier A was involved in the clinical care of the patient, also participated in the manuscript review and critical revision before submission. Haga H made the anatomopathological review and performed the immohistochemistry analysis of the lung biopsy. Carvalho CRR was involved in the manuscript review and critical revision before submission. All authors have read and approved the final manuscript.

REFERENCES 1. Kamisawa T, Funata N, Hayashi Y, Eishi Y, Koike M, Tsuruta K, et al. A new clinicopathological entity of IgG4-related autoimmune disease. J Gastroenterol. 2003;38:982-4, doi: 10.1007/s00535-003-1175-y. 2. Masaki Y, Dong L, Kurose N, Kitagawa K, Morikawa Y, Yamamoto M, et al. Proposal for a new clinical entity, IgG4-positive multiorgan lymphoproliferative syndrome: analysis of 64 cases of IgG4-related disorders. Ann Rheum Dis. 2009;68:1310-5, doi: 10.1136/ard.2008.089169. 3. Dhobale S, Bedetti C, Killian P, Ilyas M, Liput J, Jasnosz K, et al. IgG4 related sclerosing disease with multiple organ involvements and response to corticosteroid treatment. J Clin Rheumatol. 2009;15:354-7, doi: 10.1097/RHU.0b013e3181b5d631. 4. Hamed G, Tsushima K, Yasuo M, Kubo K, Yamazaki S, Kawa S, et al. Inflammatory lesions of the lung, submandibular gland, bile duct and prostate in a patient with IgG4-associated multifocal systemic fibrosclerosis. Respirology. 2007;12:455-7, doi: 10.1111/j.1440-1843.2007.01053.x. 5. Hamano H, Kawa S, Horiuchi A, Unno H, Furuya N, Akamatsu T, et al. High serum IgG4 concentrations in patients with sclerosing pancreatitis. N Engl J Med. 2001;344:732-8, doi: 10.1056/NEJM200103083441005.

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9. Yamamoto H, Yamaguchi H, Aishima S, Oda Y, Kohashi K, Oshiro Y, et al. Inflammatory myofibroblastic tumor versus IgG4-related sclerosing disease and inflammatory pseudotumor: a comparative clinicopathologic study. Am J Surg Pathol. 2009;33:1330-40, doi: 10.1097/PAS. 0b013e3181a5a207. 10. Kawassaki AM, Haga H, Dantas TCA, Musolino RS, Baldi BG et al. Adenopathy and pulmonary infiltrates in a Japanese emigrant in Brazil. Chest. 2011;139:947-52, doi: 10.1378/chest.10-0632. 11. Inoue D, Zen Y, Abo H, Gabata T, Demachi H, Kobayashi T, et al. Immunoglobulin G4â&#x20AC;&#x201C;related lung disease: CT findings with pathologic correlations. Radiology. 2009;251:260-70, doi: 10.1148/radiol.2511080965.

6. Kobayashi H, Shimokawaji T, Kanoh S, Motoyoshi K, Aida S. IgG4positive pulmonary disease. J Thorac Imaging. 2007;22:360-2, doi: 10. 1097/RTI.0b013e31813fab9f. 7. Zen Y, Kitagawa S, Minato H, Kurumaya H, Katayanagi K, Masuda S, et al. IgG4-positive plasma cells in inflammatory pseudotumor (plasma cell granuloma) of the lung. Human Pathol. 2005;36:710-7, doi: 10.1016/j. humpath.2005.05.011. 8. Zen Y, Inoue D, Kitao A, Onodera M, Abo H, Miyayama S, et al. IgG4related lung and pleural disease: a clinicopathologic study of 21 cases. Am J Surg Pathol. 2009;33:1886-93, doi: 10.1097/PAS. 0b013e3181bd535b.

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CLINICS 2011;66(11):1987-1989

DOI:10.1590/S1807-59322011001100022

CASE REPORT

The best approach for diagnosing primary sclerosing cholangitis Wellington Andraus, Luciana Haddad, Lucas Souto Nacif, Felipe D. Silva, Roberto Blasbalg, Luiz Augusto Carneiro D’Albuquerque Hospital das Clıńicas da Faculdade de Medicina da Universidade de Saõ Paulo, Gastroenterology, Saõ Paulo/SP, Brazil. Email: wellington@usp.br/wandraus@usp.br Tel.: 55 11 30618319

(c-NMR) study showed a pattern of stenosis and dilation that suggested a diagnosis of primary sclerosing cholangitis (PSC) (Figure 1). The patient underwent ERCP with papillotomy, which confirmed this diagnosis. After three days, the patient presented with a fever, leukocytosis, and chills. He developed multiple intra-hepatic abscesses. Acute cholangitis was diagnosed and successfully treated with long-term IV antibiotics. After three years, the patient experienced three more episodes of cholangitis, which were treated with IV antibiotics. He is currently on daily prophylactic treatment with ciprofloxacin and remains asymptomatic. CASE 2: A 39-year-old woman started having diarrhea, hepatomegaly, jaundice, and elevated canalicular enzymes seven years ago. An ERCP with papillotomy was performed, and the findings strongly supported the diagnosis of PSC (Figure 2). Since then, the patient has experienced at least four major attacks of acute cholangitis that necessitated inpatient treatment. She is currently receiving prophylactic ciprofloxacin daily and is asymptomatic. However, she developed hepatic cirrhosis with portal hypertension. Three months ago, she experienced upper gastrointestinal bleeding due to esophageal varices. Liver transplantation has been indicated, and her current MELD score is 17.

