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I S S N - 1807-5932 printed version I S S N - 1980-5322 online version

Volume 67 Number 12 - December/2012

Official Scientific Journal of Faculdade de Medicina and Hospital das ClĂ­nicas Universidade de SĂŁo Paulo


CLINICS Editor Mauricio Rocha-e-Silva Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Area Editors Ana Maria de Ulhoa Escobar Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Antonio Egidio Nardi Universidade Federal do Rio de Janeiro Rio de Janeiro, RJ, Brazil Anuar Ibrahim Mitre Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Ashok Agarwal The Cleveland Clinic Foundation Cleveland, Ohio, USA Berenice Bilharinho Mendonc¸a Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Bruno Zilberstein 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 Heitor Franco de Andrade Jr. Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Ivete Bedin Prado Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Joaquim Prado Moraes-Filho Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Ludhmila Abrahao Hajjar Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Fulvio Alexandre Scorza Universidade Federal de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Luı´z Eugeˆnio Garcez-Leme Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Lydia Masako Ferreira Universidade Federal de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Maria Cecı´lia Solimene Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Mauricio Etchebehere Universidade Estadual de Campinas Campinas, SP, Brazil Nelson Wolosker Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Newton Kara-Junior Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Olavo Pires de Camargo Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Oswaldo Keith Okamoto Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

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

Patricia Rieken Macedo Rocco Universidade Federal do Rio de Janeiro Rio de Janeiro, RJ, Brazil

Carlos Serrano Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Carmen Silvia Valente Barbas Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Claudia Regina Furquim de Andrade Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Edmund Chada Baracat Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Eliete Bouskela Universidade do Estado do Rio de Janeiro Rio de Janeiro, RJ, Brazil Emilia Inoue Sato Universidade Federal 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 Raul Coimbra University of California, San Diego La Jolla, CA, USA Renato Delascio Lopes Universidade Federal de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Rubens Belfort Jr. Universidade Federal de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Ruth Guinsburg Universidade Federal de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Ruy Jorge Cruz Junior University of Pittsburgh Pittsburgh, PA, USA Sandro Esteves ANDROFERT - Andrology & Human Reproduction Clinic Campinas, SP, Brazil Sergio Paulo Bydlowski Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Silvia Vanessa Lourenc¸o Faculdade de Odontologia da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Simone Appenzeller Universidade Estadual de Campinas Campinas, SP, Brazil Sophie Franc¸oise Mauricette Derchain Faculdade de Cieˆncias Me´dicas, Universidade Estadual de Campinas Campinas, SP, Brazil Suely Kazue Nagahashi Marie Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Thelma Suely Okay Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Vale´ria Aoki Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Editorial Board Abhijit Chandra King George’s Medical College Lucknow, India

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

Ernest Eugene Moore University of Colorado Denver Denver, CO, USA

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

Adamastor Humberto Pereira Universidade Federal do Rio Grande do Sul Porto Alegre, RS, Brazil

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

Euclides Ayres Castilho Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Ke-Seng Zhao Southern Medical University Guangzhou, China

Adauto Castelo Universidade Federal de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

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

Ademar Lopes Fundac¸a˜o Antoˆnio Prudente, Hospital do Caˆncer Sa˜o Paulo, SP, Brazil

Cesar Gomes Victora Faculdade de Medicina da Universidade Federal de Pelotas Pelotas, RS, Brasil

Alberto Azoubel Antunes Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Daniel Romero Mun˜oz Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Alexandre Roberto Precioso Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Edmund Neugebauer Witten/Herdecke University Witten, North Rhine - Westphalia, Germany

Andrea Schmitt University of Goettingen Goettingen, Germany Arnaldo Valdir Zumiotti

Egberto Gaspar de Moura Jr. Universidade do Estado do Rio de Janeiro Rio de Janeiro, RJ, Brazil

Fa´bio Biscegli Jatene Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Laura Cunha Rodrigues London School of Hygiene and Tropical Medicine - University of London London, UK

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

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

Hiroyuki Hirasawa Chiba University School of Medicine Chiba, Japan

Marco Martins Amatuzzi Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

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

Maria Aparecida Shikanai Yasuda Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Mauro Perretti William Harvey Research Institute London, UK


Michael Gregory Sarr Mayo Clinic Rochester, MN, USA

Pedro Puech-Lea˜o Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Milton de Arruda Martins Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Peter Libby Brigham and Women’s Hospital Boston, Boston, MA, USA

Mitchell C. Posner The University of Chicago Medical Center Chicago, IL, USA

Philip Cohen University of Houston Health Center Houston, Texas, USA

Moyses Szklo Johns Hopkins Bloomberg School of Public Health Baltimore, USA

Rafael Andrade-Alegre Santo Toma´s Hospital Republic of Panama´, Panama´

Navantino Alves Faculdade de Cieˆncias Me´dicas de Minas Gerais Belo Horizonte, MG, Brazil

Ricardo Antonio Refinetti Faculdade de Medicina da Universidade Federal do Rio de Janeiro Rio de Janeiro, RJ, Brazil

Noedir Antonio Groppo Stolf Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, 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 Sa˜o Paulo Sa˜o Paulo, SP, Brazil Tarcisio Eloy Pessoa de Barros Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Valentim Gentil Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Wagner Farid Gattaz Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Board of Governors Alberto Jose´ da Silva Duarte Aluisio Augusto Cotrim Segurado Ana Claudia Latronico Xavier Berenice Bilharinho de Mendonc¸a Carlos Roberto Ribeiro de Carvalho Clarice Tanaka Claudia Regina Furquim de Andrade Cyro Festa Neto Dalton de Alencar Fischer Chamone Daniel Romero Mun˜oz Edmund Chada Baracat Eduardo Massad Eloisa Silva Dutra de Oliveira Bonfa´ Euripedes Constantino Miguel Fa´bio Biscegli Jatene Flair Jose´ Carrilho Gerson Chadi Gilberto Luis Camanho Irene de Lourdes Noronha Irineu Tadeu Velasco Ivan Cecconello Jorge Elias Kalil

Jose´ Antonio Franchini Ramires Jose´ Antonio Sanches Jose´ Eduardo Krieger Jose´ Ota´vio Costa Auler Jose´ Ricardo de Carvalho Mesquita Ayres Lenine Garcia Branda˜o Luiz Augusto Carneiro D’Albuquerque Luiz Fernando Onuchic Magda Maria Sales Carneiro-Sampaio Manoel Jacobsen Teixeira Marcelo Zugaib Marcos Boulos Marcus Castro Ferreira Maria Aparecida Shikanai Yasuda Maria Irma Seixas Duarte Miguel Srougi Milton de Arruda Martins Nelson de Luccia Olavo Pires de Camargo Paulo Andrade Lotufo Paulo Hila´rio Nascimento Saldiva Paulo Marcelo Gehm Hoff

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

Pedro Puech-Lea˜o Remo Susanna Ricardo Ferreira Bento Ricardo Nitrini Roberto Kalil 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 Werther Brunow de Carvalho William Carlos Nahas Wilson Jacob

Editorial Assistants Nair Gomes Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Daniela Aquemi Higa Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil Ariane Maris Gomes Faculdade de Medicina da Universidade de Sa˜o Paulo Sa˜o Paulo, SP, Brazil

Editorial Office: Rua Dr. Ovı´dio Pires de Campos, 225 - 6 ˚ Andar CEP 05403-010 Sa˜o Paulo/SP Tel.: +55-11-2661-6235 Email: clinics.office@gmail.com Website: www.scielo.br/clinics Submission: http://mc04.manuscriptcentral.com/clinics-scielo 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. Sa˜o Paulo: Scientific Journal of Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, 2005Monthly Periodical: January to December ISSN 1807-5932 printed version ISSN 1980-5322 online version Formerly Revista do Hospital das Clı´nicas da FMUSP, 1946–2004. 1. Medicine-scientific production. 2. Medical Sciences I. Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o 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 and 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 German Research Foundation (DFG), which request or require deposition of the published articles that they fund into 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 prospectively registered (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 regarding 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 as a user friendly site.

clinical, and surgical research. Original studies must conform to the following format: Title page:

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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 published article. Our 2012 prices are as follows: fast track: US$ 1,500.00; original articles, review articles and rapid communications: US$ 1,100.00. Invited reviews, editorials and letters to the editors: no charge. * The exchange rate for payments in Brazil-Real is the commercial exchange rate of the day the articles is accepted. Clinics uses the Banco do Brasil currency conversion tool. 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 consent form is signed by the participating patient, other human participant, or his/her legally constituted representative. Manuscripts should be digitalized using a Word *.doc-compatible software program and submitted online in English. Authors are strongly advised to submit the manuscript in its final form to a spell check for English (US). 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 strongly advised to use abbreviations sparingly whenever possible to avoid jargon and improve the readability of the manuscript. All abbreviations must be defined the first time that they are used. Only terms or expressions that are used at least 5 times throughout the text should be abbreviated. Never use abbreviations that spell common English words, such as FUN, PIN, SCORE and SUN. Please make sure to submit your manuscript in the exact format that is 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,

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 degrees). 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.

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Abstract: Abstracts are limited to 250 words and structured into objectives, method, results, and conclusions. Citations or abbreviations (except internationally recognized abbreviations, such as weights, measures, and physical or chemical abbreviations) are not permitted. Authors are strongly encouraged not to display numerical statistical information but to merely state what is significantly different (or not) between the described parameters. Keywords: For keywords, 3–6 items from the Medical Subject Headings (MeSh) should be used. Introduction: The introduction should set the purpose of the study, provide a brief summary (not a review) of previous relevant studies, and state the new advances in the current investigation. The introduction should not include data or conclusions from the work being reported. A final sentence summarizing the novel finding to be presented is permissible. Materials and Methods: This section 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 were in accordance with the ethical standards of the responsible committee on human experimentation (institutional or regional) and with the Helsinki Declaration of 1975, which was revised in 1983. When reporting experiments on animals, indicate whether the institution’s guide, a national research council’s guide, or any national law on the care and use of laboratory animals was followed. Results: The results section should be a concise account of the new information that was discovered, with the least personal judgment. Do not repeat in text all the data in the tables and illustrations but briefly describe what these data comprise. Discussion: The discussion should include the significance of the new information and relevance of the new findings in light of existing knowledge. Only unavoidable citations should be included. Citation to review articles are not encouraged in this section. Acknowledgments: This section should be short, concise, and restricted to acknowledgments that are necessary.


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References in text: CLINICS adopts the Vancouver format. Cite references in the text with Arabic numerals in the order of appearance, within parentheses, (1) after the 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 …’’ or 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 that is 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 the first six authors followed by ‘‘et al.’’. Tables and Figures: The maximum number of tables and/or figures is six tables and/or figures. Tables: Do not 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. The tables should be numbered in order of appearance in the text, using Arabic numerals. Each table should have a title and an explanatory legend, if necessary. All tables must be referenced and succinctly described in the text. Under no circumstances should a table repeat data that are 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. Number figures consecutively using Arabic numerals in order of appearance. Upload each figure individually into the system. Figure legend(s) should be descriptive and should 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. Images must be of professional quality and uploaded as *.tiff files. Typewritten or hand-lettered notations or figures that are generated by dot matrix printers are unacceptable. Generally, figures will be reduced to fit one column of text. The actual magnification of all photomicrographs should be provided, preferably by placing a scale bar on the print. Line graphs and charts should never be sent as *.jpeg illustrations. We recommend preparing line graphs and charts as ExcelH files and copying these files into a Word *.doc sheet.

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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 will produce a first-action response in the shortest possible time and will 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 the Comments section, the authors must explain the justification for fast-track publication. Rejection by journals with a higher impact factor than ours is an acceptable reason for requesting fast-track status. However, the reviewers’ reports from the previous submission must be included in the current 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: Review articles should cover themes that are relevant to medical practice. Spontaneously submitted reviews are welcome; however, 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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Title page: As described in the Original Study section. Manuscript: Abstract, keywords and text should be arranged to cover the subject that is being reviewed. If appropriate, the method of reference collection should be described. The use of headings, subheadings, and paragraph titles is encouraged to improve clarity. Abbreviations, acknowledgments, tables and figures should be formatted as described in the Original Study section. The number of references is at the discretion of the authors. No publication fee discount is allowed for spontaneously submitted review articles that are accepted for publication.

RAPID COMMUNICATIONS:

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Title page: As described in the Original Study section. Manuscript: Rapid communications are limited to 1,500 words, not including the reference list. Authors should format rapid communications based on the subject at hand. No abstract or keywords are required. Please copy an introductory sentence into the abstract box on Page 1 of the submission procedure.

LETTERS TO THE EDITOR: Letters to the editor expressing comments or dissenting opinions concerning articles that have been recently published in CLINICS are not submitted to peer review and are published at the discretion of the editor. A letter is a single section containing untitled text concerning the article under discussion, followed by references. No publication fee is charged for this class of manuscripts. EDITORIAL: Editorials should cover broad aspects of medical or biological sciences. Such manuscripts are not submitted to peer review and are published at the discretion of the editor. No publication fee is charged for this class of manuscripts.


COMMENTARY: A commentary is an invited text with respect to an article that is being published by Clinics. No publication fee is charged for this class of manuscripts. INVITED REVIEW: These reviews are by invitation only and follow the format proposed for general reviews. No publication fee is charged for this class of manuscripts. SPECIAL ISSUE ARTICLE: Special issue articles are by invitation only and follow a specific format that is set by the editor in charge of the collection. Currently CLINICS does not accept: Case Reports, Technical Notes, Retrospective Studies, Translations and Validations of Questionnaires, and articles referring to First Demonstration in Brazil. Peer Review: Manuscripts are reviewed by at least two expert consultants. Accepted manuscripts are edited to comply with the journal’s format, remove redundancies, and 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 may 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). When the authors are satisfied that the manuscript complies with the journal format, our site should be accessed using the website www.clinics.org.br. The system will guide authors through the manuscript submission process and will 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 the submission when the submission letter reaches the office. The progress of the manuscript through the Editorial Office’s procedures will be available to authors at all times.


ISSN-1807-5932

CLINICS CONTENTS Clinics 2012 67(12):1353–1532

EDITORIAL

Molecular and genetic aspects of oncology and infectology in Clinics Mauricio Rocha-e-Silva . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1353

CLINICAL SCIENCES

Supine sleep and positional sleep apnea after acute ischemic stroke and intracerebral hemorrhage Millene R. Camilo, Regina M. F. Fernandes, Heidi H. Sander, Fernando Nobre, Taiza Santos-Pontelli, Antonio C. dos Santos, Draulio B. de Araujo, Joa˜o P. Leite, Octavio M. Pontes-Neto . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1357

The effects of one-half of a soccer match on the postural stability and functional capacity of the lower limbs in young soccer players Ricardo Kim Fukushi Yamada, Gustavo Gonc¸alves Arliani, Gabriel Peixoto Lea˜o Almeida, Andre´ Manrique Venturine, Ciro Veronese dos Santos, Diego Costa Astur, Moise´s Cohen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1361

The perception of sleep quality in kidney transplant patients during the first year of transplantation Dnyelle Souza Silva, Elisangela dos Santos Prado Andrade, Rosilene Motta Elias, Elias David-Neto, William Carlos Nahas, Manuel Carlos Martins de Castro, Maria Cristina Ribeiro de Castro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1365

Differential expression of hypoxia-inducible factor 1a in non-small cell lung cancer and small cell lung cancer Eleni Karetsi, Maria G. Ioannou, Theodora Kerenidi, Markos Minas (in memoriam), Paschalis A. Molyvdas, Konstantinos I. Gourgoulianis, Efrosyni Paraskeva . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1373

Mechanical thrombectomy with solitaire stent retrieval for acute ischemic stroke in a Brazilian population Luis Henrique de Castro-Afonso, Thiago Giansante Abud, Octa´vio Marques Pontes-Neto, Lucas Moretti Monsignore, Guilherme Seizem Nakiri, Pedro Telles Cougo-Pinto, Lı´via de Oliveira, Daniela dos Santos, Francisco A Dias, Soraia Cabette Ramos Fa´bio, Francisco Antoˆnio Coletto, Daniel Giansante Abud . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1379

Nutritional risk and anthropometric evaluation in pediatric liver transplantation Patrı´cia Zamberlan, Cla´udio Leone, Uenis Tannuri, Werther Brunow de Carvalho, Artur Figueiredo Delgado. . . . . 1387

Novel GATA5 loss-of-function mutations underlie familial atrial fibrillation Jian-Yun Gu, Jia-Hong Xu, Hong Yu, Yi-Qing Yang . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1393


The impact of asymptomatic vertebral fractures on quality of life in older community-dwelling women: the Sa˜o Paulo Ageing & Health Study Jaqueline B. Lopes, Leandro K. Fung, Caroline C. Cha, Gustavo M. Gabriel, Liliam Takayama, Camille P. Figueiredo, Rosa Maria R. Pereira . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1401

Effects of Brazilian Cardioprotective Diet Program on risk factors in patients with coronary heart disease: a Brazilian Cardioprotective Diet randomized pilot trial Bernardete Weber, Andrea Polo Galante, Angela Cristine Bersch-Ferreira, Camila Ragne Torreglosa, Vitor Oliveira Carvalho, Elivane da Silva Victor, Jose Amalth do Espı´rito-Santo, Maria Beatriz Ross-Fernandes, Rafael Marques Soares, Rosana Perim Costa, Enilda de Sousa Lara, Anna Maria Buehler, Ota´vio Berwanger . . . . . . . . . . 1407

Standard surgical treatment for benign prostatic hyperplasia is safe for patients over 75 years: Analysis of 100 cases from a high-volume urologic center Rafael Marmiroli, Alberto A. Antunes, Sabrina T. Reis, Elcio Nakano, Miguel Srougi . . . . . . . . . . . . . . . . . . . . . 1415

Plantar thermography is useful in the early diagnosis of diabetic neuropathy Luciane Fachin Balbinot, Luis Henrique Canani, Caroline Cabral Robinson, Matilde Achaval, Milton Antoˆnio Zaro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1419

The modified Hodge test is a useful tool for ruling out klebsiella pneumoniae carbapenemase Ana Paula Cury, Denise Andreazzi, Ma´rcia Maffucci, He´lio Hehl Caiaffa-Junior, Fla´via Rossi . . . . . . . . . . . . . . . 1427

The influence of anthropometric factors on postural balance: the relationship between body composition and posturographic measurements in young adults Ange´lica Castilho Alonso, Nata´lia Mariana S. Luna, Luis Mochizuki, Fa´bio Barbieri, Sileno Santos, Julia Maria D’Andre´ia Greve . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1433

Serum adenosine deaminase, catalase and carbonic anhydrase activities in patients with bladder cancer Necip Pirinc¸c¸i, I˙lhan Gec¸it, Mustafa Gu¨nes¸, Mehmet Bilgehan Yu¨ksel, Mehmet Kaba, Serhat Tanık, Halit Demir, Mehmet Aslan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1443

BASIC RESEARCHES

Antioxidant enzyme activity and malondialdehyde levels can be modulated by Piper betle, tocotrienol rich fraction and Chlorella vulgaris in aging C57BL/6 mice Nor Syahida Aliahmat, Mohd Razman Mohd Noor, Wan Junizam Wan Yusof, Suzana Makpol, Wan Zurinah Wan Ngah, Yasmin Anum Mohd Yusof . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1447

Effects of the administration of pentoxifylline and prednisolone on the evolution of portal fibrogenesis secondary to biliary obstruction in growing animals: immunohistochemical analysis of the expression of TGF- b and VEGF Wagner de Castro Andrade, Luiz Fernando Ferraz da Silva, Maria Cecilia de Mendonc¸a Coelho, Ana Cristina Aoun Tannuri, Venancio Avancini Ferreira Alves, Uenis Tannuri . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1455

Hypertonic saline solution reduces the inflammatory response in endotoxemic rats Mariana Cardillo Theobaldo, Hermes Vieira Barbeiro, Denise Frediani Barbeiro, Ricardo Petroni, Francisco Garcia Soriano. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1463

The effects of exercise modalities on adiposity in obese rats Guilherme Fleury Fina Speretta, Marisa Cristina Rosante, Fernanda Oliveira Duarte, Richard Diego Leite, Anderson Diogo de Souza Lino, Rafael Arquias Andre, Joa˜o Guilherme de Oliveira Silvestre, Heloisa Sobreiro Selistre de Araujo, Ana Claudia Garcia de Oliveira Duarte . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1469


RAPID COMMUNICATION

Does lack of improvement in the first two weeks predict treatment resistance in recent-onset psychosis? Monica Kayo, Ivson Tassell, Vivian Hiroce, Anny Menezes, Helio Elkis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1479

CASE REPORTS

Inevitable hemodialysis for treating resistant hypertension in a patient with Leriche syndrome Murvet Yilmaz, Ozlem Harmankaya Kaptanogullari, Can Caliskan, Ayse Sinangil Arar, Cuneyt Akgol, Kayhan Erturk, Abdulkadir Unsal. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1483

Association of myelodysplastic syndrome with CD5+, CD23+ monoclonal B-cell lymphocytosis Alex F. Sandes, Maria de Lourdes L. F. Chauffaille, Alberto Orfao, Graziella C. Siufi, Maria Regina R. Silva, Mihoko Yamamoto . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1487

Acute abdominal pain in a 24-year-old woman: Fitz-Hugh-Curtis syndrome associated with pyelonephritis Giorgio Di Rocco, Domenico Giannotti, Marco Collalti, Rita Mele, Stefano Pontone, Francesca Frezzotti, Adriano Redler, Gregorio Patrizi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1493

Rare association of cutaneous vasculitis, IgA nephropathy and antiphospholipid antibody syndrome with tuberculous lymphadenitis Roberto Bueno Filho, Alberto Pinto Cordeiro, Flavia Tremeschin de Almeida, Catarina Shaletich, Roberto Silva Costa, Ana Maria F. Roselino. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1497

Birt-Hogg-Dube´ syndrome: metalloproteinase activity and response to doxycycline Suzana Pinheiro Pimenta, Bruno Guedes Baldi, Ellen Caroline Toledo do Nascimento, Thais Mauad, Ronaldo Adib Kairalla, Carlos Roberto Ribeiro Carvalho . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1501

Fractures in connection with an atypical form of craniodiaphyseal dysplasia: case report of a boy and his mother Ali Al Kaissi, Robert Csepan, Jochen G. Hofstaetter, Klaus Klaushofer, Rudolf Ganger, Franz Grill . . . . . . . . . . . 1505

Challenges in patients supported with extracorporeal membrane oxygenation in Brazil Pedro Vitale Mendes, Ewandro Moura, Edzangela Vasconcelos Santos Barbosa, Adriana Sayuri Hirota, Paulo Rogerio Scordamaglio, Fabiana Maria Ajjar, Eduardo Leite Vieira Costa, Luciano Cesar Pontes Azevedo, Marcelo Park, on behalf of ECMO Group . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1511

Right coronary artery originating from the distal left circumflex artery, evaluation using three imaging techniques ¨ mer Go¨ktekin . . . . 1517 Murat Turfan, Cemalettin Aydin, Mehmet Ali Elbey, Ercan Erdog˘an, Emin Asoglu, Halil Basel, O

Glove port single-incision laparoscopic splenectomy and the treatment of its complications Erkin Ismail, Cihangir Akyol, Salim Ilksen Basceken, Utku Tantoglu, Ilgaz Kayilioglu, Atıl Cakmak . . . . . . . . . . . 1519

Congenital aneurysmal circumflex coronary artery fistula in a pregnant woman Meliza Goi Roscani, Silmeia Garcia Zanati, Pericles S. Salmazo, Fabio C. Carvalho, Claudia G. Magalha˜es, Vera T.M. Borges, Edson A. Bregagnollo, Beatriz B. Matsubara, Joa˜o Carlos Hueb . . . . . . . . . . . . . . . . . . . . . . . 1523

Successful treatment of thrombotic thrombocytopenic purpura associated with mitral valve replacement Yong Liu, Jian Zhu, Er-Ping Xi, Wei Jiang, Feng Xia, Shui-Bo Zhu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1527


Respiratory failure after lung transplantation: extracorporeal membrane oxygenation as a rescue treatment Paulo Manuel Peˆgo-Fernandes, Ludhmila Abraha˜o Hajjar, Filomena Regina Barbosa Gomes Galas, Marcos Naoyuki Samano, Alexandre Kazantzi Fonseca Ribeiro, Marcelo Park, Rodolfo Soares, Eduardo Osawa, Fabio Biscegli Jatene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1529


CLINICS 2012;67(12):1353-1356

DOI:10.6061/clinics/2012(12)01

EDITORIAL

Molecular and genetic aspects of oncology and infectology in Clinics Mauricio Rocha-e-Silva Faculdade de Medicina da Universidade de Sa˜o Paulo, Hospital das Clı´nicas, Sa˜o Paulo/SP, Brazil.

The number of papers regarding the molecular and genetic aspects of a number of different pathological entities are on the rise in Clinics. This editorial selects some of the most outstanding contributions in oncology and infectology using continuously variable ratings (1) as the selection tool. Brito et al. (2) report that hypoxia-inducible factor-1alpha is associated with a poor prognosis and that vascular endothelial growth factor-C can be used as a predictive factor in locally advanced breast cancer patients with complete pathological responses after neoadjuvant chemotherapy. Cabral et al. (3) report that the overall alterations that were observed in the repetitive DNA of actinic keratosis and squamous cell carcinoma indicate the presence of a spectrum of malignant progression. Cani et al. (4) find evidence of beta-catenin gene overexpression in the majority of adamantinomatous craniopharyngioma cases and detected a nuclear beta-catenin staining pattern regardless of the presence of a beta-catenin gene mutation, which suggests that WNT signaling activation plays an important role in the pathogenesis of adamantinomatous craniopharyngioma. Carvalho et al. (5) claim that glucose transporter 1 is a valuable immunohistochemical marker for the malignant tumors of various cell types and can be used to identify patients with such tumors using positron emission tomography scanning. Cintra et al. (6) provide evidence that CD34 microvessel density in chondrosarcoma can be helpful in predicting patient outcomes and may add to our understanding of chondrosarcoma pathogenesis. Costa et al. (7) report on a first investigation of glucosedependent insulinotropic peptide receptor expression in adrenocortical lesions without 11q13 loss of heterozygosity in MEN1 syndrome patients. Cunha et al. (8) report that the inheritance of a G allele in the interleukin-10 G/A polymorphism at position -1082 may favor a concurrent thyroid autoimmunity in differentiated thyroid carcinoma patients and that this autoimmunity may provide a better prognosis for these patients. El Moneim and Zaghloul (9) report that the loss of Ecadherin and overexpression of N-cadherin and Snail in

breast carcinomas may play a central role in the development of invasive ductal breast carcinoma and may provide a valuable reference for studying invasive ductal carcinoma progression and characterizing the biological behavior of this tumor. Estrozi and Bacchi (10) report that the proportion of neuroendocrine tumor cases among the total number of surgical pathology cases at our institution over the past 12 years is increasing. Florence et al. (11) employ the Chalkley method to quantify the microvascular area by comparing panendothelial viability (CD34) with neoangiogenesis (CD105) immunohistochemical markers and find that skin carcinogenesis depended on angiogenesis. Gerhard et al. (12) report that thyroid carcinomas show increased immunohistochemical N-myc downstream-regulated gene 1 expression compared with normal and benign thyroid lesions and are correlated with more advanced tumor stages. Levy et al. (13) report that the H/R FccRIIa131 polymorphism has no impact on treatment outcomes, including the overall response rate, overall survival time and disease-free survival time, in a Brazilian population of DLBCL patients who were treated with R-CHOP. Melotti et al. (14) report that using the immunoglobulin heavychain FR3-trad and immunoglobulin light-chain kappa Biomed protocols for clonality analysis improved diagnostic accuracy. Ozerhan et al. (15) report that fascin is heterogeneously expressed in approximately one third of colorectal carcinomas and has a significant association with lymph node metastasis, tumor stage and location, which indicates that fascin may have a role in the lymph node metastasis of colorectal carcinomas. Reis et al. (16) report that TGF-beta1 was underexpressed in prostate cancers; however, a higher expression was observed in tumors with higher Gleason scores, which suggests that TGF-beta1 expression may be a useful prognostic marker for prostate cancer. Rodrigues et al. (17) report that the XRCC1 Arg194Trp variant of the tumor protein 53, which is positively associated with Elston grade III breast tumors, may influence breast cancer development and prognosis. Samarghandian et al. (18) report that honey has antiproliferative effects on prostate cancer cells and that these effects are mainly caused by chrysin. Therefore, chrysin may be a potential compound for both cancer prevention and treatment. Further in vivo investigation is needed to support the use of chrysin in cancer therapy. Toledo et al. (19) present the first molecular analysis of non-pituitary tumors in AIP-mutated patients. The finding of AIP inactivation in adrenocortical tumors suggests that further investigation of the potential role of this recently identified tumor suppressor gene in non-pituitary tumors (particularly in tumors in

Email: mauricio.silva@hc.fm.usp.br Tel.: 11 2661 6235 Copyright ß 2012 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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CLINICS 2012;67(12):1353-1356

a nested polymerase chain reaction assay, along with clinical findings, and followed the outcomes of patients undergoing liver transplants. The authors find that few patients remain free of beta herpesviruses after liver transplantation and that nested polymerase chain reaction assays may be of limited value in monitoring cytomegalovirus and human herpesvirus 6. Costa et al. (31) report that the Pediatric Risk Of Mortality Score showed adequate discriminatory capacity and thus constitutes a useful tool for assessing the prognoses of pediatric patients who are admitted to tertiary pediatric intensive care units. Crovella et al. (32) search for the presence of HLA B*5701 in 96 HIVpositive patients who were treated with abacavir and 243 healthy subjects from northeastern Brazil to verify the percentage of HLA B*5701 allele carriers in HIV patients and in the general population. Cursino et al. (33) detect higher levels of anti-retina antibodies in uveitis patients and in a small fraction of asymptomatic patients with chronic toxoplasmosis. The presence of anti-retina antibodies in sera may be a marker of eye disease in asymptomatic patients, particularly when whole human retina extract is used in a solid-phase ELISA. Franco et al. (34) report that metallobeta-lactamases among imipenem-resistant Pseudomonas aeruginosa were detected in 30.4% of the imipenemresistant Pseudomonas aeruginosa isolates. This number may have been higher if other genes had been included. SPM-1 was the predominant enzyme found. Phenotypic tests with low kappa values could be misleading when testing for metallo-beta-lactamases, and polymerase chain reaction detection thus remains the gold standard. Gomes et al. (35) compare three different control charts (traditional Shewhart control, cumulative sum, and exponentially weighted moving average charts) to monitor the nosocomial infection rate per 1,000 patient-days and report that the ability to detect nosocomial outbreaks improved by using the information provided by all three control charts. Kebapcilar et al. (36) find that H. pylori eradication reduces the levels of pro-inflammatory cytokines, such as migration inhibitory factor and hs-CRP, and also significantly increases anti-inflammatory markers, such as fetuin-A. Marchiori et al. (37) characterize the neuroinfection profiles in a tertiary neurological ward and find that the results are similar to those observed in developed and developing countries. They claim that comparisons with the literature may be considered health assistance quality control. Nascimento et al. (38) report that the widespread use of molecular-based methods yields new insights into the etiology of the studied viral diseases, but the impact of viral etiologies on early outcomes is still unclear. NishiwakiDantas et al. (39) report that the association between vernal keratoconjunctivitis and Chlamydia trachomatis infection was confirmed by positive direct fluorescent antibody assays in 49.4% of vernal keratoconjunctivitis patients and by positive polymerase chain reactions in 20% of these patients. Olandoski et al. (40) report that patient referral to a pediatric nephrologist was late. A reduction in the number of urinary tract infections was observed with adequate treatment, but microalbuminuria and metabolic acidosis occurred frequently despite adequate management. Pacheco et al. (41) report that dermatology patients are colonized by community- and hospital-acquired Staphylococcus aureus. Half of the nosocomial Staphylococcus aureus isolates were SCCmec type IV. Despite the identification of the colonized patients, the subsequent implementation of contact precautions and

which the cAMP and the 11q13 locus are implicated) may be worthwhile. Uno et al. (20) report that O6-methylguanineDNA-methyltransferase promoter methylation status is a more reliable predictor of adjuvant therapy response and glioblastoma prognosis than MGMT protein or gene expression levels. Uno et al. (21) establish the frequency of the IDH1 mutation in a Brazilian study on glioblastoma, thereby confirming the IDH1 mutation as a genetic marker for secondary GBM and providing complementary information to help predict glioblastoma patient outcomes. Yazbek et al. (22) claim that Anti-Epstein-Barr nuclear antigen-1 antibodies did not increase the risk for rheumatoid arthritis and were not associated with the studied rheumatoid arthritis risk factors. Smoking and shared epitope alleles correlated with anti-cyclic citrullinated peptide-antibody-positive rheumatoid arthritis. Of the risk factors, only anticyclic citrullinated peptide antibodies were independently associated with rheumatoid arthritis. Zhang et al. (23) report that tumor-associated macrophages in lung adenocarcinoma have an M2-polarized subtype and are associated with poor prognoses, which may result from accelerated lymphangiogenesis and lymph node metastasis. Aikawa et al. (24) document that microsporidiosis with intestinal mucosa disruption is frequent in patients undergoing concomitant anti-tumor necrosis factor/disease-modifying anti-rheumatic drug therapy. Burns et al. (25) report that although pneumonia had little influence on pulmonary gas exchange in immature female swine, it influenced cardiac output, urine output and survival compared with healthy swine, which may lead to a diminished physiologic reserve. The authors claim that this information may be relevant in patients with subclinical infection who are stressed by hemorrhage and may partially explain why some similarly injured patients require more resuscitation efforts than others. Capelozzi et al. (26) report that in cases of H1N1 and other pulmonary infections, virallike particles can be successfully observed in lung tissue by ultrastructural examination without confirming the virus by RT-PCR in nasopharyngeal aspirates. The authors also claim that bronchioles and epithelium, rather than endothelium, are most likely the primary targets of infection. They also maintain that diffuse alveolar damage causes airway obliteration and innate immunity dysfunction, which suggests that treatment should be focused on epithelial repair. Carrilho et al. (27) report that the epidemiology, classification, and therapy selection for hepatocellular carcinoma varied among Brazilian regions, that hepatitis C infection was the most common etiology of liver cirrhosis and that chemoembolization was the most common therapy employed. Carrillo et al. (28) report that the characteristics of a population treated in the dentistry unit of the hematology-oncology service of a tertiary teaching hospital in SaËœo Paulo, Brazil were similar to those of the general Brazilian and global populations, particularly with regard to gender distributions and diagnoses. Contri et al. (29) report that the use of protease inhibitors per se does not seem to significantly interfere with the anthropometric measures, body composition and food intake of HIV-infected children and adolescents. However, this antiretroviral therapy was associated with a significant increase in triglyceride and LDL-cholesterol levels in the subjects. Costa et al. (30) simultaneously monitored active cytomegalovirus and human herpesvirus 6 infections using

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room placements, Staphylococcus aureus colonization continued to occur and colonization pressure increased. Pemphigus and other bullous diseases were associated with Staphylococcus aureus. Souza et al. (42) claim that resistance exercise safely increases the strength of older patients living with HIV and allows them to achieve the performance levels that are observed in otherwise healthy controls. These findings favor the recommendation of resistance exercise for elderly adults living with HIV-infected adults. Souza et al. (43) find that ozone therapy modulates the inflammatory response and acute lung injury in the cecal ligation/puncture infection rat model, although there is no improvement in the survival rates. Tannuri et al. (44) report that in newborns with gastroschisis, more aggressive attention to hyponatremia and hypoalbuminemia would improve patient outcomes. Teng et al. (45) explore the characteristics and outcomes of myeloma patients with chronic hepatitis virus infection and claim that these patients may be a distinct subgroup and that close monitoring of adverse hepatic events should be mandatory. Toufen et al. (46) report on the long-term follow-up of patients with swine-origin influenza A virus infection that progressed to acute respiratory distress syndrome and claim that despite the marked severity of lung disease at admission, these patients present with a late but substantial recovery over six months of follow-up.

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41. Pacheco RL, Lobo RD, Oliveira MS, Farina EF, Santos CR, Costa SF, et al. Methicillin-resistant Staphylococcus aureus (MRSA) carriage in a dermatology unit. Clinics. 2011;66(12):2071-7, http://dx.doi.org/10. 1590/S1807-59322011001200012. 42. Souza PM, Jacob-Filho W, Santarem JM, Zomignan AA, Burattini MN. Effect of progressive resistance exercise on strength evolution of elderly patients living with HIV compared to healthy controls. Clinics. 2011;66(2):261-6, http://dx.doi.org/10.1590/S1807-59322011000200014. 43. Souza YM, Fontes B, Martins JO, Sannomiya P, Brito GS, Younes RN, et al. Evaluation of the effects of ozone therapy in the treatment of intra-abdominal infection in rats. Clinics. 2010;65(2):195-202, http://dx. doi.org/10.1590/S1807-59322010000200012. 44. Tannuri AC, Sbragia L, Tannuri U, Silva LM, Leal AJ, Schmidt AF, et al. Evolution of critically ill patients with gastroschisis from three tertiary centers. Clinics. 2011;66(1):17-20, http://dx.doi.org/10.1590/S180759322011000100004. 45. Teng CJ, Liu HT, Liu CY, Hsih CH, Pai JT, Gau JP, et al. Chronic hepatitis virus infection in patients with multiple myeloma: clinical characteristics and outcomes. Clinics. 2011;66(12):2055-61, http://dx.doi.org/10.1590/ S1807-59322011001200010. 46. Toufen C, Jr., Costa EL, Hirota AS, Li HY, Amato MB, Carvalho CR. Follow-up after acute respiratory distress syndrome caused by influenza a (H1N1) virus infection. Clinics. 2011;66(6):933-7, http://dx.doi.org/10. 1590/S1807-59322011000600002.

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CLINICAL SCIENCE

Supine sleep and positional sleep apnea after acute ischemic stroke and intracerebral hemorrhage Millene R. Camilo,I Regina M. F. Fernandes,I Heidi H. Sander,I Fernando Nobre,II Taiza Santos-Pontelli,I Antonio C. dos Santos,III Draulio B. de Araujo,IV Joa˜o P. Leite,I Octavio M. Pontes-NetoI I

University of Sa˜o Paulo, Ribeira˜o Preto School of Medicine, Neurology Division, Department of Neuroscience and Behavioral Sciences, Ribeira˜o Preto/SP, Brazil. II University of Sa˜o Paulo, Ribeira˜o Preto School of Medicine, Cardiology, Ribeira˜o Preto/SP, Brazil. III University of Sa˜o Paulo, Ribeira˜o Preto School of Medicine, Cardiology, Radiology Division, Department of Internal Medicine, Ribeira˜o Preto/SP, Brazil. IV Federal University of Rio Grande do Norte, Brain Institute/Onofre Lopes University Hospital, Natal/RN, Brazil.

OBJECTIVE: Obstructive sleep apnea is frequent during the acute phase of stroke, and it is associated with poorer outcomes. A well-established relationship between supine sleep and obstructive sleep apnea severity exists in nonstroke patients. This study investigated the frequency of supine sleep and positional obstructive sleep apnea in patients with ischemic or hemorrhagic stroke. METHODS: Patients who suffered their first acute stroke, either ischemic or hemorrhagic, were subjected to a full polysomnography, including the continuous monitoring of sleep positions, during the first night after symptom onset. Obstructive sleep apnea severity was measured using the apnea-hypopnea index, and the NIHSS measured stroke severity. RESULTS: We prospectively studied 66 stroke patients. The mean age was 57.6¡11.5 years, and the mean body mass index was 26.5¡4.9. Obstructive sleep apnea (apnea-hypopnea index $5) was present in 78.8% of patients, and the mean apnea-hypopnea index was 29.7¡26.6. The majority of subjects (66.7%) spent the entire sleep time in a supine position, and positional obstructive sleep apnea was clearly present in the other 23.1% of cases. A positive correlation was observed between the NIHSS and sleep time in the supine position (rs = 0.5; p,0.001). CONCLUSIONS: Prolonged supine positioning during sleep was highly frequent after stroke, and it was related to stroke severity. Positional sleep apnea was observed in one quarter of stroke patients, which was likely underestimated during the acute phase of stroke. The adequate positioning of patients during sleep during the acute phase of stroke may decrease obstructive respiratory events, regardless of the stroke subtype. KEYWORDS: Stroke; Sleep Apnea; Supine; Positional; Intracerebral Hemorrhage. Camilo MR, Fernandes RM, Sander HH, Nobre F, Santos-Pontelli T, dos Santos AC, et al. Supine sleep and positional sleep apnea after acute ischemic stroke and intracerebral hemorrhage. Clinics. 2012;67(12):1357-1360. Received for publication on July 6, 2012; Accepted for publication on August 6, 2012 E-mail: opontesneto@fmrp.usp.br Tel.: 55 16 3602-2556

OSA and perihematoma edema in patients with hypertensive ICH (2). The severity of breathing abnormalities in the general OSA population is frequently related to the body position during sleep. The supine position influences the occurrence of sleep-disordered breathing by increasing the number and severity of apneic events (6). The collapsibility of the upper airways is higher in the supine position than in the lateral decubitus position during all sleep stages (7). Positional OSA occurs when patients exhibit an AHI while sleeping in the supine position that is at least two times higher than their lateral AHI (8). Positional OSA and supine position during sleep are frequent in ischemic stroke patients (9,10). However, the frequency of supine sleep and positional OSA within the first 24 hours after stroke and in ICH patients are controversial. Therefore, we investigated the frequency of supine sleep and positional OSA during the first night after an acute ischemic stroke or primary ICH.

INTRODUCTION Obstructive sleep apnea (OSA) is frequent during the acute phase of stroke; it occurs in 62% of patients with ischemic stroke and 59.4% of patients with primary intracerebral hemorrhage (ICH) (1,2). The impact of OSA is clinically significant after ischemic stroke because it produces early neurological deterioration, poor functional outcome, and increased long-term mortality (3,4). The apnea-hypopnea index (AHI), which measures apnea severity, is an independent predictor of mortality in these patients (5). We recently reported a relationship between

Copyright ß 2012 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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was used for categorical data, and the t test or the MannWhitney U test compared the means or the distribution of AHI values between supine and lateral positions, respectively. The percentage of the total sleep time spent in the supine position was tested for rank correlation with stroke severity (NIHSS) using the Spearmanโ€™s rank correlation coefficient (rs). All statistical analyses were performed using the SPSS software package, version 17.0 (Chicago, IL, USA).

SUBJECTS AND METHODS Patients were prospectively recruited from the Emergency Unit of our University Hospital. Subjects were eligible for the study if they were over 18 years of age and presented with their first ischemic stroke or primary ICH. The following exclusion criteria were used: orotracheal intubation; severe chronic obstructive pulmonary disease; decompensated heart failure; recent myocardial infarction; .24 hours between the onset of stroke symptoms and hospital admission; and secondary causes of ICH. Demographic data and vascular risk factors of all patients were recorded. The Ethics Committee at our institution approved this study. Written informed consent was obtained from all of the patients or their relatives. Subjects underwent a full polysomnography (PSG) during the first night after stroke symptoms onset from 11:00 PM to TM 7:00 AM using a digital system (BioLogic Sleepscan II ; Mundelein, Il., USA). The PSG did not interfere with the conventional care of the patient. System variables included six EEG channels (F3-A2, F4-A1, C3-A2, C4-A1, O1-A2 and O2-A1 of the international electrode placement system), two electro-oculographic leads, chin and bilateral anterior tibialis surface electromyograms, electrocardiogram, nasal and oral airflow, thoracic and abdominal movements and finger pulse oximetry. Sleep stages and respiratory events were scored using standard criteria (11). Apnea was defined as the absence of airflow for at least 10 seconds. Respiratory effort was maintained in obstructive apnea, but breathing movements were absent in central apnea. Mixed apnea was defined as a combination of central and obstructive apnea. Hypopnea was defined as a thoracoabdominal amplitude decrease of $50% of baseline for at least 10 seconds with an arousal or oxygen desaturation $3%. Cheyne-Stokes respiration was defined as a periodic crescendo and decrescendo breathing pattern with central apnea or hypopnea. The AHI was calculated as the average number of apnea and hypopnea episodes per hour of sleep. Sleep apnea was further classified as obstructive or central in each patient according to the type of predominating event. OSA was defined as an AHI$5 with a predominance of obstructive events (1,3). The sleep positions (e.g., prone, supine, left or right side) were recorded continuously using an electronic position sensor and were confirmed by the sleep technologist during the PSG. The subjects were not provided with instructions for their sleep position. Positional OSA was defined when AHI$5; the AHI was at least 50% lower in lateral positions than the AHI in the supine position. Possible positional OSA was defined as an AHI$5 in the supine position with no recorded sleep in lateral positions. Nonpositional OSA was defined as an AHI$5 with less than a 50% reduction in AHI in the lateral position (left or right) compared to supine, which required sleep in a nonsupine position (8,9). Certified study personnel measured stroke severity using the NIH Stroke Scale (NIHSS) at the time of study enrollment (12).

RESULTS Thirty-four of the 66 subjects (51.5%) presented with ischemic stroke, and 32 subjects (48.5%) suffered ICH. Demographics and major risk factors between the stroke subtypes are presented in Table 1. The median baseline NIHSS was 12.5 (IR: 7-17), which was significant between ICH and ischemic stroke patients (p = 0.002). The mean recorded total sleep time per stroke case was 206.9ยก93.8 minutes, and the mean total recording time was 338.1ยก69.6 minutes. The majority of sleep time was spent in the supine position in all subjects; the median percentage of total sleep time in the supine position was 100% (IR: 85.9100). The majority (66.7%) of subjects spent the entire sleep time in a supine posture. Fifty-seven patients (86.4%) spent no time sleeping in the prone position, 52 patients (78.8%) spent no time sleeping on their left side, and 49 patients (74.2%) spent no time sleeping on their right side. A significant correlation was observed between the percent of sleep time in the supine position and the NIHSS (rs = 0.5; p,0.001). A full night of diagnostic studies was obtained for all subjects. Fifty-two patients (78.8%) exhibited OSA; 42.4% of these patients suffered ischemic stroke, and 36.4% suffered ICH. The mean AHI was 29.7 (ยก26.6). Twelve patients (23.1%) exhibited positional OSA, and 6 patients (11.5%) exhibited nonpositional OSA. The other 34 patients (65.4%) exhibited OSA but spent the entire sleep period in a supine position (i.e., possible positional OSA). No differences were observed between the stroke subtypes and the presence of OSA or positional OSA (Table 2). AHI was significantly reduced when patients changed from a supine to a lateral position (p,0.001 - Table 3). Table 1 - Demographics and vascular risk factors.

Statistical analyses We collected demographic, clinical, and polysomnographic data from all study subjects. The meansยกstandard deviations (SD) or medians with interquartile ranges (IR) were calculated for numeric variables. The Chi-square test

Features*

All patients

Ischemic Stroke

ICH

p-value

Total patients Age Sex Male Female BMI Hypertension Diabetes Atrial fibrillation Hyperlipidemia Current smoker Alcohol consumption NIHSS{

66 57.6ยก11.5

34 (51.5) 58.1ยก11.3

32 (48.5) 57ยก11.8

0.69

54 (81.8) 12 (18.2) 26.6ยก4.9 58 (87.9) 16 (24.2) 6 (9.1) 31 (47) 36 (54.5) 40 (60.6)

31 (91.2) 3 (8.8) 26.6ยก5 26 (76.5) 7 (20.6) 5 (14.7) 20 (58.8) 18 (52.9) 19 (55.9)

23 (71.9) 9 (28.1) 26.5ยก4.9 32 (100) 9 (28.1) 1 (3.1) 12 (37.5) 18 (56.2) 21 (65.6)

0.99 0.005 0.47 0.20 0.08 0.80 0.42

12.5 (7-17)

9.5 (5-15)

15.5 (10.2-19.7)

0.002

0.04

ICH: Intracerebral hemorrhage; BMI: body mass index; NIHSS: National Institutes of Health Stroke Scale. * Results are expressed as the meansยกSD or number of patients (%). { Medians (interquartile ranges).

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Table 2 - Polysomnographic findings. Characteristics* No. of patients Total time recorded (min) Total sleep time recorded (min) Sleep efficiency (%) Percentage of sleep time spent supine{ Patients who spent all sleep time in a supine position Minimal oxygen saturation (%) AHI AHI$5 Obstructive apnea Central apnea Positional OSA Possible Positional OSA Nonpositional OSA

All patients

Ischemic Stroke

ICH

p-value

66 338.1¡69.6 206.9¡93.8 60.9¡22.9 100 (87.9-100) 44 (66.7) 84.7¡9.0 29.7¡26.6 54 (81.8) 52 (78.8) 2 (3.0) 12 (23.1) 34 (65.4) 6 (11.5)

34 336.6¡69.4 192.4¡95.4 57.1¡24.5 100 (78-100) 20 (58.8) 84.4¡6.5 34.7¡28.2 29 (85.3) 28 (82.4) 1 (2.9) 7 (25.0) 17 (60.7) 4 (14.3)

32 339.7¡70.8 222.4¡91 64.9¡20.8 100 (94.8-100) 24 (75) 85.1¡11.2 24.3¡24.0 25 (78.1) 24 (75.0) 1 (3.1) 5 (20.8) 17 (70.8) 2 (8.4)

0.83 0.23 0.28 0.22 0.16 0.10 0.16 0.45 0.46 0.99 0.60 0.80 0.67

ICH: intracerebral hemorrhage; AHI: apnea–hypopnea index; OSA: obstructive sleep apnea. * Results are expressed as the means¡SD or number of patients (%). { Medians (interquartile ranges).

further investigation. A recent small, randomized, controlled, two-night crossover study demonstrated that positional therapy using a pillow that was designed to prevent supine sleep was adequately tolerated by patients after ischemic stroke and reduced the absolute percentage of time in a supine position by 36% and modestly reduced sleep apnea severity (19). The limitations of our study include the small sample size and the lack of reliable information on sleep apnea severity prior to stroke. The time spent in a supine position may also be influenced by the availability of local resources and the act of performing the PSG (20). However, other multiparametric monitoring systems that are routinely used for stroke patients may exert the same influence on sleep position. In conclusion, positional sleep apnea was detected in one quarter of patients during the acute phase of ischemic stroke and ICH, but the actual frequency was likely underestimated. The adequate positioning of patients during sleep may be an alternative for reducing the impact of obstructive respiratory events during the acute phase of stroke, especially in patients with a high NIHSS, regardless of their stroke subtype. Further studies are required to examine the efficacy of positional therapy on the outcomes of OSA patients during the acute phase of stroke.

DISCUSSION We observed high frequencies of obstructive sleep apnea (78.8%) and exclusive supine positioning during sleep (66.7%) in patients with ischemic stroke or ICH. The predominance of supine posture was related to the severity of neurological deficits regardless of the stroke subtype in our patients. Supine sleep may aggravate OSA severity and contribute to hypoxemia, endothelial dysfunction, increased oxidative stress, the activation of the coagulation cascade and inflammation during the acute phase of stroke (13,14). Therefore, prolonged supine positioning during the acute phase of stroke and ICH may potentiate the negative impact of OSA on clinical outcomes. Positional OSA was confirmed in 23.1% of patients. However, this percentage may be underestimated because the majority of patients spent the entire recorded sleep time in a supine position. The frequency of possible positional OSA increases significantly (up to 65.4%). Similar results were observed in a recent study of ischemic stroke patients in which full polysomnography was performed within the first seven days after symptom onset (9). Continuous positive airway pressure (CPAP) is the standard treatment for OSA, but its efficacy depends on patient adherence, and stroke patients may have a low tolerance for CPAP (15-17). More definitive results on CPAP treatment in acute stroke patients are required. Therefore, the appropriate positioning of stroke patients is a reasonable and harmless initiative to avoid a prolonged supine decubitus position. Positional OSA may be more frequent during the acute stroke setting, and the incidence may decrease significantly over six months (18). Therefore, the potential for positional therapy to decrease OSA in ischemic stroke patients during the acute phase of stroke requires

ACKNOWLEDGMENTS This work received support from Fundac¸a˜o de Amparo a Pesquisa do Estado de Sa˜o Paulo (FAPESP), Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq) and Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior (CAPES).

AUTHOR CONTRIBUTIONS Camilo MR and Pontes-Neto OM contributed to the data collection and analysis, critical review of the results, and manuscript development. Fernandes RMF and Sander HH contributed to the polysomnography analysis and the manuscript review. Nobre F, Santos-Pontelli T, Dos Santos AC, De Araujo DB, and Leite JP participated in the critical review of the results and the manuscript.

Table 3 - AHI in supine and lateral positions in patients with positional OSA.

Mean AHI supine Mean AHI lateral p-value

All patients (n = 12)

Ischemic stroke (n = 7)

ICH (n = 5)

36.3¡26.8 6.3¡5.8 ,0.001

38.8¡31.3 7.4¡7.4 ,0.001

32.7¡22 4.9¡2.5 ,0.001

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ICH: intracerebral hemorrhage; AHI: apnea–hypopnea index.

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2. Pontes-Neto OM, Fernandes RM, Sander HH, da Silva LA, Mariano DC, Nobre F, et al. Obstructive sleep apnea is frequent in patients with hypertensive intracerebral hemorrhage and is related to perihematoma edema. Cerebrovasc Dis. 2010;29(1):36-42, http://dx.doi.org/10.1159/000255972. 3. Bassetti CL, Milanova M, Gugger M. Sleep-disordered breathing and acute ischemic stroke: Diagnosis, risk factors, treatment, evolution, and long-term clinical outcome. Stroke. 2006;37(4):967-72, http://dx.doi.org/ 10.1161/01.STR.0000208215.49243.c3. 4. Sahlin C, Sandberg O, Gustafson Y, Bucht G, Carlberg B, Stenlund H, et al. Obstructive sleep apnea is a risk factor for death in patients with stroke: A 10-year follow-up. Arch Intern Med. 2008;168(3):297-301, http://dx.doi.org/10.1001/archinternmed.2007.70. 5. Parra O, Arboix A, Montserrat JM, Quinto´ L, Bechich S, Garcı´a-Eroles L. Sleep-related breathing disorders: impact on mortality of cerebrovascular disease. Eur Respir J. 2004;24(2):267-72, http://dx.doi.org/10.1183/ 09031936.04.00061503. 6. Oksenberg A, Khamaysi I, Silverberg DS, Tarasiuk A. Association of body position with severity of apneic events in patients with severe nonpositional obstructive sleep apnea. Chest. 2000;118(4):1018-24, http://dx.doi.org/10.1378/chest.118.4.1018. 7. Penzel T, Moller M, Becker HF, Knaack L, Peter JH. Effect of sleep position and sleep stage on the collapsibility of the upper airways in patients with sleep apnea. Sleep. 2001;24(1):90-5. 8. Cartwright RD. Effect of sleep position on sleep apnea severity. Sleep. 1984;7(2):110-4. 9. Brown DL, Lisabeth LD, Zupancic MJ, Concannon M, Martin C, Chervin RD. High prevalence of supine sleep in ischemic stroke patients. Stroke. 2008;39(9):2511-4, http://dx.doi.org/10.1161/STROKEAHA.107.513572. 10. Dziewas R, Hopmann B, Humpert M, Ritter M, Dittrich R, Schabitz WR, et al. Positional sleep apnea in patients with ischemic stroke. Neurol Res. 2008;30(6):645-8, http://dx.doi.org/10.1179/174313208X289598. 11. Iber C, Ancoli-Israel S, Chesson AL, Quan SF. Respiratory Rules. The AASM Manual for the Scoring of Sleep And Associated Events: Rules, Terminology And Technical Specifications. Westchester, American Academy of Sleep Medicine, 2007.

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The effects of one-half of a soccer match on the postural stability and functional capacity of the lower limbs in young soccer players Ricardo Kim Fukushi Yamada, Gustavo Gonc¸alves Arliani, Gabriel Peixoto Lea˜o Almeida, Andre´ Manrique Venturine, Ciro Veronese dos Santos, Diego Costa Astur, Moise´s Cohen Universidade Federal de Sa˜o Paulo, Centro de Traumatologia do Esporte, Departamento de Ortopedia e Traumatologia (CETE/ DOT-EPM/UNIFESP), Sa˜o Paulo/SP, Brazil.

OBJECTIVE: Most injuries occur during the final 15 minutes of each half of a soccer match, suggesting that physical exertion may influence changes in neuromuscular control and the body’s ability to stabilize the joints of the lower extremities. The aim of this study was to analyze the effects of one-half of a soccer match on the functional capacity and stability of the lower limbs in young soccer players. METHODS: We analyzed 27 soccer players by evaluating the functional capacity of their lower limbs using the hop test protocol and their level of postural stability using the Biodex Stability System. The evaluations were performed before and after 45 minutes of game time. RESULTS: After the match, there was a decrease in the overall stability index (OSI) (F(1,23) = 5.64, p = 0.026) and the anterior-posterior stability index (APSI) (F(1,23) = 5.24,p = 0.032). In the single and triple hop tests, there was a higher functional capacity in the dominant limb compared to the non-dominant limb in the pre- and post-game comparisons. CONCLUSION: The results of this study show that there is a decrease in the stability of the lower limbs in young soccer players after a 45 minutes soccer match, but the same result was not found for the functional capacity. KEYWORDS: Postural Stability; Soccer; Injury; Balance. Yamada RK, Arliani GG, Almeida GP, Venturine AM, dos Santos CV, Astur DC, et al. The effects of one-half of a soccer match on the postural stability and functional capacity of the lower limbs in young soccer players. Clinics. 2012;67(12):1361-1364. Received for publication on May 26, 2012; First review completed on July 13, 2012; Accepted for publication on August 7, 2012 E-mail: gabriel_alm@hotmail.com Tel.: 55 11 5082-3010.

injuries occur in participants younger than 15 years of age (4). In the United States, soccer injuries among young athletes have peaked at 2 injuries per 1,000 participants (5). Most injuries occur in the final 15 minutes of each half of a soccer match, suggesting that physical exertion may influence changes in neuromuscular control and the ability to stabilize the joints of the lower limbs. One possible hypothesis for this shift could be a change in the postural stability of the lower limbs due to physical exertion (6,7). It has been theorized that muscle fatigue may alter the proprioceptive and kinesthetic properties of joints by increasing the threshold for muscle spindle firing, disrupting the afferent feedback and changing the somatosensory input, thus causing deficits in neuromuscular control. These characteristics can be visualized by the lack of postural control (6). Postural stability is a complex process that depends on proprioceptive stimuli from mechanoreceptors and vestibular and visual receptors. All of these signals are processed in the central nervous system to generate an appropriate motor response (6). Previous studies have investigated the effects of physical exertion on the functional stability and proprioception of the

INTRODUCTION Soccer is considered to be the most popular sport in the world. This sport currently has approximately 200,000 professional athletes and 240 million amateur players, of whom approximately 80% are male (1,2). Youth participation in soccer is very important in current public health programs because it increases the level of exercise and physical activity of young people. Thus, soccer is a key tool in the fight against the high rates of childhood obesity and a sedentary lifestyle (3). Soccer is among the fastest growing sports in terms of the number of young players worldwide. However, there has also been an increase in the number of injuries related to this sport among young athletes; approximately 44% of soccer

Copyright ß 2012 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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lower limbs in athletes. However, most of these studies used controlled fatigue protocols, which do not accurately reproduce the conditions of an actual soccer match. Thus, the primary objective of this study was to analyze the effects of one-half of a soccer match on the functional capacity and stability of the lower limbs in young soccer players.

MATERIALS AND METHODS Subjects We analyzed 27 soccer athletes from the men’s team at the Vila Guarani’s Team Club (mean¡standard deviation: 16¡0.83 years old, 1.7¡0.08 m, 60.5¡9.48 kg, 21.16¡2.20 body mass index (BMI), 6.5¡2.56 years of practice). The inclusion criteria for this study included soccer players who were under the age of 18, were male, trained more than three times per week for at least nine hours per week and had participated in the sport for more than two years. The exclusion criteria included goalkeepers, the occurrence of knee and/or ankle injuries in the last six months, the presence of mechanical or functional instability of the knee and/or ankle, a history of prior orthopedic knee and/or ankle surgery or spine and/or hip injuries in the last six months and the presence of cerebellar disorders. Two athletes were excluded from the study: one player had a history of an ankle sprain within the previous six months, and the other player had an anterior cruciate ligament reconstruction in his right knee. The athletes completed a questionnaire on their anthropometric characteristics, previous injuries, and soccer practice routine. The dominant lower limb was defined by the athlete as the leg that was predominantly used to kick the ball. The players were also subjected to a functional capacity evaluation of their lower limbs using a hop test protocol and an evaluation of their level of postural stability using the Biodex Stability System (Biodex Inc., Shirley, NY) before and immediately after a soccer match that lasted for 45 minutes. The athletes were evaluated during a series of 10 friendly matches held in the afternoon (3 pm) in June and July 2011. Figure 1 - Four hop tests.

Procedures Hop Tests. The athletes underwent the following tests: single hop test, triple hop test, crossover hop test, and timed hop test (Figure 1). The execution sequence for the tests of the lower limbs was randomized. Before the start of each data collection, two practice tests were conducted to familiarize the participants with the tests; these tests were then followed by three official tests for data collection. To perform the jumps, all of the participants were instructed to keep their arms crossed on their lumbar spine and jump while maintaining stability upon landing. For the single hop test, each participant jumped with one leg, attempting to jump as far as possible with a single jump. For the triple hop test, three consecutive jumps were performed with the same leg in an attempt to jump as far as possible. For the crossover hop test, the participant performed three consecutive jumps over a 15-cm line that had been marked on the floor. For the timed hop test, each participant jumped as fast as possible toward a previously determined mark 6 m away (8). In previous studies (9,10), the coefficients of interclass reliability for the tests were as follows: single hop test, .92 - .96; triple hop test, .95 - .97; crossover hop test, .93 .96; and timed hop test, .66 - .92.

Level of postural stability The Biodex Stability System (Biodex, Inc., Version 3.1, Shirley, New York, USA) was used to measure postural stability. The evaluation was performed for eight different levels of platform stability for a total of 30 seconds: level 8 was the most stable, and level 1 was the most unstable (consisting of 3.75 seconds at each level). This platform provides an objective assessment of postural stability using three indices: the overall stability index (OSI), the anteriorposterior stability index (APSI), and the medial-lateral stability index (MLSI) (Figure 2). These indices were calculated using the degree of oscillation of the platform, in which low values indicated that the individual had good stability (11). In a previous study, Salavat et al. (8) reported interclass reliability coefficients of .77 and .99 using the same methodology that was used in this study. The test protocol performed was unipodal, consisting of two periods of adaptation to the device and three consecutive assessment tests. The participants were allowed to rest for 60 seconds between the tests. The testing order was randomized (dominant 6 non-dominant), and the

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Soccer match on the stability and function Yamada RK et al.

(F(1,23) = 5.64, p = 0.026) and the anterior-posterior stability index (APSI) (F(1,23) = 5.24, p = 0.032). However, there were no significant changes in the medial-lateral stability index (MLSI) or the four hop tests (Table 1). The Bonferroni post-hoc test indicated that the dominant leg was responsible for the significant difference in the OSI (F(1,23) = 5.52, p = 0.014) and APSI (F(1,23) = 9.02, p = 0.006), whereas the non-dominant leg did not show any differences in the pre- and post-game comparisons. There was also a significant difference between the dominant and nondominant leg in both the pre- and post-game situations in the single hop test (F (1,23) = 7.15, p = 0.015) and the triple hop test (F(1,23) = 4.53, p = 0.047) (Table 1).

DISCUSSION The principal findings of this study were the decreases in the overall and anterior-posterior stability indices of the dominant lower limb in young players after a soccer match. The results from previous studies on this subject, which were conducted using various populations, have been controversial (12-14). In the elderly, some studies have shown that muscle fatigue in the lower limbs changes the sense of joint position and balance (13). Another study with a similar population showed no relationships between postural stability and performing moderate physical activity (12). The results from the current study demonstrate the importance of the intensity of the exercise on the muscles of the lower extremities when evaluating stability following exercise. Gioftsidou et al. (15) demonstrated that there was no difference in the balance of young soccer players following a training session. Thus, these authors concluded that lowerlimb muscle fatigue was likely not the cause for the increased incidence of injuries at the end of training sessions. In that study, however, the amount of time spent training was not specified, and the authors attributed the maintenance of balance following exertion to the absence of muscle fatigue after training. In contrast, our study found differences in the overall stability of these athletes following exertion. It is possible that the results were different due to the different loads that the subjects were exposed to and the

Figure 2 - An athlete performing the platform stability test.

athlete was positioned with his arms parallel to the longitudinal axis of the body while keeping his hand in contact with his thigh. The subject was instructed to keep his eyes open and fixed on a point on a white wall 1 meter from the equipment, and his knees were bent 10 ˚ to 15 ˚ while keeping his hips in a neutral position. After the three tests, the device software reported the stability index based on the degree of oscillation of the platform during the evaluations.

Statistical analyses Initially, we used a Kolmogorov-Smirnov test to verify the normality of the data. Then, we used a two-way ANOVA (2x2) with repeated measures to determine the differences between the legs (dominant and non-dominant) and between the different conditions (pre-game and post-game) for OSI, APSI, MLSI, and the four hop tests. The Bonferroni test was used for a post-hoc analysis when appropriate. A 5% level of significance was used (p,0.05), and the statistical analyses were performed using SPSS 17.0 software for Windows (Statistical Package for Social Sciences Inc., Chicago, IL, USA).

Table 1 - Means and standard deviations of the stability indices and function tests in the pre- and post-game evaluations*. Pre-Game

OSI (degrees){ APSI (degrees){ MLSI (degrees) Single Hop (m){ Triple Hop (m){ Cross Hop (m) Timed Hop (sec)

Ethics The study was submitted to and approved by the Ethical Committee at Federal University of Sa˜o Paulo under protocol number 1635/10. All of the participants provided written informed consent. *

Post-Game

Dominant

NonDominant

Dominant

NonDominant

7.84¡1.6 6.69¡1.79 4.26¡0.93 1.75¡0.19 4.92¡0.51 4.33¡0.44 2.13¡0.27

7.21¡1.37 6.06¡1.43 4.15¡1.02 1.72¡0.18 4.86¡0.42 4.38¡0.6 2.07¡0.3

7.06¡1.53 5.78¡1.56 4.09¡0.9 1.74¡0.2 4.9¡0.65 4.33¡0.64 2.09¡0.2

7.21¡1.82 5.93¡1.72 4.19¡0.91 1.69¡0.16 4.76¡0.61 4.38¡0.68 2.12¡0.33

OSI, overall stability index; APSI, anterior-posterior stability index; MLSI, medial-lateral stability index. { Significant difference (p,0.001) between the pre-game and post-game results for the dominant lower limb. { Significant difference (p,0.05) between the limbs.

RESULTS Using an ANOVA, we found a significant difference (moment 6 leg) in the overall stability index (OSI)

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bibliographic review, data tabulation, and statistical analysis. Cohen M critically revised the content of the manuscript.

different local climate conditions. In our study, all of the young players were evaluated before and after the first half of a soccer match, which was 45 minutes long, not following a training session. The study was conducted in Sa˜o Paulo, Brazil, where the normal average temperature is higher than that in Greece (20 ˚C vs. 16 ˚C), which was the location of the cited study. Other previous studies have also found that muscle fatigue of the lower extremities, particularly of the proximal muscle groups, affects postural stability (8,16). We did not find any differences in the overall stability with regard to the stability of the dominant leg compared to the nondominant leg. Similar results were reported by Thorpe and Ebersole (17) and Teixeira et al. (18), who found no difference in the balance between the dominant and nondominant legs in studies with 12 and 11 soccer players, respectively. Another important finding in our study was the difference in the functional capacity between the dominant and non-dominant legs in the pre- and post-game comparisons for the single and triple hop tests. Swearingen et al. (16) presented similar findings in a study of healthy patients of both sexes who averaged 24 years of age (19). However, in a study of healthy subjects and sports players, van der Harst et al. (20) found no differences between the dominant and non-dominant legs in single hop test and timed hop test evaluations. However, in both of these studies, the tests were not conducted both before and after exercise. Although we used four hop test evaluations, a recent study of non-surgically treated patients following the rupture of the anterior cruciate ligament demonstrated that the single hop test alone can be used as a predictor of knee functionality (21). One weakness of our study was the lack of isokinetic evaluations to determine the degree of muscle fatigue imposed on the musculature of the lower limbs in athletes post-exercise. Another limitation of our study was the lack of standardization of the player’s position. The effort of an athlete varies greatly depending on the position (for example, defender, midfielder, and forward in soccer), which can be considered a bias because the players were not exposed to the same physical stress. However, we chose to use a soccer match and evaluate the players at different positions to most closely align the characteristics of the study with how the sport is actually played. The results of this study show that there is a decrease in the stability of the lower limbs in young soccer players after a 45 minutes soccer match. Another important finding was the functional capacity of the dominant leg compared to the non-dominant leg in the pre- and post-game comparisons in the single and triple hop tests.

REFERENCES 1. Junge A, Dvorak J. Soccer injuries: A review on incidence and prevention. Sports Med. 2004;34(13):929-38, http://dx.doi.org/10.2165/ 00007256-200434130-00004. 2. Timpka T, Risto O, Bjormsjo M. Boys soccer league injuries: A community-based study of time-loss from sports participation and long-term sequelae. Eur J Public Health. 2008;18(1):19-24, http://dx.doi. org/10.1093/eurpub/ckm050. 3. Bergeron MF. Improving health through youth sports: Is participation enough? New Dir Youth Dev. 2007 Fall;(115):27-41, 6. 4. Koutures CG, Gregory AJ, , American Academy of Pediatrics. Council on Sports Medicine and Fitness. Injuries in youth soccer. Pediatrics 2010;125(2):410-4. 5. Leininger RE, Knox CL, Comstock RD. Epidemiology of 1.6 million pediatric soccer-related injuries presenting to us emergency departments from 1990 to 2003. Am J Sports Med. 2007;35(2):288-93. 6. Hiemstra LA, Lo IK, Fowler PJ. Effect of fatigue on knee proprioception: Implications for dynamic stabilization. J Orthop Sports Phys Ther. 2001;31(10):598-605. 7. Rahnama N, Reilly T, Lees A. Injury risk associated with playing actions during competitive soccer. Br J Sports Med. 2002;36(5):354-9, http://dx. doi.org/10.1136/bjsm.36.5.354. 8. Salavati M, Moghadam M, Ebrahimi I, Arab AM. Changes in postural stability with fatigue of lower extremity frontal and sagittal plane movers. Gait Posture. 2007;26(2):214-8, http://dx.doi.org/10.1016/j. gaitpost.2006.09.001. 9. Ross MD, Langford B, Whelan PJ. Test-retest reliability of 4 single-leg horizontal hop tests. J Strength Cond Res. 2002;16(4):617-22. 10. Bolgla LA, Keskula DR. Reliability of lower extremity functional performance tests. J Orthop Sports Phys Ther. 1997;26(3):138-42. 11. Schmitz R, Arnold B. Intertester and intratester reliability of a dynamic balance protocol using the Biodex stability system. J Sport Rehabil. 1998;7(2):95-101. 12. Egerton T, Brauer SG, Cresswell AG. Dynamic postural stability is not impaired by moderate-intensity physical activity in healthy or balanceimpaired older people. Hum Mov Sci. 2010;29(6):1011-22, http://dx.doi. org/10.1016/j.humov.2010.06.001. 13. Ribeiro F, Mota J, Oliveira J. Effect of exercise-induced fatigue on position sense of the knee in the elderly. Eur J Appl Physiol. 2007;99(4):379-85, http://dx.doi.org/10.1007/s00421-006-0357-8. 14. Mohammadi F, Roozdar A. Effects of fatigue due to contraction of evertor muscles on the ankle joint position sense in male soccer players. Am J Sports Med. 2010;38(4):824-8, http://dx.doi.org/10.1177/ 0363546509354056. 15. Gioftsidou A, Malliou P, Pafis G, Beneka A, Godolias G, Maganaris CN. The effects of soccer training and timing of balance training on balance ability. Eur J Appl Physiol. 2006;96(6):659-64, http://dx.doi.org/10. 1007/s00421-005-0123-3. 16. Yaggie JA, McGregor SJ. Effects of isokinetic ankle fatigue on the maintenance of balance and postural limits. Arch Phys Med Rehabil. 2002;83(2):224-8, http://dx.doi.org/10.1053/apmr.2002.28032. 17. Thorpe JL, Ebersole KT. Unilateral balance performance in female collegiate soccer athletes. J Strength Cond Res. 2008;22(5):1429-33, http://dx.doi.org/10.1519/JSC.0b013e31818202db. 18. Teixeira LA, de Oliveira DL, Romano RG, Correa SC. Leg preference and interlateral asymmetry of balance stability in soccer players. Res Q Exerc Sport. 2011;82(1):21-7. 19. Swearingen J, Lawrence E, Stevens J, Jackson C, Waggy C, Davis DS. Correlation of single leg vertical jump, single leg hop for distance, and single leg hop for time. Phys Ther Sport. 2011;12(4):194-8, http://dx.doi. org/10.1016/j.ptsp.2011.06.001. 20. van der Harst JJ, Gokeler A, Hof AL. Leg kinematics and kinetics in landing from a single-leg hop for distance. A comparison between dominant and non-dominant leg. Clin Biomech (Bristol, Avon). 2007;22(6):674-80. 21. Grindem H, Logerstedt D, Eitzen I, Moksnes H, Axe MJ, Snyder-Mackler L, et al. Single-legged hop tests as predictors of self-reported knee function in nonoperatively treated individuals with anterior cruciate ligament injury. Am J Sports Med. 2011;39(11):2347-54, http://dx.doi. org/10.1177/0363546511417085.

AUTHOR CONTRIBUTIONS Yamada RK, Arliani GG, Almeida GP, and Venturine AM were responsible for the project conception, data collection, and manuscript writing. Dos Santos CR and Astur DC were responsible for the

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DOI:10.6061/clinics/2012(12)04

CLINICAL SCIENCE

The perception of sleep quality in kidney transplant patients during the first year of transplantation Dnyelle Souza Silva,I Elisangela dos Santos Prado Andrade,II Rosilene Motta Elias,III Elias David-Neto,IV William Carlos Nahas,IV Manuel Carlos Martins de Castro,III Maria Cristina Ribeiro de CastroV I

Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, Renal Transplantation Service, Psychologist, Sa˜o Paulo/SP, Brazil. II Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, Nephrology, Sa˜o Paulo/ SP, Brazil. III Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, Dialysis Unit, Nephrology, Sa˜o Paulo/SP, Brazil. IV Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, Renal Transplantation Service, Division of Urology, Sa˜o Paulo/SP, Brazil. V Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, Heart Institute (INCOR), Renal Transplantation Service and Laboratory of Immunology, Sa˜o Paulo/SP, Brazil.

OBJECTIVE: Poor sleep quality is one of the factors that adversely affects patient quality of life after kidney transplantation, and sleep disorders represent a significant cardiovascular risk factor. The objective of this study was to investigate the prevalence of changes in sleep quality and their outcomes in kidney transplant recipients and analyze the variables affecting sleep quality in the first years after renal transplantation. METHODS: Kidney transplant recipients were evaluated at two time points after a successful transplantation: between three and six months (Phase 1) and between 12 and 15 months (Phase 2). The following tools were used for assessment: the Pittsburgh Sleep Quality Index; the quality of life questionnaire Short-Form-36; the Hospital Anxiety and Depression scale; the Karnofsky scale; and assessments of social and demographic data. The prevalence of poor sleep was 36.7% in Phase 1 and 38.3% in Phase 2 of the study. RESULTS: There were no significant differences between patients with and without changes in sleep quality between the two phases. We found no changes in sleep patterns throughout the study. Both the physical and mental health scores worsened from Phase 1 to Phase 2. CONCLUSION: Sleep quality in kidney transplant recipients did not change during the first year after a successful renal transplantation. KEYWORDS: Quality of Life; Sleep; Renal Transplantation. Silva DS, Andrade ES, Elias RM, David-Neto E, Nahas WC, Castro MC, et al. The perception of sleep quality in kidney transplant patients during the first year of transplantation. Clinics. 2012;67(12):1365-1371. Received for publication on June 6, 2012; First review completed on June 26, 2012; Accepted for publication on August 7, 2012 E-mail: dnyelle.silva@yahoo.com Tel.: 55 11 2661-8089

use, uremia, malnutrition, and anemia (10). Sleep-related disorders have become a focus of interest for research due to increasing evidence of an association between cardiovascular disturbances and sleep apnea-hypopnea syndrome (11,12). In addition, studies have suggested that sleep disorders are closely linked to physical, psychological and social well-being (13) and cognitive function (14,15). Detecting the presence of sleep disorders after successful renal transplantation and obtaining a better understanding of this outcome is thus very important due to its impact on patient HRQoL, the perception of changes in lifestyle and well-being and the adherence to treatment. Therefore, the present study was conducted to prospectively assess sleep quality and HRQoL in kidney transplant recipients at two time points after a successful transplantation. We examined patients during the initial phase (3-6 months post-transplant) and at the second year postprocedure to determine whether there had been changes in sleep quality over time after transplantation. This is a relevant issue because sleep disorders are highly prevalent in patients with chronic kidney disease and are associated

INTRODUCTION Sleep disorders are more common in patients with chronic kidney disease (CKD) undergoing dialysis (1,2) than in the general population (3). Successful renal transplantation is expected to correct most abnormalities of CKD and significantly improve the patients’ health-related quality of life (HRQoL) (4-7), but some studies have demonstrated that sleep disorders do not always improve after transplantation (6-9). The sleep quality and quality of life of kidney transplant recipients are known to be affected by several factors, such as social-demographic variables, comorbidities, psychiatric disorders, and other physical conditions, including tobacco

Copyright ß 2012 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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with increased cardiovascular risk in addition to having a negative impact on the quality of life of this population.

(HAD-D). Each item may be scored from 0 to 3, generating a maximum total score of 21 for each scale. Scores equal to or higher than 9 indicate the presence of symptoms of depression or anxiety (20).

PATIENTS AND METHODS Patients who received kidney transplants at the Renal Transplantation Service of Hospital das Clı´nicas of University of Sa˜o Paulo School of Medicine (HC/FMUSP) from January 1st to December 31st, 2008, were selected to participate in this study. Eligible patients were individuals 18 years of age or older who had undergone renal transplantation in the past three to six months, had stable renal function with creatinine clearance above 40 mL/min, and who agreed to participate in the study by signing the informed consent form. Patients who were hospitalized within 30 days of inclusion, night-shift workers, patients with active infection, patients who were previously diagnosed with depression, and patients who declined to participate in any phase of the study were excluded. At inclusion, information was collected about patient age, gender, race, educational background (years of schooling), marital and employment status and medical history. Information on the presence of comorbidities, type of donor, time on dialysis, and laboratory data was retrieved from the patients’ hospital files. All patients were interviewed on two occasions: between the third and the sixth month after transplantation and between the 12th and the 15th month after transplantation. On both occasions, the following variables were assessed: sleep quality using the Pittsburg Sleep Quality Index (PSQI); quality of life perception using the Short-Form Health Survey (SF-36); the presence of anxiety or depression using the Hospital Anxiety and Depression (HAD) scale; and patient self-care ability using the Karnofsky scale.

Karnofsky scale The Karnofsky scores were used to assess physical health status as follows: 100–normal; 90-minor signs or symptoms of disease; 80-normal activity with effort; 70–unable to carry out normal activity; 60-requires occasional assistance; 50– requires considerable assistance; 40-disabled; 30-severely disabled; 20-hospital admission necessary; 10-moribund. Patients were considered as ‘‘rehabilitated’’ when the Karnofsky score was 70 to 100, ‘‘able to care for personal needs’’ when the score was 60, ‘‘requiring considerable assistance’’ when the score was 30 to 50, and ‘‘requiring hospital admission’’ when the score was 10 to 20 (21).

Statistics The Kolmogorov-Smirnov test was used to verify data normality. Student’s t test was used to compare group averages. Whenever the data normality assumption was rejected, the non-parametric Mann-Whitney test was used. To compare group behavior at each timepoint, we used the paired t test for normally distributed data and the nonparametric Wilcoxon test for non-normally distributed data. The non-parametric McNemar test was used to compare the classification data between Phase 1 and Phase 2.

Ethics This study was approved by the local Research Ethics Committee. Before inclusion, patients received detailed verbal and written information about the study objectives and procedures and signed the informed consent form.

Quality of sleep The PSQI is a self-administered questionnaire that evaluates sleep quality over the past month. It contains 19 questions regarding subjective sleep quality, latency, duration, efficacy, disorders, use of sleep medications, and daily sleep problems. Each component is assessed on a 0 to 3 scale, generating a global score between 0 and 21; higher scores indicate lower sleep quality. The PSQI identifies two groups of patients: poor sleepers (PSQI.5) and good sleepers (PSQI,5). A global score .5 indicates that the person is a poor sleeper and has severe difficulties in at least two areas or moderate difficulties in more than three areas (16-18). In this study, patients with a score equal to or below 5 were considered as having normal sleep patterns.

RESULTS In 2008, 163 renal transplantation procedures were performed at the HCFMUSP, 156 of which were successful. Seventy-six patients met the study inclusion criteria and were included. The details of the inclusion process are shown on Figure 1. The study population was 60% white, with a mean age of 42¡12 years and similar proportions of male and female subjects. Recipients of transplants from living donors represented 59% of the population; the most frequently used immunosuppressive regimen was the triple combination of tacrolimus, mycophenolate mofetil, and prednisone. Table 1 shows a detailed profile of the study sample. Renal function and hematological parameters remained stable throughout the study period, but a significant increase in body mass index (BMI) was observed between the two assessments (Table 2). There was a significant increase in the proportion of patients who were able to work, from 13 (21.7%) in Phase 1 to 28 (46.7%) in Phase 2 (p = 0.006).