INTRODUCTION Primary sclerosing cholangitis (PSC) is a chronic cholestatic syndrome with autoimmune features and is associated with other immunological diseases such as autoimmune pancreatitis and inflammatory bowel disease. In addition to primary biliary cirrhosis, PSC is one of the most common chronic cholestatic liver diseases.1 The diagnosis of PSC is a challenge because patients are asymptomatic or display unspecific symptoms. Patients experience fatigue, pruritus, and jaundice. In addition, symptom onset is not associated with age. The diagnosis of PSC is typically achieved after a complication such as cholangitis or hepatic dysfunction occurs. Patients may experience periods of remission. Laboratory tests show cholestatic profiles with high levels of alkaline phosphatases (APs), gamma-glutamyltransferases (GGTs) and, occasionally, greater bilirubin levels. Definitive diagnosis is achieved using imaging methods to assist in the differential diagnosis.2 Endoscopic retrograde cholangiography (ERC) has been considered the gold-standard diagnostic method for PSC.1 However, ERC has its inherent risks such as acute pancreatitis, cholangitis, and duodenum perforation.3 Moreover, in a patient with biliary chronic disease, the loss of the Oddi sphincter barrier may lead to acute complications and chronic problems such as recurrent cholangitis, liver abscesses, and reduced liver function.4 Magnetic resonance imaging (MRI) is an attractive option for PSC diagnosis. Many studies have reported that MRI has similar sensitivity to ERC for PSC and has the remarkable advantages of being less invasive and less likely to cause complications.5 In this case report, we present two patients with PSC who had serious complications after ERC with recurrent cholangitis and are on the waiting list for a liver transplant. CASE 1: A 51-year-old male patient with a previous diagnosis of inflammatory bowel disease (IBD) and complaints of chronic fatigue and pruritus was referred to our hospital. Liver function tests showed normal transaminase levels and a two-fold increase in the alkaline phosphatase and gamma-glutamyl transpeptidase levels. A cholangio-NMR

DISCUSSION The etiology of PSC remains elusive. PSC is related to other autoimmune diseases, such as IBD and autoimmune pancreatitis (AP). Genetic studies have demonstrated the expression of the haplotypes HLA A1-B8-DR3, DR-6, and DR-2 in some cases of PSC. HLA DR-4 expression is related to protective effects that are not specific for PSC. The lack of specificity of these markers remains a challenge in PSC diagnosis.6 PSC affects more men than women (251). Early clinical findings are nonspecific. Fatigue, anorexia, and arthralgia are common symptoms. Jaundice and pruritus are not rare and are troublesome for the patients.2 Laboratory tests have shown a typical cholestatic profile with elevated alkaline phosphatase and gamma-glutamyl transpeptidase levels. Occasionally, slight elevations of transaminases and bilirubin have been noted. Several auto-antibody studies have indicated elevated p-ANCA and anti-nuclear antibodies. However, elevated auto-antibodies are unspecific for PCS. 2,6 Imaging studies are fundamental to the investigation of any cholestatic disease. The intra- and extra-hepatic biliary

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An alternative approach for diagnosing PSC Andraus W et al.

CLINICS 2011;66(11):1987-1989

(EUS) and cholangio-NMR jeopardize the former pivotal role of ERCP as the gold-standard technique for the diagnosis of PSC. Ciorcilan et al. have analyzed the ERCPassociated complications in patients with cholestatic diseases and have concluded that this method is indicated only when diagnosis is not possible with EUS or cholangio-NMR or in patients that require therapeutic intervention.8 Some studies have shown that ERCP may be dangerous for patients with PSC.9,10 In patients with PSC, liver function may be impaired after ERCP. Beuers et al. have demonstrated that cholestasis worsened after ERCP in 53% of patients with PSC. A bilirubin level that exceeds 1.0 mg/dl in elderly patients is a risk factor for complications.4 In recent years, cholangio-NMR has undergone a significant technical evolution and is a preferred tool to investigate cholestatic disease. Vitellas et al. have compared the efficacies of ERCP and cholangio-NMR in the diagnosis of PSC and concluded that c-NMR defines peripheral biliary lesions more effectively than ERCP.11 Textor et al. have concluded that the sensitivity and specificity of c-NMR are similar to those of ERCP in the diagnosis of PSC and has fewer complications than ERCP.12 Talwalkar et al. have performed a prospective analysis of cost-effectiveness and concluded that c-NMR is more valuable than ERCP as a diagnostic procedure for cholestatic diseases.13 However, ERCP remains a valuable therapeutic method to manipulate the biliary tree (removal of sludge and stones and the dilation of the duct). The cases described in the current report emphasize a rare but potentially dangerous complication following ERCP in patients with PSC: acute and recurrent cholangitis (RC). Nevertheless, there are other similar clinical complications. Analysis of liver transplantation explants showed that bacteriobilia is more frequent in patients with PSC than in patients with PBC.14 Moreover, ERCP increases colonization by pathogenic flora, which may originate from the endoscope itself.15 Furthermore, when performing ERCP in patients with cholestatic disease, a wide papillotomy may be necessary for complete contrast wash-out of the biliary tree. Afterward, duodenal-biliary reflux may occur, leading to chronic biliary contamination.16,17 These data support the idea that chronic cholangitis is similar to the recurrent events of acute cholangitis and may lead to the deterioration of global liver function in patients with cholestatic diseases. Therefore, the contamination of a diseased biliary tree should be avoided whenever possible. ERCP with papillotomy may complicate cholestatic liver disease. Because other non-invasive tests such as cholangioNMR are currently widely available, we believe that ERCP has a limited role in the diagnosis of PSC and should be indicated only for therapeutic purposes in patients with PSC.