Health-related quality of life (HRQoL) Quality of life was evaluated using the Short-Form Health Survey (SF-36), which consists of eight sub-scales: physical function, role limitation attributable to physical problems, bodily pain, perception of general health, social function, vitality, role limitation attributable to emotional problems, and mental health. The sub-scales are presented as scores between 0 and 100, with higher scores indicating a better health status (19).

Sleep quality and health-related quality of life outcomes after renal transplantation We observed poor sleep quality in 22 (36.7%) and 23 (38.3%) patients in Phases 1 and 2 of the study, respectively. The mean PSQI showed no significant change from Phase 1 to Phase 2 (5.4 vs. 5.8; p = 0.319).

Hospital anxiety and depression (HAD) The HAD scale consists of 14 items, of which seven evaluate anxiety (HAD-A) and seven evaluate depression

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Figure 1 - Study enrollment flowchart.

Most SF-36 domains also failed to show significant differences between Phase 1 and 2. However, the vitality (p = 0.015) and role emotional (p = 0.015) domains showed significant differences at follow-up (Figure 2). The presence of anxiety and/or depression symptoms and self-care ability, as measured on the Karnofsky scale, were not significantly different at the two time points, as shown in Table 2. When questioned about their perceived health improvements from the previous year, 93.3% and 85% of the patients reported improvements in Phase 1 and Phase 2 of the study, respectively.

Influence of sleep quality on health-related quality of life and self-care ability after renal transplantation When we categorized the patients as poor sleepers (PSQI.5) or good sleepers (PSQI,5) according to the PSQI, we found no significant difference between these groups with respect to demographics, clinical and laboratory data, or renal transplantation parameters (Table 3). However, we found that the body mass index was higher in the poor sleepers group, in which 17.4% of the patients required sleep medication (Table 3).

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DISCUSSION

Table 1 - Patient characteristics. Male – n (%) Age (years) (mean/SD) White Years of education - n (%) ,8 8-12 Undergraduate/graduate degree Non-smoking – n (%) Marital status – n (%) Single/widowed/divorced Married Immunosuppressive regimen – n (%) Tacrolimus Prednisone Mycophenolate mofetil Comorbidity- n (%) Hypertension Diabetes mellitus Donor – n (%) Living Deceased

There have been a number of cross-sectional studies on sleep quality and sleep disorders (2,6,7,9,10,23-29), but none have assessed sleep quality longitudinally after transplantation. To the best of our knowledge, this study is the first to conduct a sequential assessment of sleep quality in kidney transplant recipients, including the late post-transplantation phase. Our findings showed no significant change in sleep quality from the early postoperative period to one year after transplantation. We found a high prevalence of poor sleepers [PSQI.5] at the first time point, and the prevalence remained unchanged in the second assessment. We found no difference in the mean PSQI between the two assessments. This study only included patients with adequate graft function and excluded patients with a current diagnosis of depression or recent hospitalization because those factors might have had a negative influence on patient sleep quality and quality of life and thus increase the heterogeneity of the sample. Therefore, we conclude that despite a successful transplantation, sleep quality did not improve over the study period. We found no time-dependent change in most quality of life parameters between the two assessments. However, there was a significant difference in the vitality and role emotional scores, suggesting a worsening of these domains over time. Previous cross-sectional studies on sleep quality and quality of life found low scores for these domains in transplant recipients compared with other CKD patients and controls (9,10,22,24). When we compared good sleepers (PSQI,5) and poor sleepers (PSQI.5), we found that the poor sleepers had lower scores in the SF-36 domains, particularly the role physical and social function domains. We found that 10% of patients exhibited depressive symptoms in each phase, while anxiety symptoms were observed in 10% of patients in Phase 1 and 15% of patients in Phase 2. These values are lower than those found in other studies, i.e., 22 to 39% (32) for depressive symptoms and 10 to 22% for clinical depression (31). This difference may be explained by our exclusion of patients with clinical depression at baseline and/or by our use of a symptom screening tool (HAD). Our study was not designed to diagnose anxiety or depression, but because we used a screening tool, it could be expected that these symptoms would be reported by some patients. The association of anxiety and depression symptoms with poor sleep quality in kidney transplant recipients has been reported previously (30-33), but our study failed to show such an association. Some reports consider biological and clinical factors to be significant predictors of the SF-36 physical component score, even though these factors can only explain part of the observed changes (5,33,34). However, no correlation has been identified between these variables and the mental health component of the SF-36. Based on these findings, researchers have suggested that comparisons should consider patient age because this variable affects perceived quality of life. To date, no studies have assessed additional personal, environmental and/or clinical factors that might influence the patient perception of quality of life after renal transplantation. In our study, we also analyzed the correlation between BMI and sleep quality. Sleep disorders, such as sleep apnea,

31 (51.7) 42/12 36 (60.0) 32 (52.4) 20 (33.3) 8 (13.3) 57 (95.0) 27 (45.0) 33 (55.0) 57 (95.0) 60 (100.0) 49 (81.7) 33 (55.0) 11 (18.3) 35 (58.3) 25 (41.7)

The poor sleepers group showed worse role physical (p = 0.034), social functioning (p = 0.040), and Karnofsky index (p = 0.031) scores, even in the early post-transplantation period. A more frequent use of benzodiazepines and anti-anxiety medication and higher BMI values were observed in the poor sleepers group (Table 4). We also found an inverse correlation between BMI and Karnofsky score; among poor sleepers, patients with higher body mass index values had lower Karnofsky index values. During Phase 1 of the study, labor activity was a significant driver of good sleep quality (p = 0.018). The use of benzodiazepines or anti-anxiety medication was associated with worse SQ in both phases (p,0.001). We found no correlation between napping or smoking and SQ. Table 2 - Clinical and laboratory data, sleep quality and HRQoL in each study phase. Phase 1

Serum creatinine(mg/dl) Hematocrit Hemoglobin (g/L) BMI Physical functioning Role physical Bodily pain General health Vitality Social functioning Role emotional Mental health Depression (HAD) Anxiety (HAD) Karnofsky PSQI

Phase 2

3-6m Tx

12-15m Tx

MeaniSD

MeaniSD

n = 60

n = 60

1.3¡0.3

1.3¡0.4

0.783*

42.0¡6.8 13.5¡2.3 25.5¡5.3 88¡11.6 73.3¡33.4 74.1¡16.5 47.8¡17.6 76¡16.7 76¡28.1 67.2¡36.5 72.8¡20.6 3.1¡3.4 4.9¡3.2 92.5¡8.9 5.4¡4.0

41.7¡4.9 13.5¡1.7 26.4¡5.8 87¡16.6 79.5¡12.6 71.3¡23.9 46.3¡16.8 70.9¡19.3 68.2¡32.3 54.4¡37.3 71.2¡18 3.6¡3.6 5.8¡3.7 93.5¡8.2 5.8¡3.9

0.546* 0.929* , 0.001* 0.723** 0.172** 0.422** 0.363** 0015** 0.156** 0.015** 0.610** 0.432** 0.113** 0.326** 0.319**

p

(*) paired t test; (**) Wilcoxon non-parametric testBMI, body mass index; HAD, hospital anxiety and depression; PSQI, Pittsburgh sleep quality index; SF36 –domains.

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Sleep quality after renal transplantation Silva DS et al.

Figure 2 - Comparison of SF-36 domain and PSQI scores in Phase 1 vs. Phase 2.

are expected to be associated with high BMI and neck circumference (12). Although our study was designed to assess sleep quality in general and not sleep disorders, we found a correlation between body mass index and sleep deprivation. Perceived self-care ability was measured on the Karnofsky scale, and we found a negative correlation between poor sleep quality and Karnofsky score. Previous

studies support these findings, showing improvements in the Karnofsky and Disease Impact scores after transplantation (35,36). We could therefore assume that the degree of independence, as measured by the patient’s self-care ability, may be important for sleep quality. In our study, patients who were employed reported significantly better sleep quality early after transplantation compared to the patients who did not work. This result suggests that working may be important to achieving good sleep quality, although it should be noted that only a few patients were actively working at that time. Some authors believe that occupation and social reintegration are directly linked to good sleep quality and quality of life (4,5,33,37). Their studies support our findings that having an occupation may be associated with good sleep quality in kidney transplant recipients. Due to the logistic limitations of including recipients of deceased donor transplants, our study did not assess patients prior to transplantation; therefore, we cannot compare the pre-transplantation period with the other two time points.

Table 3 - Clinical and social characteristics versus sleep quality. PSQI

Age (years) Gender Ethnicity Lives with partner Employment status Smoker Medication Diurnal somnolence Donor Serum creatinine (mg/dL) Hemoglobin (g/L) Hematocrit BMI Diabetes Hypertension Dialysis (months)

Mean ¡ SD Female Male Caucasian Other Yes No

Living Deceased

p

# 5 (n = 37)

. 5(n = 23)

40.08¡11.86 16 (43.2%) 21 (56.8%) 15 (40.5%) 22 (59.5%) 21 (56.8%) 16 (43.2%) 11 (29.7%)

46.00¡11.66 13 (56.5%) 10 (43.5%) 9 (39.1%) 14 (60.9%) 12 (52.2%) 11 (42.8%) 2 (8.7%)

0.064(1) 0.317(2)

1 (2.7%) 0 (0.0%) 18 (48.7%)

2 (8.7%) 4 (17.4%) 15 (65.2%)

0.552(3) 0.018(3) 0.210(2)

24 (65%) 13 (35.1%) 1.32¡0.37

11 (45.%) 12 (52.1%) 1.41¡0.36

13.5¡2.8

13.5¡1.4

42.1¡8.1 24.42¡4.66 5 (13.5%) 18 (48.7%) 27.38¡27.85

41.8¡4.1 27.30¡5.98 6 (26.1%) 15 (65.2%) 38.91¡39.23

0.914(2) 0.729(2) 0.105(3)

Table 4 - Relationship between HRQoL and sleep quality after renal transplantation.

(3)

0.462 0.322(1)

Physical functioning Bodily pain Vitality Role emotional Role physical General health Social functioning Mental health Depression (HAD) Anxiety (HAD) Karnofsky

(1)

0.899

0.856(1) 0.041(1) 0.306(3) 0.210(2) 0.220(4)

(1) Student’s t test; (2) chi-square test; (3) Fisher’s exact test; (4) MannWhitney non-parametric test. Data are presented as mean¡SD or n (%) as appropriate.

*

1369

PSQI , 5

PSQI . 5

p*

90.1¡10.6

84.7¡12.7

0.053

76.2¡16.3 77.5¡16.4 71.1¡37.8 80.4¡30.1 45.6¡17.5 82.0¡24.5

70.7¡16.6 73.4¡17.4 60.8¡34.3 61.9¡36.0 51.4¡17.5 66.3¡31.1

0.147 0.350 0.173 0.034 0.476 0.040

74.9¡19.2 2.8¡3.1 4.7¡3.1 94.3¡8.6

69.3¡22.7 3.4¡3.6 5.1¡3.3 89.5¡8.7

0.340 0.513 0.591 0.031

Mann-Whitney non-parametric test.Data are presents as Mean¡SD. Hospital Anxiety Depression.


Sleep quality after renal transplantation Silva DS et al.

CLINICS 2012;67(12):1365-1371

11. Skobel E, Kaminski R, Breuer C, Topper R, Reffelmann T, Schwarz ER. [Remission of nocturnal pathological respiratory patterns after orthotopic heart transplantation. A case report and overview of current status of therapy]. Med Klin (Munich). 2000;95(12):706-11. 12. Elias RM, Castro MC, de Queiroz EL, Abensur H, Romao JE, Jr., LorenziFilho G. Obstructive sleep apnea in patients on conventional and short daily hemodialysis. Am J Nephrol. 2009;29(6):493-500, http://dx.doi. org/10.1159/000178941. 13. Kutner NG, Zhang R, Huang Y, Bliwise DL. Patient-reported sleep difficulty and cognitive function during the first year of dialysis. Int Urol Nephrol. 2008;40(1):203-10, http://dx.doi.org/10.1007/s11255-007-9188-8. 14. Kutner NG. Promoting functioning and well-being in older CKD patients: review of recent evidence. Int Urol Nephrol. 2008;40(4):1151-8, http://dx.doi.org/10.1007/s11255-008-9469-x. 15. Molnar MZ, Novak M, Szeifert L, Ambrus C, Keszei A, Koczy A, et al. Restless legs syndrome, insomnia, and quality of life after renal transplantation. J Psychosom Res. 2007;63(6):591-7, http://dx.doi.org/ 10.1016/j.jpsychores.2007.06.007. 16. Backhaus J, Junghanns K, Broocks A, Riemann D, Hohagen F. Test-retest reliability and validity of the Pittsburgh Sleep Quality Index in primary insomnia. J Psychosom Res. 2002;53(3):737-40, http://dx.doi.org/10. 1016/S0022-3999(02)00330-6. 17. Bertolazi AN, Fagondes SC, Hoff LS, Dartora EG, Miozzo IC, de Barba ME, et al. Validation of the Brazilian Portuguese version of the Pittsburgh Sleep Quality Index. Sleep Med. 2011;12(1):70-5, http://dx. doi.org/10.1016/j.sleep.2010.04.020. 18. Buysse DJ, Reynolds CF 3rd, Monk TH, Berman SR, Kupfer DJ. The Pittsburgh Sleep Quality Index: a new instrument for psychiatric practice and research. Psychiatry Res. 1989;28(2):193-213. 19. Campolina AG, Ciconelli RM. [SF-36 and the development of new assessment tools for quality of life]. Acta Reumatol Port. 2008;33(2):127-33. 20. Marcolino JA, Mathias LA, Piccinini Filho L, Guaratini AA, Suzuki FM, Alli LA. Hospital Anxiety and Depression Scale: a study on the validation of the criteria and reliability on preoperative patients. Rev Bras Anestesiol. 2007;57(1):52, http://dx.doi.org/10.1590/S0034-709420 07000100006. 21. Cunqueiro JMG, Cortes MJG, Foronda J, Borrego JF, Perales MCS, del Barrio PP, et al. Health-related quality of life in elderly patients in haemodialysis. Nefrologia: publicacion oficial de la Sociedad Espanola Nefrologia. 2003;23(6):528-37. 22. Beecroft JM, Zaltzman J, Prasad R, Meliton G, Hanly PJ. Impact of kidney transplantation on sleep apnoea in patients with end-stage renal disease. Nephrol Dial Transpl. 2007;22(10):3028-33, http://dx.doi.org/10.1093/ ndt/gfm309. 23. Sabbatini M, Crispo A, Pisani A, Gallo R, Cianciaruso B, Fuiano G, et al. Sleep quality in renal transplant patients: a never investigated problem. Nephrol Dial Transpl. 2005;20(1):194-8, http://dx.doi.org/10.1093/ndt/ gfh604. 24. Bohlke M, Marini SS, Rocha M, Terhorst L, Gomes RH, Barcellos FC, et al. Factors associated with health-related quality of life after successful kidney transplantation: a population-based study. Qual Life Res. 2009;18(9):1185-93, http://dx.doi.org/10.1007/s11136-009-9536-5. 25. Rodrigues CLD, Marson O, Tufic S, Kohlmann O, Guimaraes SM, Togeiro P, et al. Relationship among end-stage renal disease, hypertension, and sleep apnea in nondiabetic dialysis patients. American journal of hypertension. 2005;18(2):152-7. 26. Eryavuz N, Yuksel S, Acarturk G, Uslan I, Demir S, Demir M, et al. Comparison of sleep quality between hemodialysis and peritoneal dialysis patients. International urology and nephrology. 2008;40(3):78591, http://dx.doi.org/10.1007/s11255-008-9359-2. 27. Kachuee H, Ameli J, Taheri S, Assari S, Riahipour F, Khedmat H, et al. Sleep quality and its correlates in renal transplant patients. Transplantation proceedings. 2007;39(4):1095-7, http://dx.doi.org/10.1016/j.transproceed. 2007.04.001. 28. Mavanur M, Sanders M, Unruh M. Sleep disordered breathing in patients with chronic kidney disease. Indian Journal of Medical Research. 2010;131(2):277-84. 29. Sabbatini M, Pisani A, Crispo A, Nappi R, Gallo R, Cianciaruso B, et al. Renal transplantation and sleep: a new life is not enough. J Nephrol. 2008;Suppl 13:S97-101. 30. Karaminia R, Tavallaii SA, Lorgard-Dezfuli-Nejad M, Lankarani MM, Mirzaie HH, Einollahi B, et al. Anxiety and depression: A comparison between renal transplant recipients and hemodialysis patients. Transplantation proceedings. 2007;39(4):1082-4, http://dx.doi.org/10.1016/ j.transproceed.2007.03.088. 31. Noohi S, Khaghani-Zadeh M, Javadipour M, Assari S, Najafi M, Ebrahiminia M, et al. Anxiety and depression are correlated with higher morbidity after kidney transplantation. Transplantation proceedings. 2007;39(4):1074-8, http://dx.doi.org/10.1016/j.transproceed.2007.04.002. 32. Gregorio MAPS, Rodriguez AM, Bernal JP. Psychological differences of patients and relatives according to post-transplantation anxiety. Span J Psychol. 2008;11(1):250-8. 33. Prihodova L, Nagyova I, Rosenberger J, Roland R, van Dijk JP, Groothoff JW. Impact of personality and psychological distress on health-related

One relevant contribution of our study was to show that the prevalence of poor sleep quality remains high after successful renal transplantation and that clinicians should pay attention to subjective factors associated with sleep quality and quality of life even after the first year posttransplantation. The assessment of sleep quality in routine post-transplantation follow-up might contribute significantly to the identification and treatment of these disorders. Additionally, social reintegration and a return to professional activities seem to contribute to improved sleep quality in the first year after renal transplantation. Determining the influence of the transplantation team on late post-procedural psychological, emotional, and social aspects may help to improve adherence to therapy and patient perception of sleep quality and quality of life. Further studies should address possible interventions, such as identifying patients at risk of emotional and psychosocial conflicts, that might influence treatment outcomes and sleep quality while maintaining the focus on the multidisciplinary approach of renal transplantation.

ACKNOWLEDGMENTS The study was funded by CAPES.

AUTHOR CONTRIBUTIONS Silva DS was responsible for the study design and execution. Andrade ESP translated and reviewed the manuscript. Elias RM, David-Neto E, Nahas WC, and Castro MC, reviewed the manuscript. Castro MC designed the study.

REFERENCES 1. Iliescu EA, Coo H, McMurray MH, Meers CL, Quinn MM, Singer MA, et al. Quality of sleep and health-related quality of life in haemodialysis patients. Nephrol Dial Transplant. 2003;18(1):126-32, http://dx.doi.org/ 10.1093/ndt/18.1.126. 2. Iliescu EA, Yeates KE, Holland DC. Quality of sleep in patients with chronic kidney disease. Nephrology, dialysis, transplantation: official publication of the European Dialysis and Transplant Association European Renal Association. 2004;19(1):95-9, http://dx.doi.org/10.1093/ ndt/gfg423 3. 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;328(17):1230-5, http://dx.doi.org/10.1056/NEJM199304293281704. 4. Reimer J, Franke GH, Lutkes P, Kohnle M, Gerken G, Philipp T, et al. [Quality of life in patients before and after kidney transplantation]. Psychother Psychosom Med Psychol. 2002;52(1):16-23, http://dx.doi.org/ 10.1055/s-2002-19662. 5. Molnar MZ, Novak M, Mucsi I. Sleep disorders and quality of life in renal transplant recipients. Int Urol Nephrol. 2009;41(2):373-82, http:// dx.doi.org/10.1007/s11255-009-9527-z. 6. Rodrigue JR, Mandelbrot DA, Hanto DW, Johnson SR, Karp SJ, Pavlakis M. A cross-sectional study of fatigue and sleep quality before and after kidney transplantation. Clin Transplant. 2011;25(1):E13-21, http://dx. doi.org/10.1111/j.1399-0012.2010.01326.x. 7. Kovacs AZ, Molnar MZ, Szeifert L, Ambrus C, Molnar-Varga M, Szentkiralyi A, et al. Sleep disorders, depressive symptoms and healthrelated quality of life–a cross-sectional comparison between kidney transplant recipients and waitlisted patients on maintenance dialysis. Nephrol Dial Transplant. 2011;26(3):1058-65, http://dx.doi.org/10.1093/ ndt/gfq476. 8. Koyama H, Fukuda S, Shoji T, Inaba M, Tsujimoto Y, Tabata T, et al. Fatigue is a predictor for cardiovascular outcomes in patients undergoing hemodialysis. Clin J Am Soc Nephrol. 2010;5(4):659-66, http://dx. doi.org/10.2215/CJN.08151109. 9. Ameli J, Kachuee H, Assari S, Rasta VR, Khoddami-Vishte HR, et al. Does etiology of end-stage renal disease affect sleep quality in kidney transplant recipients? Transplant Proc. 2007;3(4):1091-4, http://dx.doi. org/10.1016/j.transproceed.2007.03.011. 10. Eryilmaz MM, Ozdemir C, Yurtman F, Cilli A, Karaman T. Quality of sleep and quality of life in renal transplantation patients. Transplant Proc. 2005;37(5):2072-6, http://dx.doi.org/10.1016/j.transproceed.2005.03.084.

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Sleep quality after renal transplantation Silva DS et al. 36. Jofre R, Lopez-Gomez JM, Moreno F, Sanz-Guajardo D, Valderrabano F. Changes in quality of life after renal transplantation. American Journal of Kidney Diseases. 1998;32(1):93-100, http://dx.doi.org/10.1053/ajkd. 1998.v32.pm9669429. 37. Matas AJ, Halbert RJ, Barr ML, Helderman JH, Hricik DE, Pirsch JD, et al. Life satisfaction and adverse effects in renal transplant recipients: a longitudinal analysis. Clin Transplant. 2002;16(2):113-21, http://dx.doi. org/10.1034/j.1399-0012.2002.1o126.x.

quality of life in kidney transplant recipients. Transplant International. 2010;23(5):484-92, http://dx.doi.org/10.1111/j.1432-2277.2009.01003.x. 34. Weng LC, Dai YT, Huang HL, Chiang YJ. Self-efficacy, self-care behaviours and quality of life of kidney transplant recipients. J Adv Nurs. 2010;66(4):828-38, http://dx.doi.org/10.1111/j.1365-2648.2009.05243.x. 35. Valderrabano F, Jofre R, Lopez-Gomez JM. Quality of life in end-stage renal disease patients. Am J Kidney Dis. 2001;38(3):443-64, http://dx.doi. org/10.1053/ajkd.2001.26824.

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DOI:10.6061/clinics/2012(12)05

CLINICAL SCIENCE

Differential expression of hypoxia-inducible factor 1a in non-small cell lung cancer and small cell lung cancer Eleni Karetsi,I Maria G. Ioannou,II Theodora Kerenidi,I Markos Minas (in memoriam), Paschalis A. Molyvdas,III Konstantinos I. Gourgoulianis,I Efrosyni ParaskevaIII I University of Thessaly, Medical School, Department of Respiratory Medicine, Biopolis, Larissa, Greece. II University of Thessaly, Medical School, Department of Pathology, Biopolis, Larissa, Greece. III University of Thessaly, Medical School, Department of Physiology, Biopolis, Larissa, Greece.

OBJECTIVES: The aim of this study was to compare the expression of hypoxia-inducible factor 1a and vascular endothelial growth factor in small cell lung cancer and subtypes of non-small cell lung cancer and examine their relationships with clinicopathologic factors, response to treatment and survival. METHODS: We examined samples obtained by bronchial endoscopic biopsy from 55 patients with inoperable lung cancer (16 with adenocarcinoma, 17 with squamous cell carcinoma, and 22 with small cell lung cancer). Hypoxiainducible factor 1a and vascular endothelial growth factor were detected using immunohistochemistry. The diagnosis, treatment, and follow-up of patients were conducted according to the standard practice. RESULTS: A significant difference (p = 0.022) in hypoxia-inducible factor 1a expression was observed between nonsmall cell lung cancer (75.8% positive) and small cell lung cancer (45.5% positive). The frequency of hypoxiainducible factor 1a nuclear expression was 88.2% in squamous cell carcinoma, 62.5% in adenocarcinoma, and 45.5% in small cell lung cancer. A significant correlation was observed between hypoxia-inducible factor 1a and vascular endothelial growth factor expression (Fisher’s exact test, p = 0.001) when all types of lung cancer were examined, either collectively or separately. CONCLUSIONS: The expression of hypoxia-inducible factor-1a differs significantly between subtypes of lung cancer. These findings could help elucidate the biology of the different types of non-operable lung carcinomas and have implications for the design of new therapeutic approaches for lung cancer. KEYWORDS: Hypoxia-Inducible Factor 1a; Lung Cancer; Vascular Endothelial Growth Factor; Small Cell Lung Cancer; Non-small Cell Lung Cancer. Karetsi E, Ioannou MG, Kerenidi T, Molyvdas PA, Gourgoulianis KI, Paraskeva E. Differential expression of hypoxia-inducible factor 1a in non-small cell lung cancer and small cell lung cancer. Clinics. 2012;67(12):1373-1378. Received for publication on July 24, 2012; First review completed on August 1, 2012; Accepted for publication on August 14, 2012 E-mail: fparaskeva@med.uth.gr Tel.: 30 2410685559

types of lung cancer have distinct histological features and biological behavior, which influence the treatment and prognosis of lung cancer patients (1). It is therefore important to understand the molecular profiles of the different types of lung cancer to improve the repertoire of therapies used for their treatment. The transcriptional activator hypoxia-inducible factor 1 (HIF-1) is a key mediator of the cellular response to hypoxia. HIF-1 consists of a regulatory alpha subunit and a constitutive beta subunit and controls the expression of many genes involved in angiogenesis, erythropoiesis, glucose uptake, cell metabolism, apoptosis, invasion and metastasis (2-4). The importance of HIF-1a and its target gene vascular endothelial growth factor (VEGF) as biomarkers and therapeutic targets in cancer therapy is currently emerging (3,5,6). Increased expression of HIF-1a has been observed in several human cancers, including NSCLC (7-9), and is, in some cases, associated with a poor prognosis (10,11). In

INTRODUCTION Lung cancer is the primary type of cancer responsible for patient death worldwide. Lung cancer is divided into two major subtypes, non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). NSCLC is further divided into four histological subtypes - adenocarcinoma, squamous cell carcinoma, large cell carcinoma, and NSCLC NOS (not otherwise specified) - of which adenocarcinoma and squamous cell carcinoma are the most common and represent approximately 80% of all cases. The different

Copyright ß 2012 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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addition, the expression of HIF-1a in SCLC has been recently reported (12). These findings highlight the possible use of HIF-1a as a prognostic factor for the outcome of lung cancer patients. Moreover, biological agents against VEGF have been approved for clinical use as a first-line treatment in several cancer types, including advanced NSCLC (13,14). Consequently, HIF-1a could be considered an additional target for the treatment of lung cancer in cases in which its expression is elevated. In this study, we evaluated the expression of HIF-1a and VEGF in bronchial biopsies from patients with inoperable NSCLC and SCLC and examined the correlation between these markers, the different cancer types, clinicopathologic factors, response to treatment, and survival. This comparative analysis could assist with the clarification of the unique HIF-1a and VEGF expression profiles in SCLC and subtypes of NSCLC and assist with their evaluation as prognostic factors and therapeutic targets.

Table 1 - Characteristics and response to treatment of patients with SCLC and NSCLC. SCLC

Gender (male) Age (years) #65 years .65 years Smoking status S NS/ES Weight loss ($5%) Performance status N 0 N 1 Stage N III N IV T T1-T2 T3-T4 N N0-N1 N2-N3 M M0 M1 Response to treatment N PD, SD N PR, CR

MATERIALS AND METHODS Patients and Tissue Samples This study was performed on 216 consecutive patients who underwent bronchoscopy for diagnostic purposes in the Respiratory Medicine Department of the Medical School of the University of Thessaly in Larissa between May and December 2008. Of these patients, 134 were excluded from the study because they did not have endobronchial findings. Of the rest that underwent bronchial endoscopic biopsy, eight patients were excluded because they had operable lung cancer, 14 patients were excluded because their bronchial endoscopic biopsy showed histological types other than SCLC or NSCLC, and five patients were excluded because data were missing in their medical records. The remaining 55 patients (52 men and three women), who were diagnosed with SCLC or NSCLC (16 with adenocarcinoma, 17 with squamous cell carcinoma, and 22 with SCLC) and had not received any treatment, were evaluated. The study protocol was approved by the local ethics committee, and all subjects gave their written informed consent. Subject characteristics are presented in Table 1. Tumor staging was based on the staging system of the American Joint Committee on Cancer (AJCC), Revised Definition of TNM classification. Only patients with inoperable lung cancer were included (24 patients with stage III and 31 patients with stage IV). The mean age of patients was 62.8¡9.8 years. The treatment and follow-up of patients were conducted according to the standard practice. The surveillance included follow-up visits every three months during the first and second years and every six months during third to fifth years. Patients with incomplete medical records or inadequate follow-up were excluded from study. Overall survival was calculated from the day of first-line treatment initiation to the day of the last follow-up (death or follow-up visit). All histological samples were obtained using bronchoscopy techniques for diagnostic purposes and were fixed in 10% buffered formalin, processed, and embedded in paraffin. All biopsies were retrieved from the Pathology Department of the University of Thessaly.

NSCLC

N = 22

N = 33

21 (95.5%) 60.7¡10.0 16 (72.7%) 6 (27.3%)

31 (93.9%) 64.2¡9.5 17 (51.5%) 16 (48.5%)

16 (72.7) 6 (27.3) 9 (40.9%)

20 (60.6%) 13 (39.4%) 12 (36.4%)

12 (54.5%) 10 (45.5%)

17 (51.5%) 16 (48.5%)

8 (36.4%) 14 (63.6%)

16 (48.5%) 17 (51.5%)

7 (31.8%) 15 (68.2%)9

11 (33.3%) 22 (66.7%)

4 (18.2%) 18 (81.8%)

11 (33.3%) 22 (66.7%)

8 (36.4%) 14 (63.6%)

16 (48.5%) 17 (51.5%)

7 14

21 12

p-value

0.808 0.198

0.354

0.734 0.825

0.375

0.907

0.216

0.375

0.034

Data were obtained at the time of diagnosis, prior to the initiation of any treatment. Data are presented as the mean¡S.E.M with the p-value. N = number of patients. Abbreviations: NSCLC, non-small cell lung cancer; SCLC, small cell lung cancer; S, smokers; NS, non-smokers; ES, ex-smokers; PD, progressive disease; SD, stable disease; PR, partial response; CR, complete response. Bold letters indicate statistically significant differences.

clone JH121 (Neomarkers, UK, dilution 1:50) as previously described (15). Briefly, the assessment of HIF-1a immunoreactivity scores was based on the percentage of cells showing positive nuclear immunostaining in the whole tumor area of the section. The percentage of tumor cells with cytoplasmic VEGF staining was recorded to assess VEGF reactivity. HIF-1a and VEGF expression was considered positive when immunostaining was observed in at least 10% of the tumor cells.

Statistical analysis Statistical analysis was performed using the SPSS software package, version 16.0 (SPSS Inc., Chicago, IL, USA). Spearman’s rank correlation test was used to assess the relationship between ordinal variables. A linear regression analysis was used to study the association between HIF-1a and VEGF expression. The association between HIF-1a and VEGF was assessed using HIF-1a as a continuous variable. Pair-wise comparisons were performed with the nonparametric Mann-Whitney U test. For all tests, p#0.05 was considered statistically significant.

RESULTS

HIF-1a and VEGF immunohistochemistry and evaluation of immunostaining

Detection of HIF-1a in NSCLC and SCLC

HIF-1a was detected using the monoclonal antibody 54/ HIF-1a (BD Transduction Laboratories, San Diego, dilution 1:20), and VEGF was detected using the primary antibody

After immunohistological examination, 75.8% (n = 33) of NSCLC cases (i.e., 88.2% (n = 17) of squamous cell carcinoma cases and 62.5% (n = 16) of adenocarcinoma cases) and

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45.5% (n = 22) of SCLC cases were scored as HIF-1a positive (Figure 1). The percentage of HIF-1a-positive cases was significantly higher in NSCLC compared to SCLC (p = 0.022, Figure 1B).

adenocarcinoma) and 16 cases (72.7%) of SCLC (Figures 2A and 2B). VEGF immunoreactivity showed no association with the histological types of lung cancer. A significant correlation was observed between HIF-1a with VEGF expression when all types of lung cancer were included in the analysis (R2 = 0.52, p,0.001, Fig. 2C). In addition, further analysis of the lung cancer subtypes revealed a significant correlation between HIF-1a and VEGF expression in squamous cell carcinoma (R2 = 0.80, p,0.001), adenocarcinoma (R2 = 0.45, p,0.001), and SCLC (R2 = 0.59, p,0.001).

Detection of VEGF and correlation with HIF-1a in NSCLC and SCLC Cytoplasmic VEGF immunostaining was detected in 76.4% of all cases. The VEGF-positive biopsies were observed in 26 cases (78.8%) of NSCLC (i.e., 14 cases (82%) of squamous cell carcinoma and 12 cases (75%) of

Figure 1 - HIF-1a immunostaining in lung carcinoma specimens. A) Strong nuclear and weak cytoplasmic HIF-1a staining in squamous cell carcinoma, HIF-1a nuclear staining in adenocarcinoma cells and HIF-1a nuclear staining in SCLC (immunoperoxidase staining, original magnification 6400). B) Percentage of HIF-1a-positive samples in NSCLC compared to SCLC samples (p = 0.022). C) Percentage of HIF-1a-positive samples in squamous cell carcinoma, adenocarcinoma and SCLC.

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Figure 2 - VEGF immunoreactivity in lung carcinoma specimens. A) Diffuse cytoplasmic staining in many malignant cells in squamous cell carcinoma, adenocarcinoma and SCLC (immunoperoxidase staining, original magnification 6200). B) Percentage of VEGF-positive samples in squamous cell carcinoma, adenocarcinoma and SCLC. C) Linear regression analysis of the percentage of HIF-1a-positive cells with VEGF-positive cells (r2: correlation coefficient).

VEGF expression and overall survival for any of the histologic subtypes (data not shown).

Relationships between HIF-1a and VEGF expression and clinicopathologic factors, response to treatment and survival

DISCUSSION

We observed no significant association between HIF-1a or VEGF expression and various clinical factors, including age, smoking habit, performance status, weight loss, stage, and N (lymph nodes) or M (metastases) stage or response to treatment in adenocarcinoma, squamous cell carcinoma or SCLC (Table 2). However, the analysis revealed a significant positive association between T stage and HIF-1a expression (p = 0.003) and VEGF expression (p = 0.008) in adenocarcinoma but not squamous cell carcinoma or SCLC (Table 2). We observed no significant associations between HIF or

Individual lung cancer patients respond differently to chemotherapy and have different survival rates. This variability is related to the lung cancer histological subtype and its individual biological characteristics (Table 1). Therefore, it is important to improve our knowledge of the pathophysiology and molecular profiles of the different histological types of lung cancer, thus allowing personalization of the available therapies and the design of new, efficient ones. In this respect, understanding the contribution of

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Hypoxia-inducible factor 1a in lung cancer Karetsi E et al.

Table 2 - The relationships between HIF-1a and VEGF expression and clinicopathologic factors in three types of lung cancer. Squamous carcinoma HIF-1a N Age #65 years .65 years Smoking status S NS/ES

+

p-value

N

0.63 10 5

0.21 10 4

0.21 7 8

0.76 6 8

0.33 5 10 0.64

0.21 10 4

0.92

0.60 7 7

0.19

0.35

0.91 3 13

0.57 3 7

0.24 8 3

0.92 5 11

3 7

3 9 0.79

7 2

0.095

0.074

0.33

0.07 4 12

5 5

6 6

4 6

0.57

0.008

0.42 3 7

0.48 8 8

3 7

1 11

0.59 9

0.69

0.35

0.003

0.87

0.34

5 5

3 9

0 10

5 10

10 5

0.33 4 6

0.69 12 4

3 0.25

0.07 4 12

0.14 2 8

p-value 0.69

0.48

0.55

7 5

N

12 4

8 2

4 0.30

p

VEGF+

0.48

0.55

0.79

4 6

N

+

8 2

7 5

4 0.45

p-value

HIF-1a

0.77

0.18

0.93

8 6

N

+

7 5

5 5

5 0.92

p-value

SCLC

VEGF

0.51

0.76

0.26

8 7

N

+

5 5

8 6

6

HIF-1a

0.45

0.78

8 7

p-value

6 8

9 6

Weight loss $5% Perform.status 0 1 Stage III IV T T1-T2 T3-T4 N N0-N1 N2-N3 M M0 M1 Response to treatment PD, SD PR, CR

VEGF

0.11 6 9

Adenocarcinoma +

0.24 7 9

N = patient number. Abbreviations: SCLC, small cell lung cancer; S, smokers; NS, non-smokers; ES, ex-smokers; PD, progressive disease; SD, stable disease; PR, partial response; CR, complete response. Bold letters indicate statistically significant differences.

VEGF, the major angiogenic factor, and its key transcriptional regulator, HIF-1, is of great importance. Unfortunately, most of the studies performed to date on the expression of HIF-1a and VEGF in lung cancer concern operable NSCLC, and mainly, the adenocarcinoma subtype (16,17). In contrast, few studies have evaluated the expression of these factors in subtypes of operable NSCLC (8) or bronchial biopsies of SCLC (12). Because the majority of lung cancer patients are diagnosed at an advanced stage of the disease and carry inoperable tumors, it is very important to diagnose their tumor molecular profile using specimens obtained from less invasive methods, such as bronchoscopy. To the best of our knowledge, the present study is the first to compare the expression of the angiogenic and pro-metastatic factors HIF1a and VEGF in bronchial biopsies obtained from inoperable adenocarcinoma, squamous cell carcinoma and SCLC (Figure 1A). We detected convincing HIF-1a immunoreactivity in slightly less than half of the SCLC samples. A significantly higher incidence of HIF-1a expression was observed in NSCLC (squamous cell carcinoma and adenocarcinoma) compared to SCLC (Figure 1B). We also observed a higher frequency of HIF-1a-positive squamous cell carcinoma cases than adenocarcinoma cases (Figure 1C). However, VEGF expression was equally high in subtypes of NSCLC and SCLC (Figure 2B), and there was a correlation between HIF-1a and VEGF expression in all types of lung cancer collectively (Figure 2C) as well as in each type of lung cancer separately.

Interestingly, we found biopsies that were positive for VEGF staining and negative for HIF-1a expression. In HIF-1anegative, VEGF-positive cases, it is possible that HIF-1aindependent pathways are responsible for the induction of VEGF. VEGF transcription in these samples might be controlled by HIF-2, which contains the HIF-2a isoform (3,6). The relative importance of the HIF 1a and 2a subunits in different tissues and cell types is still under intense study (18). The expression of HIF-2a has been previously shown in early operable NSCLC specimens (7), but there are no data concerning SCLC or subtypes of NSCLC. In HIF-1a-negative, VEGF-positive adenocarcinoma or SCLC cases, HIF-2a or other molecules and pathways may be responsible for VEGF induction. In the present study, we found that the expression of both HIF-1a and VEGF was associated with T stage in adenocarcinoma. A previous report also indicated that HIF-1a expression in operable non-small cell lung cancer was marginally associated with T stage (7). We found no association between HIF-1a or VEGF expression and response to treatment or survival for the different types of lung cancer. This could be in part due to the relatively small number of biopsies examined. Previous studies have also reported a diverse degree of association between HIF-1a expression and the survival of lung cancer patients (7,8,12), while HIF-1a-positive, VEGFpositive SCLC biopsies were associated with poor survival (12). In conclusion, this study demonstrated that HIF-1a was differentially expressed across lung cancer subtypes and that HIF-1a and VEGF had varying associations with

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8. Lee CH, Lee MK, Kang CD, Kim YD, Park DY, Kim JY, et al. Differential Expression of Hypoxia Inducible Factor-1 alpha and Tumor Cell Proliferation Between Squamous Cell Carcinomas and Adenocarcinomas Among Operable Non-Small Cell Lung Carcinomas. J Korean Med Sci. 2003;18(2):196-203. 9. Zhang H, Zhang Z, Xu Y, Xing L, Liu J, Li J, et al. The expression of hypoxia inducible factor 1-alpha in lung cancer and its correlation with P53 and VEGF. J Huazhong Univ Sci Technolog Med Sci. 2004;24(2):1247. 10. Giatromanolaki A, Koukourakis MI, Sowter HM, Sivridis E, Gibson S, Gatter KC, et al. BNIP3 Expression Is Linked with Hypoxia-Regulated Protein Expression and with Poor Prognosis in Non–Small Cell Lung Cancer. Clin Cancer Res. 2004;10(16):5566-71, http://dx.doi.org/10. 1158/1078-0432.CCR-04-0076. 11. Swinson D, Jones JL, Cox G, Richardson D, Harris AL, O’Byrne KJ. Hypoxia-inducible factor-1 in non small cell lung cancer: Relation to growth factor, protease and apoptosis pathways. Int J Cancer. 2004;111(1):43-50, http://dx.doi.org/10.1002/ijc.20052. 12. Ioannou M, Papamichali R, Kouvaras E, Mylonis I, Vageli D, Kerenidou T, et al. Hypoxia Inducible Factor-1alpha and Vascular Endothelial Growth Factor in Biopsies of Small Cell Lung Carcinoma Lung. 2009;187(5):321-9. 13. Pakkala S, Ramalingam SS. Combined Inhibition of Vascular Endothelial Growth Factor and Epidermal Growth Factor Signaling in Non–SmallCell Lung Cancer Therapy. Clin Lung Cancer. 2009;10(Suppl 1):S17-23, http://dx.doi.org/10.3816/CLC.2009.s.003. 14. Sandler A, Gray R, Perry MC, Brahmer J, Schiller JH, Dowlati A, et al. Paclitaxel-carboplatin alone or with bevacizumab for non-small-cell lung cancer. N Engl J Med. 2006;355(24):2542-50, http://dx.doi.org/10.1056/ NEJMoa061884. 15. Ioannou M, Mylonis I, Kouvaras E, Papamichali R, Daponte A, Paraskeva E, et al. Validated analysis of HIF-1a expression in cancer cells using a controlled and comparative immunoassay. Oncol Rep. 2010;24(1):161-9. 16. Felip E, Taron M, Rosell R, Mendez P, Queralt C, Ronco MS, et al. Clinical significance of hypoxia-inducible factor-1a messenger RNA expression in locally advanced non-small-cell lung cancer after platinum agent and gemcitabine chemotherapy followed by surgery. Clin Lung Cancer. 2005;6(5):299-303, http://dx.doi.org/10.3816/CLC.2005.n.009. 17. Kim SJ, Rabbani ZN, Dewhirst MW, Vujaskovic Z, Vollmer RT, Schreiber E-G, et al. Expression of HIF-1a, CA IX, VEGF, and MMP-9 in surgically resected non-small cell lung cancer. Lung Cancer. 2005;49(3):325-35, http://dx.doi.org/10.1016/j.lungcan.2005.03.036. 18. Keith B, Johnson RS, Simon MC. HIF1alpha and HIF2alpha: sibling rivalry in hypoxic tumour growth and progression. Nat Rev Cancer. 2012;12(1):9-22.

clinicopathological markers in different lung cancer subtypes. It also highlights the importance of using small bronchial biopsy specimens to evaluate the expression of important molecular markers, which can contribute to optimal therapy design for patients with advanced inoperable lung cancer.

ACKNOWLEDGMENTS This work was supported by a grant from the Hellenic Thoracic Society.

AUTHOR CONTRIBUTIONS Karetsi E conceived the study, performed the experiments, analyzed the data and wrote the manuscript. Ioannou MG performed the experiments and analyzed the data. Kerenidi T analyzed the data. Minas M analyzed the data. Molyvdas PA and Gourgoulianis KI conceived the study and wrote the manuscript. Paraskeva E conceived and designed the study and wrote the manuscript.

REFERENCES 1. Hammerschmidt S, Wirtz H. Lung cancer: current diagnosis and treatment. Dtsch Arztebl Int. 2009;106(49):809-18; quiz 819-20. 2. Harris AL. Hypoxia - a key regulatory factor in tumour growth. Nat Rev Cancer 2002;2(1):38-47, http://dx.doi.org/10.1038/nrc704. 3. Pouyssegur J, Dayan F, Mazure NM. Hypoxia signalling in cancer and approaches to enforce tumour regression. Nature. 2006;441(7092):437-43, http://dx.doi.org/10.1038/nature04871. 4. Semenza GL. Hypoxia-inducible factors in physiology and medicine. Cell. 2012;148(3):399-408, http://dx.doi.org/10.1016/j.cell.2012.01.021. 5. Melilo G. Targeting hypoxia cell signaling for cancer therapy. Cancer and Metastasis Reviews. 2007;26(2):341-52, http://dx.doi.org/10.1007/ s10555-007-9059-x. 6. Poon E, Harris AL, Ashcroft M. Targeting the hypoxia-inducible factor (HIF) pathway in cancer. Expert Rev Mol Med. 2009;11:e26, http://dx. doi.org/10.1017/S1462399409001173. 7. Giatromanolaki A, Koukourakis MI, Sivridis E, Turley H, Talks K, Pezzella F, et al. Relation of hypoxia inducible factor 1alpha and 2alpha in operable non-small cell lung cancer to angiogenic/molecular profile of tumours and survival. British Journal of Cancer. 2001;85(6):881-90, http://dx.doi.org/10.1054/bjoc.2001.2018.

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DOI:10.6061/clinics/2012(12)06

CLINICAL SCIENCE

Mechanical thrombectomy with solitaire stent retrieval for acute ischemic stroke in a Brazilian population Luis Henrique de Castro-Afonso,I Thiago Giansante Abud,I Octa´vio Marques Pontes-Neto,II Lucas Moretti Monsignore,I Guilherme Seizem Nakiri,I Pedro Telles Cougo-Pinto,II Lı´via de Oliveira,I Daniela dos Santos,I Francisco A Dias,II Soraia Cabette Ramos Fa´bio,II Francisco Antoˆnio Coletto,II Daniel Giansante AbudI I University of Sa˜o Paulo, Medical School of Ribeira˜o Preto, Division of Interventional Neuroradiology, Ribeira˜o Preto/SP, Brazil. II University of Sa˜o Paulo, Medical School of Ribeira˜o Preto, Division of Neurology, Ribeira˜o Preto/SP, Brazil.

OBJECTIVE: Large vessel occlusion in acute ischemic stroke is associated with low recanalization rates under intravenous thrombolysis. We evaluated the safety and efficacy of the Solitaire AB stent in treating acute ischemic stroke. METHODS: Patients presenting with acute ischemic stroke were prospectively evaluated. The neurological outcomes were assessed using the National Institutes of Health Stroke Scale and the modified Rankin Scale. Time was recorded from the symptom onset to the recanalization and procedure time. Recanalization was assessed using the thrombolysis in cerebral infarction score. RESULTS: Twenty-one patients were evaluated. The mean patient age was 65, and the National Institutes of Health Stroke Scale scores ranged from 7 to 28 (average 17¡6.36) at presentation. The vessel occlusions occurred in the middle cerebral artery (61.9%), distal internal carotid artery (14.3%), tandem carotid occlusion (14.3%), and basilar artery (9.5%). Primary thrombectomy, rescue treatment and a bridging approach represented 66.6%, 28.6%, and 4.8% of the performed procedures, respectively. The mean time from symptom onset to recanalization was 356.5¡107.8 minutes (range, 80-586 minutes). The mean procedure time was 60.4¡58.8 minutes (range, 14-240 minutes). The overall recanalization rate (thrombolysis in cerebral infarction scores of 3 or 2b) was 90.4%, and the symptomatic intracranial hemorrhage rate was 14.2%. The National Institutes of Health Stroke Scale scores at discharge ranged from 0 to 25 (average 6.9¡7). At three months, 61.9% of the patients had a modified Rankin Scale score of 0 to 2, with an overall mortality rate of 9.5%. CONCLUSIONS: Intra-arterial thrombectomy with the Solitaire AB device appears to be safe and effective. Large randomized trials are necessary to confirm the benefits of this approach in acute ischemic stroke. KEYWORDS: Acute Ischemic Stroke; Mechanical Thrombectomy; Stent Retrieval; Thrombolysis. Castro-Afonso LH, Abud TG, Pontes-Neto OM, Monsignore LM, Nakiri GS, Cougo-Pinto PT, et al. Mechanical thrombectomy with solitaire stent retrieval for acute ischemic stroke in a Brazilian population. Clinics. 2012;67(12):1379-1386. Received for publication on June 11, 2012; First review completed on August 10, 2012; Accepted for publication on August 14, 2012 E-mail: dgabud@fmrp.usp.br Tel.: 55 16 3602-2640

treatment (4). Although national initiatives aimed at improving stroke care have been implemented (5), few national studies have examined intra-arterial recanalization therapies for acute ischemic stroke. Intravenous thrombolysis using a recombinant tissue plasminogen activator (TPA) for arterial recanalization is the standard treatment for acute ischemic stroke (6,7). Recanalization is strongly associated with clinical outcomes (8). Intravenous thrombolysis has not been associated with high recanalization rates in large vessel occlusions (9,10). In this setting, intra-arterial thrombectomy with retrievable stent devices has reportedly achieved higher recanalization rates and shorter recanalization times (11-28). The aim of this study was to assess the safety and efficacy of the Solitaire AB device (ev3, Irvine, CA, USA) in acute ischemic stroke patients who were prospectively evaluated and treated at the Clinics Hospital of the Ribeira˜o Preto

INTRODUCTION Ischemic stroke is the third leading cause of death and the leading cause of long-term disability worldwide (1); it is the leading cause of mortality in Brazil (2). An increased national incidence of stroke is expected, and preventive and acute medical care is needed (3). Despite the rising occurrence of strokes and stroke-related deaths, the adult Brazilian population has demonstrated an alarming lack of knowledge about stroke prevention, recognition, and

Copyright ß 2012 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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Figure 1 - (a) Digital subtraction angiography (DSA) of the right common carotid artery ([RCCA] arterial phase, oblique view) shows an occlusion of the distal right internal carotid artery (RICA) (black arrow); (b) a road map of the RICA (oblique view) shows a guiding catheter in the RICA (arrowhead); the distal tip of the microcatheter (white arrow) and the microwire have crossed the occluded portion of the RICA and moved through the thrombi and into the right middle cerebral artery (RMCA) (black arrow); (c) a road map of the RICA (oblique view) shows the terminal tip of microcatheter (white arrow) distal to the occlusion site; (d) a road map of the RICA (oblique view) shows the terminal tip of the microcatheter (white arrow) and the terminal radiopaque marks of a 6x30 mm Solitaire AB device (ev3, Irvine, CA, USA), which indicate the start of deployment (white arrowhead); (e) a road map of the RICA (oblique view) in which the proximal stent radiopaque mark is seen ahead of the distal tip of microcatheter (white arrowhead), indicating the full deployment of the stent into the RMCA and the distal RICA; (f) a control DSA of the RCCA (arterial phase, oblique view) that was performed after the stent retrieval shows that the distal RICA, right anterior cerebral artery (RACA) and RMCA branches have been completely opened; (g) a picture shows the Solitaire AB (ev3, Irvine, CA, USA) and the removed clot.

Medical School, SaËœo Paulo, Brazil. The literature on intraarterial recanalization therapies was reviewed and compared to the results of the present study.

circulation stroke or within nine hours for posterior circulation stroke. The arterial occlusion site was assessed with either combined transcranial color-coded duplex (TCCD) and carotid ultrasound (US) or brain computed tomography angiography (CTA). When vessel imaging was unavailable, a score$10 on the National Institutes of Health Stroke Scale (NIHSS), coma, tetraparesis and locked-in syndrome were used as the proximal occlusion criteria.

METHODS Patient Population We prospectively followed a group of patients who were treated for acute ischemic stroke with the Solitaire AB device (ev3, Irvine, CA, USA) by the same stroke team from June 2011 to January 2012. The study was approved by the ethics committee at our institution. The patients or their legal representatives signed the consent forms that were previously approved by the institutional review board. The patients were selected for intra-arterial recanalization treatment according to our institution’s acute stroke protocol for recanalization strategies. Initially, the patients were assessed for eligibility for intravenous thrombolysis using the National Institutes of Neurological Disorders and the European Cooperative Stroke Study 3 trial criteria (1,2). The endovascular recanalization procedures have three modalities: rescue treatment, a bridging approach, and primary thrombectomy. All of the patients who were eligible for standard intravenous thrombolysis were considered for rescue intra-arterial therapy if they exhibited a persistent proximal occlusion at the end of intravenous fibrinolysis. Likewise, the patients were considered for bridging intraarterial therapy if they presented with a tandem carotid occlusion or basilar occlusion, which are known to have low recanalization rates with intravenous TPA (9,10). The patients who were ineligible for intravenous fibrinolysis were treated with primary thrombectomy. Patients were excluded from intra-arterial therapy if they presented with hypoattenuation in more than one-third of the middle cerebral artery territory on brain computed tomography (CT) or if intra-arterial recanalization could not be performed within eight hours of symptom onset for anterior

Patient assessment and follow-up The stroke severity was assessed using the NIHSS upon admission. A brain CT scan was quickly performed to determine the brain tissue injury pattern. When available, TCCD and carotid US were also performed in the acute phase to assess the artery occlusion site. If visualizing the intracranial arteries was not possible using TCCD, a CTA scan was performed. A second and third brain CT scan were obtained immediately after thrombolysis and between 24 and 48 hours after treatment, respectively. All of the patients were kept under observation until discharge and were followed up three months after their strokes. The patient outcomes were assessed by certified examiners at discharge (NIHSS) and the three-month follow-up (modified Rankin Scale, mRS). The time from the symptom onset to the recanalization and procedure times were recorded. The procedure time began with the groin puncture and ended at the instant of recanalization. Recanalization was assessed using the thrombolysis in cerebral infarction score (TICI) (29). The number of Solitaire AB device (ev3, Irvine, CA, USA) passes was recorded.

Rescue treatment Standard intravenous thrombolysis was performed with a 0.9 mg/kg dose of TPA (with a maximum total dose of 90 mg), which was delivered as an initial bolus of 10% with the remaining 90% infused within 60 minutes. The patients

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Figure 2 - (a) DSA of the left common carotid artery (LCCA), arterial phase, frontal view, shows a proximal occlusion of M1 segment of left middle cerebral artery (LMCA) (black arrow); (b) DSA of left interna carotid artery (LICA), arterial phase, frontal view, shows a Solitaire AB (ev3, Irvine, CA, USA) deployed into the M1 segment and promptly flow restoration of LMCA (black arrow); (c) Final control DSA of the LCCA, arterial phase, frontal view, shows the complete recanalization of the LMCA; (d, e, f) road maps of LICA, frontal views, show (d) the microwire crossed the occluded LMCA (black arrow), (e) the terminal tip of the microcatheter (black arrow) placed beyond the thrombi into the M1 segment of the LMCA and (f) terminal radiopaque marks of a 4x20 mm Solitaire AB device (ev3) deployed into the occluded site of LMCA; (g, h, i) Brain CT scans, (g) performed on presentation revealing no signs of hemorrhage or ischemic injury, (h) performed immediately after intra-arterial thrombectomy showing contrast medium enhance in the left middle cerebral artery territory and (i) performed 48 hours after intra-arterial thrombectomy showing contrast medium cleared and slight parenchymal hypoattenuation signs.

were monitored using NIHSS assessment and continuous TCCD. An urgent rescue thrombectomy was performed 60 minutes after the bolus if a persistent proximal occlusion was visible on TCCD or the NIHSS score did not improve by$4 points (if TCCD was unavailable).

Neurointerventional procedure For the thrombectomy, the patient was transferred to the angiography suite, where the intubation was performed. The patient’s blood pressure was carefully monitored throughout the anesthesia induction and the procedure to maintain a minimum pressure of 180 mm Hg (systolic arterial pressure) or 80 mm Hg (mean arterial pressure). If there were no complications, the patient was extubated immediately after the procedure. All of the procedures were performed using the femoral artery approach. An intravenous bolus of 5,000 IU of standard heparin was administered after the puncture if intravenous TPA was not previously infused. If TPA was infused prior to the endovascular procedure, no heparin was administered after the femoral puncture. A 7-Fr guiding catheter (Guider Softip; Boston Scientific, Natick, MA) or a 6-Fr guiding catheter (Neuron; Penumbra, Alameda, CA) was introduced through a femoral sheath into the internal carotid artery or the most navigable vertebral artery (related to the ischemic vascular territory). The guiding catheter was continuously perfused with a solution of 40 mg of verapamil hydrochloride diluted in 1,000 mL of physiological saline (0.9%). Frontal, oblique,

Bridging approach The patients who presented with carotid tandem occlusion or basilar artery occlusion associated with coma, tetraparesis or locked-in syndrome within 4.5 hours of the symptom onset were placed on intravenous fibrinolysis using TPA at a dose of 0.6 mg/kg (15% as a bolus and the remaining 85% as an infusion after 30 minutes), and the angiography suite was prepared for an urgent thrombectomy.

Primary thrombectomy Primary thrombectomy was indicated for those patients with acute ischemic stroke who were ineligible for intravenous fibrinolysis and those patients presenting between 4.5 and 8 hours after the onset of an anterior circulation occlusion or between 4.5 and 9 hours after the onset of a posterior circulation occlusion.

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syringe. Successful recanalization was defined as a TICI score of 3 or 2b in all of the treatable vessels. If the treatable vessel was not opened to a minimum of TICI 2b after a maximum of eight passes of the thrombectomy device, the treatment was considered to be a failure. No intra-arterial fibrinolytics were administered, even if the recanalization attempt was unsuccessful. The groin punctures were closed with an Angio-Seal (St. Jude Medical, St. Paul, MN).

Endpoints, statistical analysis and literature review We collected clinical and radiological data from all of the study subjects. The mean and standard deviation (SD) were calculated for the numeric variables, and Fisher’s exact test was used for the categorical data. All of the statistical analyses were performed with SPSS version 20.0 (Chicago, IL). We searched PubMed for studies published through May 1, 2012 that evaluated retrieval devices for acute ischemic stroke and large intra-arterial therapy for acute ischemic stroke trials. The percentage of recanalization, percentage of mRS scores#2 at 3 months, symptomatic hemorrhage rates and mortality rates in the present study were compared to those in the earlier large trials.

RESULTS Twenty-one patients were evaluated. Vessel imaging (TCCD and US or CTA) was performed on all of the subjects. The mean patient age was 65 years, and the patients’ NIHSS scores ranged from 7 to 28 (average 17, SD¡6.36) on presentation. Vessel occlusions were located in the middle cerebral artery (61.9%), distal internal carotid artery (14.3%), tandem carotid occlusion (14.3%), and basilar artery (9.5%). Primary thrombectomy, rescue treatment and the bridging approach were performed in 66.6%, 28.6%, and 4.8% of the procedures, respectively. The mean time from symptom onset to recanalization was 356.5¡107.8 (SD) minutes (range, 80–586 minutes). The mean procedure times were 60.4¡58.8 (SD) minutes (range, 14–240 minutes), and 45.7¡35.7 minutes for the successful procedures. We obtained an overall recanalization rate (TICI scores of 2b and 3) of 90.4% and a symptomatic intracranial hemorrhage rate of 14.2%. The number of device passes ranged from 1 to 8 (average 3.1, SD¡2.4). There were no device fractures or arterial dissections. The NIHSS scores at discharge ranged from 0 to 25 (average 6.9, SD¡7.0). At three months, 61.9% of the patients had an mRS score#2. The overall mortality rate was 9.5%. One (4.7%) patient died from pneumonia after being discharged. Symptomatic hemorrhagic complications were observed in three patients (14.2%), one of whom (4.7%) had a subarachnoid hemorrhage and died during hospitalization. The other two patients (9.4%) presented with hemorrhagic transformation and were completely dependent on caregivers at the three-month follow-up. Contrast medium enhancement was observed in 12 cases (57.1%). The patients’ clinical and technical data are summarized in Table 1. Figures 1, 2 and 3 illustrate procedures with patients 12, 14 and 20, respectively. We reviewed the literature and compared our results with the results from 17 studies that investigated retrieval devices for treating acute stroke (11-27). We investigated the recanalization rate, recanalization time, procedure time, symptomatic intracranial hemorrhage rate, 90-day mortality rate, average patient NIHSS upon presentation, and study outcomes. Overall, the results were similar (Table 2).

Figure 3 - (a, b) a DSA of (a) the right vertebral artery (RVA) and (b) the left vertebral artery (LVA) (white arrows), arterial phase, frontal views, shows an occlusion of the proximal basilar artery (BA) (white arrowheads); (c) a DSA of the LCCA (frontal view) shows the BA (white arrow) filled by the left posterior communicating artery; (d, e) a Road map of the RVA (frontal view) shows (d) the microwire (white arrow) crossing the occluded section of the BA and (e) the distal tip of the microcatheter (white arrow) advanced by the microwire through the thrombi; (f) a DSA of the BA (frontal view) shows the contrast medium being injected through the microcatheter distal to the thrombi in the BA (white arrow); (g) the distal (white arrow) and proximal (white arrowhead) radiopaque marks of the stent (ev3, Irvine, CA, USA) reveal its full deployment into the distal RVA and BA; (h, i) A final control DSA of the RVA (arterial phase, [h] frontal and [i] lateral views) shows the complete recanalization of the BA.

and lateral angiography series were completed to determine the cervical vessel that was related to the ischemic territory of the brain and to define the occluded intracranial vessels. If a proximal carotid or vertebral occlusion was identified, a Wallstent or Express stent, respectively, (both by Boston Scientific Target, Fremont, CA, USA) was used to perform an angioplasty stenting procedure. After stenting, the guiding catheter was placed into the cervical artery, and a manual thrombus aspiration was performed. After this step, or if no proximal cervical artery occlusion was identified, a 0.021-inch Rebar 18 or a 0.027-inch Rebar 27 microcatheter (ev3, Irvine, CA, USA) was navigated distal to the point of occlusion over a 0.014-inch steerable microwire (Transend EX Platinum; Boston Scientific Target). The microwire was exchanged with a 4620 mm or 6630 mm Solitaire AB device (ev3, Irvine, CA, USA). The device was left in place for 3 to 5 minutes. Then, the microcatheter was advanced to recoat the first third of the Solitaire AB device (ev3, Irvine, CA, USA). The fully deployed Solitaire AB device and the delivery microcatheter were gently pulled back as a single unit and recovered via manual aspiration of the guiding catheter; this procedure was performed with a 60 mL

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79/F 69/M 79/M 58/F 68/M 75/M 59/M 86/F 71/M 61/M 67/M 42/M 80/M 49/M 57/F 81/F 85/F 81/M 48/M 33/F 37/F

Age/Gender

7 20 21 12 12 19 18 14 26 24 20 20 18 26 16 28 19 8 8 7 15

1 3 8 0 4 15 7 NA 3 1 25 0 17 6 4 20 10 4 2 4 4

NIHSS on discharge N N N N N N N Y N N Y N N N N N Y N N N N

Symptomatic intracranial hemorrhage Y Y Y N N Y Y (a) Y N (b) N Y Y N Y (b) Y N Y Y

0 2 5 1 2 6 4 6 2 2 5 0 5 2 2 4 5 2 1 2 2

Proximal LMCA Proximal LMCA Proximal LMCA Proximal LMCA Proximal LMCA Proximal RMCA Right tandem Proximal RMCA Proximal LMCA Right tandem Right tandem Distal RICA Proximal RMCA Proximal LMCA Distal RICA Distal LICA Proximal RMCA BA Proximal RMCA BA Proximal LMCA

CT scan constrast enhance mRS - 3 months Artery site occluded (c) (c) 412 365 (c) 329 336 (c) 360 157 447 367 320 162 586 415 268 452 481 300 304

Recanalization time (minutes) 40 106 57 20 16 29 51 46 20 97 240 43 160 16 140 81 16 22 27 14 29

Procedure time (minutes)

R M R M M R B M M M M R M M M R M M M M R

Treatment modality

1 6 2 3 1 1 1 5 2 4 8 2 8 1 5 7 2 4 1 2 1

No of device passes

3 3 3 3 3 3 3 2b 3 3 0 3 0 3 3 2b 3 3 3 3 3

TICI posttreatment

(NA): not available; (a): subarachnoid hemorrhage; (b): symptomatic intra-parenchimal hemorrhage; (c): cases that represented wake-up stroke, thus without accurate onset time; (Y) yes, (N) no; (M) primary mechanical thrombectomy; (R) rescue treatment; (BA) bridging approach.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

Patient

NIHSS on presentation

Table 1 - Clinical and technical data of patients.

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Table 2 - The main studies assessing the use of retrieval devices for treating acute ischemic stroke (searched in PubMed through May 1, 2012).

Author - Year

Symptomatic Mean time from 90-day intracranial onset to mortality n hemorrhage n 3 month recanalization (%) (%) mRS#2 n (%) (minutes)

Sample

Mean NIHSS baseline

20 22 7 17 50 141

19 19.4 18.8 19 14.7 18

2 2 1 0 5

10 14 26 25

19 16.5 21.4 14

2 (20) 4 (29) NR 3 (12)

NR 6 (45) 11 (42.3) 15 (60)

3 1 5 2

8 18 17 26 48 60

18.3 21 20.5 16 17.4 18

0 (0) 3 (16.6) 2 (11.7) NR NR 7 (12)

0 5 1 2

14 523 21

13.6 17.9 17

1 (7.1) 38 (13.4) 3 (14.2)

Castanho et al. 2010 Roth et al. 2010 Naiak et al. 2010 Brenkfeld et al. 2011 Costalat et al. 2011 Mendes Pereira et al. 2011 Rohde et al. 2011 Fesl et al. 2011 Miteff et al. 2011 Mohlenbruch et al. 2011 Park et al. 2011 Stampfl et al. 2011 Cohen et al. 2012 Mpotsaris et al. 2012 Parrilla et al. 2012 San Roma´n et al. 2012 Menom et al. 2012 Total Castro-Afonso et al. 2012

(10) (9) (14.2) (0) (10) NR

9 11 4 7 27 77

(45) (50) (57) (43) (54) (54.6)

4 (20) 4 (18.1) NR NR 6 (12) 29 (20.5)

Mean Mean procedure number of TICI 2b, 3 time (minutes) device passes n(%)

422 277 244 NR 377 NR

50.0 NR 84.0 52.5 54.0 45

1.4 1.7 1.8 1.3 2.0 NR

18 20 4 16 44 121

(90) (90.9) (57) (94.1) (88) (86)

(30) (8.3) (19.2) (8)

260.7 267.0 476.4 237

88.7 29.0 95.6 54

3.0 NR NR 2.0

10 13 11 22

(100) (93) (42) (88)

4 (50) 6 (33.3) 15 (88.2) 10 (38.4) NR 27 (45)

(0) (27.7) (5.8) (7.6) NR 17 (28)

221.7 289.2 299.1 327 409.3 302

41.5 48.3 45 NR 121.3 80

1.5 NR 2.3 2.1 NR NR

8 16 16 18 48 44

(100) (88.8) (94.1) (69.2) (100) (73.3)

8 (57.1) 465 (50.1) 13 (61.9)

2 (14.2) 81 (17.9) 2 (9.5)

NR 316.3 356.5

84 64.8 60.1

NR 1.9 3.1

Comparing the outcomes of the present study with the results of six large trials assessing intra-arterial treatment for stroke, (28,30-34) our study obtained a higher recanalization rate than the PROACT-II, MERCI, IMSII and Multi-MERCI trials. However, our recanalization results were similar to those of the Penumbra pivotal stroke trial and the primary results of the SWIFT trial. The outcome (mRS scores#2 at 3 months) and three-month mortality rate results in our study were significantly improved over the results found in the MERCI, Multi-MERCI, and Penumbra pivotal stroke trials. No differences were observed in the symptomatic intracranial hemorrhage. The outcomes of the different trials are presented in Table 3.

12 (85.7) 441 (84.3) 19 (90.4)

AB stent (ev3, Irvine, CA, USA). In addition, a significant proportion of patients exhibited a slight or no long-term disability. Our overall results in the Brazilian population are in accordance with previous studies using retrievable stents (11-27). The new retrievable intracranial stents have shown superior results in large vessel occlusions when compared with other recanalization devices and techniques. The higher retrievable stent recanalization rates observed in the context of acute ischemic stroke could be attributed to a device design that is technically simpler, promotes rapid recanalization, removes the clot, and avoids the deployment of a definitive stent, which may lead to rethrombosis and necessitate using a dual antiplatelet regime (11-27). Although the preliminary trials assessing retrievable stents have shown promising technical and clinical results, more data regarding the use of retrievable stents in acute ischemic stroke will be available in the near future. The results of four large studies assessing the Solitaire device (ev3, Irvine, CA, USA) are expected soon: the STAR Trial (Solitaire FR Thrombectomy for Acute Revascularization - NCT01327989),

DISCUSSION A high recanalization rate (90.4%) without an accompanying increase in the number hemorrhagic complications was observed in this prospective series of patients with acute stroke caused by a proximal artery occlusion and treated with intra-arterial thrombectomy with the Solitaire

Table 3 - The outcomes of the present study compared to the results from 6 large trials assessing intra-arterial treatment for acute ischemic stroke.

Sample (*), n Recanalization, n (%) Symptomatic intracranial hemorrhage, n (%) 3-month mortality, n (%) 3-month mRS#2, n (%)

PROACT-II

MERCI

IMSII

Multi-MERCI

Penumbra pivotal stroke trial

SWIFT trial (SolitaireTM arm)

121 80 (66.1) p = 0.037 11 (9.0) p = 0.436 30 (24.7) p = 0.161 48 (39.6) p = 0.093

141 68 (48.2) p,0.001 11 (7.8) p = 0.396 61 (43.2) p = 0.003 39 (27.6) p = 0.005

55 33 (60.0) p = 0.013 8 (14.5) p = 0.999 13 (23.6) p = 0.212 37 (67.2) p = 0.788

164 112 (68.2) p = 0.041 16 (9.7) p = 0.458 56 (34.1) p = 0.024 59 (35.9) p = 0.031

125 102 (81.6) p = 0.530 14 (11.2) p = 0.713 41 (32.8) p = 0.037 52 (41.6) p = 0.010

58 48 (88.9) p = 0.499 1 (1.7) p = 0.055 10 (17.2) p = 0.499 32 (58.2) p = 0.619

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Mechanical thrombectomy with Solitaire stent Castro-Afonso LH et al.

the IMSIII Trial (Interventional Management of Stroke III NCT00359424), the EXTEND-IA (Extending the Time for Thrombolysis in Emergency Neurological Deficits NCT00887328), and the THRACE trial (Trial and Cost Effectiveness Evaluation of Intra-arterial Thrombectomy in Acute Ischemic Stroke - NCT01062698). The main limitations of the present study are the lack of a control group and the small sample size. Therefore, our conclusions concerning the safety and efficacy of the procedure are limited. The one-year study duration also precluded an analysis of the local learning curve. Nevertheless, the initial reports regarding the safety and effectiveness of new devices and technologies are usually the first critical step in the rapidly evolving field of endovascular intervention. Despite the small sample size, the use of the Solitaire AB device (ev3, Irvine, CA, USA) in a Brazilian population provided good technical and clinical results. Our results underscore the importance of a combined stroke team and the enrollment of patients under a strict stroke protocol. Considering the recent efforts to organize an acute stroke network in Brazil (5), we believe that this study could serve as a preliminary ischemic stroke treatment model and thus motivate the government, hospitals and public health administrators to widely support and promote acute stroke care for the Brazilian people. Intra-arterial thrombectomy with the Solitaire AB device (ev3, Irvine, CA, USA) appears to be safe and effective and provided a high recanalization rate when used with a strict stroke protocol. Large randomized trials are necessary to confirm the benefits of this treatment approach to acute ischemic stroke.

7. 8. 9.

10.

11.

12.

13. 14. 15.

16.

17.

AUTHOR CONTRIBUTIONS 18.

Castro-Afonso LH guaranteed the integrity of the entire study, contributed to the study concepts and design, definition of intellectual content, literature research, clinical studies, data acquisition and analysis, and statistical analysis. Abud DG guaranteed the integrity of the entire study, contributed to the study concepts and design, definition of intellectual content, clinical studies, and data acquisition and analysis. Pontes-Neto OM and Cougo-Pinto PT contributed to the study concepts and design, data acquisition and analysis, and statistical analysis. Abud TG contributed to the definition of intellectual content, data acquisition and analysis, and statistical analysis. Nakiri GS contributed to the study concepts, data acquisition and analysis, and statistical analysis. Oliveira L, Monsignore LM, Santos D, Dias FA, Fa´bio SR, Coletto FA contributed to the data acquisition and analysis.

19.

20.

21.

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22.

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27. Menon BK, Kochar P, Ah-Seng A, Almekhlafi MA, Modi J, Wong JH et al. Initial experience with a self-expanding retrievable stent for recanalization of large vessel occlusions in acute ischemic stroke. Neuroradiology. 2012;54(2):147-54, http://dx.doi.org/10.1007/s00234-010-0835-x. 28. SWIFT trialists. Primary Results of the SOLITAIRETM With the Intention for Thrombectomy (SWIFT). Annual Stroke Association’s International Stroke Conference (ISC); 2012 February 3; New Orleans, USA. 29. Higashida R, Furlan A, Roberts H, Tomsick T, Connors B, Barr J, et al. Trial design and reporting standards for intra- arterial cerebral thrombolysis for acute ischemic stroke. J Vasc Interv Radiol. 2003;14:493-S494, http://dx. doi.org/10.1097/01.RVI.0000064855.87207.C5. 30. The Interventional Management of Stroke (IMS) II Study. IMS II Trial Investigators. Stroke. 2007;38(7):2127-35. 31. Smith WS, Sung G, Starkman S, Saver JL, Kidwell CS, Gobin YP, et al. Safety and efficacy of mechanical embolectomy in acute ischemic stroke:

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DOI:10.6061/clinics/2012(12)07

CLINICAL SCIENCE

Nutritional risk and anthropometric evaluation in pediatric liver transplantation Patrı´cia Zamberlan, Cla´udio Leone, Uenis Tannuri, Werther Brunow de Carvalho, Artur Figueiredo Delgado Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, Instituto da Crianc¸a (ICr), Sa˜o Paulo/SP, Brazil.

OBJECTIVE: To analyze the nutritional status of pediatric patients after orthotopic liver transplantation and the relationship with short-term clinical outcome. METHOD: Anthropometric evaluations of 60 children and adolescents after orthotopic liver transplantation, during the first 24 hours in a tertiary pediatric intensive care unit. Nutritional status was determined from the Z score for the following indices: weight/age, height/age or length/age, weight/height or weight/length, body mass index/age, arm circumference/age and triceps skinfold/age. The severity of liver disease was evaluated using one of the two models which was adequated to the patients’ age: 1. Pediatric End-stage Liver Disease, 2. Model for End-Stage Liver Disease. RESULTS: We found 50.0% undernutrition by height/age; 27.3% by weight/age; 11.1% by weight/height or weight/ length; 10.0% by body mass index/age; 61.6% by arm circumference/age and 51.0% by triceps skinfold/age. There was no correlation between nutritional status and Pediatric End-stage Liver Disease or mortality. We found a negative correlation between arm circumference/age and length of hospitalization. CONCLUSION: Children with chronic liver diseases experience a significant degree of undernutrition, which makes nutritional support an important aspect of therapy. Despite the difficulties in assessment, anthropometric evaluation of the upper limbs is useful to evaluate nutritional status of children before or after liver transplantation. KEYWORDS: Nutritional Assessment; Anthropometry; Liver Transplantation; Children; Pediatric Intensive Care Unit. Zamberlan P, Leone C, Tannuri U, Carvalho WB, Delgado AF. Nutritional risk and anthropometric evaluation in pediatric liver transplantation. Clinics. 2012;67(12):1387-1392. Received for publication on July 16, 2012; First review completed on August 6, 2012; Accepted for publication on August 14, 2012 E-mail: patrı´cia.zamberlan@icr.usp.br / pzamberlan@uol.com.br Tel.: 55 11 2661-8558

undernutrition in the hospital. This condition is especially prevalent in developing countries. Nutritional therapy is one of the most important procedures in the management of liver disease and should be considered an essential adjunct to clinical therapies, especially when the patient is a candidate for liver transplantation (4). Orthotopic liver transplantation (OLT) revolutionized the management of liver disease from the results achieved with the technique, as well as improved understanding of the indications, contraindications, and problems associated with the technique (5). The indications for and clinical outcomes of OLT depend on the patient’s nutritional status. As undernutrition is known to be associated with a greater risk of post-operative complications and higher mortality rates in patients with liver disease (6,7), this correlation is also believed to be present in patients undergoing liver transplantation (8). Recipients with undernutrition were found to be at greater risk for increased operative blood loss, have longer stays in the intensive care unit, and have higher mortality rates and higher total hospital charges after liver transplantation (8,9). In the immediate phase after liver transplantation, protein catabolism is markedly increased, as demonstrated by the excretion of large amounts of urinary nitrogen (10).

INTRODUCTION The liver is the largest and most important metabolic organ, playing a pivotal role in integrating several biochemical pathways of carbohydrate, fat, protein, and vitamin metabolism (1,2). Thus, children with chronic liver disease are at high risk for developing undernutrition, with important prognostic implications. The pathogenesis of undernutrition in chronic liver diseases is multifactorial and includes a reduction in nutrient and caloric intake, anorexia and dietary restrictions, impaired intestinal absorption, abnormalities in nutrient metabolism (carbohydrate, lipid, and protein), and increased proinflammatory cytokine levels, resulting in a hypermetabolic state (1,3). Due to several metabolic alterations that characterize end-stage liver disease, most adult and pediatric liver transplantation candidates present with

Copyright ß 2012 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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0.1 cm. Children were placed in the supine position for the measurement. In children older than three years, height was obtained with a wooden stadiometer, which was accurate to the nearest 0.1 cm. In children whose condition prevented the use of conventional measuring techniques (i.e., mechanical ventilation, vasoactive drugs), height was predicted from measurements of the distance between the knee and the ankle, with the child in the supine position. The length between the heel and the anterior surface of the leg at the knee (femoral condyle) was measured using a pediatric anthropometer. The following equations proposed by Chumlea et al. (16) were used: a) Caucasian girls = 43.21 + (2.15 6 distance knee/ankle); b) African-American girls = 46.59 + (2.02 6distance knee/ ankle); c) Caucasian boys = 40.54 + (2.22 6 distance knee/ankle); and African-American boys = 39.60 + (2.18 6 distance knee/ ankle). Using the data for W and H or L, we obtained the following indices: W/age (A), H/A or L/A, W/H or W/L. We calculated body mass index for age (BMI/A) using the following equation: BMI = W (kg)/H2 (m). AC was measured with metric tape marked at increments of 0.5 cm. Measurements were taken at the midpoint of the distance between the acromion and the olecranon, with the arm extended along the body. TST was obtained using a Lange Skinfold Caliper (Cambridge Scientific Industries, Cambridge, MD) with a constant pressure of 10 g/mm2 on the contact surface. The measurement was taken on the back of the arm, parallel to the longitudinal axis, at the midpoint between the acromion and olecranon. We used the average of three consecutive measurements. Nutritional status was determined from the Z score (Z) for the following indices: W/A, H/A or L/A, W/H or W/L, BMI/A, AC/A, and TST/A. We used reference values from the World Health Organization (WHO) (17). WHO Anthro (version 3.1)/2010 and WHO Anthro Plus/2007 were used for the calculations. Anthropometric evaluation is important for classifying nutritional status and is correlated with the nutritional condition prior to transplantation. Although the post-operative state may influence the nutritional condition, we believe that this influence is not sufficient to change the nutritional classification (undernutrition or not). The severity of liver disease was evaluated by the extent of Pediatric End-stage Liver Disease (PELD), which indicates the need for a liver transplantation. The extent of PELD is a numerical value applied to children under 12 years old and considers the outcomes of laboratory tests such as bilirubin, albumin, international normalized ratio (INR) and stunting based on gender, weight and height. For patients over 12 years old, the Model for End-stage Liver Disease (MELD) was used to quantify the urgency of OLT. The population was analyzed for age at admission, sex, type of donor (cadaveric or living-relative donor), mortality, discharge and cause of liver disease. The results were tabulated in an ExcelH spreadsheet. The mean, median and standard deviation (SD) Z were obtained for all anthropometric indicators (W/A, H/A or L/A, W/H, BMI/A, AC/A and TST/A) as well as PELD and length of stay in the PICU.

Multiple techniques have been proposed to detect undernutrition in patients with liver disease (11-14). Nevertheless, it is well known that some common nutritional parameters can be misleading in advanced liver disease because of water retention and ascites (11), compromised protein synthesis and coexisting alterations in renal function (12). Although there is no gold standard for the assessment of nutritional status in patients with liver disease, anthropometric measurements, such as arm muscle circumference and triceps skinfold, have been utilized in large groups of patients, mainly in those awaiting OLT, and proved to be useful in identifying muscle and fat depletion. In the current study, the nutritional status of a group of pediatric patients was prospectively analyzed immediately after OLT, and the relationship between nutritional status and short-term clinical outcomes (mortality and length of stay in the pediatric intensive care unit) were analyzed.

MATERIALS AND METHODS For this descriptive and prospective study, the participants were selected from infants, pre-school children, school children, and adolescents sequentially admitted to the pediatric intensive care unit (PICU) of Instituto da Crianc¸a, Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sao Paulo, Brazil, immediately after OLT between January 2006 and December 2009. This tertiary PICU has 20 beds for clinical and surgical patients. The third author (UT) performed or supervised all operations. The Ethical Committee of the institution approved the study protocol. Children with liver disease were followed in the outpatient Pediatric Surgery Unit, where the OLT was indicated. The unit serves patients from various regions of the country with different clinical conditions. There is no prescribed nutritional program for these patients; only obvious nutritional deficiencies are treated. We performed anthropometric nutritional evaluations for 60 patients after deceased donor or living-relative donor OLT, during the first 24 hours in the PICU. The study was approved by the Research and Ethics Committee of the institution. Anthropometric nutritional assessment was carried out during the first 24 hours after admission following OLT. To minimize the possibility of errors, all measurements were performed by the first author of the study (PZ) using the same method described in a previous study (15). The assessment included weight (W), height (H) or length (L), arm circumference (AC), and triceps skinfold thickness (TST). Weight was measured with a scale that was calibrated for accuracy before each use. Children over 16 kg were weighed standing up, and infants were weighed using a scale accurate to the nearest 5 g. Children and adolescents (six cases) who could not be weighed independently were held by an adult (a parent or evaluator) while being weighed. The child’s weight was obtained by subtracting the weight of the adult from the total weight of the child and adult. In general, children and adolescents were extubated in the first 24 hours allowing them to be weighed more easily. Length was measured in children up to three years of age using a pediatric anthropometer accurate to the nearest

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The distribution of anthropometric indicators in the study population was considered normal by the KolmogorovSmirnov test. Anthropometric indicators of nutritional status and their relationship with disease severity (PELD/MELD), mortality and PICU stay were analyzed using correlation and linear regression analyses with Pearson coefficient calculations. Data were considered statistically significant at p,0.05, and a 95% confidence interval was used for measures of central tendency. We used SPSS 12.01 to perform the calculations.

Table 2 - Mean (¡SD) and median of anthropometric indicators (Z) of children and adolescents assessed after liver transplant.

RESULTS

on the methods used and countries involved (18,19). Children who are admitted to the hospital, mainly in the ICU, are at a high risk of developing undernutrition, particularly children with an underlying disease and severe clinical condition (higher Pediatric Risk of Mortality Score) (20-22). Undernutrition is associated with increased morbidity and mortality in children, including a higher risk of infections due to poor immune defense, wound-healing problems, and reduced gut function, and longer dependency on mechanical ventilation and longer hospital stays (19). Roggero et al. (23) found that the predominant factors that influence undernutrition in children with chronic liver disease are age for infants and the degree of hepatic failure for older children. Frequently, undernutrition is associated with higher incidence of infection, surgical complications, and a lower survival rate after liver transplantation. With the exception of patients with fulminant hepatic failure, most candidates for human OLT present with significant undernutrition, especially in developing countries. Nutritional deficiencies usually occur prior to clinical signs of hepatic insufficiency. Assessing the nutritional status of patients with chronic liver disease is difficult and controversial because the parameters utilized to perform the evaluation are frequently altered by organomegaly, ascites, and edema. Furthermore, patients with end-stage liver disease (ESLD) exhibit significant impairment in hepatocyte synthetic function, which can result in depressed albumin, prealbumin, and transferrin serum levels, thus impairing an adequate nutritional diagnosis (2,9,24). There is no diagnostic ‘‘gold standard’’ for assessing nutritional status in patients with liver disease. No single criterion should be neglected when assessing nutritional status in these patients. The assessment must be as complete as possible and should include the following components: medical and dietary history, subjective global assessment, anthropometric measurements, biochemical parameters and indices, and a more complex body composition analysis (24,25). No controversy currently exists regarding the importance of nutritional status as an important predictor of post-transplant

The study population consisted of 60 patients evaluated in the PICU after OLT, with a median age of 26 months (2.8 years). The predominant age group was comprised of children in pre-school and children who were less than two years old; most of these children were female. The median length of PICU stay was seven days, and the average PELD/MELD value was 24. Table 1 shows the main characteristics of this population. The mean, median and standard deviation of Z anthropometric indicators are shown in Table 2, and the frequency of malnutrition in children is shown in Table 3. There was no correlation between nutritional status assessed by BMI/A and PELD/MELD (r = 0.05792; p = 0.66) and mortality (p = 0.92), despite the high mortality rate, which was 18% (11 children) overall and 16% to 20% among malnourished children (Table 4). We did not find a correlation between nutritional status assessed by BMI/A (r = -0.03242), H/A (r = -0.2005) and W/A (r = -0.1975) and the length of stay in the PICU. This correlation was found only with AC/A (r = - 0.2976), as shown in Figure 1.

DISCUSSION Hospital protein-energy undernutrition was first described in 1980. Current studies report variable percentages depending Table 1 - Main characteristics for study subjects. Population

n

%

Age ,2 years 2 a ,5 years 5 a ,10 years 10 a 20 years Sex Male Female Kind of donor Deceased donation Living donor Diagnoses Biliary atresia (AVB) Cirrhosis Deficiency of ornithine transcarbomilase Cholesterol ester storage disease Autoimmune hepatitis Chronic hepatitis Fulminant hepatitis Hepatoblastoma Alagille syndrome Tyrosinemia

29 17 8 6

48.4 28.3 13.3 10.0

22 38

36.6 63.4

36 24

60 40

37 5.0 1.0

61.7 8.3 1.7

1.0 1.0 2.0 6.0 1.0 4.0 2.0

1.7 1.7 3.3 10.0 1.7 6.6 3.3

Indicator

Mean

Median

SD

H/A or L/A W/A H/W or H/L BMI/A AC/A TST/A

-1.69 -0.88 0.03 0.23 -2.81 -1.99

-2.02 -0.80 0.23 0.31 -2.88 -1.78

1.97 1.73 1.46 1.59 1.98 1.75

Table 3 - Frequency of malnutrition in 60 children after liver transplantation, according to the different indicators (Z) used in the anthropometric screening.

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Indicator

Cutoff

Patients (%)

H/A W/A W/H or W/L BMI/A AC/A TST/A

#2SD #2SD #2SD #2SD #2SD #2SD

50 27.3 11.1 10 61.6 51


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parameter because edema, ascites and hepatosplenomegaly overestimate nutritional status (1,23). Nonetheless, we found that 27.3% of children and adolescents were malnourished, as evaluated by W/A. The degree of height deficits for age is generally considered to be indicative of chronic undernutrition (24). Half of the patients studied had z-scores of -2.02 SD for H/A or L/A, demonstrating chronic impairment of nutritional status, although 48% of children were less than 2 years old. Roggero et al. (23) also found deficits in H/A in children under 1 year waiting for liver transplants, showing that growth can be impaired in children with liver disease who are candidates for OLT even at early ages. H/A can be a useful index in assessing these children. W/H and BMI, which is generally used to detect acute undernutrition, as well as W/A, may underestimate the degree of undernutrition in children with chronic liver disease (29). This statement is supported by our findings: W/H identified only 11% of children as malnourished and BMI/A identified 10% of children as malnourished. A similar overall result was also found in critically ill patients, in whom edema and fluid retention are usually present. Body weight is increased by edema, resulting in underestimation of under nutrition. In these studies, the minority of patients was considered malnourished by W/H or BMI/A (30-32). Skinfold measurements and MAC are considered useful parameters with which to assess subcutaneous fat and muscle mass (25) and can help to identify children with chronic under nutrition. In these cases, skinfolds and circumferences are considered to be more accurate measurements than height because variations in these parameters appear earlier than changes in height. However, peripheral edema may falsely increase such measurements (24). To minimize factors contributing to error in anthropometric assessment, such as edema, measurements of skinfold thickness in the upper body are indicated because edema is more readily accumulated in the lower body (24). Furthermore, it is recommended that measurements be performed by the same individual. The results of our study showed that body muscle mass and fat mass values were reduced more markedly. Despite the possible bias, we found undernutrition in almost 70% of

Table 4 - Frequency of mortality among malnourished and well-nourished children assessed by H/A and AC/A after liver transplantation. Indicator H/A AC/A

Malnourished (Z#-2)

Well-nourished (Z$-2)

6/30 (20%) 6/37 (16.2%)

5/30 (16.6%) 5/23 (21.7%)

outcomes and the benefits of therapeutic nutritional improvement (25). The main objective of the present study was to evaluate the nutritional status of children and adolescents immediately after liver transplantation and to determine the relationship with short-term clinical outcomes (mortality and length of stay in the PICU). Nutritional status was measured immediately after transplant; this parameter primarily reflects the pre-operative condition in these patients, who usually have major fluid overload and frequently have positive balance during surgery as a consequence of the provision of fluids and blood products. However, in this study, even with fluid overload, there was a high percentage of undernutrition demonstrating that this condition has a high prevalence among patients awaiting or undergoing OLT (deceased donation and living donor). Easily applicable techniques include anthropometric measurements in liver transplant candidates, and current guidelines for enteral nutrition recommend the use of anthropometry parameters to assess the nutritional status of these patients, despite the limitations of this technique. In our study, we used anthropometry to assess children and adolescents after OLT. We found 50.0% undernutrition by H/A; 27.3% by W/A; 11.1% by W/H or W/L; 10.0% by BMI/A; 61.6% by AC/A, and 51.0% by TST/A. These results confirm that undernutrition is common in children with liver diseases waiting for or undergoing OLT, as demonstrated by previous studies (6,23,26,27), and that prevalence of undernutrition varies according to the index used. Israels et al. (28) evaluated 128 pediatric patients with cancer using anthropometric indexes and found 59.3% malnourished children by muscular arm circumference (MAC) and TST compared to 17.2% diagnosed by W/H. Body weight is not a sensitive

Figure 1 - Correlation between stay of hospitalization in PICU and Z-score AC/A of children after liver transplantation.

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Anthropometric evaluation liver transplantation Zamberlan P et al. 5. Gonza´lez JCM, Herna´ndez MVC. Transplante hepa´tico. Implicaciones nutricionales. Nutr Hosp. 2008;23(Suppl 2):S34-40. 6. Moukarzel AA, Najm I, Vargas J, McDiarmid SV, Busuttil RW, Ament ME. Effect of nutritional status on outcome of orthotopic liver transplantation in pediatric patients. Transplant Proc. 1990;22(4):1560-3. 7. Millwala F, Nguyen GC, Thuluvath PJ. Outcomes of patients with cirrhosis undergoing non-hepatic surgery: risk assessment and management. World J Gastroenterol. 2007;13(30):4056-63. 8. Pikul J, Sharpe MD, Lowndes R, Ghent CN. Degree of preoperative malnutrition is predictive of postoperative morbidity and mortality in liver transplant recipients. Transplantation. 1994;57(3):469-72, http://dx. doi.org/10.1097/00007890-199402150-00030. 9. Stephenson GR, Moretti EW, El-Moalem H, Clavien PA, Tuttle-Newhall JE. Malnutrition in liver transplant patients: pre operative subjective global assessment is predictive of outcome after liver transplantation. Transplantation. 2001;72(4):666-70, http://dx.doi.org/10.1097/00007890200108270-00018. 10. Shanbhogue RL, Bistrian BR, Jenkins RL, Randall S, Blackburn GL. Increased protein catabolism without hypermetabolism after human orthotopic liver transplantation. Surgery. 1987;101(2):146-9. 11. McCullough AJ, Mullen KD, Kalhan SC. Measurements of total body and extracellular water in cirrhotic patients with and without ascites. Hepatology. 1991;14(6):1102-8, http://dx.doi.org/10.1002/hep.1840140626. 12. Pirlich M, Selberg O, Bo¨ker K, Schwarze M, Mu¨ller MJ. The creatinine approach to estimate skeletal muscle mass in patients with cirrhosis. Hepatology. 1996;24(6):1422-7, http://dx.doi.org/10.1002/hep.510240620. 13. Plauth M, Merli M, Kondrup J, Weimann A, Ferenci P, Muller MJ, et al. ESPEN guidelines for nutrition in liver disease and transplantation. Clin Nutr. 1997;16(2):43-55, http://dx.doi.org/10.1016/S0261-5614(97)80022-2. 14. Selberg O, Bo¨ttcher J, Tusch G, Pichlmayr R, Henkel E, Muller MJ. Identification of high-and low-risk patients before liver transplantation: a prospective cohort study of nutritional and metabolic parameters in 150 patients. Hepatology. 1997;25(3):652-7, http://dx.doi.org/10.1002/hep. 510250327. 15. Zamberlan P, Delgado AF, Leone C, Feferbaum R, Okay TS. Nutritional therapy in a pediatric intensive care unit: indications, monitoring and complications. JPEN J Parenter Enteral Nutr. 2011;35(4):523-9, http://dx. doi.org/10.1177/0148607110386610. 16. Chumlea WC, Guo SS, Steinbaugh ML. Prediction of stature from knee height for black and white adults and children with application to mobility- impaired or handicapped persons. J Am Diet Assoc. 199;94(12):1385-8, 1391; quiz 1389-90. 17. World Health Organization 2007. Who Child Growth Standards. http:// www.who.int/childgrowth/en/. Accessed 2 Sep 2011. 18. Delgado AF, Okay TS, Leone C, Nichols B, Del-Negro GM, Costa-Vaz FA. Hospital malnutrition and inflammatory response in critically ill children and adolescents admitted to a tertiary intensive care unit. Clinics. 2008;63(3):357-62, http://dx.doi.org/10.1590/S1807-59322008000300012. 19. Joosten KF, Hulst JM. Malnutrition in pediatric hospital patients: current issues. Nutrition. 2011;27(2):133-7, http://dx.doi.org/10.1016/j.nut.2010.06.001. 20. Pawellek I, Dokoupil K, Koletzko B. Prevalence of malnutrition in paediatric hospital patients. Clin Nutr. 2008;27(1):72-6, http://dx.doi.org/10.1016/j. clnu.2007.11.001. 21. Costa GA, Delgado AF, Ferraro A, Okay TS. Application of the Pediatric Risk of Mortality Score (PRISM) score and determination of mortality risk factors in a tertiary pediatric intensive care unit. Clinics. 2010;65(11):108792, http://dx.doi.org/10.1590/S1807-59322010001100005. 22. Batista TP, Sabat BD, Melo PSV, Miranda LEC, Fonseca-Neto OCL, Amorim AG, et al. Impact of MELD allocation policy on survival outcomes after liver transplantation: a single-center study in northeast Brazil. Clinics. 2011;66(1):57-64, http://dx.doi.org/10.1590/S1807-59322011000100011. 23. Roggero P, Cataliotti E, Ulla L, Stuflesser S, Nebbia G, Bracaloni D, et al. Factors influencing malnutrition in children waiting for liver transplants. Am J Clin Nutr. 1997;65(6):1852-7. 24. Romaccioni V, Soriano HE, Arumugan R, Klish WJ. Nutritional aspects of chronic liver disease and liver transplantation in children. J Pediatr Gastroenterol Nutr. 2000;30(4):361-7, http://dx.doi.org/10.1097/00005176200004000-00003. 25. Campos ACL, Matias JEF, Coelho JCU. Nutritional aspects of liver transplantation. Curr Opin Clin Nutr Metab Care. 2002;5(3):297-307, http://dx.doi.org/10.1097/00075197-200205000-00010. 26. Sokol RJ, Stall C. Anthropometric evaluation of children with chronic liver disease. Am J Clin Nutr. 1990;52(2):203-8. 27. Shepherd RW, Chin SE, Cleghorn GJ, Patrick M, Ong TH, Lynch SV, et al. Malnutrition in children with chronic liver disease accepted for liver transplantation: clinical profile and effect on outcome. J Paediatr Child Health. 1991;27(5):295-9, http://dx.doi.org/10.1111/j.1440-1754.1991.tb02541.x. 28. Israe¨ls T, Chirambo C, Caron HN, Molyneux EM. Nutritional status at admission of children with cancer in Malawi. Pediatr Blood Cancer. 2008;51(5):626-8, http://dx.doi.org/10.1002/pbc.21697. 29. Figueiredo F, Dickson ER, Pasha T, Kasparova P, Therneau T, Malinchoc M, et al. Impact of nutritional status on outcomes after liver transplantation. Transplantation. 2000;70(9):1347-52, http://dx.doi.org/ 10.1097/00007890-200011150-00014.

patients by AC/A and more than half of patients by TST/A. We found similar results in a previous study, which utilized measurements in the arm and showed higher accuracy in identifying malnutrition among 90 children and adolescents in a tertiary pediatric intensive care unit, 90% of whom had underlying disease (15). Anthropometric evaluation was useful for classifying nutritional status and followed these patients even with the physiological changes common after OLT. It is assumed that undernutrition increases mortality after liver transplantation (2,6). Among the patients of the current study, 18.4% died, and although we have not found any association between nutritional status and mortality, the high incidence of undernutrition may have caused a negative impact on mortality (Table 4). Figueiredo et al. (29) investigated the impact of nutritional status on outcomes after liver transplantation. They prospectively studied the predictive value of preoperative nutritional status on the adverse outcomes in 53 patients that received OLT and concluded that none of the nutritional parameters (body cell mass, anthropometry, subjective global assessment) was associated with increased risk of mortality or global resource utilization. Although no study has demonstrated that preoperative undernutrition is an independent predictor of perioperative mortality, an increased need for blood transfusions, prolonged ICU stays, longer duration of hospitalization and, consequently, higher hospital costs for liver transplantation have been reported (32). We found an association between nutritional status, as determined by AC/A and length of stay in the PICU, showing that malnutrition negatively influences the immediate postoperative OLT and that it indirectly increases hospital costs. Our findings show that patients with chronic liver diseases exhibit substantial undernutrition. This condition is known as a basic indicator of surgical risk, making nutritional support an important aspect of therapy for children with chronic diseases (33), such as end-stage liver disease, and/or waiting for an OLT. Despite the difficulties in assessing and technical limitations, we recommend that a simple, inexpensive, easily reproducible, and noninvasive technique, such as anthropometric evaluation of the upper limbs, is useful for assessment of nutritional status. Serial anthropometric measurements are recommended to evaluate the effects and adequacy of nutritional interventions.

AUTHOR CONTRIBUTIONS Zamberlan P carried out dates and helped to draft the manuscript. Leone C performed data analyses and statistical analysis. Tannuri U and Carvalho WB participated in the coordination of the study and critically revised it. Delgado AF participated in design, coordination of the study and helped to draft the manuscript.

REFERENCES 1. Sanchez AJ, Aranda-Michel J. Nutrition for the liver transplant patient. Liver Transpl. 2006;12(9):1310-6, http://dx.doi.org/10.1002/lt.20894. 2. Shetty AK, Schmidt-Sommerfeld E, Udall Jr JN. Nutritional aspects of liver disease in children. Nutrition. 1999;15(9):727-9. 3. Merli M, Giusto M, Gentili F, Novelli G, Ferretti G, Riggio O, et al. Nutritional status: its influence on the outcome of patients undergoing liver transplantation. Liver Int. 2010;30(2):208-14, http://dx.doi.org/10. 1111/j.1478-3231.2009.02135.x. 4. Cleghorn G. The role of basic nutritional research in pediatric liver disease: An historical perspective. J Gastroenterol Hepatol. 2009; 24(Suppl 3):S93-6, http://dx.doi.org/10.1111/j.1440-1746.2009.06078.x.

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32. Descheˆnes M, Villeneuve JP, Dagenais M, fenyves D, Lapointe R, PomierLayrargues G, et al. Lack of relationship between preoperative measures of the severity of cirrhosis and short-term survival after liver transplantation. Liver Transpl Surg. 1997;3(5):532-7, http://dx.doi.org/10.1002/lt.500030509. 33. Skillman HE. Monitoring the efficacy of a PICU nutrition therapy protocol. JPEN J Parenter Enteral Nutr. 2011;35(4):445-6, http://dx.doi. org/10.1177/0148607111409046.

30. Huang YC, Yen CE, Cheng CH, Jih KS, Kan MN. Nutritional status of mechanically ventilated critically ill patients: comparison of different types of nutritional support. Clin Nutr. 2000;19(2):101-7, http://dx.doi. org/10.1054/clnu.1999.0077. 31. Ravasco P, Camilo ME, Gouveia-Oliveira A, Adam S, Brum G. A critical approach to nutritional assessment in critically ill patients. Clin Nutr. 2002;21(1):73-7, http://dx.doi.org/10.1054/clnu.2001.0508.

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DOI:10.6061/clinics/2012(12)08

CLINICAL SCIENCE

Novel GATA5 loss-of-function mutations underlie familial atrial fibrillation Jian-Yun Gu,I Jia-Hong Xu,I Hong Yu,I Yi-Qing YangII I Tongji University School of Medicine, Tongji Hospital, Department of Cardiology, Shanghai/China. II Department of Cardiology and Cardiovascular Research, Shanghai Chest Hospital, Medical College of Shanghai Jiaotong University, Shanghai / China.

OBJECTIVE: This study aimed to identify novel GATA5 mutations that underlie familial atrial fibrillation. METHODS: A total of 110 unrelated patients with familial atrial fibrillation and 200 unrelated, ethnically matched healthy controls were recruited. The entire coding region of the GATA5 gene was sequenced in 110 atrial fibrillation probands. The available relatives of the mutation carriers and 200 controls were subsequently genotyped for the identified mutations. The functional effect of the mutated GATA5 was characterized using a luciferase reporter assay system. RESULTS: Two novel heterozygous GATA5 mutations (p.Y138F and p.C210G) were identified in two of the 110 unrelated atrial fibrillation families. These missense mutations cosegregated with AF in the families and were absent in the 400 control chromosomes. A cross-species alignment of GATA5 protein sequence showed that the altered amino acids were completely conserved evolutionarily. A functional analysis revealed that the mutant GATA5 proteins were associated with significantly decreased transcriptional activation when compared with their wild-type counterpart. CONCLUSION: The findings expand the spectrum of GATA5 mutations linked to AF and provide novel insights into the molecular mechanism involved in the pathogenesis of atrial fibrillation, suggesting potential implications for the early prophylaxis and personalized treatment of this common arrhythmia. KEYWORDS: Atrial Fibrillation; Transcriptional Factor; GATA5; Genetics; Reporter Gene. Gu JY, Xu JH, Yu H, Yang YQ. Novel GATA5 loss-of-function mutations underlie familial atrial fibrillation. Clinics. 2012;67(12):1393-1399. Received for publication on July 31, 2012; First review completed on August 14, 2012; Accepted for publication on August 14, 2012 E-mail: xu_jiahong@sina.com / yang99yang66@hotmail.com Tel.: 86 21 56051080

with AF averages 5% per year, 2 to 7 times that of individuals without AF (6). Additionally, when transient ischemic attacks and clinically ‘‘silent’’ strokes documented by brain imaging are included, the rate of cerebral ischemia accompanying nonvalvular AF exceeds 7% per year (7). Among the patients in the Framingham Heart Study with rheumatic heart disease and AF, the stroke risk was increased 17-fold compared with age-matched controls (8). Given the frequency of comorbidities, AF imposes a huge economic burden on both the individual and society as a whole, and the economic burden for society is expected to increase in the near future due to the aging population (9). Despite the high prevalence and clinical significance of AF, its underlying molecular etiology remains largely unknown. AF can occur in the context of various structural heart diseases or systemic disorders, including ischemic heart disease, valvular heart disease, congenital heart disease, cardiomyopathy, cardiac surgery, pericarditis, myocarditis, congestive heart failure, essential hypertension, diabetes mellitus, hyperthyroidism, and electrolyte imbalance (1,10,11). However, in 30 to 45% of the cases, especially among those younger than 60 years old, AF occurs without any known risk factors, a condition known as ‘‘lone AF’’. At least 15% of patients have a positive family history and are considered to have familial AF (12). Accumulating evidence

INTRODUCTION Atrial fibrillation (AF) is the most common form of cardiac arrhythmia seen in clinical practice, accounting for approximately one-third of hospitalizations for cardiac rhythm disturbances. The prevalence of AF increases markedly with advancing age, ranging from 1 to 2% of the general population to 5 to 15% of individuals in their 80s (1). The lifetime risk of developing AF is approximately 25% in individuals aged 40 years or older (2). The chaotic heart rhythm is responsible for a variety of symptoms, including palpitations, dizziness, and shortness of breath, and it is associated with degraded quality of life, reduced exercise capacity, cognitive dysfunction, tachycardiomyopathy, thromboembolic strokes, congestive heart failure, and even death (1,3,4). The mortality rate of patients with AF is approximately two times that of subjects with a normal sinus rhythm (5). The rate of ischemic stroke among patients

Copyright ß 2012 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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a normal ECG. In addition, paroxysmal AF was defined as AF that lasted more than 30 seconds and terminated spontaneously. Persistent AF was defined as AF that lasted more than seven days and required either pharmacologic therapy or electrical cardioversion for termination. AF that was refractory to cardioversion or that was allowed to continue was classified as permanent.

from epidemiological studies highlights the familial aggregation of AF and the increased risk of AF among the close relatives of patients with AF, strongly suggesting a hereditary determinant for AF (13-15). Following genomewide linkage analysis with polymorphic genetic markers, specific susceptibility loci for AF have been mapped to human chromosomes 10q22, 6q14–16, 11p15.5, 5p15, 10p11– q21, and 5p13, and AF-causing mutations in two genes, KCNQ1 on chromosome 11p15.5 and NUP155 on chromosome 5p13, have been identified and characterized (16). Analyses of candidate genes and genome-wide association studies have revealed a long list of AF-associated genes, including KCNE2, KCNH2, KCNA5, KCNJ2, KCNN3, GJA1, GJA5, NPPA, ZFHX3, and SCN5A (16). Nevertheless, AF demonstrates substantial genetic heterogeneity, and the genetic defects that lead to AF in an overwhelming majority of patients have not yet been identified. It is now well established that abnormal embryological development of the myocardial sleeves clothing the systemic venous tributaries and the pulmonary veins at their junctions with the atrial chambers is a major anatomic defect associated with AF (17). Recent studies underline the pivotal role of several transcription factors, including NKX2-5, GATA4, GATA5, and GATA6, during cardiogenesis (18), and mutations in NKX2-5, GATA4, and GATA6 have been causally implicated in the pathogenesis of AF (19-24). GATA5 is a member of the GATA family of transcription factors, and its expression and functions overlap with those of GATA4 and GATA6 during cardiac development, especially the synergistic regulation of target gene expression with NKX2-5. This relationship provides a logical rationale for screening GATA5 as a candidate gene associated with familial AF (25).

Genetic studies Genomic DNA from all participants was extracted from peripheral venous blood lymphocytes using the Wizard Genomic DNA Purification Kit (Promega, Madison, WI, USA). The entire coding region and the flanking splice junction sites of GATA5 were sequenced in 110 unrelated index patients with familial AF, and GATA5 genotyping was conducted for the available relatives of mutation carriers and 200 ethnically matched unrelated healthy controls to determine the presence of the mutations identified in the probands. The reference genomic DNA sequence of GATA5 was obtained from GenBank (accession No. NT_011362). The primer pairs used to amplify the coding exons and exon/intron boundaries of GATA5 using polymerase chain reaction (PCR) were designed as previously described using Primer 3 software (http://frodo.wi. mit.edu), (26). PCR was performed using HotStar Taq DNA Polymerase (Qiagen GmbH, Hilden, Germany) on a PE 9700 Thermal Cycler (Applied Biosystems, Foster, CA, USA). The amplicons were purified with a QIAquick Gel Extraction Kit (Qiagen), and both strands of each PCR product were sequenced using a BigDyeH Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and an ABI PRISM 3130 XL DNA Analyzer (Applied Biosystems). The DNA sequences were analyzed with the DNA Sequencing Analysis Software v5.1 (Applied Biosystems). The variant was validated by resequencing an independent PCRgenerated amplicon from the subject, and it met our quality control thresholds with a call rate greater than 99%.

MATERIALS AND METHODS Ethics statement This study was performed in compliance with the ethical principles of the revised Declaration of Helsinki (Somerset West, Republic of South Africa, 1996). The research protocol was reviewed and approved by the local institutional ethics committee, and written informed consent was obtained from all participants prior to the study.

Alignment of multiple GATA5 protein sequences Multiple GATA5 protein sequences across various species were aligned using the online program MUSCLE, Version 3.6 (http://www.ncbi.nlm.nih.gov/).

Construction of the recombinant pcDNA3.1hGATA5 expression plasmid

Study participants A cohort of 110 unrelated probands with familial AF was identified among the Chinese Han population. The available relatives of the probands who harbored the identified GATA5 mutations were also included. A total of 200 ethnically matched unrelated healthy individuals were recruited as controls. Peripheral venous blood specimens and clinical data, including medical records and electrocardiogram (ECG) and echocardiography reports, were collected. The study subjects were clinically classified using a consistently applied set of definitions (12). Briefly, AF was diagnosed when ECGs demonstrated no P waves and irregular R-R intervals, regardless of clinical symptoms. Lone AF was defined as AF that occurred in individuals less than 60 years of age without evidence of other cardiac or systemic diseases after physical examination, ECG, transthoracic echocardiogram, and extensive laboratory tests. Familial AF was designated when lone AF occurred in one or more first-degree relatives of the proband. Relatives were classified as ‘unaffected’ if they were asymptomatic and had

Human fetal cardiac tissue specimens were previously collected and preserved in RNAlater RNA stabilization reagent (Qiagen). Total RNA was prepared using an RNeasy Protect Mini Kit (Qiagen). Reverse transcription was performed with an oligo(dT)20 primer using SuperScript III reverse transcriptase (Invitrogen, Carlsbad, CA, USA). The full-length wild-type human GATA5 cDNA, including partial 5’- and 3’-untranslated regions, was PCR amplified using pfuUltra high-fidelity DNA polymerase (Stratagene, La Jolla, CA, USA). The following primer pairs were used for the specific amplification of the GATA5 transcript: forward, 5’-GTA, GCT, AGC, CAC, CGC, CGT, GCC, CTG, CCG-3’, and reverse, 5’-GAT, GCG, GCC, GCT, GTT, CCC, CTG, ACA, TGG, GC-3’. A 1296-base pair PCR fragment was digested with the endonucleases NheI and NotI (TaKaRa, Dalian, Liaoning, China). The digested product was fractionated using 1.5% agarose gel electrophoresis, purified using a QIAquick Gel Extraction Kit (Qiagen), and

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then subcloned into pcDNA3.1 (Promega) to generate the eukaryotic expression vector, pcDNA3.1-hGATA5.

Table 1 - Baseline clinical characteristics of the 110 probands with familial atrial fibrillation.

Site-directed mutagenesis

Parameter

The identified mutation was introduced into wildtype GATA5 using a QuickChange II XL Site-Directed Mutagenesis Kit (Stratagene) with a complementary pair of primers. The mutant was sequenced to confirm the desired mutation and to exclude any other sequence variations.

Male Age of onset Paroxysmal atrial fibrillation on presentation Progression to permanent atrial fibrillation History of cardioversion History of pacemaker Resting heart rate (bpm) Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) Body mass index (kg/m2) Left atrial dimension (mm) left ventricular ejection fraction Fasting blood glucose (mmol/L) Total cholesterol (mmol/L) Triglycerides (mmol/L) Medications Amiodarone Warfarin Beta blocker Calcium channel blocker Digoxin

Reporter gene assay The atrial natriuretic factor (ANF)-luciferase reporter construct (ANF(-2600)-Luc), which contains the 2600-bp 5’flanking region of the ANF gene, was graciously provided by Dr. Ichiro Shiojima from Chiba University School of Medicine (Chiba-shi, Chiba, Japan). HEK-293 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum. The ANF(-2600)-Luc reporter construct and an internal control reporter plasmid pGL4.75 (hRluc/CMV, Promega), were used in transient transfection assays to examine the transcriptional activation activity of the GATA5 mutant. HEK-293 cells were transfected with 0.4 mg of wild-type or mutant pcDNA3.1hGATA5, 0.4 mg of the ANF(-2600)-Luc reporter construct, and 0.04 mg of the pGL4.75 control reporter vector using PolyFect Transfection Reagent (Qiagen). For the cotransfection experiments, 0.2 mg of wild-type pcDNA3.1-hGATA5, 0.2 mg of mutant pcDNA3.1-hGATA5, 0.4 mg of ANF(-2600)Luc, and 0.04 mg of pGL4.75 were used. Firefly luciferase and Renilla luciferase activities were measured using the Dual-Glo luciferase assay system (Promega) 48 h after transfection. The activity of the ANF promoter is presented as the fold activation of Firefly luciferase relative to Renilla luciferase. A minimum of three independent experiments were performed for wild-type and mutant GATA5.

n or mean

% or range

65 45.3 82 24 72 7 75.6 125.2 82.8 22.1 38 0.6 4.6 3.5 1.2

59 22–59 75 22 65 6 50–160 90–136 60–88 20–24 26–40 0.5–0.7 3.6–5.8 3.2–5.5 0.7–1.6

68 62 16 23 26

62 56 15 21 24

thymine to adenine transversion in the second nucleotide of codon 138 (c.413ART), resulting in a tyrosine (Y) to phenylalanine (F) substitution at amino acid 138 (p.Y138F), was identified in the proband from Family 1. A thymine to guanine transversion in the first nucleotide of codon 210 (c.628TRG), corresponding to a cysteine (C) to glycine (G) substitution at amino acid 210 (p.C210G), was discovered in the proband from Family 2. The sequence chromatograms showing the detected heterozygous GATA5 mutations of c.413ART and c.628TRG in contrast to control sequences are shown in Figure 1. The two variants were not observed in the 400 control alleles nor were they found in the NCBI SNP database (http://www.ncbi.nlm.nih.gov/SNP). Genetic scans of each family showed that the variation was present in all affected living family members but absent in the unaffected family members who were examined. An analysis of the pedigrees demonstrated that in each family, the mutation cosegregated with AF, which was transmitted in an autosomal dominant pattern with complete penetrance. The pedigrees of the two families are illustrated in Figure 2. The phenotypic characteristics and genetic screening results for the affected family members are summarized in Table 2.

Statistical analysis The data are expressed as the mean ¡ SD. Continuous variables were tested for normality of distribution, and Student’s unpaired t-test was used to compare numeric variables between two groups. The categorical variables were compared between two groups using Pearson’s chi-squared test or Fisher’s exact test, when appropriate. A two-sided pvalue less than 0.05 was considered statistically significant.

RESULTS Clinical characteristics of the study population

Alignments of GATA5 protein sequences from multiple species

A cohort of 110 unrelated index patients with familial AF was enrolled, clinically evaluated and compared with 200 ethnically matched, unrelated healthy individuals. None of the subjects had traditional risk factors for AF. There were no significant differences between the proband and control groups in baseline characteristics, including age, gender, body mass index, blood pressure, fasting blood glucose, serum lipid levels, left atrial dimension, left ventricular ejection fraction, and heart rate at rest. The baseline clinical characteristics of the 110 probands with familial AF are listed in Table 1.

A cross-species alignment of GATA5 protein sequences showed that the altered amino acids were completely conserved evolutionarily (Figure 3), suggesting that these amino acids are functionally important.

Transcriptional activity of the GATA5 mutants Wild-type GATA5, Y138F-mutant GATA5, and C210Gmutant GATA5 increased the transcriptional activity of the ANF promoter approximately 13-fold, 5-fold, and 2-fold, respectively. When wild-type GATA5 was coexpressed with the same amount of Y138F-mutant GATA5 or C210Gmutant GATA5, the transcriptional activity of the ANF promoter was increased approximately 7-fold and 4-fold, respectively. These results reveal that each of the two

GATA5 mutations Two heterozygous missense mutations in GATA5 were identified in two of 110 unrelated probands. The total population prevalence of GATA5 mutations based on the proband cohort was approximately 1.82%. Specifically, a

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Figure 1 - Sequence electropherograms showing the GATA5 mutations in contrast with their corresponding controls. The arrow indicates the heterozygous A/T (Figure 1A) and T/G (Figure 1B) nucleotides in the probands from Families 1 and 2, respectively, (mutant) or the homozygous A/A (Figure 1A) and T/T (Figure 1B) nucleotides in the corresponding controls (wild-type). The square denotes the nucleotides comprising a GATA5 codon.

GATA5 mutations is associated with significantly reduced activation activity when compared with wild-type GATA5 (Figure 4).

GATA5 is responsible for AF in these families. Given recently reported GATA5 mutations in a larger cohort of AF patients (26), GATA5 mutations may be a relatively common molecular defect associated with the pathogenesis of AF. GATA transcription factors are a group of zinc finger– containing, DNA-binding proteins characterized by their ability to bind to the consensus DNA sequence "GATA". In vertebrates, the GATA family comprises six members (GATA1–6), of which GATA4–6 are expressed in various mesoderm- and endoderm-derived tissues, especially in the embryonic heart (18). The GATA5 gene maps to human chromosome 20q13.33 by fluorescence in situ hybridization and encodes a 397–amino acid protein (27). An alignment of GATA5 with GATA4 suggests that the structural domains associated with GATA5 function consist of two transcriptional activation domains (TAD1: 1–49; TAD2: 107–154), two adjacent zinc fingers (ZF1: 187–212; ZF2: 242–266), and one nuclear localization signal (NLS: 226–296). The two TADs are both important to the transcriptional activity of GATA5. The C-terminal ZF is essential for DNA sequence recognition and binding to the consensus motif, whereas the Nterminal ZF is crucial for the stability and sequence specificity of protein-DNA binding and transcriptional activation by GATA factors. Most of the protein–protein interactions of GATA factors are mediated by their Cterminal ZF. The NLS sequence is associated with the subcellular trafficking and distribution of GATA5. The p.Y138F and p.C210G GATA5 mutations identified in this study are located in the TAD2 and ZF1, respectively; thus, they may affect the transcriptional activity of GATA5. It has been demonstrated that GATA5 is an upstream regulator of multiple genes expressed during embryogenesis,

DISCUSSION In the present study, we report two novel heterozygous missense mutations of GATA5 identified in two AF families. In each family, the mutant allele is present in all of the affected living family members but absent in the unaffected relatives who were examined and in the 400 control chromosomes. A cross-species alignment of GATA5 protein sequences demonstrated that the altered amino acids are completely conserved evolutionarily. The functional analysis demonstrated that the mutant GATA5 proteins are associated with significantly decreased transcriptional activation. Therefore, it is very likely that functionally impaired

Figure 2 - The pedigrees of the families with atrial fibrillation. The families are designated as Family 1 and Family 2. Family members are identified by generation and number. Squares indicate male family members; circles, female members; a symbol with a slash, a deceased member; closed symbols, affected members; open symbols, unaffected members; arrows, probands; ‘‘+’’, carriers of the heterozygous mutations; and ‘‘2’’, non-carriers.

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Novel GATA5 mutations in AF Gu JY et al.

Table 2 - Phenotypic characteristics and the GATA5 mutation status of the affected pedigree members. Subject Information

Identity Family 1 I-2 II-1 II-6 III-1 Family 2 I-1 II-3 III-2

Phenotype

Electrocardiogram

Echocardiogram

Gender

Age at time of study (years)

Age at first diagnosis of AF (years)

AF (classification)

QRS interval (ms)

QT / QTc

LAD (mm)

LVEF (%)

F M F F

75 52 46 26

44 38 42 25

Permanent Paroxysmal Persistent Paroxysmal

98 92 90 88

398/417 350/458 368/405 372/437

40 35 32 28

60 68 65 64

M M F

72a 50 24

45 32 24

Permanent Paroxysmal Paroxysmal

102 98 90

362/420 414/443 356/452

42 35 30

58 66 62

Genotype GATA5 mutations Y138F +/– +/– +/– +/– C210G N/A +/– +/–

Note: AF = atrial fibrillation; F = female; M = male; N/A = not available or not applicable; LAD = left atrial dimension; LVEF = left ventricular ejection fraction; QT = QT interval; QTc = corrected QT interval. + indicates present and – denotes absent. a Age at death.

development of the pulmonary venous myocardium. The pulmonary venous vessel is surrounded by a layer of myocardium known as the pulmonary myocardial sleeve, which is believed to be responsible for the initiation and maintenance of AF by several potential arrhythmogenic mechanisms that favor reentry, including enhanced intrinsic pacemaker activity, anisotropic arrangement of the myocardial fibers and slowed conduction (28-30). Genetic-labeling lineage-tracing studies have demonstrated that NKX2-5 is expressed in the atria and pulmonary myocardium and is important in the localization of the sinoatrial node during embryogenesis. NKX2-5 may act as a repressor of the sinoatrial node lineage gene program, limiting pacemaker activity to the sinoatrial and atrioventricular nodes. When NKX2-5 expression decreased in a hypomorphic model, the pulmonary cardiomyocytes switched to connexin40-negative, HCN4-positive cells, a nodal-like phenotype with pacemaker activity (30). In NKX2-5 knockout mouse

including the genes that encode atrial natriuretic factor (ANF), brain natriuretic peptide, a myosin heavy chain, b myosin heavy chain, and cardiac troponin C and I (18). Hence, the functional effect of the identified GATA5 mutations can be explored by analyzing the transcriptional activity of the ANF promoter in cells transfected with a GATA5 mutant or its wild-type counterpart. In this study, the functional characteristics of the novel GATA5 mutations identified in our familial AF patients (p.Y138F and p.C210G) were investigated using transcriptional activity assays, and the significantly reduced transcriptional activity was observed following the expression of the mutant proteins. These findings indicate that dysfunctional GATA5 is potentially an alternative pathological mechanism involved in AF, although the functional roles of the recently reported AFassociated GATA5 mutations remain to be addressed (26). The findings that dysfunctional GATA5 confers susceptibility to AF may be partially attributable to the abnormal

Figure 3 - Alignment of the GATA5 protein sequence of multiple species. The amino acids altered as a result of the described mutations (p.Y138 and p.C210) are completely conserved evolutionarily.

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2.

3. 4.

5.

6.

Figure 4 - Functional defects associated with GATA5 mutations. Activation of the ANF-luciferase reporter in HEK-293 cells by wildtype GATA5 (WT), mutant Y138F or mutant C210G, alone or in combination. Expression of the mutant proteins results in significantly reduced transactivation of the ANF promoter. Experiments were performed in triplicate, and means and standard deviations are shown. ** and * indicate p,0.0005 and p,0.001, respectively, when compared with wild-type GATA5.

7.

8. 9.

embryos, HCN4 was overexpressed in the whole embryonic heart, whereas connexin40 expression was suppressed and ectopic pacemaker cells were observed throughout the heart tube (31). In humans, AF has been observed as an isolated phenotype or as a part of compound phenotypes in patients carrying NKX2-5 mutations (32-34). Therefore, as a transcriptional cooperative partner of NKX2-5 (29), GATA5, when loss-of-function mutations occur, may contribute to the altered formation of the pulmonary myocardium sleeve and the shift of the pulmonary myocardium to a sinoatrial node–like phenotype, hence creating an electrophysiological substrate for AF. Some downstream genes are upregulated by GATA5, and mutations in several target genes have been associated with AF, including the ANF genes (35). Therefore, it is likely that mutated GATA5 predisposes affected individuals to AF by downregulating the expression of these target genes. In conclusion, the present investigation links GATA5 lossof-function mutations to familial AF and provides novel insights into the molecular mechanisms involved in the pathogenesis of AF. Furthermore, these results have potential implications for early prophylactic therapies and personalized treatment for AF.

10.

11.

12.

13.

14.

15.

16. 17. 18.

ACKNOWLEDGMENTS We are deeply thankful to the participants for their devotion to the study. This work was funded in part by grants from the National Natural Science Fund of China (81070153) and the Shanghai Pujiang Program (09PJ1410400).

19.

20.

AUTHOR CONTRIBUTIONS Gu JY, Xu JH, and Yu H contributed to the experimental design, the clinical and experimental research, the analysis and interpretation of the data, and manuscript writing. Yang YQ contributed to the study design, the analysis and interpretation of the data, and the initial drafting and review of the manuscript.

21.

22. 23.

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CLINICS 2012;67(12):1401-1406

DOI:10.6061/clinics/2012(12)09

CLINICAL SCIENCE

The impact of asymptomatic vertebral fractures on quality of life in older community-dwelling women: the Sa˜o Paulo Ageing & Health Study Jaqueline B. Lopes, Leandro K. Fung, Caroline C. Cha, Gustavo M. Gabriel, Liliam Takayama, Camille P. Figueiredo, Rosa Maria R. Pereira Faculdade de Medicina da Universidade de Sa˜o Paulo, Disciplina de Reumatologia, Sa˜o Paulo/SP, Brazil.

OBJECTIVES: The aim of this study was to investigate the impact of asymptomatic vertebral fractures on the quality of life in older women as part of the Sao Paulo Ageing & Health Study. METHODS: This study was a cross-sectional study with a random sample of 180 women 65 years of age or older with or without vertebral fractures. The Quality of Life Questionnaire of the European Foundation for Osteoporosis was administered to all subjects. Anthropometric data were obtained by physical examination, and the body mass index was calculated. Lateral thoracic and lumbar spine X-ray scans were obtained to identify asymptomatic vertebral fractures using a semi-quantitative method. RESULTS: Women with asymptomatic vertebral fractures had lower total scores [61.4(15.3) vs. 67.1(14.2), p = 0.03] and worse physical function domain scores [69.5(20.1) vs. 77.3(17.1), p = 0.02] for the Quality of Life Questionnaire of the European Foundation for Osteoporosis compared with women without fractures. The total score of this questionnaire was also worse in women classified as obese than in women classified as overweight or normal. High physical activity was related to a better total score for this questionnaire (p = 0.01). Likewise, lower physical function scores were observed in women with higher body mass index values (p,0.05) and lower physical activity levels (p,0.05). Generalized linear models with gamma distributions and logarithmic link functions, adjusted for age, showed that lower total scores and physical function domain scores for the Quality of Life Questionnaire of the European Foundation for Osteoporosis were related to a high body mass index, lower physical activity, and the presence of vertebral fractures (p,0.05). CONCLUSION: Vertebral fractures are associated with decreased quality of life mainly physical functioning in older community-dwelling women regardless of age, body mass index, and physical activity. Therefore, the results highlight the importance of preventing and controlling asymptomatic vertebral fractures to reduce their impact on quality of life among older women. KEYWORDS: Quality Of Life (QOL); Vertebral Fractures; Physical Activity; Body Mass Index (BMI); Old Women. Lopes JB, Fung LK, Cha CC, Gabriel GM, Takayama L, Figueiredo CP, et al. The impact of asymptomatic vertebral fractures on quality of life in older community-dwelling women: the Sa˜o Paulo Ageing & Health Study. Clinics. 2012;67(12):1401-1406. Received for publication on June 11, 2012; First review completed on July 27, 2012; Accepted for publication on August 19, 2012 E-mail: rosamariarp@yahoo.com Tel.: 55 11 3061-7213

has been used as a measure complementary to bone mineral density to evaluate and monitor the burden of osteoporosis on a patient’s daily life (3). There are several instruments that can be used to assess the quality of life of individuals with osteoporosis, including the Osteoporosis Assessment Questionnaire, the Quality of Life Questionnaire for Osteoporosis (OPTQoL), the Osteoporosis Quality of Life Questionnaire, and the Questionnaire of the European Foundation for Osteoporosis (QUALEFFO) (4-7). The QUALEFFO, a specific tool used to evaluate subjects with vertebral fractures and that includes questions on pain, physical functioning, social functioning, general health perception and mental functioning, has been shown to be repeatable and consistent (7-8). Vertebral fractures are the most frequent osteoporotic fractures, occurring in at least 30% of the elderly population

INTRODUCTION Health-related quality of life (HRQoL) is a multidimensional concept that defines a person’s health based on specific aspects, such as physical, emotional, and social functioning and general welfare (1). The assessment of HRQoL consists of an evaluation of the degree to which these aspects are decreased by symptoms, incapacities, and limitations caused by disease (2). The assessment of HRQoL

Copyright ß 2012 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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between the two individuals was reached. The agreement between the assessments of the two individuals was 96%, and the Kappa coefficient was 0.82. Vertebral fractures were classified using a Genant SQ approach (19). Each identified fractured vertebra was assigned a grade based on the Genant SQ scale, where mild (grade 1) corresponds to a 2025% reduction in the anterior, middle, and/or posterior height; moderate (grade 2) corresponds to a 26-40% reduction in any height; and severe (grade 3) corresponds to a reduction of over 40% in any height.

(9), and have important clinical implications (10-14). These fractures are associated with increased risks of new osteoporotic fractures and mortality, especially in older women (10-11). Only one-third of vertebral fractures are symptomatic; therefore, patients may be unaware of their presence. Indeed, in studies based on the radiographic screening of populations, the incidence of all vertebral fractures has been estimated to be three times higher than the incidence of hip fractures, and only 30% of people with vertebral fractures were found to have received medical attention (12). Women with vertebral fractures can also experience decreased HRQoL due to physical limitations and psychosocial disabilities (13-14). Some studies have assessed the impact of vertebral fractures on HRQoL in older women in many countries (15,16), but few such epidemiological studies have been conducted in Brazil. Moreover, most of the studies conducted in this country have been performed in ambulatory or institutionalized individuals (17,18). Therefore, this study evaluated the impact of vertebral fractures on the quality of life of healthy, communitydwelling women aged 65 years or older using the QUALEFFO.

Anthropometry The height (without shoes) of each participant was measured to the nearest 0.1 cm with a wall-mounted stadiometer. The weight of each participant (without shoes and wearing only light clothing) was measured to the nearest 0.25 kg using a double-beam balance scale. The body mass index (BMI) was calculated by dividing the participant’s weight (kilograms) by her height squared (square meters), and the subjects were categorized using the following cutoff points proposed by the World Health Organization (WHO): normal weight = BMI,25; overweight = 25#BMI,30; and obese = BMI$30 (20).

Other variables and definitions SUBJECTS AND METHODS

Information regarding health, lifestyle and risk factors for osteoporosis/fractures was obtained through individual interviews. Women who had had two or more falls in the last 12 months were defined as chronic fallers (21). Physical activity was classified as (a) low, does not even perform housework; (b) moderate, performs regular housework, walks irregularly, and gardens; and (c) high, performs regular physical activity aside from her daily routine at least twice a week for 30 min (22). Regarding concomitant diseases, those mentioned at the time of the interview were noted, as well as those diagnosed during the physical examination. Systemic arterial hypertension (SAH) was defined as a history of hypertension with the use of antihypertensive drugs or a systolic blood pressure.140 mmHg and/or diastolic blood pressure. 90 mmHg, which was measured with a standard sphygmomanometer with the subject seated for at least 5 minutes prior to the measurement (23). Participants taking oral hypoglycemic agents or insulin or those with fasting blood glucose levels$126 mg/dL were considered to be diabetic (24).

Population This study was performed using the framework of the Sa˜o Paulo Ageing & Health Study (SPAH), which was a population-based, cross-sectional study (9). Based on the size sample calculation, 180 women were randomized and included. The inclusion and exclusion criteria were the same as those of the core study (9). Specifically, only well-functioning elderly women were recruited to participate. All of the individuals were apparently healthy and showed no evidence of malabsorption, chronic diarrhea, hepatic disease, severe chronic diseases, or cancer. Current or previous bisphosphonate use was also an exclusion criterion (9).

Quality of life assessment Quality of life was assessed through individual interviews using the validated QUALEFFO with 41 questions covering five domains: pain (5 questions), physical functioning (17 questions), social functioning (sevn questions), general health perception (three questions), and mental functioning (nine questions) (7). The total score for each domain was obtained by summing the scores of all questions for that domain and submitting this sum to a linear transformation to a scale ranging from 0 to 100, where 0 corresponds to the worst HRQoL and 100 to the best HRQoL. The interviewers were blinded to the presence of vertebral fractures.

Bone mineral density (BMD) assessment The BMD was measured by dual X-ray absorptiometry (DXA) using Hologic densitometry equipment (Hologic Inc. Bedford, MA, USA, Discovery model) in the following regions: lumbar spine, femoral neck, and total femur. All BMD measurements were performed by the same experienced technologist. Anatomically abnormal vertebrae were excluded from the analysis of the lumbar spine only if they were clearly abnormal and were not assessable within the resolution of the system or if there was a difference in the Tscore of more than 1.0 between the vertebra in question and adjacent vertebrae, as recommended by the International Society for Clinical Densitometry (ISCD) (25). The precision of the BMD measurements was determined based on standard ISCD protocols (26). We calculated the least significant change with 95% confidence to be 0.033 g/cm2 for the spine, 0.047 g/cm2 for the femoral neck, and 0.039 g/cm2 for the total femur.

Assessment of vertebral fractures Radiographs of the lumbar and thoracic spine centered on L2 and T7, respectively, were obtained for all participants, with 40" between the tube and the film. All of the obtained images provided good visibility of the T4 to L4 segment. The identification of vertebral fractures was performed by two individuals with experience in the field of analyzing vertebral fractures. They were blinded to each other’s assessments, and when the results conflicted, a consensus

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According to the classification criteria of the ISCD (International Society of Clinical Densitometry), the lowest T-score among the three sites (lumbar spine, femoral neck, and total femur) was used to classify each participant as having osteoporosis, osteopenia, or normal bone density. Thus, the individuals with T-scores that were 2.5 standard deviations or more below the scores for healthy controls for the peak bone mass were diagnosed with osteoporosis, individuals with T-scores between 2.5 and 1.0 standard deviations below the scores for healthy controls were diagnosed with osteopenia, and individuals with T-scores greater than 1.0 standard deviation below the scores for healthy controls were classified as normal (26).

and statistically significant variables (p,0.05) were retained in the final model. All tests were performed using STATA 10.0.

Statistical analysis

Forty-one women (22.8%) had vertebral fractures. The demographic, anthropometric and clinical data for all participants in the study, grouped based on the presence (Vertebral Fracture) or absence of vertebral fractures (No Vertebral Fracture), are shown in Table 1. There were no significant differences with respect to the mean BMI or the percentage of Caucasian individuals between the groups (p.0.05) (Table 1). Regarding the BMI classification, 22.8% and approximately 38% of the subjects were classified as normal and obese, respectively, with no significant difference between the two groups (p.0.05). A tendency of older age in the vertebral fracture group was observed (p = 0.057). The frequencies of hypertension (p = 0.224), diabetes (p = 0.672), hypothyroidism (p = 0.723), and two or more concomitant diseases (p = 0.216) and the average number of medications used (p = 0.497) were comparable between the groups. Interestingly, the Vertebral Fracture group contained a higher frequency of women defined as chronic fallers than the No Vertebral Fracture group (64.7 vs. 32.1%, p = 0.017). As expected, the Vertebral Fracture group had a higher frequency of osteoporosis (73.2 vs. 51.1%, p = 0.012) and a

Ethics This study was conducted in compliance with the ethical principles of the Helsinki Declaration (2008) and local applicable laws and regulations. This study was approved by the Local Ethics and Research Committee (Research Protocol 1110/07).

RESULTS

The sample size of 180 was based on a standard deviation of 15% (27) for the total QUALEFFO score and a two-sided 5% significance level. The study had 95% power to detect a difference of 10 points in the total QUALEFFO score. The results for the quantitative variables are expressed as the mean (standard deviation), and results for the qualitative variables are described by the absolute and relative (%) frequencies. Demographic characteristics and the QUALEFFO results were compared between women with and without fractures using the Mann-Whitney-Wilcoxon test for quantitative variables and the chi-square test for qualitative variables. The associations between the QUALEFFO scores and potential determinants of quality of life were assessed using the Wilcoxon rank-sum test or the Kruskal-Wallis test. Correlations between continuous variables and the QUALEFFO questionnaire data were tested using the Spearman correlation coefficient (rs). Generalized linear models with gamma distributions and logarithmic link functions were performed to determine the influence of the vertebral fractures on the QUALEFFO score. Variables with a statistical significance better than 0.1 (p,0.1) in the bivariate tests were included in these models,

Table 1 - Demographic, anthropometric, and clinical data of the Vertebral Fracture (VF) and No Vertebral Fracture (NVF) groups.

Age, years Caucasian individuals, n (%) Body Mass Index, kg/m2 Classification according to BMI Normal, n (%) Overweight, n (%) Obese, n (%) Currently smoking, n (%) Physical activity level, n (%) Low Moderate High Chronic faller, n (%) SAH, n (%) Diabetes, n (%) Hyperthyroidism, n (%) Two or more concomitant diseases Number of medications Densitometric criteria (ISCD) Osteopenia, n (%) Osteoporosis, n (%)

All (n = 180)

Vertebral Fracture (n = 41)

No Vertebral Fracture (n = 139)

p-value VF vs. NVF

75.2 (4.5) 121 (67.2) 28.8 (4.9)

76.3 (5.4) 27 (65.9) 29.1 (5.1)

74.8 (4.2) 94 (67.6) 28.7 (4.8)

0.057 0.832 0.572

44 67 67 18

(22.8) (37.6) (37.6) (10)

9 16 16 3

(22.0) (39.0) (39.0) (7.3)

8 120 52 28 126 44 14 128 1.5

(4.4) (66.7) (28.9) (40) (74.6) (24.4) (8.3) (71.1) (1.4)

3 (7.3) 27 (65.9) 11 (26.8) 11 (64.7) 25 (83.3) 9 (22.0) 2 (6.7) 26 (63.4) 1.7(1.4)

5 (3.6) 93 (66.9) 41 (29.5) 17 (32.1) 101 (72.7) 35 (25.2) 12 (8.6) 102 (73.4) 1.5(1.4)

0.017 0.224 0.672 0.723 0.216 0.497

61 (33.9) 101 (56.1)

8 (19.5) 30 (73.2)

53 (38.1) 71 (51.1)

0.027 0.012

Data are presented as the mean (standard deviation) or n (percentage). SAH = Systemic Arterial Hypertension.

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35 51 51 15

(25.6) (37.2) (37.2) (10.8)

0.896 0.515

0.586


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lower frequency of osteopenia (19.5 vs. 38.1%, p = 0.027) than the No Vertebral Fracture group. The results for each domain of the QUALEFFO and the total QUALEFFO score in both groups are shown in Table 2. The total QUALEFFO score was lower in the Vertebral Fracture group than in the No Vertebral Fracture group [61.4 (15.4) vs. 67.1 (14.2), p = 0.031]. Likewise, the physical function domain score of the QUALEFFO was worse in the Vertebral Fracture group compared with the No Vertebral Fracture group [69.5 (20.1) vs. 77.3 (17.1), p = 0.018]. No difference was observed regarding the other QUALEFFO domains (pain, social functioning, health perception, and mental functioning) (Table 2). The total QUALEFFO score was inversely related to BMI (rs = -0.21, p = 0.005) and weight (rs = -0.22, p = 0.009). The total QUALEFFO score was worse in women classified as obese than in those classified as overweight or normal [61.7 (15.4) vs. 66.4 (13.8) vs. 70.8 (13.5), respectively, p = 0.008]. A lower total QUALEFFO score was also observed in women with low physical activity than in those with moderate or high activity [51.8 (19.1) vs. 64.8 (14.3) vs. 70.3 (13.2), respectively, p = 0.010] (Table 3). Similarly, the physical function domain of the QUALEFFO was inversely related to the BMI (rs = -0.24, p = 0.001). In this domain, women classified as obese were found to have lower scores than women classified as overweight or normal [70.7 (19.3) vs. 76.1 (16.1) vs. 81.4 (17.4), respectively, p = 0.002]. Finally, lower scores for the physical function domain were found among women with low physical activity compared with those with moderate or high activity [49.6(24.9) vs. 75.1(18.1) vs. 80.7(12.6), respectively, p = 0.002] (Table 3). A generalized linear model with gamma distributions and logarithmic link functions was developed to identify patient characteristics that were related to the total and physical function domain scores of the QUALEFFO. Variables with a p,0.10 in the univariate analysis (age, BMI classification, physical activity, diabetes, presence of at least one fracture) were included as independent variables. The presence of obesity was negatively associated with the total QUALEFFO score (p = 0.001). A high physical activity level was positively associated with the total QUALEFFO score (p = 0.001). The presence of at least one fracture was associated with a worse total QUALEFFO score, independent of age, BMI classification and physical activity level (p = 0.030). Likewise, the presence of at least one fracture was negatively associated with the physical function domain, independent of these same variables (p = 0.041) (Table 4).

DISCUSSION This study was the first study conducted in Brazil that specifically assessed the impact of vertebral fractures on the quality of life in older, community-dwelling women using a specific questionnaire. In this study, we demonstrated that the presence of vertebral fractures in this population is related to worse HRQoL, particularly with respect to physical functioning. The major advantage of the present study is the homogenous selection of community-dwelling women, unlike previous studies in which individuals were recruited from clinics or from populations included in clinical trials. These latter populations represent sick individuals with a high risk of fractures. Studies showing worse HRQoL in patients with vertebral fractures have been published in several countries (15,16). There are only two studies evaluating the HRQoL in patients with vertebral fractures in Brazil, but neither was specific to older community-dwelling individuals (17,18). The first of these two other studies was performed in 55 outpatient women divided into three groups: 1- women without osteoporosis, 2- women without osteoporosis and no vertebral fractures, 3- women with osteoporosis and vertebral fractures. In that study, the quality of life was assessed with the SF-36, and no difference was found among the three groups. One of the reasons for this finding was the inclusion of only women who were able to perform the spirometric tests, resulting in the exclusion of women in worse conditions who would most likely belong to the fracture group (17). Later, de Oliveira et al. evaluated the quality of life in ambulatory women with osteoporosis and found similar results for those who had vertebral fractures and those who did not. However, that study was not designed to assess the impact of vertebral fractures on quality of life, and the number of women with fractures was too small to enable an accurate assessment (18). The assessment of quality of life in relation to the QUALEFFO pain domain was similar in women with and without vertebral fractures in our study. Some studies have found that the pain domain is worse in women with fractures; however, the patients included in those studies were recruited based on clinical symptoms related to fractures and were compared with those without back pain (28). Vertebral fractures do not always manifest with symptoms and are often diagnosed based on radiographs taken for other reasons. Gehlbach et al. evaluated the chest radiographs of older women who had been hospitalized for several causes, and they found that only a few of the vertebral fractures present had been previously identified

Table 2 - QUALEFFO questionnaire data: total score and score for each domain for the Vertebral Fracture (VF) and No Vertebral Fracture (NVF) groups. QUALEFFO Total Domains Pain Physical function Social function Health Perception Mental Function

All (n = 180)

Vertebral Fracture (n = 41)

No Vertebral Fracture (n = 139)

p-value VF vs. NVF

65.8 (14.7)

61.4 (15.4)

67.1 (14.2)

0.031

68.8 75.5 32.9 49.0 69.6

63.1 69.5 31.7 44.5 67.3

70.50 (27.7) 77.3 (17.1) 33.2 (14.6) 50.2 (21.7) 70.3 (18.1)

0.115 0.018 0.438 0.118 0.379

(27.5) (18.1) (14.8) (21.9) (18.4)

(26.7) (20.1) (15.6) (22.3) (19.3)

Data are presented as the mean (standard deviation).

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Table 3 - Association between categorical variables and the quality of life questionnaire scores (QUALEFFO: Total score and physical function domain score). Variables

QUALEFFO Total score

p-value

QUALEFFO Physical Function

p-value

70.8 (13.5) 66.4 (13.8) 61.7 (15.4)

0.008

81.4 (17.4) 76.1 (16.1) 70.7 (19.3)

0.002

Classification according to BMI Normal Overweight Obese Currently smoking Yes No Physical activity level Low Moderate High Hypertension Yes No Diabetes Yes No Osteoporosis Yes No Severity of vertebral fracture No Fracture Grade 1 Grade 2 or 3 Chronic faller Yes No

63.7 (14.2) 66.0 (14.7)

0.419

51.8 (19.1) 64.8 (14.3) 70.3 (13.2)

0.010

72.1 (14.1) 75.9 (18.4) 49.6 (24.9) 75.1 (18.1) 80.7 (12.6)

0.159

0.002

68.6 (15.0) 69.9 (16.6)

0.534

75.2 (17.1) 76.4 (20.8)

0.317

62.1 (14.2) 70.0 (14.7)

0.052

72.1 (17.4) 76.6 (18.2)

0.073

67.7 (14.7) 69.8 (15.9)

0.346

67.1 (14.2) 58.6 (16.1) 62.5 (15.3)

0.073

63.3 (13.9) 66.3 (14.8)

0.230

73.2 (17.2) 77.3 (18.6) 77.3 (17.1) 68.4 (17.8) 69.9 (21.0) 72.3 (17.8) 76.1 (18.1)

0.027

0.053

0.223

Data are presented as the mean (standard deviation).

with osteoporosis (18,31-32). It is important to highlight the fact that both obesity and a sedentary lifestyle are preventable factors and can be controlled by a change in lifestyle. Some authors observed that physical exercise is associated with a better quality of life (33) and have demonstrated that a home exercise program for women with vertebral fractures (60 min/d, 3x/week for 12 months) significantly improves the HRQoL (34). A study performed at our institution in osteoporosis patients showed that an exercise program improved the quality of life in these women (35). Therefore, our data reinforce the need for all older women to be advised about the benefits of exercise. These women should be encouraged to exercise regularly to reduce their BMI and improve their general welfare. Our study is the first in Brazil to conduct a thorough assessment of the relationship between quality of life and vertebral fractures. An important characteristic of this study was the use of standardized and reliable instruments for both the vertebral fracture assessment (Genant semi-quantitative method) and the HRQoL assessment (QUALEFFO). Although the QUALEFFO has not been validated in Brazil, which is a limitation of the present study, this tool is the most frequently used for assessing quality of life in osteoporosis and was recommended for the investigation of vertebral fracture subjects in multicentric studies that included centers in Brazil (36). This questionnaire is more sensitive in detecting differences between groups, and it provides a better discrimination between individuals with and without vertebral fractures compared with generic HRQOL instruments, such as the SF-36, particularly with respect to physical functioning, which is significantly affected in these patients (8). In conclusion, this study demonstrated the negative impact of vertebral fractures on the quality of life in older women, independent of other factors such as BMI and

by clinicians (29). Indeed, in our previous study performed in Brazil, 29.4% subjects had vertebral fractures, and none of these patients had prior knowledge of their vertebral fractures (9). As observed in other studies, the mental function, social function, and health perception domains were not significantly different between women with and without fractures (30). The relatively small differences between the groups with and without fractures may be the result of the acceptance of poor health conditions due to the natural expectation of physical decline in older women (13). In our study, we found that obesity and low physical activity were associated with lower quality of life. Other authors have reported that higher BMI and a sedentary lifestyle are factors that influence the quality of life in patients Table 4 - Generalized linear models. QUALEFFO

Independent Variables

Coefficient p-value

Total Age Classification according to BMI* Physical activity level Diabetes Vertebral Fracture

-0.01 -0.07

0.055 0.001

0.10 -0.05 -0.08

0.001 0.208 0.030

Age Classification according to BMI* Physical activity level Diabetes Vertebral Fracture

-0.01 -0.07

0.060 0.002

0.13 -0.04 -0.10

,0.001 0.569 0.041

Physical Function

*

BMI = body mass index.

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15. Adachi JD, Loannidis G, Berger C, Joseph L, Papaioannou A, Pickard L et al. Canadian Multicentre Osteoporosis Study (CaMos) Research Group: The influence of osteoporotic fractures on health-related quality of life in community-dwelling men and women across Canada. Osteoporos Int. 2001;12(11):903-8, http://dx.doi.org/10.1007/ s001980170017. 16. Adachi JD, Adami S, Gehlbach S, Anderson FA Jr, Boonen S, Chapurlat RD et al. Impact of prevalent fractures on quality of life: baseline results from the global longitudinal study of osteoporosis in women. Mayo Clin Proc. 2010;85(9):806-13, http://dx.doi.org/10.4065/mcp.2010.0082. 17. Lombardi I Jr, Oliveira LM, Monteiro CR, Confessor YQ, Barros TL, Natour J. Evaluation of physical capacity and quality of life in osteoporotic women. Osteoporos Int. 2004;15(1):80-5, http://dx.doi. org/10.1007/s00198-003-1512-2. 18. de Oliveira Ferreira N, Arthuso M, da Silva R, Pedro AO, Pinto Neto AM, Costa-Paiva L. Quality of life in women with postmenopausal osteoporosis: correlation between QUALEFFO 41 and SF-36. Maturitas. 2009;62(1):85-90, http://dx.doi.org/10.1016/j.maturitas.2008.10.012. 19. Genant HK, Wu CY, van Kuijk C, Nevitt MC. Vertebral fracture assessment using a semiquantitative technique. J Bone Miner Res. 1993;8(9):1137-48. 20. World Health Organization. Physical status: the use and interpretation of 16. anthropometry. Genebra: World Health Organization; 1995. 21. Schwartz AV, Villa ML, Prill M, Kelsey JA, Galinus JA, Delay RR et al. Falls in older Mexican-American womem. J Am Geriatr Soc. 1999;47(11):1371-8. 22. Fitti JE, Kovar M. The Supplement on Aging to the 1984 National Health Interview Survey. Vital Health Stat 1. 1987;(21):1-115. 23. Chobanian AV, Bakris GL, Black HR, Cushman WC, Green LA, Izzo JL Jr, et al. The seventh report of the Joint National Committee on Prevention, Detection, Evaluation and Treatment of high blood pressure: the JNC 7 report. JAMA. 2003;289(19):2560-72, http://dx.doi.org/10. 1001/jama.289.19.2560. 24. American Diabetes Association. Diagnosis and classification of diabetes mellitus. Diabetes Care. 2006;29:S43-8. 25. Lewiecki EM, Gordon CM, Baim S, Leonard MB, Bishop NJ, Bianchi ML et al. International Society for Clinical Densitometry 2007 Adult and Pediatric Official Positions. Bone. 2008;43(6):1115-21, http://dx.doi.org/ 10.1016/j.bone.2008.08.106. 26. Shepherd JA, Lu Y, Wilson K, Fuerst T, Genant H, Hangartner TN, et al. Cross-calibration and Minimum Precision Standards for Dual-Energy Xray Absorptiometry: The 2005 ISCD Official Positions. J Clin Densitom. 2006;9(1):31-6, http://dx.doi.org/10.1016/j.jocd.2006.05.005. 27. Romagnoli E, Carnevale V, Nofroni I, D’Erasmo E, Paglia F, De Geronimo S, et al. Quality of life in ambulatory postmenopausal women: the impact of reduced bone mineral density and subclinical vertebral fractures. Osteoporos Int. 2004;15(12):975-80, http://dx.doi.org/10.1007/ s00198-004-1633-2. 28. Cook DJ, Guyatt GH, Adachi JD, Clifton J, Griffith LE, Epstein RS et al. Quality of life issues in women with vertebral fractures due to osteoporosis. Arthritis Rheum. 1993;36(6):750-6, http://dx.doi.org/10. 1002/art.1780360603. 29. Gehlbach SH, Bigelow C, Heimisdottir M, May S, Walker M, Kirkwood JR. Recognition of vertebral fracture in a clinical setting. Osteoporos Int. 2000;11(7):577-82, http://dx.doi.org/10.1007/s001980070078. 30. Oleksik A, Lips P, Dawson A, Minshall ME, Shen W, Cooper C et al. Health-related quality of life in postmenopausal women with low BMD with or without prevalent vertebral fractures. J Bone Miner Res 2000;15(7):1384-92. 31. Heo M, Allison DB, Faith MS, Zhu S, Fontaine KR. Obesity and quality of life: mediating effects of pain andcomorbidities. Obes Res. 2003;11(2):209-16, http://dx.doi.org/10.1038/oby.2003.33. 32. Marchesini G, Solaroli E, Baraldi L, Natale S, Migliorini S, Visani F et al. Health-related quality of life in obesity: the role of eating behaviour. Diabetes Nutr Metab. 2000;13(3):156-64. 33. Moriyama CK, Oneda B, Bernardo FR, Cardoso CG Jr, Forjaz CL, Abrahao SB, et al. A randomized, placebo-controlled trial of the effects of physical exercises and estrogen therapy on health-related quality of life in postmenopausal women. Menopause. 2008;15(4 Pt 1):613-8, http://dx. doi.org/10.1097/gme.0b013e3181605494. 34. Papaioannou A, Adachi JD, Winegard K, Ferko N, Parkinson W, Cook RJ, et al. Efficacy of home-based exercise for improving quality of life among elderly women with symptomatic osteoporosis-related vertebral fractures. Osteoporos Int. 2003;14(8):677-82, http://dx.doi.org/10.1007/ s00198-003-1423-2. 35. Madureira MM, Bonfa´ E, Takayama L, Pereira RM. A 12-month randomized controlled trial of balance training in elderly women with osteoporosis: improvement of quality of life. Maturitas. 2010;66(2):20611, http://dx.doi.org/10.1016/j.maturitas.2010.03.009. 36. Hadji P, Zanchetta JR, Russo L, Recknor CP, Saag KG, McKiernan FE, et al. The effect of teriparatide compared with risedronate on reduction of back pain in postmenopausal women with osteoporotic vertebral fractures. Osteoporos Int. 2012;23(8):2141-50, http://dx.doi.org/10.1007/ s00198-011-1856-y.

physical activity. Although the clinical relevance of vertebral fractures is well established, these results are important for assessing the burden of this disease and reinforce the need to reduce the underdiagnosis and undertreatment of these fractures. The results of this study also highlight the need for awareness of the importance of maintaining proper weight and promoting changes in lifestyle through physical activity and dietary control.

ACKNOWLEDGMENTS This study was supported by Fundac¸a˜o de Amparo e Pesquisa do Estado de Sa˜o Paulo (FAPESP) # 03/09313-0; Conselho Nacional de Cieˆncia e Tecnologia (CNPQ) #305691/2006-6 (RMRP) and #119601/2009-5 (LF); Federico Foundation Grants (RMRP); and Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior (CAPES) (JBL, CPF).

AUTHOR CONTRIBUTIONS Lopes JB and Pereira RM contributed to the study conception and design. Lopes JB, Fung LK, Cha CC, Gabriel GM, and Figueiredo CP assisted the recruitment of patients. Lopes JB, Fung LK, Cha CC, Gabriel GM, Fung LK, and Figueiredo CP acquired the data. Lopes JB, Fung LK, and Pereira RM contributed to the analysis and interpretation of the data. Lopes JB and Pereira RM prepared the manuscript.

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DOI:10.6061/clinics/2012(12)10

CLINICAL SCIENCE

Effects of Brazilian Cardioprotective Diet Program on risk factors in patients with coronary heart disease: a Brazilian Cardioprotective Diet randomized pilot trial Bernardete Weber, Andrea Polo Galante, Angela Cristine Bersch-Ferreira, Camila Ragne Torreglosa, Vitor Oliveira Carvalho, Elivane da Silva Victor, Jose Amalth do Espı´rito-Santo, Maria Beatriz Ross-Fernandes, Rafael Marques Soares, Rosana Perim Costa, Enilda de Sousa Lara, Anna Maria Buehler, Ota´vio Berwanger Hospital do Corac¸a˜o (IEP–HCor), Research Institute, Sa˜o Paulo/SP, Brazil.

OBJECTIVE: To evaluate the effectiveness of the Brazilian Cardioprotective Diet Program in reducing blood pressures, fasting glucose levels and body mass indices in patients with established atherothrombotic disease. METHOD: This randomized controlled pilot trial included outpatients who were over 45 years of age with atherothrombotic cardiovascular disease. Group A, who received the Brazilian Cardioprotective Diet Program, had weekly sessions with dietitians. Groups B and C received the usual dietary therapy that is given to patients with cardiovascular diseases as proposed by the Brazilian guidelines. This diet had the same nutrient profile as that given to Group A, but it was customized by the integration of typical Mediterranean foods. The difference between Groups B and C was the number of sessions with the dietitian. Group B received weekly sessions, while group C only had monthly sessions. ClinicalTrials.gov: NCT 01453166. RESULTS: There was a greater reduction in systolic (7.8%) and diastolic (10.8%) blood pressures in Group A compared with Group B (2.3% and 7.3%), and Group C (3.9% and 4.9%, respectively). Fasting glucose decreased by 5.3% and 2% in Groups A and B, respectively. Fasting glucose increased by 3.7% in Group C. The BMIs decreased by 3.5% and 3.3% in Groups A and B, respectively. Group C did not present with any changes in BMI. However, none of these data showed statistical differences between the groups, which is methodologically acceptable in pilot trials. CONCLUSIONS: The Brazilian Cardioprotective Diet Program seems to be more effective in reducing blood pressures, fasting glucose levels, weights and BMIs in patients with previous cardiovascular disease compared with the diet that has been proposed by the Brazilian guidelines. KEYWORDS: Diet; Cardiovascular Diseases; Risk factors; Prevention. Weber B, Galante AP, Bersch-Ferreira AC, Torreglosa CR, Carvalho VO, Victor ES, et al. Effects of Brazilian Cardioprotective Diet Program on risk factors in patients with coronary heart disease: a Brazilian Cardioprotective Diet randomized pilot trial. Clinics. 2012;67(12):1407-1414. Received for publication on July 6, 2012; First review completed on August 10, 2012; Accepted for publication on August 20, 2012 E-mail: bweber@hcor.com.br Tel.: 55 11 3053-6611

It is a consensus that the Mediterranean diet is effective and possibly the most appropriate dietary intervention for the prevention and treatment of CVD (2,3,4,5,6). This diet is characterized by a low saturated fat intake in addition to a high consumption of vegetables, fish, and olive oil and a moderate consumption of wine (5). Nevertheless, adherence to this diet seems to be an obstacle in successfully controlling cardiovascular risk factors. Thus, cultural adaptation seems to be the most appropriate means for its management in countries outside of the Mediterranean region (6). In Brazil, the Mediterranean diet differs vastly from local customs, and this factor is seemingly related to low adherence to the diet (7). Brazil is a developing country with a wide range of cultures and important socio-economic limitations. Additionally, significantly increased mortality and morbidity from CVD have been observed, which compromises the government’s finances. Brazil also possesses a rich variety of natural

INTRODUCTION Cardiovascular diseases (CVD) represent the leading causes of mortality and morbidity in developed countries worldwide, including Brazil (1). The control of cardiovascular risk factors plays an important role in patients’ quality of life and survival rates. The World Health Organization has stated that over three-quarters of all CVD deaths may be avoided with adequate changes in lifestyle, including diet and exercise (2-3).

Copyright ß 2012 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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and nutritional resources that are potentially beneficial in the treatment of CVD (8,9). Nevertheless, no studies have explored the putative benefits of food resources that are part of the Brazilian culture in comparison with the standard diet that has been proposed by the Brazilian guidelines in the prevention and treatment of cardiovascular diseases. The aim of this randomized pilot trial was to evaluate the Brazilian Cardioprotective Diet Program (a Brazilian version of an accessible dietary therapy for cardiovascular diseases) in reducing blood pressures, fasting glucose levels, and body mass indices (BMIs) in patients with established atherothrombotic disease.

of an accessible dietary therapy for cardiovascular diseases and weekly counseling with dieticians. The patients in Group B received the dietary therapy that was proposed by the Brazilian guidelines for cardiovascular diseases and also attended weekly counseling sessions with dietitians. Group C received the same dietary intervention as Group B, but the patients were counseled monthly.

Study interventions The nutrient profiles of the diets were based on the Brazilian guidelines for cardiovascular disease treatment (10,11,12). The diets contained 50-60% of energy from carbohydrates, 15% from proteins, and 25-35% from fats. In addition, 20-30 g/d of fiber and 2,000 mg/d of sodium were recommended. The concentrations of saturated, monounsaturated, and polyunsaturated fatty acids were ,7%, ,20%, and ,10%, respectively. The total dietary energy intake was adjusted only for patients with a baseline BMI .25 kg/m2. The main difference between the Brazilian Cardioprotective Diet Program, which involves a Brazilian version of an accessible dietary therapy for cardiovascular diseases, and the usual dietary therapy that is prescribed for cardiovascular diseases was the consideration of energy density. The Brazilian Cardioprotective Diet Program helped the patients to avoid high energy density foods (.1 kcal/g), thus allowing them to eat more and consume fewer calories. As they made the right food choices, they felt less restricted, aiding in the improvement of adherence. In general, the Brazilian Cardioprotective Diet Program features nutritional recommendations that are feasible for the Brazilian population. Accordingly, the diet composition allows for the easy access and full use of foods, in addition to the prioritization of regional foods that are culturally accepted by the patients (rice, bean, soy oil, and Brazilian fruits and vegetables). Group A participated in the Brazilian Cardioprotective Diet Program. This group attended weekly session with dietitians. These were conducted in person, by phone or in a gourmet shop. During attendance at the gourmet shop, the patients received tips for eating in restaurants, instructions on label reading and a list of typical Brazilian recipes that were adjusted for nutrients and energy densities. This nutritional prescription protocol was developed by dietitians from our hospital. Groups B and C received the usual dietary therapy that is prescribed to patients with cardiovascular diseases as proposed by the Brazilian guidelines. This diet had the same nutrient profile as that which was presented in Group A but was customized by the integration of typical Mediterranean foods (e.g., olives, olive oil, chestnuts, walnuts, almonds, hazelnuts, peanuts, and cold water fish). The difference between groups B and C was the number of sessions with the dietitian. Group B received weekly sessions that were conducted in person or by telephone, and Group C had monthly sessions that were conducted in person. Anthropometric measurements and 24-h dietary recalls were performed by well-trained dietitians. The patients described their average portion sizes for food items in terms of household measurements, the standard weights of food items and validated food portion photographs of known weights. The Nutriquanti diet system (SaËœo Paulo, SP, Brazil) (13), which is an online computer program that is based on

METHODS Trial design We conducted a randomized (concealed) controlled pilot trial to evaluate the effects of the Brazilian Cardioprotective Diet Program on blood pressures, BMIs, and fasting glucose levels in patients with established atherothrombotic disease. The trial was designed by the Steering Committee and registered at Clinicaltrials.gov (NCT01453166). The study protocol was approved by the ethics committee of our institution. All patients provided informed consent prior to participation.