Figure 1 - Cholangio-NMR showing intrahepatic bile-duct strictures that are consistent with a diagnosis of PSC.

tree must be thoroughly examined to achieve a differential diagnosis.1,2,5 In the past, ERCP and percutaneous cholangiography have been widely used. ERCP is considered the gold-standard diagnostic technique for PSC because the documentation of detailed biliary anatomy that is achieved with this method is informative. However, ERCP is associated with complications, including biliary contamination, cholangitis and pancreatitis.7 New conservative methods to assess biliary anatomy such as endoscopic ultrasound

AUTHOR CONTRIBUTIONS Andraus W wrote the manuscript. Haddad L was responsible for the English revisions and writing. Nacif LS and Silva FD were responsible for the data collection. Blasbalg R was responsible for the radiology revisions. Dâ&#x20AC;&#x2122;Albuquerque LAC was responsible for the manuscript revisions.

REFERENCES 1. Angulo P, Lindor KD. Primary sclerosing cholangitis. Hepatology. 1999;30:325-32, doi: 10.1002/hep.510300101. 2. Schrumpf E, Boberg KM. Primary sclerosing cholangitis: challenges of a new millenium. Dig Liver Dis. 2000;32:753-5, doi: 10.1016/S15908658(00)80350-3.

Figure 2 - Endoscopic retrograde cholangiopancreatography showing intra- and extrahepatic bile-duct strictures and dilations.

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An alternative approach for diagnosing PSC Andraus W et al. 11. Vitellas KM, El-Dieb A, Vaswani KK, Bennett WF, Tzalonikou M, Mabee C, et al. MR cholangiopancreatography in patients with primary sclerosing cholangitis: interobserver variability and comparison with endoscopic retrograde cholangiopancreatography. AJR Am J Roentgenol. 2002;179:399-407. 12. Textor HJ, Flacke S, Pauleit D, Keller E, Neubrand M, Terjung B, et al. Three-dimensional magnetic resonance cholangiopancreatography with respiratory triggering in the diagnosis of primary sclerosing cholangitis: comparison with endoscopic retrograde cholangiography. Endoscopy. 2002;34:984-90, doi: 10.1055/s-2002-35830. 13. Talwalkar JA, Angulo P, Johnson CD, Petersen BT, Lindor KD. Costminimization analysis of MRC versus ERCP for the diagnosis of primary sclerosing cholangitis. Hepatology. 2004;40:39-45, doi: 10.1002/hep.20287. 14. Rosch T, Triptrap A, Born P, Ott R, Weigert N, Frimberger E, et al. Bacteriobilia in percutaneous transhepatic biliary drainage: occurrence over time and clinical sequelae. A prospective observational study. Scand J Gastroenterol. 2003;38:1162-8, doi: 10.1080/00365520310003549. 15. Pohl J, Ring A, Stremmel W, Stiehl A. The role of dominant stenoses in bacterial infections of bile ducts in primary sclerosing cholangitis. Eur J Gastroenterol Hepatol. 2006;18:69-74, doi: 10.1097/00042737200601000-00012. 16. Bordas JM, Elizalde I, Llach J, Mondelo F, Bataller R, Teres J. Biliary reflux due to sphincter of Oddi ablation: a new pathogenetic explanation for long-term major biliary symptoms after endoscopic-sphincterotomy. Endoscopy. 1996;28:642, doi: 10.1055/s-2007-1005568. 17. Mandryka Y, Klimczak J, Duszewski M, Kondras M, Modzelewski B. [Bile duct infections as a late complication after endoscopic sphincterotomy]. Pol Merkur Lekarski. 2006;21:525-7.

3. Moff SL, Kamel IR, Eustace J, Lawler LP, Kantsevoy S, Kalloo AN, et al. Diagnosis of primary sclerosing cholangitis: a blinded comparative study using magnetic resonance cholangiography and endoscopic retrograde cholangiography. Gastrointest Endosc. 2006;64:219-23, doi: 10.1016/j.gie. 2005.12.034. 4. Beuers U, Spengler U, Sackmann M, Paumgartner G, Sauerbruch T. Deterioration of cholestasis after endoscopic retrograde cholangiography in advanced primary sclerosing cholangitis. J Hepatol. 1992;15:140-3, doi: 10.1016/0168-8278(92)90026-L. 5. Angulo P, Pearce DH, Johnson CD, Henry JJ, LaRusso NF, Petersen BT, et al. Magnetic resonance cholangiography in patients with biliary disease: its role in primary sclerosing cholangitis. J Hepatol. 2000;33:5207, doi: 10.1034/j.1600-0641.2000.033004520.x. 6. Maggs JR, Chapman RW. Sclerosing cholangitis. Curr Opin Gastroenterol. 2007;23:310-6, doi: 10.1097/MOG.0b013e32805867e6. 7. Ang TL, Fock KM, Ng TM, Teo EK, Chua TS, Tan JY. Clinical profile of primary sclerosing cholangitis in Singapore. J Gastroenterol Hepatol. 2002;17:908-13, doi: 10.1046/j.1440-1746.2002.02835.x. 8. Ciocirlan M, Ponchon T. Diagnostic endoscopic retrograde cholangiopancreatography. Endoscopy. 2004;36:137-46, doi: 10.1055/s-2004-814181. 9. Enns R, Eloubeidi MA, Mergener K, Jowell PS, Branch MS, Baillie J. Predictors of successful clinical and laboratory outcomes in patients with primary sclerosing cholangitis undergoing endoscopic retrograde cholangiopancreatography. Can J Gastroenterol. 2003;17:243-8. 10. Christensen M, Hendel HW, Rasmussen V, Hojgaard L, Schulze S, Rosenberg J. Endoscopic retrograde cholangiopancreatography causes reduced myocardial blood flow. Endoscopy. 2002;34:797-800, doi: 10. 1055/s-2002-34270.