Study population We included outpatients who were over 45 years of age with established or previous atherothrombotic CVD occurring in the past 10 years and who were at high CVD risk. Atherothrombotic CVD was defined as follows: coronary heart disease (stable or unstable angina pectoris, a history of myocardial infarction or acute coronary syndrome, a prior coronary revascularization procedure, a history of abdominal segmental wall motion as shown by cardiac echocardiography or a fixed segmental defect in the radionuclide imaging test), ischemic cerebrovascular disease (ischemic stroke or transient ischemic attack), or peripheral vascular disease (prior peripheral revascularization procedure or amputation due to vascular disease). The patients also had at least one of the following risk factors: diabetes mellitus (serum glucose $100 mg/dl), hypertension (systolic blood pressure $130 mm Hg or diastolic blood pressure $85 mm Hg), smoking, dyslipidemia (low density lipoprotein (LDL) $160 mg/dL, triglycerides $150 mg/dL, or total cholesterol (CT) $200 mg/dL), family coronary artery disease history, asymptomatic carotid disease or BMI .25 kg/m2 for adults and .28 kg/ m2 for seniors. We excluded patients with neurocognitive or psychiatric conditions (as defined by the clinical trial investigators), pregnant or lactating women, patients with hepatic impairment or renal insufficiency, and patients with a life expectancy of less than six months (e.g., those with metastatic malignancies).

Randomization and Allocation Concealment After providing written informed consent, the patients were randomized in a 1:1:1 ratio to receive one of three dietary interventions (A, B, or C). The allocation concealment was guaranteed by using sealed and opaque envelopes that were numbered sequentially. The patients who were allocated to Group A joined the Brazilian Cardioprotective Diet Program, which involves a Brazilian version

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A Brazilian Cardioprotective Diet Trial Weber B et al.

Figure 1 - Flow diagram of the participants in the study.

the Brazilian food database, was used to calculate the Brazilian Cardioprotective Diet Program and dietary assessments. Data quality control was guaranteed by weekly meetings between the investigators and dieticians.

and nutrition goals were reviewed. The phone interviews lasted approximately 15 minutes and included just the time that was necessary to assess the 24-hour dietary recall.

Outcomes Study procedures

The primary outcome of this pilot trial was the changes in blood pressures that occurred after 12 weeks of adherence to the Cardioprotective Diet Program. The secondary outcomes of this pilot trial were improved BMIs and fasting glucose levels. Considering that this was a pilot trial, we chose one well-established biochemical, hemodynamic, and anthropometric parameter as the endpoint.

Anthropometric measurements and laboratory tests were performed at baseline and after 6 and 12 weeks. At each contact with the dietitian (weekly or monthly), the dietary intake profile was assessed by a 24-hour dietary recall. The first nutritional session lasted for 60 minutes. The follow-up counseling sessions lasted for 30 minutes once the teaching

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Table 1 - Baseline characteristics of participants. Variables

Group A

B

(n = 42) Male sex, No. (%) Age, mean¡SD, y Weight, mean¡SD, kg High, mean¡SD, cm BMI, mean¡SD, kg/m2 CAD, No. (%) Ischemic stroke/transient ischemic attack, No. (%) PAD, No. (%) Hypertension, No. (%) Diabetes mellitus, No. (%) Smoking, No. (%) Dyslipidemia, No. (%) CAD family history, No. (%) Asymptomatic carotid disease, No. (%) Overweight, No. (%) Hypoglycemic drugs, No. (%) Antihypertensive drugs, No. (%) Hypolipidemic drugs, No. (%)

31 62 80.6 167 28.9 41 1 3 35 17 6 37 32 2 27 16 39 35

(73.8%) (7) (14.6) (9) (3.9) (100.0%) (2.4%) (7.3%) (85.4%) (41.5%) (14.6%) (90.2%) (78.0%) (4.9%) (64.3%) (39.0%) (95.1%) (85.4%)

(n = 41) 26 65 88.4 169 30.9 40 2 1 30 15 5 34 28 5 30 15 37 35

(63.4%) (8) (16.0) (10) (4.5) (97.6%) (4.9%) (2.4%) (73.2%) (36.6%) (12.2%) (82.9%) (68.3%) (12.2%) (75.0%) (36.6%) (92.5%) (87.5%)

C (n = 39) 24 62 84.3 168 30.0 37 1 1 32 16 5 33 27 6 30 16 37 36

(61.5%) (11) (15.9) (10) (4.7) (97.4%) (2.6%) (2.6%) (84.2%) (42.1%) (13.2%) (86.8%) (71.1%) (15.8%) (78.9%) (42.1%) (97.4%) (94.7%)

CAD: coronary artery disease; PAD: peripheral arterial disease.

difference of 7 mm Hg in the systolic and diastolic blood pressures with 80% statistical power and a two-tailed p-value of 5%, we sought to include 50 patients in each of the three groups.

Biochemical analysis The blood samples were collected and handled according to hospital routines. Fasting glucose was determined by an enzymatic colorimetric dry chemistry method (OrthoClinical Diagnostics VITROS 5.1) at the Center for Laboratory Medicine at the Hospital do Corac¸a˜o.

Statistical analyses All analyses followed the ‘‘intention to treat’’ principle. To evaluate the effects of the diet interventions, we used an analysis of variance (ANOVA). In cases of significant group effects, we used multiple comparisons with Bonferroni corrections to locate the differences. All tests were performed with the SPSS statistical software or STATA SE 11 for Windows (College Station, USA). Statistical significance was defined as p,0.05.

Blood pressures and weights Blood pressures were assessed by an auscultatory method following the recommendations of the American Heart Association (14). The patients were seated comfortably with their backs supported, and their upper arms were free of constrictive clothing. Arms were supported at heart level, and the mercury columns were deflated at 2–3 mm/sec. The first and last audible sounds were recorded as the systolic and diastolic pressures, respectively. Two readings were performed at intervals of at least 1 minute, and the average was taken as the patient’s blood pressure. If there was a difference of greater than 5 mm Hg between the first and second readings, additional (one or two) readings were obtained, and the average of these multiple readings was used (17). To assess the weights, we used a digital scale with a maximum capacity of 150 kg. The patients dressed in as little clothing as possible and removed objects that could interfere with measurements. Heights were measured using a stadiometer with a bilateral scale from 35 to 213 cm and a resolution of 0.1 cm. BMIs were calculated by dividing the patients’ current weights by the square of their heights in meters and classified according to WHO recommendations (15).

Quality Control Due to the type of intervention in this pilot trial, it was not possible to blind the investigators and participants. To improve quality control, the laboratory technicians who performed the biochemical tests were not aware of which group the patients had been allocated to. Data quality control was guaranteed by weekly contacts with investigators, and general feedback was provided at investigators’ meetings. Moreover, two independent investigators evaluated the database, and anthropometric measurements were performed twice to ensure the reliability of the data.

RESULTS Baseline Characteristics This trial enrolled 117 patients from September to December 2011 in a cardiology hospital (Figure 1). The baseline characteristics, such as the medication intakes, were similar between the groups (Table 1). More than 70% of the patients presented with histories of hypertension and/or dyslipidemia in addition to reporting the frequent use of antihypertensive and hypolipidemic drugs. The

Sample size Considering this pilot trial, the sample size was set to compare the groups in relation to changes in the systolic and diastolic blood pressures following dietary intervention. We anticipated a standard deviation of 14 mm Hg in the systolic or diastolic blood pressures. To detect a

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*

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1,370.08¡327.67 65.29¡16.33 19.0 171.70¡41.32 50.1 48.99¡14.35 32.1 14.78¡5.10 9.7 13.90¡4.13 9.1 14.94¡5.22 9.8 178.00¡63.26 18.80¡5.95 2432.82¡674.82

1,574.12¡523.43 71.40¡31.82 18.1 191.99¡63.01 48.7

56.46¡22.14 32.2 18.05¡8.29 10.3

16.68¡7.89 9.5

16.18¡6.09 9.2

224.08¡159.56 20.53¡9.52 2803.44¡1048.14

3 Months

Differences between groups B and C using one-way ANOVA (p,0.05).

Total Caloric Intake Protein (g/d) Protein (% total calorie intake) Carbohydrate (g/d) Carbohydrate (% total calorie intake) Total fat (g/d) Total fat (% total calorie intake) Saturated Fat (g/d) Saturated Fat (% total calories intake) Monounsaturated Fatty Acids (g/d) Monounsaturated Fatty Acids (% total calories intake) Polyunsaturated Fat (g/d) Polyunsaturated Fat (% total calories intake) Cholesterol (mg/d) Dietary fiber (g/d) Sodium (mg/d)

Baseline

Group A

230.02¡91.75 17.06¡7.35 2641.3¡910.49

14.64¡5.87 8.2

9.3

16.74¡7.22

58.88¡23.81 32.9 20.18¡9.45 11.3

3 Months

232.99¡72.12 16.46¡4.49 2473.58¡571.87

14.09¡4.61 8.9

10.0

15.89¡4.82

53.88¡15.20 34.1 17.48¡4.92 11.0

1,420.59¡273.11 77.33¡17.18 21.7 157.55¡32.61 44.4

Group B

1,607.05¡481.33 81.13¡25.78 20.2 191.12¡67.51 47.5

Baseline

Table 2 - Individual daily caloric intake at baseline and three months.

253.04¡148.67 23.43¡13.72 3384.78¡1860.60

18.33¡9.67 8.8

9.2

19.27¡7.77

72.82¡38.18 35.0 23.41¡13.88 11.3

1,867.99¡767.94 86.26¡37.13 18.4 218.60¡108.82 46.8

Baseline

3 Months

188.26¡89.68 19.37¡8.84 2666.05¡858.61

15.47¡6.68 9.8

9.7

15.03¡5.93

53.20¡17.53 33.8 15.33¡5.98 9.7

1,417.30¡386.99 68.60¡22.71 19.3 168.16¡57.90 47.4

Group C

0.62 0.03* 0.05*

0.1 -

-

0.24

0.32 0.92 -

0.69 0.11 0.25 -

p-value Baseline

0.06

0.11 ,0.01

,0.01 0.02 ,0.01

0.03

0.4 0.03 0.05

0.13

0.07 0.26 0.14

0.39

0.18

0.05

0.05 ,0.01

0.28

0.15

0.34 ,0.01

,0.01

0.05 0.13

Time x Group 0.17 0.05

Group ,0.01 ,0.01

Time

p-value between diets

CLINICS 2012;67(12):1407-1414 A Brazilian Cardioprotective Diet Trial Weber B et al.


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Figure 2 - Mean and standard deviation of outcomes in Groups A, B, and C.

baseline characteristics were well balanced between the groups, except in the case of asymptomatic carotid disease. Participation in all visits, phone calls and contact with the dietitians was evaluated in Groups A and B due to the similarities in the number of contacts (weekly sessions, which in Group C were monthly sessions). The attendance of patients in Group A at the meetings with the dieticians was higher than that in Group B (88.1% versus 78%, respectively). The analyses of the dietary recalls that were collected during the intervention period show that the caloric intakes decreased between the initial and final assessments in a similar manner in all three groups. The consumption of total cholesterol, saturated fatty acids, and sodium also decreased (Table 2). The ingestion of all nutrients showed lower variabilities following the intervention period, indicating greater homogeneity in consumption.

DISCUSSION This is the first study to investigate the clinical effects of a diet that is based on widely available foods in the Brazilian culture on cardiovascular risk factors. The main finding of this randomized pilot trial was that Group A, who received the Brazilian Cardioprotective Diet Program, showed greater decreases in blood pressures, serum glucose levels, and BMIs compared with Groups B and C, who received the dietary therapy that has been proposed by the Brazilian guidelines for CVD. Among the various dietary interventions, there is a consensus that the Mediterranean diet is effective and possibly more appropriate for the prevention and treatment of CVD (2,7). This diet is characterized by the low intake of saturated fat, high consumption of vegetables, fish, olive oil, and moderate consumption of wine (8). Despite the remarkable effects of the Mediterranean diet, there is some controversy regarding its administration in countries outside of the Mediterranean region. The Lyon Diet Heart Study showed no differences between patients that followed the Mediterranean diet and those that followed common eating habits in the secondary prevention of CAD. This study investigated the total cholesterol levels (650 vs. 617 mmol/l; p.0.05), LDLs (452 vs. 418 mmol/l; p.0.05), HDLs (116 vs. 128 mmol/l; p.0.05), triglycerides (215 vs. 185 mmol/l; p.0.05), weights (74.2 vs. 75.6 kg; p.0.05), and systolic (119 vs. 126 mm Hg; p.0.05) and diastolic (74 vs. 78 mm Hg; p.0.05) blood pressures of patients during a period of three years of follow-up (16). Our pilot study, which was conducted

Effects on risk factors Considering our primary outcome, there was a greater reduction in the systolic (7.8%) and diastolic (10.8%) blood pressures in Group A compared with Groups B (2.3% and 7.3%) and C (3.9% and 4.9%, respectively). Considering our secondary outcome, the fasting glucose levels decreased by 5.3% and 2% in Groups A and B, respectively. In Group C, they increased by 3.7%. The BMIs decreased by 3.5% and 3.3% in Groups A and B, respectively. The BMIs in Group C did not change (Figure 2). Nevertheless, none of these data showed statistical differences between the groups, which is methodologically acceptable in pilot trials (Table 3).

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0.25

,0.01

0.55

0.1

0.49

0.45

0.07

,0.01 0.08

0.08

0.04

,0.01 0.69

0.07

0.33

,0.01

124 (119;129) 78 (75;82) 84 (79;90) 30 (28;31) 104 (97;111)

126 (119;133) 76 (72;79) 86 (81;91) 30 (29;31) 102 (95;109)

128 (123;132) 81 (78;84) 84 (79;90) 30 (28;32) 108 (98;118)

126 (119;132) 76 (73;79) 84 (78;89) 30 (28;31) 113 (101;125)

123 (116;130) 77 (73;80) 84 (79;89) 30 (28;31) 112 (103;120)

0.72

0.21

during a brief 12-week period, showed a trend of decreasing blood pressures, BMIs, and fasting plasma glucose levels; these factors were not evaluated in the Lyon study. Moreover, the only Brazilian study that evaluated the influence of the Mediterranean diet in secondary prevention showed no differences in BMIs, systolic or diastolic blood pressures, or fasting glucose levels (10). However, studies that have adapted the Mediterranean diet to the tastes and cultural habits of the local population observed greater effectiveness in reducing cardiovascular risk factors. An Indian study showed that after one year of dietary intervention, blood glucose levels decreased by 1.36 mmol/l, systolic blood pressures decreased by 13.4 mm Hg, and diastolic blood pressures decreased by 9.3 mm Hg (17). A Swedish study (NORDIET) that also adapted the Mediterranean dietary habits for patients in the primary prevention program observed reductions in systolic blood pressures by 9.5 mm Hg and weights by 14.4 kg (18). In our pilot study, the Brazilian Cardioprotective Diet Program seemed to be more efficient than the diet intervention that was proposed by the Brazilian guidelines. We believe that a cardioprotective diet including foods that are widely available in Brazil played a key role in our results. Our findings are of potentially great importance to public health in our country, considering the promising cost/benefit relationship (19,20,21). The financial costs of the foods were not assessed in this pilot study; however, the diet that was proposed by the Brazilian guidelines to control cardiovascular risk factors, which involves components of the Mediterranean diet, is costly for a major proportion of the Brazilian population. Thus, we propose a new intervention with potentially low costs and high feasibility in Brazil. The efficacy of the Brazilian Cardioprotective Diet Program is substantiated by the fact that the diet that has been proposed by the Brazilian guidelines is not widely available nor is it in accordance with the Brazilian culture. Despite our encouraging results, we cannot generalize and recommend the implementation of a cardioprotective diet following the same format as in this pilot study in all Brazilian regions. Our country has continental dimensions and presents with multicultural characteristics that should be considered when interpreting our data and prescribing diets. For the improvement of cardioprotective diets and to contemplate the cultural diversities of the various regions of our country, a multicenter trial is needed. However, the foundation for the Brazilian Cardioprotective Diet has been established.

117 (111;124) 74 (70;78) 79 (74;84) 28 (27;30) 107 (97;116) 120 (114;126) 75 (71;79) 80 (75;85) 29 (27;30) 103 (96;111)

Study limitations This study had some limitations; we did not assess the patients’ dietary adherences, we only assessed their adherences to the intervention programs. Nevertheless, we observed that the numbers of absences were higher in Group B than in Group A, which was the group following the Brazilian Cardioprotective Diet Program. Our randomization process did not include a web-based automated randomization system, which could represent a methodological disadvantage. Moreover, this study did not find statistically significant differences between groups, which is acceptable in pilot trials. We believe that the statistical significance will be evident when the Brazilian Cardioprotective Diet Program is assessed in a larger population over all regions of Brazil.

Systolic blood pressure (mm Hg) Diastolic blood pressure (mm Hg) Weight (kg) BMI (kg/m2) Fasting glycemia (mg/dl)

127 (123;130) 83 (80;85) 81 (76;85) 29 (28;30) 113 (99;126)

(n = 41) (n = 40) (n = 43)

129 (124;134) 82 (78;85) 88 (83;93) 31 (30;32) 104 (97;11)

(n = 38) (n = 37) (n = 39) (n = 38) (n = 34)

12 weeks Moment

6 weeks

(n = 40)

0.98

0.33

Time x Group Group 6 weeks Baseline 12 weeks 6 weeks

A Brazilian Cardioprotective Diet Trial Weber B et al.

Baseline

Baseline

B A

Table 3 - Laboratory characteristics at baseline and following three-month intervention period.

C

12 weeks

p-value Baseline

Time

p-value between diets

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The Brazilian Cardioprotective Diet Program seems to be more effective in reducing blood pressures, fasting glucose levels, weights, and BMIs in patients with previous cardiovascular diseases compared with the diet that was proposed by the Brazilian guidelines. The Brazilian Cardioprotective Diet Program can be considered as an alternative for Brazilian patients with cardiovascular risk factors; however, it will be necessary to test this intervention on a larger scale.

8.

9.

10.

ACKNOWLEDGMENTS The Brazilian Cardioprotective Diet Trial is funded by the Brazilian Ministry of Health (Programa Hospitais de Excelencia a Servic¸o do SUS). The sponsor had no role in the analyses, study design or decision to publish these results.

11.

AUTHOR CONTRIBUTIONS

13.

12.

Weber B, Galante AP and Berwanger O conceived, designed and coordinated the study, and helped to draft the manuscript. Carvalho VO, Bersch-Ferreira AC, Buehler AM, Torreglosa CR, Espı´rito-Santo JA, RossFernandes M, Soares RM, Lara ES and Costa RP participated in the study design and helped to draft the manuscript. Victor ES performed the statistical analysis. All the authors read and approved the final version of the manuscript.

14.

15.

REFERENCE

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1. World Health Organization. Mortality estimates by cause, age, and sex for the year 2008. 2011. Geneva: WHO. 2. Hooper L, Griffiths E, Abrahams B, Alexander W, Atkins S, Atkinson G, et al. Dietetic guidelines: diet in secondary prevention of cardiovascular disease (first update, June 2003). J Hum Nutr Diet. 2004;17(4):337-49, http://dx.doi.org/10.1111/j.1365-277X.2004.00533.x. 3. Appel LJ, Sacks FM, Carey VJ, Obarzanek E, Swain JF, Miller ER, III, et al. Effects of protein, monounsaturated fat, and carbohydrate intake on blood pressure and serum lipids: results of the Omni Heart randomized trial. JAMA. 2005;294(19):2455-64, http://dx.doi.org/10.1001/jama.294. 19.2455. 4. Parikh P, McDaniel MC, Ashen MD, Miller JI, Sorrentino M, Chan V, et al. Diets and cardiovascular disease: an evidence-based assessment. J Am Coll Cardiol. 2005;45:1379-87, http://dx.doi.org/10.1016/j.jacc. 2004.11.068. 5. Willett WC, Sacks F, Trichopoulou A, Drescher G, Ferro-Luzzi A, Helsing E, et al. Mediterranean diet pyramid: a cultural model for healthy eating. Am J Clin Nutr. 1995;61(6 Suppl):1402S-6S. 6. Bellisle F. Infrequently asked questions about the Mediterranean diet. Public Health Nutr. 2009;12(9A):1644-7, http://dx.doi.org/10.1017/ S1368980009990498. 7. Thomazella MC, Go´es MF, Andrade CR, Debbas V, Barbeiro DF, Correia RL, et al. Effects of high adherence to mediterranean or low-fat diets in

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medicated secondary prevention patients. Am J Cardiol. 2011;108 (11):1523-9, http://dx.doi.org/10.1016/j.amjcard.2011.07.008. Maranha˜o PA, Kraemer-Aguiar LG, de Oliveira CL, Kuschnir MC, Vieira YR, Souza MG, et al. Brazil nuts intake improves lipid profile, oxidative stress and microvascular function in obese adolescents: a randomized controlled trial. Nutr Metab (Lond). 2011;8(1):32, http://dx.doi.org/10. 1186/1743-7075-8-32. Guerra JF, Magalha˜es CL, Costa DC, Silva ME, Pedrosa ML. Dietary ac¸ai modulates ROS production by neutrophils and gene expression of liver antioxidant enzymes in rats. J Clin Biochem Nutr. 2011;49(3):188-94, http://dx.doi.org/10.3164/jcbn.11-02. Sposito AC, Caramelli B, Fonseca FAH, Bertolami MC. IV Diretriz Brasileira sobre Dislipidemias e Prevenc¸a˜o da Aterosclerose: Departamento de Aterosclerose da Sociedade Brasileira de Cardiologia. Arq. Bras. Cardiol. 2007;88:suppl1, http://dx.doi.org/10.1590/S0066-782X2007000700002. Sociedade Brasileira de Cardiologia; Sociedade Brasileira de Hipertensa˜ o; Sociedade Brasileira de Nefrologia. VI Diretrizes Brasileiras de Hipertensa˜o. Arq Bras Cardiol. 2010;95(supl.1): 1-51. Sociedade Brasileira de Cardiologia. I Diretriz Brasileira de Diagno´stico e Tratamento da Sı´ndrome Metabo´lica. Arq Bras Cardiol. 2005;84(supl. 1):1-28. Galante AP. Desenvolvimento e validac¸a˜o de um me´todo computadorizado para avaliac¸a˜o do consumo alimentar, preenchido por indivı´duos adultos utilizando a Web. Tese de doutorado. Universidade de Sa˜o Paulo;2007. Pickering TG, Hall JE, Appel LJ, Falkner BE, Graves JW, Hill MN, et al. Recommendations for blood pressure measurement in humans: an AHA scientific statement from the Council on High Blood Pressure Research Professional and Public Education Subcommittee. J Clin Hypertens (Greenwich). 2005;7(2):102-9, http://dx.doi.org/10.1111/j.1524-6175. 2005.04377.x. BMI classification - WHO http://apps.who.int/bmi/index.jsp?intro Page=intro_3.html. de Lorgeril M, Salen P, Martin JL, Monjaud I, Delaye J, Mamelle N. Mediterranean diet, traditional risk factors, and the rate of cardiovascular complications after myocardial infarction: final report of the Lyon Diet Heart Study. Circulation. 1999;99:779-85, http://dx.doi.org/10. 1161/01.CIR.99.6.779. Singh RB, Rastogi SS, Verma R, Laxmi B, Singh R, Ghosh S, et al. Randomised controlled trial of cardioprotective diet in patients with recent acute myocardial infarction: results of one year follow up. BMJ. 1992;304(6833):1015-9, http://dx.doi.org/10.1136/bmj.304.6833.1015. Adamsson V, Reumark A, Fredriksson IB, Hammarstro¨m E, Vessby B, Johansson G, et al. Effects of a healthy Nordic diet on cardiovascular risk factors in hypercholesterolaemic subjects: a randomized controlled trial (NORDIET). J Intern Med. 2011;269:150-9, http://dx.doi.org/10.1111/j. 1365-2796.2010.02290.x. Solimene MC. Coronary heart disease in women: a challenge for the 21st century. Clinics. 2010;65:99-106, http://dx.doi.org/10.1590/S180759322010000100015. Torres MR, Ferreira Tda S, Nogueira Lde P, do Nascimento DC, Sanjuliani AF. Dietary counseling on long-term weight loss in overweight hypertensive patients. Clinics. 2011;66(10):1779-85. Azevedo CH, Wajngarten M, Prete AC, Diament J, Maranha˜o RC. Simultaneous transfer of cholesterol, triglycerides, and phospholipids to high-density lipoprotein in aging subjects with or without coronary artery disease. Clinics. 2011;66(9):1543-8.


CLINICS 2012;67(12):1415-1418

DOI:10.6061/clinics/2012(12)11

CLINICAL SCIENCE

Standard surgical treatment for benign prostatic hyperplasia is safe for patients over 75 years: Analysis of 100 cases from a high-volume urologic center Rafael Marmiroli, Alberto A. Antunes, Sabrina T. Reis, Elcio Nakano, Miguel Srougi Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, General Surgery, Division of Urology, Sa˜o Paulo, SP/Brazil.

OBJECTIVES: In this study, we aimed to determine the complications of standard surgical treatments among patients over 75 years in a high-volume urologic center. METHODS: We analyzed 100 consecutive patients older than 75 years who had undergone transurethral prostatic resection of the prostate or open prostatectomy for treatment of benign prostatic hyperplasia from January 2008 to March 2010. We analyzed patient age, prostate volume, prostate-specific antigen level, international prostatic symptom score, quality of life score, urinary retention, co-morbidities, surgical technique and satisfaction with treatment. RESULTS: Median age was 79 years. Forty-eight patients had undergone transurethral prostatic resection of the prostate, and 52 had undergone open prostatectomy. The median International Prostatic Symptom Score was 20, the median prostate volume was 83 g, 51% were using an indwelling bladder catheter, and the median prostatespecific antigen level was 5.0 ng/ml. The most common comorbidities were hypertension, diabetes and coronary disease. After a median follow-up period of 17 months, most patients were satisfied. Complications were present in 20% of cases. The most common urological complication was urethral stenosis, followed by bladder neck sclerosis, urinary fistula, late macroscopic hematuria and persistent urinary incontinence. The most common clinical complication was myocardial infarction, followed by acute renal failure requiring dialysis. Incidental carcinoma of the prostate was present in 6% of cases. One case had urothelial bladder cancer. CONCLUSIONS: Standard surgical treatments for benign prostatic hyperplasia are safe and satisfactory among the elderly. Complications are infrequent, and urethral stenosis is the most common. No clinical variable is associated with the occurrence of complications. KEYWORDS: Prostatic Hyperplasia; Prostatectomy; Transurethral Resection of Prostate; Aged; Quality of Life, Complications. Marmiroli R, Antunes AA, Reis ST, Nakano E, Srougi M. Standard surgical treatment for benign prostatic hyperplasia is safe for patients over 75 years: Analysis of 100 cases from a high-volume urologic center. Clinics. 2012;67(12):1415-1418. Received for publication on June 12, 2012; First review completed on July 26, 2012; Accepted for publication on August 20, 2012 E-mail: antunesuro@uol.com.br Tel.: 55 11 3255-6372

Assuming that by the year 2030, 20% of the population of the United States will be older than 65 years (2), the rise in the incidence of lower urinary tract symptoms (LUTS) due to BPH makes it a public health question. LUTS involve an estimated annual impact of $1.1 billion of direct costs (excluding outpatient pharmaceuticals) and indirect costs of approximately 38 million hours of lost productivity by these patients (3). In recent decades, several new methods for treating BPH have been developed, both pharmaceutical and surgical. Several minimally invasive procedures are still favored by the international community due to their lower complication rates. However, it is necessary to remember that the perceived efficacy and long-term durability of these therapies remain to be proven; so far, the gold-standard treatments are still open prostatectomy (OP) and transurethral resection of the prostate (TURP) (4).

INTRODUCTION Benign prostatic hyperplasia (BPH) represents an increase in the total number of stromal and epithelial cells within the prostate gland. It is associated with bothersome lower urinary tract symptoms that affect the individual’s quality of life and interfere with day-to-day activities. BPH is now one of the most common diseases in the elderly. According to histological studies, more than 50% of men will face this diagnosis by the age of 60 (90% by the age of 85) (1).

Copyright ß 2012 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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Because BPH is associated with old age, OP and TURP are usually performed in patients with various comorbidities. Some studies have confirmed a direct effect of comorbidity on perioperative morbidity and mortality after these procedures (5,6). However, OP and TURP patient populations, especially in multicenter studies and meta-analyses with large numbers of patients, are generally approximately 65 to 75 years old (7,8). A better understanding of the clinical characteristics of patients over 75 years old is important for preventing complications and improving clinical outcomes. In this study, we aimed to determine the complications (both clinical and urological) of standard surgical treatments among patients over 75 years in a high-volume urologic center.

Incidental carcinoma of the prostate was present in 6% of cases (5% T1a and 1% T1b). One case had incidental urothelial bladder cancer. Even though patients who presented complications had larger prostates, higher PSA levels and higher rates of hypertension, diabetes and coronary disease, no variable was statistically associated with the occurrence of complications (Table 1).

DISCUSSION This study demonstrates that traditional surgical treatments for BPH can be performed in patients older than 75 years with acceptable complication rates and good functional results. No variable was statistically associated with the occurrence of complications; therefore, no comorbidity taken alone can be considered an absolute contra-indication for these procedures. With the increase in the number of aging males in most populations, an improved understanding of the clinical characteristics of this selected population is important for improving surgical outcomes. On average, patients with BPH who are candidates for surgical treatment are older than before. Choi et al. (18) compared the characteristics of patients who underwent surgery in 1985 to 1989, in 1995 to 1999 and in 2005 to 2009. The mean ages of the men were 65.4, 65.9 and 69.3 years, respectively. Likewise, the prevalence of hypertension, a history of surgery, and "other complications" (e.g., stroke, cancer and cardiac conditions) increased significantly over time. The prevalence of hypertension increased from 22% in the first period to 43% in the last, and the prevalence of diabetes increased from 8% to 13%. Due to the relative morbidity of TURP and OP, minimally surgical alternatives have been proposed for the treatment of BPH. Among these alternatives, transurethral needle ablation of the prostate with radiofrequency and transurethral microwave thermotherapy (TUMT) have been frequently performed in the United States. Their main advantage is the fact that they are performed under local anesthesia in an outpatient fashion. However, systematic reviews of the literature have concluded that they do not achieve the same level of efficacy as the classic methods with respect to any subjective or objective variable. Further, their efficacy declines in the long term, with a significantly higher rate of secondary treatment than encountered with classic methods [20-22].

MATERIALS AND METHODS We analyzed a selected group of 100 consecutive patients older than 75 years who had undergone TURP or OP for BPH from January 2008 to March 2010. Exclusion criteria in this study were previous surgical treatment for BPH, diagnosis of prostate cancer and suspected neurogenic bladder. Preoperatively, all patients were subjected to anamnesis focused on urinary symptoms, according to the International Prostate Symptom Score (IPSS) and QOL (Quality Of Life) gradation. We also analyzed the available information from digital rectal examinations, prostate ultrasounds, prostate-specific antigen (PSA) levels, use of an indwelling bladder catheter due to urinary retention, presence of co-morbidities (arterial hypertension, diabetes and coronary diseases) and surgical technique. To analyze treatment outcome, patients were asked if they were satisfied with the treatment results. Urologic and non-urologic complications were recorded. It is important to note that all patients preoperatively attended an evaluation with specialists; patients who were considered to be at high risk of complications (cardiological or other) after an analysis of their global health status and comorbidities were not treated with surgery. Statistical analysis was performed using SPSSH 19.0 software for WindowsH (IBM Incorporated, Armonk, New York), and significance was defined as p,0.05. All data are presented as median (range) unless specified otherwise. We determined the risk factors for complications using the chisquare and Student’s-t tests.

RESULTS The median patient age was 79 years (75 to 91). Fortyeight patients were subjected to TURP and 52 to OP. Hypertension, diabetes and coronary disease were present in 69%, 24% and 26% of the cases, respectively. Median preoperative IPSS was 20 (8 to 31), and 51% of patients were using an indwelling bladder catheter. Median prostate volume was 83 g (24 to 417), and median PSA level was 5.0 ng/ml (0.2 to 60). After a median follow-up period of 17 months (1 to 40), 83% of patients were satisfied with the treatment. Overall, complications were present in 20% of cases. Regarding urological complications, 10% presented urethral stenosis, 2% had bladder neck sclerosis, 2% had urinary fistula, 2% had late macroscopic hematuria and 2% had persistent urinary incontinence. Among clinical complications, 1% presented acute renal failure requiring dialysis, and 2% presented myocardial infarction. One patient died due to infarction.

Table 1 - Patient characteristics according to the incidence of complications.

Age (years) IPSS QoL score Prostate volume (g) PSA (ng/ml) Urinary retention Hypertension Diabetes Coronary disease Surgery TURP OP

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Complications (n = 20)

No complications (n = 80)

79.3 15.8 4.1 124.8 8.8 55% 80% 30% 40%

79.7 19.6 4.1 92.2 7.8 52% 66% 22% 22%

17% 23%

83% 77%

p-value 0.72 0.14 0.86 0.20 0.66 0.80 0.23 0.48 0.11 0.42


CLINICS 2012;67(12):1415-1418

Benign prostatic hyperplasia and elderly Marmiroli R et al.

The relatively small number of patients included and the lack of a control group of patients less than 65 years old are known limitations of this study. It is also important to note that the study only included men who were considered to be adequate candidates for surgery after clinical evaluation. This fact may constitute a possible selection bias, as the complication rates were likely reduced because we did not perform surgical therapy in patients with more adverse clinical conditions. According to the data presented in this study, complications are infrequent, and standard surgical treatments for the treatment of BPH are safe for elderly patients. No clinical variable was associated with the occurrence of complications. In our study, most patients were satisfied after surgery.

Laser therapies are also surgical alternatives that might be considered for high-risk surgical patients. Among these, the photoselective vaporization of the prostate with the GreenLight laser and holmium laser enucleation of the prostate are the two most performed methods. Both techniques are associated with shorter catheter time and hospital stay. Studies with a longer follow-up are necessary to establish the role of laser techniques in the surgical treatment of BPH. Regarding patients’ expectations about prostate surgery, Zwergel et al. (12), in a study with a follow-up of more than 15 years after TURP reported satisfaction rates of 79% satisfied, 12% neutral, and 9% dissatisfied with their micturition, which is similar to our data (83% satisfied). Reich et al. (11), in a multicentric prospective evaluation that involved 10,654 patients, reported a 27.5% rate of preoperative catheterization. In our study, 51% of patients were using an indwelling bladder catheter. This high rate can be justified by our patients’ larger prostates and previous acute urinary retention episodes. We think that the issue of preoperative catheterization may be related not only to public health and educational issues but also to cultural barriers that lead to a delayed search for medical help and, after receiving medical help, a delay in undergoing a proper urological evaluation. The rates of urological complications in this study were 10% urethral stenosis, 2% bladder neck sclerosis, 2% urinary fistula, 2% late macroscopic hematuria and 2% persistent urinary incontinence. Varkarakis et al. (13), in a Greek study that analyzed complications after OP in 232 patients, identified long-term complications of bladder neck contraction in 3.3%, urethral strictures in 0.6% and meatal stenosis in 1.3% of the patients. In the study by Ahyai et al, consistent with our study, the most common urological late complication was urethral stenosis, which they observed in 4.1% of patients. Our co-morbidity rates are slightly higher than others. In comparing our rates to the recent study of 1,878 patients by Hammarsten and Hogstedt (8), we noted higher proportions of patients with hypertension [69% vs. 46.1%], diabetes (24% vs. 18.2%) and cardiac disorders (26% vs. 17%). Additionally, with a median age of 79 years, our patients were ten years older. Due in part to their younger and healthier patients, Hammarsten and Hogstedt reported a 5.8% complication rate, which is quite lower than we found (20%). Ahyai et al. (7), in a meta-analysis of 27 studies (including 23 randomized controlled studies) with a total of 2,247 patients, reported an overall complication rate of 32.4%. In the consulted literature, postoperative death due to coronary disease ranges from 0.05 to 1%. These rates are consistent with our data. No patient in our series developed postoperative urinary incontinence. This finding is important because our group previously demonstrated that patients older than 70 years have twice the probability of post-procedural incontinence. Additionally, the chances of bladder dysfunction, which may be the isolated cause of incontinence in approximately 25% of patients, rises 5.3% for each year added to patient age (20). Incidental carcinoma of the prostate was found in 6% of cases. (5% T1a and 1% T1b). Previous studies have reported finding cancer in 7% to 14% of cases following nononcological surgery (16-18).

AUTHOR CONTRIBUTIONS Marmiroli R, Reis ST and Nakano E contributed to the medical records, literature review and manuscript writing. Antunes AA contributed to the medical records, literature review, manuscript writing and statistical analysis. Srougi M contributed to the literature review and manuscript writing.

REFERENCES 1. McConnell JD, Barry MJ, Bruskewitz RC. Clinical Practice Guidelines, Number 8: Agency for Health Care Policy and Research. Rockville, MD: US Department of Health and Human Services; 1994. Benign prostatic hyperplasia: diagnosis and treatment. AHCPR publication no. 940582. Gavrilov LA, Heuveline P: Aging of population. In: Demeny P, McNicoll G, editors. The Encyclopedia of Population. New York: Macmillan;2003. 2. Wei JT, Calhoun E, Jacobsen SJ. Urologic Diseases in America Project: benign prostatic hyperplasia. J Urol. 2005;173:1256-61, http://dx.doi. org/10.1097/01.ju.0000155709.37840.fe. 3. Kacker R, Williams SB. Endourologic Procedures for Benign Prostatic Hyperplasia Review of Indications and Outcomes. Urol J. 2011;8:1716. 4. Concato J, Horwitz RI, Feinstein AR, Elmore JG, Schiff SF. Problems of comorbidity in mortality after prostatectomy. JAMA. 1992;267:1077-82, http://dx.doi.org/10.1001/jama.1992.03480080047025. 5. Holman CD, Wisniewski ZS, Semmens JB, Rouse IL, Bass AJ. Mortality and prostate cancer risk in 19,598 men after surgery for benign prostatic hyperplasia. BJU Int. 1999;84(1):37-42. 6. Ahyai SA, Gilling P, Kaplan SA, Kuntz RM, Madersbacher S, Montorsi F, et al. Meta-analysis of Functional Outcomes and Complications Following Transurethral Procedures for Lower Urinary Tract Symptoms Resulting from Benign Prostatic Enlargement. Eur Urol. 2010;58(3):384-97, http://dx.doi.org/10.1016/j.eururo.2010.06.005. 7. Hong JY, Yang SC, Ahn S, Kil HK. Preoperative Comorbidities and Relationship of Comorbidities With Postoperative Complications in Patients Undergoing Transurethral Prostate Resection. J Urol. 2011;185(4):1374-8, http://dx.doi.org/10.1016/j.juro.2010.11.086. 8. Hammarsten J, Hogstedt B. Hyperinsulinaemia as a risk factor for developing benign prostatic hyperplasia. Eur Urol. 2001;39(2):151-8, http://dx.doi.org/10.1159/000052430. 9. Michel MC, Heemann U, Schumacher H, Mehlburger L, Goepel M. Association of hypertension with symptoms of benign prostatic hyperplasia. J Urol. 2004;172(4 Pt 1):1390-3. 10. Reich O, Gratzke C, Bachmann A, Seitz M, Schlenker B, Hermanek P, et al. Morbidity, mortality and early outcome of transurethral resec-tion of the prostate: a prospective multicenter evaluation of 10,654 patients. J Urol. 2008;180(1):246-9. 11. Zwergel U, Wullich B, Lindenmeir U, Rohde V, Zwergel T. Long-term results following transurethral ressection of the prostate. Eur Urol. 1998;33(5):476–80, http://dx.doi.org/10.1159/000019638. 12. Varkarakis I, Kyriakakis Z, Delis A, Rotogerou V, Delivelioti C. Longterm results of open transvesical prostatectomy from a contemporary series of patients. Urology. 2004;64(2):306–10, http://dx.doi.org/10. 1016/j.urology.2004.03.033. 13. Simforoosh N, Abdi H, Kashi AH, Zare S, Tabibi A, Danesh A, et al. Open Prostatectomy Versus Transurethral -Resection of the Prostate, Where Are We Standing in the New Era? Urol J. 2010;7(4):262-9. 14. Jones JS, Follis HW, Johnson JR. Probability of finding T1a and T1b (incidental) prostate cancer during TURP has decreased in the PSA era.

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dysfunction. Int Braz J Urol. 2011;37(3):380-6, http://dx.doi.org/10. 1590/S1677-55382011000300012. 19. Bouza C, Lo´pez T, Magro A, Navalpotro L, Amate JM. Systematic review and meta-analysis of Transurethral Needle Ablation in symptomatic Benign Prostatic Hyperplasia. BMC Urol. 2006;6:14, http://dx.doi.org/ 10.1186/1471-2490-6-14. 20. Metcalfe C, Poon KS. Long-term Results of Surgical Techniqu es and Procedures in Men with Benign Prostatic Hyperplasia. Curr Urol Rep. 2011;12(4):265–73, http://dx.doi.org/10.1007/s11934-011-0193-1. 21. Kaye JD, Smith AD, Badlani GH, Lee BR, Seideman BA, Ost MC. HighEnergy Transurethral Thermotherapy with CoreTherm Approaches Transurethral Prostate Resection in Outcome Efficacy: A MetaAnalysis. J Endourol. 2008;22(4):713-8, http://dx.doi.org/10.1089/end. 2007.0039.

Prostate Cancer Prostatic Dis. 2009;12(1):57-60, http://dx.doi.org/10. 1038/pcan.2008.14. Martino P, Palazzo S, Battaglia M, Lucarelli G, Selvaggi FP. Incidental prostatic cancer: repeat TURP or biopsy? Urol Int. 2004;73(3):193-7, http://dx.doi.org/10.1159/000080826. Dellavedova T, Ponzano R, Racca L, Minuzzi F, Dominguez M. Prostate cancer as incidental finding in transurethral resection. Oncologic Urology Arch. Esp. Urol. 2010;63(10):855-61. Choi SY, Kim TH, Myung SC, Moon YT, Kim KD, Kim YS, et al. Impact of changing trends in medical therapy on surgery for benign prostatic hyperplasia over two decades. Korean J Urol. 2012;53(1):23-8, http://dx. doi.org/10.4111/kju.2012.53.1.23. Bruschini H, Simonetti R, Antunes AA, Srougi M. Urinary incontinence following surgery for BPH: the role of aging on the incidence of bladder

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DOI:10.6061/clinics/2012(12)12

CLINICAL SCIENCE

Plantar thermography is useful in the early diagnosis of diabetic neuropathy Luciane Fachin Balbinot, Luis Henrique Canani, Caroline Cabral Robinson, Matilde Achaval, Milton Antoˆnio Zaro Universidade Federal do Rio Grande do Sul, Instituto de Cieˆncias Ba´sicas da Sau´de, Laborato´rio de Histofisiologia Comparada, Porto Alegre/RS, Brazil.

OBJECTIVES: This study evaluated plantar thermography sensitivity and specificity in diagnosing diabetic polyneuropathy using cardiac tests (heart rate variability) as a reference standard because autonomic small fibers are affected first by this disease. METHODS: Seventy-nine individuals between the ages of 19 and 79 years old (28 males) were evaluated and divided into three groups: control (n = 37), pre-diabetics (n = 13) and type 2 diabetics (n = 29). The plantar images were recorded at baseline and then minutes after a provocative maneuver (Cold Stress Test) using an infrared camera that is appropriate for clinical use. Two thermographic variables were studied: the thermal recovery index and the interdigital anisothermal technique. Heart rate variability was measured in a seven-test battery that included three spectral indexes (in the frequency domain) and four Ewing tests (the Valsalva maneuver, the orthostatic test, a deep breathing test, and the orthostatic hypotension test). Other classically recommended tests were applied, including electromyography (EMG), Michigan inventory, and a clinical interview that included a neurological physical examination. RESULTS: Among the diabetic patients, the interdigital anisothermal technique alone performed better than the thermal recovery index alone, with a better sensitivity (81.3%) and specificity (46.2%). For the pre-diabetic patients, the three tests performed equally well. None of the control subjects displayed abnormal interdigital anisothermal readouts or thermal recovery indices, which precluded the sensitivity estimation in this sample of subjects. However, the specificity (70.6%) was higher in this group. CONCLUSION: In this study, plantar thermography, which predominately considers the small and autonomic fibers that are commonly associated with a sub-clinical condition, proved useful in diagnosing diabetic neuropathy early. The interdigital anisothermal test, when used alone, performed best. KEYWORDS: Thermography; Diabetic Neuropathy; Cardiac Autonomic Neuropathy; Small Fibers Neuropathy. Balbinot LF, Canani LH, Robinson CC, Achaval M, Zaro MA. Plantar thermography is useful in the early diagnosis of diabetic neuropathy. Clinics. 2012;67(12):1419-1425. Received for publication on June 30, 2012; First review completed on July 24, 2012; Accepted for publication on August 24, 2012 E-mail: luciane.balbinot@gmail.com Tel.: 55 51 33083624

validated inventories, are valued in specialized diabetes centers; nevertheless, the screening for subclinical cases has low sensitivity, as demonstrated by the absence of symptoms or signs of distal symmetrical polyneuropathy. Small fiber neuropathy may occur at any stage of diabetes, including during pre-diabetes. It is often painful, even in the absence of abnormalities, which are measured using conventional standard neurophysiological tests. The discrepancy between clinical presentation and test results could delay the correct diagnosis and appropriate treatment (8,10-12). Many diagnostic methods can assess small fibers; however, no single test can make this diagnosis. A battery of sensitive, reproducible and specific tests covering the somatic and autonomic systems is recommended (5,11,12). These tests include cardiovascular monitoring, sudomotor testing, pupillary responses, quantitative sensory tests, laser evoked potentials and thermography (12). Using such techniques has proven useful not only for diagnosis but also for guiding adequate therapy and

INTRODUCTION Peripheral neuropathy is a common complication in diabetes mellitus (DM) that leads to serious functional disability; however, its evaluation has not been standardized (1-7). Electromyography with nerve conduction assessment is the neurophysiological test that confirms the distal symmetric polyneuropathy that is typical of diabetes, and it is the standard protocol in most centers that specialize in neuropathy (6-9). However, this test does not evaluate small fibers, is minimally invasive, requires expertise, and is time consuming (5,6). The clinical aspects, including

Copyright ß 2012 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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relationship with cardiac and somatic autonomic nerve fibers in diabetes (18,23).

optimizing the follow-up. Infrared computerized thermography is a method of visualization, documentation and measurement of the infrared rays along the human body. According to Stefan-Boltzmann, this emission is proportional to the temperature of the skin and is directly related to blood flow in the cutaneous vessels (13,14,15). This method offers some advantages over others because it is completely non-contact, is painless and does not generate inputs beyond the equipment itself. In diabetes, vascular activity in the extremities, especially in the feet, may be indicative of autonomic neuropathy. Because vasomotor tone is regulated by fibers from the neurovegetative sympathetic system, its dysfunction could be associated with varying temperature patterns (16-19). Peripheral neurovegetative sympathetic nerve degeneration in advanced neuropathy damages the neurogenic control mechanisms that regulate capillary and arteriovenous (AV) shunt flow, which leads to an increase in the AV shunt in the feet of patients with diabetic neuropathy (20-23). These shunts are maintained in the constricted state by neurovegetative sympathetic tone. Losing this tone because of sympathetic neuropathy results in the shunt opening and the deviation of blood flow from the skin (20-23). Because of the great vulnerability that small fibers have to metabolic changes associated with hyperglycemia, injuries to these fibers may present earlier and with less marked changes than sensory-motor neuropathies (24,25). Although diabetic neuropathies are classified as diabetic microangiopathic complications, it is now known that their pathophysiological mechanism is multifactorial, and there is sufficient evidence that small-fiber polyneuropathy and even cardiac autonomic neuropathy may precede diabetes (i.e., during the pre-diabetic condition) (26-29). Cardiovascular reflex tests are standard for clinical autonomic evaluation because of their good sensitivity, specificity, and reproducibility; these tests are also noninvasive and well standardized. However, these tests require a well-trained evaluator and considerable evaluation time. Cardiovascular reflex tests can identify cardiac autonomic neuropathy even at the subclinical stage (29-36). This type of neuropathy can be subdivided into subclinical (predominantly functional and reversible) and clinical (structural neuronal changes are present). The subclinical subtype can only be diagnosed by tests and may occur as early as the time of the initial diabetes diagnosis. The clinical subtype is symptomatic and occurs during more advanced disease stages (35). The autonomic nerves are affected in various clinical diabetic neuropathy subtypes. In the most common type (typical polyneuropathy: symmetric, distal, and predominantly sensory), there is a strong correlation between the progressive damage of the somatic and autonomic fibers. Indeed, 50% of diabetic patients with polyneuropathy have asymptomatic cardiac autonomic neuropathy, while 100% of patients with symptomatic cardiac autonomic neuropathy have polyneuropathy (3,37). The prevalence of cardiac autonomic neuropathy progressively increases in direct proportion to age, diabetes duration, and poor glucose control. The aim of this study was to evaluate plantar thermography sensibility and specificity in diabetic patients with polyneuropathy at the diagnosis using cardiac tests (heart rate variability) as a reference standard because the autonomic small fibers are first affected, and there is a close

MATERIALS AND METHODS This study was performed from March 2010 to August 2011. The principles of the Declaration of Helsinki (38) were applied, and all of the patients provided informed written consent. The study was approved by the Ethics Committee of the Hospital de Clı´nicas de Porto Alegre (HCPA), RS, Brazil, decision No. 09-446, January 2010.

Subjects Seventy-nine individuals aged between 19 and 79 years (28 males) were evaluated and divided into three groups: controls (n = 37), pre-diabetics (n = 13), and type 2 diabetics (n = 29). The patients with pre-diabetes and diabetes were referred to the study from the Endocrinology and Prediabetes Out-clinic Unit of the Hospital de Clı´nicas de Porto Alegre (Porto Alegre, Brazil). Diabetes and pre-diabetes were defined according to the American Diabetes Association (ADA) criteria (9). The control group consisted of volunteers that did not fit the criteria for type 2 diabetes or pre-diabetes. Smokers and subjects with other conditions that could potentially cause neuropathies, such as hypothyroidism and alcoholism, or a condition in which the thermal plantar images could mimic neuropathy, such as lumbosacral radiculopathy, were excluded. Ischemic heart disease or stroke patients were also excluded because of the heart rate variability tests were used.

Data collection and instrumentation The data collection was conducted at a controlled laboratory temperature of 23¡0.5 ˚C and a relative humidity of 50¡5%. All of the tests occurred on the same day and lasted 1.5 hours per participant.

Clinical evaluation Clinical data, including age, gender, body mass index, and arterial systolic and diastolic pressure, were collected on the evaluation day. The clinical diagnosis of distal symmetric polyneuropathy was assessed using the Michigan inventory (39), a neuropathy score that consists of an inspection of foot deformities or ulcers and a brief neurological examination. We used the Achilles reflex test, a vibration sensitivity test on the hallux dorsum with a 128 Hz tuning-fork and a test for tactile sensibility using 10 g of nylon monofilament (SoryH, BauruSP-Brazil) on the plantar aspect of the hallux. The Michigan inventory was considered to be positive for neuropathy when four or more points (of a possible ten) were scored.

Heart rate variability Both the ADA and the American Academy of Neurology (AAN) (40,41) recommend the following protocol: three Ewing tests (the deep breathing test, Valsalva maneuver, and orthostatic test) at the time of a type 2 diabetes diagnosis and five years after a type 1 diabetes diagnosis and repeated annually thereafter. These three tests performed together have good reproducibility and specificity above 91%; the deep breathing and orthostatic tests have 93% sensitivity, and the Valsalva maneuver has 98% sensitivity. In addition, a spectral analysis was performed for heart rate variability, which is a result of sympathetic and parasympathetic balance at the sinus node. The method

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consists of seven parameters that are evaluated together, including a three-band spectral analysis of heart rate variability and four Ewing tests (the Valsalva maneuver, orthostatic test, deep breathing test and orthostatic hypotension test); this method has 98% sensitivity and 100% specificity (35). To evaluate the presence of cardiac autonomic neuropathy, heart rate variability tests were performed. These tests comprised three spectral indices (in the frequency domain) and four Ewing tests (35), including the Valsalva maneuver, orthostatic test, deep breathing test, and orthostatic hypotension test. The electrocardiogram was recorded (particularly the QRS complex) using electrocardiography equipment (NeurosoftH, Ivanovo, Russia) and software that was created for heart rate variability analysis (Poly-SpectrumH). A questionnaire concerning autonomic dysfunction symptoms was given following the protocol described by Rolim et al. (35). The presence of incipient cardiac autonomic neuropathy was defined as having two abnormal tests (98% specificity), and established neuropathy was diagnosed when three tests were abnormal (100% specificity) (35). In this study, we considered patients to be either positive or negative for cardiac autonomic neuropathy.

Plantar thermography The plantar infrared images were recorded using three thermal infrared cameras (the PV320T Electrophysics, IRI 4010 IRYSIS, and T400 Flir) with thermal sensitivities of 0.08 and 0.01 ˚C, a full-scale spectral range between 7-12 and 8-14 m and a maximum error of 2%. The protocol for infrared images followed the recommendations of the American Academy of Thermology (42). Notably, caffeinated drinks or other vasoactive substances were suspended for at least eight hours prior to testing, and all prescribed medications were suspended 12 hours prior to testing. The participants were acclimated for 15 minutes in the examination room by lying on a stretcher with bare legs and feet and no surface contact. The plantar infrared image was recorded at baseline followed by the provocative maneuvers using the cold stress test. This test consists of immersing the feet, which are protected with thin plastic, for 60 seconds in water at 15 ˚C. After 10 minutes, a new plantar infrared image was recorded to evaluate the thermal recovery index. To calculate the recovery index, the average temperatures of 10 regions of interest with similar dimensions were used: the hallux, 1st, 3rd, and 5th metatarsal heads and heel on both soles, as shown in Equation 1.  TRI~

Figure 1 - (A) Plantar thermographic image in a diabetic patient, showing Interdigital Anisothermal (the white arrow shows the different colors in toes meaning DT $ 0.4 ˚C). (B) Plantar thermographic image in a control subject, with regular thermal distribution, without Interdigital Anisothermal (color gradient of toes considered normal, DT , 0.4 ˚C).

  x100 TaB LhazL1mhzL3mhzL5mhzLhezRhazR1mhzR3mhzR5mhzRhe 10 Ta100 CST LhazL1mhzL3mhzL5mhzLhezRhazR1mhzR3mhzR5mhzRhe 10

In this study, which is based on specific literature, an interdigital anisothermal result or a thermal recovery index of ,90% or .100% after the cold stress test were considered (alone or in combination) to be positive for early neuropathy (small fibers and neurovegetative sympathetic fibers with vasomotor functions) (42-50).

Electromyography

TRI: thermal recovery Index; TaB: the average basal temperature of the 10 ROI ( ˚C); Ta10’CST: the average temperature 10 minutes after the cold stress test of the 10 ROI ( ˚C); L: left foot; R: right foot; ha: hallux; 1 mh: 1st metatarsal head; 3 mh: 3rd metatarsal head; 5 mh: 5th metatarsal head; he: heel.

The neuropathy screening protocol included the functional assessment of motor and sensory nerves of the four segments, as well as a myography using a needle electrode in suspected cases of axonal injury or root involvement (3,5-8). To record and analyze the data, a two channel electromyogram (NeurosoftH, Ivanovo, Russia) and dedicated software (NeuroMepH) were used. This method classifies the neuropathy into its various manifestations (i.e., mononeuropathies, multiple mononeuropathy and polyneuropathy). In this study, EMG was considered to be positive when it demonstrated distal symmetric polyneuropathy and was typical of

The interdigital anisothermal was assessed and was considered to be positive when thermal gradients (DT) $0.4 ˚C existed between any of the toes 10 minutes after the cold stress test (41-49). Figure 1 shows two examples of the interdigital anisothermal test.

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Table 1 - Patient characteristics. Variables*

DM (n = 29)

Pre-DM (n = 13)

b

Age (years) Gender (Female/Male) Time of diagnosis (years) Body mass index (kg/m2) Systolic BP (mmHg) Diastolic BP (mmHg)

b

56.8¡12.6 10/3 1 (1-2) 30.0¡2.8)b 130.5¡6.2b 83.5¡6.3ab

55.9¡9.4 20/9 5 (2-9) 27.1¡4.0ab 131.7¡8.0b 85.9¡7.9b

Control (n = 37)

p-value

a

45.1¡14.9 21/16 25.3(3.8)a 121.7(9.0)a 79.7(8.8)a

0.001 0.350 ,0.001 ,0.001 ,0.001 0.012

BP: Blood Pressure. * Mean¡standard deviation or median (25th percentile to 75th percentile). a,b Same letters do not differ by Tukey’s post hoc test at the 5% significance level.

diabetic neuropathy (5,7,51,52). The evaluator was blinded to the other test results.

had abnormal interdigital anisothermal results. The DM group presented with the highest number of abnormal results from the neuropathy diagnosis tests (heart rate variability, Michigan inventory, and EMG), as reported in Table 2. Although we observed a greater number of positive results for neuropathy diagnosis, only 6.9% (n = 2) of the patients from the DM group presented with a positive Michigan inventory test, thus identifying clinical neuropathy by the loss of protective foot sensation. Cardiac autonomic neuropathy (diagnosed using the heart rate variability method) was the reference used to estimate the sensitivity and specificity of the plantar infrared thermography. The interdigital anisothermal test and the thermal recovery index, alone or in combination, were used to identify the subjects with cardiac autonomic neuropathy in the three groups (Table 3). In the diabetic subjects, the interdigital anisothermal test alone performed better than the thermal recovery index, and both had better sensitivity (81.5%) and specificity (46.2%). For the prediabetic patients, all three tests performed equally well (sensitivity 80.0%, specificity 25.0%). None of the control subjects had abnormal results for the interdigital anisothermal test or the thermal recovery index, which would have precluded the sensitivity estimation in this sample of subjects. However, the specificity was higher in the control subjects (70.6%). Because the interdigital anisothermal test performed best, the clinical data regarding the results of this test are depicted in Table 4. In the pre-diabetic patients, the clinical data were significantly different between the subjects who presented or did not present with interdigital anisothermal results. The subjects in the pre-DM group who presented

Statistical analysis The categorical variables were described by absolute and relative frequencies, and the continuous variables were described by the mean and standard deviation (SD) or the median and interquartile range. The cutoff point for infrared thermography, which was represented by the thermal recovery index in this study using heart rate variability as a reference standard, was obtained by creating a receiver operating characteristic (ROC) curve. The area under the curve was used to estimate the accuracy of the method. The Kappa coefficient was assessed to analyze the agreement between the thermographic methods versus heart rate variability. The association between the categorical variables was assessed using the chi-squared test or Fishers exact test. The means were compared using Student’s t-test or a oneway analysis of variance (ANOVA) with Tukey’s post-hoc test. In asymmetric cases, a Mann-Whitney U-test was applied. A significance level of 5% was adopted, and all of the statistical analyses were performed using SPSS (Statistical Package for the Social Sciences) version 18.0.

RESULTS The analyses were separated by group because the groups were not homogeneous, with the exception of gender. The control subjects were younger (p = 0.001) and had a lower body mass index than the pre-diabetic subjects (p,0.001). The controls also had lower blood pressure values than the diabetic and pre-diabetic subjects (systolic, p,0.001, diastolic, p = 0.012), as shown in Table 1. For the three glycemic statuses, the pre-DM group presented with the highest thermal recovery index and

Table 3 - Comparison of the methods. Comparisons

Table 2 - Absolute and relative frequencies of neuropathy test results according to glycemic status. DM (n = 29)

Test

Pre-DM (n = 13)

DM Group (n = 29) HRV6TRI HRV6IDA HRV6both (TRI+IDA) Pre-DM Group (n = 13) HRV6TRI HRV6IDA HRV6both (TRI+IDA) Control Group (n = 37) HRV6TRI HRV6IDA HRV6both (TRI+IDA)

Control (n = 37)

Abnormal Normal Abnormal Normal Abnormal Normal TRI (%) IDA (%) TRI+AID (%) HRV (%) Michigan (%) EMG (%)

22 20 15 16 2 16

(75.9) (69.0) (51.7) (55.2) (6.9) (55.2)

7 (24.1) 9 (31.0) 14 (48.3) 13 (44.8) 27 (93.1) 13 (44.8)

10 (76.9) 10 (76.9) 10 (76.9) 5 (38.5) 0 (0) 2 (15.4)

3 (23.1) 14 (37.8) 23 (62.2) 3 (23.1) 13 (35.1) 24 (64.9) 3 (23.1) 13 (35.1) 24 (64.9) 8 (61.5) 3 (8.1) 34 (91.9) 13 (100) 0 (0.0) 37 (100) 11 (84.6) 0 (0.0) 37(100)

TRI: thermal recovery index; IDA: interdigital anisothermal, HRV: heart rate variability, (Michigan inventory); EMG: electromyography.

% abnormal Sensibility (%) Specificity (%)

55.2675.9 55.2669.0 55.2651.7

75.0 81.3 56.3

23.1 46.2 53.8

38.5676.9 38.5676.9 38.5676.9

80.0 80.0 80.0

25.0 25.0 25.0

8.1637.8 8.1635.1 8.1627.0

-

58.8 61.8 70.6

HRV: heart rate variability; TRI: thermal recovery index, IDA: interdigital anisothermal.

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Thermography in diabetic neuropathy Balbinot LF et al.

Table 4 - Interdigital anisothermal clinical characteristics. Variables*

Age (years) Gender (Female/ Male) Time of diagnosis (years) BMI (kg/m2) SBP (mmHg) DBP (mmHg) *

DM (n = 29)

Pre-DM (n = 13)

Control (n = 37)

Abnormal

Normal

p-value

Abnormal

Normal

p-value

Abnormal

Normal

p-value

56.1¡8.8 14/6

55.4¡11.1 6/3

0.876 1.000

60.7¡10.9 9/1

43.7¡9.1 K

0.032 0.108

46.4¡16.4 8/5

44.3¡14.3 12/12

0.695 0.744

5.5 (2.3-9.5)

2 (1-10.5)

0.216

1.5 (0.9-2.0)

1 (1-1)

0.469

-

-

-

27.2¡4.4 133.5¡5.9 87.0¡5.5

26.9¡3.3 127.8¡10.9 83.3¡11.7

0.825 0.170 0.393

29.7¡3.0 129.7¡6.7 83.5¡11.7

31.1¡2.2 133.3¡2.9 83.3¡7.6

0.467 0.393 0.970

26.2¡4.6 121.2¡10.8 80.0¡11.7

24.8¡3.2 121.9¡8.1 79.6¡7.1

0.290 0.799 0.893

Mean (SD) or median (25th percentile to 75th percentile). BMI: body mass index; SBP: systolic blood pressure; DBP: diastolic blood pressure. Level of significance = 5%.

In this study, plantar thermography proved useful in the early diagnosis of diabetic neuropathy, particularly the small and autonomic fibers that are commonly associated with a sub-clinical condition. Future research studying the thermal recovery index for diabetes duration would be useful, particularly when comparing the density of nerve fibers using skin biopsy and Quantitative Sensitive Test (QST) to demonstrate a low sensitivity to heat. In addition, it would be interesting to compare the potential amplitude of the sural nerve, when present, with the thermographics variable studied here (interdigital anisothermal and thermal recovery index), as demonstrated by Shun et al. (61). One limitation of this study is that it is a cross-sectional study that involves a sensitive test. Therefore, follow-up will be needed to assess the development of more advanced forms of neuropathy and diabetic complications.

with an abnormal plantar thermography result were older than those who presented with a normal interdigital thermographic pattern.

DISCUSSION The presence of interdigital anisothermal using a simplified plantar thermographic study appears to be the most appropriate diagnostic test for the early diagnosis of neuropathy in the patients with DM and pre-DM. Because of its sensitivity and specificity, in addition to its convenience and timing, interdigital anisothermal assessed by plantar thermography seems to be most suitable for diabetic neuropathy screening programs. Its application is simple using the proposed protocol, and it is cost-effective because it requires no additional supplies. It is important to identify diabetic neuropathy early to prevent secondary complications, such as neuropathic pain and diabetic foot (10,11,52,53). Unfortunately, because of economic concerns and the lack of technological resources, a consensus of international associations dedicated to treating and preventing diabetes has advocated only using clinical examination as a population screening method (56-59). In this study, the clinical application of a validated clinical inventory (Michigan inventory) did not identified neuropathy even in cases with positive EMG, which would certainly include late cases of neuropathy. Plantar thermography is a new, non-invasive method that can be included in neuropathy screening programs, thereby increasing the sensitivity for proper diagnosis. The current method for the early diagnosis of diabetic neuropathy is a battery of tests, including methods capable of assessing thick and thin nerve fibers, particularly the autonomic nerve fibers (5,12). The two types of thermographic tests (thermal recovery index after the cold stress test and the interdigital anisothermal test) demonstrated differences in patterns between the three groups. These patterns reflect the sympathetic nerve fibers with vasomotor tone function and its effect on the skin temperature of the feet (15,17,18). Another benefit provided by plantar thermography for chronic patients, such as those with DM, is the possibility of following the evolutionary aspects of the disease because thermography allows for functional imaging (60). Because of the strict control of risk factors, such as glycemic control, cutaneous vasomotor functionality of the feet can be monitored periodically using this method.

ACKNOWLEDGMENTS This work received financial support from CAPES and CNPq.

AUTHOR CONTRIBUTIONS Balbinot LF was responsible for designing the experiments, data acquisition, writing and review of the manuscript. Canani LH was responsible for technical support with the clinical aspects of diabetes, neuropathy, paper redaction, and final paper review. Robinson CC was responsible for data acquisition, writing an review of the manuscript. Achaval M was responsible for technical support concerning diabetes and thermoregulatory control and review of the manuscript. Zaro MA was responsible for technical support with the infrared thermography and manuscript review.

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Thermography in diabetic neuropathy Balbinot LF et al. recovery of locomotion after sciatic nerve crush in rats. Clinics. 2011;66(7):1259-66, http://dx.doi.org/10.1590/S1807-59322011000700023. 61. Shun CT, Chang YC, Wu HP, Hsieh SC, Lin WM, Lin YH, et al. Skin denervation in type 2 diabetes: Correlations with diabetic duration and functional impairments. Brain. 2004;127(Pt 7):1593-605, http://dx.doi. org/10.1093/brain/awh180.

59. Olaleye D, Perkins BA, Bril V. Evaluation of three screening tests and a risk assessment model for diagnosing peripheral neuropathy in the diabetes clinic. Diabetes Research and Clinical Practice. 2001;54(2):11528, http://dx.doi.org/10.1016/S0168-8227(01)00278-9. 60. Sacharuk VZ, Lovatel GA, Ilha J, Marcuzzo S, Pinho AS, Xavier LL, et al. Thermographic evaluation of hind paw skin temperature and functional

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DOI:10.6061/clinics/2012(12)13

CLINICAL SCIENCE

The modified Hodge test is a useful tool for ruling out klebsiella pneumoniae carbapenemase Ana Paula Cury,I Denise Andreazzi,II Ma´rcia Maffucci,III He´lio Hehl Caiaffa-Junior,III Fla´via RossiI I Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, Pathology Department, Microbiology Laboratory, DLC–LIM 03, Sa˜o Paulo/ SP, Brazil. II Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, Pathology Department, Telemedicine Laboratory, Sa˜o Paulo/SP, Brazil. III Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, Pathology Department, Molecular Biology Laboratory, DLC–LIM 03, Sa˜o Paulo/SP, Brazil.

OBJECTIVE: Enterobacteriaceae bacteria harboring Klebsiella pneumoniae carbapenemase are a serious worldwide threat. The molecular identification of these pathogens is not routine in Brazilian hospitals, and a rapid phenotypic screening test is desirable. This study aims to evaluate the modified Hodge test as a phenotypic screening test for Klebsiella pneumoniae carbapenemase. METHOD: From April 2009 to July 2011, all Enterobacteriaceae bacteria that were not susceptible to ertapenem according to Vitek2 analysis were analyzed with the modified Hodge test. All positive isolates and a random subset of negative isolates were also assayed for the presence of blaKPC. Isolates that were positive in modified Hodge tests were sub-classified as true-positives (E. coli touched the ertapenem disk) or inconclusive (distortion of the inhibition zone of E. coli, but growth did not reach the ertapenem disk). Negative results were defined as samples with no distortion of the inhibition zone around the ertapenem disk. RESULTS: Among the 1521 isolates of Enterobacteriaceae bacteria that were not susceptible to ertapenem, 30% were positive for blaKPC, and 35% were positive according to the modified Hodge test (81% specificity). Under the proposed sub-classification, true positives showed a 98% agreement with the blaKPC results. The negative predictive value of the modified Hodge test for detection was 100%. KPC producers showed high antimicrobial resistance rates, but 90% and 77% of these isolates were susceptible to aminoglycoside and tigecycline, respectively. CONCLUSION: Standardizing the modified Hodge test interpretation may improve the specificity of KPC detection. In this study, negative test results ruled out 100% of the isolates harboring Klebsiella pneumoniae carbapenemase2. The test may therefore be regarded as a good epidemiological tool. KEYWORDS: Modified Hodge Test; KPC; Carbapenemase; Ertapenem. Cury AP, Andreazzi D, Maffucci M, Caiaffa-Junior HH, Rossi F. The modified Hodge test is a useful tool for ruling out klebsiella pneumoniae carbapenemase. Clinics. 2012;67(12):1427-1431. Received for publication on August 3, 2012; First review completed on August 24, 2012; Accepted for publication on August 28, 2012 E-mail: anapaulacury@gmail.com Tel.: 55 11 2661-6136/6348

identified on transferable plasmids, giving these genes the potential to disseminate to other genera and species (4). Currently, KPC producers are found all over the world, with reports from Greece (5), Israel (6), France (7), China (8), and South America (4). In Brazil, KPC was first described in the northwestern region in 2006 and has become an important mechanism of resistance to carbapenems since then (9-11). In our institution, carbapenem resistance among Enterobacteriaceae was only sporadically noted prior to 2005. However, starting in 2008, KPC producers became an epidemiological challenge. The early recognition of KPC-producing isolates has become necessary due to the clinical and epidemiological implications of the presence of KPC. The routine laboratory detection of KPC is a challenge, especially for laboratories without molecular diagnostic resources, as is common in Brazilian hospitals. The modified Hodge test (MHT) is a phenotypic screening test for carbapenemases that is used for epidemiological

INTRODUCTION Carbapenems are one of the last remaining antimicrobial treatments for multidrug-resistant Enterobacteriaceae, and resistance to this class of antibiotics is becoming a threat worldwide (1). Impermeability due to porin loss concomitant with the hyperproduction of cephalosporinases is often the cause of carbapenem resistance, but serine betalactamases, such as Klebsiella pneumoniae carbapenemase (KPC), are spreading fast and becoming prevalent worldwide (1-3). The genes encoding the KPC enzyme are usually flanked by transposon-related sequences that have been

Copyright ß 2012 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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purposes, and its use is currently proposed by the Clinical and Laboratory Standards Institute (CLSI) (12). The MHT is easy to perform, but divergent specificity values have been reported, and false-positive results are a concern (13,14). The aim of this study was to evaluate the performance of a new proposal for interpreting the modified Hodge test and detect the blaKPC gene among Enterobacteriaceae isolates, which were collected in our 2500–bed hospital, that exhibited ertapenem MICs $1 mg/ml.

All isolates with positive and indeterminate modified Hodge test results were submitted to molecular detection of the blaKPC gene by PCR. Isolates with negative test results were also randomly chosen for molecular KPC testing. The primers used for PCR amplification and the reaction conditions were described previously (15). The PCR products were visualized by electrophoresis on 2% agarose gels stained with ethidium bromide and visualized with UV light. Two samples of blaKPC amplicons (859 bp) produced by PCR were selected and submitted to DNA sequencing with a BigDye Terminator cycle sequencing kit v3.1 (Applied Biosystems Inc., Foster City, CA) in an MJ Research PTC-200 DNA Engine thermal cycler (Bio-Rad Laboratories, Waltham, USA). The sequencing reaction products were purified by ethanol precipitation, separated, and analyzed using an ABI Prism 3100 genetic analyzer (ABI, Foster City, USA) following the manufacturer’s protocols. The sequenced products were compared using software available at the National Center for Biotechnology Information website (http://blast.ncbi.nlm.nih. gov). The antibiograms for KPC producers and non-producers were analyzed. The MIC50 and MIC90 values were calculated using Whonet version 5.4. The sensitivity, specificity, positive predictive values (PPVs), and negative predictive values (NPVs) were calculated according to the method of Ilstrup (16).

MATERIALS AND METHODS From April 2009 to July 2011, a total of 1521 Enterobacteriaceae isolates that were not susceptible to ertapenem (ENSEs), with MICs $1 mg/ml from any clinical specimen, were selected from among the patient cultures of the Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo. When more than one isolate was recovered from a patient, the second isolate was only included if it was another species or the same species with a different susceptibility pattern. Identification and antimicrobial susceptibility testing were performed using a Vitek2 system (bioMerieux Marcy-L’ile, France) with the AST-N105 and AST-N104 cards. Categorical interpretations were performed according to CLSI M100–S21 (12). For colistin, the CLSI breakpoints for P. aeruginosa were the reference for categorical interpretation, and the U.S. Food and Drug Administration (FDA) breakpoints were used for tigecycline. All isolates were tested by the MHT according to the CLSI recommendations and quality control. The results of the modified Hodge test were categorized according to the CLSI recommendations as follows: negative when there was no distortion of the inhibition zone around the ertapenem disk, positive when any distortion of the E. coli ATCC 25922 (strain indicator) inhibition zone was noted around the ertapenem disk, and indeterminate when the inhibition of E. coli ATCC 25922 growth around the streak (tested strain) was evidenced by a clear area. All isolates with positive modified Hodge test results were classified using a new sub-classification proposed by our group: A) a result was considered a true-positive when E. coli ATCC 25922 touched the ertapenem disk; and B) a result was considered inconclusive when there was distortion of the E. coli ATCC 25922 inhibition zone around the ertapenem disk, but the bacteria did not touch the edge of the disk. See Figure 1 for the modified Hodge test subclassification definition in our study.

RESULTS We analyzed 1521 Enterobacteriaceae isolates that were not susceptible to ertapenem, of which 30% (458) were positive for blaKPC. Among the blaKPC-positive isolates, 93% were Klebsiella pneumoniae. The modified Hodge test results are summarized according to the species distribution and molecular detection of blaKPC in Table 1. The modified Hodge test was positive in 35.5% of Enterobacteriaceae isolates that were not susceptible to ertapenem (540), and according to our proposed sub-classification, 71% (386) of those isolates showed true-positive results, and 29% (154) had inconclusive results. The true-positive results showed 98% agreement with the molecular KPC results, whereas the level of agreement for the inconclusive results was only 51%. The modified Hodge test results according to the sensitivity, specificity and predictive values for KPC detection are shown in Table 2. The modified Hodge test had a 100% negative predictive value. Specimens yielding KPC-producing isolates were primarily urine samples (37%), rectal swabs (17%), blood samples (10%), and catheters (10%). The antimicrobial susceptibility profile of the KPC-producing isolates revealed high resistance rates to all cephalosporins, aztreonam (98.9%), piperacillin/tazobactam (99.3%), and fluoroquinolones (92%). Amikacin and gentamycin had susceptibility rates of 95% and 88%, respectively. Tigecycline had an 8% susceptibility rate, and colistin had an MIC50 of 2 mg/l and MIC90 of 4 mg/l. The meropenem results for KPC isolates from the automated system indicated that 50% were resistant, but the disk method indicated that 70% of isolates were resistant (data not shown). The KPC non-producing isolates also had high rates of resistance to all cephalosporins, aztreonam, piperacillin/tazobactam, and fluoroquinolones, and the detailed information is summarized in Table 3. The DNA sequencing of the KPC-producing isolates confirmed that they carried the KPC-2 variant.

Figure 1 - Modified Hodge test (MHT) with ertapenem disks. Interpretation of the MHT results proposed in this study: A) truepositive MHT, B) inconclusive MHT and C) negative MHT.

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Modified Hodge test for KPC detection. Cury AP et al.

Table 1 - Distribution of 1521 Enterobacteriaceae isolates that were not susceptible to ertapenem (ENSE) according to the modified Hodge test (MHT) results and the presence of blaKPC. MHT resultsa N˚ ENSE

blaKPC Positive

(191) (111) (16) (22) (8) (1) (0) (4) (2) (0) (0) (0) (355)

191 111 16 22 8 1 0 4 2 0 0 0 355 (100%)

0 0 0 0 0 0 0 0 0 0 0 0 0 (0)

K. pneumoniae E. cloacae E. aerogenes E. coli S. marcescens C. koseri C. freundii E. asburiae K. oxytoca Total K. pneumoniae E. cloacae E. aerogenes E. coli E. gergoviae E. asburiae S. marcescens Total

(354) (10) (3) (6) (6) (1) (1) (2) (2) (386) (111) (26) (10) (2) (2) (1) (2) (154)

1 3 0 0 3 0 0 0 0 7 (2%) 40 24 10 0 2 0 0 76 (49%)

353 7 3 6 3 1 1 2 2 379 (98%) 71 2 0 2 0 1 2 78 (51%)

E. cloacae E. coli K. pneumoniae Total

(4) (1) (1) (6)

3 1 1 5 (86%)

1 0 0 1 (14%)

K. pneumoniae E. cloacae E. aerogenes E. coli S. marcescens P. mirabilis M. morgannii C. freundii E. gergoviae K. oxytoca P. stuartii P. vulgaris Total

Negative 975 (64.1%)

True-positiveb 386 (71%)

Positive 540 (35.5%)

Inconclusiveb 154 (29%)

Indeterminate 6 (0.4%)

Total: 1521 a

N˚ ENSE (blaKPC tested)

blaKPC Negative

Organism

481 293 85 62 15 12 8 7 7 2 2 1 975

(901)

443

458

Positive, indeterminate, and negative results were defined according to the recommendations of the Clinical and Laboratory Standards Institute. True-positive and inconclusive results were defined according to this study’s proposal (see Figure 1).

b

not susceptible to ertapenem has a low specificity but is a starting point to identify isolates harboring carbapenemases. The modified Hodge test is relatively easy to perform in standard microbiology laboratories, and the standardization of the interpretation of the results may improve the specificity of this test for KPC detection. Infections caused by KPC-producing isolates are now a concern in our institution, and 30% of the Enterobacteriaceae isolates that were not susceptible to ertapenem were KPC positive in this study period. The majority of those isolates (37%) were from urine cultures, but the positivity rate for KPC among blood isolates was proportionally higher (data not shown). The molecular detection of blaKPC is the gold standard for diagnosis, but the majority of standard laboratories in our country do not have the resources necessary to perform this test on a routine basis. Phenotypic tests should be made available to detect KPC early or rule it out. The modified Hodge test may detect the presence of carbapenemases, but it is not specific for KPC and may have false-positive results due to non-carbapenemase enzymes, such as AmpC and/or extended-spectrum beta-lactamases (ESBLs), combined with

DISCUSSION The presence of blaKPC deserves special attention because isolates harboring this plasmid gene are usually multidrug resistant, limiting the therapeutic options available and causing a high mortality rate (17). KPC represents an epidemiological challenge for infection control groups because it can spread very quickly, and there are limited treatment options available (18). The early detection of such isolates is critical. Screening for KPC producers by identifying Enterobacteriaceae isolates that are Table 2 - Performance of the modified Hodge test (MHT) for KPC detection. MHT True-positive Inconclusive Positivea

N˚ Sensitivity Specificity Isolates (%) (%) 386 154 540

100 100 100

98 82 81

PPV (%)

NPV (%)

98 51 85

100 100 100

a True-positive plus inconclusive results. PPV - positive predictive value, NPV – negative predictive value.

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Table 3 - In vitro activity of antimicrobial agents tested against 901 ENSE isolates. KPC producers (458)

Antibiotica

PTZ Ceftazidime Cefotaxime Cefepime Aztreonam Ertapenem Imipenem Meropenem Amikacin Gentamicin Ciprofloxacin SUT Colistinb Tigecyclinec

KPC non-producers (443)

%R

%I

%S

MIC50

MIC90

%R

%I

%S

MIC50

MIC90

99.3 82.9 94.7 54.0 98.9 100 69.8 50 3.1 11 92 94 32 8

0.7 5.6 3.7 23.5 0.3 0 16.4 7 1.5 1 1 0 0 15

0 11.5 1.5 22.5 0.8 0 13.8 43 95.4 88 7 6 68 77

128 64 64 64 64 8 8 2 2 1 4 16 0.5 2

128 64 64 64 64 8 16 16 16 16 4 16 16 4

93.3 91 95.9 68.7 93.4 99.5 22.5 27.9 17 48.3 79.5 60.4 11.7 11

2.1 2 0.7 4.6 0.8 0.5 8 3.6 3 2.3 2.5 0 0.5 13

4.6 7 3.4 26.7 5.8 0 69.5 68.4 80 49.4 18 39.6 87.8 76

128 64 64 64 64 4 1 0.25 2 8 4 16 0.5 1

128 64 64 64 64 8 8 8 64 16 4 16 16 8

a

Minimum inhibitory concentration (MIC) breakpoint in mg/L, as determined by the Vitek2H system. Breakpoint for P. aeruginosa according to the Clinical and Laboratory Standards Institute. U.S. Food and Drug Administration (FDA) breakpoints. SUT: trimethoprim/sulfamethoxazole; PTZ: piperacillin/tazobactam; ENSE: Enterobacteriaceae not susceptible to ertapenem; KPC: Klebsiella pneumoniae carbapenemases; R: resistant; I: intermediate; S: susceptible.

b c

porin loss. Positive results could also be due to other carbapenemases. In the literature, the specificity of the modified Hodge test differs because some authors analyzed data for distinct bacteria groups, such as non-fermenters and Enterobacteriaceae, and detected different carbapenemases, such as NDM (13). In our study, the modified Hodge test was evaluated only for Enterobacteriaceae bacteria and KPC producers, which may explain the high level of agreement with the molecular results. The proposed positive modified Hodge test category had 98% agreement with the molecular blaKPC results, highlighting the good positive predictive value of KPC detection among Enterobacteriaceae when a standardized method for interpretation is in place. Seven isolates showed truepositive results and were negative for blaKPC, but an accurate analysis of the reason for these discrepancies was limited because no tests were performed for other carbapenemases. Of the isolates in the inconclusive group, 49% were negative for blaKPC, and these negative strains most likely produced CTX-M or hyperproduced AmpC in association with porin loss. Another possibility is that these isolates produced carbapenemases other than KPC. The indeterminate result, as proposed by the CLSI, does not have a clear microbiological or clinical interpretation in the literature and should be the focus of future studies. Enterobacteriaceae bacteria that not susceptible to ertapenem but have a negative modified Hodge test result are not KPC producers. Although tests other than the modified Hodge test, such as the aminophenylboronic acid and dipicolinic acid tests, may also be options for the phenotypic screening for carbapenemases (19), the supplies necessary for these tests are not routinely available in the majority of Brazilian laboratories. The antibiograms of the 458 KPC isolates generated using the automated system revealed multidrug resistance and showed that 43% of isolates were susceptible to meropenem (MIC #1 mg/ml). The meropenem susceptibility rates were not reproducible by TREK microdilution panels (Cleveland, OH) or the disk diffusion method (OXOID) (data not shown), with 45% very major errors (VMEs) for this drug. Corroborating

these findings, the literature contains reports of unacceptable VME levels for meropenem when KPC isolates were tested using automated systems. Microbiologists and clinicians should be aware of the current problems regarding false susceptibility results for meropenem among KPC isolates when only automated systems are used to screen isolates (20). Regarding the colistin and tigecycline Vitek results, our internal validation process did not show major discrepancies with the E-test (data not shown), and published studies have not reported major problems with these drugs (21). Every laboratory should perform its own validation of automated systems when CLSI breakpoints are in place. The modified Hodge test results for Enterobacteriaceae using standardized readings are a useful phenotypic and epidemiological tool to identify KPC isolates while waiting for molecular results. Negative modified Hodge test results ruled out all KPC isolates in our institution in an efficient and timely manner.

AUTHOR CONTRIBUTIONS Cury AP designed and conducted the study, and was also responsible for the microbiological procedures, data collection, analysis and interpretation, and manuscript drafting. Andreazzi D contributed to the interpretation and presentation of the results, statistical analysis and manuscript review. Maffucci M and Caiaffa-Junior HH conducted the molecular biological procedures. Rossi F mentored Cury AP, conducted the analysis of the results and was also responsible for the manuscript writing, review and approval.

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Modified Hodge test for KPC detection. Cury AP et al.

Agents Chemother. 2005;49(7):3018-20, http://dx.doi.org/10.1128/AAC. 49.7.3018-3020.2005. Villegas MV, Lolans K, Correa A, Suarez CJ, Lopez JA, Vallejo M, et al. First detection of the plasmid-mediated class A carbapenemase KPC-2 in clinical isolates of Klebsiella pneumoniae from South America. Antimicrob Agents Chemother. 2006;50(8):2880-2, http://dx.doi.org/10.1128/AAC.00186-06. Maltezou HC, Giakkoupi P, Maragos A, Bolikas M, Raftopoulos V, Papahatzaki H, et al. Outbreak of infections due to KPC-2-producing Klebsiella pneumoniae in a hospital in Crete (Greece). J Infect. 2009;58(3):213-9, http://dx.doi.org/10.1016/j.jinf.2009.01.010. Navon-Venezia S, Chmelnitsky I, Leavitt A, Schwaber MJ, Schwartz D, Carmeli Y. Plasmid-mediated imipenem-hydrolyzing enzyme KPC-2 among multiple carbapenem-resistant Escherichia coli clones in Israel. Antimicrob Agents Chemother. 2006;50(9):3098-101, http://dx.doi.org/ 10.1128/AAC.00438-06. Naas T, Nordmann P, Vedel G, Poyart C. Plasmid-mediated carbapenem-hydrolyzing beta-lactamase KPC in a Klebsiella pneumoniae isolate from France. Antimicrob Agents Chemother. 2005;49(10):4423-4, http:// dx.doi.org/10.1128/AAC.49.10.4423-4424.2005. Wei ZQ, Du XX, Yu YS, Shen P, Chen YG, Li LJ. Plasmid-mediated KPC2 in a Klebsiella pneumoniae isolate from China. Antimicrob Agents Chemother. 2007;51(2):763-5, http://dx.doi.org/10.1128/AAC.01053-06. Pavez M, Mamizuka EM, Lincopan N. Early dissemination of KPC-2producing Klebsiella pneumoniae strains in Brazil. Antimicrob Agents Chemother. 2009;53(6):2702, http://dx.doi.org/10.1128/AAC.00089-09. Monteiro J, Santos AF, Asensi MD, Peirano G, Gales AC. First report of KPC2-producing Klebsiella pneumoniae strains in Brazil. Antimicrob Agents Chemother. 2009;53(1):333-4, http://dx.doi.org/10.1128/AAC.00736-08. Andrade LN, Curiao T, Ferreira JC, Longo JM, Climaco EC, Martinez R, et al. Dissemination of blaKPC-2 by the spread of Klebsiella pneumoniae clonal complex 258 clones (ST258, ST11, ST437) and plasmids (IncFII, IncN, IncL/M) among Enterobacteriaceae species in Brazil. Antimicrob Agents Chemother. 2011;55(7):3579-83, http://dx.doi.org/10.1128/AAC. 01783-10. Clinical and Laboratory Standards Institute. Performance Standards for antimicrobial susceptibility testing. CLSI document Wayne, PA, USA. 2011;M100-S21.

13. Girlich D, Poirel L, Nordmann P. Value of the modified Hodge test for detection of emerging carbapenemases in Enterobacteriaceae. J Clin Microbiol. 2012;50(2):477-9, http://dx.doi.org/10.1128/JCM.05247-11. 14. Carvalhaes CG, Picao RC, Nicoletti AG, Xavier DE, Gales AC. Cloverleaf test (modified Hodge test) for detecting carbapenemase production in Klebsiella pneumoniae: be aware of false positive results. J Antimicrob Chemother. 2010;65(2):249-51, http://dx.doi.org/10.1093/jac/dkp431. 15. Castanheira M, Sader HS, Jones RN. Antimicrobial susceptibility patterns of KPC-producing or CTX-M-producing Enterobacteriaceae. Microb Drug Resist. 2010;16(1):61-5, http://dx.doi.org/10.1089/mdr.2009.0031. 16. Ilstrup DM. Statistical methods in microbiology. Clin Microbiol Rev. 1990;3(3):219-26. 17. Tumbarello M, Viale P, Viscoli C, Trecarichi EM, Tumietto F, Marchese A, et al. Predictors of Mortality in Bloodstream Infections Caused by KPC-Producing Klebsiella pneumoniae: Importance of Combination Therapy. Clin Infect Dis. 2012;55(7):943:50. 18. Zarkotou O, Pournaras S, Tselioti P, Dragoumanos V, Pitiriga V, Ranellou K, et al. Predictors of mortality in patients with bloodstream infections caused by KPC-producing Klebsiella pneumoniae and impact of appropriate antimicrobial treatment. Clin Microbiol Infect. 2011;17(12):1798-803, http://dx.doi.org/10.1111/j.1469-0691.2011.03514.x. 19. Pasteran F, Mendez T, Rapoport M, Guerriero L, Corso A. Controlling false-positive results obtained with the Hodge and Masuda assays for detection of class a carbapenemase in species of enterobacteriaceae by incorporating boronic Acid. J Clin Microbiol. 2010;48(4):1323-32, http:// dx.doi.org/10.1128/JCM.01771-09. 20. Bulik CC, Fauntleroy KA, Jenkins SG, Abuali M, LaBombardi VJ, Nicolau DP, et al. Comparison of meropenem MICs and susceptibilities for carbapenemase-producing Klebsiella pneumoniae isolates by various testing methods. J Clin Microbiol. 2010;48(7):2402-6, http://dx.doi.org/ 10.1128/JCM.00267-10. 21. Lat A, Clock SA, Wu F, Whittier S, Della-Latta P, Fauntleroy K, et al. Comparison of polymyxin B, tigecycline, cefepime, and meropenem MICs for KPC-producing Klebsiella pneumoniae by broth microdilution, Vitek 2, and Etest. J Clin Microbiol. 2011;49(5):1795-8, http://dx.doi.org/ 10.1128/JCM.02534-10.

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CLINICS 2012;67(12):1433-1441

DOI:10.6061/clinics/2012(12)14

CLINICAL SCIENCE

The influence of anthropometric factors on postural balance: the relationship between body composition and posturographic measurements in young adults Ange´lica Castilho Alonso,I Nata´lia Mariana S. Luna,I Luis Mochizuki,I Fa´bio Barbieri,II Sileno Santos,I Julia Maria D’Andre´ia GreveI I

Faculdade de Medicina da Universidade de Sa˜o Paulo, Department of Orthopedics and Traumatology, Laboratory for the Study of Movement, Sa˜o Paulo/ SP, Brazil. II Faculdade de Medicina da Universidade de Sa˜o Paulo, Department of Radiology, Sa˜o Paulo/SP, Brazil.

OBJECTIVE: The aim of the present study was to evaluate the influence of anthropometric characteristics and gender on postural balance in adults. One hundred individuals were examined (50 males, 50 females; age range 20-40 years). METHODS: The following body composition measurements were collected (using bone densitometry measurements): fat percentage (% fat), tissue (g), fat (g), lean mass (g), bone mineral content (g), and bone mineral density (g/cm2). In addition, the following anthropometric measurements were collected: body mass (kg), height (cm), length of the trunk-cephalic region (cm), length of the lower limbs (cm) and length of the upper limbs (cm). The following indices were calculated: body mass index (kg/m2), waist-hip ratio and the support base (cm2). Also, a postural balance test was performed using posturography variables with open and closed eyes. RESULTS: The analysis revealed poor correlations between postural balance and the anthropometric variables. A multiple linear regression analysis demonstrated that the whole group (female and male) height explained 12% of the medial-lateral displacement, 10% of the speed of oscillation, and 11% of the displacement area. The length of the trunk-cephalic length explained 6% of the displacement in the anteroposterior direction. With eyes closed, the support base and height explained 18% of the medial displacement, and the lateral height explained 10% of the displacement speed and 5% of the scroll area. CONCLUSION: Measured using posturography, the postural balance was only slightly influenced by the anthropometric variables, both with open and closed eyes. Height was the anthropometric variable that most influenced postural balance, both in the whole group and separately for each gender. Postural balance was more influenced by anthropometric factors in males than females. KEYWORDS: Assessment; Postural Balance; Anthropometry; Sensorimotor Performance; Young Adult. Alonso AC, Luna NM, Mochizuki L, Barbieri F, Santos S, D’Andre´ia Greve JM. The influence of anthropometric factors on postural balance: the relationship between body composition and posturographic measurements in young adults. Clinics. 2012;67(12):1433-1441. Received for publication on July 22, 2012; First review completed on August 27, 2012; Accepted for publication on August 30, E-mail: angelicacastilho@msn.com Tel.: 55 11 3938-4889

reliability of the quantitative evaluations. In clinical practice, this lack of consensus impedes using these tests as a safe tool for assessing the risk of falls and the results of therapeutic interventions (1-8). Studies using various assessment tools in various populations have shown that as body mass increases, balance worsens. Studies have been conducted on groups of prepubescent children and adolescents (9,10), adults (1115), and elderly people (16,17) who were obese or extremely obese, and in all of these populations, body mass influenced postural stability. Evaluations that were performed on stable surfaces with individuals who were overweight or with normal body mass indices (BMI) have shown that balance does not appear to be affected in such situations (6,18,19). However, in situations that combine instability (20) and extreme BMI, postural balance worsens (10).

INTRODUCTION Many balance assessment methods exist, including simple observations, clinical tests, scales, posturographic measurements and integrated assessment systems of greater complexity. They all have advantages and limitations and can produce different results with multiple interpretations. This diversity is worsened by the lack of consensus regarding which individual characteristics (particularly anthropometric factors) must be controlled to ensure the

Copyright ß 2012 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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CLINICS 2012;67(12):1433-1441

A number of questions remain unanswered in the literature. Do anthropometric factors interfere with postural balance in young adults with normal or slightly higher BMIs? Can body composition better explanation for the variations encountered, and should these variables be considered during balance assessments? The aim of the present study was to evaluate the influence of anthropometric characteristics and gender on postural balance in irregularly active adults placed in an erect semistatic position standing on two feet with the eyes open and closed.

METHODS The study evaluated 100 males and females aged 20-40 years who were irregularly physically active. The participants provided written informed, and the study was approved by the Faculdade de Medicina da Universidade de SaËœo Paulo (no. 1256/06). The following inclusion criteria were applied: no history of injury to or surgery on the lower limbs and trunk, irregularly active over the last six months, as defined by the International Physical Activity Questionnaire, the absence of disease or functional impairment of the auditory, vestibular and proprioceptive systems, and no current use of medications that might alter postural balance. Patients who were unable to carry out the postural balance tests were excluded. The characteristics of the individuals who participated in the study are described in Table 1. The anthropometric measurements were made in accordance with the ISAK standard (21). The BMI (kg/m2) and the waist-hip ratio (cm) were calculated. The support base area (cm2) was evaluated with the individual placed in an upright position and standing on both feet with a

Figure 1 - AccuSwayPlus portable force platform.

comfortable separation but without exceeding the shoulder width. This distance was recorded on a piece of paper and served as the baseline for all of the tests. To calculate the support base area, the formula described by Chiari et al. was used (18). Body composition was assessed using bone densitometry with a dual energy X-ray absorptiometry (DEXA) on a LUNAR-DPX apparatus (Madison Corporation, USA). The postural balance assessment (posturography) was performed on a portable force platform (AccuSway Plus, AMTIH, MA, USA) (Figure 1). The data were gathered and stored using the Balance ClinicH software, configured to a frequency of 100 Hz with a fourth-order Butterworth filter and a cutoff frequency of 10 Hz. All of the subjects assumed

Table 1 - Characteristics of the study population (anthropometric and posturographic). Variables Age (years) Anthropometrics Height (cm) Body mass (kg) BMI (kg/m2) Upper-limb length (cm) Trunk-cephalic length (cm) Lower-limb length (cm) Support base area (cm2) % fat Soft tissue (g) Fat (g) Lean mass (g) Bone mineral composition (g) Bone mineral density (g/cm2) Waist-hip ratio (cm) Posturographic measurements (log10) Eyes open Mediolateral displacement (cm) Anteroposterior displacement (cm) Sway velocity (cm/s) Displacement area (cm2) Eyes closed Mediolateral displacement (cm) Anteroposterior displacement (cm) Sway velocity (cm/s) Displacement area (cm2)

Whole group Mean (SD) N = 100

Female group Mean (SD) N = 50

Male group Mean (SD) N = 50

27.2 (5.7)

26.4 (5.1)

28.0 (6.1)

168.8 69.9 24.3 168.9 89.9 79.0 322.3 30.2 67231.5 20297.1 46934.4 2774.6 1198.0 81.7

(9.5) (14.3) (3.6) (11.9) (4.4) (6.7) (59.8) (10.1) (13911.0) (8029.9) (11888.3) (551.9) (92.3) (7.6)

161.8 61.2 23.2 160.3 87.6 74.3 306.0 37.3 58997.9 22483.4 36514.6 2347.5 1142.0 77.9

(6.8) (10.9) (3.7) (8.3) (3.3) (4.4) (56.7) (6.6) (10745) (7515) (4963) (333) (67.9) (7.6)

175.8 78.6 25.3 177.4 83.6 83.6 338.6 23.1 75465.2 18110.9 57354.3 3201.7 1254.0 86

(6.2) (11.8) (3.3) (8.3) (5.3) (5.3) (58.9) (7.7) (11711) (8002.6) (6271.7) (363.5) (78.8) (0.5)

-0.685 -0.421 -0.130 0.140

(0.154) (0.128) (0.097) (0.243)

-0.716 -0.429 -0.153 0.106

(0.14) (0.13) (0.09) (0.25)

-0.653 -0.412 -0.107 0.173

(0.16) (0.11) (0.09) (0.23)

-0.612 -0.332 0.008 0.306

(0.161) (0.148) (0.110) (0.259)

-0.629 -0.328 -0.008 0.294

(0.17) (0.17) (0.10) (0.28)

-0.594 -0.337 0.026 0.317

(0.15) (0.12) (0.10) (0.23)

Legend: cm - centimeters; kg - kilograms; g - grams; cm2 – square centimeters; % - percentage; BMI - body mass index; SD - standard deviation.

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CLINICS 2012;67(12):1433-1441

Anthropometric factors postural balance Alonso AC et al.

a standing position on two feet, with their arms suspended alongside their bodies and their eyes fixed on a point that was located one meter away. Three measurements were made with the eyes open and three with the eyes closed (60 seconds each). The arithmetic means of the results were calculated from the three tests conducted under each condition. The following parameters were used to measure the stability of the subjects with their eyes open (EO) and eyes closed (EC): the root mean square of the displacements from the center of pressure (COP) in the mediolateral (XSD) and anteroposterior planes (YSD), the mean velocity calculated from the total displacement of the COP in all directions (VAvg) and the elliptical area encompassing 95% of the displacement from the COP.

Whole group ‘‘Eyes open’’

N N

Height explained 12% of the mediolateral displacement, 10% of the sway velocity and 11% of the displacement area. Trunk-cephalic length explained 6% of the anteroposterior displacement. ‘‘Eyes closed’’

N N

Height and support base area explained 18% of the mediolateral displacement. Trunk-cephalic length explained 10% of the displacement velocity and 5% of the displacement area.

Statistical analysis The data were stored and analyzed using the SPSS 17.0 software (IBM, Chicago, USA). The Kolmogorov-Smirnov test was used to ascertain whether the continuous variables presented normal distributions; the variables that did not present normal distributions were transformed into log10. Pearson’s correlation coefficient was used to assess the correlations between the dependent variables (the posturographic parameters) and the independent variables (the anthropometric measurements and age) in the whole population and by gender. A linear regression model analysis was performed by selecting all of the variables that presented p#0.20 in the correlation coefficient analysis. These variables were then ranked from the lowest to highest p-value. A multiple modeling process using stepwise forward selection was conducted, and the variables were added to the model one by one, according to their ranking. The variables with p#0.05 were kept in the model.

Female group ‘‘Eyes open’’

N

Height and bone mineral density explained 16% of the anteroposterior displacement.

‘‘Eyes closed’’

N N

Upper-limb length explained 15% of the mediolateral displacement. Age explained 5% of the anteroposterior displacement.

Male group ‘‘Eyes open’’

N N

RESULTS Correlation analysis The correlation coefficients between the postural balance variables and the anthropometric variables in the whole group (male and female) were divided according to gender under the ‘‘eyes open’’ condition, as shown in Table 2. Whole group: Height, trunk-cephalic length and bone mineral composition significantly correlated with all of the balance variables. Female group: None of the anthropometric variables correlated with all of the balance variables. Male group: Height was the only variable that was significantly correlated with all of the balance variables. The correlation coefficients between the postural balance variables and the anthropometric variables in the whole group (male and female) were divided by gender under the ‘‘eyes closed’’ condition, as shown in Table 3. Whole group and male group: Height was the only variable that was significantly correlated with all of the balance variables. Female group: None of the anthropometric variables were correlated with all of the balance variables.

Height explained 14% of the mediolateral displacement and 15% of the sway velocity. Lean mass explained 18% of the anteroposterior displacement and 18% of the displacement area.

‘‘Eyes closed’’

N N N N

Height and support base area explained 28% of the mediolateral displacement. Lean mass explained 10% of the anteroposterior displacement. Lower-limb length and waist-hip ratio explained 26% of the sway velocity. Mean mass and support base area explained 25% of the sway area.

DISCUSSION Age is not an anthropometric variable, but it is an important factor in assessing postural balance. However, it was not important in the present study of young adults, and this finding was consistent with other studies (5,13,22,23). Among the women with EC, older age correlated with greater anteroposterior sway and explained 5% of the performance. Hue et al. (13) have stated that under challenging conditions, increased age worsens balance. When vision is suppressed, greater participation is required from other body systems (e.g., sensory-motor and vestibular),

Regression analysis The regression analyses of the anthropometric variables in relation to the postural balance variables in the whole group with the eyes open and closed are described in Table 4.

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1436

-0.26 -0.04 -0.17 0.16 0.01

-0.23 0.20 -0.10 0.31 0.26

-0.01 (0.91)

0.24 (0.08)

0.12 (0.21)

0.26 (0.08)*

(0.06) (0.76) (0.23) (0.24) (0.93)

-0.19 (0.17)

-0.13 (0.17)

(0.019)* (0.038)* (0.30) (0.001)* (0.008)*

0.18 (0.20)

(0.85) (0.08) (0.74) (0.41) (0.78)

0.32 (0.001)*

0.02 0.24 -0.04 -0.11 0.04

0.06 (0.65)

(0.37) (0.000)* (0.03)* (0.81) (0.005)*

Female group r (p)

0.21 (0.02)*

0.09 0.36 0.21 0.02 0.28

Whole group r (p) (0.52) (0.004)* (0.09) (0.79) (0.01)*

(0.82) (0.10) (0.75) (0.007)* (0.03)*

0.12 (0.39)

0.01 (0.93)

0.03 0.23 0.04 0.37 0.29

-0.21 (0.13)

0.32 (0.02)*

0.18 (0.20)

0.09 0.40 0.23 0.03 0.33

Male group r (p)

Mediolateral displacement (log10)

(0.63) (0.005) * (0.01) * (21) (0.03) *

0.07 (0.43)

0.007 (0.94)

-0.03 (0.73) 0.24 (0.01) * 0.10(0.30) 0.21 (0.03) * 0.22 (0.02) *

0.02 (0.79)

0.17 (0.08)

0.27 (0.006) *

0.04 0.28 0.24 0.12 0.20

(0.92) (0.01)* (0.61) (0.63) (0.13)

(0.33) (0.56) (0.86) (0.12) (0.16)

0.08 (0.95)

-0.15 (0.27)

-0.14 0.08 -0.02 0.22 0.19

0.006 (0.96)

0.19 (0.18)

0.32 (0.01)*

-0.01 0.35 0.07 -0.06 0.21

Female group r (p) (0.52) (0.02)* (0.001)* (0.01)* (0.08)

(0.21) (0.001)* (0.03)* (0.001)* (0.01)*

0.10 (0.48)

0.07 (0.63)

0.17 0.44 0.29 0.45 0.35

0.012 (0.93)

0.16 (0.24)

0.24 (0.09)

0.09 0.32 0.45 0.34 0.24

Male group r (p)

Anteroposterior displacement (log10) Whole group r (p)

(0.009)* (0.10) (0.16) (0.004)* (0.05)*

0.18 (0.06)

0.02 (0.81)

-0.26 0.16 -0.14 0.28 0.19

-0.09 (0.36)

0.29 (0.003)*

(0.84) (0.54) (0.22) (0.08) (0.46)

(0.10) (0.29) (0.16) (0.83) (0.26)

-0.12 (0.39)

-0.25 (0.07)

-0.23 -0.15 -0.20 -0.02 -0.16

-0.12 (0.40)

0.01 (0.93)

0.11 (0.42)

-0.02 0.08 -0.17 -0.24 0.10

Female group r (p)

(0.71) (0.18) (0.82) (0.02)* (0.27)

0.24 (0.09)

-0.07 (0.60)

-0.05 0.19 0.03 0.31 0.15

-0.21 (0.11)

0.32 (0.01)*

0.16 (0.25)

(0.35) (0.003)* (0.17) (0.91) (0.05)*

Male group r (p) 0.13 0.40 0.19 -0.01 0.27

Sway velocity (log10)

(0.37) (0.001)* (0.11) (0.57) (0.002)*

0.24 (0.01)*

0.09 0.33 0.15 -0.05 0.29

Whole group r (p)

Pearson’s coefficient (r); * p#0.05. Legend: cm - centimeters; kg - kilograms; g - grams; cm2 - square centimeters; % - percentage; BMI - body mass index.

Age (years) Height (cm) Mass (kg) BMI (kg/m2) Upper-limb length (cm) Trunk-cephalic length (cm) Lower-limb length (cm) Support base area (cm2) % fat Soft tissue (g) Fat (g) Lean mass (g) Bone mineral composition (g) Bone mineral density (g/ cm2) Waist-hip ratio (cm)

Variables

-0.10 (0.48)

0.20 (0.15)

0.17 (0.08)

0.09 (0.49)

-0.12 (0.39)

(0.15) -0.21 (0.13) (0.02)* 0.008 (0.95) (0.960) -0.11 (0.44) (0.006)* 0.18 (0.19) (0.01)* 0.10 (0.45)

0.04 (0.67)

-0.14 0.23 -0.00 0.27 0.24

-0.087 (0.38)

0.28 (0.004)*

0.22 (0.11)

(0.47) 0.003 (0.98) (0.00)* 0.33 (0.01)* (0.02)* 0.001 (0.99) (0.54) -0.12 (0.40) (0.007)* 0.16 (0.25) 0.24 (0.01)*

0.07 0.35 0.23 0.06 0.26

Female group r (p)

(0.45) (0.003)* (0.009)* (0.18) (0.01)*

(0.60) (0.009)* (0.20) (0.001)* (0.02)*

0.13 (0.35)

0.009 (0.95)

0.07 0.36 0.18 0.44 0.32

0.16 (0.26)

0.31 (0.02)*

0.20 (0.16)

0.10 0.41 0.36 0.19 0.33

Male group r (p)

Displacement area (log10) Whole group r (p)

Table 2 - Correlations between balance and the anthropometric variables in the whole group and by gender, with eyes open.

Anthropometric factors postural balance Alonso AC et al. CLINICS 2012;67(12):1433-1441


1437

0.12 (0.39)

0.25 (0.07)

0.07 (0.47)

0.25 (0.01) *

(0.40) (0.39) (0.92) (0.08) (0.08)

-0.12 0.12 0.013 0.24 0.24

-0.12 0.22 0.01 0.24 0.22

(0.22) (0.02)* (0.87) (0.01)* (0.02)*

0.003 (0.98)

-0.15 (0.12)

0.38 (0.005)*

(0.90) (0.001)* (0.39) (0.75) (0.003)*

0.32 (0.001)*

-0.01 0.44 0.12 -0.04 0.40

0.24 (0.09)

(0.30) (0.000)* (0.02)* (0.69) (0.001)*

Female group r (p)

0.21 (0.03) *

0.10 0.35 0.22 0.04 0.32

Whole group r (p) (0.19) (0.004)* (0.06) (0.60) (0.03)*

(0.90) (0.06) (0.57) (0.005)* (0.12)

0.23 (0.09)

-0.09 (0.51)

-0.01 0.26 0.08 0.39 0.22

-0.38 (0.006)*

0.32 (0.02)*

0.14 (0.30)

0.18 0.40 0.26 0.07 0.30

Male group r (p)

Mediolateral displacement (log10)

(0.28) (0.56) (0.42) (0.53) (0.70)

(0.98) (0.38) (0.54) (0.54) (0.98)

0.02 (0.81)

-0.11 (24)

-0.002 0.08 0.06 0.06 -0.002

0.02 (0.79)

0.02 (0.81)

0.05 (0.60)

0.10 0.05 0.08 0.06 0.03

Whole group r (p) (0.05)* (0.52) (0.79) (0.64) (0.70)

(0.38) (0.86) (0.63) (0.72) (0.81)

0.04 (0.76)

-0.18 (0.20)

-0.12 -0.02 -0.06 0.05 -0.03

0.04 (0.78)

-0.03 (0.83)

0.16 (0.26)

-0.27 0.09 -0.03 -0.06 0.05

Female group r (p) (0.56) (0.27) (0.01)* (0.04)* (0.36)

(0.59) (0.01)* (0.12) (0.01)* (0.40)

0.07 (0.62)

-0.06 (0.65)

0.07 0.33 0.22 0.34 0.12

0.03 (0.82)

0.17 (0.23)

-0.002 (0.99)

0.08 0.15 0.33 0.29 0.13

Male group r (p)

Anteroposterior displacement (log10)

(0.36) (0.001)* (0.08) (0.84) (0.003)*

(0.06) (0.07) (0.54) (0.01)* (0.09)

0.18 (0.06)

0.03 (0.71)

-0.18 0.17 -0.06 0.25 0.16

-0.02 (0.82)

0.32 (0.001)*

0.16 (0.09)

0.09 0.31 0.17 -0.02 0.29

Whole group r (p) (0.30) (0.21) (0.53) (0.18) (0.06)

(0.38) (0.63) (0.366 (0.73) (0.44)

-0.07 (0.62)

-0.18 (0.19)

-0.12 -0.06 -0.13 0.04 -0.11

-0.01 (0.94)

0.10 (0.47)

0.16 (0.24)

-0.14 0.17 -0.09 -0.19 0.26

Female group r (p)

0.32 (0.02)*

0.02 (0.89)

(0.93) (0.05)* (0.54) (0.004)* (0.09)

-0.12 (0.38)

0.46 (0.001)*

0.05 (0.70)

-0.01 0.27 0.08 0.39 0.24

Whole group r (p)

(0.59) (0.06) (0.54) (0.08) (0.17)

0.16 (0.09)

-0.02 (0.83)

-0.05 0.18 0.06 0.17 0.13

-0.09 (0.35)

0.20 (0.04)*

0.16 (0.10)

(0.25) (0.04)* (0.67) (0.74) (0.07)

(0.39) (0.63) (0.94) (0.25) (0.40)

0.14 (0.30)

-0.03 (0.79)

-0.12 0.06 -0.009 0.16 0.12

0.02 (0.88)

0.18 (0.18)

0.21 (0.13)

-0.16 0.29 0.06 -0.04 0.25

Female group r (p)

(0.29) (0.01)* (0.01)* (0.17) (0.05)*

(0.75) (0.01)* (0.21) (0.002)* (0.14)

0.21 (0.13)

-0.08 (0.54)

0.04 0.35 0.17 0.42 0.21

-0.26 (0.06)

0.30 (0.03)*

0.12 (0.40)

0.15 0.35 0.35 0.19 0.27

Male group r (p)

Displacement area (log10)

(0.07) -0.002 (0.98) (0.001)* 0.25 (0.01)* (0.05)* 0.18 (0.07) (0.64) 0.06 (0.50) (0.05)* 0.21 (0.03)*

Male group r (p) 0.25 0.44 0.27 0.06 0.26

Sway velocity (log10)

Pearson’s coefficient (r); * p # 0.05. Legend: cm - centimeters; kg - kilograms; g - grams; cm2 - square centimeters; % - percentage; BMI - body mass index.

Age (years) Height (cm) Mass (kg) BMI (kg/m2) Upper-limb length (cm) Trunk-cephalic length (cm) Lower-limb length (cm) Support base area (cm2) % fat Soft tissue (g) Fat (g) Lean mass (g) Bone mineral composition (g) Bone mineral density (g/cm2) Waist-hip ratio (cm)

Variables

Table 3 - Correlations between balance and the anthropometric variables in the whole group and by gender, with eyes closed.

CLINICS 2012;67(12):1433-1441 Anthropometric factors postural balance Alonso AC et al.


Variables

1438

r2

–

-0.001 (,0.001) -0.001 (0.01)

+0.011 (,0.001) +0.006 (,0.001) +0.010 (,0.001)

-

-

-

-

-

-

b (p)

Bone mineral density

-0.001 (0.04)

+00.001 (,0.001) -

-

b (p)

Support base area

+20.396 (0.01)

-

+0.004 (,0.001) +0.007(0.01)

+0.007 (,0.001) -

+0.003 (0.001) +0.009 (,0.001)

+0.008 (0.006) -

b (p)

Trunk-cephalic length

+0.006 (,0.001)

b (p)

r adjusted; * p # 0.05. Legend: b - beta value.

Whole group Eyes open Mediolateral displacement Anteroposterior displacement Sway velocity Displacement area Eyes closed Mediolateral displacement Anteroposterior displacement Sway velocity Displacement area Female group Eyes open Anteroposterior displacement Eyes closed Mediolateral displacement Anteroposterior displacement Male group Eyes open Mediolateral displacement Anteroposterior displacement Sway velocity Displacement area Eyes closed Mediolateral displacement Anteroposterior displacement Sway velocity Displacement area

Group condition

Height

-

-

-

-

b (p)

Age

0.004 (,0.001) 0.009 (0.05) -

-

-

-

-

b (p)

Upper-limb length

+60.788 (0.01) +10.731 (,0.001)

-

-

-

-

-

b (p)

Lean mass

+0.009 (,0.001) -

-

-

-

-

-

-

b (p)

Lower-limb length

+0.537 (0.03) -

-

-

-

-

-

-

b (p)

Waist-hip ratio

r2

0.28 0.10 0.26 0.25

0.14 0.18 0.15 0.18

00.15 0.05

0.16

0.10 0.05

0.18

0.12 0.06 0.10 0.11

adjust

Table 4 - Linear regression analysis on postural balance and the anthropometric variables for the whole group and per gender, with eyes open and closed.

Anthropometric factors postural balance Alonso AC et al. CLINICS 2012;67(12):1433-1441


CLINICS 2012;67(12):1433-1441

Anthropometric factors postural balance Alonso AC et al.

Lean mass explained 18% of the anteroposterior displacement and displacement area among the men. The greater quantity of lean mass with greater development of the musculature among the men was most likely the factor responsible for this effect, in addition to the influence of height. The postural balance among the men may be more dependent on the action of joint and muscle effectors, which might also explain the greater activity. Under the EC condition, lean mass explained 10% of the anteroposterior displacement and, together with the support base area, 25% of the displacement area. The complexity and multiplicity of postural control may explain this correlation (i.e., the greater the lean mass and the smaller the support base area, the greater the displacement and sway area), but these movements are capable of balancing an individual. These findings should not be viewed as a worsening of balance and a risk of falling but rather as one of the strategies used to maintain the center of pressure within the area of stability. The greater the waist-hip ratio, the worse the postural balance in the mediolateral direction (under both conditions evaluated and in the whole sample). In the male group with EC, the greater the waist-hip ratio, the greater the sway velocity. The waist-hip ratio and the upper-limb length explained 26% of the postural balance, and this result was similar to the findings of Menegoni et al. (14). It is possible that a concentration of fat mass in the chest and abdomen (android shape) increases the load on the hips, thereby explaining the greater displacement in the mediolateral direction. A centripetal fat distribution changes the center of mass, which ends up being greater in android than in gynoid shapes. The bone mineral density and height explained 16% of the postural balance in the anteroposterior direction. Bone mineral density has been correlated with loading and impact on bones, and it is reasonable to assume that this correlation would have some influence on balance. Winters and Snow (24) correlated bone mineral density with anthropometric variables and found an interrelation between these variables, but they reported that it did not influence postural balance. Bone mineral composition was shown to have a positive correlation with all of the variables in the whole group and in the male group, except for sway velocity with the EO. With the EC, there was a positive correlation with the mediolateral displacement in the whole group. Lower bone mineral density and bone mineral composition values combined with poor balance increases the risk of fractures from falls, but no relationship between these measurements and balance was found in the present study nor in the literature. The smaller the support base area, the greater the mediolateral sway in the male group with EC. A larger support base area increased the balance and decreased the postural control activity, and these findings were consistent with those of other authors (18,25-27). The support base area and height explained 18% of the variation in balance in the mediolateral direction in the whole group and 28% of that in the male group. The relationship between height and support base area in the male group (whose measurements were larger than those in the female group) can be understood through the inverse relationship between postural stability and the height of the center of gravity. Widening the support base area decreases

and this factor may explain the need for greater adjustments to maintain balance. With greater body mass and soft-tissue mass (sum of the lean and fat masses), there was greater mediolateral sway (EO and EC) and anteroposterior sway and displacement area (EO) in the whole group. A separate evaluation of the genders indicated that these variables only correlated among males, which may indicate that the greater body mass in men interfered more with balance than it did in women. This finding was consistent with other studies (6,18,19). This weak correlation, which was observed for some variables and under some conditions, may indicate that semi-static balance among individuals with normal body composition and BMIs does not depend on body mass and soft-tissue mass. In a more challenging situation (EC), the displacement velocity was greater in the male group, which may be associated with the greater male body mass (14). There was a weak positive correlation between BMI and anteroposterior displacement in the male group (EO and EC). Although this result corroborates the findings from studies of individuals with normal BMIs (6,18,19), it contradicts the findings of Greve et al. (20) who demonstrated that there was a moderate to strong correlation between BMI and balance among young adults on an unstable platform and those of Singh et al. (15) who stated that under extreme conditions (BMI.40 kg/m2), balance becomes impaired during prolonged activities. There is a consensus that obesity worsens balance, but Winters and Snow (24) and Mainenti et al. (17) have demonstrated that DEXA and bioimpedance are important for settling controversies because of the less refined nature of body mass and BMI measurements. The fat percentage was negatively correlated to the mediolateral sway and the displacement velocity in the whole group with eyes open. The fat measurement in grams was only correlated with the anteroposterior movements in the male group. There are few studies of body composition variables for comparative purposes. In the female group, there was no correlation between the fat mass measurements and the balance parameters, either with EO or with EC. This outcome differed from the findings of Mainenti et al. (17) who observed that elderly women with greater fat mass exhibited worse performance. Winters and Snow (24) reported that 31% of balance variations in premenopausal women who were evaluated on a multidirectional platform was caused by variations in fat mass. Assessments on unstable surfaces require greater motor control and may be more sensitive to variations in body composition than static evaluations (20). Greater lean mass correlated with significantly greater postural control in relation to all of the balance variables with EO and the mediolateral direction and velocity with EC. These results were observed for the whole group. The male group showed correlations with all of the variables, with EO and EC; however, there were no correlations in the female group. It is possible that this behavioral difference occurred because of differences in body composition or because the women were more skillful in postural control because of habits, footwear and adaptation to a lower level of lean mass. The women may have developed other strategies for maintaining balance that depended less on body composition.

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CLINICS 2012;67(12):1433-1441

the distance from the center of gravity to the base and improves stability. There is a consensus in the literature, which was also observed in the present study, that increased height worsens balance (5,13,16). Height showed a positive correlation with all of the balance variables in the whole group and in the male group with the EO, as well as with anteroposterior displacement in the female group with the EO and EC. In the whole group and in the male group with EC, height correlated with mediolateral displacement, velocity and area. In the regression analysis, height explained half of the variation in balance. Berger et al. (28) have stated that ankle displacement and the response of the gastrocnemius increased with increasing height. Allardy et al. (29) and Lee and Lin (30) have reported that ectomorph individuals presented greater postural sway than that shown by endomorph and mesomorph individuals; the authors attributed this difference to a higher center of mass. The greater height in the male group may explain the greater influence of this parameter on balance, in comparison with the female group. The lengths of the upper and lower limbs showed positive correlations with mediolateral displacement, sway velocity and displacement with both EO and EC. In the male group, the upper-limb length and the waist-hip ratio together explained 26% of the balance. In the female group with the EC, there was a correlation between mediolateral displacement and the upper-limb length, which explained 15% of the balance. Molikova et al. (6) have reported that in flexedknee positions (30 ˚ and 60 ˚), the upper limbs exerted a greater influence on the postural balance. The limbs generally follow the format of the body because taller individuals tend to have longer limbs, and a similar correlation can be derived from this association. A greater upper-limb length is correlated with a greater distance between the center of mass and the support base area, similarly to height (5,6,16). The greater the trunk-cephalic length, the worse the balance in relation to all of the variables in the whole group with the EO. With the EC, the mediolateral displacement is possibly related to height (16). In the female group, there was a positive correlation between balance and anteroposterior displacement, which may have been related to the gynoid shape and longer trunk; this difference made balance more difficult for the women because more adjustments were required. In our study population of healthy and normal young adults, the anthropometric parameters had little influence on balance. It does may not be necessary to take anthropometric variables into consideration in studies of static balance using posturography in such populations, with the exception of height. There are methodological limitations caused by the particular multifactorial characteristics of balance. Systems that make integrated assessments of vision, labyrinth activity, and the neuromotor responses associated with posturography and center-of-pressure analysis may be more appropriate for assessing balance in the evaluations of other systems and in assessing situations that are more challenging. Postural balance measured using posturography was little influenced by anthropometric variables, both with the eyes open and closed.

Height was the anthropometric variable that most influenced postural balance in the whole group and grouped by gender. Postural balance was more influenced by anthropometric factors in the male group.

AUTHOR CONTRIBUTIONS Alonso AC conceived the project and participated in the construction of all the phases. Luna MN and Santos S assisted in the data collection and in the manuscript drafting. Mochizuki L assisted in the data analysis and in the final correction of the manuscript. Barbieri F conducted the densitometry examinations and assisted in the data interpretation. D’Andre´a Greve JM organized the work.

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bioelectrical impedance and stabilometric signals in elderly Brazilian women. Clinics. 2011;66(9):1513-8. Chiari L, Rocchi L, Capello A. Stabilometric parameters are affected by anthropometry and foot placement. Clin Biomech. 2002;17:666-77, http://dx.doi.org/10.1016/S0268-0033(02)00107-9. Bankoff ADP, Bekedorf RG, Schmidt A, Ciol P, Zanai CA. Ana´lise do equilı´brio corporal esta´ticoatrave´s de um baropodoˆmetro eletroˆnico. Rev Conexo˜es. 2006;4(2):19-29. Greve J, Alonso A, Bordini ACPG, Camanho GL. Correlation between body mass index and postural balance. Clinics. 2007;62(6):717-20, http:// dx.doi.org/10.1590/S1807-59322007000600010. Lohman TG, Roche AF, Martorell R. Anthropometric standartization reference manual. Champaign IL: USA, Human Kinectics Books 1988, 177p. Vieira TMM, Oliveira LF, Nadal J. An overview of age-related changes in postural control during quiet standing tasks using classical and modern stabilometric descriptors. J Electromyogr Kinesiol. 2009;19(6):e513-9, http://dx.doi.org/10.1016/j.jelekin.2008.10.007. Cavalheiro GL, Almeida MFS, Pereira AAP, Andrade AO. Study of agerelated changes in postural control during quiet standing through Linear Discriminant Analysis. BioMedical Engineering. 2009;8(35):1-13.

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CLINICS 2012;67(12):1443-1446

DOI:10.6061/clinics/2012(12)15

CLINICAL SCIENCE

Serum adenosine deaminase, catalase and carbonic anhydrase activities in patients with bladder cancer Necip Pirinc¸c¸i,I I˙lhan Gec¸it,I Mustafa Gu¨nes¸,I Mehmet Bilgehan Yu¨ksel,II Mehmet Kaba,III Serhat Tanık,III Halit Demir,IV Mehmet AslanV I

Yuzuncu Yıl University, Faculty of Medicine, Department of Urology, Van, Turkey. II Celal Bayar University, Faculty of Medicine, Department of Urology, Manisa, Turkey. III Ministry of Health, Regional Training and Research Hospital, Clinic of Urology, Van, Turkey. IV Yuzuncu Yıl University, Faculty of Science and Art, Department of Chemistry, Van, Turkey. V Yuzuncu Yıl University, Faculty of Medicine, Department of Internal Medicine, Van, Turkey.

OBJECTIVES: The relationship between adenosine deaminase and various cancers has been investigated in several studies. However, serum adenosine deaminase activity and carbonic anhydrase and catalase activities in patients with bladder cancer have not previously been reported. Therefore, the aim of this study was to measure serum adenosine deaminase, carbonic anhydrase and catalase activities in patients with bladder cancer. MATERIALS AND METHODS: Forty patients with bladder cancer and 30 healthy controls were enrolled in the study. Serum adenosine deaminase, carbonic anhydrase and catalase activities were measured spectrophotometrically. RESULTS: Serum adenosine deaminase, carbonic anhydrase and catalase activities were significantly higher in patients with bladder cancer than controls (all significant, p,0.001). CONCLUSIONS: These markers might be a potentially important finding as an additional diagnostic biochemical tool for bladder cancer. KEYWORDS: Adenosine Deaminase; Bladder Cancer; Carbonic Anhydrase; Catalase. Pirinc¸c¸i N, Gec¸it I, Gu¨nes¸ M, Kaba M, Tanık S, Demir H, et al. Serum adenosine deaminase, catalase and carbonic anhydrase activities in patients with bladder cancer. Clinics. 2012;67(12):1443-1446. Received for publication on August 20, 2012; First review completed on September 11, 2012; Accepted for publication on October 19, 2012 E-mail: necippirincci@hotmail.com Tel.: 90 (432) 215 0473

the reversible hydration of carbon dioxide to carbonic acid. CAs have recently become a target of intensive research into carcinogenesis and tumor invasion (4). The relationship between CA and bladder cancer has been investigated in several studies (5-8). In the purine metabolic pathway, adenosine deaminase (AD) is an important aminohydrolase. AD catalyzes the conversion reaction of adenosine to inosine and deoxyadenosine to deoxyinosine (9). Its main physiologic activity is related to lymphocytic proliferation and differentiation (10). The relationship between adenosine deaminase and various cancers has been investigated in several clinical studies, but the results are conflicting. Although enzyme activity was found to be increased in some cancerous tissue (11-13) in several studies, it was typically decreased in various neoplastic tissues (14,15). In addition, Watanabe et al. showed the activity of curcumin in an animal bladder cancer model, which most likely acted via the regulation of nuclear factor-kappa B and p53. Curcumin decreased tumor cell proliferation. Therefore, curcumin is a good choice for treating superficial bladder cancer in clinical trials (16). To the best of our knowledge, serum AD, CA, and CAT activities in patients with bladder cancer have not previously been reported. Therefore, the aim of this study was to simultaneously investigate serum AD, CA, and CAT activities in patients with bladder cancer.

INTRODUCTION Bladder cancer is a common tumor of the urinary tract. It is the fourth most common type of cancer in men in the United States. The most common risk factors for bladder cancer are exposure to industrial carcinogens, cigarette smoking, male gender and, possibly, diet (1). The most common type of bladder cancer develops from the urothelium and is known as transitional cell carcinoma (2). Some defense mechanisms in the body prevent the development of free radicals and the damage they cause. One of the most important antioxidant enzymes is superoxide dismutase (SOD), which catalyzes the dismutation of superoxide anion into hydrogen peroxide (H2O2), which is removed by catalase (CAT) and glutathione peroxidase (GSH-PX). CAT is a well-known plasma antioxidant enzyme (3). Carbonic anhydrase (CA) is a member of the alpha-family of carbonic anhydrases of zinc metalloenzymes that catalyze

Copyright ß 2012 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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MATERIALS AND METHODS

Table 1 - Demographic characteristics of the two groups in this study.

Subjects This prospective cross-sectional study included 40 male patients who were newly diagnosed (preoperative) with bladder cancer (mean age of 65.32¡3.4 years) between January 2011 and October 2011 and treated at the Department of Urology at the University Hospital of Yuzuncu Yil. All patients were lifetime non-smokers and free of drug, alcohol, or antioxidant supplement consumption and any metabolic disease. None of the patients had any other significant disease or malignancies except for bladder cancer, and only newly diagnosed patients (preoperative) with no prior chemotherapeutic treatment were included in this study. Controls consisted of 30 healthy males (mean age of 60.82¡5.2 years) randomly selected from a group of healthy non-smoking volunteers with no history of previous disease or drug or alcohol consumption. The patient and control groups were of similar socioeconomic status. Regarding tumor staging, 30 patients were diagnosed with a non-invasive tumor (Ta–T1), and 10 patients were diagnosed with an invasive tumor (T2–T4). Patients were classified into three groups with respect to tumor grading: 24 patients had well-differentiated tumors (G1), 10 had intermediate tumors (G2), and six had poorly differentiated (G3) tumors. The superficial urothelial papillary tumors were graded according to the 2004 World Health Organization (WHO) grading system as low-grade and high-grade papillary neoplasms. The study protocol was performed in accordance with the Helsinki Declaration as revised in 1989. All participants were informed about the study protocol, and written consent was obtained from each participant.

Parameters

Control (n = 30)

Patients (n = 40)

p-value

60.82¡5.2 22.56¡2.43

65.32¡3.4 23.12¡1.62

ns ns

Age (years) Body mass index (kg/m2) Values are the mean¡SD; ns = non-significant.

Statistical analysis The results are expressed as the mean¡standard deviation. Continuous variables were compared using Student’s t test. Nonparametric continuous variables were compared using a Kruskal–Wallis one-way analysis of variance with a Mann–Whitney U-test post-hoc analysis. The results were considered to be statistically significant when the p-value was less than 0.05. The data were analyzed using the SPSSH for Windows computing program (version 11.0).

RESULTS The demographic and clinical data of the bladder cancer and control groups are shown in Table 1. There were no statistically significant differences between bladder cancer patients and controls with respect to age and body mass index (p.0.05) (Table 1). AD, CA, and CAT activities were detected in all samples. The mean activities of the enzymes are summarized in Table 2. In the healthy controls, the serum AD, CA, and CAT mean values of activities were 16.4¡0.4, 0.65¡0.19, and 1.15¡0.04 U/L, respectively (Table 2). In patients with bladder cancer, the serum AD, CA, and CAT mean values of activities were 26.1¡2.1, 0.96¡0.20, and 1.85¡0.07 U/L, respectively (Table 2). The serum AD, CA, and CAT mean values of activities were significantly higher in patients with bladder cancer than in healthy controls (all significant; p,0.001) (Table 2).

Blood collection Blood samples were obtained in the morning after 12 h of fasting. Blood samples were collected into empty tubes and immediately stored on ice at 4 ˚C. The serum was then separated from the cells by centrifugation at 3,000 rpm for 10 min. Serum samples used for the measurement of AD, CA, and CAT levels were stored at -20 ˚C until they were used.

DISCUSSION In this study, we analyzed serum CA and CAT levels in patients with bladder cancer. In addition, we studied the activity of serum AD, which is an important aminohydrolase in purine metabolism, in patients with bladder cancer. Additionally, we aimed to determine the relationship between these enzyme activities in patients with bladder cancer. We observed that serum AD, CA, and CAT levels were significantly higher in patients with bladder cancer than in healthy controls. To the best of our knowledge, no other studies have examined serum AD, CA, and CAT levels in

Measurement of adenosine deaminase activity Serum AD activity was estimated spectrophotometrically by the method of Giusti, which is based on the direct measurement ammonia formation, which occurs when AD is in the presence of excess adenosine (17). The results were expressed as units per liter (U/L).

Measurement of carbonic anhydrase enzyme activity Serum CA activity was assayed by CO2 hydration. The hydration of CO2 was measured using the method of Rickli and Wilbur-Anderson, with bromothymol blue as the indicator (18). The results were expressed as units per liter (U/L).

Table 2 - Serum adenosine deaminase, carbonic anhydrase and catalase activities in the two groups in this study. Parameters

Measurement of catalase activity

Adenosine deaminase (U/L) Carbonic anhydrase (U/L) Catalase (U/L)

Serum CAT activity was measured using H2O2 as a substrate (19). The degradation of H2O2 was monitored at 240 nm for 5 min using a spectrophotometer, and enzyme activity was expressed in units per liter of serum (U/L) at 25 ˚C.

Values are the mean¡SD. Significant, p,0.001.

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Control (n = 30) Patients (n = 40) 16.4¡0.4 0.65¡0.19 1.15¡0.04

26.1¡2.1 0.96¡0.20 1.85¡0.07

p-value ,0.001 ,0.001 ,0.001


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compared with adjacent cancer-free tissues and control bladder tissues. In contrast, Bayraktar et al. (31) recently reported that patients with bladder cancer had increased CAT levels. Several studies have shown decreased CAT levels in patients with lung cancer (27,28). Moreover, Corrocher et al. (29) reported decreased CAT activity in human hepatoma and suggested that the antioxidant defense system of hepatocellular carcinoma cells was severely impaired. In contrast, Bayraktar et al. (31) recently reported that patients with bladder cancer had increased CAT levels. The higher CAT activity observed in this study supports previous observations that the enzymatic antioxidant defense mechanism is not impaired in tumor cells and tissue, although this was not a universal characteristic of neoplastic cells. In the present study, we found significantly increased CAT activity in the serum of bladder cancer patients. Our data clearly demonstrate that CAT activity is increased in patients with bladder cancer, and thus, our results are not in agreement with Durak et al. (13). However, our results are in agreement with Bayraktar et al. (31). CAs are involved in several physiological and biological processes in humans. CA is not only a highly active enzyme but is also involved in cell-cell adhesion and cell proliferation (32), and it plays different roles in various tissues. Species can produce many different CA isozymes, some of which act in the cytosol, while others are membrane bound. For example, in humans, there are three cytosolic isozymes (CA-I, II, and III), five membrane-bound isozymes, and several related proteins that lack catalytic activity. CA-I is found primarily in erythrocytes. The human CA-II isozyme is widely distributed, and it has been identified in erythrocytes and the brain, eyes, and kidneys (4). Carbonic anhydrase IX (CAIX) is a hypoxia-inducible member of the carbonic anhydrase family that regulates intracellular pH, cell proliferation, cell adhesion, and tumor progression. Studies have demonstrated that high CAIX expression yields an aggressive tumor phenotype and a poor prognosis in many cancer types. However, with regard to bladder cancer, there have been only a few reports on a limited number of patients, and those reports produced inconclusive results (5,7,8). CAs are key enzymes that regulate acidbase homeostasis under both normal and pathological conditions (33). CA is abundantly expressed as a direct consequence of hypoxia in numerous cancers (34), and its activity has been documented in some cancerous tissues (5,7,8). However, there is limited information regarding serum CA activity in patients with bladder cancer (6). In the present study, we found significantly increased CA activity in the serum of bladder cancer patients. The current study is the first to investigate serum AD, CA, and CAT activities in patients with bladder cancer. These markers might be potentially important as an additional biochemical diagnostic tool for bladder cancer. Further investigations in a larger cohort of patients with bladder cancer are needed to provide definitive data about the prognostic role of AD, CA, and CAT activities.

patients with bladder cancer. Increased serum AD, CA, and CAT activities may play a role in the pathogenesis of bladder cancer. Therefore, we believe that serum AD, CA, and CAT levels may be an informative prognostic marker for bladder cancer. The activities of AD, CAT, and CA in patients with bladder tumors can also be measured in the urine. The follow-up to determine recurrence, particularly after the treatment of bladder tumors, may benefit from determining the activities of this enzyme before cystoscopy. AD, which catalyzes the reaction in which adenosine is deaminated to inosine, has been accepted as an important enzyme in the maturation, proliferation, differentiation, and function of T lymphocytes (20). As an indicator of cellular immunity, the plasma activity of this enzyme has been suggested to be increased in diseases that cause a cellmediated immune response, such as cancer (21). Some studies have confirmed the value of estimating AD in the diagnosis and follow-up of patients with tuberculosis (22). Recent studies have focused on the diagnostic value of AD activity in typical (10,23) and atypical (24) pneumonia. AD activity was increased in cancerous tissues and cells compared to noncancerous tissue in several studies (11-13). Some authors, however, found low lymphocyte AD activities in cancer patients (14,15). Sufrin et al. (13) found that the AD levels in lymphocytes from patients with bladder cancer were elevated with transitional cell carcinoma and correlated with stage, activity, clinical course, and tumor resection but not tumor grade. These researchers also found higher erythrocyte AD activities in the same patients and suggested that lymphocyte AD levels might be a sensitive indicator of bladder carcinoma. The high AD activity determined in cancerous bladder tissue reflected accelerated purine turnover and high salvage pathway activity (12). Similarly, Durak et al. (13) found increased AD activity in cancerous bladder tissues compared with adjacent cancerfree tissues and control bladder tissues. Our results are in agreement with those studies (12,13). In contrast, Lal et al. (25) demonstrated that the increase in serum AD activity was directly related to the cancer stage, indicating that the increase was directly proportional to the primary tumor mass. Moreover, Nishihara et al. (26) reported that patients with lung cancer had elevated AD levels and that serum AD levels were significantly reduced in patients with lung cancer following surgery and radiotherapy. In contrast, Dasmahapatra et al. (14) and Kojima et al. (15) found low lymphocyte AD activities in head and neck cancer patients and gastric cancer patients, respectively. They suggested that low lymphocyte AD activities might be a more sensitive indicator of suppressed cellular immunity. In the present study, we observed that serum AD activity was significantly higher in patients with bladder cancer than in healthy controls. Increased AD activity might provide a selective advantage for cancer cells to grow and develop more rapidly. We speculate that increased AD activity might be a result of the leakage of the enzyme from primary tumor cells. CAT is highly expressed in some tissues and protects cells against the excessive formation of reactive oxygen species. CAT prevents the accumulation of H2O2 formed during oxygen transport. Although there is accumulating evidence that CAT activity is suppressed in cancerous tissues (13,2729), some authors found unchanged or increased activity in some tumor tissues (30,31). Durak et al. (13) found decreased CAT activity in cancerous bladder tissues

AUTHOR CONTRIBUTIONS Pirinc¸c¸i N conceived and designed the study and was also responsible for the draft of the manuscript. Gec¸it I was responsible for the data collection, critical revision and important intellectual content of the manuscript. Gu¨nes¸ M was responsible for the data collection. Yu¨ksel MB was responsible for the statistical analysis. Kaba M and Tank S were

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17. Giusti G. Adenosine deaminase. In: Bergmeyer MV, editor. Methods of enzymatic analysis. New York: Academic Press, 1974. p. 1092-8. 2nd ed. 18. Rickli EE, Ghazanfar SAS, Gibbons BH, Edsall JT. Carbonic anhydrase from human erytrocytes. Preparation and properties of two enzymes. J Biol Chem. 1964;239:1065-78. 19. Aebi H. Catalase. In: Bergmayer HU, editor. Methods of enzymatic analysis. New York: Academic Press Inc 1974; p. 673-7. 20. Pacheco R, Martinez-Navio JM, Lejeune M, Climent N, Oliva H, Gatell JM, et al. CD26, adenosine deaminase, and adenosine receptors mediate costimulatory signals in the immunological synapse. Proc Natl Acad Sci. USA. 2005;102(27):9583-8, http://dx.doi.org/10.1073/pnas.0501050102. 21. Piras MA, Gakis C, Budroni M, Andreoni G. Adenosine deaminase activity in pleural effusions: an aid to differential diagnosis. Br Med J. 1987;2(6154):1751-2. 22. Collazos J, Espana P, Mayo J, Martı´nez E, Izquierdo F. Sequential evaluation of serum adenosine deaminase in patients treated for tuberculosis. Chest. 1998;114(2):432-5, http://dx.doi.org/10.1378/chest. 114.2.432. 23. Klockars M, Kleemola M, Leinonen M, Leinonen M, Koskela M. Serum adenosine deaminase in viral and bacterial pneumonia. Chest. 1991;99(3):623-6, http://dx.doi.org/10.1378/chest.99.3.623. 24. Molinos L, Fernandez R, Dominguez MJ, Riesgo C, Escudero C, Martinez J. Adenosine deaminase activity in the aetiological diagnosis of community-acquired pneumonia. Scand J Infect Dis. 1997;29(3):287-90. 25. Lal H, Munjial SK, Wig U, Saini AS. Serum enzymes in head and neck cancer III. J Laryngol Otol. 1987;101(10):1062-5, http://dx.doi.org/10. 1017/S0022215100103226. 26. Nishihara H, Akedo H, Okada H, Hattori S. Multienzyme patterns of serum adenosine deaminase by agar gel electrophoresis: An evaluation of the diagnostic value in lung cancer. Clin Chim Acta. 1970;30(2):251-8. 27. Cobanoglu U, Demir H, Duran M, S¸ehitogullari A, Mergan D, Demir C. Erythrocyte catalase and carbonic anhydrase activities in lung cancer. Asian Pac J Cancer Prev. 2010;11(5):1377-82. 28. Korotkina RN, Matskevich GN, Devlikanova Ash, Vishnevskii AA, Kunitsyn AG, Karelin AA. Activity of glutathione-metabolizing and antioxidant enzymes in malignant and benign tumors of human lungs. Bull Exp Biol Med. 2002;133(6):606-8, http://dx.doi.org/10.1023/ A:1020206514239. 29. Corrocher R, Casaril M, Bellisola G, Gabrielli GB, Nicoli N, Guidi GC, et al. Severe impairment of antioxidant system in human hepatoma. Cancer. 1986;58(8):1658-62, http://dx.doi.org/10.1002/1097-0142(19861015)58:8,1658 ::AID-CNCR2820580814.3.0.CO;2-7. 30. Nakada T, Akiya T, Koike H, Katayama T. Superoxide dismutase activity in renal cell carcinoma. Eur Urol. 1988;14(1):50-5. 31. Bayraktar N, Kilic S, Bayraktar MR, Aksoy N. Lipid peroxidation and antioxidant enzyme activities in cancerous bladder tissue and their relation with bacterial infection: a controlled clinical study. J Clin Lab Anal. 2010;24(10):25-30, http://dx.doi.org/10.1002/jcla.20356. 32. Sly WS, Hu PY. Human carbonic anhydrases and carbonic anhydrase deficiencies. Annu Rev Biochem. 1995;64:375-401, http://dx.doi.org/10. 1146/annurev.bi.64.070195.002111. 33. Sayir F, Kavak S, Meral I, Demir H, Cengiz N, C ¸ obanog˘lu U. Effect of crush and axotomy of phrenic nerves on oxidative stress in diaphragm muscle of rats. Muscle Nevre. 2012;45(3):412-5, http://dx.doi.org/10. 1002/mus.22312. 34. Ivanov S, Liao SY, Ivanova A, Danilkovitch-Miagkova A, Tarasova N, Weirich G, et al. Expression of hypoxia-inducible cell surface transmembrane carbonic anhydrases in human cancer. Am J Pathol. 2001; 158(3):905-19.

responsible for the data collection. Demir H conceived and designed the study and was responsible for the critical revision and important intellectual content of the manuscript. Aslan M was responsible for the administrative support.

REFERENCES 1. Macvicar AD. Bladder cancer staging. BJU Int. 2000;86 Suppl 1:111-22. 2. Sharma S, Ksheersagar P, Sharma P. Diagnosis and treatment of bladder cancer. Am Family Physician. 2009;80(7):717-23. 3. Jimenez P, Piazuelo E, Sanchez MT, Ortego J, Soteras F, Lanas A. Free radicals and antioxidant systems in reflux esophagitis and Barrett’s esophagus. World J Gastroenterol. 2005;11(18):2697-703. 4. Demir C, Demir H, Esen R, Atmaca M, Tagdemir E. Erythrocyte catalase and carbonic anhydrase activities in acute leukemias. Asian Pac J Cancer Prev. 2010;11(1):247-50. 5. Malentacchi F, Vinci S, Melina AD, Kuncova J, Villari D, Giannarini G, et al. Splicing variants of carbonic anhydrase IX in bladder cancer and urine sediments. Urol Oncol. 2012;30(3):278-84. 6. Hyrsl L, Zavada J, Zavadova Z, Kawaciuk I, Vesely S, Skapa P. Soluble form of carbonic anhydrase IX (CAIX) in transitional cell carcinoma of urinary tract. Neoplasma. 2009;56(4):298-302, http://dx.doi.org/10.4149/ neo_2009_04_29. 7. Klatte T, Seligson DB, Rao JY, Yu H, de Martino M, Kawaoka K, et al. Carbonic anhydrase IX in bladder cancer: a diagnostic, prognostic, and therapeutic molecular marker. Cancer. 2009;115(7):1448-58, http://dx .doi.org/10.1002/cncr.24163. 8. Hussain SA, Palmer DH, Ganesan R, Hiller L, Gregory J, Murray PG, et al. Carbonic anhydrase IX, a marker of hypoxia: correlation with clinical outcome in transitional cell carcinoma of the bladder. Oncol Rep. 2004;11(5):1005-10. 9. Gulec M, Akin H, Yuce H, Ergin E, Elyas H, Yalc¸in O, et al. Adenosine deaminase and xanthine oxidase activities in bladder washing fluid from patients with bladder cancer: a preliminary study. Clin Biochem. 2003;36(3):193-6, http://dx.doi.org/10.1016/S0009-9120(02)00452-6. 10. Akyol O, Gokbulut I, Koksal N, Akin H, Ozyurt H, Yildirim Z. The activities of purine catabolizing enzymes in plasma and bronchial washing fluid in patients with lung cancer and pneumonia. Clin Biochem. 2001;34(3):251-4, http://dx.doi.org/10.1016/S0009-9120(01)00203-X. 11. Camici M, Tozzi MG, Allegrini S, Del Corso A, Sanfilippo O, Daidone MG, et al. Purine salvage enzyme activity in normal and neoplastic human tissues. Cancer Biochem Biophys. 1990;11(3):201-9. 12. Sufrin G, Tritsch GL, Mittelman A, Murphy GP. Adenosine deaminase activity in patients with carcinoma of the bladder. J Urol. 1978;119(3):343-6. 13. Durak I, Perk H, Kavutc¸u M, Canbolat O, Akyol O, Bedu¨k Y. Adenosine deaminase, 5’nucleotidase, xanthine oxidase, superoxide dismutase, and catalase activities in cancerous and noncancerous human bladder tissues. Free Radic Biol Med. 1994;16(6):825-31, http://dx.doi.org/10.1016/08915849(94)90199-6. 14. Dasmahapatra KS, Facs HZH, Dasmahapatra A, Suarez S. Evaluation of adenosine deaminase activity in patients with head and neck cancer. J Surg Res. 1986;40(4):368-73, http://dx.doi.org/10.1016/0022-4804(86)90201-5. 15. Kojima O, Majima T, Uehara Y, Yamane T, Fujita Y, Takahashi T, et al. Alteration of adenosine deaminase levels in peripheral blood lymphocytes of patients with gastric cancer. Jpn J Surg. 1985;15(2):130-3. 16. Watanabe FT, Chade DC, Reis ST, Piantino C, Dall’ Oglio MF, Srougi M, et al. Curcumin, but not Prima-1, decreased tumor cell proliferation in the syngeneic murine orthotopic bladder tumor model. Clinics. 2011; 66(12):2121-4, http://dx.doi.org/10.1590/S1807-59322011001200019.

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DOI:10.6061/clinics/2012(12)16

BASIC RESEARCH

Antioxidant enzyme activity and malondialdehyde levels can be modulated by Piper betle, tocotrienol rich fraction and Chlorella vulgaris in aging C57BL/6 mice Nor Syahida Aliahmat, Mohd Razman Mohd Noor, Wan Junizam Wan Yusof, Suzana Makpol, Wan Zurinah Wan Ngah, Yasmin Anum Mohd Yusof Department of Biochemistry, Faculty of Medicine, Universiti Kebangsaan, Malaysia, Jalan Raja Muda Abdul Aziz, Malaysia

OBJECTIVE: The aim of this study was to determine the erythrocyte antioxidant enzyme activity and the superoxide dismutase, catalase, glutathione peroxidase, and plasma malondialdehyde levels in aging mice and to evaluate how these measures are modulated by potential antioxidants, including the tocotrienol-rich fraction, Piper betle, and Chlorella vulgaris. METHOD: One hundred and twenty male C57BL/6 inbred mice were divided into three age groups: young (6 months old), middle-aged (12 months old), and old (18 months old). Each age group consisted of two control groups (distilled water and olive oil) and three treatment groups: Piper betle (50 mg/kg body weight), tocotrienol-rich fraction (30 mg/kg), and Chlorella vulgaris (50 mg/kg). The duration of treatment for all three age groups was two months. Blood was withdrawn from the orbital sinus to determine the antioxidant enzyme activity and the malondialdehyde level. RESULTS: Piper betle increased the activities of catalase, glutathione peroxidase, and superoxide dismutase in the young, middle, and old age groups, respectively, when compared to control. The tocotrienol-rich fraction decreased the superoxide dismutase activity in the middle and the old age groups but had no effect on catalase or glutathione peroxidase activity for all age groups. Chlorella vulgaris had no effect on superoxide dismutase activity for all age groups but increased glutathione peroxidase and decreased catalase activity in the middle and the young age groups, respectively. Chlorella vulgaris reduced lipid peroxidation (malondialdehyde levels) in all age groups, but no significant changes were observed with the tocotrienol-rich fraction and the Piper betle treatments. CONCLUSION: We found equivocal age-related changes in erythrocyte antioxidant enzyme activity when mice were treated with Piper betle, the tocotrienol-rich fraction, and Chlorella vulgaris. However, Piper betle treatment showed increased antioxidant enzymes activity during aging. KEYWORDS: Aging; Antioxidant Enzymes; Piper Betle; Tocotrienol-Rich Fraction; Chlorella Vulgaris. Aliahmat NS, Noor MR, Yusof WJ, Makpol S, Ngah WZ, Yusof YA. Antioxidant enzyme activity and malondialdehyde levels can be modulated by Piper betle, tocotrienol rich fraction and Chlorella vulgaris in aging C57BL/6 mice. Clinics. 2012;67(12):1447-1454. Received for publication on July 18, 2012; First review completed on August 13, 2012; Accepted for publication on August 13, 2012 E-mail: anum@medic.ukm.my Tel.: 603-92897297

species (ROS) such as superoxide anions (O22), hydrogen peroxide (H2O2) and hydroxyl radicals (OH2) (3). These cytotoxic species can cause irreversible oxidative damage to biologic molecules, such as lipids, proteins, and DNA in the cells, which affect enzyme activity and membrane function (4,5). Fortunately, the accumulation of ROS is controlled in vivo by a wide spectrum of non-enzymatic antioxidant systems, such as bilirubin; glutathione (GSH); vitamins A, C, and E; and defense-related enzymes such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) (6). The activity of antioxidant enzymes in response to the accumulation of ROS is equivocal, and both increased and decreased activities have been reported during the aging

INTRODUCTION Free oxygen radicals have been proposed as important causative agents of aging. The theory of aging associated with the accumulation of free radicals, which damage cells and tissues, was suggested by Harman (1,2). The univalent reduction of molecular oxygen results in reactive oxygen

Copyright Ă&#x; 2012 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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was conducted at the Department of Biochemistry, Faculty of Medicine, Universiti Kebangsaan Malaysia (UKM), Kuala Lumpur, Malaysia. The mice were randomly divided into three main groups according to their age. These groups were 6-month-old (young) mice, 12-month-old (middle-aged) mice, and 18month-old (old) mice. Each group was further divided into four groups, which were two control and three treatment groups. Each group consisted of eight mice. The mice in the control group for the PB and the CV groups were given distilled water, and the mice in the control group for the TRF group were given olive oil. Both controls were given to rats by gavage. Mice in the treatment groups were given PB (50 mg/kg body weight), CV (50 mg/kg body weight), and TRF (30 mg/kg body weight) each day via gavage (forced feeding). Each treatment was administered for two months for each age group. For each age group, blood was collected from the mice, after the two-month treatment period to analyze antioxidant enzymes and oxidative stress marker analysis (SOD, GPx, CAT and MDA). Blood was collected from the orbital sinus and maintained on ice in tubes containing heparin (an anticoagulant) followed by centrifugation at 3,000 x g for 15 min at 4 ˚C. The top yellow plasma layer was pipetted without disturbing the white buffy layer and was stored at -20 ˚C for later analysis of MDA. The white buffy layer (leukocytes) was removed. The erythrocytes (red blood cells) were washed with normal saline and centrifuged at 3,000 x g for 15 min at 4 ˚C, which was repeated four times until the supernatant was clear. The erythrocytes were aliquoted into several microcentrifuge tubes and stored at -20 ˚C for later determination of antioxidant enzyme activity.

processes (4). However, if this defense mechanism fails to control the increased levels of ROS, oxidative stress occurs and is capable of injuring membrane lipids, proteins and nucleic acids (7). Increased ROS react with polyunsaturated fatty acids to induce the release of toxic and reactive aldehyde metabolites, such as MDA, which is one of the end products of the lipid peroxidation process (8,9). Lipid peroxidation is associated with aging and a variety of chronic health diseases, such as cancer and atherosclerosis (10-12). Numerous studies suggest that antioxidants can prevent the oxidation of various macromolecules, such as DNA, proteins, and lipids, thus preventing the aging process and increasing the lifespan of the organism (13,14). In this study, we investigated three types of antioxidants: Piper betle (PB), the tocotrienol-rich fraction (TRF), and Chlorella vulgaris (CV). Piper betle L. (Piperaceae) or Sireh leaves have a strong, pungent aromatic flavor and are widely used as masticatory agents in Asia. PB has a high antioxidant capacity and contains several bioactive compounds, such as hydroxychavicol, eugenol, chavibetol, and allylpyrocatechol (15,16). PB also has significant antioxidant activity and can modulate antioxidant enzyme activity, such as that of SOD, GPx, and CAT, in radiation-induced lipid peroxidation in Swiss albino mice (17,18). Tocotrienols extracted from crude palm oil consist mainly of a mixture of a, b, c, and d tocotrienols and some a tocopherols; this extract is referred to as the tocotrienol-rich fraction (TRF). Treatment with the TRF results in lifespan extension and a reduction in the age-dependent accumulation of protein carbonyls in Caenorhabditis elegans (19). The TRF also prevents oxidative damage by inhibiting protein and lipid peroxidation in rat liver microsomes (20). Chin et al. (21) reported that long-term supplementation with the TRF can modulate antioxidant enzymes, such as SOD, GPx, and CAT, in older humans, reduce protein damage and improve levels of advanced glycosylation end (AGE) products. Chlorella vulgaris, a unicellular green algae, has been widely used as a food additive (22) and has nutritive value (23) in addition to other medicinal benefits. An in vitro study revealed the antitumor effects of CV, which inhibits the proliferation of HepG2 liver cancer cells and induces apoptosis (24). CV reduced the number of tumors in ethionine-induced liver cancer in rats and modulated the antioxidant enzyme activity and blood malondialdehyde (MDA) levels (25). Although many studies have demonstrated the antioxidant potential of PB, TRF, and CV, limited studies have addressed their effects in association with aging. Therefore, we embarked on this study to evaluate whether PB, TRF, and CV can modulate oxidative stress that is induced by the accumulation of ROS and MDA during aging in mice.

Preparation of PB extract Dried PB leaves were purchased from Ethno Resources Company (Sungai Buloh, Selangor, Malaysia). The identification and voucher number (UKMB 29768) of the plants was obtained from Herbarium, UKM, Bangi. The dried PB leaves were grounded into powder, and a 10% concentration of PB was prepared. Aqueous PB extractions were prepared using a Soxhlet Extractor (Eyela, Japan) according to the method of Pin et al. (26) with some modifications. Finally, the aqueous PB extract was dried into a powdered form using a freeze dryer (Labconco, Kansas City, MO, USA). The powdered PB was diluted in distilled water to yield a concentration of 50 mg/kg body weight before being used throughout the experiment.

Preparation of the TRF The TRF (70%) was purchased from Golden Hope Bioganic (Selangor, Malaysia). The TRF consists of atocopherol (149.2 mg/g), a- tocotrienol (164.7 mg/g), btocotrienol (48.6 mg/g), c-tocotrienol (213.2 mg/g), and dtocotrienol (171.9 mg/g). The TRF was diluted in olive oil to yield a concentration of 30 mg/kg body weight before being used throughout the experiment. This concentration was used based on our earlier laboratory findings that long-term supplementation of 30 mg/kg body weight of palm oil vitamin E reduced elevated levels of MDA in rats during aging (27).

METHODS Animal model and experimental design A total of 120 male C57BL/6 inbred mice were purchased from bioLASCO, Taiwan Co., Ltd (Taipei, Taiwan). The mice were maintained in polycarbonate cages placed in a room with a controlled temperature, controlled humidity and a 12 h light-dark cycle. The study was approved by the Animal Ethics Committee of the Faculty of Medicine, UKM (FP/ BIOK/2008/YASMIN/13-FEB/216-FEB-2008-DEC-2010) and

Preparation of CV The stock CV Beijerinck (strain 072) was obtained from the University of Malaya Algae Culture Collection (UMACC,

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decompose H2O2 in phosphate buffer, at pH 7.0, in 1 sec of reaction.

Malaysia) and was grown in Bold Basal Media (BBM) (12-h light-dark cycle). The algae slurry was centrifuged three times at 3,000 rpm for 10 min at 4 ˚C to separate the algae from the medium. The pelleted algae were then diluted in distilled water at a concentration of 50 mg/kg body weight before being used throughout the experiment. This concentration was used based on our previous study in rats in which 50 mg/kg body weight of CV exhibited sufficient anti-tumor and antioxidant effects in liver cancer-induced rats (25).

Measurement of hemoglobin The hemoglobin levels were determined through the quantitative determination of cyanmethemoglobin in the blood. Ferricyanide oxidizes oxyhemoglobin to methemoglobin, and cyanide converts methemoglobin to cyanmethemoglobin. The absorbance measurements were recorded at 540 nm. The cyanmethemoglobin reagent (Eagle Diagnostic, St. Louis, USA) contains a surfactant to promote rapid hemolysis and to accelerate the formation of cyanmethemoglobin. The reaction was completed in 3 min.

Measurement of antioxidant enzyme activity SOD activity was measured following the method proposed by Beyer & Fridovich (28). The assay mixture contained 50 mmol/l potassium phosphate buffer at a pH of 7.8, 0.1 mmol/l EDTA, 9.9 mmol/l L-methionine, 5.761025 mol/ l nitro blue tetrazolium (NBT), and 2.561022% (w/v) Triton X100. Approximately 0.02 ml of the sample (red blood cells) was added to 1.0 ml of the assay mixture. Riboflavin (0.01 ml of 4.4%) was added last to initiate the reaction. The reduction of NBT was measured at 560 nm in a Shimadzu-UV 160A Spectrometer (Kyoto, Japan). The SOD activity was expressed in units of enzyme/gram hemoglobin. One unit of SOD corresponds to the enzyme concentration required to inhibit the chromogen produced (NBT) by 50% in 1 min under standard conditions. GPx activity was measured according to the method proposed by Paglia & Valentine (29). The activity was assayed with a coupled enzyme system in which the oxidized GSH reduction was coupled to nicotinamide adenine dinucleotide phosphate (NADPH) oxidation by GSH reductase. The oxidation of NADPH was measured spectrometrically at 340 nm. The assay mixture contained 0.05 mol/l phosphate buffer at a pH of 7.0, 5 mmol/l EDTA, 2 mmol/l NaN3, 1 mmol/l GSH, 0.2 mmol/l NADPH, and 4 mg GSH reductase. The sample (0.02 ml) was added to 2.88 ml of the assay mixture. The reaction was initiated by the addition of 0.1 ml of 2.5 mmol/l H2O2. A blank assay with buffer instead of a sample was used to correct for any non-enzymatic oxidation of GSH and NADPH by peroxide. GPx activity was expressed in milliunits/milligram hemoglobin. One specific unit is defined as 1 mmol of NADPH needed with 1 g of hemoglobin to be converted to NADP in 1 min of reaction. CAT activity was measured according to the method proposed by Aebi (30). In the ultraviolet range, H2O2 shows a continual increase in absorption with a decreasing wavelength. The decomposition of H2O2 can be followed directly by the decrease in the absorbance at 240 nm. The difference in absorbance (DA240) per unit time is a measure of CAT activity. The reagents used were a phosphate buffer (50 mmol/l, pH 7.0) and 30 mmol/l H2O2 in a phosphate buffer, which was prepared fresh before each assay. Hemolysate was obtained by adding 0.1 ml of red blood cells to 0.4 ml of distilled water. A 1:500 dilution of this concentrated hemolysate was prepared with phosphate buffer immediately before the assay was performed. The reaction was initiated by the addition of 1 ml of 30 mmol/l H2O2 to 2 ml of diluted hemolysate. A blank assay with buffer instead of substrate and 2 ml of hemolysate was used to correct for any non-enzymatic reaction. The absorbance was observed for approximately 30 sec. The CAT activity is defined in specific units/milligram hemoglobin. One unit of CAT corresponds to the amount of enzyme needed to

Measurement of plasma MDA The LPO product, MDA, was quantified using the method proposed by Pilz et al. (31) with some modifications by Sim et al. (32). The principle of the test is based on the derivation of MDA with 2,4-dinitrophenylhydrazine and its subsequent conversion into pyrazole and hydrazone derivatives, which are then separated using high-performance liquid chromatography (HPLC). This method allows for a more specific estimation of MDA compared to the thiobarbituric acid reactive substances (TBARS) method. HPLC analyses were performed on a LC-10 AT VP Shidmadzu (Kyoto, Japan) liquid chromatography system equipped with a diode array detector and an auto injection valve. An a-bond C18 125A column (3.96150 mm) with a 5 mm particle size (Alltech, Deerfield, Illinois, USA) was used. A Shidmadzu Class-VP software system controlled the equipment and was utilized for data processing. Elution was performed isocratically with a mixture of 0.2% (v/v) acetic acid in MilliQ water and acetonitrile (62:38) (v/v) at a flow rate of 0.6 ml/min at room temperature. The chromatograms were acquired at 310 nm.