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DOI:10.1590/S1807-59322011001100023

CASE REPORT

Primary Sjo¨gren’s syndrome in children: Is a family approach indicated? Barbara S. Longhi,I Simone Appenzeller,II Maraisa Centeville,I,III Reinaldo J. Gusma˜o,IV Roberto MariniI I Universidade Estadual de Campinas (UNICAMP), Faculty of Medical Science State, Department of Pediatrics, Campinas/SP, Brazil. II Universidade Estadual de Campinas (UNICAMP), Department of Medicine, Division of Rheumatology, Campinas/SP, Brazil. III Universidade Estadual de Campinas (UNICAMP), Faculty of Medical Science, Post-Graduate Program in Child and Adolescent Health, Campinas/SP, Brazil. IV Universidade Estadual de Campinas (UNICAMP), Pediatric Otorhinoloaryngology Unit, Campinas/SP, Brazil.

Email: appenzellersimone@yahoo.com Tel.: 55 19 3289-1818

patient was negative for RF and antinuclear antibodies (ANA), but positive for anti-Ro. Antibodies against La, double-stranded DNA (anti-dsDNA), and Sm were also negative. Her white blood cell counts (WBCs) and ESR were normal, and no hypergammaglobulinemia was observed. A minor salivary gland biopsy showed diffuse lymphocytic infiltration. Patient 2 - During medical evaluation, the mother of the patient mentioned that her other 8-year-old daughter also had a two-year history of recurrent parotid gland swelling. The patient denied having dry mouth, dry eyes, or any systemic symptoms. Her physical exam was unremarkable. The patient had a normal Schirmer’s test and did not have any evidence of chronic sialadenitis upon sialography. Her laboratory test results revealed that she was positive for RF, ANA, Ro, and La antibodies as well as negative for antidsDNA and anti-Sm. The patient displayed normal WBC and ESR, but exhibited polyclonal hypergammaglobulinemia. A salivary gland biopsy revealed lymphocytic sialadenitis with more than four lymphoid foci (4 mm2 in size), which was compatible with SS. Patient 3 - The mother was 28 years in age and complained of persistent dry mouth, photophobia, and dry eyes after two years of her daughters’ follow-up. Her physical exam was unremarkable. She was negative for RF, positive for ANA, and anti-Ro, had a normal WBC and ESR, and presented no hypergammaglobulinemia. Ophthalmological examination revealed positive results both on Schirmer’s test and the Rose Bengal test. Her salivary gland scintigraphy revealed deficient drainage of her left parotid gland, and her salivary gland biopsy showed no evidence of diffuse lymphocytic infiltration.

INTRODUCTION Sjo¨grens syndrome (SS) is a chronic autoimmune disease primarily affecting the lachrymal and salivary glands with varying degrees of systemic involvement. SS can be isolated (primary SS or pSS) or associated with other autoimmune diseases, such as rheumatoid arthritis, systemic lupus erythematosus, or scleroderma (secondary SS). pSS predominantly affects middle-aged women and is uncommon in childhood.1-4 In addition to being underdiagnosed, clinical manifestations are often different in children when compared to adults. Recurrent parotid swelling is a common feature observed in children with pSS (37, 5– 100%).2 Oral and ocular involvement, however, is present in a minority of children, especially at the onset of disease (23– 80%).2 Pathologic and laboratory findings are similar to those found in adults and include characteristic lymphocytic infiltration of the exocrine glands, the presence of hypergammaglobulinemia, an elevated erythrocyte sedimentation rate (ESR), and the presence of autoantibodies (such as anti-Ro/SSA, anti-La/SSB, antinuclear antibody (ANA), and rheumatoid factor (RF)4-6). Although pSS is an autoimmune disease, familial incidence of pSS is infrequently reported.6-8 We report the occurrence of pSS in two siblings and in their mother and review the literature on multiplex pSS families.

CASE REPORTS Patient 1 - A 9-year-old girl with a one-year history of bilateral recurrent enlargement of the parotid gland was sent to the rheumatology unit. She denied having dry mouth, dry eyes, or any systemic symptoms. Upon examination, she was healthy with a weight in the 75th percentile and a height in the 90th percentile. Her physical examination was unremarkable, except for the presence of a bilateral, painful parotid enlargement. The physician also identified sialadenitis upon sialography and a positive Schirmers test upon ophthalmological evaluation. The

DISCUSSION pSS is a well-known autoimmune disease that predominantly affects middle-aged women. pSS is diagnosed following the Revised International Classification Criteria for Sjo¨gren’s Syndrome from the American-European Group and includes six items: (I) presence of ocular symptoms, (II) presence of oral symptoms, (III) evidence of keratoconjunctivitis sicca, (IV) focal sialadenitis upon minor salivary gland biopsy, (V) instrumental evidence of salivary gland involvement, and (VI) presence of SSA or SSB autoantibodies. Diagnosis of pSS requires 4 of the six criteria.5,9 As there are no specific diagnostic criteria for pSS in children, and the adult criteria are often used. The

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Table 1 - Case reports of familial Sjo¨gren’s Syndrome. Reference Coverdale

Cases 20

Bloch et al. Camus et al.22 Koivukagas et al.23 Simila S et al.24 Besana et al.25 Ostuni et al.26 Houghton et al.6 Bolstad et al.27 Boling et al.15 Lichtenfeld et al.8 Sa´bio et al.19

Comment

Father and daughter Mother and daughter Mothers of two patients Mother and daughter

Both had KS and intermittent parotid gland enlargement Association with familial scleroderma Two sisters Association with achalasia of the esophagus Two siblings KS(1,2), X(1,2), achalasia (1,2), gastric hyposecretion (1,2) Identical twins Bilateral dacryiadenitis; KS Identical twins PS(1,2), KS(1) Dizygotic twins PS (1,2), X (1), LIP (1) Monozygotic twins (1,2) and the mother (3)X, KS, salivary gland dysfunction (1,2); X, KS, dry nose, dry skin, dry cough, RP (3) Two siblings Hemolytic anemia, no association with HLA-DR3 Four siblings Association with primary salivary gland lymphoma Dizygotic siblings (1,2,3) and the mother (4) X and KS (1,2,3,4), PS (1)

Ks: keratoconjunctivitis sicca; X: xerostomia, PS: parotid swelling; RP: Raynaud’s phenomenon.

diagnosis of childhood SS may be difficult because the classical diagnostic criteria that are successfully used for adults are often not fulfilled by children at disease onset. The onset of disease may not only be different in children, but also characterized by nonspecific symptoms. Frequently, salivary gland enlargement or recurrent parotitis is observed; sicca symptoms are rarely referred.2 Laboratory signs are present early during the course of disease and include hypergammaglobulinemia, positive tests for anti-SS-A and/or anti-SSB, a high titer of speckled antinuclear antibodies, and/or the presence of rheumatoid factor.10 pSS is rare in children and adolescents.4,6,11 During a three-year study, Cimaz et al.2 collected data on 40 patients from ten different centers worldwide. The most common manifestation reported in their study was recurrent parotid swelling (72.5%) at the onset of disease, whereas ocular and/or oral symptoms were only found in a minority of children. Other clinical signs and symptoms observed at disease onset were arthritis (10%), fever (10%), fatigue (7.5%), and submandibular swelling (5%). Similar results were observed in other reports.6,12 The differential diagnosis of recurrent parotitis is broad in children and includes mechanical, infectious, malignant, and inflammatory etiologies.6 It is important to note that in both the children in this case study, recurrent parotitis was the initial clinical manifestation of pSS. Therefore, recurrent parotitis should alert the clinician to the possibility of pSS.6,12 Familial cases of pSS are rare (Table 1).6,8,19-27 Lichtenfeld et al. described one patient with SS in whom a primary parotid gland lymphoma subsequently developed.8 Moreover, two of four siblings had evidence of SS, and the authors suggested that genetic influences may facilitate the development of SS. Houghton et al. reported pSS in dizygotic adolescent twins; recurrent parotitis was the initial clinical manifestation in both parents.6 The authors proposed that pSS is likely a polygenic disorder resulting from several genes interacting with environmental factors. Some studies of pSS showed that it has a strong association with HLA-DR3.6,13,14 However, recent studies have suggested that pSS is a complex, polygenic disorder sharing common genetic determinants with related autoimmune diseases, such as systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA).14 Recent

advances in SLE and RA provide valuable insights into the potential genetic complexity of SS.14 Several families with multiplex SS have also been described,6,15,16 and relatives often have other autoimmune diseases, such as SS (12%), autoimmune thyroid disease (AITD) (14%), RA (14%), and SLE (5%–10%).17,18 In conclusion, although pSS is a complex genetic disorder, familial cases of SS are rarely reported. In children, pSS may be underdiagnosed because of the lack of classic sicca symptoms. However, pSS should be included in the differential diagnosis of recurrent parotid swelling, and when diagnosed, siblings should be evaluated.

ACKNOWLEDGEMENTS Fundaçaõ Amparo a` Pesquisa Estado Saõ Paulo-Brasil (FAPESP 2008/ 02917-0, Conselho Nacional Pesquisa Desenvolvimento-Brasil CNPq (300447/2009-4).

AUTHOR CONTRIBUTIONS Longhi BS, Marini R, Appenzeller S, Centeville M, and Gusma˜o RJ were responsible for the revision of the charts and manuscript preparation.

REFERENCES 1. Civilibal M, Canpolat N, Yurt A, Kurugoglu S, Erdamar S, Bagci O, et al. A Child With Primary Sjo¨gren Syndrome and a Review of the Literature. Clinical Pediatrics. 2007;46:738-42, doi: 10.1177/0009922807301945. 2. Cimaz R, Casadei A, Rose C, Bartunkova J, Sediva A, Falcini F, et al. Primary Sjo¨gren syndrome in paediatric age: a multicentre survey. Eur J Pediatr. 2003;162:661-5, doi: 10.1007/s00431-003-1277-9. 3. Stiller M, Golder W, Do¨ring E, Biedermann T. Primary and secondary Sjo¨gren’s syndrome in children—a comparative study. Clin Oral Invest. 2000;4:176-82, doi: 10.1007/s007840000070. 4. Nikitakis NG, Rivera H, Lariccia C, Papadimitriou JC, Sauk JJ. Primary Sjo¨gren syndrome in childhood: report of a case and review of the literature. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2003;96:42-7, doi: 10.1016/S1079-2104(03)00159-8. 5. Fox R. Sjo¨gren’s syndrome. Lancet. 2005;366:321-31, doi: 10.1016/S01406736(05)66990-5. 6. Houghton KM, Cabral DA, Petty RE, Tucker LB. Primary Sjo¨gren syndrome in dizygotic adolescent twins: one case with lymphocytic interstitial pneumonia. J Rheumatol. 2005;32:1603-6. 7. Doni A, Brancato R, Bartolettl L, Berni G. La famlliarita della malattia di Sjbgren. Riv Crit Clln Med. 65:750:1965. 8. Lichtenfeld JL, Kirschner RH, Wiernik PH. Familial Sjogren’s syndrome with associated primary salivary gland lymphoma. Am J Med. 1976;60:286–92, doi: 10.1016/0002-9343(76)90439-3. 9. Vitali C, Bombardieri S, Jonsson R, Moutsopoulos HM, Alexander EL, Carsons SE, et al. European Study Group on Classification Criteria for

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10.