Standard and sample preparation for MDA A standard of 1,1,3,3-tetraetoxypropane (TEP) was used. A stock solution of MDA was obtained as follows: 25 ml TEP was dissolved in 100 ml of deionized water to yield a 1 mmol/l stock solution. The MDA was prepared by hydrolysis of 1 ml TEP stock solution in 50 ml of 1% sulfuric acid and was incubated for 2 h at room temperature. The solution was stored at 40 ˚C and used within four weeks. The resulting MDA standard of 20 nmol/ml was further diluted with 1% sulfuric acid to yield different concentrations from 1 to 20 nmol/ml of MDA. An aliquot of 250 ml diluted standard or plasma was placed in a 1.5-ml Eppendorf tube, and 50 ml of 6 M aqueous sodium hydroxide was added. This mixture was incubated in a 60 ˚C water bath for 30 min to achieve the alkaline hydrolysis of the protein-bound MDA. The protein was precipitated by adding 125 ml of 35% (v/v) perchloric acid, and the mixture was centrifuged at 60006g for 10 min. The entire volume of the supernatant was transferred to an Eppendorf vial and mixed with 50 ml 2,4-dinitrophenylhydrazine prepared as a 5 mM solution in 2 M hydrochloric acid. Finally, this reaction mixture was incubated for 30 min at room temperature and was protected from light. An aliquot of 40 ml of this reaction mixture was injected into the HPLC system. The retention time for the MDA was detected at the 8th minute, and the area under the peak represented the amount of MDA in 40 ml of the reaction mixture. A serial

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concentration of the standard was generated as a reference curve.

When treated with CV, no significant changes were observed for SOD activity for all age groups (p.0.05), while GPx activity was increased significantly in the middle age group (p,0.05). In contrast, CAT activity decreased significantly in the young age group (p,0.05) when compared with the control group (Figure 3). Lipid peroxidation levels in plasma during aging in response to PB, TRF, and CV treatments. MDA is a measurement for lipid peroxidation of cell membranes. We noted no significant changes in MDA for all age groups in both distilled water and olive oil control groups and the PB treatment groups. However, CV treatment reduced lipid peroxidation in all age groups (p,0.05). The TRF treatment reduced lipid peroxidation in the middle-aged group (p,0.05) when compared with the control group (Figure 4).

Statistical analysis The results are expressed as the means¡standard error of the mean (SEM) and were analyzed using a one-way ANOVA with Statistical Package for Social Sciences (SPSS) version 13.0. p,0.05 was accepted as statistically significant.

RESULTS SOD, GPx and CAT activities in the erythrocytes during aging in response to PB treatment In the distilled water control group, the SOD activity decreased significantly (p,0.05) in the old age group, while no significant changes were observed for GPx and CAT activities (p.0.05) as the mice aged. When treated with PB, SOD activity increased in the old age group (p,0.05), and the GPx activity increased significantly in the middle and old age groups (p,0.05). In contrast, CAT activity was significantly increased in the young age group (p,0.05) when compared with the distilled water control group (Figure 1). SOD, GPx, and CAT activities in the erythrocytes during aging in response to TRF treatment. Total SOD and GPx activities decreased significantly in the old age group (p,0.05), while no significant changes were observed in the CAT activity (p.0.05) in the olive oil control group (olive oil was used as the vehicle for the TRF). When treated with the TRF, SOD activity decreased significantly in the middle and old age groups (p,0.05), while no significant changes were found in the GPx and CAT activities (p.0.05) when compared with the control group (Figure 2). SOD, GPx, and CAT activities in the erythrocytes during aging in response to CV treatment.

DISCUSSION Aging is normally associated with an increased level of reactive oxygen species (ROS), which cause severe damage to cells and tissues. An imbalance between the formation and elimination of ROS and the development of oxidative stress plays a vital role in age-associated diseases, such as cancer, Alzheimer’s, and heart disease (33,34). In the present study, we investigated three plant extracts (PB, TRF, and CV) in association with aging. These extracts possess certain degrees of antioxidant potential by scavenging ROS (25,35,36). Without any antioxidant treatment, we noted that SOD activities decreased with age in mice in both control groups (water and olive oil). Anderson et al. (37) reported an age-related decrease in SOD activity in human erythrocytes. The inactivation of SOD is also associated with aging in rats (38). The decreased SOD activity could be explained by an irreversible inactivation of SOD by its product, H2O2, in a concentration-dependent manner (39).

Figure 1 - Effect of Piper betle (PB) on SOD, GPx and CAT levels in the erythrocytes of mice with different ages. Values are the mean ¡ S.E.M. for 8 mice. a-c: significantly different (p,0.05) from the control at 6 M, 12 M, and 18 M, respectively. d-f: significantly different (p,0.05) from PB at 6 M, 12 M, and 18 M, respectively. Superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT), hemoglobin (Hb), 6 month old (6 M), 12 month old (12 M), and 18 month old (18 M).

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Piper betle modulates antioxidant enzymes Aliahmat NS et al.

Figure 2 - Effect of the tocotrienol-rich fraction (TRF) on erythrocyte SOD, GPx and CAT levels in mice with different ages. Values are the mean ¡ S.E.M. for 8 mice. a-c: significantly different (p,0.05) from the control at 6 M, 12 M, and 18 M, respectively. d-f: significantly different (p,0.05) from TRF at 6 M, 12 M, and 18 M, respectively.

H2O2 is usually removed by catalase and GPx in erythrocytes; however, the enzyme levels decreased with age (40). This decrease was also observed in our study, where GPx activity decreased with age, while CAT activity did not change with age. Apparently, CAT and GPx function in

parallel pathways (21). A decrease in GPx’s H2O2 salvaging activity is usually compensated for by CAT. Conversely, Inal et al. found that GPx and CAT activities increased during aging in healthy older adults (4). Chin et al. reported that GPx activity increased while CAT activity decreased in

Figure 3 - Effect of Chlorella vulgaris (CV) on erythrocyte SOD, GPx and CAT levels in mice with different ages. Values are the mean ¡ S.E.M. for 8 mice. a-c: significantly different (p,0.05) from the control at 6 M, 12 M and 18 M, respectively. d-f: significantly different (p,0.05) from CV at 6 M, 12 M, and 18 M, respectively.

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Figure 4 - Effect of PB, TRF, and CV on the plasma malondialdehyde (MDA) level in mice with different ages. Values are the mean ยก S.E.M. for 8 mice. a-c: significantly different (p,0.05) from the control at 6 M, 12 M, and 18 M, respectively. d-f: significantly different (p,0.05) from TRF at 6 M, 12 M, and 18 M, respectively. g-i: significantly different (p,0.05) from PB at 6 M, 12 M, and 18 M, respectively. j-l: significantly different (P,0.05) from CV at 6 M, 12 M, and 18 M, respectively.

The results from the present study concur with the findings of earlier investigation, in which PB elevated the antioxidant enzyme status in radiation-induced lipid peroxidation of Swiss albino mice (18). However, with TRF treatment, we noted a significant increase in SOD activity only in the old age group, while no changes were noted for the GPx or the CAT activity. One plausible explanation is that TRF is a powerful antioxidant that strongly scavenges most of the free radicals that accumulate in the body, thereby compensating the endogenous antioxidant potential in mice (21). However, CV treatment had no effect on SOD activity with age; conversely, it increased GPx activity in the middle-aged group and decreased CAT activity in the young group. No significant changes in MDA were observed for either the controls or the PB treatment for all age groups. CV treatment reduced lipid peroxidation in all age groups. This result is in agreement with our previous finding that CV has a protective effect in rats induced with liver cancer by reducing elevated MDA levels (25). The TRF decreased the elevated MDA level in the middle-aged group, but no changes were found in the old age group. This result is in agreement with a previous study by Chin et al. (21), who found that long-term supplementation of TRF by older, healthy individuals did not reduce MDA levels compared with non-supplemented individuals. Conversely, long-term supplementation of TRF (60 mg/kg body weight) in rats reduced lipid peroxidation during aging (from six months up until 24 months) (45). PB and TRF appeared to have a lesser effect on lipid peroxidation compared to CV. This result may occur because the accumulation of oxidized proteins during aging is most likely linked to an age-related decline of antioxidant enzyme activity, whereas lipid peroxidation is less sensitive to the aging process (46). In conclusion, PB, TRF, and CV, which are rich in antioxidants, can modulate the antioxidant enzymes activities

healthy older adults (21). These equivocal results in agerelated antioxidant enzyme activity could be due to methodology, environmental and subject variation in both animal and human studies (4). Human aging is affected by both genetic and lifestylerelated factors, such as diet and exercise. Nutrients can affect the rate of aging by altering the type and quantity of proteins (41), which alter the oxidative status of individuals (42). Dietary supplementation rich in antioxidants can reduce oxidative damage or boost the antioxidant system. In this study, we found that supplementation with PB, TRF, and CV as exogenous antioxidants improved the endogenous antioxidant function associated with removing accumulated ROS and improving the balance between ROS and antioxidants. The most effective dose of PB on certain biological activities ranges from concentrations of 1 mg/kg to 1,000 mg/kg in experimental studies using mice and rats (18,43,44). We decided to use a lower dose of 50 mg/kg body weight in mice. This decision was based on two studies that noted that low doses of PB (1 mg/kg, 5 mg/kg, and 10 mg/kg) significantly modulated SOD, GPx, and CAT activity in radiation-induced lipid peroxidation in Swiss albino mice (18). This dose was considered toxicologically safe when fed orally to ICR mice when investigating antimalarial effects of PB (44). Chitra & Vidya (43) reported that high doses of PB extract (.200 mg/kg) produced toxic effects in the erythrocytes of experimental mice. The administration of PB (50 mg/kg body weight) to mice markedly increased the levels of antioxidant enzymes by increasing CAT activity at a young age, increasing GPx activity in middle-aged mice, and increasing both SOD and GPx activities in the old age group. These results support the antioxidant potential of PB. PB treatment helps to produce more antioxidant enzymes, especially during old age, to cope with the overload of oxygen radicals and H2O2.

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Piper betle modulates antioxidant enzymes Aliahmat NS et al. 19. Adachi H, Ishii N. Effects of tocotrienols on life span and protein carbonylation in Caenorhabditis elegans. J Gerontol A Biol Sci Med Sci. 2000;55(6):280-5, http://dx.doi.org/10.1093/gerona/55.6.B280. 20. Kamat JP, Sarma HD, Devasagayam TPA, Nesaretnam K, Basiron Y. Tocotrienols from palm oil as effective inhibitors of protein oxidation and lipid peroxidation in rat liver microsomes. Mol Cell Biochem. 1997;170(1-2):131-8, http://dx.doi.org/10.1023/A:1006853419214. 21. Chin SF, Ibahim J, Makpol S, Abdul Hamid NA, Abdul Latiff A, Zakaria Z, et al. Tocotrienol Rich Fraction Supplementation Improved Lipid Profile and Oxidative Status in Healthy Older Adults: A Randomized Controlled Study. Nutr Metab (Lond). 2011;8(1):42, http://dx.doi.org/ 10.1186/1743-7075-8-42. 22. Hasegawa T, Kimura Y, Hiromatsu K, Kobayashi N, Yamada A, Makino M, et al. Effect of hot water extract of Chlorella vulgaris on cytokine expression patterns in mice with murine acquired immunodeficiency syndrome after infection with Listeria monocytogenes. Immunopharmacology. 1997;35(3):273-82, http://dx.doi.org/10.1016/ S0162-3109(96)00150-6. 23. Mohd Yusof YA, Hassan Basari J, Mukti NA, Sabuddin R, Muda AR, Sulaiman S, et al. Fatty acids composition of microalgae Chlorella vulgaris can be modulated by varying carbon dioxide concentration in outdoor culture. African J of Biotech. 2011;10(62):13536-42. 24. Mohd Yusof YA, Md Saad S, Makpol S, Shamaan NA, Wan Ngah WZ. Hot water extract of Chlorella vulgaris induced DNA damage and apoptosis. Clinics. 2010;65(12):1-7. 25. Suhaniza S, Nor Aripin S, Wan Zurinah WN, Yasmin Anum MY. Chemopreventive effect of Chlorella vulgaris in choline deficient diet and ethionine induced liver carcinogenesis in rats. Int J Cancer Res. 2006;2(3):234-41. 26. Pin KY, Chuah TG, Choong TSY, Abdull Rashih A, Rasadah MA, Laws CL. 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Measurement of free and bound malondialdehyde in plasma by high-performance liquid chromatography as the 2,4-dinitrophenylhydrazine derivative. J Chromatogr B Biomed Sci Appl. 2000;742(2):315-25, http://dx.doi.org/10.1016/S03784347(00)00174-2. 32. Sim SA, Salonikas C, Naidoo D, Wilcken DEL. Improved method for plasma malondialdehyde measurement by high-performance liquid chromatography using methyl malondialdehyde as an internal standard. J Chromatogr B Analyt Technol Biomed Life Sci. 2003 5;785(2):337-44, http://dx.doi.org/10.1016/S1570-0232(02)00956-X. 33. Waris G, Ahsan H. Reactive oxygen species: role in the development of cancer and various chronic conditions. J Carcinog. 2006;5:14, http://dx. doi.org/10.1186/1477-3163-5-14. 34. Moreira PI, Duarte AI, Santos MS, Rego AC, Oliveira CR. An integrative view of the role of oxidative stress, mitochondria and insulin in Alzheimer’s disease. J Alzheimers Dis. 2009;16(4):741-6. 35. 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Free Rad Biol Med. 1988;5(5-6):3359, http://dx.doi.org/10.1016/0891-5849(88)90105-0. 40. Alejendro DB, Martha SB, Nestor OB. Superoxide dismutase, catalase and glutathione peroxidase activities in human blood: influence of sex, age, and cigarette smoking. Clin Biochem. 1997;30(6):449-54. 41. Papet I, Dardevet D, Sornet C, Be´chereau F, Prugnaud J, Pouyet C, et al. Acute phase protein levels and thymus, spleen and plasma protein synthesis rates differ in adult and old rats. J Nutr. 2003;133(1):215-9. 42. Friel JK, Widness JA, Jiang T, Belkhode SL, Rebouche CJ, Ziegler EE. Antioxidant status and oxidant stress may be associated with vitamin E

of SOD, GPx, and CAT with different patterns during aging. CV had a greater effect compared with PB and TRF in reducing lipid peroxidation during aging.

ACKNOWLEDGMENTS The authors wish to express their sincere gratitude to the National University of Malaysia (UKM) for full financial support of this project (UKM-GUP-SK-07-21-201). The authors are also grateful to the staff of the Department of Biochemistry for their technical support.

AUTHOR CONTRIBUTIONS Nor Syahida A was involved in the experimental work, analysis and interpretation of the data and drafting of the manuscript. Mohd Razman MN and Wan Junizam WY were involved in the experimental work, especially the animal handling and the interpretation of the data for the MDA levels. Yasmin Anum MY, Suzana M and Wan Zurinah WN were involved in the acquisition, interpretation and critical analysis of the data and the final revision of the manuscript.

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DOI:10.6061/clinics/2012(12)17

BASIC RESEARCH

Effects of the administration of pentoxifylline and prednisolone on the evolution of portal fibrogenesis secondary to biliary obstruction in growing animals: immunohistochemical analysis of the expression of TGF- b and VEGF Wagner de Castro Andrade, Luiz Fernando Ferraz da Silva, Maria Cecilia de Mendonc¸a Coelho, Ana Cristina Aoun Tannuri, Venancio Avancini Ferreira Alves, Uenis Tannuri Faculdade de Medicina da Universidade de Sa˜o Paulo, Pediatric Surgery Division, Laboratory of Pediatric Surgery (LIM-30) and Laboratory of Hepatic Pathology (LIM-14), Sa˜o Paulo/SP, Brazil

OBJECTIVE: During the neonatal and infancy periods, some chronic liver diseases may lead to progressive hepatic fibrosis, which is a condition that can ultimately result in the loss of organ function and severe portal hypertension necessitating hepatic transplantation. In a previous report, pharmacological interventions were demonstrated to modulate hepatic fibrosis induced by bile duct ligation in young rats. The administration of pentoxifylline or prednisolone, or the combination of both, resulted in reduced fibrogenesis in portal spaces. The objectives of the present study were to evaluate the expression of transforming growth factor b and vascular endothelial growth factor after bile duct ligation in young rats and to assess the effect of those same drugs on cytokine expression. METHODS: In this experimental study, 80 young rats (21 or 22 days old) were submitted either to laparotomy and common bile duct ligation or to sham surgery. The animals were allocated into four groups according to surgical procedure, and the following treatments were administered: (1) common bile duct ligation + distilled water, (2) sham surgery + distilled water, (3) common bile duct ligation + pentoxifylline, or (4) common bile duct ligation + prednisolone. After 30 days, a hepatic fragment was collected from each animal for immunohistochemical analysis using monoclonal antibodies against transforming growth factor b and vascular endothelial growth factor. Digital morphometric and statistical analyses were performed. RESULTS: The administration of pentoxifylline reduced the transforming growth factor b-marked area and the amount of transforming growth factor b expressed in liver tissue. This effect was not observed after the administration of prednisolone. There was a significant reduction in vascular endothelial growth factor expression after the administration of either drug compared with the non-treatment group. CONCLUSIONS: The administration of pentoxifylline to cholestatic young rats resulted in the diminished expression of transforming growth factor b and vascular endothelial growth factor in liver tissue. The administration of steroids resulted in the diminished expression of vascular endothelial growth factor only. These pathways may be involved in hepatic fibrogenesis in young rats submitted to bile duct ligation and exposed to pentoxifylline or prednisolone. KEYWORDS: Liver Fibrogenesis; Experimental Cholestasis; Steroids; Pentoxifylline; TGFb; VEGF. Andrade WC, da Silva LF, Coelho MC, Tannuri AC, Alves VA, Tannuri U. Effects of the administration of pentoxifylline and prednisolone on the evolution of portal fibrogenesis secondary to biliary obstruction in growing animals: immunohistochemical analysis of the expression of TGF- b and VEGF. Clinics. 2012;67(12):1455-1461. Received for publication on June 14, 2012; First review completed August 8, 2012; Accepted for publication on August 15, 2012 E-mail: wwwag@uol.com.br Tel.: 55 11 3061-7246

INTRODUCTION Cholestatic disorders are responsible for chronic hepatic failure in a significant number of patients during infancy. In the neonatal period, the most frequent cholestatic disease requiring liver transplantation is biliary atresia, a condition for which the pathogenesis is not yet fully understood but that is absolutely fatal if left untreated (1).

Copyright ß 2012 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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Figure 1 - Digitized slide microphotography showing Anti-VEGF immunohistochemical staining. From left to right: CBDL (100x magnification), PTX (200x magnification), and PRED (200x magnification).

Since the 1970s, some authors have suggested that the administration of corticosteroids may promote better late outcomes of biliary atresia in children who undergo Kasai’s portoenterostomy (2-4). In recent years, some results of this new strategy have been published, but the available data still do not clearly demonstrate a difference in jaundice-free survival and a reduced need for early liver transplantation (5-7). Other potentially antifibrogenic drugs have also been experimentally tested in animals, including pentoxifylline (PTX), a phosphodiesterase inhibitor already used in clinical practice for the treatment of arterial insufficiency (8-10). Some studies have demonstrated the inhibition of inflammatory cytokines, such as tumor necrosis factor a, transforming growth factor b, and interleukins (ILs) 1, 6, and 8 (11,12). Other studies have failed to demonstrate this effect

in animal models (9,13). Therefore, no consensus exists regarding the role of PTX in the inflammatory and fibrogenic cascades. Our group has been investigating the effects of antifibrogenic drugs over the last few years, with a particular focus on their effects in growing animals. We reported our initial results in 2009 after developing a model to study portal fibrosis secondary to biliary obstruction in young rats (1415). We concluded that hepatic fibrosis induced by bile duct ligation in young rats could be modulated by pharmacologic interventions. The administration of pentoxifylline or prednisolone (PRED), or the combination of both, resulted in diminished collagen-filled areas in the portal spaces. We have continued this work and now present an immunohistochemical analysis of the expression of transforming

Figure 2 - Ratio between the anti-TGFb-stained area (mm2) and total area (mm2): PTX x CBDL, p = 0.032; PRED x CBDL, p = 0.089. The numbers in the graphic are outliers.

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Figure 3 - Integrated optical density (IOD). Anti-TGFb stained slides: PTX x CBDL, p = 0.032; PRED x CBDL, p = 0.231. The numbers in the graphic are outliers.

After 30 days, cardiorespiratory arrest was induced by the inhalation of anesthesia (ether gas chamber at a high concentration). The animals were rapidly weighed and submitted to midline incision for the harvesting of a 1-cm3 hepatic fragment from the left lobe.

growth factor b (TGFb) and vascular endothelial growth factor (VEGF) after bile duct ligation in young rats and the influence of these drugs on cytokine expression.

METHODS This study was approved by the Ethical Committee for Research Project Analysis of our institution and was conducted according to international guidelines regarding the use of laboratory animals. All operative procedures were performed by the same surgeon.

Histological analysis The hepatic fragments were processed according to standard techniques (3-mm-thick sections of paraffin-embedded material) to obtain two slides for each animal. The slides were included in 3-aminopropyl-trietoxi-silano, and immunohistochemical reactions followed the biotin-streptavidin-peroxidase protocol. The primary antibodies were as follows: monoclonal anti-mouse TGFb IgG (sc-52893, Santa Cruz Biotechnology, USA) and monoclonal anti-mouse VEGF IgG (sc-7269, Santa Cruz Biotechnology, Santa Cruz, CA, USA). The secondary antibody was the Vectastain anti-mouse ABCkitTM (Vector Laboratories, Burlingame, CA, USA) for both reactions. The protein block was accomplished with methanol/azide for the TGFb slides and with the NovocastraTM system (Leica Microsystems, Milton Keynes, England, UK) for the VEGF reactions. The slides were analyzed under a Leica DMR microscope and digitized with Panoramic Scan MidiTM (3DHistech, Hungary Software, Budapest, Hungary).

Surgical procedures Young (21- to 22-day-old) Wistar rats were submitted to common bile duct ligation (CBDL) as described in a previous report (15). Under ether anesthesia, a median laparotomy was performed, and the common bile duct was ligated and divided twice using 6.0 monofilament nylon ligation. The sham surgery (SHAM) consisted of the laparotomy and exposure of the hepatic hilum without duct ligation. The animals were randomly allocated into four groups (20 animals per group) according to the surgical procedure and were administered a solution as follows: 1) CBDL + distilled water; 2) SHAM + distilled water; 3) CBDL + PTX 10 mg/kg per day; or 4) CBDL + PRED 3 mg/kg per day. The solutions were administered once a day via nasogastric tubing under sedation. The drug doses per kilogram were similar to those used in humans in the clinical setting.

Morphometric analysis Aleatory areas were delineated on each digitized slide and processed with the aid of the software Image Pro-Plus 4.5.0.29 Windows XP version (Media Cybernetics Inc.). After

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Figure 4 - Ratio between the anti-VEGF-stained area (mm2) and total area (mm2): PTX x CBDL, p,0.001; PRED x CBDL, p,0.001. The number in the graphic is an outlier.

image acquisition, the software was calibrated to automate the cytokine (TGFb or VEGF) identification. Several measurements were performed: total area, cytokine-marked area (area +), integrated optical density (IOD; antibody color intensity measure, which reflects the amount of cytokine present in the tissue), antibody distribution or proportion (area +/total area), and mean density (IOD/area +).

VEGF expression: All groups exhibited VEGF expression in hepatocytes, endothelial and hepatic stellate cells and the biliary epithelium. Only fibrous areas were not stained (Figure 1).

Statistical analysis

Morphometric analysis

4. PTX: less intense expression in hepatocytes and endothelial cells than in the PRED group.

Comparisons of the data between groups were performed using the software SPSS version 18.0 for Windows (SPSS Inc., Chicago, IL). As the data had a normal distribution, the analysis of variance and Tukey’s post hoc test were applied to test for differences between groups. The hypothesis of sample equality was rejected for p,0.05.

TGFb expression Validating the findings of the descriptive analysis, the administration of pentoxifylline (PTX) to growing rats reduced the size of the cytokine-marked area (Figure 2) and the amount of TGFb expressed in liver tissue (Figure 3), as reflected by the integrated optical density (IOD). These effects were not observed after the administration of prednisolone (Figures 2 and 3).

RESULTS Descriptive analysis TGFb expression

VEGF expression The reduction in the expression of VEGF after the administration of both drugs was quite remarkable. The distribution of this cytokine (Figure 4) was reduced in the PTX and PRED groups relative to the CBDL group (p,0.001). Although the decrease in the absolute amount of cytokine in the PRED group, as revealed by the IOD index, did not reach statistical significance (p = 0.195, Figure 5) compared with the CBDL group, when transformed into the mean density (ratio

1. Sham: no hepatocyte presentation, expression in endothelial and hepatic stellate cells; 2. CBDL: mild expression in periportal hepatocytes, no expression in the biliary epithelium; 3. PRED: irregular hepatocyte expression in different areas of the hepatic lobule, clear presentation in endothelial and hepatic stellate cells;

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Figure 5 - Integrated optical density (IOD). Anti-VEGF-stained slides: PTX x CBDL, p,0.001; PRED x CBDL, p = 0.195.

between the IOD and positive area), a significant decrease was revealed (p = 0.001, Figure 6).

downregulate the production of IL-1, IL-6, and tumor necrosis factor a (all proinflammatory cytokines) (26). In a previous report (15), we revealed that the administration of a steroid and pentoxifylline to recently weaned rats submitted to biliary obstruction could promote a reduction in the collagen-filled areas in liver tissue, indicating a potential mechanism for the pharmacological modulation of cholestasis-induced portal fibrosis. We believe that the use of growing animals, rather than adults, as experimental models may more accurately represent the histological abnormalities found in newborns or children affected by cholestasis (14). The exact mechanisms involved in portal biliary fibrosis in young animals are not completely understood. The present investigation was conducted to contribute to the elucidation of the following specific question: how is hepatic collagen deposition secondary to biliary obstruction affected by the administration of steroids and PTX? Among other cytokines, TGFb and VEGF appear to actively participate in the processes of inflammatory cell recruitment and hepatic fibrogenesis and the mediation of the adaptive proliferative response of cholangiocytes to cholestasis (27-29). Are these cytokines affected by the administration of steroids and PTX? In this model, we were able to demonstrate, through immunohistochemical analyses, that VEGF expression was substantially reduced following exposure to steroids and PTX. However, only PTX appeared to affect the liver tissue expression of TGFb. We failed to demonstrate a significant

DISCUSSION Regarding the treatment of biliary atresia in children, clinicians have been empirically prescribing steroids immediately post-Kasai surgery since the 1980s (16-18). In the wake of progressive liver disease following bile duct obstruction, steroids are hypothesized to suppress inflammation and promote bile flow (19), but recent clinical reports have been controversial (20,21) and have failed to clearly demonstrate an actual long-term benefit (i.e., transplant-free survival) of the adjuvant use of steroids after portoenterostomy. Another medication included in this study was pentoxifylline (PTX), a potentially antifibrogenic drug (9-13,22). Classified as a nonspecific phosphodiesterase inhibitor, this drug has already been incorporated into the clinical setting for the treatment of chronic occlusive arterial disease because of its oxygen transport-enhancing effect. As phosphodiesterases regulate the intracellular levels of cyclic nucleotides (cyclic adenosine monophosphate and cyclic guanosine monophosphate), their inhibition can affect several processes, such as apoptosis, muscle contraction, cellular differentiation, migration, and proliferation (23-25). Specifically regarding hepatic fibrogenesis, since 1993, there have been some publications demonstrating that PTX can decrease the activation and proliferation of stellate cells and

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Figure 6 - Mean density (ratio between the IOD and cytokine-marked area). Anti-VEGF-stained slides: PTX x CBDL, p,0.001; PRED x CBDL, p = 0.001. The number in the graphic is an outlier.

2. Carceller A, Blanchard H, Alvarez F, St-Vil D, Bensoussan AL, Di Lorenzo M. Past and future of biliary atresia. J Pediatr Surg. 2000;35(5):717-20, http://dx.doi.org/10.1053/jpsu.2000.6034. 3. Karrer FM, Lilly JR. Corticosteroid therapy in biliary atresia. J Pediatr Surg. 1985;20:693-5, http://dx.doi.org/10.1016/S0022-3468(85)80026-9. 4. Muraji T, Higashimoto Y. The improved outlook for biliary atresia with corticosteroid therapy. J Pediatr Surg. 1997;32(7):1103-7, http://dx.doi. org/10.1016/S0022-3468(97)90408-5. 5. Kobayashi H, Yamataka A, Koga H, Okazaki T, Tamura T, Urao M, et al. Optimum prednisolone usage in patients with biliary atresia postportoenterostomy. J Pediatr Surg. 2005;40(2):327-30, http://dx.doi.org/ 10.1016/j.jpedsurg.2004.10.017. 6. Escobar MA, Jay CL, Brooks RM, West KW, Rescorla FJ, Molleston JP, et al. Effect of corticosteroid therapy on outcomes in biliary atresia after Kasai portoenterostomy. Journal of pediatric surgery. 2006;41(1):99-103; discussion 99-. 7. Stringer MD, Davison SM, Rajwal SR, McClean P. Kasai portoenterostomy: 12-year experience with a novel adjuvant therapy regimen. Journal of pediatric surgery. 2007;42(8):1324-8, http://dx.doi.org/10. 1016/j.jpedsurg.2007.03.026. 8. Oberti F, Pilette C, Rifflet H, Maiga MY, Moreau A, Gallois Y, et al. Effects of simvastatin, pentoxifylline and spironolactone on hepatic fibrosis and portal hypertension in rats with bile duct ligation. Journal of hepatology. 1997;26(6):1363-71, http://dx.doi.org/10.1016/S0168-8278 (97)80473-4. 9. Peterson TC, Neumeister M. Effect of pentoxifylline in rat and swine models of hepatic fibrosis: role of fibroproliferation in its mechanism. Immunopharmacology. 1996;31(2-3):183-93, http://dx.doi.org/10.1016/ 0162-3109(95)00048-8. 10. Ward A, Clissold SP. Pentoxifylline. A review of its pharmacodynamic and pharmacokinetic properties, and its therapeutic efficacy. Drugs. 1987;34(1):50-97. 11. Raetsch C, Jia JD, Boigk G, Bauer M, Hahn EG, Riecken EO, et al. Pentoxifylline downregulates profibrogenic cytokines and procollagen I expression in rat secondary biliary fibrosis. Gut. 2002;50(2):241-7, http:// dx.doi.org/10.1136/gut.50.2.241.

difference in this expression after the administration of prednisolone. The impairment in the pathways of these two cytokines after the administration of PTX to these growing animals may explain the slightly more accentuated reduction in collagen deposition observed in our previous report compared with the PRED group. Obviously, other cells, cytokines, signaling pathways, and mechanisms are involved in a process as complex as hepatic fibrogenesis. Therefore, subsequent experimental and clinical studies should be conducted to confirm the benefits of exposing jaundiced children to these medications, with the aim of reducing hepatic fibrosis and the need for liver transplantation.

ACKNOWLEDGMENTS We thank FAPESP (Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo) for providing financial support (grant #2009/07225-2).

AUTHOR CONTRIBUTIONS Andrade WC designed and conceived the study, performed the experiments, conducted the analyses, and contributed to the discussion. Silva LF and Coelho MC conducted the analyses and contributed to the discussion. Tannuri AC, Alves VA, and Tannuri U contributed to the discussion.

REFERENCES 1. Sokol RJ, Mack C. Etiopathogenesis of biliary atresia. Semin Liver Dis. 2001;21(4):517-24, http://dx.doi.org/10.1055/s-2001-19032.

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12. Schandene´ L, Vandenbussche P, Crusiaux A, Ale`gre ML, Abramowicz D, Dupont E, et al. Differential effects of pentoxifylline on the production of tumor necrosis factor-alpha (TNFa) and interleukin-6 (IL-6) by monocytes and T cells. Immunology. 1992;76(1):30-4. 13. Tarc¸in O, Avsar K, Demirtu¨rk L, Gu¨ltepe M, Oktar BK, Ozdog˘an OC, et al. In vivo inefficiency of pentoxifylline and interferon-alpha on hepatic fibrosis in biliary obstructed rats: assessment by tissue collagen content and prolidase activity. J Gastroenterol Hepatol. 2003;18(4):437-44, http://dx.doi.org/10.1046/j.1440-1746.2003.03004.x. 14. Gibelli NE, Tannuri U, de Mello ES, Rodrigues CJ. Bile duct ligation in neonatal rats: Is it a valid experimental model for biliary atresia studies. Pediatr Transplant. 2009;13(1):81-7, http://dx.doi.org/10.1111/j.13993046.2008.00947.x. 15. Andrade WdeC, Tannuri U, da Silva LF, Alves VA. Effects of the administration of pentoxifylline and prednisolone on the evolution of portal fibrogenesis secondary to biliary obstruction—an experimental study in growing animals. J Pediatr Surg. 2009;44(11):2071–7, http://dx. doi.org/10.1016/j.jpedsurg.2009.05.020. 16. Meyers RL, Book LS, O’Gorman MA, Jackson WD, Black RE, Johnson DG, et al. High dose steroids, ursodeoxycholic acid, and chronic intravenous antibiotics improve bile flow after Kasai procedure in infants with biliary atresia. J Pediatr Surg. 2003;38(3):406-11, http://dx. doi.org/10.1053/jpsu.2003.50069. 17. Rhen T, Cidlowski JA. Antiinflammatory action of glucocorticoids--new mechanisms for old drugs. N Engl J Med. 2005;353(16):1711-23, http:// dx.doi.org/10.1056/NEJMra050541. 18. Muraji T, Nio M, Ohhama Y, Hashimoto T, Iwanaka T, Takamatsu H, et al. Postoperative corticosteroid therapy for bile drainage in biliary atresia: a nationwide survey. J Pediatr Surg. 2004;39(12):1803-5, http:// dx.doi.org/10.1016/j.jpedsurg.2004.08.019. 19. Miner PB, Gaito JM. Bile flow in response to pharmacologic agents. Biochem Pharmacol. 1979;28(7):1063-6, http://dx.doi.org/10.1016/00062952(79)90304-6.

20. Suzuki T, Hashimoto T, Kondo S, Sato Y, Hussein MH. Evaluating patients’ outcome post-Kasai operation: a 19-year experience with modification of the hepatic portoenterostomy and applying a novel steroid therapy regimen. Pediatr Surg Int. 2010;26(8):825-30, http://dx. doi.org/10.1007/s00383-010-2637-y. 21. Sarkhy A, Schreiber RA, Milner RA, Barker CC. Does adjuvant steroid therapy post-Kasai portoenterostomy improve outcome of biliary atresia? Systematic review and meta-analysis. Can J Gastroenterol. 2011;25(8):440-4. 22. Windmeier C, Gressner AM. Pharmacological aspects of pentoxifylline with emphasis on its inhibitory actions on hepatic fibrosis. Gen Pharmacol. 1997;29:181-96, http://dx.doi.org/10.1016/S0306-3623 (96)00314-X. 23. Bender AT, Beavo JA. Cyclic nucleotide phosphodiesterases: molecular regulation to clinical use. Pharmacol Rev. 2006;58:488-520, http://dx.doi. org/10.1124/pr.58.3.5. 24. Conti M, Beavo JA. Biochemistry and physiology of cyclic nucleotide phosphodiesterases: essential components in cyclic nucleotide signaling. Ann Rev Biochem. 2007;76:481-511, http://dx.doi.org/10.1146/annurev. biochem.76.060305.150444. 25. Omori K, Kotera J. Overview of PDEs and their regulation. Circ Res 2007;100(3):306-27. 26. Peterson T. Pentoxifylline prevents fibrosis in an animal model and inhibits platelet-derived growth factor driven proliferation of fibroblast. Hepatology. 1993;17(3):486-93. 27. Housset C. Biliary epithelial cell response to cholestasis. J Hepatol. 2000;32:14-15, http://dx.doi.org/10.1016/S0168-8278(00)80436-5. 28. Kinnman N, Housset C. Peribiliary myofibroblasts in biliary type fibrosis. Front Biosci. 2002;7:d496-503, http://dx.doi.org/10.2741/ kinnman. 29. Gaudio E, Barbaro B, Alvaro D, Glaser S, Glaser S, Francis H, Ueno Y, et al. Vascular endothelial growth factor stimulates rat cholangiocyte proliferation via an autocrine mechanism. Gastroenterology. 2006;130(4):1270-82, http://dx.doi.org/10.1053/j.gastro.2005.12.034.

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DOI:10.6061/clinics/2012(12)18

BASIC RESEARCH

Hypertonic saline solution reduces the inflammatory response in endotoxemic rats Mariana Cardillo Theobaldo, Hermes Vieira Barbeiro, Denise Frediani Barbeiro, Ricardo Petroni, Francisco Garcia Soriano Faculdade de Medicina da Universidade de Sa˜o Paulo, Emergency Medicine Division, Sa˜o Paulo/SP, Brazil.

OBJECTIVE: Volume replacement in septic patients improves hemodynamic stability. This effect can reduce the inflammatory response. The objective of this study was to evaluate the effect of 7.5% hypertonic saline solution versus 0.9% normal saline solution for volume replacement during an inflammatory response in endotoxemic rats. METHODS: We measured cytokines (serum and gut), nitrite, and lipid peroxidation (TBARS) as indicators of oxidative stress in the gut. Rats were divided into four groups: control group (C) that did not receive lipopolysaccharide; lipopolysaccharide injection without treatment (LPS); lipopolysaccharide injection with saline treatment (LPS +S); and lipopolysaccharide injection with hypertonic saline treatment (LPS +H). Serum and intestine were collected. Measurements were taken at 1.5, 8, and 24 h after lipopolysaccharide administration. RESULTS: Of the four groups, the LPS +H group had the highest survival rate. Hypertonic saline solution treatment led to lower levels of IL-6, IL-10, nitric oxide, and thiobarbituric acid reactive substances compared to 0.9% normal saline. In addition, hypertonic saline treatment resulted in a lower mortality compared to 0.9% normal saline treatment in endotoxemic rats. Volume replacement reduced levels of inflammatory mediators in the plasma and gut. CONCLUSION: Hypertonic saline treatment reduced mortality and lowered levels of inflammatory mediators in endotoxemic rats. Hypertonic saline also has the advantage of requiring less volume replacement. KEYWORDS: LPS; Hypertonic Solution; Interleukin; Oxidative Stress; Intestine. Theobaldo MC, Barbeiro HV, Barbeiro DF, Petroni R, Soriano FG. Hypertonic saline solution reduces the inflammatory response in endotoxemic rats. Clinics. 2012;67(12):1463-1468. Received for publication on May 30, 2012; First review completed on July 10, 2012; Accepted for publication on August 19, 2012 E-mail: gsoriano@usp.br Tel.: 55 11 30617227

adhesion molecules (I-CAM-1 and E-selectin), cyclooxygenase- 2, and inducible NO synthase (4). It has been shown that MAPK is functionally associated with the transcription factor NF-kB. In the absence of MAPK, the in vivo release of inflammatory mediators is down-regulated because NF-kB dependent activation is compromised (5). Hypertonic saline has been shown to reduce the activity of MAPK (6,7). The exacerbated inflammatory condition can induce cell necrosis, leading to organ dysfunction and death (2,3,8). The gut is one of the most important organs involved in sepsis, and it is the largest interface between the individual and his/her environment. An intact intestinal barrier is essential for good health and the prevention of various diseases. The intestinal barrier has various immunological and nonimmunological components (4). Lung damage is a frequent cause of respiratory failure and implies the use of mechanical ventilation, which is also associated with a high risk of death in septic patients (4). The beneficial effects of hypertonic saline were first shown by Velasco et al. in an experiment on hemorrhagic shock (9). Hypertonic saline limits local and end-organ injury in experimental pancreatitis and reduces mortality (9) by altering circulating plasma volume, reducing levels of

INTRODUCTION Sepsis syndrome is the leading cause of death for patients in the intensive care unit (1). The syndrome occurs as a consequence of inappropriate immune activation due to bacteria and bacterial components released during infection (2). Host receptors recognize distinct bacterial components and initiate the inflammatory response. The bacterial cell wall component lipopolysaccharide (LPS) is one of the most potent known inducers of the inflammatory response and employs Toll-like receptor 4 (TLR4) to exert its effects (3). The mechanism by which LPS induces endotoxic shock is related to its capacity to activate the NF-kB family of transcription factors, which enables the expression of several critical genes involved in the pathogenesis of septic shock: TNF-a, interleukins (IL-1b, IL-2, IL-6, and IL-8),

Copyright ß 2012 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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dihydrochloride in water and 1% sulfanilamide in 5% concentrated H3PO4; 1:1). The optical density at 550 nm (OD 550, corrected for absorbance at 650 nm) was measured using a Spectramax microplate reader (Molecular devices, Minnesota, USA). Nitrite concentrations were calculated using an OD 550 and a standard solution of sodium nitrite prepared in phosphate-buffered saline (PBS).

trypsinogen, preventing acinar necrosis, and reducing levels of inflammatory cytokines and pancreatic infection. Our group showed that hypertonic saline protects the lung (10) and liver (11) from injury after pancreatitis by modulating the expression and activity of several proteins (12). Previous studies in hypovolemic shock and pancreatitis indicated that infusion of hypertonic saline reduced the production of pro-inflammatory cytokines and increased the production of anti-inflammatory cytokines, such as IL-10 (13-17). Studies using hypertonic saline in the setting of sepsis have reported hemodynamic improvements, which lead to better tissue perfusion and reduced necrosis and inflammation. A recent clinical trial using hypertonic saline (7.5% NaCl) noted an improvement in cardiac contractility and vascular tone in patients receiving hypertonic saline compared to normal saline (18). The aim of the present study was to assess the impact of hypertonic saline on the systemic inflammatory response and on the gut in experimental endotoxemia.

MDA assay MDA formation was utilized to quantify lipid peroxidation in the tissues and was measured as thiobarbituric acidreactive material. Tissues were homogenized (100 mg/mL) in a 1.15% KCl buffer. Two hundred microliters of the homogenate was added to a reaction mixture consisting of 1.5 mL of 0.8% thiobarbituric acid, 200 mL of 8.1% sodium dodecyl sulfate, 1.5 mL of 20% acetic acid (pH 3.5), and 600 mL of distilled water. The mixture was then heated at 90 ˚C for 45 min. After cooling to room temperature, the samples were cleared by centrifugation (10,000 g for 10 min), and their absorbances were measured at 532 nm using 1,1,3,3tetramethoxypropane as an external standard. The level of lipid peroxides was expressed as nmol MDA/mg protein (Bradford assay). TNF-a, IL-6, and IL-10 — The plasma concentrations and tissue levels of immunoreactive murine TNF-a (DY410), IL-6 (DY406) and IL-10 (DY417) were determined using commercially available enzyme-linked immunoabsorbent assays (ELISA) according to the manufacturer’s protocol (R&D Systems, Minneapolis, MN).

MATERIALS AND METHODS All procedures were performed according to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. The study protocol was approved by the Research Ethics Committee of the University of Sa˜o Paulo, School of Medicine, Hospital das Clı´nicas.

Endotoxemic induction Male Wistar rats weighing 253.9¡26.2 g were randomly assigned to four main groups: 1 - endotoxemic - LPS group (n = 10), induced by a single intraperitoneal injection of LPS (10 mg/kg) (extracted from Escherichia coli, serotype 026:B6; Sigma-Aldrich Canada, Ltd., Oakville, Ontario, Canada); 2 endotoxemia treated with a single dose of hypertonic solution 7.5% (4 ml/kg) (LPS +H); 3 - group treated with a single dose of saline solution 0.9% (34 ml/kg) (LPS +S); and 4 - control group that did not receive LPS (C). The control group (C) was used to obtain baseline values. The LPS+S and LPS+H groups were treated 15 minutes after LPS injection. Samples of serum and intestine were collected 1.5, 8, and 24 h after LPS injection. The animals were anesthetized with chloral hydrate (20 mg/ml).

Tissue preparation Frozen tissue (100 mg) was pulverized in liquid nitrogen. Samples were homogenized in NP40 buffer containing 135 mM NaCl, 20 mM Tris (pH 8.0), 10% glycerol, and proteolytic enzyme inhibitors (40 ug/mL phenylmethylsufonylfluoride 1 mM; Sigma, St, Louis, MO). After separating the debris by centrifugation for 30 min at 10,000 rpm, the supernatants were preserved, and the protein concentrations were calculated using the Bradford method (Bio Rad, Hercules, CA). Samples were stored at –80 ˚C until assayed.

Statistical analysis All values were expressed as means ¡ standard errors of the mean (SEM). The analyses were performed using InStat Statistical Software (GraphPad, La Jolla, CA, USA). Comparisons between the experimental groups were performed using analysis of variance. A Tukey test was used as a post hoc test to compare individual groups. A p-value less than 0.05 was considered to be significant. A log-rank test was used to analyze survival.

Experimental protocols For biochemical measurements, 90 rats were made endotoxemic by LPS injection, and 18 were sham. The measurements from the plasma and gut were taken 1.5, 8, and 24 h after LPS injection. Blood samples were obtained by cardiac puncture. Harvested samples from the gut (ileum) were immediately frozen in liquid nitrogen. Tissue samples were stored at 270˚C until they were assayed for cytokines, as detailed below. For survival experiments, 50 animals were exposed to LPS, and their mortality was then recorded every 8 h for 24 h.

RESULTS The values in the sham group were the same along the time course of the study. Therefore, we pooled and used these data as time zero for the control group. Plasma cytokine concentrations are illustrated in Figure 1. The time points chosen for this analysis (1.5, 8, and 24 h after LPS injection) correspond to the peak and relevant time course of circulating cytokine levels in this model. LPS induced an increase in plasma levels of TNF-a, IL-6, and IL-10 compared to the control group. Plasma levels of TNF-a did not change following administration of normal saline or hypertonic saline during the entire study. However, treatment with hypertonic

Measurements Tissue nitrite. Tissue nitrate and nitrite levels were measured as an index of NO production. First, nitrate in the sample was reduced to nitrite by incubation with nitrate reductase (610 mU/mL) and NADPH (170 mM) at room temperature for 3 h. After 3 h, the nitrite concentration in the samples was measured using the Griess reaction by adding 100 mL of Griess reagent (0.1% naphthalethylenediamine

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Figure 2 - Inflammatory profile - Cytokine concentrations (pg/ml) in gut: TNF-a (a), IL-10 (b), and IL-6 (c). Wistar male rats were challenged with LPS (LPS - white) and treated with SH (LPS+H black) and SS (LPS+S - gray). The levels of TNF- a, IL-10 and IL-6 were measured at 1.5, 8, and 24 h after the administration of LPS in the gut. *p,0.05 when compared to the LPS group in the same period. #p,0.05 when compared to the LPS+S group in the same period. Data are means¡SEM.

Figure 1 - Inflammatory profile - Cytokine concentrations (pg/ml) in serum: TNF-a (a), IL-10 (b), and IL-6 (c). Wistar male rats were challenged with LPS (LPS - white) and treated with SH (LPS+H - black) and SS (LPS+S - gray). The levels of TNF- a, IL-10 and IL-6 were measured at 1.5, 8 and 24 h after the administration of LPS in serum. *p,0.05 when compared to the LPS group in the same period. #p,0.05 when compared to the LPS+S group in the same period. Data are means¡SEM.

Alterations in the gut due to oxidative stress were further investigated by measuring malondialdehyde (MDA) formation (Figure 3) and nitric oxide (NOx) (Figure 4) levels. Hypertonic saline treatment reduced the levels of MDA in the gut. Hypertonic saline significantly reduced MDA levels compared to normal saline at 24 h (from 14.05¡¡1.40 nmol/mg to 2.12¡0.48 nmol/mg at 24 h), despite having lower levels of MDA at baseline. For nitric oxide only, hypertonic saline treatment as associated with significant reduction at 1.5 h (NOx, from 33.35¡9.95 mmol/mg to 7.97¡2.60 mmol/ml at 1.5 h). Based on these data on MDA and nitric oxide, hypertonic saline reduced the negative impact of oxidative stress on the gut. The results of the survival experiments are shown in Figure 5. Rats in the LPS group started to die at 8 h. Rats that received normal saline (Figure 5) started to die 16 h after LPS administration, with a mortality rate approaching 40% at 24 h post-LPS. The mortality rate at the end of the experiment was similar between the normal saline group and the LPS group. Rats that received hypertonic saline started to die at 16 h and had a mortality rate of 15%. The onset of death was markedly delayed in rats receiving volume treatment. Rats treated with hypertonic saline had a statistically significant survival advantage on the log-rank test.

saline did induce a significant decrease in plasma levels of the pro-inflammatory cytokine IL-6 (from 11.58¡1.07 ng/ml to 1.84¡0.91 ng/ml at 1.5 h), and the anti-inflammatory cytokine IL-10 (from 475.08¡106.80 pg/ml to 65.89¡ 10.31 ng/ml). The cytokine levels in the gut are illustrated in Figure 2. LPS induced high levels of all measured cytokines compared to the control group. Volume infusion was characterized by lower tissue levels of cytokines compared to the LPS group. Hypertonic saline treatment led to an increase in TNF-a levels in the first time period measured compared to the LPS group. However, at 24 h, hypertonic saline was effective in reducing TNF-a levels compared to the LPS and the LPS+S groups (TNF-a, from 3.72¡0.33 pg/mg to 2.48¡0.40 pg/mg at 24 h). Hypertonic saline treatment reduced IL-6 (from 94.44¡33.01 pg/mg to 53.97¡8.51 pg/mg at 1.5 h) and IL10 (from 6.57¡0.76 pg/mg to 2.42¡0.68 pg/mg at 8 h) levels more than the LPS group.

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septic shock, it is likely that their downregulation was a central mechanism underlying the overall beneficial effects of hypertonic saline. In general, it is presumed that these mediators exert detrimental effects when extremely high levels are present. However, it has been proposed that complete inhibition of these cytokines in sepsis is detrimental because an appropriate level of cytokines is necessary for fighting bacteria (20). Hypertonic saline was able to reduce proinflammatory cytokine levels but did not completely inhibit cytokine activity. This outcome enabled an appropriate amount of physiologic cytokine activity. Another feature of the LPS group was the high tissue and plasma levels of the anti-inflammatory cytokine IL-10. IL-10 levels were also reduced by hypertonic saline infusion. In addition, IL-10 has been shown to be crucial in preventing the overproduction of proinflammatory cytokines during systemic inflammation (21). However, this outcome did not occur in our study. Hypertonic saline decreased IL-10 and consistently reduced inflammation after LPS administration. These findings are consistent with previous studies that report the deleterious effects of increased IL-10 production in animal models of septic shock. Indeed, IL-10 was reported to suppress lymphocyte proliferation and lymphokine production during sepsis, and the administration of anti-IL-10 monoclonal antibodies has been shown to improve the survival of mice after Cecal Ligation and Puncture (CLP) (22) and in a two-hit model of EQ CLP followed by intratracheal instillation of Pseudomonas (23). The adequate levels and balance between proinflammatory and anti-inflammatory cytokines can reduce self-damage and maintain enough activity against bacteria to protect animals from exaggerated inflammation, bacterial proliferation, and invasion (20). In addition, it has been proposed that PMN activation and recruitment to various organs (especially the lung) were triggered by factors contained in mesenteric lymph tissue (24), which suggests a critical link between gut dysfunction, PMN activation, and distant organ injury in shock.

Figure 3 - Oxidative stress - MDA concentrations (nmol/mg of protein) in the intestine. Wistar male rats were challenged with LPS (LPS - white); 15 minutes later, they were treated with SH (LPS+H black) and SS (LPS+S - gray). The levels of MDA were measured in the intestine. *p,0.05 vs. LPS in the same period; & p,0.05 vs. LPS and HS in the same period. n = 10 mice per group. Data are meansยกSEM.

DISCUSSION In this article, we investigated the effects of hypertonic saline in an experimental endotoxemic model. Hypertonic saline-treated animals had reduced levels of TNF, IL-6, IL10, nitric oxide and superoxide in the plasma and gut. In addition, we showed that hypertonic saline treatment resulted in a higher survival rate compared to no treatment or normal saline treatment. Previous data from our laboratory and other investigators indicate that hypertonic saline has in vivo and in vitro anti-inflammatory effects. These effects are thought to be mediated, at least in part, by MAPK. It is also suggested that hypertonic saline acts mostly through cytoskeleton changes (14,19). The data presented in our study confirm and expand on these previous findings by showing that hypertonic saline has major beneficial effects in a model of endotoxemia.

Effects of hypertonic saline on the production of nitric oxide and oxidative stress during endotoxemia

Effects of hypertonic saline on the production of cytokines during endotoxemia LPS induced a massive systemic inflammatory response, evidenced by the high levels of tissue and plasma TNF-a and IL-6. Because both TNF-a and IL-6 are proximal mediators of

Reactive oxygen species and reactive nitrogen species are produced during the inflammatory process of sepsis and

Figure 5 - Survival curve - The effect of treatment with SH or SS on the mortality of the endotoxemic rat challenged with intraperitoneal LPS (10 mg/kg) 15 min before the treatment. LPS+H group treated with SH; LPS+S treated with SS; LPS without treatment. n = 10 mice per group.

Figure 4 - Oxidative stress - Nitrite concentrations (mmol/mg of protein) in the intestine. Wistar male rats were challenged with LPS (LPS white) and treated with SH (LPS+H - black) and SS (LPS+S - gray). The levels of nitrite were measured in the intestine. *p,0.05 vs. LPS in the same period. n = 10 mice per group. Data are meansยกSEM.

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endotoxemia (3,8,25). The high affinity between NO and O2leads to the rapid production of peroxynitrite (ONOO-), which forms acidic peroxynitrous (ONOOH). Peroxynitrous is an unstable chemical species that decomposes into two free radicals: nitrite (NO2-) and hydroxyl radical (OH-) (25). This oxidant scenario can induce important organ damage, causing dysfunction (26,27). Volume replacement reduced nitric oxide production in our study. Hypertonic saline resulted in significant nitric oxide reduction early after the onset of endotoxemia. Despite the advantages of hypertonic saline, this protective effect is not exclusive of hypertonic saline and can be related to the hemodynamic improvement produced by volume infusion. However, the data on lipid peroxidation indicated a more intense reduction with hypertonic treatment compared to normal saline, and this effect lasted for the entire study period.

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Effects of hypertonic saline on survival after endotoxemia

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We found that hypertonic saline significantly improved the survival of endotoxemic rats. The effects of hypertonic saline were observed in a post-treatment strategy. Although both types of volume replacement were effective in modulating the inflammatory response, the group treated with hypertonic saline had a better oxidant profile and improved survival rate. Altogether, these data indicate that hypertonic saline may represent a valuable approach for reducing systemic inflammation, reducing oxidative stress and improving outcomes in clinical endotoxemic shock. Several distinct strategies have been previously reported to improve survival in animals challenged with endotoxemia or sepsis. The steroid hormone dehydroepiandrosterone reduced short-term mortality, an effect associated with a reduction of TNF-a release and an improvement in the activity of T cell immunity (28). Finally, neutralization of macrophage migration inhibitory factor (MIF) by anti-MIF antibodies was shown to produce a marked increase in survival, even when the treatment was delayed up to 8 h after CLP. This study defined the critical role of MIF in the pathogenesis of septic shock (29). Although it is difficult to compare the results of the aforementioned studies with our data, it is worth mentioning that hypertonic saline was effective in our severe model of LPS-induced shock. The LPS dose used in this study killed almost 35% of the control group. Our study provides evidence that hypertonic saline, beyond its immune-modulatory effects, also affords protection from several important pathophysiologic alterations associated with endotoxemia, such as oxidant stress.

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ACKNOWLEDGMENTS This study was supported by FAPESP-09/03338-7 and 06/00443-6 and CNPQ-470744/2004-9.

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AUTHOR CONTRIBUTIONS

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Theobaldo MC performed experiments and measurements and analyzed results. Barbeiro HV performed experiments and measurements. Barbeiro DF performed experiments and measurements. Petroni R performed experiments and measurements. Soriano FG developed the project, analyzed results, and wrote the manuscript.

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DOI:10.6061/clinics/2012(12)19

BASIC RESEARCH

The effects of exercise modalities on adiposity in obese rats Guilherme Fleury Fina Speretta,I Marisa Cristina Rosante,I Fernanda Oliveira Duarte,II Richard Diego Leite,III Anderson Diogo de Souza Lino,I Rafael Arquias Andre,I Joa˜o Guilherme de Oliveira Silvestre,I Heloisa Sobreiro Selistre de Araujo,II Ana Claudia Garcia de Oliveira DuarteI I Federal University of Sa˜o Carlos (UFSCar), Department of Physical Education, Center of Biological and Health Sciences, Laboratory of Nutrition and Metabolism Applied to Exercise, Sa˜o Carlos/SP, Brazil. II Federal University of Sa˜o Carlos (UFSCar), Department of Physiological Sciences, Laboratory of Biochemistry and Molecular Biology, Sa˜o Carlos/SP, Brazil. III State University of Rio de Janeiro, Physiological Sciences Department, Laboratory for Clinical and Experimental Research in Vascular Biology (BioVasc), Rio de Janeiro, RJ/Brazil.

OBJECTIVE: The aim of the present study was to evaluate the effect of both swimming and resistance training on tumor necrosis factor-alpha and interleukin-10 expression, adipocyte area and lipid profiles in rats fed a high-fat diet. METHODS: The study was conducted over an eight-week period on Wistar adult rats, who were divided into six groups as follows (n = 10 per group): sedentary chow diet, sedentary high-fat diet, swimming plus chow diet, swimming plus high-fat diet, resistance training plus chow diet, and resistance training plus high-fat diet. Rats in the resistance training groups climbed a vertical ladder with weights on their tails once every three days. The swimming groups swam for 60 minutes/day, five days/week. RESULTS: The high-fat diet groups had higher body weights, a greater amount of adipose tissue, and higher tumor necrosis factor-alpha expression in the visceral adipose tissue. Furthermore, the high-fat diet promoted a negative change in the lipid profile. In the resistance training high-fat group, the tumor necrosis factor-alpha expression was lower than that in the swimming high-fat and sedentary high-fat groups. Moreover, smaller visceral and retroperitoneal adipocyte areas were found in the resistance training high-fat group than in the sedentary high-fat group. In the swimming high-fat group, the tumor necrosis factor-alpha expression was lower and the epididymal and retroperitoneal adipocyte areas were smaller compared with the sedentary high-fat group. CONCLUSION: The results showed that both exercise modalities improved the lipid profile, adiposity and obesityassociated inflammation in rats, suggesting their use as an alternative to control the deleterious effects of a high-fat diet in humans. KEYWORDS: Obesity; Adipocytokines; Exercise; Visceral Fat. Speretta GF, Rosante MC, Duarte FO, Leite RD, Lino AD, Andre RA. et al. The effects of exercise modalities on adiposity in obese rats. Clinics. 2012;67(12):1469-1477. Received for publication on August 14, 2012; Accepted for publication on August 19, 2012 E-mail: gsperetta@gmail.com Tel.: 55 16 3301-6481

homeostasis (1) through the secretion of numerous cytokines and adipokines (2,3). Furthermore, there is evidence that adipocyte size may be an important determinant of inflammatory cytokine secretion, including such cytokines as tumor necrosis factor-alpha (TNF-alpha), interleukin-6, and interleukin-1b (2-4). Additionally, several diseases have been associated with low-grade inflammation, such as insulin resistance, dyslipidemia, type II diabetes, and cardiovascular disease (5-9). TNF-alpha seems to have an important role in the physiopathology of insulin resistance by inducing a diminished expression of glucose transporter 4 (GLUT-4) and decreasing the phosphorylation of insulin receptor substrate-1 (IRS-1) and specific insulin receptors (10,11). Furthermore, Bradley et al. demonstrated that a hyperlipidemic diet (46% fat) increased the expression of TNF-alpha

INTRODUCTION There is a growing concern regarding adipose tissue accumulation, as white adipose tissue is considered the most important fat depot associated with a chronic, lowgrade pro-inflammatory metabolic state (1). Studies have demonstrated that metabolic signals from specialized cells (e.g., adipocytes, macrophages, and T-cells) initiate the inflammatory responses that induce alterations in metabolic

Copyright ß 2012 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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and other pro-inflammatory cytokines in adipose tissue in mice. Moreover, the results demonstrated that low-grade inflammation is associated with higher insulin, leptin and glucose levels (12). In this sense, there is great interest in studying strategies to prevent or attenuate the effects of adipose tissue accumulation and the low-grade systemic inflammation associated with obesity (1,6,9,11). Several anti-inflammatory cytokines have been studied due to their role in the modulation of pro-inflammatory cytokines (e.g., interleukin-10 and adiponectin). Interleukin-10 (IL-10), produced mainly by macrophages and lymphocytes (11), can significantly inhibit both the expression and synthesis of proinflammatory cytokines and adipokines (13,14). Exercise training is considered an important environmental factor associated with body weight regulation, and this training has been shown to decrease chronic, low-grade systemic inflammation in humans (15-17) and animals (12). Thus, increasing physical activity has become an important aspect of a non-pharmacological strategy to control obesity and weight gain (18-20). Our research group has already demonstrated that intermittent swimming exercise is more efficient than continuous swimming exercise in decreasing adiposity in rats fed a high-fat diet (18). Bradley et al. (12) showed that voluntary wheel running could decrease visceral white adipose tissue inflammation in high-fat diet- induced obesity in mice. Moreover, the literature has demonstrated that treadmill exercise can minimize inflammation even in non-obese rats (21,22). Among the exercise interventions investigated, resistance training has been shown to be effective in decreasing the adipose tissue depots in rats (23-25). However, the direct effect of resistance training, with or without a high-fat diet, on adipose tissue inflammation in obesity remains unclear. In addition, there is a lack of studies comparing aerobic exercise and resistance training protocols in obese rats. Therefore, our hypothesis was that both resistance training and swimming exercise could alter the deleterious effects of a high-fat diet on adipose tissue, parameters of inflammation, and the lipid profile. Due to the significant differences in the volume and intensity of exercise modalities reported in the literature, the role of each one could be different. The aim of the present study was to evaluate the effect of both swimming and resistance training exercise on TNF-alpha and IL-10 expression, adipocyte area and lipid profiles in rats fed a high-fat diet.

Diets The experimental groups received the standard rat chow diet (MP-77; Primor, Sa˜o Paulo, Brazil) in a pellet form, which contains 23 g of protein, 49 g of carbohydrates, 4 g of total fat, and 5 g of fiber per 100 g of diet. The high-fat diet was composed of standard rat chow plus peanuts, milk chocolate, and sweet biscuits in a proportion of 3:2:2:1. This diet contained 20 g of protein, 20 g of total fat, 48 g of carbohydrate, and 4 g of fiber per 100 g of diet. The caloric density of the diets was determined with an adiabatic calorimeter (IKA-C400), and the values were as follows: 5.12 kcal/g for the high-fat diet and 4.07 kcal/g for the chow diet (27). All components of the high-fat diet were ground and blended. All the high-fat diet groups received a high-fat diet for three weeks before the training period and then throughout the training period (28).

Experimental groups The rats were randomly distributed into the following six experimental groups (ten animals in each group): (I) sedentary chow (Sed-C); (II) sedentary high-fat diet (SedHF); (III) swim plus chow (Swim-C); (IV) swim plus high-fat diet (Swim-HF); (V) resistance training plus chow (RT-C); and (VI) resistance training plus high-fat diet (RT-HF). The sedentary groups (Sed-C and Sed-HF) were kept in their cages during the experimental period without any type of exercise. The groups that underwent training (Swim-C, Swim-HF, RT-C, and RT-HF) performed eight weeks of their respective exercise modalities. The training started at the same time for all groups.

Exercise protocols Swimming. The rats were adapted to the water before the training began. The adaptation consisted of swimming for 30 minutes, once per day for five days, in water at a temperature of 31¡1 ˚C. After adaptation, the rats were trained by swimming for 60 minutes/day, five days a week for eight weeks, with a constant overload equivalent to 5% of their body weight (29). The water tanks were 50 cm in height and 30 cm in diameter, and the overload (lead fish sinkers) was attached to the animal’s chest using an appropriate vest.

Resistance training The eight-week resistance training was performed once every three days with one week of familiarization. After this adaptation period, the animals were submitted to the experimental protocol consisting of 8-12 dynamic movements (reps) per climb, as previously described (30) and adapted (25). The maximum load was determined by the following procedure. Each animal performed an initial climb carrying a load corresponding to 75% of the animal’s body weight. Then, additional weight was added in 30-g increments until the rat could not climb the entire length of the ladder. The highest load successfully carried over the entire length of the ladder was considered the rat’s maximal carrying capacity for that training session. The next training sessions consisted of four ladder climbs at 50, 75, 90, and 100% of the rat’s previous maximum carrying capacity, as determined in the previous session. For the subsequent five ladder climbs, an additional weight was added in 30-g increments until a new maximum carrying capacity was determined. If

METHODS Animals Sixty male Wistar rats (Rattus novergicus var. albinus, Rodentia, Mammalia) (90 days old) from the breeding colony of the Federal University of Sa˜o Carlos (UFSCar), SP, Brazil, with an initial weight of 300¡17.31 g, were used. The animals were kept in individual polypropylene cages (30x20x13 cm), with food and water provided ad libitum, at a controlled temperature and humidity of 22-24 ˚C and 5060%, respectively, and a 12-h light/dark cycle. All animal procedures were performed according to the principles of the USA Guide for the Care and Use of Laboratory Animals (26). The study was approved by the Committee of Experimental Animals (protocol no. 031/2009) at the Federal University of Sa˜o Carlos.

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Figure 1 - Body weight evolution. All values are presented as means¡standard error of the mean; *p#0.05 comparing before chow vs. highfat diet (HF) 3 weeks before the training period (A) and during training period (B, C and D); #comparing trained groups vs. sedentary groups during training period (B, C and D) (n = 10 each group). C, chow; HF, high-fat diet; Sed-C, sedentary chow; Sed-HF, sedentary high-fat diet; Swin-C, swimming chow; Swim-HF, swimming high-fat diet; RT-C, resistance training chow; RT-HF, resistance training high-fat diet.

necessary, a stimulus with tweezers was applied to the animal’s tail to initiate movement.

Adipocyte area determination Fragments (100 mg) of RET EPI, and VISC adipose tissues were separated and fixed in 0.2 M collidine buffer (pH 7.4) containing 2% osmium tetroxide at 37 ˚C. After 48 hours, the samples were washed with warmed saline and spread on a plate as described previously (32). The adipocyte area was measured in different cells from the same tissue (EPI, RET, and VISC) approximately 50 times using image analysis software Image-Pro Plus KS-300 (Carl Zeiss, Oberkochen, Germany) and expressed in terms of mm2. The cells were randomly chosen, and the person analyzing the images was blinded to the group assignments.

Measurements Body weight and food intake were recorded daily.

Tissue collection Animals were euthanized by decapitation 48 hours after the last training session. Retroperitoneal (RET), epididymal (EPI), visceral (VISC), and subcutaneous (SUBC) white adipose tissues were immediately removed, weighed and stored at – 80˚C for further analysis. The adipose tissue collection was performed according to the descriptions of Cinti (31). Blood was also collected, centrifuged (3000 RPM (1157 g) for 10 min at 4˚C), and stored at –80˚C.

Quantitative PCR Total RNA was extracted from visceral adipose tissues using TRIZOLH (Invitrogen Corporation, Carlsbad, California) according to the manufacturer’s instructions. RNA quantification was performed using a NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). Absorbance of the RNA samples was quantified at 260 and 280 nm, and the 260/280 ratio was calculated. The samples exhibited a 260/280 ratio .1.9, which was selected as

Analytical methods The concentrations of total triacylglycerols (TGLs), total cholesterol (TC), and high-density lipoprotein (HDL) were determined enzymatically (Laborlab, Sa˜o Paulo, Brazil), with sensitivities of 0.7 mmol6L–1, 0.14 mmol6L–1, and 0.5 mmol6L–1, respectively. All assays were performed in duplicate, with a coefficient of variation ,0.05.

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Table 1 - The effects of exercise and diet on food consumption, caloric intake and maximum workload during the experiment period.

SED-C SED-HF SWIM-C SWIM-HF RT-C RT-HF

Food Consumption (g)

Caloric intake (Kcal)

2348.20¡74.78 2260¡69.70 2301¡63.36 2041.77¡61.79a,b 2232.40¡55.61 1961.22¡63.49a,b,c

9557.17¡304.37 11571,2¡357.30a 9368.73¡257.89 10453.90¡316.38a,b 9085.86¡226.37 10041.45¡325.08a,b

Table 2 - Epididymal, retroperitoneal and visceral adipocyte areas. EPI (mm2) SED-C SED-HF SWIM-C SWIM-HF RT-C RT-HF

Week 1

Week 8

600.66¡17.75 599.08¡19.55

1451.11¡35.14d 1403.33¡37.97d

VISC (mm2)

14805.38¡125.16 6832.32¡92.77 28945.10¡102.23a 12578.13¡56.90a 10523.88¡47.44b 6609.82¡31.64 23777.72¡327.28a,b 12715.79¡114.35a c b,c 14442.78¡80.33 8301.50¡45.20 > 19408.60¡117.48a,b,c 11013.45¡63.21a,b,c

All values are presented as the means ¡ standard error of the mean; a p#0.05 comparing the normal chow diet vs. high-fat diet groups; b p#0.05 comparing the sedentary vs. trained groups; c p#0.05 comparing the swimming vs. resistance training groups (n = 5 in each group). Abbreviations used: EPI, epididymal; RET, retroperitoneal; VISC, visceral; Sed-C, sedentary chow diet; Sed-HF, sedentary high-fat diet; Swim-C, swimming chow diet; Swim-HF, swimming high-fat diet; RT-C, resistance training chow diet; RT-HF, resistance training high-fat diet.

Maximum workload (g)

RT-C RT-HF

8921.53¡62.45 21935.42¡130.23a 8026.83¡20.29b 21147.42¡103.34a,b 13298.14¡56.30b,c 18.238.62¡76.58a,b,c

RET (mm2)

All values are presented as the mean ¡ standard error of the mean; a p#0.05 comparing the groups fed a normal chow diet vs. the groups fed a high-fat diet; b p#0.05 comparing the sedentary vs. training groups; c p#0.05 comparing the swimming vs. resistance training groups; d p#0.05 comparing week 1 vs. week 8 (n = 10 in each group). Abbreviations used: Sed-C, sedentary chow diet; Sed-HF, sedentary high-fat diet; Swim-C, swimming chow diet; Swim-HF, swimming high-fat diet; RT-C, resistance training chow diet; RT-HF resistance training high-fat diet.

normal chow diet groups in weeks 1, 2, and 3. (Figure 1A; p = 0.003, p = 0.032, and p = 0.003, respectively). After the eight-week training period, all groups treated with the highfat diet (Sed-HF, Swim-HF, and RT-HF) had a higher body weight compared with their respective normal chow diet groups (Sed-C, Swim-C, and RT-C) (Figure 1B, 1C, 1D; p,0.05). Nevertheless, the trained HF groups (Swim-HF and RT-HF) had a lower body weight than the Sed-HF group at the end of the training period (p,0.05). There was no statistically significant difference between the sedentary groups (Sed-C and Sed-HF) with regard to food consumption (g) (p = 0.34) at the end of the experimental period (a three-week diet and an eight-week training period). The trained high-fat diet groups consumed a lower amount of food compared with the normal chow diet groups (p,0.05). Moreover, the Swim-HF group consumed a greater amount of food (p = 0.002) than the RT-HF group (Table 1). The caloric intake (Kcal) was higher in all of the HF groups compared with the C groups (Table 1; p,0.05). The Swim-HF and RT-HF groups had lower caloric intakes compared with the Sed-HF group (p = 0.01 and p,0.001, respectively). There was no statistically significant difference in the interaction between diet and exercise related to either food consumption or caloric intake.

an indicator of RNA purity. The samples were treated with amplification-grade DNAse (Deoxyribonuclease I) (Invitrogen Corporation, Carlsbad, California). cDNA was synthesized using oligo (dT) primers with the Advantage RT-for-PCR kit (Promega Corporation, Madison, WI USA). PCR amplifications were quantified using SYBR Green PCR (Promega Corporation) using a Rotor-Gene 3000 Real-Time Thermal Cycler (Corbett Research, Sydney, Australia). The primers used in this study were as follows: TNF-alpha (Fwd: 5GGCTCCCTCTCATCAGTTCCA-3, Rev: 5-CGCTTGGTGGTTTGCTACGA-3), and IL-10 (Fwd: 5-CAGCTGCGACGCTGTCATCGA-3, Rev: 5-GCAGTCCAGTAGATGCCGGGTG 3). The results were normalized to the housekeeping gene GAPDH (Fwd: 5-GGTGGAGAGCACCAAGACAGA-3, Rev: 5-GCCGGAGTCGACAATGATG-3). The following primers are available on the NCBI website: TNF-alpha (NM_012675.3), IL-10 (NM_012854.2), and GAPDH (NM_017008.3).

Statistical analysis All data are presented as the mean ¡ standard error of the mean (SEM). The statistical analyses were initially performed using the Kolmogorov-Smirnov normality test and the homoscedasticity test (Bartlett criterion). The interaction between the effects of diet and exercise on all variables was analyzed by two-way ANOVA. One-way ANOVA and the independent t-test were also used to compare the sedentary groups with the swimming and resistance training groups, as well as the high-fat diet groups with the chow groups. Fisher’s post-hoc test was applied in the event of a significant (p,0.05) F ratio. The software package used was StatisticaH 6.1 (Tulsa, OK, USA).

Maximum workload The maximum workload capacity of the resistance training groups (RT-C and RT-HF) increased throughout the training period (Table 1). The workloads were higher in weeks four and eight compared with week one (p,0.001) in both resistance training groups (p,0.001).

Relative white adipose tissue weight and adipocyte area The mean areas of adipocytes in the epididymal, retroperitoneal and visceral adipose tissues of the Sed-HF group were larger (p,0.001) than those of the Sed-C group (Table 2). Similar results were observed between the trained high-fat diet groups (Swim-HF and RT-HF) and the trained normal chow groups (Swim-C and RT-C) (p,0.005). Moreover, regardless of the diet, all adipocyte areas were smaller in the swimming groups compared with the sedentary groups (p,0.001), with the exception of the visceral adipocyte area in both the swimming high-fat diet

RESULTS Body weight, food consumption and caloric intake Before starting the diet (week 0), both the normal chow diet (C) and high-fat diet (HF) groups had similar body weights (Figure 1A, p = 0.06). However, the high-fat diet groups exhibited higher body weights compared with the

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more effective in increasing anti-inflammatory adipokine (IL-10) expression than the resistance training. The swimming groups (Swim-C and Swim-HF) exhibited higher expression levels of IL-10 than the Sed-C, Sed-HF, RT-C, and RT-HF groups (p,0.05), except the Swim-HF group, compared with the Sed-HF group (Figure 3A). Additionally, the Swim-C group exhibited significantly higher levels than the Swim-HF group (p = 0.001). However, the resistance training groups exhibited a lower expression level of the pro-inflammatory adipokine TNF-alpha. TNF-alpha expression levels were lower in the trained high-fat groups (SwimHF, p = 0.01, and RT-HF, p,0.001) compared with the Sed-HF groups. Nevertheless, the Swim-HF groups exhibited significantly higher expression than the RT-HF group (Figure 3B; p,0.001). The Swim-HF and Sed-HF groups

group (p = 0.14) and the swimming chow group (p = 0.55). The resistance training chow diet group (RT-C) had larger epididymal and visceral adipocyte areas than the sedentary (Sed-C) and swimming (Swim-C) groups (p,0.001). In addition, the retroperitoneal adipocyte area was larger in the RT-C group than in the Swim-C group (p,0.001). All adipocyte areas were smaller in the resistance training highfat diet group (RT-HF) compared with the sedentary highfat diet group (Sed-HF) and the swimming high-fat diet group (Swim-HF) (p,0.001). All high-fat diet groups exhibited significantly higher relative epididymal, retroperitoneal, visceral, and subcutaneous adipose tissue weights compared with the normal chow diet groups (Figures 2A and 2B; p,0.05). The relative weight of the epididymal tissue was lower in the Swim-HF (p = 0.03) and RT-HF (p = 0.04) groups compared with the Sed-HF group. Additionally, the Swim-C group had a lower relative epididymal weight compared with the Sed-C group (p = 0.03). Nevertheless, the relative visceral weights were higher in the Swim-HF group than in the sedentary (p = 0.009) and resistance training (p,0.001) high-fat diet groups.

Gene expression There was an interaction between diet and exercise with regard to TNF-alpha (p,0.001), but not IL-10 (p = 0.53), expression. The swimming exercise training seemed to be

Figure 3 - IL-10 (A) and TNF-alpha expression (B). All values are presented as meansยกstandard error of the mean; ap#0.05 comparing normal chow diet vs high-fat diet groups; bp#0.05 comparing sedentary vs trained groups; cp#0.05 comparing swimming vs resistance training groups (n = 5 each group). IL-10, interleukin-10; TNF-alpha, tumor necrose factor-alpha; Sed-C, sedentary chow diet; Sed-HF, sedentary high-fat diet; Swim-C, swimming chow diet; SwimHF, swimming high-fat diet; RT-C, resistance training chow diet; RTHF resistance training high-fat diet.

Figure 2 - Relative weight of adipose tissue. All values are presented as meansยกstandard error of the mean; ap#0.05 comparing normal chow diet vs high-fat diet groups; bp#0.05 comparing sedentary vs trained groups; cp#0.05 comparing swimming vs resistance training groups (n = 10 each group). A, EPI, epididymal; RET, retroperitoneal; B, VISC, visceral; SUBC, subcutaneous; SED, sedentary; SWIM, swimming; RT, resistance training.

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mice (12,33-35). Specifically, these diets have been shown to impair glucose metabolism, stimulate abnormal glucose production, and promote both insulin resistance and a chronic, low-grade pro-inflammatory metabolic state (12,27). In the present study, we used the diet previously described by Estadella et al. (27). Our research group has demonstrated that this particular diet can induce weight gain and promote deleterious effects on adiposity, fatty liver development, and the lipid profile in rats (18,28). Moreover, our study demonstrated that a high-fat diet can induce a chronic, low-grade pro-inflammatory metabolic state in rats. Our results showed that the high-fat diet exerted a strong influence on body weight at three weeks. Previous studies have demonstrated that a few weeks of a high-fat diet are sufficient to induce body weight gain, fat accumulation and lipid profile alterations in rats (12,18,27,36). Additionally, the body weights of all the high-fat diet groups, both sedentary and trained, were significantly increased until the end of the experiment compared with the body weights of the chow diet groups. However, the trained groups had a lower body weight than the sedentary groups. Our results are in agreement with the other studies in the literature reporting that exercise alone does not promote a significant loss of body weight in humans and animals (12,15). Therefore, changes in diet are more related to body weight changes than exercise (15). Lower food consumption was associated with an increased caloric intake in the HF groups. These results also suggest that rats are sensitive to both energy and the high-fat contents of diets with regard to the controlling of their food intake by satiety (37,38). Moreover, our data showed that both the swimming and resistance training groups had a lower food consumption and caloric intake compared with the sedentary groups. Previous studies showed similar results (18,35). Furthermore, Ropelle et al. (34) demonstrated that exercise increases the expression of IL-6 and IL-10 in the hypothalamus, which increases insulin and leptin sensitivity through the reduction of IkappaB kinase (IKKb) and inhibition of endoplasmic reticulum (ER) stress, culminating in a decrease in food intake. Thus, the increase in energy expenditure through exercise training and the lower caloric intake could have contributed to the lower body weight gain found in both of the trained HF groups (Swim-HF and RT-HF). In the current study, a high-fat diet also promoted higher relative weights of all adipose tissues and larger adipocyte areas of the epididymal, retroperitoneal, and visceral adipose tissues. Other studies have demonstrated the same results (18,27,39,40). In the trained groups fed a high-fat diet, the relative weight of the epididymal adipose tissue was lower compared with that of the sedentary group fed the same diet. Moreover, high-volume/moderate-intensity swimming was not able to reduce the visceral adipocyte area. Our findings are consistent with other studies that did not observe a significant decrease in either visceral tissue relative weight or adipocyte area with continuous exercise in rats fed a high-fat diet (18,36). These results suggest a higher free fatty acid (FFA) mobilization from other adipose tissues compared with the visceral adipose tissue during exercise. These results could be associated with the load that was used in the swimming exercise in our study (5% of the rat’s body weight). This intensity of exercise is considered to correspond to the transition between aerobic and anaerobic

exhibited higher levels (p,0.001) of TNF-alpha expression compared with the respective normal chow diet groups (Swim-C and Sed-C). However, there was no significant difference between the RT-HF and RT-C groups with regard to TNF-alpha expression (p = 0.69).

Lipid parameters All exercised groups exhibited lower levels of total cholesterol and triacylglycerols (TGLs) and higher values of HDL than the sedentary groups (p,0.05), with the exception of the Swim-HF group, which exhibited TGL values similar to the Sed-HF group (Table 3). Moreover, the TGL levels in the Swim-HF group were higher than those in the RT-HF group (p = 0.001), and they were also higher in the Sed-HF group than in the Sed-C group (p = 0.01). Furthermore, higher values (p = 0.02) of total cholesterol were found in the Swim-HF group compared with the respective normal chow diet group (Swim-C). When the trained groups were compared, the Swim-C group was found to have lower values (p = 0.02) than the RT-C group. The HDL values were higher in the Swim-HF group compared with the Swim-C (p = 0.04) and RT-HF (p,0.001) groups.

DISCUSSION Our study showed that both swimming and resistance training can alter the deleterious effects of a high-fat diet in rats. However, this study adds some new findings related to the training modalities. The exercise protocols have significant differences both in intensity and volume. In this sense, low-volume/high-intensity resistance training was effective in controlling the expression of TNF-alpha in the visceral adipose tissue, controlling body weight, minimizing the increase on the retroperitoneal and visceral adipocyte areas, and producing significant changes in the lipid profile. On the other hand, high-volume/moderateintensity swimming was effective in increasing the expression of IL-10 in the visceral adipose tissue, controlling body weight, reducing the epididymal and retroperitoneal adipocyte areas, and inducing positive effects on the lipid profile. In the literature, different high-fat diets are found to induce a positive energy balance and obesity in rats and

Table 3 - Total serum cholesterol, HDL and triacylglycerol levels. Total Cholesterol (mg/dl) SED-C SED-HF SWIM-C SWIM-HF RT-C RT-HF

51.05¡3.06 57.83¡2.34 39.61¡1,36b 46.94¡4.22a,b 48.22¡1.62b,c 47.22¡2.41b

HDL (mg/dl) 13.85¡0.68 13.77¡0.66 15.85¡1.21b 19.85¡0.85a,b 18.69¡0.62b 17.87¡1.01b,c

TGL (mg/dl) 73.66¡4.22 88.94¡6.71a 62.72¡4.30b 87.83¡6.44 65.27¡4.32b 66.94¡5.15b,c

All values are presented as the mean ¡ standard error of the mean. a p#0.05 comparing the normal chow diet vs. high-fat diet groups; b p#0.05 comparing the sedentary vs. trained groups; c p#0.05 comparing the swimming vs. resistance training groups (n = 10 in each group). Abbreviations used: HDL, high-density lipoprotein; TGL, triacylglycerol; Sed-C, sedentary chow diet; Sed-HF, sedentary high-fat diet; Swim-C, swimming chow diet; Swim-HF, swimming high-fat diet; RT-C, resistance training chow diet; RT-HF resistance training high-fat diet.

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metabolism (lactate threshold) in rats, as previously described by Gobatto et al. (29). The corresponding intensity of exercise promotes a substantial increase in catecholamine levels (41); visceral adipose tissue is very sensitive to catecholamines due to a higher number of alpha and beta adrenergic receptors (42). In light of these facts, we speculate that the load used by the obese rats during swimming was not enough to promote a significant increase in catecholamine levels, resulting in a smaller mobilization of visceral adipose tissue during the swimming exercise. Recently, Araujo et al. (33) demonstrated that the load corresponding to the maximum steady-state lactate level (the gold standard for the identification of the anaerobic threshold) in obese rats is 6% of the rats’ body weights. Therefore, it seems that the load that we used for obese rats is a limitation to our study. The main purpose of the current study was to analyze the inflammatory response of both high-volume/moderate-intensity swimming exercise and low-volume/high-intensity resistance training in rats fed a high-fat diet. In this sense, our results suggest an important role of both types of exercise in regulating the low-grade inflammatory status in obesity. The high-fat diet induced a higher (six-fold) TNF-alpha expression level in visceral adipose tissue. Both high-fat diet trained groups (SwimHF and RT-HF) exhibited a lower expression of this proinflammatory adipokine compared with the Sed-HF group. These findings are in accordance with the literature (1,12,43), where studies have shown a decrease in both TNF-alpha expression in adipose tissue and serum TNF-alpha levels after aerobic training. Bradley et al. (12) demonstrated that voluntary exercise could decrease TNF-alpha expression and improve insulin sensitivity in obese mice through a high-fat/highsucrose diet. Even with a lower training volume, the highintensity resistance training seemed to be more effective in controlling the TNF-alpha expression compared with the swimming exercise. According to Gregor and Hotamisligil (1), TNF-alpha is more highly expressed in hypertrophied adipocytes than in normal size adipocytes, and this finding could support the results in the current study showing that the resistance training promoted smaller visceral adipocyte areas. TNF-alpha plays a key role in the development of insulin resistance, type II diabetes, atherosclerosis and metabolic syndrome, mainly by activating the c-jun N-terminal kinase (JNK) and inhibiting the k kinase and the protein kinase R (PKR) (1). Thus, our results suggest that low-volume/high-intensity resistance training could be an important intervention strategy for both the prevention and treatment of the inflammatory state of obesity and associated chronic diseases. On the other hand, high-volume/moderate-intensity swimming exercises promoted higher IL-10 expression levels in rats fed the chow diet and the high-fat diet, while resistance training was not able to induce the same adaptations. Our results are in agreement with previous studies (34,44) that observed an increase in IL-10 expression in obese and lean rats through aerobic exercises. IL-10 is an important immunoregulatory cytokine with multiple biological effects. In the cytoplasm, it has been shown that IL-10 blocks nuclear factor kappa B (NF-kB) at two levels, namely by suppressing the inhibitor of k kinase (IKK) activity and NF-kB DNA binding activity (45). Therefore, low levels of IL-10 have been observed in obesity, metabolic syndrome and type II diabetes (11). Moreover, Ropelle et al. (34) observed that the intra-hypothalamic infusion of

recombinant IL-10 blocked IKK/NF-kB signaling and endoplasmic reticulum (ER) stress while restoring serinethreonine kinase (Akt) and the signal transducer and activator of transcription-3 (STAT3) phosphorylation, promoting a re-balance in the energy intake in obese animals. According to Waters et al. (46), the increase in IL-10 expression seems to be an important protective factor against insulin resistance, atherosclerotic plaque instability and acute coronary ischemia. Our results also confirmed the evidence that a high-fat diet promotes changes in the lipid profile (18,27). Furthermore, the high-fat diet modified the serum lipid profile by increasing triacylglycerol (TGL) and total cholesterol (TC) levels. Studies in the literature have shown that the lipid profile alteration is strongly associated with metabolic syndrome (47), insulin resistance and nonalcoholic fatty liver disease (48). The current results showed that both the swimming and resistance training groups had higher HDL levels compared with sedentary groups, regardless of the diet administered. Furthermore, the resistance training yielded lower TGL levels. In contrast, several studies have reported a decrease in TGL levels after aerobic exercise training (18,47,48). These effects could be due to the higher levels of TNF-alpha expression in the swimming groups compared with the resistance training groups after eight weeks of exercise in the current study. The effect of TNF-alpha in reducing fatty acid oxidation in hepatocytes and skeletal muscle through the induction of protein phosphatase 2C and the suppression of AMPactivated protein kinase (AMPK) has been previously demonstrated (49). Thus, the reduced rates of fatty acid oxidation are accompanied by an increased accumulation of bioactive lipids, such as TGLs (49). In conclusion, the effect of different exercise modalities on low-grade inflammation and obesity has attracted much interest. In the present study, both resistance training and swimming positively affected the inflammation parameters, body weight, adipocyte area and lipid profile in obese rats. These data support the finding that both exercise modalities seem to be effective in controlling the effects of the consumption of a high-fat diet. However, the influence of each modality on the analyzed parameters seems to be different. Therefore, we can suggest that concurrent training, i.e., swimming exercise at a high volume/moderate intensity along with resistance training at a low volume/ high intensity, could be a great choice for the prevention and/or treatment of obesity. Furthermore, despite the fact that exercise has been shown to alter various pathways associated with obesity, many questions still remain unanswered. Thus, more studies are needed in this field to evaluate the exercise-induced changes associated with obesity and chronic low-grade inflammation.

ACKNOWLEDGMENTS The authors thank Mr. Jose Alves da Silva for laboratory technical assistance. Financial support was provided by the Consellho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico [CNPq] and the Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior (CAPES), Brazil.

AUTHOR CONTRIBUTIONS Speretta GF and Rosante MC conceived and designed the study, drafted the manuscript, analyzed and interpreted the data, critically revised the manuscript for intellectual content, and approved the final version of the

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Schottelius AJ, Mayo MW, Sartor RB, Baldwin AS, Jr. Interleukin-10 signaling blocks inhibitor of kappaB kinase activity and nuclear factor kappaB DNA binding. J Biol Chem. 1999;274(45):31868-74, http://dx.doi. org/10.1074/jbc.274.45.31868.

manuscript. Duarte FO, Leite, RD, Lino AD, Andre RA, Silvestre JG, Selistre de Araujo HS, and Duarte AC drafted the manuscript, interpreted the data and critically revised the manuscript for intellectual content.

REFERENCES 1. Gregor MF, Hotamisligil GS. Inflammatory Mechanisms in Obesity. Annu Rev Immunol. 2010 Apr 5. 2. Wellen KE, Hotamisligil GS. Obesity-induced inflammatory changes in adipose tissue. J Clin Invest. 2003;112(12):1785-8. 3. Hajer GR, van Haeften TW, Visseren FL. Adipose tissue dysfunction in obesity, diabetes, and vascular diseases. Eur Heart J. 2008;29(24):2959-71, http://dx.doi.org/10.1093/eurheartj/ehn387. 4. Otto TC, Lane MD. Adipose development: from stem cell to adipocyte. Crit Rev Biochem Mol Biol. 2005;40(4):229-42, http://dx.doi.org/10. 1080/10409230591008189. 5. Foster-Schubert KE, Cummings DE. Emerging therapeutic strategies for obesity. Endocr Rev. 2006;27(7):779-93. 6. Petersen AM, Pedersen BK. The anti-inflammatory effect of exercise. J Appl Physiol. 2005;98(4):1154-62, http://dx.doi.org/10.1152/ japplphysiol.00164.2004. 7. Mokdad AH, Ford ES, Bowman BA, Dietz WH, Vinicor F, Bales VS, et al. Prevalence of obesity, diabetes, and obesity-related health risk factors, 2001. JAMA. 2003;289(1):76-9, http://dx.doi.org/10.1001/jama.289. 1.76. 8. Tock L, Prado WL, Caranti DA, Cristofalo DM, Lederman H, Fisberg M, et al. Nonalcoholic fatty liver disease decrease in obese adolescents after multidisciplinary therapy. Eur J Gastroenterol Hepatol. 2006;18(12):12415, http://dx.doi.org/10.1097/01.meg.0000243872.86949.95. 9. Walsh NP, Gleeson M, Shephard RJ, Woods JA, Bishop NC, Fleshner M, et al. Position statement. Part one: Immune function and exercise. Exerc Immunol Rev. 2011;17:6-63. 10. Costa JV, Duarte JS. [Adipose tissue and adipokines]. Acta Med Port. 2006;19(3):251-6. 11. Arslan N, Erdur B, Aydin A. Hormones and cytokines in childhood obesity. Indian Pediatr. 2010;47(10):829-39, http://dx.doi.org/10.1007/ s13312-010-0142-y. 12. Bradley RL, Jeon JY, Liu FF, Maratos-Flier E. Voluntary exercise improves insulin sensitivity and adipose tissue inflammation in dietinduced obese mice. Am J Physiol Endocrinol Metab. 2008;295(3):E58694, http://dx.doi.org/10.1152/ajpendo.00309.2007. 13. Choi KM, Ryu OH, Lee KW, Kim HY, Seo JA, Kim SG, et al. Serum adiponectin, interleukin-10 levels and inflammatory markers in the metabolic syndrome. Diabetes Res Clin Pract. 2007;75(2):235-40, http:// dx.doi.org/10.1016/j.diabres.2006.06.019. 14. Volp AC, Alfenas Rde C, Costa NM, Minim VP, Stringueta PC, Bressan J. [Inflammation biomarkers capacity in predicting the metabolic syndrome]. Arq Bras Endocrinol Metabol. 2008;52(3):537-49. 15. Vieira VJ, Valentine RJ, Wilund KR, Antao N, Baynard T, Woods JA. Effects of exercise and low-fat diet on adipose tissue inflammation and metabolic complications in obese mice. Am J Physiol Endocrinol Metab. 2009;296(5):E1164-71, http://dx.doi.org/10.1152/ajpendo.00054.2009. 16. Pedersen BK. The anti-inflammatory effect of exercise: its role in diabetes and cardiovascular disease control. Essays Biochem. 2006;42:105-17, http://dx.doi.org/10.1042/bse0420105. 17. Woods JA, Vieira VJ, Keylock KT. Exercise, inflammation, and innate immunity. Neurol Clin. 2006;24(3):585-99, http://dx.doi.org/10.1016/j. ncl.2006.03.008. 18. Sene-Fiorese M, Duarte FO, Scarmagnani FR, Cheik NC, Manzoni MS, Nonaka KO, et al. Efficiency of intermittent exercise on adiposity and fatty liver in rats fed with high-fat diet. Obesity (Silver Spring). 2008;16(10):2217-22, http://dx.doi.org/10.1038/oby.2008.339. 19. Saris WH, Blair SN, van Baak MA, Eaton SB, Davies PS, Di Pietro L, et al. How much physical activity is enough to prevent unhealthy weight gain? Outcome of the IASO 1st Stock Conference and consensus statement. Obes Rev. 2003;4(2):101-14. 20. Ciolac EG, Greve JM. Exercise-induced improvements in cardiorespiratory fitness and heart rate response to exercise are impaired in overweight/obese postmenopausal women. Clinics. 2011;66(4):583-9. 21. Gomez-Merino D, Drogou C, Guezennec CY, Chennaoui M. Effects of chronic exercise on cytokine production in white adipose tissue and skeletal muscle of rats. Cytokine. 2007;40(1):23-9, http://dx.doi.org/10. 1016/j.cyto.2007.07.188. 22. Sakurai T, Izawa T, Kizaki T, Ogasawara JE, Shirato K, Imaizumi K, et al. Exercise training decreases expression of inflammation-related adipokines through reduction of oxidative stress in rat white adipose tissue. Biochem Biophys Res Commun. 2009;379(2):605-9, http://dx.doi.org/10. 1016/j.bbrc.2008.12.127. 23. Pighon A, Paquette A, Barsalani R, Chapados NA, Yasari S, Doucet E, et al. Substituting food restriction by resistance training prevents liver and body fat regain in ovariectomized rats. Climacteric. 2009;12(2):15364, http://dx.doi.org/10.1080/13697130802447074.

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46. Waters KA, Mast BT, Vella S, de la Eva R, O’Brien LM, Bailey S, et al. Structural equation modeling of sleep apnea, inflammation, and metabolic dysfunction in children. J Sleep Res. 2007;16(4):388-95, http://dx.doi.org/10.1111/j.1365-2869.2007.00614.x. 47. Touati S, Meziri F, Devaux S, Berthelot A, Touyz RM, Laurant P. Exercise Reverses Metabolic Syndrome in High Fat Diet-Induced Obese Rats. Med Sci Sports Exerc. 2011;43(3):398-407.

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RAPID COMMUNICATION

Does lack of improvement in the first two weeks predict treatment resistance in recent-onset psychosis? Monica Kayo, Ivson Tassell, Vivian Hiroce, Anny Menezes, Helio Elkis Schizophrenia Program (PROJESQ), Institute of Psychiatry, Faculdade de Medicina da Universidade de Sa˜o Paulo, Sa˜o Paulo/ SP, Brazil. Email: monica.kayo@usp.br Tel.: 55 11 2661 7808

the included patients were regularly taking AP drugs upon entering the study. The treatment followed the IPAP algorithm, which states that patients should be treated in monotherapy with either a first-generation (FGA) or second-generation antipsychotic (SGA) for at least two 4- to 6-week trials. The patients who failed to respond to these two trials were considered to be resistant to treatment and were eligible for clozapine. The subjects were assessed with the Positive and Negative Syndrome Scale (PANSS) (11) at baseline and at weeks 2, 4, 6, 8, and 12. The PANSS ratings were performed by three blinded and independent psychiatrists. The scale showed high reliability (intraclass correlation coefficient = 0.865).

INTRODUCTION Randomized controlled trials among patients with schizophrenia have shown that ,20% improvement in the first two weeks of treatment predicts nonresponse after 12 weeks. The findings have been consistent for patients treated with both conventional (1) and second-generation antipsychotic (AP) drugs (2). However, despite the lack of evidence regarding the length of time that clinicians should pursue one treatment regimen, most psychiatric textbooks and practice guidelines suggest that patients should be treated for at least four to six weeks with one AP (3-5) before switching to another (6). Large pragmatic trials with chronic patients, such as the Clinical Antipsychotic Trials of Intervention Effectiveness (CATIE) (7), have found no benefit from switching AP drugs (8,9). No studies have considered less chronic patients, such as patients with recent-onset schizophrenia (i.e., less than five years of illness duration). We investigated responses to APs and switching strategies in patients with recent-onset schizophrenia. We used the IPAP algorithm, which is a well-defined algorithm for treating schizophrenia (4,10).

Subjects Patients between ages 18 and 45 years who met the DSMIV TR criteria (12) for schizophrenia or schizoaffective disorders were included in the study. The patients had #5 years of illness and an exacerbation of symptoms with a minimum PANSS total score of 60 and a minimum Clinical Global Impression-Severity (CGI-S) score of 4. The exclusion criteria were the presence of cognitive disorders of neurological etiology, a history of refractoriness to AP (i.e., previous or active use of clozapine), and active substance abuse.

METHODS Study Design The present study was a pilot, single-center, open, randomized trial. The trial was conducted in an outpatient setting at the Institute of Psychiatry (IPq) at the Hospital das Clı´nicas at the University of Sa˜o Paulo Medical School, Sa˜o Paulo/SP, Brazil. All of the subjects signed informed consent forms. The study was approved by the local ethics committee (protocol 0802/08) and was registered at Clinicaltrials.gov: NCT01016145. The study was conducted in accordance with the Declaration of Helsinki (1975, revised in 1989). The subjects were randomized to receive either first-generation antipsychotics (FGAs) or secondgeneration antipsychotics (SGAs). Once assigned to the FGA or SGA groups, the choice of drug within the class was left to the discretion of each treating psychiatrist. None of

Pharmacotherapy The following APs were allowed in the study: FGAs

N N N

haloperidol, 5-10 mg/day. chlorpromazine, 25-800 mg/day trifluoperazine, 5-10 mg/day

SGAs

N N N N N

Copyright ß 2012 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.

olanzapine, 5-20 mg/day. risperidone, 1-6 mg/day quetiapine, 25 a 800 mg/day ziprasidone, 80-160 mg/day aripiprazole, 15-30 mg/day

Objectives The primary aim of the study was to assess treatment resistance among patients with recent-onset schizophrenia

No potential conflict of interest was reported.

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Statistical analyses

Table 1 - Patient baseline characteristics (n = 20).

The demographic and outcome variables (response/ nonresponse) were compared between the groups using ttests, Chi-squared test, and Fischer’s exact tests. To compare the overall treatment effect over time, a repeated measures analysis of variance (ANOVA) was used; treatment was the between-group factor, and time was the within-subject factor. An analysis of covariance was performed to evaluate the treatment response at two weeks, and baseline PANSS was used as a covariate. The significance level was set at 0.05.

Characteristic Male/Female (n) Paranoid schizophrenia (n) Catatonic schizophrenia (n) Schizoaffective disorder (n) Age at screening (years, mean¡SD) Duration of untreated psychosis (years, mean¡SD) Illness duration (years, ¡SD) Total PANSS score (mean ¡SD) CGI severity (mean¡SD)

10/10 17 1 2 30.05¡8.06 1.65¡2.66 3.25¡3.14 94.16¡21.99 5.35¡0.75

CGI: clinical global impression. PANSS: positive and negative syndrome scale.

RESULTS Forty-nine subjects were screened for study eligibility, and 22 were included and randomized. All of the patients had recent-onset schizophrenia or schizoaffective disorder and were not taking APs when they were included in the study. The baseline characteristics of all the participants are described in Table 1. Twelve patients were randomized to receive SGAs, and 10 were randomized to receive FGAs (Figure 1). One patient withdrew consent after being randomized but before receiving treatment, and another patient could not be assessed because of catatonic mutism; therefore, we analyzed the baseline data from 20 patients (Table 1). Two patients withdrew from the study because they did not have a caregiver to transport them to the visits (n = 2), and one patient was excluded because of gastric cancer. All of the

or schizoaffective disorder. The study tested the hypothesis that no improvement in the PANSS score after two weeks of treatment was associated with nonresponse at 12 weeks. The secondary objectives were to evaluate the effectiveness of switching antipsychotic drugs after four weeks of treatment and to compare the response rate between the FGAs and SGAs.

Response definition Response was defined as a $30% decrease in the PANSS score at any time in the study. Improvement (in the first two weeks) was defined as a $20% decrease in the PANSS score.

Figure 1 - A patient allocation flow diagram. FGA: First-generation antipsychotic; SGA: second-generation antipsychotic.

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Treatment response in schizophrenia Kayo M et al.

Figure 2 - PANSS – the mean change from baseline. PANSS: positive and negative syndrome scale, n = 17, *p,0.05 vs. baseline, **p,0.01 vs. baseline.

dropouts occurred in the SGA group before they had received treatment. Ultimately, the data from 17 patients were analyzed (7 SGA and 10 FGA patients). After 12 weeks, 13 patients responded to treatment and 4 did not. The number of responders was similar in each group (FGA = 7, SGA = 6, p = 1.0). The mean PANSS reduction from baseline was also similar between the groups. Independent sample t-tests demonstrated that the FGA and SGA groups were comparable in the patient demographic characteristics and treatment responses throughout the study. Because more than 50% of the patients switched their APs (7 from FGA and 5 from SGA), and there was no observable difference between the SGA and FGA groups, we conducted a pooled analysis of all the patients to assess the correlation between the 2-week improvements and 12week responses. At 12 weeks, the mean PANSS change was 38.74%. This improvement corresponded to a mean decrease of 35 points (Figure 2). Repeated measures ANOVA showed the significant effect of time; there was an accumulated response rate over time. Five of the 13 responders showed no responses until 4 weeks. Pairwise comparisons between the baseline scores and weeks 2, 4, 6, 8, and 12 showed a significant change in the mean PANSS scores starting at week 4 (p,0.05). A Chi-squared test between the lack of improvement at 2 weeks (at least 20% improvement in the PANSS score) showed no relationship to the 12-week nonresponse (x2 = 0.60, df = 1, p = 0.57). The subjects who failed to respond by the end of the study had lower baseline PANSS scores than the responders (79.25 vs. 91.23, p = 0.02); however, the ANCOVA analysis showed that a lack of improvement at two weeks failed to predict treatment nonresponse at 12 weeks (F = 1.907, p = 0.192). Twelve of the 20 patients switched APs. One patient switched from an SGA to an FGA after experiencing an allergic reaction to a single dose of risperidone. The other AP switches occurred because of a lack of efficacy. In the patients who switched, 75% responded by the end of the study, but this rate was not significant when compared to the patients who did not switch (p = 1.00).

DISCUSSION We found that lack of improvement in the first two weeks does not predict nonresponse at 12 weeks. Even the patients who did not exhibit a minimal PANSS improvement of 20% at two weeks had responded by 12 weeks. Previous reports have shown that early nonresponse was predictive of refractoriness; however, such analyses were based on patients with chronic and possibly already refractory schizophrenia (1,2). As observed in chronic patients (8,9), switching APs was of little benefit in our study. However, the small sample size limits the generalizability of our findings. Physicians tend to switch antipsychotics for different reasons. Physicians are willing to wait longer for some drugs than for others because the onset of efficacy is not exact for all antipsychotics (e.g., risperidone acts faster than olanzapine) (13). D2/3 receptors are occupied by the AP in a few hours, but the clinical response may not occur for weeks (14,15); this delayed onset of a clinical response can be explained by the fact that a stable blockade of those receptors may be necessary before a sustained clinical effect is observed (15). Because this is a pilot study with a small sample of recentonset patients, caution is needed in interpreting the results. Our study found that lack of improvement in the first two weeks does not predict treatment resistance in recent-onset psychosis.

ACKNOWLEDGMENTS The authors thank nursing staff members Norma Aparecida da Silva and Luis Antonio da Silva for their kind collaboration in collecting samples and biometrics. The authors declare no conflicts of interest and have received no outside funding.

AUTHOR CONTRIBUTIONS Kayo M and Elkis H were responsible for the study concept, design, data analysis and interpretation. Kayo M was also responsible for executing the project and writing the first draft of the manuscript. Tassell I, Hiroce VY, and Menezes AK were raters and contributed to the data analysis and interpretation.

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7. Lieberman JA, Stroup TS, McEvoy JP, Swartz MS, Rosenheck RA, Perkins DO, et al. Effectiveness of antipsychotic drugs in patients with chronic schizophrenia. N Engl J Med. 2005 22;353(12):1209-23. 8. Essock SM, Covell NH, Davis SM, Stroup TS, Rosenheck RA, Lieberman JA. Effectiveness of switching antipsychotic medications. Am J Psychiatry. 2006;163(12):2090-5, http://dx.doi.org/10.1176/appi.ajp.163.12.2090. 9. Rosenheck R, Cramer J, Xu W, Thomas J, Henderson W, Frisman L, et al. A comparison of clozapine and haloperidol in hospitalized patients with refractory schizophrenia. New England Journal of Medicine. 2009;337(12):809-15. 10. Elkis H, Meltzer HY. [Refractory schizophrenia]. Rev Bras Psiquiatr. 2007;29 Suppl 2:S41-7. 11. Kay SR, Fiszbein A, Opler LA. The positive and negative syndrome scale (PANSS) for schizophrenia. Schizophr Bull. 1987;13(2):261-76. 12. American Psychiatric Association. Diagnostic and Statistical Manual of Mental Disorders. 4th edition ed. Washington DC: American Psychiatric Press; 1994. 13. Hatta K, Sato K, Hamakawa H, Takebayashi H, Kimura N, Ochi S, et al. Effectiveness of second-generation antipsychotics with acute-phase schizophrenia. Schizophr Res. 2009;113(1):49-55, http://dx.doi.org/10. 1016/j.schres.2009.05.030. 14. Kapur S, Agid O, Mizrahi R, Li M. How antipsychotics work-from receptors to reality. NeuroRx. 2006;3(1):10-21, http://dx.doi.org/10. 1016/j.nurx.2005.12.003. 15. Pani L, Pira L, Marchese G. Antipsychotic efficacy: relationship to optimal D2-receptor occupancy. Eur Psychiatry. 2007;22(5):267-75, http://dx.doi.org/10.1016/j.eurpsy.2007.02.005.

REFERENCES 1. Correll CU, Malhotra AK, Kaushik S, McMeniman M, Kane JM. Early prediction of antipsychotic response in schizophrenia. Am J Psychiatry. 2003;160(11):2063-5, http://dx.doi.org/10.1176/appi.ajp.160.11.2063. 2. Kinon BJ, Chen L, Ascher-Svanum H, Stauffer VL, Kollack-Walker S, Zhou W, et al. Early response to antipsychotic drug therapy as a clinical marker of subsequent response in the treatment of schizophrenia. Neuropsychopharmacology. 2010;35(2):581-90, http://dx.doi.org/10. 1038/npp.2009.164. 3. Buchanan RW, Kreyenbuhl J, Kelly DL, Noel JM, Boggs DL, Fischer BA, et al. The 2009 schizophrenia PORT psychopharmacological treatment recommendations and summary statements. Schizophr Bull. 2010;36(1):71-93, http://dx.doi.org/10.1093/schbul/sbp116. 4. Elkis H. Treatment-resistant schizophrenia. Psychiatr Clin North Am. 2007;30(3):511-33, http://dx.doi.org/10.1016/j.psc.2007.04.001. 5. Lehman AF, Lieberman JA, Dixon LB, McGlashan TH, Miller AL, Perkins DO, et al. Practice guideline for the treatment of patients with schizophrenia, second edition. Am J Psychiatry. 2004;161(2 Suppl):156. 6. Suzuki T, Uchida H, Watanabe K, Nomura K, Takeuchi H, Tomita M, et al. How effective is it to sequentially switch among Olanzapine, Quetiapine and Risperidone? A randomized, open-label study of algorithm-based antipsychotic treatment to patients with symptomatic schizophrenia in the real-world clinical setting. Psychopharmacology (Berl). 2007;195(2):285-95.

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CASE REPORT

Inevitable hemodialysis for treating resistant hypertension in a patient with Leriche syndrome Murvet Yilmaz,I Ozlem Harmankaya Kaptanogullari,I Can Caliskan,II Ayse Sinangil Arar,III Cuneyt Akgol,III Kayhan Erturk,I Abdulkadir UnsalIII I

Bakirkoy Dr. Sadi Konuk Training and Research Hospital, Department of Nephrology, Istanbul, Turkey. II Sisli Etfal Training and Research Hospital, Department of Radiology, Istanbul, Turkey. III Sisli Etfal Training and Research Hospital, Istanbul, Department of Nephrology, Turkey. Email: murvetyilmaz@hotmail.com Tel.: 90 532 6469726

The patient’s brachial, radial, ulnar, and femoral pulses were palpable bilaterally; however, his dorsalis pedis, tibialis posterior and popliteal pulses were absent. There was no evidence of atrophic or ischemic changes in the lower extremities. Carotid bruits were heard on both sides of the neck, and an audible bruit was noted over the left renal artery. His blood pressure was 190/90 mmHg, and grade-2 hypertensive retinopathy was found. Blood chemistry analysis revealed a urea level of 108 mg/ dl and a creatinine level of 3.6 mg/dl; serum triglycerides, cholesterol, glucose, HbA1c, and other biochemical parameters were within normal limits. The tests for AT III and proteins C and S were negative. Ultrasonography revealed that the right kidney was 95.6 mm, with a cortex width of 13.8 mm; the left kidney was 69.9 mm, with a cortex width of 8.8 mm. The echogenicity was increased in both kidneys. Color Doppler ultrasonography did not detect blood flow in either renal artery. A 40-60% carotid stenosis was diagnosed bilaterally. Doppler ultrasonography revealed high resistance and normal triphasic waveforms in the bilateral subclavian, axillary, brachial, radial and ulnar arteries. Magnetic resonance angiography (MRA) revealed aortoiliac occlusive disease (Leriche syndrome), concurrent with severe bilateral stenosis of the renal and common iliac arteries. MRA also demonstrated significant collateral circulation (Figure 1). Tc99m DTPA perfusion scintigraphy revealed a delay in the initiation of perfusion and a deterioration of function in both kidneys, particularly in the left kidney (Figure 2). A two-dimensional transthoracic echocardiography revealed mild left atrial enlargement (LAd: 4.3 cm) and concentric left ventricular hypertrophy.

INTRODUCTION Leriche syndrome, also referred to as aortoiliac occlusive disease, results from thrombotic occlusion of the abdominal aorta immediately above the site of its bifurcation (1). The characteristic symptoms include intermittent bilateral claudication with ischemic pain and absent or diminished femoral pulses, along with pallor, coldness and fatigue of both lower extremities, and inability to maintain penile erection (1). Risk factors include hyperlipidemia, hypertension, diabetes mellitus, and smoking (2). Physical examination reveals weak or absent femoral and distal pulses. Pathologically, the most common cause of Leriche syndrome has been found to be luminal narrowing of the abdominal aorta and/or iliac arteries by atherosclerotic obstruction (3). The syndrome typically begins at the distal aorta or common iliac artery origins and slowly progresses proximally and distally over time (4), rarely affecting the visceral or renal arteries (5). In this paper, we report a patient with Leriche syndrome who suffered from bilateral renal artery occlusion-induced resistant hypertension and could not be treated by revascularization procedures or angiotensin-converting enzyme (ACE) blockade. Eventually, maintenance hemodialysis (HD) was initiated, with accompanying renin-angiotensin blockers, to control the hypertension.

CASE DESCRIPTION A 53-year-old male patient was admitted to the outpatient clinic with a history of uncontrolled hypertension despite antihypertensive treatment, consisting of diltiazem 120 mg qd, amlodipine 10 mg qd, and doxazosin 4 mg qd. The patient stated that he suffered from bilateral calf and buttock claudications with a 100-m walk as well as erectile dysfunction. He had been a heavy smoker (40 pack-years) but never experienced angina. He did not suffer from diabetes or hypercholesterolemia.

Clinical course The patient was hospitalized due to uncontrolled hypertension. A balloon angioplasty and stenting procedure via the axillary artery was attempted. However, the aortography revealed complete occlusion of the abdominal aorta immediately below the superior mesenteric artery (SMA), and the renal arteries could not be observed (Figure 3). An attempt to introduce the catheter into the renal arteries using a 0.035 inch nitinol guide was unsuccessful; thus, the procedure was terminated. By-pass grafting was discussed with the patient, with an emphasis on the high risk of the procedure and the potentially limited beneficial effects. Valsartan (at a dosage of 80 mg/day) was added to the current antihypertensive therapy. A very close follow-up was indicated. A dramatic drop in blood pressure was

Copyright ß 2012 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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Figure 1 - Magnetic resonance angiography of the abdominal vessels, which reveals total occlusion of the abdominal aorta just below superior mesenteric artery. The lower extremities and the abdomen are perfused by means of the collateral arteries.

Because the progression of the disease is slow and collaterals develop, limb-threatening ischemic disease does not tend to occur (8). In our patient, because arterial perfusion of the lower extremities was achieved via arterial collaterals, there were no atrophic or ischemic changes in the lower extremities. Leriche syndrome typically begins at the distal aorta or common iliac artery origins and slowly progresses proximally and distally over time. This progression is quite variable but may ultimately extend to the level of the renal arteries or result in complete aortic occlusion (4). Proximal propagation of an occlusive distal aortic thrombus to the suprarenal level is rare (9). Bergen and Trippel (10) suggested that aortic thrombosis may progress to involve one or both renal arteries in 3-15% of patients with distal aortic occlusion. As it progresses, the thrombosis may gradually encroach upon a renal artery and cause increasingly severe hypertension. The patient in this case had uncontrolled blood pressure and bilateral renal artery occlusion, with an occlusion of the abdominal aorta below the SMA. MRA was indicated for imaging the renal vasculature; however, the risk of nephrogenic systemic fibrosis was quite high for this patient. The possible benefits and drawbacks were first discussed extensively among the attending doctors, and it was decided that the potential benefits outweighed the risks. Afterward, the risks were discussed with the patient, and only after having his verbal consent,

observed the next day; however, the renal function continued to deteriorate, with urea and creatinine levels escalating up to 298 and 8.5 mg/dl, respectively. The valsartan was withdrawn, which resulted in a decrease in urea and creatinine levels to 234 and 4.6 mg/dl, respectively. As soon as the valsartan was stopped, the patient’s blood pressure again increased up to 200/110 mmHg, despite conventional antihypertensive treatment without ACE blockade. Considering that ACE blockade improved the patient’s uncontrolled hypertension but deteriorated kidney function, the patient was enrolled in a maintenance hemodialysis program. An ACE-inhibitor (ramipril 2.5 mg/day) was initiated, which resulted in a dramatic improvement in blood pressure. He was discharged in good clinical condition with no subjective complaints and was placed on a thrice-weekly hemodialysis regimen.

DISCUSSION Leriche syndrome, which is characterized by the triad of claudication, decreased pulses due to aortoiliac occlusion and impotence, mostly occurs in men and is generally diagnosed between 40 and 60 years of age (6,7). The occlusion is usually due to atherosclerotic plaques in the aorta with associated thrombi.

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Resistant hypertension in a patient with Leriche syndrome Yilmaz M et al.

Figure 2 - A Tc99m DTPA perfusion scintigraphy revealed a delay in the initiation of perfusion and deterioration of function for both kidneys, affecting left kidney particularly.

the MRA was performed using Dotarem (gadoterate meglumine), which is least likely to cause nephrogenic systemic fibrosis (11). Revascularization of the renal arteries should be considered a radical treatment modality in all cases of renal artery stenosis; renal angioplasty combined with stenting is the preferred procedure (12-14). Because successful revascularization via the brachial or axillary artery has been reported in the literature (15-17), we attempted to place a stent in the renal arteries bilaterally via the axillary artery; however, the procedure was unsuccessful because the catheter could not be introduced into the occluded segment. Another option may be to place a by-pass graft between the proximal and distal segments of the stenotic region. However, this option was abandoned because the patient did not give consent for the intervention when he was informed about the risks of the operation. At this time point, we were faced with the dilemma of either leaving the patient with the risks of hypertension, which was resistant to conventional antihypertensive medications, or administering an ACE blockade medication, despite the risks of acute kidney injury superimposed on chronic renal failure. After discussing the options with the patient, we decided to administer the angiotensin-II receptor blocker valsartan at a dosage of 80 mg/day. His blood pressure decreased rapidly to 110/70 mmHg; however, acute renal function deterioration superimposed on chronic renal failure developed. This acute renal dysfunction could be due to nonspecific effects of hypotension,

Figure 3 - An AP view of the abdominal aorta by aortography. The distal portion of the a. mesenterica superior (SMA) is completely occluded. The proximal of the SMA, the coeliac truncus and its branches can be seen clearly.

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2. Frederick M, Newman J, Kohlwes J. Leriche syndrome. J Gen Intern Med. 2010;25(10):102-4. 3. Patel M, Waxman P, Rozenblit A, Tafreshi M, Alexander LL. An unusual angiographic appearance of aorto-iliac disease presenting as the Leriche syndrome. J Natl Med Assoc. 1985;77(1):57-61. 4. Takigawa M, Akutsu K, Kasai S, Tamori Y, Yoshimuta T, Higashi M, et al. Angiographic documentation of aortoiliac occlusion in Leriche’s syndrome. Can J Cardiol. 2008;24(7):568. 5. Ferral Hector, Lorenz Jonathan. Interventional Radiology (RadCases). NY. 2010;105-6. 6. Erskine JM, Gerbode FL, French SW 3rd, Hood RM. Surgical treatment of thrombotic occlusion of aorta and iliac arteries; the Leriche syndrome. AMA Arch Surg. 1959;79(1):85-93, http://dx.doi.org/10.1001/archsurg.1959.0432 0070089015. 7. Leriche R, Morel A. The syndrome of thrombotic obliteration of the aortic bifurcation. Ann Surg. 1948;127(2):193-206, http://dx.doi.org/10.1097/ 00000658-194802000-00001. 8. www.learningradiology.com/archives06/COW%20201-Leriche% 20Syndrome/lerichecorrect.htm - 26k. 9. Sequera JC, Beckmann CF, Levin DC. AJR. 1979;132(5):773-6. 10. Bergan JJ, Trippel OH. Management of juxtra-renal aortic occlusion. Arch Surg. 1963;87:230-8, http://dx.doi.org/10.1001/archsurg.1963.01310140038010. 11. Nortier JL, del Marmol V. Nephrogenic systemic fibrosis—the need for a multidisciplinary approach. Nephrol Dial Transplant. 2007;22(11):3097101, http://dx.doi.org/10.1093/ndt/gfm430. 12. Dorros G, Jaff M, Mathiak L, Dorros II, Lowe A, Murphy K, et al. Fouryear follow-up of Palmaz-Schatz stent revascularization as treatment for atherosclerotic renal artery stenosis. Circulation. 1998;98(8):642-7, http://dx.doi.org/10.1161/01.CIR.98.7.642. 13. Morganti A. Renal angioplasty: better for treating hypertension or for rescuing renal function? J Hypertens 1999;17(12Pt 1):1659-65, http://dx. doi.org/10.1097/00004872-199917120-00001. ¨ nal IA, Yavuzkir M, Dag˘li N, Karaca I, Celiker H, et al. Effect of 14. Ilkay E, Gu renal artery stenting on renal function in patients with ischemic nephropathy. Jpn Heart J. 2004;45(4):637-45, http://dx.doi.org/10.1536/jhj.45.637. 15. Shiraishi J, Higaki Y, Oguni A, Inoue M, Tatsumi T, Azuma A, et al. Transradial renal artery angioplasty and stenting in a patient with Leriche syndrome. Int Heart J. 2005;46(3):557-62, http://dx.doi.org/10. 1536/ihj.46.557. 16. Jian W, Tajima H, Murata S, Abe Y, Hakozaki K, Kumazaki T, et al. Renal artery stenosis in a patient with Leriche syndrome: brachial artery access for stent placement. Radiat Med. 2004;22(1):49-51. 17. Kaukanen ET, Manninen HI, Matsi PJ, So¨der HK. Brachial artery access for percutaneous renal artery interventions. Cardiovasc Intervent Radiol. 1997;20(5):353-8, http://dx.doi.org/10.1007/s002709900167. 18. Hricik DE, Dunn MJ. Angiotensin-converting enzyme inhibitor-induced renal failure: causes, consequences, and diagnostic uses. J Am Soc Nephrol. 1990;1(6):845-58.

which may have caused a sudden deterioration in kidney perfusion. In contrast, the dysfunction may have been due to ACE inhibition-related acute renal failure due to bilateral renal artery stenosis. The incidence of significant azotemia complicating ACE inhibition therapy in patients with renovascular disease is uncertain. ACE inhibitor-induced renal failure has been reported in 23% of patients with bilateral renal artery stenosis and in 38% of patients with only one kidney with a stenotic artery (18). This problem is usually reversed by stopping the ACE blockade, although some patients may suffer from permanent renal failure. After stopping the valsartan, the previous clinical/laboratory findings (i.e., hypertension and moderately high levels of serum urea and creatinine) reversed. At this time point, the initiation of maintenance hemodialysis was inevitable. This strategy provided the opportunity to control volume overload while also applying an ACE blockade to satisfactorily control blood pressure. This patient deserves special mention because the only way to manage his condition was to initiate HD and prescribe an ACE inhibitor. The patient was discharged with well-controlled hypertension, but he would need thrice-weekly HD for the rest of his life. In conclusion, in patients with Leriche syndrome and bilateral renal artery occlusion, hemodialysis accompanied by antihypertensive therapy with a renin-angiotensin blocker may be inevitable if renal revascularization is not an option.

AUTHOR CONTRIBUTIONS Yilmaz M followed up and treated the patient and drafted the manuscript. Kaptanogullari OH and Erturk K performed data collection and wrote the manuscript. Caliskan C performed the radiological examinations. Arar AS, Akgol C, and Unsal A followed-up and treated the patient.

REFERENCES 1. Lee WJ, Cheng YZ, Lin HJ. Leriche syndrome. Int J Emerg Med. 2008;1(3):223.

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DOI:10.6061/clinics/2012(12)22

CASE REPORT

Association of myelodysplastic syndrome with CD5+, CD23+ monoclonal B-cell lymphocytosis Alex F. Sandes,I Maria de Lourdes L. F. Chauffaille,I Alberto Orfao,II Graziella C. Siufi,I Maria Regina R. Silva,III Mihoko YamamotoI I

Universidade Federal de Sa˜o Paulo (UNIFESP), Disciplina de Hematologia e Hemoterapia, Sa˜o Paulo/SP, Brazil. II University of Salamanca, Servicio General de Citometria, Department of Medicine and Cancer Research Center (IBMCC-USAL/CSIC), Salamanca, Spain. III Universidade Federal de Sa˜o Paulo (UNIFESP), Pathology Department, Sa˜o Paulo/SP, Brazil. Email: alex.sandes@bol.com.br / yamamoto@unifesp.br Tel.: 55 11 5576-4240

was hypercellular and showed increased blast cells (9%), marked dysplastic abnormalities in the erythroid, granulocytic and megakaryocytic lineages and no ring sideroblasts. The BM biopsy showed mild reticulin fibrosis without the presence of lymphoid aggregates or infiltrates. Cytogenetic analysis was inconclusive. A diagnosis of refractory anemia with excess blasts-1 was established according to the WHO classification and the patient received supportive therapy consisting of red blood cell transfusions. Five months later, the patient’s myeloblasts had increased to 15% (BM) and an abnormal karyotype (46,XX,del(9) (q22) [20]) was detected. Immunophenotyping of BM cells was performed using a large panel of monoclonal antibodies in a four-color combination to analyze precursor cells, granulocytic, monocytic and erythroid BM compartments (5) (Table 1). Multiparameter flow cytometry confirmed the increased number of myeloblasts (Figure 1) with an aberrant (CD7+, CD56+), immature (CD34+, CD117+, HLA-DR+) myeloid (CD13+, CD33+) phenotype. No CD34+ B-cell precursors were detected and phenotypic abnormalities were identified in the maturing neutrophils (e.g., aberrant CD13/CD11b expression) and in the monocytic (e.g., CD2+, CD56+) and erythrocytic (e.g., CD71lo) compartments (Figure 1). In addition, 8% of the marrow cells were mature T-lymphocytes and 9% were mature B-lymphocytes that presented aberrant CD25 and CD22dim expression. A detailed study was performed due to this B-cell phenotype, which demonstrated that 88% of Bcells were monoclonal, surface kappa light chain restricted and a CD5+, CD19+, CD23+ and CD25+ phenotype (Figure 2). Further analysis of the peripheral blood (PB) demonstrated the presence of monoclonal B-lymphocytes with a CD19+, CD20dim, CD22dim, CD5+, CD23+, CD79bdim, CD25+, CD43+ and CD38+ phenotype, lacking in reactivity to FMC7, sIgM, CD10, CD103, and CD11c. The absolute PB Blymphocyte count was 3.06109/L and a diagnosis of CD5+CD23+ MBL was made. The patient was treated for MDS with daunorubicin and cytarabine, with no response and died six months later with AML and the persistence of the PB monoclonal B-cell population (26109/L). The reported case followed the guidelines of the local ethics committees and the Helsinki Declaration. This patient was included in a large study on MDS (6) and informed consent was obtained after the study was approved by the Institutional Review Board of UNIFESP (Brazil).

INTRODUCTION Myelodysplastic syndromes (MDS) are clonal disorders characterized by bone marrow (BM) failure and an increased risk of transformation into acute myeloid leukemia (AML). Typically, MDS patients are elderly and are already anemic, leucopenic and/or thrombocytopenic upon presentation. Despite their myeloid origin, many MDS cases show abnormalities in B-cells, usually related to a decrease in the B-cell production and/or an increase in apoptosis (1,2). Such alterations may be due to an increased production demand for essential hematopoietic cells, such as red cells and neutrophils. Alternatively, a blockade on B-cell maturation might occur (2). A low level of monoclonal B-cells (,56109/L) in otherwise healthy individuals characterizes a condition called monoclonal B-cell lymphocytosis (MBL). The overall reported frequency of MBL is between 0.5% and 12% of adults, depending on the age of the population and the sensitivity of the flow cytometry approach used to detect the B-cell clones (3,4). MBL is more frequently observed among the relatives of patients with familial chronic lymphocytic leukemia (CLL) and in individuals exposed to toxic environments. Although coexistence of CLL and AML or CML has been sporadically reported, the actual frequency of MBL in association with other hematological neoplasias remains to be established.

CASE DESCRIPTION Here, we report a 61-year-old female patient diagnosed with both MDS and MBL. She complained of a three-month history of fatigue associated with sustained anemia and thrombocytopenia detected on a routine blood test. Splenomegaly or lymphadenomegaly were not observed. Her hemoglobin level was 3.8 g/dL, her absolute white blood cell (WBC) count was 7.66109/L (neutrophils 68%; eosinophils 1%; lymphocytes 23%; monocytes 9%) and her platelet count was 346109/L. Hyposegmented neutrophils were observed on blood film examination. The BM aspirate

Copyright ß 2012 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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1.2:1. Most cases corresponded to patients with MDS and CLL (19/31) and no significant association with specific subtypes of MDS was observed. Florensa et al. showed a frequency of 1% of B-CLPD in a series of 1198 MDS patients (CLL 0.5%, lymphoplasmacytic lymphoma 0.4% and multiple myeloma 0.1%) (7). At present, the general consensus is that these associations may occur randomly (8-10). In line with this idea, we found only one case report supporting the existence of an ontogenic association between both disorders: trisomy 8 was detected in 55% of CD13+ neutrophils and in 13% of CD19+/CD5+ B lymphocytes in a case with MDS and systemic vasculitis, suggesting a common stem cell precursor had generated the two neoplastic cell populations (11). Low numbers of circulating monoclonal B-cells in otherwise healthy individuals has been investigated in the last ten years. A MBL diagnosis is confirmed by the presence of ,56109/L monoclonal B-cells associated with a normal physical examination and negative history for lymphoproliferative disease, as observed in our case (12). The progression rate to CLL has been described as approximately 1-2% of MBL cases per year (12). MBL has been observed in a significant number of healthy individuals and in, particular, in elderly people. Despite its frequency, the association of MBL in patients with other hematological neoplastic diseases (e.g., MDS) remains to be established.

Table 1 - Monoclonal antibody reagents used for the immunophenotypic characterization of myelodysplastic syndrome (modified from Matarraz S. et al., Leukemia 2008;22:1175-83) (5).

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

FITC

PE

PerCP-Cy5.5

APC

HLA-DR HLA-DR CD11b CD36 CD15 CD2 CD65 CD71 CD61 CD22 nTdT cCD3 CD19

CD117 CD123 CD13 CD64 CD16 CD56 NG2 Glycophorin-A CD33 CD25 cMPO CD7 CD79a

CD45 CD45 CD45 CD45 CD45 CD45 CD45 CD45 CD45 CD45 CD45 CD45 CD45

CD34 CD34 CD34 CD34 + CD14 CD34 CD34 CD34 CD34 CD34 CD34 CD34 CD34 CD34

DISCUSSION Association of MDS and B-chronic lymphoproliferative disorders (B-CLPD) is an uncommon finding that has been sporadically described (Table 2). We found that 31 cases had been reported since 1974. The median age at presentation was 72 years (range: 49-95) and the male-to-female ratio was

Figure 1 - Immunophenotypic analysis of bone marrow cell compartments: the blast cells (orange) are CD34+ and CD117+ with partial expression of CD7; the erythroblasts (purple) present glycophorin-A and low expression of CD71; the maturing granulocytes (blue) demonstrate an anomalous maturation pattern of CD13/CD11b expression, with increased levels of CD13 and CD11b during intermediate maturation stages; the monocytes (green) express aberrant CD2.

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MDS and monoclonal B-cell lymphocytosis Sandes AF et al.

Figure 2 - Immunophenotypic analysis of bone marrow B-cells: monoclonal B-lymphocytes (green) are CD19+, CD5+, CD45++ and presents dim k light chain restriction. Residual polyclonal B-lymphocytes are illustrated in red. The same immunophenotypic B-cells were detected in the peripheral blood.

To the best of our knowledge, this is the first report describing the association of MDS and CD5+with CD23+MBL. Interestingly, despite the presence of a monoclonal B-cell population, no CD34+B-cell precursors were identified in the patient’s BM, as usually occurs in MDS cases. Caballero et al. reported a patient with AML associated with CLL in which the progression of CLL disease was observed after treatment for AML and remission was achieved (13). In contrast, the lymphoid clone remained stable in our case, without change after therapy during the follow-up. MDS develops in a multistep way. An increasing number of accumulated genetic abnormalities lead to ineffective hematopoiesis. In addition, it has been noted that immune dysfunction in MDS could also contribute to the development of cytopenia in some groups of patients. Accumulating evidence has demonstrated the association of MDS and autoimmune manifestations, T-cell mediated myelosuppression and cytokine-induced cytopenia (14,15). Immunosuppressive therapy in selected MDS patients results in high rates of hematological recovery with improved survival, especially in young patients and in the presence of HLA-DR15 (16). In addition, autoimmune complications are well recognized in CLL, occurring in

10% to 25% of patients at some time during the course of the disease. Autoimmunity in CLL predominantly targets blood constituents, most commonly red cells. The association of MBL and immune dysfunction is uncertain, although Mittal et al. (17) reported a high prevalence (20%) of CLL phenotype lymphocytes in 31 patients with autoimmune disorders (AIHA, idiopathic thrombocytopenic purpura and Evans’ Syndrome), suggesting the importance of these clones in the pathogenesis of autoimmune blood disorders. For the reasons stated above, the coexistence of a monoclonal B-cell disorder with MDS deserves special interest. This is particularly true because multiparameter flow cytometry immunophenotyping is the method of choice to detect MBL. Although numerous reports had described the immunophenotypic abnormalities in MDS, it has only recently begun to be applied during the routine work-up for the diagnosis and prognosis of potential MDS cases (18). Moreover, our case illustrates the importance of well-designed flow cytometry panels, capable of analyzing all hematopoietic cell populations because more than one neoplastic disorder may be present in the same case. We suggest the addition of one screening tube in the MDS investigational panel, containing CD19, CD5, anti-k and l

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Table 2 - Previous reports of MDS and B-chronic lymphoproliferative disorders. References

N (31)

Age y/Gender

N. of cases Subtype of CLPD

Light Chain

MDS subtype*

1

71/M

CLL

NR

RARS

1

66/M

CLL

NR

RARS

47, XY, +mar [25]/46, XY (10) 46, XY

1

74/F

l

CMML

NR

1

79/F

Lymphocytic Lymphoma CLL+MM

l (CLL)

RARS

46,XX

1

80/F

PLL

l

RA

NR

5

2 B-NHL; 3 CLL CLL

NR; NR k (3 CLL) NR

RAEB; 2 RA; 2 RARS RAEB

Normal in all cases

1

81.4 (67-95)/ 1 M, 4 F 72/M

1

49/M

CLL

k

RAEB

1

60/M

LPL

k

CMML

46, XY, -4, +der(4), -12, +der (12) NR

1

89/F

B-NHL

k

RARS

NR

11

6 CLL, 5 LPL

NR

CLL

k

3 RA, 2 RARS, 2 RAEB, 4 CMML RCMD

NR

1

79.3 (59-86)/ 8 M, 3 F 85/F

1

69/F

CLL

k

RAEB

Myeloid clone: 46, XX, del(13)/ Lymphoid clone: 47, XX, +12 46,XX

1

67/M

CLL

NR

RARS

NR

1

66/M

CLL

k

RCMD

1

76/F

B-NHL

l

RAEB

46, XY, add(1),del(11) (10)/47, XY, +3 (4) Myeloid clone: +13 (FISH)

1

63/M

CLL

k

RCMD

1. Papayannis AG et al. Br J Haematol. 1974;28:125-129. 2. Escribano LE et al. Sangre. 1977;22:639-645 3. Manoharan A et al. Br J Haematol. 1981; 48:111-116. 4. Greenberg BR et al. Br J Haematol. 1983;53:125-133. 5. Camba L et al. J Clin Pathol. 1985;38:297-300. 6. Copplestone JA et al. Br J Haematol. 1986;63:149-159 7. Bracey AW et al. Am J Hematol. 1989;30:174-180. 8. Bastion Y et al. Leukemia. 1991;5:1006-1009. 9. Shulze R et al. Clin Investig. 1992;70:1082-1084 10. Uematsu M et al. Int J Hematol. 1995;62:45-51. 11. Florensa L et al. Leuk Lymphoma. 1996;23:609-612. 12. Sylvester LS et al. Leuk Res. 1997;21:619-621. 13. Mitterbauer G et al. Ann Hematol. 1997;74:193-197. 14. Lai R et al. Am J Clin Pathol. 1999;111:373-378. 15. Mossafa H et al. Leuk Lymphoma. 2001;41:337-341. 16. Cauwelier B et al. Leuk Lymphoma. 2001;41:337-341 17. Aviv H et al. Leuk Lymphoma. 2004;45:1279-1283.

Cytogenetic

46, XY, t(1;7), -7, +8

46, XY, +12, del (14)(q21) (1)/47, XY,+8 (1)/46, XY [23]

*

All cases were reclassified according to the WHO criteria; N - number of cases; NR–not reported; CLPD–Chronic lymphoproliferative disorder; NHL–NonHodgkin Lymphoma; LPL–Lymphoplasmacytic Lymphoma; PLL–Prolymphocytic Leukemia; MDS–Myelodysplastic Syndrome; RARS–refractory anemia with ring sideroblasts; CMML–chronic myelomonocytic leukemia; RA–refractory anemia; RCMD- refractory cytopenia with multilineage dysplasia; RAEB– refractory anemia with excess blasts. APC, allophycocyanin; FITC, fluorescein isothiocyanate; PE, phycoerythrin; PerCP, peridinin chlorophyll protein.

light chains, which could be expanded when necessary. Widespread use of flow cytometry in the routine evaluation of MDS patients will potentially contribute to defining the actual frequency of the association between MBL and MDS and it may provide new insights into the association of these disorders.

2.

3. 4.

ACKNOWLEDGMENTS This report was supported by grants from FAPESP (proc no. 05/57792-0) and FADA (Unifesp). AFS was supported by CNPq (proc n.142968/20064) and CAPES (proc.n. PDEE BEX 1025/05-8).

5.

AUTHOR CONTRIBUTIONS Sandes AF and Chauffaille ML attended the patient and provided clinical data. Silva MR performed the bone marrow anatomopathological study, and Chauffaille ML was responsible for the cytogenetic experiments and analysis. Sandes AF and Siufi GC were responsible for data acquisition and flow cytometric immunophenotyping analysis. Sandes AF, Orfao A and Yamamoto M planned the study, interpreted the data and drafted the manuscript. All co-authors approved the final version of the manuscript.

6.

7.

8.

REFERENCES 1. Ogata K, Kishikawa Y, Satoh C, Tamura H, Dan K, Hayashi A. Diagnostic application of flow cytometric characteristics of CD34+ cells

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in low-grade myelodysplastic syndromes. Blood. 2006;108(3):1037-44, http://dx.doi.org/10.1182/blood-2005-12-4916. Ribeiro E, Matarraz SS, de Santiago M, Lima CS, Metze K, Giralt M, et al. Maturation-associated immunophenotypic abnormalities in bone marrow B-lymphocytes in myelodysplastic syndromes. Leuk Res. 2006;30(1):9-16, http://dx.doi.org/10.1016/j.leukres.2005.05.019. Marti G, Abbasi F, Raveche E, Rawstron AC, Ghia P, Aurran T, et al. Overview of monoclonal B-cell lymphocytosis. Br J Haematol 2007; 139(5):701-8, http://dx.doi.org/10.1111/j.1365-2141.2007.06865.x. Nieto WG, Almeida J, Romero A, Teodosio C, Lo´pez A, Henriques AF, et al. Increased frequency (12%) of circulating chronic lymphocytic leukemia-like B-cell clones in healthy subjects using a highly sensitive multicolor flow cytometric approach. Blood. 2009;114(1):33-7, http://dx. doi.org/10.1182/blood-2009-01-197368. Matarraz S, Lopez A, Barrena S, Fernandez C, Jensen E, Flores J, et al. The immunophenotype of different immature, myeloid and B-cell lineage-committed CD34+ hematopoietic cells allows discrimination between normal/reactive and myelodysplastic syndrome precursors. Leukemia. 2008;22(6):1175-83, http://dx.doi.org/10.1038/leu.2008.49. Sandes AF, Yamamoto M, Matarraz S, Chauffaille MdL, Quijano S, Lopez A, et al. Altered immunophenotypic features of peripheral blood platelets in myelodysplastic syndromes. Haematologica. 2012;97(6):895902, http://dx.doi.org/10.3324/haematol.2011.057158. Florensa L, Vallespi T, Woessner S, Domingo A, Crespo N, Rozman M, et al. Incidence and characteristics of lymphoid malignancies in untreated myelodysplastic syndromes. Leuk Lymphoma. 1996;23(5-6):609-2, http:// dx.doi.org/10.3109/10428199609054871. Aviv H, Tang D, Das K, Harrison JS, Hameed M, Varma M. Simultaneous appearance of trisomy 8 and trisomy 12 in different cell populations in a patient with untreated B-cell chronic lymphocytic leukemia and myelodysplasia. Leuk Lymphoma. 2004;45(6):1279-83, http://dx.doi.org/10.1080/10428190310001638869.


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MDS and monoclonal B-cell lymphocytosis Sandes AF et al. 14. Pinheiro RF, Silva MR, Chauffaille ML. The 5q- syndrome and autoimmune phenomena: report of three cases. Leuk Res. 2006;30(4):50710, http://dx.doi.org/10.1016/j.leukres.2005.08.025. 15. Chamuleau ME, Westers TM, van Dreunen L, Groenland J, Zevenbergen A, Eeltink CM, et al. Immune mediated autologous cytotoxicity against hematopoietic precursor cells in patients with myelodysplastic syndrome. Haematologica. 2009;94(4):496-506, http://dx.doi.org/10.3324/ haematol.13612. 16. Sloand EM, Wu CO, Greenberg P, Young N, Barrett J. Factors affecting response and survival in patients with myelodysplasia treated with immunosuppressive therapy. J Clin Oncol. 2008;26(15):2505-11, http:// dx.doi.org/10.1200/JCO.2007.11.9214. 17. Mittal S, Blaylock MG, Culligan DJ, Barker RN, Vickers MA. A high rate of CLL phenotype lymphocytes in autoimmune hemolytic anemia and immune thrombocytopenic purpura. Haematologica. 2008;93(1):151-2, http://dx.doi.org/10.3324/haematol.11822. 18. Loken MR, van de LA, Ogata K, Orfao A, Wells DA. Flow cytometry in myelodysplastic syndromes: report from a working conference. Leuk Res. 2008;32(1):5-17, http://dx.doi.org/10.1016/j.leukres.2007.04.020.

9. Cauwelier B, Nollet F, De Laere E, Van Leeuwen M, Billiet J, Criel A, et al. Simultaneous occurrence of myelodysplastic syndrome and monoclonal B lymphocytes with a different clonal origin. Leuk Lymphoma. 2002;43(1):1913, http://dx.doi.org/10.1080/10428190210205. 10. Sylvester LS, Nowell PC, Bonner H, Moreau L, Moore JS. Concurrent diagnosis of chronic lymphocytic leukemia and myelodysplastic syndrome. Leuk Res. 1997;21(7):619-21, http://dx.doi.org/10.1016/S01452126(97)00017-9. 11. Billstrom R, Johansson B, Strombeck B, el Rifai W, Larramendy M, Olofsson T, et al. Clonal CD5-positive B lymphocytes in myelodysplastic syndrome with systemic vasculitis and trisomy 8. Ann Hematol 1997;74(1):37-40. 12. Marti G, Abbasi F, Raveche E, Rawstron AC, Ghia P, Aurran T, et al. Overview of monoclonal B-cell lymphocytosis. Br J Haematol 2007;139(5):701-8, http://dx.doi.org/10.1111/j.1365-2141.2007.06865.x. 13. Caballero MD, Gonzalez M, Canizo MC, Orfao A, Nieto MJ, San Miguel JF. Concomitant chronic lymphocytic leukemia (CLL) and acute myeloid leukemia. Complete remission of CLL achieved with high-dose cytosine arabinoside. Leukemia. 1992;6(8):856-8.

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Acute abdominal pain in a 24-year-old woman: FitzHugh-Curtis syndrome associated with pyelonephritis Giorgio Di Rocco, Domenico Giannotti, Marco Collalti, Rita Mele, Stefano Pontone, Francesca Frezzotti, Adriano Redler, Gregorio Patrizi ‘‘Sapienza’’ University of Rome, Department of Surgical Sciences, Rome/IT. Email: stefano.pontone@uniroma1.it Tel.: 00390649972446

globular, distended, and barely palpable, particularly in the right upper quadrant, with torpid peristalsis. Murphy’s and Blumberg’s signs were positive, but Rovsing’s and McBurney’s signs were negative. Her blood test results were as follows: Hb 12.7 g/dL, WBC 16,020, neutrophils 87.6%, PLT 194,000, INR 1.05, ratio 1, urea 16 mg/dL, fibrinogen 858 mg/ dL, creatinine 1.1 mg/dL, ALT 23 IU/L, AST 32 IU/L, amylase 20 IU/L, total bilirubin 1.35 mg/dL, direct bilirubin 0.91 mg/dL, sodium 136 mEq/L, potassium 3.48 mEq/dL, LDH 281 IU/L, CRP 30.27 mg/dL, ferritin 450 ng/ mL, and ESR 82 mm/h. A physical-chemical examination of her urine showed rare lower urinary tract epithelial cells, rare urothelial cells, rare erythrocytes, and 3-4 leukocytes per field. Chest Xray showed the accentuation of broncho-vascular markings and mild obliteration of the sinuses bilaterally. An abdominal radiograph showed no free air or hydroaeric levels below the diaphragm. Despite the negative results of the blood tests, we suspected that the pain had a hepatobiliary cause. We therefore performed liver ultrasonography, which showed an enlarged liver, no dilation of the intra- or extra-hepatic bile ducts, and a gallbladder with thickened walls and a multilayered appearance, which was similar to the findings in inflammatory disorders. Gallstones were not observed in the lumen of the gallbladder, which had a layer of perihepatic and pericholecystic effusion (Figure 1). A focal area of unclear nature with a lack of homogeneity was observed in the middle layer of the left kidney (Figures 2 and 3). The patient was transferred to our department for further investigation. MRI confirmed the ultrasonographic results described previously and more clearly demonstrated that the alteration of the left kidney was pyelonephritis. The patient was examined by a gynecologist, who noted abundant leucorrhoea and vulvovaginal erythema and therefore performed trans-vaginal ultrasonography, which showed a normal uterus and endometrium and ovarian polycystosis. Vaginal and cervical swabs were collected to identify pathogenic microorganisms; these swabs were positive for Candida albicans and Ureaplasma urealyticum. Gene amplification by PCR was positive for Chlamydia trachomatis. Finally, we came to a conclusive diagnosis of pelvic inflammatory disease (PID) and perihepatitis resulting in cholecystitis caused by C. trachomatis, which constituted FHC syndrome. Previous antibiotic treatment with piperacillin and tazobactam was replaced by a single intravenous dose of 2 g of azithromycin, which led to a gradual decrease in pain and inflammation markers within approximately 72 hours. After three weeks, the patient was in good general condition, with no pain on palpation in the right

INTRODUCTION Fitz-Hugh-Curtis syndrome is an inflammatory disease of the liver capsule that occurs as a complication of pelvic inflammatory disease in approximately 25% of cases. The most common etiological agents are Chlamydia trachomatis and Neisseria gonorrhoeae. Classically, it manifests as acute right upper quadrant pain, which can sometimes be confused with pyelonephritis or a primitive condition of hepatobiliary origin or the digestive tract. A correct diagnosis is often difficult. Ultrasonography and computed tomography (CT) may be helpful in the differential diagnosis of other forms of peritonitis localized in the right abdominal quadrants, and serology, urine cultures and cervical swabs are generally used to isolate the responsible organism. The differential diagnosis could include Chilaiditi syndrome (interposition of a colonic segment between the liver and diaphragm with occlusion) or exudative peritonitis from salpingitis. We describe the clinical case of a 24year-old Caucasian woman who presented with acute right upper quadrant pain, positive Murphy’s sign, neutrophilic leukocytosis, and fever.

CASE DESCRIPTION We report the clinical case of a 24-year-old Caucasian woman who was treated in November 2011. She presented with pain in the right upper quadrant associated with fever and neutrophilic leukocytosis. She was a light smoker, nulliparous, and sexually active. In the previous two years, she had taken estrogen-progestin oral therapy for hypertrichosis associated with a polycystic ovary. She reported recurrent episodes of vaginitis in the last year. The patient presented to our hospital due to pain in the right upper abdominal quadrant that had lasted for three days, fever, abdominal distention, and obstipation of feces and gases associated with an episode of vomiting. These symptoms appeared to coincide with her menstrual cycle. The patient was hemodynamically stable, and her abdomen appeared

Copyright ß 2012 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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Figure 3 - The focal area of pyelonephritis in the left kidney on color Doppler imaging.

Figure 1 - Perihepatic and pericholecystic effusion.

hypochondriac region, negative swab results and normalization of the ultrasound and laboratory results.

(2,6,7). These microorganisms can travel from the pelvis to infect the surface capsule of the liver or the spleen via the peritoneal fluid (4,8). The symptoms are varied, ranging from simple non-specific pain in the hypogastric region, dysuria and dyspareunia to excessive vaginal secretions (3,9). Although most cases show a normal number of leukocytes, neutrophilic leukocytosis occurred in our case. Liver enzymes are typically normal or only mildly elevated, and ESV and CRP are elevated in almost all cases. The standard examination for the diagnosis is gene amplification by PCR, which has replaced the conventional bacterial cultures and allows for the isolation of microorganisms using cervical, urethral, rectal and throat swabs. Diagnostic tools play a key role in the diagnosis of FHC syndrome, and in particular, ultrasonography and CT are often conclusive, although in our case, MRI was used in consideration of the young age of the patient (10). The final diagnosis is made by integrating several laboratory and instrumental tests to exclude other diseases that occur with acute right upper quadrant pain. We would like to emphasize the particular association between acute left pyelonephritis with urine cultures positive for C. trachomatis and FHC syndrome. In recent years, there has been a progressive increase in the incidence of this syndrome, which is likely due to the continuous improvement of diagnostic techniques. The treatment is pharmacological, and if properly performed after a correct diagnosis, it leads to complete resolution of the clinical case, reducing the duration of hospitalization. Otherwise, incorrect diagnosis in the acute phase followed by inadequate antibiotic treatment may cause the disease to become chronic, with subsequent further complications; in this case, only surgical treatment by laparotomy or laparoscopy can lead to the correct diagnosis and therapy.

DISCUSSION Classically, pain in the right upper quadrant of the abdomen occurs as a result of hepatobiliary diseases, such as gallstones, cholecystitis, hepatitis, hepatic or sub-phrenic abscess, and herpes zoster (1,2). Right upper quadrant pain, with or without hypogastric consequences, is due to FitzHugh-Curtis (FHC) syndrome in 12-25% of cases, which is characterized by the inflammation of Glisson’s capsule concomitant with pelvic inflammatory disease (1,3). FHC syndrome manifests as perihepatitis in association with pelvic inflammatory disease. The syndrome occurs predominantly in young women, who complain of severe right upper quadrant pain during the acute phase that sometimes simulates pleurisy (1,4). In the chronic phase, numerous viscero-visceral and viscero-parietal adhesions are formed, initiating at Glisson’s capsule; such cases are diagnosed using explorative laparotomy or laparoscopy (3,5). In the literature, approximately 12-25% of pelvic inflammatory disease cases occur concomitantly with FHC. The frequently involved etiological agents are of bacterial origin, and N. gonorrhoeae and C. trachomatis are among the most common

AUTHOR CONTRIBUTIONS Di Rocco G and Giannotti D designed the study and were also responsible for the manuscript writing. Patrizi G was responsible for data analysis and interpretation. Mele R, Collalti M and Frezzotti F were responsible for data collection and management. Pontone S and Redler A were responsible for the critical revision of the manuscript for important intellectual content.

REFERENCES 1. Rivero-Sa´nchez L, Lo´pez-Soriano EM, Guarner-Aguilar L. Fitz-HughCurtis syndrome: abdominal pain in women of 26 years old. Rev Esp

Figure 2 - The focal area of pyelonephritis in the left kidney.

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2.

3.

4. 5.

A Fitz-Hugh-Curtis variation Di Rocco G et al. 6. Le Moigne F, Lamboley JL, Vitry T, Salamand P, Milou F, Farthouat P. Usefulness of contrast-enhanced CT scan for diagnosis of Fitz-HughCurtis syndrome. Gastroenterol Clin Biol. 2009;33(12):1176-8, http://dx. doi.org/10.1016/j.gcb.2009.09.002. 7. Woo SY, Kim JI, Cheung DY, Cho SH, Park SH, Han JY, Kim JK. Clinical outcome of Fitz-Hugh-Curtis syndrome mimicking acute biliary disease. World J Gastroenterol. 2008;14(45):6975-80, http://dx.doi.org/10.3748/ wjg.14.6975. 8. Olson S, McComb PF. Formation of Fitz-Hugh Curtis adhesions due to perihepatitis associated with salpingitis. J Obstet Gynaecol Can. 2010; 32(6):533-4. 9. MecLean AB. Fitz-Hugh-Curtis syndrome. J Obstet Gynaecol. 2008; 28(3):259-60, http://dx.doi.org/10.1080/01443610802042993. 10. Wang CL, Guo XJ, Yuan ZD, Shi Q, Hu XH, Fang L. Radiologic diagnosis of Fitz-Hugh-Curtis syndrome. Chin Med J (Engl). 2009 20;122(6):741-4.

Enferm Dig. 2011;103(10):546-8, http://dx.doi.org/10.4321/S1130-0108 2011001000009. Huang HH, Tsai CM, Tyan YS. Unusual cause should be kept in mind of abdominal pain in female patient. Fitz-Hugh-Curtis syndrome. Gastroenterology 2011;140(3):e7-8, http://dx.doi.org/10.1053/j.gastro. 2010.02.067. Ricci P, Lema R, Sola´ V, Ferna´ndez C, Fabres C, Ferna´ndez E, Pardo J. Fitz-Hugh-Curtis syndrome: Three cases of incidental diagnosis during laparoscopy. J Obstet Gynaecol. 2008;28(3):352-4, http://dx.doi.org/10. 1080/01443610802058411. Hong DG, Choi MH, Chong GO, Yi JH, Seong WJ, Lee YS, et al. FitzHugh-Curtis Syndrome: single centre experiences. J Obstet Gynaecol. 2010;30(3):277-80, http://dx.doi.org/10.3109/01443610903576282. Burton E, McKeating J, Stahlfeld K. Laparoscopic management of a small bowel obstruction of unknown cause. JSLS. 2008;12(3):299-302.

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DOI:10.6061/clinics/2012(12)24

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Rare association of cutaneous vasculitis, IgA nephropathy and antiphospholipid antibody syndrome with tuberculous lymphadenitis Roberto Bueno Filho,I Alberto Pinto Cordeiro,I Flavia Tremeschin de Almeida,II Catarina Shaletich,III Roberto Silva Costa,III Ana Maria F. RoselinoI I

University of Sa˜o Paulo, School of Medicine of Ribeira˜o Preto, Department of Medical Clinics, Division of Dermatology, Ribeira˜o Preto/SP, Brazil. University of Sa˜o Paulo, School of Medicine of Ribeira˜o Preto, Department of Medical Clinics, Division of Dermatology, Biomedical, Laboratory of Molecular Biology, Ribeira˜o Preto/SP, Brazil. III University of Sa˜o Paulo, School of Medicine of Ribeira˜o Preto, Hospital of Clinics, Department of Pathology, Ribeira˜o Preto/SP, Brazil. II

Email: amfrosel@fmrp.usp.br Tel.: 55 16 3602-2447

which had recently increased in number and size. She had a history of headaches and seizures, an ischemic stroke five years earlier (resulting in facial motor sequelae) and five pregnancies, which consisted of four normal deliveries and one abortion at 22 years of age. On examination, there were cervical adenomegalies with bulky and coalescing lymph nodes (the largest measuring 3 cm in diameter) and crusted lesions on the dorsal feet and tips of the toes with purulent exudates and interdigital maceration (Figure 1). The peripheral sensitivity test yielded normal results. Laboratory tests showed hypochromic anemia with microcytosis (hemoglobin: 11.0 g/dL; NR: 12.0-15.5 g/dL), increased inflammatory activity (ESR: 30 mm/1st hour; NR: ,10 mm/1st hour; C-reactive protein: 3.46 mg/dL; NR: up to 0.5 mg/dL; alpha1-acid glycoprotein: 156 mg/dL; NR: 50-120 mg/dL), increased levels of immunoglobulins (IgA: 1,070 mg/dL; NR: 134-297 mg/dl; IgG: 1,900 mg/dL; NR: 770-1,510 mg/dl; IgM: 222 mg/dL; NR: 67-208 mg/dl), positive autoantibodies (antinucleolar antibody (ANA)-positive 1:100 with nucleolar pattern; lupus anticoagulant (PIL and dRVVT)positive; anticardiolipin IgG: 14.9 GPL/mL (weak positive); NR: up to 14.0 GPL/ml; p-ANCA-positive), urinary disorders (microscopic hematuria: 25-30 RBCs/field; NR: 35 RBCs/field; urinary erythrocytes: 90% total dysmorphic cells, 19% acanthocytes; NR: up 4% acanthocytes; proteinuria: 255 mg/24 h; NR: up to 150 mg/24 h), and intradermal reaction test positive for Tb (PPD: 45 mm with necrosis). Cervical, thoracic and abdominal computed tomographies showed cervical adenomegaly (up to 1.8 cm in length) with central necrosis, a left axillary 2.3-cm lymph node, and several retroperitoneal lymph nodes (up to 0.9 cm in length). A cranial MRI showed a cerebral infarction on the left parietal region and lacunar infarctions in the region of capsular nuclei. Histopathology showed the following: (1) cervical lymph node - chronic granulomatous lymphadenitis with caseous necrosis; (2) fifth left toe - focal granulomatous in addition to leukocytoclastic vasculitis and direct immunofluorescence (DIF) strongly positive for anti-fibrinogen serum (3+) on capillary walls; and (3) renal biopsy - focal and segmental sclerosis with mild focal and chronic tubulointerstitial damage, characterized by mesangial deposition of IgA on DIF, which may correspond to primary IgA nephropathy (Berger’s Disease) or Henoch-Scho¨nlein purpura (Figure 2).

INTRODUCTION Mycobacterium tuberculosis infections can be associated with several immune mechanisms. Some of these mechanisms, such as vasculitis associated with tuberculosis, are rare and can challenge dermatologists when making differential diagnoses. The association between pulmonary tuberculosis (Tb) and vasculitis was first described by Parish and Rhodes (1) in 1967. These associations fall into three main types: pulmonary Tb/ cutaneous leukocytoclastic vasculitis (CLV), pulmonary Tb/ Henoch-Scho¨nlein purpura (HSP) and pulmonary Tb/vasculitis secondary to rifampicin (2-7). The existence of circulating immune complexes in pulmonary Tb has been demonstrated, and the levels of these complexes are related to disease activity. The mechanism of vascular damage is attributed to immune complexes rather than to direct damage caused by M. tuberculosis (8,9). Another differential diagnosis is CLV due to rifampicin therapy (7). In cases of Tb-related vasculitis, skin lesions usually improve following the administration of a specific Tb treatment; an anti-inflammatory therapy is not required (2-5,10). Immune complexes are also responsible for renal injury, which is associated with increased levels of immunoglobulins (mainly IgA against the A-60 antigen of M. tuberculosis), and mesangial deposition, which leads to the activation of the alternative complement and lecithin pathways, resulting in glomerular damage (IgA nephropathy) (11). Finally, Tb is associated with the development of anti-phospholipid antibody syndrome (APS) by inducing the production of autoantibodies (18,19). The association of tuberculous lymphadenitis, CLV, IgA nephropathy and APS in a single patient has not been reported yet.

CASE DESCRIPTION A 45-year-old woman from Ribeira˜o Preto (northeastern region of Sa˜o Paulo State, Brazil) presented to our clinic with painful necrotic lesions on both feet, mainly on the toes,

Copyright ß 2012 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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Figure 1 - Clinical presentation of a patient with cutaneous vasculitis. The upper figures show progressive necrotic lesions on the dorsal feet, mainly on the toes. The lower figures show the amputation of the left fifth toe and the improvement of vasculitic skin lesions following RIPE treatment.

PCR with DNA that was extracted from paraffin blocks of lymph node and skin biopsies confirmed Mycobacterium sp. only in the lymph node (Figure 3). The final diagnosis consisted of Tb lymphadenitis, CLV, primary IgA nephropathy and APS. Ciprofloxacin (500 mg) was prescribed twice per day for ten days, and Fluconazole (150 mg) was prescribed once per week for eight weeks for the secondary infection and interdigital maceration on the feet. Thereafter, aspirin (200 mg daily) and warfarin (for anticoagulation) were prescribed. The patient was evaluated by physicians in the Departments of Neurology, Ophthalmology, Nephrology and Infectious Diseases. Tb treatment was initiated with RIPE (rifampicin, isoniazid, pyrazinamide and ethambutol). Because an adverse drug reaction to pyrazinamide was observed, administration of this medication was suspended. Because of the worsening necrosis on her toes, the patient’s left fifth toe was amputated with no complications. The patient finished the Tb treatment, which was followed by weight recovery,

Figure 3 - 2% agarose gel. PCR was performed with specific primers for Mycobacterium sp. (17), whose amplicon is 383 bp. Columns: 1 and 7, 100-bp marker; 2, negative control (without DNA); 3, paraffin biopsy of skin vasculitis; 4, paraffin sample of a cervical lymph node; 5, culture of M. avium complex; and 6, culture of M. tuberculosis. PCR was positive for M. tuberculosis culture and the cervical lymph node (site of infection) and negative for the skin (hypersensitivity vasculitis).

normalization of lymph node size, absence of new vasculitic lesions and significant improvement of previous skin lesions. ANA and PIL were negative, but glomerular hematuria and proteinuria remained positive.

DISCUSSION There are few reported cases of an association between Tb and CLV (nine cases) and Tb and IgA nephropathy (six cases) (2-6,13,14). Only 10% of CLV cases are attributed to

Figure 2 - Histopathology of lymph node (A), skin (B) and kidney (C) biopsies. A. Chronic granulomatous lymphadenitis with caseous necrosis without microorganisms (magnification: 20x and 40x) B. Leukocytoclastic and focal granulomatous vasculitis (objectives: 20x and 40x) C. Renal biopsy contained 13 glomeruli, 12 normal and one with a focal segmental lesion. DIF showed diffuse and global mesangial deposits of IgA (objective: 40x).