11. 12.

13.

14.

15.

16.

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Primary Sjo¨gren’s syndrome in children Longhi BS et al.

Sjo¨ gren’s Syndrome. Classification criteria for Sjo¨ gren’s syndrome: a revised version of the European criteria proposed by the American-European Consensus Group. Ann Rheum Dis. 2002;61:554-8, doi: 10.1136/ard.61.6.554. Bartunkova´ J, Sediva´ A, Vencovsky J, Tesar V. Primary Sjo¨gren syndrome in Children and adolescents: Proposal for diagnostic criteria. Clin Exp Rheumatol. 1999;17:381-86. Anaya J, Ogawa N, Talal N. Sjo¨gren’s syndrome in childhood. J Rheumatol. 1995;22:1152-8. Houghton K, Mah W-L, Maleeson P, Cabral D, Petty R, Tucker L. Primary Sjo¨gren syndrome in Children and adolescents: are current diagnostic criteria applicable? [abstract}. Canadian Rheumatology Association Meeting. Lake Louise, Alberta, Canada, February 25-28. J Rheumatol. 2004;31:1436. Foster H, Stephenson A, Walker D, Cavanagh G, Kelly C, Griffiths I. Linkage studies of HLA and Primary Sjo¨gren syndrome in multicase families. Arthritis Rheum. 1993;36:473-84, doi: 10.1002/art.1780360407. Cobb BL, Lessard CJ, Harley JB,Moser KL. Genes and Sjo¨ gren’s Syndrome. Rheum Dis Clin N Am. 2008;34:847–868, doi: 10.1016/j.rdc. 2008.08.003. Boling EP, Wen J, Reveille JD, Bias WB, Chused TM, Arnett FC. Primary Sjogren’s syndrome and autoimmune hemolytic anemia in sisters. A family study. Am J Med. 1983;74:1066–71. Sestak AL, Shaver TS, Moser KL, Neas BR, Harley J B. Familial aggregation of lupus and autoimmunity in an unusual multiplex pedigree. J Rheumatol. 1999;26:1495–9. Reveille JD. The molecular genetics of systemic lupus erythematosus and Sjogren’s syndrome. Curr Opin Rheumatol. 1992;4:644–56.

18. Reveille JD, Wilson RW, Provost TT, Bias WB, Arnett FC. Primary Sjogren’s syndrome and other autoimmune diseases in families. Prevalence and immunogenetic studies in six kindreds. Ann Intern Med. 1984;101:748–56. 19. Sabio JM, Milla E, Jimenez-Alonso J. A multicase family with primary Sjogren’s syndrome. J Rheumatol. 2001;28:1932–4. 20. Coverdale H. Some unusual cases of Sjogren’s syndrome. Br J Ophthalmol. 1948;32:669-73, doi: 10.1136/bjo.32.9.669. 21. Bloch KJ, Buchanan WW, Wohl MI, Bunim JJ. Sjogren’s syndrome: a clinical pathological and serological study of sixty-two cases. Medicine (Battlmore). 1965;44:187-231, doi: 10.1097/00005792-196505000-00001. 22. Camus JP, Emerit I, Reinhart P, Guillien P, Crouzet J, Fourot J. Sclerodermie familiale avec syndrome de Sjbgren et anomalies lymphocytaires et chromosomiques. Ann Med lnterne (Paris). 1970;121:149. 23. Koivukagas T, Simila S, Heikkinen E, Wasz-Hochert 0. Sjogren’s syndrome and achalasia of the cardia in two siblings. Pediatrics. 1973;51:943. 24. Simila S, Kokkonen J, Kaski M. Achalasia sicca – juvenile Sjogren’s syndrome with achalasia and gastric hyposecretion. Eur J Pediatr. 1978;129:175-81, doi: 10.1007/BF00442161. 25. Besana C, Salmaggi C, Pellegrino C, Pierro L, Vergani S, Faravelli A, et al. Chronic bilateral dacryo-adenitis in identical twins: a possible incomplete form of Sjogren syndrome. Eur J Pediatr. 1991;150:652–5, doi: 10. 1007/BF02072627. 26. Ostuni PA, Ianniello A, Sfriso P, Mazzola G, Andretta M, Gambari PF. Juvenile onset of primary Sjogren’s syndrome: report of 10 cases. Clin Exp Rheumatol. 1996;14:689-93. 27. Bolstad AI, Haga HJ, Wassmuth R, Jonsson R. Monozygotic twins with primary Sjogren’s syndrome. J Rheumatol. 2000;27:2264–6.

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DOI:10.1590/S1807-59322011001100024

CASE REPORT

Unusual presentation of multiple aneurysms of the ascending aorta Sergio Francisco Santos Jr.,I Marcelo Luiz Peixoto Sobral,II Anderson da Silva Terrazas,II Gilmar Geraldo Santos,II Noedir A. G. StolfII,III I

Santa Casa de Itabuna, Cirurgia Cardiovascular, Itabuna/BA, Brazil. II Hospital Beneficeˆncia Portuguesa de Saõ Paulo, Cirurgia Cardiovascular, Saõ Paulo/ SP, Brazil. III Faculdade de Medicina da Universidade de Saõ Paulo, Saõ Paulo/SP, Brazil. Email: sergio.cardiaca@gmail.com Tel.: 55 11 3505-5262

of the medial layer of the vessel wall, all of which are noninflammatory in nature.9 Medial degeneration does not uniformly involve the ascending aorta.8,9 The convexity of the vessel looks more damaged because of more severe medial necrosis, greater loss of smooth muscle cells and apoptosis and greater elastic fiber fragmentation. These alterations play an important role in the development of aortic aneurysms because they participate in the matrix remodeling process resulting in the synthesis of extracellular proteins such as collagen, elastin, and proteoglycans.8

INTRODUCTION At the beginning of the twentieth century, Carrell and Guthrie were the first to use homografts to reconstruct dilated vessels, starting a new era in aorta surgery.1 In 1952, Cooley and DeBakey conducted the first successful ascending aorta intervention without cardiopulmonary bypass in the resection of a sacciform aneurysm by aortorrhaphy.2 In 1956, the same authors performed the first successful replacement of the ascending aorta using cardiopulmonary bypass.3 Since then, aortic surgery has developed quickly because of advances in cardiopulmonary bypass, postoperative care, and surgical techniques that reduce the mortality rates of these procedures.4,5 At present, less invasive techniques, such as endovascular interventions, are performed, especially in thoracic and thoracoabdominal aortic disease, depending on factors such as whether the patients would be at high risk during conventional procedures.6,7 Recent studies and case reports have shown the possibility of performing endovascular procedures in the ascending aorta, which seems to be a promising approach.7 Concerning ascending aorta aneurysms, there are several surgical approaches depending on the level of the disease of the aortic valve, aortic root, sinotubular junction and the ascending aorta. These issues are relevant in the context of congenital diseases such as Marfans syndrome,7 EhlersDanlos syndrome,8 congenital aortic valve malformations, and acquired aortic valve diseases.9 It is accepted that apoptosis is the major mechanism for the control of cell density in developing physiological and pathological conditions affecting smooth muscle cells. It was shown that death signals may be triggered outside the cells by cytokine pathways and stress mechanical forces.8 Today, it is known that when there is aortic dilation, there is also cystic medial necrosis. This histological abnormality is characterized by a triad of noninflammatory smooth muscle cell loss, the fragmentation of elastic fibers and the accumulation of basophilic ground substance within cell-depleted areas

CASE REPORT We report the case of a 24-year-old man who presented progressive precordial pain and dyspnea that had worsened in the last six months prior to the consultation with no previous history of arterial hypertension, dyslipidemia, smoking habits, or infectious diseases, such as syphilis or immunodeficiency syndrome. The physical examination showed no signs of congenital syndromes and revealed only severe systolic murmur in the aortic focus. Preoperative examinations were conducted as routine. Laboratory tests as well as the chest radiograph were unchanged. The electrocardiogram showed sinus rhythm, incomplete right bundle branch block and moderate left ventricular hypertrophy. The echocardiogram revealed aortic stenosis caused by a thickened aortic valve, which determined a gradient of 65 mmHg, left ventricular ejection fraction of 0.63, ascending aortic diameter of 30 mm, and systolic and diastolic diameters of 32 and 48 mm, respectively. Then, we proposed the surgery for aortic valve replacement. The procedure was performed by median sternotomy. After the pericardiotomy, we noticed an uncommon aspect of the ascending aorta, with an irregular surface and thinned dilated areas with soft superficial texture (Figure 1). Aortic cannulation was performed above and medial to the innominate artery, and right atrial cannulation was performed through the right atrial appendage. A left ventricular vent was placed via the right superior pulmonary vein. After total heparinization, cardiopulmonary bypass was then established, the aorta was cross-clamped and a transverse aortotomy was performed. The internal aspect of the vessel was thickened, with multiple focal cavities of different sizes corresponding to the external dilatations; no thrombi were found (Figure 2).

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Figure 1 - External macroscopic aspect of the ascending aorta segment.

Antegrade cold blood cardioplegic solution was infused every twenty minutes. The three thickened aortic leaflets, as well as the anterolateral convexity and anteromedial concavity of the aorta were excised, leaving the posterior one third, macroscopically normal, to serve as support tissue to the vascular prosthesis that would be placed. Samples were submitted for histological study, and the procedure was completed. The prosthesis implanted was a mechanical no. 21; a Dacron tube prosthesis (no. 24) was positioned in replacement of the ascending aorta above the coronary ostia. Deairing was performed through a catheter placed in the tube; the aortic cross-clamp was removed. As the heartbeats became effective, the cardiopulmonary bypass was ended, and the surgery was finalized as usual. The patient was sent to the intensive care unit, where he remained for one day. He was then discharged by the tenth day in good health. He recovered and has lived until the present moment without complications. The histopathological findings were ascending aorta with areas of intimal fibrosis with dystrophic calcification consistent with arteriosclerosis. Areas of medial cystic degeneration with saccular dilations were observed to be diffusely distributed; inflammation was absent (Figure 3-A,B,C). The

Figure 3 - A) Aorta segment on the level of the little saccular dilations on the aneurysmatic wall showing a severe lack of smooth muscle cells (Tricromic of Masson, 50x). B) ‘‘Proteoglycan lakes’’ are related to the areas of widespread loss of elastic fibers (Alcian Blue, 200x). C) Intense elastic fiber fragmentation area (Verhoeff, 200x).

aortic leaflets were fibrous with myxomatous degeneration and dystrophic calcification. The loss of smooth muscle cells was markedly important, as was the deposition of extracellular matrix following the degeneration. Angiotomography of the aorta was performed postoperatively and showed no other injuries in the aorta. Furthermore, the prosthetic graft in the ascending aorta and the mechanic valve prosthesis in the aortic position were properly positioned (Figure 4). Some laboratory tests were performed in order to try to identify infectious diseases, such as syphilis and HIV, and autoimmune diseases, such as vasculitis, with no positive results.

DISCUSSION Figure 2 - Internal macroscopic aspect of the ascending aorta segment. Note the multiple focal cavities of different sizes corresponding to the external dilations.

These related microscopic changes determine the thinness of the aortic wall; however, these changes do not explain

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Multiple aneurysms of the ascending aorta Santos SF et al.

which limits the determination of this diseases etiopathogenesis. We decided to perform an ascending aorta replacement, which was not indicated by classical aneurysm definitions. Although the largest diameter was 30 mm, not more than 1.5 times the size of the normal aorta, failure to intervene could cause further complications in the future, such as greater dilations, thromboembolic events, aortic dissection or aortic rupture.

AUTHOR CONTRIBUTIONS Santos Jr SF, surgeon assistant, was responsible for the literature researches and manuscript writing. Sobral MLP, surgeon, was also responsible for the literature researches. Terrazas AS, training surgeon, was responsible for the literature researches. Santos GG, co-chief of surgical team, and literature advisor. Stolf NAG, chief of surgical team, literature advisor, and reviewer.

REFERENCES 1. Brinster DRi, Rizzo RJi, Bolman RMi III. Ascending Aortic Aneurysms. In: Cohn LH, ed. Cardiac Surgery in the Adult. New York: McGraw-Hill. 2008;1223-50. 2. Cooley DA, De Bakey ME. Surgical considerations of intrathoracic aneurysms of the aorta and great vessels. Ann Surg. 1952;135:660-680, doi: 10.1097/00000658-195205000-00010. 3. Cooley DA, DeBakey ME. Resection of entire ascending aorta in fusiform aneurysm using cardiac bypass. JAMA. 1956;162:1158â&#x20AC;&#x201C;9. 4. Cohn LH, Rizzo RJ, Adams DH, Aranki SF, Couper GS, Beckel N, et al. Reduced Mortality and Morbidity for Ascending Aortic Aneurysm Resection Regardless of Cause. Ann Thorac Surg. 1996;62:463-8, doi: 10. 1016/0003-4975(96)00280-9. 5. Cooley DA. Aortic aneurysm operations: past, present, and future. Ann Thorac Surg. 1999;67:1959-62, doi: 10.1016/S0003-4975(99)00393-8. 6. Almeida RMS, Leal JC, Saadi EK, Braile DM, Rocha AST, Volpiani G, et al. Thoracic endovascular aortic repair: a Brazilian experience in 255 patients over a period of 112 months. Interact Cardiovasc Thorac Surg. 2009;8:524-8, doi: 10.1510/icvts.2008.192062. 7. Palma JH, Gaia DF, Guilhen JS, Buffolo E. Endovascular treatment of chronic type A dissection. Interact Cardiovasc Thorac Surg. 2008;7:164-6, doi: 10.1510/icvts.2007.165027. 8. Agozzino L, Sante` P, Ferraraccio F, Accardo M, De Feo M, De Santo LS, et al. Ascending aorta dilatation in aortic valve disease: morphological analysis of medial changes. Heart Vessels. 2006;21:213-20, doi: 10.1007/ s00380-005-0891-z. 9. Bonderman D, Gharehbaghi-Schnell E, Wollenek G, Maurer G, Baumgartner H, Lang IM. Mechanisms Underlying Aortic Dilatation in Congenital Aortic Valve Malformation. Circulation. 1999;99:2138-43.

Figure 4 - Angiotomographic construction in three dimensions of the aorta and its branches. No evidence of further injury. Proper positioning of the aortic vascular Dacron graft and the aortic mechanic valve prosthesis.

why this thinning happened in an irregular fashion, causing cavities rather than the uniform dilation that would be expected. The cited histopathological findings confirm cystic medial degeneration, amply described in the medical literature, as the main alteration in those without defined syndromes that could cause modifications in the connective tissues. Some infectious diseases may cause aneurysms of the ascending aorta; however, in these cases, fibrotic thickening and aortic dilation commonly occur. In this case, postoperative examinations were performed but did not identify diseases such as syphilis or potential causes of autoimmune vasculitis. Poststenotic dilation of the ascending aorta often occurs in chronic cases and displays a uniform enlargement pattern. Unfortunately, we did not have the chance to proceed with immunohistochemical studies of the surgical samples,

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DOI:10.1590/S1807-59322011001100025

ERRATA CLINICS 2010;65(1):107-109 RUBINSTEIN-TAYBI SYNDROME: A FEMALE PATIENT WITH A DE NOVO RECIPROCAL TRANSLOCATION T(2; 16)(Q36.3; P13.3) AND DYSGRANULOPOIESIS CASE DESCRIPTION Correction: The karyotype had a clerical error. The correct karyotype is 46,XX,t(2;16)(p11.2, p13.3), thus, the patient presents one translocation between the short arms of chromosomes 2 and 16.

1999