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considered an etiologic factor in aPL production and the triggering of APS (19). In the literature, there is a single report of concomitant CLV, Sweet syndrome, cutaneous polyarteritis nodosa and cervical adenopathy caused by M. fortuitum (20). The association of Tb, focal granulomatous, CLV and IgA nephropathy in a single patient has not been reported. This case is complex, demonstrating rare manifestations of an endemic disease in Brazil, and serves as a warning to dermatologists to be cautious in the differential diagnosis of patients with vasculitic presentations.

drugs and infections, and 61% are considered idiopathic cases. Considering the existence of circulating immune complexes in pulmonary Tb and the relationship between immune complex levels and disease activity, the damage mechanism that has been proposed for this type of vasculitis is the deposition of immune complexes that are formed by antibodies against antigens of the bacterium on the vascular wall rather than direct aggression by the bacterium (8,9). Up to 56% of Tb patients have circulating immune complexes, and there is evidence of increased immunoglobulin levels in these patients, mainly IgA and IgG, as was observed in our patient. These immunoglobulins are produced against the A-60 antigen of the mycobacterium, leading to the formation of immune complexes (11). Another differential diagnosis is the onset of vasculitis following rifampicin therapy, as demonstrated in some case reports, where CLV occurred due to this medication (7). However, our patient had skin lesions before using this drug. In cases of Tb-related vasculitis, skin lesions improve with RIPE treatment alone; no specific anti-inflammatory therapy is required (2-7,10,15). In 1985, Cohen and Rosenstein (12) described a case of an association between Tb and IgA nephropathy in which renal involvement improved after Tb treatment. In this case, HSP needed to be considered. HSP is a type of systemic vasculitis that is more common in children, and both renal involvement, defined as IgA nephropathy, and palpable purpura appear in 50% of cases (6,13,14). Some specialists defend the theory that Berger’s disease is a restricted form of HSP, and there have been five reported cases of HSP associated with Tb. However, our patient presented with CLV with antifibrinogen, not IgA, deposition in the capillary walls, which rules out a diagnosis of HSP. In addition, PCR with primers specific to Mycobacterium (17) was performed in cervical lymph node and skin samples, which confirmed the etiology of Mycobacterium sp. in the lymph node sample but not in the skin sample. The DNA that was extracted from the paraffin skin sample was amplified with keratin primers, confirming its integrity (data not shown). These results strongly suggest a hypersensitivity form of CLV. None of the case reports of Tb associated with CLV or Tb associated with IgA nephropathy in the literature included descriptions of positive autoantibodies. In our patient, ANA, p-ANCA and lupus anticoagulant (PIL and dRVVT) tests were positive, and there was a prior medical history of ischemic stroke (confirmed by MRI). These findings reinforce the diagnosis of an autoimmune disease and lead us to a diagnosis of APS secondary to Tb. The production of anti-phospholipid antibodies (aPLs) could have either an autoimmune or infectious origin. The latter origin does not involve anti-b2-glycoprotein I (antib2GPI) activity and usually does not cause thrombosis. However, there have been recently described cases of lepromatous leprosy patients with genetically determined anti-b2GPI activity followed by thrombosis (18). By inducing a specific cellular immune response and secondary antibody production (as noted by the strong positive PPD and increased immunoglobulin levels), tuberculosis stimulates the production of autoantibodies (including aPLs) and procoagulant factors, such as plasma fibrinogen (factor I), factor VIII and D-dimers. Thus, M. tuberculosis can be

AUTHOR CONTRIBUTIONS Bueno Filho R contributed to the writing, literature review, data interpretation, and data and picture collection and was clinically responsible for the patient. Cordeiro AP contributed to the collection of pictures and data and was clinically responsible for the patient. Almeida FT was involved in data collection and was responsible for the PCR experiment. Shaletich C was involved in the review and data interpretation processes. Costa RS was involved in the review, data interpretation, and writing processes. Roselino AM was involved in the review, writing, and data interpretation processes and was the coordinator of the Granulomatous Skin Diseases Clinic and coordinator of the Biomolecular Laboratory of Dermatology.

REFERENCES 1. Parish WE, Rhodes EL. Bacterial antigens and aggregated gamma globulin in the lesions of nodular vasculitis. Br J Dermatol. 1967;79(3):131-47. 2. Ekenstam E, Callen JP. Cutaneous leukocytoclastic vasculitis. Clinical and laboratory features of 82 patients seen in private practice. Arch Dermatol. 1984;120(4):484-9, http://dx.doi.org/10.1001/archderm.1984.016504000660 14. 3. Kim HM, Park YB, Maeng HY, Lee SK. Cutaneous leukocytoclastic vasculitis with cervical tuberculous lymphadenitis: a case report and literature review. Rheumatol Int. 2006;26(12):1154-7, http://dx.doi.org/ 10.1007/s00296-006-0152-1. 4. Sais G, Vidaller A, Jucgla` A, Peyrı´ J. Tuberculous lymphadenitis presenting with cutaneous leucocytoclastic vasculitis. Clin Exp Dermatol. 1996;21(1):65-6, http://dx.doi.org/10.1111/j.1365-2230.1996.tb00018.x. 5. Carvalho M, Dominoni RL, Senchechen D, Fernandes AF, Burigo IP, Doubrawa E. Cutaneous leukocytoclastic vasculitis accompanied by pulmonary tuberculosis. J Bras Pneumol. 2008;34(9):745-8. 6. Han BG, Choi SO, Shin SJ, Kim HY, Jung SH, Lee KH. A case of HenochScho¨nlein purpura in disseminated tuberculosis. Korean J Intern Med. 1995;10(1):54-9. 7. Iredale JP, Sankaran R, Wathen CG. Cutaneous vasculitis associated with rifampicin therapy. Chest. 1989;96(1):215-6, http://dx.doi.org/10.1378/ chest.96.1.215. 8. Johnson NMcI, McNicol MW, Burton Kee EJ, Mowbray JF. Circulating immune complexes in tuberculosis. Thorax. 1981;36(8):610-7, http://dx. doi.org/10.1136/thx.36.8.610. 9. Brostoff J. Immune complexes in the spectrum of tuberculosis. Tubercle. 1981;62(3):169-73, http://dx.doi.org/10.1016/0041-3879(81)90002-7. 10. Chan CH, Chong YW, Sun AJ, Hoheisel GB. Cutaneous vasculitis associated with tuberculosis and its treatment. Tubercle. 1990;71(4):297-300. 11. Alifano M, Sofia M, Mormile M, Micco A, Mormile AF, Del Pezzo M, et al. IgA immune response against the mycobacterial antigen A60 in patients with active pulmonary tuberculosis. Respiration. 1996;63(5):2927, http://dx.doi.org/10.1159/000196563. 12. Berger J. IgA glomerular deposits in renal disease. Transplant Proc. 1969;1(4):939-44. 13. De Siati L, Paroli M, Ferri C, Muda AO, Bruno G, Barnaba V. IgA Nephropathy and Pulmonary Tuberculosis. Ann Diagn Pathol. 1999; 3(5):300-3, http://dx.doi.org/10.1016/S1092-9134(99)80026-4. 14. Cohen AJ, Rosenstein ED. IgA nephropathy associated with disseminated tuberculosis. Arch Intern Med 1985;145(3):554-6, http://dx.doi. org/10.1001/archinte.1985.00360030206036. 15. Jennette JC, Falk RJ. Small vessels vasculitis. N Engl J Med. 1997;337(21):1512-23. 16. Singh SP, Misra GC, Prusty PK, Das RK. Tubercular lymphadenitis with purpura. J Indian Med Assoc 1986;84(8):247-9. 17. Brisson-Noel A, Aznar C, Chureau C, Nguyen S, Pierre C, Bartoli M, et al. Diagnosis of tuberculosis by DNA amplification in clinical practice

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19. Naithani R, Agrawal N, Choudhary VP. Deep venous thrombosis associated with tuberculosis. Blood Coagul Fibrinolysis. 2007;18(4):37780, http://dx.doi.org/10.1097/MBC.0b013e3280d942b4. 20. Chen HH, Hsiao CH, Chiu HC. Successive development of cutaneous polyarteritis nodosa, leucocytoclastic vasculitis and Sweet’s syndrome in a patient with cervical lymphadenitis caused by Mycobacterium fortuitum. Br J Dermatol. 2004;151(5):1096-100.

evaluation. Lancet 1991;338(8763):364-6, http://dx.doi.org/10.1016/ 0140-6736(91)90492-8. 18. Brochado MJ, Figueiredo JF, Mendes-Junior CT, Louzada-Junior P, Kim OM, Roselino AM. Correlation between beta-2-glycoprotein I gene polymorphism and anti-beta-2 glycoprotein I antibodies in patients with multibacillary leprosy. Arch Dermatol Res. 2010;302(8):583-91, http:// dx.doi.org/10.1007/s00403-010-1032-9.

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DOI:10.6061/clinics/2012(12)25

CASE REPORT

Birt-Hogg-Dube´ syndrome: metalloproteinase activity and response to doxycycline Suzana Pinheiro Pimenta,I Bruno Guedes Baldi,I Ellen Caroline Toledo do Nascimento,II Thais Mauad,II Ronaldo Adib Kairalla,I Carlos Roberto Ribeiro CarvalhoI I

Faculdade de Medicina da Universidade de Sa˜o Paulo, Heart Institute (InCor), Pulmonary Division, Sa˜o Paulo/SP, Brazil. Universidade de Sa˜o Paulo, Department of Pathology, Sa˜o Paulo/SP, Brazil.

II

Faculdade de Medicina da

Email: spp3847@yahoo.com.br Tel.: 55 11 8232-6007

and pleurodesis by videothoracoscopy were performed, and the patient was diagnosed with lymphangioleiomyomatosis (LAM). She was referred to our institution in 2006 to participate in a doxycycline treatment protocol. All patients with a diagnosis of LAM who were enrolled in this protocol were submitted to lung function evaluation and ELISA-based quantitative serum and urinary MMP-2 and -9 analysis (R&D Systems; Minneapolis, MN, USA) before and after doxycycline treatment (11). After six months of receiving doxycycline (100 mg/day), the patient noticed resolution of the exertional dyspnea and improvement in exercise tolerance. After treatment, DLCO and forced vital capacity (FVC) showed increases from 16.8 to 19.7 mL/min/mmHg and 2.87 to 3.12 L, respectively. The forced expiratory volume in the first second (FEV1), RV and RV/TLC values pre- and post-doxycycline were, respectively, 2.36 and 2.35 L, 2.23 and 1.51 L, and 0.45 and 0.32 (Table 1). The MMP blockade induced by doxycycline was effective, resulting in a reduction in serum MMP-9 levels from 143 to 36 ng/mL, and urinary levels of MMP-9 became untraceable (55 pg/mL before doxycycline). MMP-2 levels were not detectable before or after treatment. During follow-up, the patient presented with soft and pedunculated papules on her neck and upper thorax. The biopsy of these lesions was compatible with acrochordons. The patient’s family medical history also revealed other cases of cystic lung disease (Figure 2). The lung biopsy specimen was reviewed in 2007. Histological analysis of the lung tissue revealed several cystic areas with an emphysematous aspect in the lung parenchyma, mostly in the subpleural region. Cyst walls were formed by collapsed alveolar parenchyma or slightly thickened pleural tissue. LAM cells were not observed in the cyst walls, and monoclonal antibody HMB-45 (human melanoma black-45), S100 protein and alpha smooth muscle actin were also not observed (Figure 3). Based on the family history, the presence of cutaneous lesions (acrochordons), the CT findings and the lung histological review, the diagnosis of BHDS was established. The positive response to doxycycline treatment, which had never been demonstrated in BHDS, and the association between the loss-of-function mutation in folliculin and pulmonary extracellular matrix degradation (6), led us to evaluate MMP behavior in lung tissue. Immunohistochemical analysis in the patient’s lung tissue revealed a large number of MMP-9-positive cells, mostly

INTRODUCTION Birt-Hogg-Dube´ syndrome (BHDS) is a rare, inherited autosomal-dominant genodermatosis caused by mutations in the folliculin gene (FLCN), which is located within the chromosomal band 17p11.2 (1). Patients with BHDS are prone to fibrofolliculomas, trichodiscomas, and acrochordons on the face, neck, and upper trunk; renal tumors; pulmonary cysts; and spontaneous pneumothorax (2-4). The pathogenesis of lung cyst formation and pneumothorax remains unclear, but studies have shown that folliculin is strongly expressed in lung fibroblasts and macrophages. Folliculin mutations may alter cytokines and proteases, which are important in maintaining extracellular matrix (ECM) integrity, leading to an inflammatory response and matrix degradation with subsequent remodeling (5,6). The pulmonary architecture depends on interactions between collagen and elastin fibers in the ECM, which maintain alveoli wall integrity (7). The overexpression of metalloproteinases (MMPs), regardless of whether they are associated with the suppression of tissue inhibitors of metalloproteinases (TIMPs), leads to matrix breakdown, tissue destruction and cystic lesions (7,8). Because there is currently no treatment for BHDS, we decided to describe our experience treating one BHDS patient with doxycycline, which is an MMP inhibitor (9) that has been previously used to treat cystic lung disease (10).

Case Description A 44-year-old female non-smoker complained of mild dyspnea upon exertion in 2004 and presented with a spontaneous pneumothorax in 2005. Chest computed tomography (CT) demonstrated bilateral thin-walled cystic lesions (Figure 1). Pulmonary functional tests (PFTs) showed normal carbon monoxide diffusion capacity (DLCO = 81% of the predicted value), lung volume, and expiratory flow rate, but an increased residual volume (RV) and total lung capacity (TLC) ratio (RV/TLC = 0.45) were found, as shown in Table 1. An abdominal CT was also normal. A lung biopsy

Copyright ß 2012 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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Figure 1 - High-resolution chest CT. A) A CT scan showing thin-walled, air-filled cystic lesions, including one dominant cyst (665 cm). B) In addition to the cystic lesions, posterior pleural thickening due to previous pleurodesis is observed on the right side.

Table 1 - Pulmonary function tests performed before and during doxycycline treatment and 18 months after doxycycline interruption. Before doxy Pulmonary function variables FVC - L (% pred) FEV1 - L (% pred) FEV1/ FVC TLC - L (% pred) RV/TLC (% pred) DLco - mL/min/mmHg (% pred)

2.87 (82) 2.36 (87) 0.82 4.87 (94) 0.45 (137) 16.84 (81)

During doxy

After doxy

3 months

6 months

18 months

3.03 (83) 2.34 (79) 0.77 x x x

3.12 (85) 2.35 (80) 0.76 4.74 (93) 0.32 (97) 19.7 (80)

2.33 (70) 2.21 (80) 0.95 x x x

FVC: forced vital capacity; FEV1: forced expiratory volume in the first second; TLC: total lung capacity; RV: residual volume; DLCO: carbon monoxide diffusion capacity.

macrophages and neutrophils, in the cyst wall, whereas the adjacent lung parenchyma presented scattered MMP-9positive cells along the alveolar walls (figure 3). MMP-2 staining showed scattered positive inflammatory cells in the lung parenchyma with no specific cyst staining. During the subsequent months, the patient developed gastric intolerance symptoms, leading to the interruption of doxycycline therapy. Spirometry performed 18 months after doxycycline interruption revealed a decrease in lung volume and expiratory flow rates (Table 1) with worsening of the pulmonary symptoms.

DISCUSSION

In LAM, lung cyst development is affected by MMP-2 and -9 upregulation in LAM cells (abnormal smooth muscle-like cells) rather than in vascular and bronchiolar smooth muscle cell (14,17). High serum MMP-9 levels are also present in the blood of patients with LAM (17). Zhe et al. described the downregulation of TIMPs, especially TIMP-3, followed by MMP-2 and -14 overexpression, in LAM cells (18). LCH is characterized by irregularly dilated alveolar spaces and degraded elastic fibers that are surrounded by granulomatous lesions. An immunohistochemical study showed MMP-2 expression in the epithelial basement membranes of these damaged areas and collagen type IV impairment (12,19).

In a non-smoking female who develops spontaneous pneumothorax and presents with cysts in the thoracic high resolution CT, LAM is a strong diagnostic possibility. However, other cystic lung disorders, such as BHDS, may have the same presentation. In the past several years, lung cyst pathogenesis has been widely studied in pulmonary cystic diseases, such as LAM, Langerhans cell histiocytosis (LCH) and cystic lung light chain deposition disease (CL-LCDD). MMPs appear to have an important role in the pathogenesis of cyst formation (8,12-14). These proteins belong to a family of proteolytic enzymes and are classified according to their specific substrates into gelatinases (MMP-2 and -9), interstitial collagenases (MMP-1, -8 and -13) and stromelysins (MMP3, -7 and -10). These enzymes are mainly responsible for ECM remodeling, but they also affect cell migration, angiogenesis and pulmonary immunity (7,15,16).

Figure 2 - Pedigree of the family. Square = male member, circle = female member; solid symbol = pneumothorax, open symbol = no pneumothorax; slash through symbol = cystic lesions on lung CT; * our patient.

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Doxycycline in Birt-Hogg-Dube´ syndrome Pimenta SP et al.

Figure 3 - Histopathological analysis of cystic lung lesions. A) A large subpleural cyst within the lung parenchyma. The cyst formed by the collapse of the alveolar walls with a connective tissue lining (H&E staining; 40x magnification). B) Details of a smaller cyst stained with an anti-alpha smooth muscle actin antibody. There are no positive LAM cells in the cyst wall. Adjacent blood vessel walls express actin. V = vessel, C = cyst (400x magnification). C) Details of a subpleural cyst wall with a large number of MMP-9-positive cells, mostly macrophages and neutrophils, in the cyst wall (400x magnification). D) Normal lung parenchyma adjacent to the cyst wall, showing scattered MMP-9-positive cells (arrow) in the alveolar walls (400x magnification).

macrophages and neutrophils, located in the cyst wall. However, the lung parenchyma adjacent to the cyst wall presented only a few scattered MMP-9-positive cells. These findings suggest that MMP-9, as in other lung cystic disorders, may be implicated in BHDS cyst development. Based on the improvement in pulmonary function and the decrease in urinary and serum MMP-9 levels observed in our patient, doxycycline, a known inhibitor of tissue MMPs, may represent a promising therapy for BHDS.

Pulmonary involvement is especially rare in light chain deposition disease (LCDD), which is a systemic disorder characterized by diffuse monoclonal nonamyloid light chain deposits. There are two types of lung impairment: nodular and CL-LCDD. In CL-LCDD, giant macrophagic cells located around light chain deposits express MMP-2, -9, and -14, which can degrade the elastic network (8,20). BHDS, an inherited and rare disorder caused by an FLCN mutation, presents with dermatologic and pulmonary involvement and usually manifests during the third or fourth decade of life (2,3). Patients with BHDS have an increased risk of renal cell carcinoma, colorectal neoplasia (3,21) and parotid oncocytomas (22). Abnormal pleuropulmonary findings include lung cysts, pleural blebs and spontaneous pneumothorax (4). Histopathological studies are very limited, especially studies investigating cystic lung pathogenesis. Butnor and Guinee (23) described nonspecific features in the lung biopsies of two BHDS patients, showing intraparenchymal air-filled spaces surrounded by normal parenchyma or a thin fibrous wall. Lung tissue adjacent to the cysts appeared normal. In our patient, we observed subpleural cysts within the lung parenchyma, formed by collapsed alveolar walls with a connective tissue lining. In contrast to the descriptions of other cystic lung diseases, where a histopathological substrate was present during pulmonary cystic growth, we found lesions resembling emphysema surrounded by normal lung tissue in this BHDS case. MMP behavior has not been previously described in BHDS. In our pulmonary immunohistochemical analysis, MMP-9 was expressed in a large number of cells, mostly

ACKNOWLEDGMENTS We would like to thank Shari Anne El-Dash for the language revision.

AUTHOR CONTRIBUTIONS Pimenta SP diagnosed the patient, wrote the case presentation and contributed to the literature review. Baldi BG contributed to the literature review. Kairalla RA diagnosed the patient and contributed to the presentation of the case. Nascimento ECT and Mauad T contributed to the histopathological diagnosis. Carvalho CRR coordinated the manuscript and was responsible for the final review. All authors read and approved the final manuscript.

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Dermatol. 1999;135(10):1195-202, http://dx.doi.org/10.1001/archderm. 135.10.1195. Toro JR, Pautler SE, Stewart L, Glenn GM, Weinreich M, Toure O, et al. Lung cysts, spontaneous pneumothorax, and genetic associations in 89 families with Birt-Hogg-Dube syndrome. Am J Respir Crit Care Med. 2007;175(10):1044-53, http://dx.doi.org/10.1164/rccm.200610-1483OC. Frohlich BA, Zeitz C, Matyas G, Alkadhi H, Tuor C, Berger W, et al. Novel mutations in the folliculin gene associated with spontaneous pneumothorax. Eur Respir J. 2008;32(5):1316-20, http://dx.doi.org/10. 1183/09031936.00132707. Kalhan R, Yeldandi AV, Jain M. A 48-year-old woman with skin lesions, renal masses, and spontaneous pneumothorax. Chest. 2007;131(2):624-7, http://dx.doi.org/10.1378/chest.06-0559. Elkington PT, Friedland JS. Matrix metalloproteinases in destructive pulmonary pathology. Thorax. 2006;61(3):259-66, http://dx.doi.org/10. 1136/thx.2005.051979. Colombat M, Caudroy S, Lagonotte E, Mal H, Danel C, Stern M, et al. Pathomechanisms of cyst formation in pulmonary light chain deposition disease. Eur Respir J. 2008;32(5):1399-403, http://dx.doi.org/10.1183/ 09031936.00132007. Chung AW, Yang HH, Radomski MW, van Breemen C. Long-term doxycycline is more effective than atenolol to prevent thoracic aortic aneurysm in marfan syndrome through the inhibition of matrix metalloproteinase-2 and -9. Circ Res. 2008;102(8):e73-85, http://dx.doi. org/10.1161/CIRCRESAHA.108.174367. Moses MA, Harper J, Folkman J. Doxycycline treatment for lymphangioleiomyomatosis with urinary monitoring for MMPs. N Engl J Med. 2006;354(24):2621-2, http://dx.doi.org/10.1056/NEJMc053410. Pimenta SP, Baldi BG, Acencio MM, Kairalla RA, Carvalho CR. Doxycycline use in patients with lymphangioleiomyomatosis: safety and efficacy in metalloproteinase blockade. J Bras Pneumol. 2011;37(4):424-30, http://dx.doi.org/10.1590/S1806-37132011000400003. Hayashi T, Rush WL, Travis WD, Liotta LA, Stetler-Stevenson WG, Ferrans VJ. Immunohistochemical study of matrix metalloproteinases and their tissue inhibitors in pulmonary Langerhans’ cell granulomatosis. Arch Pathol Lab Med. 1997;121(9):930-7. Hayashi T, Fleming MV, Stetler-Stevenson WG, Liotta LA, Moss J, Ferrans VJ, et al. Immunohistochemical study of matrix metalloproteinases

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(MMPs) and their tissue inhibitors (TIMPs) in pulmonary lymphangioleiomyomatosis (LAM). Hum Pathol. 1997;28(9):1071-8, http://dx.doi. org/10.1016/S0046-8177(97)90061-7. Matsui K, Takeda K, Yu ZX, Travis WD, Moss J, Ferrans VJ. Role for activation of matrix metalloproteinases in the pathogenesis of pulmonary lymphangioleiomyomatosis. Arch Pathol Lab Med. 2000;124(2):267-75. Sato H, Takino T, Okada Y, Cao J, Shinagawa A, Yamamoto E, et al. A matrix metalloproteinase expressed on the surface of invasive tumour cells. Nature. 1994;370(6484):61-5, http://dx.doi.org/10.1038/370061a0. Jackson C. Matrix metalloproteinases and angiogenesis. Curr Opin Nephrol Hypertens. 2002;11(3):295-9, http://dx.doi.org/10.1097/ 00041552-200205000-00005. Odajima N, Betsuyaku T, Nasuhara Y, Inoue H, Seyama K, Nishimura M. Matrix metalloproteinases in blood from patients with LAM. Respir Med. 2009;103(1):124-9, http://dx.doi.org/10.1016/j.rmed.2008.07.017. Zhe X, Yang Y, Jakkaraju S, Schuger L. Tissue inhibitor of metalloproteinase-3 downregulation in lymphangioleiomyomatosis: potential consequence of abnormal serum response factor expression. Am J Respir Cell Mol Biol. 2003;28(4):504-11, http://dx.doi.org/10.1165/rcmb.20020124OC. Fukuda Y, Basset F, Soler P, Ferrans VJ, Masugi Y, Crystal RG. Intraluminal fibrosis and elastic fiber degradation lead to lung remodeling in pulmonary Langerhans cell granulomatosis (histiocytosis X). Am J Pathol. 1990;137(2):415-24. Bhargava P, Rushin JM, Rusnock EJ, Hefter LG, Franks TJ, Sabnis SG, et al. Pulmonary light chain deposition disease: report of five cases and review of the literature. Am J Surg Pathol. 2007;31(2):267-76. Khoo SK, Giraud S, Kahnoski K, Chen J, Motorna O, Nickolov R, et al. Clinical and genetic studies of Birt-Hogg-Dube syndrome. J Med Genet. 2002;39(12):906-12, http://dx.doi.org/10.1136/jmg.39.12.906. Maffe A, Toschi B, Circo G, Giachino D, Giglio S, Rizzo A, et al. Constitutional FLCN mutations in patients with suspected Birt-HoggDube syndrome ascertained for non-cutaneous manifestations. Clin Genet. 2011;79(4):345-54, http://dx.doi.org/10.1111/j.1399-0004.2010. 01480.x. Butnor KJ, Guinee DG, Jr. Pleuropulmonary pathology of Birt-HoggDube syndrome. Am J Surg Pathol. 2006;30(3):395-9.


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DOI:10.6061/clinics/2012(12)26

CASE REPORT

Fractures in connection with an atypical form of craniodiaphyseal dysplasia: case report of a boy and his mother Ali Al Kaissi,I,II Robert Csepan,II Jochen G. Hofstaetter,III Klaus Klaushofer,I Rudolf Ganger,II Franz GrillII I

Hanusch Hospital of WGKK and AUVA Trauma Centre Meidling, First Medical Department, Ludwig Boltzmann Institute of Osteology, Viena, Austria. Orthopaedic Hospital of Speising, Paediatric Department, Vienna, Austria. III Medical University of Vienna, Vienna General Hospital, Department of Orthopaedic Surgery, Vienna/Austria. II

Email: ali.alkaissi@osteologie.at Tel.: +43 01 80 182

for 23 hours per day until the age of one year. At that time, no skull radiographs were taken, and the pediatricians considered his case to be a deformational non-syndromic plagiocephaly. His subsequent course of development included motor delay, although other developmental skills were within the normal range. There was no history of visual, auditory or other neurological symptoms. There was no history of anorexia, weight loss or muscular hypotonia. Examination at the age of two years showed a pleasant boy with mild and unnoticeable facial dysmorphic features, with frontal bossing and slight facial asymmetry associated with incomplete auricular folding. Leontiasis ossea and or Cushingoid facies/macrocephaly, retarded growth and muscular hypotonia were not evident (Table 1). Neurological, ophthalmological, and auditory evaluations were normal, and there was no history of seizures or nonmotor developmental delay. Audiometric evaluation excluded peripheral hearing loss. Ophthalmological examination revealed normal vision with no strabismus or other eye problems. Musculo-skeletal examination revealed relative ligamentous hyperlaxity associated with patellar instability. Thoraco-lumbar scoliosis of 32 degrees was evident. A skeletal survey was conducted at the age of two years. A lateral skull radiograph showed marked cranial osteosclerosis and hyperostosis; the facial bones and base of the skull were severely hyperostosed. An anteroposterior skull radiograph showed facial bone hyperostosis (Figure 1). An anteroposterior radiograph of the thorax showed thick, broad and sclerosed ribs; the medial two-thirds of the clavicles were hyperostosed; thoraco-lumbar scoliosis of 32 degrees was present secondary to hemivertebrae along T810, and only 11 ribs were present (Figure 2A). Anteroposterior radiographs of the pelvis and the femora revealed diaphyseal hyperostosis associated with distal diaphyseal fracture of the left femur and a stress/fatigue fracture of the mid-diaphysis of the right femur (Figure 2B). An anteroposterior hand radiograph showed diaphyseal widening associated with hyperostosis and dysplastic terminal phalanges (increased cortex of the tubular bones) (Figure 3). CT imaging was planned to further delineate additional abnormalities. Axial reformatted CT scans revealed bilateral narrowing of the optic canal (Figure 4). Other investigations included an abdominal ultrasound and metabolic tests to examine calcium, phosphorous, and

INTRODUCTION Craniodiaphyseal dysplasia is a severe disorder characterized by distinctive facial dysmorphisms, including prominent zygomatic bones, broadening of the center of the face, hypertelorism, a small upturned tip of the nose and massive hyperostosis and sclerosis of the skull and facial bones. The sclerosis is severe and is referred to as ‘‘leontiasis ossea’’ (leonine faces), and the bone deposition causes progressive stenosis of the craniofacial foramina (1-5). The skull and facial hyperostosis result in an increasing head circumference, paranasal bossing, nasal bridge distortion and an alteration in the orbital alignment. Radiographic features consist of osteosclerosis and hyperostosis of the skull and facial bones and, to a lesser degree, the diaphyses of the tubular bones. There is often secondary optic atrophy and deafness, and epilepsy and mental retardation have both been reported (6,7). Familial osteoectasia with macrocranium and hyperphosphatasia are features encountered in juvenile Paget’s disease (juvenile deforming cortical hyperostosis) (7). Genotypical diseases of familial osteoectasia are characterized by Cushingoid facies, limb deformities (from generalized osteoporosis) and retarded growth (8,9).

CASE DESCRIPTION Patient I A two-year-old boy was referred to our department because of a distal diaphyseal fracture of the left femur. He was the first child of non-consanguineous parents. At birth, his weight, length and OFC were at approximately the 25th percentile. His mother was a 26-year-old primagravida with no history of spontaneous abortions, stillbirths or neonatal mortalities who was married to a 27-year-old unrelated man. At birth, the child presented with plagiocephaly for which he was treated with a cranial band (Care band); he wore this

Copyright ß 2012 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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Table 1 - Comparison between features observed in our patients and those seen in patients with craniodiaphyseal dysplasia (CDD) and idiopathic hyperphosphatasia (IH). Deformity Facial features Cranial radiograph

Spine Thoracic cage Limbs

Metaphyses Alkaline phosphatase

Our patient

CDD

IH

Near normal Homogenized skull base and calvarial hyperostosis

Leontiasis ossea Severe skull base hyperostosis

No platyspondyly Wide and hyperostosed ribs Diaphyseal sclerosis

Increased density of the vertebral arches Diffuse sclerosis of the clavicles and, to a lesser degree, the ribs Diaphyseal sclerosis

Cushenoid Fluffy structure of the widened diploe similar to Pagets disease with zones of patchy sclerosis Decreased vertebral height Wide ribs

Normal 5 times greater than normal

Normal 5 times greater than normal

vitamin B metabolism, which were normal; normal karyograms for the parents and the proband were also obtained. Nevertheless, the alkaline phosphatase level was five times greater than normal in the proband. Hormonal investigations included thyroid, adrenocorticotropic and growth hormones, all of which were normal. We treated the diaphyseal fractures via spica casting.

Curved long bones with characteristic cylindrical appearance due to excessive cortical widening, especially at the concavity of the diaphyses with fusiform expansion and possible obliteration of the medullary canal. Cystic appearance in some 5-20 times greater than normal

DISCUSSION Craniotubular dysplasias such as CDD are characterized by severe condensing hyperostosis of the craniofacial skeleton and the diaphyses of the long bones, along with short stature, mental retardation and an unfavorable prognosis. The main clinical feature is macrocephaly (dolichocephaly) (1-4). The characteristic leonine appearance is due to marked thickening of the cranial, nasal, and maxillary bones (5); other features include a high forehead, prominent zygomatic bones, broadening of the center of the face, hypertelorism, exophthalmos, strabismus, a small upturned tip of the nose, a prominent jaw, an open-mouth nasal obstruction, a prominent mandible, dental malocclusion, and compression of the cranial nerves leading to progressive loss of vision and hearing. Distinctive radiographic features include marked thickening and sclerosis of the cranial and facial bones with obliteration of the paranasal sinuses and mastoid cells. There is also increased density of the vertebral arches and thickening and diffuse sclerosis of the clavicles and, to a lesser extent, the ribs. In

Patient II A 26-year-old-woman (the mother of patient I) had a history of a diaphyseal fracture of the right femur a few years prior with appropriate operative treatment. On examination, she manifested mild facial features similar to those seen in patient I, including frontal bossing and a wide frontal area with a depressed nasal bridge and a large nose. She also presented with incomplete folding of the external ears, similar to that noted in patient I. Her family history was unremarkable. An anteroposterior lower extremity (retrospective) radiograph showed mild diaphyseal hyperostosis associated with a mid-diaphyseal fracture and locked intramedullary nailing (Figure 5).

Figure 1 - Lateral skull and AP radiographs show marked cranial osteosclerosis and hyperostosis. The facial bones and base of the skull are severely hyperostosed.

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Figure 2 - (A) Anteroposterior radiography of the thorax shows thick, broad and sclerosed ribs. The medial two-thirds of the clavicles are hyperostosed; thoraco-lumbar scoliosis of 32 degrees secondary to hemivertebrae along T8-10 is observed; in addition, only 11 ribs are present. (B) Anteroposterior radiography of the pelvis and the femora show diaphyseal hyperostosis with an associated distal diaphyseal fracture of the left femur and a stress/fatigue fracture of the mid-diaphysis of the right femur.

the pelvis, there is some sclerosis of the sacroiliac joints. In the limbs, a homogeneous opacity of the diaphyses of the long bones, which have a cylindrical appearance due to cortical thickening, is often observed. The small tubular bones of the hands and feet are also involved (6,7). Idiopathic hyperphosphatasia (IH) is a metabolic bone disease characterized by increased bone resorption by osteoclasts due to an increase their number, size and resorbing activity. The disease starts with a lytic phase, which is followed by compensatory increased activity of osteoblasts (sclerotic phase). The bone turnover rate progressively increases, reaching up to 20 times the normal

levels. This results in increased bone formation, in which the resulting bone is weaker highly vascular and disorganized (9). Increased alkaline phosphatase does not imply that the disease is due to excess enzyme; in fact, the cause of the lack of primary bone transformation into mature lamellar bone is unknown. Idiopathic hyperphosphatasia is characterized by increased serum alkaline phosphatase [5-20 times greater than normal]. Increased acid phosphatase is also observed, but it is less marked and is an inconsistent finding (10). A diagnosis of IH is ultimately made through imaging and laboratory findings. Radioisotope bone scanning is the most sensible method for detecting early lesions. The disease etiology remains uncertain, but genetic, environmental and viral infections are considered the most likely causes (11). The differential diagnosis of our patient includes other forms of cranio-tubular dysplasias, such as CDD, craniometaphyseal dysplasia, Camurati-Engelmann disease, Worth

Figure 3 - Anteroposterior hand radiographs show diaphyseal widening associated with hyperostosis and dysplastic terminal phalanges (increased cortex of the tubular bones).

Figure 4 - Axial CT scanning shows progressive and bilateral narrowing of the optic canal.

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cranial hyperostosis describes an enlargement of the affected skull bones, while in cranial sclerosis, the bones appear more radio-opaque but are of normal size. Because high bone mass can arise from either reduced bone resorption or increased bone formation, the sclerosing bone disorders can be subdivided into osteoclast and osteoblastrelated entities. Increased bone mass is often a diagnostic challenge. In the absence of an obvious hereditary context, osteocondensation secondary to a concomitant pathology, particularly to a neoplastic disorder, should be excluded (12). Kim et al. recently discovered mutations c.61G.A (Val21Met) and c.61G.T (Val21Leu) in two children with CDD and concluded that CDD is the most severe form of sclerotic bone disease, and it is part of a spectrum of disease caused by mutations in SOST (13). None of the abovementioned reports described femoral fractures as a presenting abnormality in patients with CCD. The blood supply of the femoral shaft is from both endosteal and periosteal blood vessels. The endosteal supply is typically derived from two nutrient vessels that enter the femur from a posteromedial direction. The periosteal capillaries supply the outer 25%-30% of cortical bone and are most prominent in the areas of muscular attachments to the femoral shaft. These two circulatory systems, together with the metaphyseal complex of vessels, are interconnected to provide a strong vascular supply that allows rapid fracture repair (14,15). Cranio-tubular dysplasias include a wide variety of symptoms and vary in their age of onset from infancy to childhood. Interestingly, neither the proband nor his mother showed the typical characteristic phenotypic features of CDD, IH or any other form of cranio-tubular dysplasias/dysostosis. Finally, we wish to stress that an etiological understanding of patients with unusual or atypical phenotypic and skeletal abnormalities, in combination with an assimilation of the natural history of the disease, are the cornerstones that allow the orthopedic surgeons, radiologists, physicians and neurologists to intervene on the affected areas and provide appropriate care for the patient.

ACKNOWLEDGMENTS We wish to thank our colleagues at the European Skeletal Dysplasia Panel and Prof. Andrea Superti-Furga Leenaards, Professor of Pediatrics, University of Lausanne, Medecin-chef and Centre Hospitalier Universitaire Vaudois (CHUV) for their continuous help and support. Accredited site for registration: Our clinical research is registered with the Medical University of Vienna (EK Nr.921/2009).

Figure 5 - Anteroposterior lower extremity (retrospective) radiographs show mild diaphyseal hyperostosis associated with middiaphyseal fractures and locked intramedullary nailing.

type endosteal hyperostosis, Van Buchem disease, sclerosteosis, Lenz-Majewski syndrome, hyperostosis, dysplasia, and IH (7). We postulate that our current patient and his mother manifest a variant of cranio-tubular dysplasia that is consistent with but not diagnostic for CDD. Sclerosing bone disorders can be subdivided according to their clinical and radiological presentations, the primary affected cell type and the cellular pathways involved. Osteoclast-rich osteopetrosis and related disorders have been related in most cases to mutations in genes required for osteoclast function. More recently, osteoclast-poor forms of osteoporosis have been described as being connected to factors that govern osteoclast differentiation. Osteosclerosis and hyperostosis in the long bones refer to trabecular and cortical thickening, respectively. Similarly,

AUTHOR CONTRIBUTIONS Kaissi AA, Ganger R, Grill F drafted the manuscript and analyzed the data. Csepan R and Klaushofer K participated in the coordination of the study. Hofstaetter JG participated in the coordination and design. All authors have read and approved the final version of the manuscript.

REFERENCES 1. Halliday J. A rare case of bone dystrophy. Br J Surg. 1949;37(145):52-63. 2. Gorlin RJ, Spranger J, Koszalka MF. Genetic craniotubular bone dysplasias and hyperostoses: a critical analysis. BDOAS. 1969;5(4):79-95. 3. Brueton LA, Winter RM. Cardiodiaphyseal dysplasia. J Med Genet. 1990;27(11):701-6, http://dx.doi.org/10.1136/jmg.27.11.701. 4. Gorlin RJ. Craniotubular bone disorders. Pediatr Radiol. 1994;24(6):392406, http://dx.doi.org/10.1007/BF02011904. 5. De Souza O. Leontiasis ossea. Porto Alegre (Brazil), Faculdade de Medic Rev. Dos Cursos. 1927;13:47.

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Craniodiaphyseal dysostosis Kaissi AA et al. 12. Josse RG, Hanley DA, Kendler D, Ste Marie LG, Adachi JD, Brown J. Diagnosis and treatment of Paget’s disease of bone. Clin Invest Med. 2007;30(5):210-23. 13. Marie-Christine de Vernejoul and Uwe Kornak. Heritable sclerosing bone disorders Presentations and new molecular mechanisms. Ann N Y Acad Sci. 2010;1192:269-77, http://dx.doi.org/10.1111/j.1749-6632.2009.05244.x. 14. Kim SJ, Bieganski T, Sohn YB, Kozlowski K, Seme¨nov M, Okamoto N, et al. Identification of signal peptide domain SOST mutations in autosomal dominant craniodiaphyseal dysplasia. Hum Genet. 2011;129(5):497-502, http://dx.doi.org/10.1007/s00439-011-0947-3. 15. Aiello LC, Dean C. An Introduction to Human Evolutionary Anatomy. London: Academic Press; 1990. 16. Loder RT, O’Donnell PW, Feinberg JR. Epidemiology and mechanisms of femur fractures in children. J Pediatr Orthop. 2006;26(5):561-6.

6. Kirkpatrick DB, Rimoin DL, Kaitila I, et al. The craniotubular bone modelling disorders: a neurosurgical introduction to rare skeletal dysplasias with cranial nerve compression. Surg Neurol. 1977;7(4):221-32. 7. Spranger JW, Brill PW, Poznanski A. 2000. Bone dysplasias. An atlas of genetic disorders of skeletal development. 2nd ed. New York, Oxford University Press, 2002. 8. Lancu TC, Almagor G, Friedman E, Hardoff R, Front D. Chronic familial hyperphosphatasemia. Radiology. 1978;129(3):669-76. 9. Moncrieff AA. A chronic progressive osteopathy with hyperphosphatasia. Proc R Soc. Med. 1962;55:238-9. 10. Moncrieff AA. A chronic progressive osteopathy with hyperphosphatasia. 1962. Proc R Soc. Med. 55:238-9. 11. Joshi SR, Ambbore S, Butala N, Patvardhan M,Kulkarni M, Pai B, et al. Paget’ disease fromWestern India. J Assoc Physicians India. 2006;54:535-8.

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DOI:10.6061/clinics/2012(12)27

CASE REPORT

Challenges in patients supported with extracorporeal membrane oxygenation in Brazil Pedro Vitale Mendes,I,III Ewandro Moura,I Edzangela Vasconcelos Santos Barbosa,I,II Adriana Sayuri Hirota,I,II Paulo Rogerio Scordamaglio,IV Fabiana Maria Ajjar,IV Eduardo Leite Vieira Costa,II,III Luciano Cesar Pontes Azevedo,I,III Marcelo Park,I,III, on behalf of ECMO GroupI,II,III I Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, Intensive Care Unit, Emergency Department, Sa˜o Paulo/SP, Brazil. II Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo, Respiratory Intensive Care Unit, Sa˜o Paulo/SP, Brazil. III Hospital Sı´rio Libaneˆs, Intensive Care Unit, Sa˜o Paulo/SP, Brazil. IV Hospital Geral de Guarulhos, Intensive Care Unit, Guarulhos/SP, Brazil.

Email: mpark@uol.com.br Tel.: 55 11 2661-6457

with a peak inspiratory pressure of 20 cmH2O, a positive end-expiratory pressure of 10 cmH2O, a respiratory rate of 10 breaths per minute and an FiO2 of 0.3. After stabilization, the patient was transferred to Hospital das Clı´nicas 27 km away in an ICU ambulance transport. The entire process, from initial call to arrival at Hospital das Clı´nicas, lasted 10 hours. The clinical and arterial blood gas data during the ICU stay are shown in Table 1. A chest radiograph is shown in Figure 1a. Twenty-four hours after arrival at Hospital das Clı´nicas, the patient’s pupils became bilaterally dilated, and he lost brainstem reflexes in the absence of sedation, a clinical finding compatible with brain death. An apnea test was performed while the patient was normothermic with a mean arterial blood pressure of 86 mmHg by setting the ECMO to provide a pulse oximetry of at least 90% with the lowest sweeper flow setting. Mechanical ventilation was withdrawn, and continuous oxygen was supplied through the tracheal tube to prevent hypoxia. Close monitoring for respiratory movements was performed for 10 minutes. An arterial blood analysis was performed before and after the test to detect an increase in PaCO2 (Table 2). The neurological tests were repeated six hours later according to the Brazilian laws regulating the diagnosis of brain death. Transcranial Doppler ultrasonography indicated cerebral circulatory arrest, and computed tomography showed diffuse brain edema, brainstem herniation, and multiple foci of hemorrhage (Figure 2). Thus, ICU support was withdrawn.

INTRODUCTION After the influenza A H1N1 epidemics, the use of extracorporeal membrane oxygenation (ECMO) has increased worldwide. The goal of respiratory ECMO support is to improve hypoxemia and hypercapnia, allowing protective mechanical ventilation to avoid further ventilator-associated lung injury (1). The current literature supports improved outcomes using ECMO in severe lung injury patients. In Brazil, few hospitals are able to provide respiratory ECMO support, and there is no transfer system (2). The aim of this manuscript was to present and discuss two cases of severe respiratory failure supported with ECMO.

CASE DESCRIPTION 1 A previously healthy 27-year-old man was admitted to a tertiary hospital in Guarulhos, Sa˜o Paulo, with a diagnosis of severe community-acquired pneumonia. Three days later, his clinical status deteriorated, and mechanical ventilation was initiated due to severe hypoxemic respiratory failure. Prone positioning and alveolar recruitment were attempted without success. Pneumothorax with a bronchopleural fistula complicated the clinical status, and the ECMO team from Hospital das Clı´nicas, Sa˜o Paulo, Brazil, was called. A team consisting of one physician, one intensive care fellow, one registered respiratory therapist, and one registered nurse was sent to assess the patient. During the evaluation, the patient was found to have sustained pulse oximetry of 52%, an arterial partial pressure of oxygen (PaO2) of 43 mmHg and an arterial partial pressure of CO2 (PaCO2) of 142 mmHg, with an inspired fraction of oxygen (FiO2) of 1 and optimized mechanical ventilation. There were no signs of hemodynamic compromise. Venous-venous ECMO support was initiated. The initial parameters were set at an oxygen flow (sweeper) of 6 L/min and a blood flow of 6 L/ min. Ventilation was adjusted to pressure control mode,

CASE DESCRIPTION 2 A 42-year-old female with a previous history of illicit drug use was admitted to a tertiary hospital in Guarulhos, Sa˜o Paulo, at 10 days postpartum with the diagnosis of community-acquired pneumonia. Five days later, she developed respiratory failure requiring mechanical ventilation and was transferred to the ICU. Two days later, her respiratory status deteriorated, with persistent hypoxemic and hypercapnic respiratory failure, and the ECMO response team from Hospital das Clı´nicas was called. The patient had a peripheral oxygen saturation of 80% and a PaCO2 of 80 mmHg without hemodynamic compromise. Mechanical ventilation was set at a PEEP of 10 cmH2O, an FiO2 of 1, and a peak pressure of

Copyright ß 2012 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.

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Table 1 - Clinical data and arterial blood gases (Case 1). Data Mechanical ventilation Ventilatory mode Peak pressure (min– max) - cmH2O PEEP (min–max) cmH2O £ FIO2 (minmax) ¥ Respiratory rate (min– max) - breaths/min ECMO J Blood flow (min–max) - L/min Sweeper flow (min– max) - L/min FIO2 Routine blood gas PaO2 - mmHg PaCO2 - mmHg SBE - mEq/L * pH Patient data SAS (min–max)# Lung injury score " Total SOFA score 1 Respiratory SOFA Cardiovascular SOFA Hematological SOFA Hepatic SOFA Neurological SOFA Renal SOFA

Pre-ECMO

Day 1

Day 2

Table 2 - Arterial blood gases during the apnea test in patient 1.

Day 3

2 ˚ test

1 ˚ test PCV

I

PCV 25

I

PCV 22

I

PCV 24

I

12 0.5 20–54 3.3–6.4

10 0.5 20-22 4.49-4.79

10 0.6 20-24 4.58-7.9

10

6-10

8

1.0

1.0

1.0

48.5 40.3 8.4 7.51

44.8 44.3 -6.5 7.27

57 22.6 -13.3 7.30

1 2.75 8 4 0 0 2 2 0

1 2.5 18 4 4 0 2 4 4

1 2.5 19 4 2 3 2 4 4

Pre pH 7.303 67.0 mmHg PaO2 50.3 mmHg PaCO2 Saturation 90.5% Bicarbonate 24.3 mmol/L SBE* -2.6 mmol/L Lactate 21 mg/dL *

Post

Pre

Post

7.072 61.2 mmHg 87.3 mmHg 77.1% 24.9 mmol/L -7.5 mmol/L 24 mg/dL

7.319 100.3 mmHg 44.4 mmHg 97.0% 22.3 mmol/L -3.9 mmol/L 25 mg/dL

7.141 66.3 mm Hg 73.0 mm Hg 84.2% 24.3 mmol/L -6.3 mmol/L 24 mg/dL

SBE denotes standard base excess.

Clı´nicas, lasted four hours. The chest radiograph acquired in our hospital is shown in Figure 1b. During the first two days after cannulation, the patient was sedated with propofol and fentanyl, with a SedationAgitation Scale ranging from 2 to 5 due to drug abstinence. Ventilation was set in pressure support mode with a positive-end expiratory pressure of 10 cmH2O, a pressure support of 8 cmH2O, and an FiO2 of 0.3. The ECMO settings were adjusted to achieve a PaO2.50 mmHg. The patient improved gradually (Table 3), and after seven days of extracorporeal support, she was awake and cooperative. Daily weaning tests from ECMO support were conducted in accordance with our criteria for ECMO discontinuation. ECMO was stopped nine days after initiation. Mechanical ventilation was discontinued two days later. The patient was discharged from the hospital 33 days after admission with no functional disability.

*

SBE denotes standard base excess. SAS denotes the Sedation-Agitation Scale. SOFA denotes sequential organ failure assessment. This score is used to diagnose and quantify organ failure, and it ranges from 0 to 24. " The lung injury score is Murray’s score, which quantifies the severity of lung injury based on the respiratory compliance, PEEP, number of quadrants infiltrated on a chest X-ray and PaO2/FIO2 ratio. I PCV denotes pressure-controlled ventilation. £ PEEP denotes positive end-expiratory pressure. ¥ FiO2 denotes the inspiratory fraction of oxygen. J ECMO denotes extracorporeal membrane oxygenation. # 1

DISCUSSION

35 cmH2O. Venous-venous ECMO support was initiated. Her blood gas parameters improved after ECMO support, and the patient was transferred by helicopter. The entire process, from the initial call to arrival at Hospital das

The use of ECMO in adult patients has increased after the influenza A H1N1 outbreak. During the H1N1 epidemics, two observational studies reported a mortality rate of 20% with ECMO support compared to the expected 50% mortality in conventionally supported patients (3,4). Additionally, in patients with severe lung injury, the CESAR trial showed that 63% of patients supported with an ECMO-including strategy survived without disability for six months compared to 47% of those allocated to conventional support (1).

Figure. 1A - Chest X-Ray after installation of ECMO in patient 1. 1B: Chest X-Ray after installation of ECMO in patient 2

Figure 2 - A e B: CT scan demonstrating diffuse cerebral edema with ventricular and sulcal effacement in case 1.

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Guarulhos. In the first case, ECMO was started several days after intubation, while the second patient was cannulated early after the diagnosis of severe respiratory failure. Because there is no organized referral system between our services, it is reasonable to presume that the first successful transport may have encouraged faster contact for the second patient. As previously reported by Forrest et al. (6), we believe that the development of a referral and transfer program is associated with faster ECMO initiation, safer transportation and, possibly, a higher rate of survival. We propose the algorithm described in Figure 3 to create a transfer program for ECMO patients. As described in this report, earlier ECMO initiation is most likely associated with higher rates of survival. Therefore, we believe that a low threshold of hypoxemia despite optimal therapy should be sufficient to trigger an ECMO specialist consultation. The classical criteria for ECMO initiation should not be used as a trigger for consultation because it may adversely hinder support. In the presence of critical but reversible lung injury with no contraindication for extracorporeal support, an ECMO team (composed of two physicians and one registered nurse) would be responsible for the evaluation of the patient on location and, if indicated, ECMO support initiation. Because ECMO is a highly complex technique that involves high costs, constant personnel training and high complication rates (8), referral centers should be reserved for tertiary care centers only.

Table 3 - Clinical data and arterial blood gases (Case 2). Data Mechanical ventilation Ventilatory mode Peak pressure (min–max) - cmH2O PEEP (min–max) cmH2O £ FIO2 (min–max) ¥ Respiratory rate (min– max) - breaths/min ECMO J Blood flow (min–max) L/min Sweeper flow (min– max) - L/min FIO2 Routine blood gas PaO2 - mmHg PaCO2 - mmHg SBE - mEq/L * pH Patient data SAS (min – max)# Lung injury score " Total SOFA score 1 Respiratory SOFA Cardiovascular SOFA Hematological SOFA Hepatic SOFA Neurological SOFA Renal SOFA

Pre-ECMO

Day 1

Day 2

PCVI

PCVI 25 10 0.6

PCVI 25 15 0.3

PSV2 25 15 0.3

PSV2 20 13 0.6

15-21 3.02-

19-28 4.486.62

16-36 4.05-

14-42 3.67-

5.33

6

5.08

3.84

6–7

1.0

3-4.5

0.5

1.0

1.0

52 33 +6.1 7.41

79.8 57.3 +6.6 7.38

3-5 2.75 8 4 2 0 0 2 0

4 2.5 4 4 0 0 0 0 0

1.0 51.4 40.6 -0.6 7.39 2 3 6 4 0 0 0 2 0

66.3 42.4 +1.9 7.41 2–6 2.5 6 4 0 0 0 2 0

Day 5 Day 10

Brain death diagnosis during ECMO support Neurological injury is common in ECMO-supported patients. It is uncertain whether the extracorporeal support may precipitate injury or if the patient’s morbid condition is the only responsible factor (9). A study of 295 patients from the ELSO registry for whom ECMO was used to support cardiopulmonary resuscitation reported brain death in 28% of non-survivors (10) More recently, ECMO support to maintain potential organ donors has been used to increase the donor pool (11). Performing an apnea test in ECMO-supported patients is a major challenge because ECMO support is able to maintain normal blood gases in apneic patients. In case 1, we decided to disconnect the patient from the ventilator and then set the sweeper flow to the lowest level necessary to achieve a pulse oximetry of 90%. Respiratory movements were monitored, and blood gas analyses were obtained before and 10 minutes after a stable sweeper flow was initiated to detect an increase in the carbon dioxide partial pressure. Muralidharan et al. (12) reported three cases of possible brain death in ECMO patients in which the apnea test was not performed due to the lack of standardized procedures. However, they suggested that patients should remain connected to the ventilator under continuous positive airway pressure with a PaCO2 between 35– 45 mmHg. Subsequently, they suggested that the sweeper flow be set to the minimum value compatible with normal pulse oximetry before the test. We adopted a similar procedure. ECMO is a specialized technique and a lifesaving measure when other rescue therapies have failed. The treatment of patients under ECMO support must be performed in referral centers with a trained ECMO team. The transfer of patients seems plausible and safe. Despite recent advances and continuous training, there are still

*

SBE denotes standard base excess. SAS denotes the Sedation-Agitation Scale. 1 SOFA denotes sequential organ failure assessment. This score is used to diagnose and quantify organ failure, and it ranges from 0 to 24. " The lung injury score is Murray’s score, which quantifies the severity of lung injury based on the respiratory compliance, PEEP, number of quadrants infiltrated on a chest X-ray and PaO2/FIO2 ratio. I PCV denotes pressure-controlled ventilation. 2 PSV denotes pressure-support ventilation £ PEEP denotes positive end-expiratory pressure. ¥ FiO2 denotes the inspiratory fraction of oxygen. J ECMO denotes extracorporeal membrane oxygenation. #

Patient transfer Most studies involving transfer to referral centers during ECMO opted to transfer the patient in conventional mechanical ventilation before starting ECMO support (1,3). However, this type of transferinvolves considerable risk, and deaths have been described during the process (1). Conversely, previously published data show the safety and feasibility of the inter-hospital transport of critically ill adults under ECMO support (5,6). After implementing a protocol for the safe transport of adults on ECMO, Forrest et al. described the transportation of 40 patients without deaths or major morbidity (6). In another study, transportation under ECMO support was associated with fewer episodes of hypoxia compared to patients transported under conventional ventilation (7). In both of our cases, we chose to initiate ECMO support before patient transfer to assure safer transport. There were no adverse events during transportation between the hospitals. The optimal timing for ECMO initiation is not well established. Both of the patients described in this report were transferred from the same hospital in the city of

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Figure 3 - (Adapted from 6.) Indications and contraindications for consultation for ECMO support. PCV denotes Pressure controlled ventilation; NO denotes nitric oxide and RM denotes Recruitment maneuver. 2. Extracorporeal life support organization (ELSO). Extracorporeal life support bed status map: checked on 03/04/2012. http://www elso med umich edu/Maps html 2012. 3. Noah MA, Peek GJ, Finney SJ, Griffiths MJ, Harrison DA, Grieve R, et al. Referral to an Extracorporeal Membrane Oxygenation Center and Mortality Among Patients With Severe 2009 Influenza A(H1N1). JAMA. 2011;306(15):1659, http://dx.doi.org/10.1001/jama.2011.1471. 4. Davies A, Jones D, Bailey M, Beca J, Bellomo R, Blackwell N, et al. Extracorporeal Membrane Oxygenation for 2009 Influenza A(H1N1) Acute Respiratory Distress Syndrome. JAMA. 2009;302(17):1888-95. 5. Forrest P, Cheong JY, Vallely MP, Torzillo PJ, Hendel PN, Wilson MK et al. International retrieval of adults on extracorporeal membrane oxygenation support. Anaesth Intensive Care. 2011;39(6):1082-5. 6. Forrest P, Ratchford J, Burns B, Herkes R, Jackson A, Plunkett B, et al. Retrieval of critically ill adults using extracorporeal membrane oxygenation: an Australian experience. Intensive Care Med. 2011;37(5):824-30, http://dx.doi.org/10.1007/s00134-011-2158-8. 7. Foley DS, Pranikoff T, Younger JG, Swaniker F, Hemmila MR, Remenapp RA et al. A review of 100 patients transported on extracorporeal life support. ASAIO J. 2002;48(6):612-9, http://dx.doi.org/10.1097/ 00002480-200211000-00007. 8. Sidebotham D, McGeorge A, McGuinness S, Edwards M, Willcox T, Beca J. Extracorporeal membrane oxygenation for treating severe cardiac and respiratory disease in adults: Part 1—overview of extracorporeal membrane oxygenation. J Cardiothorac Vasc Anesth. 2009;23(6):886-92, http://dx.doi.org/10.1053/j.jvca.2009.08.006. 9. Mateen FJ, Muralidharan R, Shinohara RT, Parisi JE, Schears GJ, Wijdicks EF. Neurological injury in adults treated with extracorporeal membrane

many uncertainties in the management of this specific population. Conflicts of interests: The authors received ECMO membranes as a donation from MAQUET Cardiopulmonary of Brazil.

AUTHOR CONTRIBUTIONS Mendes PV, Moura E, Park M, Barbosa EV, Hirota AS, Scordamaglio PR, Ajjar FM, Costa EL and Azevedo LC provided medical support to the patients. Mendes PV and Moura E wrote the manuscript. Barbosa EV, Hirota AS, Scordamaglio PR and Ajjar FM were responsible for data collection. Costa EL, Azevedo LC and Park M critically reviewed the manuscript.

REFERENCES 1. Peek GJ, Mugford M, Tiruvoipati R, Wilson A, Allen E, Thalanany MM, et al. Efficacy and economic assessment of conventional ventilatory support versus extracorporeal membrane oxygenation for severe adult respiratory failure (CESAR): a multicentre randomised controlled trial. Lancet. 2009;374(9698):1351-63, http://dx.doi.org/10.1016/S0140-6736 (09)61069-2.

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oxygenation. Arch Neurol. 2011;68(12):1543-9, http://dx.doi.org/10. 1001/archneurol.2011.209. 10. Thiagarajan RR, Brogan TV, Scheurer MA, Laussen PC, Rycus PT, Bratton SL. Extracorporeal membrane oxygenation to support cardiopulmonary resuscitation in adults. Ann Thorac Surg. 2009;87(3):778-85, http://dx.doi.org/10.1016/j.athoracsur.2008.12.079. 11. Ke HY, Lin CY, Tsai YT, Lee CY, Hong GC, Lee CH et al. Increase the donor pool: transportation of a patient with fatal head injury supported with extracorporeal membrane oxygenation. J Trauma. 2010;68(3):E87-8. 12. Muralidharan R, Mateen FJ, Shinohara RT, Schears GJ, Wijdicks EF. The challenges with brain death determination in adult patients on extracorporeal membrane oxygenation. Neurocrit Care. 2011;14(3):4236, http://dx.doi.org/10.1007/s12028-011-9516-9.

Marcelo Park Eduardo Leite Vieira Costa Alexandre Toledo Maciel Pedro Vitale Mendes Leandro Utino Taniguchi Fernanda Maria Queiroz Silva Andre´ Luiz de Oliveira Martins Edzangela Vasconcelos Santos Barbosa Raquel Oliveira Nardi Michelle de Nardi Igna´cio Cla´udio Cerqueira Machtans Wellington Alves Neves Adriana Sayuri Hirota Marcelo Brito Passos Amato Guilherme de Paula Pinto Schettino Carlos Roberto Ribeiro Carvalho

APPENDIX Participants of ECMO Group: Luciano Cesar Pontes Azevedo

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DOI:10.6061/clinics/2012(12)28

CASE REPORT

Right coronary artery originating from the distal left circumflex artery, evaluation using three imaging techniques Murat Turfan,I Cemalettin Aydın,II Mehmet Ali Elbey,III Ercan Erdog˘an,I Emin Asoglu,I Halil Basel,II ¨ mer Go¨ktekinI O I Bezmialem University, Faculty of Medicine, Department of Cardiology, Istanbul/Turkey. II Bezmialem University, Faculty of Medicine, Department of Cardiovascular Surgery, Istanbul/Turkey. III Dicle University, Faculty of Medicine, Department of Cardiology, Adnan Menderes Bulvarı Fatih, Istanbul/ Turkey.

Email: turphan@gmail.com Tel.: 90 505 3197199

groove. The vessel terminated near the right sinus of Valsalva (Figure 2). Right coronary arteriography could not be performed because the right coronary ostium was not present, which was also confirmed by aortography (Figure 3). The patient underwent mitral valve surgery, and during surgery, the right coronary artery ostium was not observed (Figure 4).

INTRODUCTION An isolated single coronary artery is extremely rare, with an incidence of 0.024% to 0.066% in the general population (1), and many patients with coronary artery anomalies are asymptomatic. However, some of these anomalies can lead to sudden death. In this report, we describe a rare case using three different imaging methods.

CASE DESCRIPTION A 58-year-old man presented with exertional dyspnea and chest pain. His physical examination was normal, except for an apical, third-degree systolic murmur. The ECG showed normal sinus rhythm without any ischemic changes. Transthoracic echocardiography showed severe mitral valve insufficiency, and the left ventricular chamber size was in the normal range. The patient was then evaluated using 64-slice multidetector computed tomography of the coronary arteries, which revealed an anomalous single coronary artery arising from the left sinus of Valsalva and the absence of the right coronary artery (RCA). The left circumflex coronary artery (LCx) was the dominant vessel. It appeared to continue, without significant stenosis, beyond the atrioventricular groove up to the level normally occupied by the RCA (Figure 1). Mid-grade stenoses were observed in the middle segment of the left anterior descending coronary artery (LAD). The patient was symptomatic, and it was therefore decided that mitral valve surgery should be performed. Selective coronary angiography showed a normal left main coronary artery originating from the left sinus of Valsalva. Left coronary arteriography showed 90% stenosis in the mid portion of the LAD, and the left circumflex artery was a dominant vessel, with an aberrant branch from the distal left circumflex artery that crossed the crux and continued to the right atrioventricular

Figure 1 - In the CT image, the LCx was the dominant vessel. It appeared to continue beyond the atrioventricular groove up to the level normally occupied by the RCA.

Copyright ß 2012 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.

DISCUSSION Lipton et al. classified single coronary arteries into nine patterns according to the site of origin and the anatomical distribution of the branch of the coronary artery (2). Our

No potential conflict of interest was reported.

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Figure 4 - During surgery, the right coronary artery ostium was not observed.

case had an anomalous coronary artery (L-1 subtype) that originated from the left sinus of Valsalva, branched from the LCx in a normal fashion, and then continued in the atrioventricular groove up to the level of the RCA.

Figure 2 - The LCx was a dominant vessel, with an aberrant branch from the distal left circumflex artery that crossed the crux and continued to the right atrioventricular groove.

AUTHOR CONTRIBUTIONS Turfan M was responsible for the manuscript writing and review. Aydin C, Elbey MA and Basel H were responsible for the patient diagnosis. Erdogan E was responsible for the data analysis. Asoglu E was responsible for the literature search and Goktekin O was responsible for the manuscript content and editing.

REFERENCES 1. Desmet W, Vanhaecke J, Vrolix M, Van de Werf F, Piessens J, Willems J, et al. Isolated single coronary artery: a review of 50,000 consecutive coronary angiographies. Eur Heart J. 1992;13(12):1637-40. 2. Lipton MJ, Barry WH, Obrez I, Silverman JF, Wexler L. Isolated single coronary artery: diagnosis, angiographic classification, and clinical significance. Radiology. 1979;130(1):39-47.

Figure 3 - No right coronary ostium was observed using aortography.

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DOI:10.6061/clinics/2012(12)29

CASE REPORT

Glove port single-incision laparoscopic splenectomy and the treatment of its complications Erkin Ismail, Cihangir Akyol, Salim Ilksen Basceken, Utku Tantoglu, Ilgaz Kayılıoglu, Atıl Cakmak Ankara University School of Medicine, Department of General Surgery, Ankara/Turkey. Email: cihangirakyol@gmail.com Tel.: 0 312 508-2435

(Figures 1 and 2). One 12-mm and two 5-mm trocars were placed through incisions made in the glove fingers. Following CO2 insufflation, adhesions around the gastrosplenic ligament, which were caused by the previous peptic ulcer surgery, were dissected using an ultrasonic dissector. The spleen was suspended with a SILS clinch 36 grasper (Covidien, Mansfield, MA, USA) and dissected from its ligaments with the help of a harmonic dissector. After the hilum of the spleen was exposed, it was cut using an EndoGIA II stapler (60-mm long, 2.5-mm staples, Auto Suture; US Surgical, Norwalk, CT) (Figure 3). The spleen was placed in a 15-mm bag (EndoCatch II, Autosuture, Covidien, Mansfield, MA, USA) (Figure 4), crushed, and removed from the abdominal cavity through the ALEXIS port. The procedure took 45 min. No intraoperative complications occurred during surgery. The patient developed tachycardia on the second postoperative day, and her hemoglobin level decreased. Computed tomography showed a hematoma initiating where the spleen had been removed and extending to the pelvis. The patient then underwent a second surgery. With the patient in the same position, another surgical glove port was made, and the abdomen was explored. Bleeding was discovered at the vascular stapler line. The bleeding was sutured laparoscopically. After the intra-abdominal hematoma was drained, the abdominal cavity was irrigated. Finally, a drain was placed at the previous location of the spleen. The patient was discharged from the hospital on the third postoperative day. The pathology report showed congested splenectomy material consistent with ITP.

INTRODUCTION Recent advances in surgical techniques have trended toward minimally invasive procedures. Currently, a laparoscopic approach has become the gold standard for splenectomy because it is an effective, reliable technique requiring a shorter hospitalization period with fewer surgical complications, less morbidity, and better esthetic results (1). However, as the number of ports increases in laparoscopy, there is an increase in the incidence of morbidity, including port entrance hernias and infection, internal organ injury, poorer esthetic results, and most importantly, bleeding. Morbidity resulting from the use of multiple ports has prompted the development of techniques using fewer ports (2). Herein, we demonstrate the applicability of glove port single-incision laparoscopy, with a review of the surgical literature.

CASE DESCRIPTION A 33-year-old female who initially presented with spontaneous nosebleeds was diagnosed with immune thrombocytopenic purpura (ITP) and had been followed for two years. After the patient developed steroid resistance, a splenectomy was planned. She had previously undergone surgery for a perforated peptic ulcer. A physical examination identified a midline incision scar above the abdomen. Ultrasonography indicated that the spleen was of normal size. No intravenous antibiotics were administered preoperatively. The patient was treated with cortisol for the surgery.

DISCUSSION Single-port laparoscopy has been adapted to many surgical procedures (2-8). Other studies have demonstrated that laparoscopic splenectomy can be performed using only one incision (8-11). The advantages of the surgical glove port technique compared to the single-port technique include its ease of placement and use of inexpensive surgical equipment (12,13). The cost difference between these two techniques is an important factor, particularly in developing countries. Additionally, the surgical glove port technique ensures a safer entry while placing the port, and the surgeon has more mobility with the glove port technique than with the single-port technique. However, manipulations performed laparoscopically through a single port are more difficult compared with a standard laparoscopic approach. More trocars can be placed through the glove fingers by making wider incisions as necessary. In this case, because of

SURGICAL TECHNIQUE With the patient in the 30 ˚ right lateral decubitus position, the abdominal cavity was entered through a 22-mm incision parallel to the skin folds at the left midclavicular line. A surgical glove port was formed using an extra-small ALEXIS wound protector (Applied Medical, Rancho Santa Margarita, CA, USA) and a size 7.5 standard surgical glove

Copyright ß 2012 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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Figure 3 - Cutting the hilum of the spleen using an Endo-GIA II stapler. Figure 1 - Glove port preparation.

Figure 4 - The spleen was placed in a 15-mm bag, crushed, and removed from the abdominal cavity through the ALEXIS port.

Figure 2 - The glove port.

previous peptic ulcer surgery, an incision in the hypochondrium was preferred to a transumbilical entry. Consequently, the adhesions resulting from the previous operation posed no problems. A transumbilical entry may be a better esthetic choice in patients with no intraabdominal adhesions. We believe that a transumbilical incision would not cause any technical difficulties. Another advantage of the surgical glove port compared with the standard laparoscopic splenectomy is that there is no need for a new incision or to enlarge the existing incision to remove the spleen from the abdomen. The disadvantages of this technique include punctures in the glove or ALEXIS port and gas leakage during the surgery. However, these problems are easily resolved during the procedure. In our case, the subsequent hemorrhage was easily treated using the glove-port technique. Although most new techniques are typically more expensive than established techniques, the glove port technique uses less costly, more widely available surgical equipment (12).

REFERENCES 1. Habermalz B, Sauerland S, Decker G, Delaitre B, Gigot JF, Leandros E, et al. Laparoscopic splenectomy: The clinical practice guidelines of the European Association for Endoscopic Surgery (EAES). Surg Endosc. 2010;22(4):821-48. 2. Romanelli JR, Earle DB. Single-port laparoscopic surgery: an overview. Surg Endosc. 2009;23(7):1419-27, http://dx.doi.org/10.1007/s00464-0090463-x. 3. Henckens T, Van de Putte D, Van Renterghem K, Ceelen W, Pattyn P, Van Nieuwenhove YJ. Laparoendoscopic single-site gastrectomy for a gastric GIST using double-bended instruments. J Laparoendosc Adv Surg Tech A. 2010;20(5):469-71, http://dx.doi.org/10.1089/lap.2009. 0391. 4. Podolsky ER, Curcillo PG 2nd. Single port access (SPA) surgery—a 24month experience. J Gastrointest Surg. 2010;14(5):759-67. 5. Fearon KC, Ljungqvist O, Von Meyenfeldt M, Revhaug A, Dejong CH, Lassen K, et al. Enhanced recovery after surgery: A consensus review of clinical care for patients undergoing colonic resection. Clin Nutr. 2005;24(3):466-77, http://dx.doi.org/10.1016/j.clnu.2005.02.002. 6. Rao PP, Bhagwat SM, Rane A. The feasibility of single-port laparoscopic cholecystectomy: A pilot study of 20 cases. HPB (Oxford). 2008;10(5):33640, http://dx.doi.org/10.1080/13651820802276622. 7. Reavis KM, Hinojosa MW, Smith BR, Nguyen NT. Single laparoscopic incision transabdominal surgery sleeve gastrectomy. Obes Surg. 2008;18(11):1492-94, http://dx.doi.org/10.1007/s11695-008-9649-x. 8. Targarona EM, Pallares JL, Balague C, Luppi CR, Marinello F, Herna´ndez P, et al. Single incision approach for splenic diseases: a preliminary report on a series of 8 cases. Surg Endosc. 2010;24(9):2236-40, http://dx.doi.org/10.1007/s00464-010-0940-2. 9. Barbaros U, Dinc¸c¸ag˘ A. Single incision laparoscopic splenectomy: the first two cases. J Gastrointest Surg. 2009;13(8):1520-3, http://dx.doi.org/ 10.1007/s11605-009-0869-8.

AUTHOR CONTRIBUTIONS Erkin I was the primary surgeon. Cihangir A was the second surgeon and was responsible for writing the manuscript. Basceken SI was the assistant surgeon and co-writer. Tantoglu U and Kayılıoglu I were co-writers. Cakmak A is the head of the Laparoscopic Surgery Unit and co-writer.

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Glove Port Laparoscopic Splenectomy Ismail E et al. 12. Hayashi M, Asakuma M, Komeda K, Miyamoto Y, Hirokawa F, Tanigawa N. Effectiveness of a surgical glove port for single port surgery. World J Surg. 2010;34(10):2487-9. 13. Ishida H, Okada N, Ishibashi K, Ohsawa T, Kumamoto K, Haga N. Single-incision laparoscopic-assisted surgery for colon cancer via a periumbilical approach using a surgical glove: initial experience with 9 cases. Int J Surg. 2011;9(2):150-4.

10. Misawa T, Sakamoto T, Ito R, Shiba H, Gocho T, Wakiyama S, et al. Single-incision laparoscopic splenectomy using the ‘‘tug-exposure technique’’ in adults: results of ten initial cases. Surg Endosc. 2011;25(10):3222-7, http://dx.doi.org/10.1007/s00464-011-1697-y. 11. Srikanth G, Wasim MD, Sajjad A, Shetty N. Single-incision laparoscopic splenectomy with innovative gastric traction suture. J Minim Access Surg. 2011;7(1):68-70.

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CLINICS 2012;67(12):1523-1525

DOI:10.6061/clinics/2012(12)30

CASE REPORT

Congenital aneurysmal circumflex coronary artery fistula in a pregnant woman Meliza Goi Roscani, Silmeia Garcia Zanati, Pericles S. Salmazo, Fabio C. Carvalho, Claudia G. Magalha˜es, Vera T.M. Borges, Edson A. Bregagnollo, Beatriz B. Matsubara, Joa˜o Carlos Hueb Universidade Estado de Sa˜o Paulo (UNESP), Botucatu Medical School, Department of Internal Medicine, Botucatu/SP, Brazil. Email: meliza10@hotmail.com Tel.: 55 14 3882-2969

pregnancy showed normal pulmonary artery pressure and no significant increase in oxygen saturation in the right side of the heart. Coronary angiography revealed a markedly dilated tortuous circumflex coronary artery connected to the right ventricle, indicating a coronary artery fistula (Figure 2). Aspirin (100 mg/day) and endocarditis prophylaxis were initiated. A cesarean section was performed at the 37th week of pregnancy without complications, and the health of the newborn was satisfactory. Six months after delivery, surgical treatment was recommended to correct the fistula. However, the patient refused treatment and was lost to follow-up. Two years later, she returned in the third trimester of pregnancy. The delivery occurred without complications, and the patient did not agree to treatment. She remains asymptomatic under clinical management.

INTRODUCTION Coronary artery fistula (CAF) is an abnormal communication between an epicardial coronary artery and a single or multiple cardiac chambers, large vessels or other vascular structures (1). Although it is a rare condition, it is the most frequent congenital coronary anomaly (2), and it accounts for 0.2-0.4% of congenital cardiac anomalies. Approximately 50% of pediatric coronary vasculature anomalies are coronary artery fistulae. Small fistulae remain clinically silent and are recognized during routine echocardiography or at autopsy. Larger fistulae enlarge progressively over time, and complications, such as congestive heart failure, arrhythmias, infectious endocarditis, aneurysm formation, rupture, and death, are more likely to arise in older patients. The most common feature is a right coronary artery fistula connecting to the right ventricle. Reports on the clinical repercussions of CAFs in pregnancy are uncommon (3,4). We describe a pregnant woman with a congenital aneurysmal circumflex coronary artery fistula to the right ventricle and discuss the appropriate management for this situation.

DISCUSSION We discussed an asymptomatic young pregnant woman with the rare condition of an aneurysmal circumflex coronary artery connecting to the right ventricle. Congenital CAFs are defined as an abnormal precapillary communication between one or several normally originating coronary arteries and any cardiac vein, chamber or large thoracic vessel that bypasses the myocardial capillary bed (1). Fistulae arise from the right coronary artery (60%) or left anterior descending artery (30%) and terminate in the right side of the heart (90%). The most frequent termination sites, in descending order, are the right ventricle, right atrium, coronary sinus, and pulmonary vasculature. The circumflex coronary artery is rarely affected (2). CAFs are isolated in 55% to 80% of cases and associated with other congenital heart diseases in 20% to 45% (1). The most frequently associated anomalies are tetralogy of Fallot, atrial septal defects, patent ductus arteriosus, ventricular septal defects, and pulmonary atresia with an intact ventricular septum. The natural history of a coronary fistula is unknown. There is a consensus that this anomaly does not typically produce symptoms before the age of 30 years (5), and in some cases, only a continuous murmur is audible, with no other signs. However, if the CAF is sufficiently large to cause a considerable left-to-right shunt, symptoms of angina, syncope, dizziness and fatigue may be present and typically occur after the fourth decade of life (6). Electrocardiography (ECG) is normal in approximately 50% of cases and shows left or right hypertrophy in the

CASE DESCRIPTION A 23-year-old woman was admitted at the general hospital for a prenatal examination in her 10th week of pregnancy. She was asymptomatic, and a physical examination found that her blood pressure was 110/80 mmHg, her heart rate was 80 bpm, and her respiration rate was 18 ipm. Heart auscultation revealed a grade 2 continuous murmur along the left sternal border. There were no signs of congestive heart failure. Electrocardiography showed normal sinus rhythm and no specific changes in the ST segment or T wave. Transthoracic echocardiography showed a slightly enlarged left ventricle and a markedly dilated left coronary artery. Additionally, there was an abnormal flow to the right ventricle, suggesting the diagnosis of a coronary fistula to a right cardiac chamber (Figure 1). Cardiac catheterization performed in the second trimester of

Copyright ß 2012 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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Figure 1 - A transthoracic echocardiogram showing the markedly dilated left coronary artery (left) and an abnormal flow to the right ventricle, suggesting the diagnosis of a coronary fistula to a right cardiac chamber (right).

other 50%. Atrial fibrillation and an ischemic pattern are rarer manifestations (5). Chest x-rays are typically normal, but cardiomegaly may be present if there is a large left-to-right shunt (5). Typically, two-dimensional and color Doppler echocardiography are not useful for establish a definitive diagnosis or determining the course of the coronary fistula; however, these methods are useful for demonstrating the dilation of the affected coronary, the related chamber enlargement and the drainage site. Transesophageal echocardiography (TEE) is better than transthoracic echocardiography for identifying the precise origin and drainage site (1,5). Cardiac catheterization is considered the gold standard for the diagnosis of CAF and should be performed to

determine the hemodynamic significance of the lesion and provide a detailed angiographic assessment of the abnormal anatomy (1,5,7). It is recommended that endocarditis prophylaxis and aspirin be provided to prevent thrombosis for all patients (8). Asymptomatic patients with small fistulae should be monitored clinically for signs of growth and increasing flow. Typically, lesions enlarge progressively and warrant operative repair, either by transcatheter or surgical techniques. Both techniques are associated with an excellent prognosis, and the choice of the procedure depends on the morphology of the fistula. Patients with large and tortuous fistulae, multiple openings, or significant aneurysmal dilatation may not be optimal candidates for transcatheter closure, and a potentially curative surgical treatment is preferred (9,10). Older patients with large fistulae that are not tortuous or aneurysmal may benefit from transcatheter closure. Studies on CAFs have not emphasized the hazards of this condition when it is associated with pregnancy, although this association may cause overt heart failure, which may occur due to volume overload (3). Fistula dissection and rupture are described as rare (11). The optimal time for intervention in pregnant women is not clear. An asymptomatic patient should be treated using a multidisciplinary approach during the prenatal period. If symptoms of myocardial ischemia are present, surgical intervention should be performed during pregnancy. In the present case, although the patient was asymptomatic, we recommended surgery after her cesarean section based on studies emphasizing that the incidence of symptoms and complications in aneurysmal arteries increases with age, including coronary thrombosis causing myocardial infarction and angina and symptomatic cardiomyopathy. Therefore, corrective treatment should be performed prior to these events. In conclusion, we describe a rare case of the discovery of an aneurysmal circumflex artery in pregnancy and advocate that the approach to management and therapeutic strategy

Figure 2 - Coronary angiography revealing a markedly dilated tortuous circumflex coronary artery connected to the right ventricle, indicating a coronary artery fistula.

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Fistula in pregnancy Roscani MG et al.

be based on symptoms, pathological changes and possible complications.

4.

AUTHOR CONTRIBUTIONS

5.

Roscani MG wrote the manuscript. Zanati SG helped with the literature review. Carvalho FC and Bregagnollo EA performed the hemodynamic study and assisted in obtaining the figures. Matsubara BB helped with the literature review and reviewed the manuscript. Hueb JC performed the echocardiogram and assisted in obtaining the figures. Salmazo PS helped with the literature and assisted in obtaining the figures.

6. 7. 8.

REFERENCES

9.

1. Chirantan V, Mangukia MBBS. Coronary artery fistula. Ann Thorac Surg. 2012;93(6):2084-92. 2. Levin DC, Fellows KE, Abrams HL. Hemodynamically significant primary anomalies of the coronary arteries. Angiographic aspects. Circulation. 1978;58(1):25-34. 3. Tay SM, Ong BC, Tan SA. Cesarean section in a mother with uncorrected congenital coronary to pulmonary artery fistula. Canadian journal of

10. 11.

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anaesthesia = Journal canadien d’anesthesie. 1999;46(4):368-71, http:// dx.doi.org/10.1007/BF03013230. Hipona FA. Congenital coronary arterial fistula to a persistent left superior vena cava. The American journal of roentgenology, radium therapy, and nuclear medicine. 1966;97(2):355-8. Qureshi SA. Coronary arterial fistulas. Orphanet J Rare Dis. 2006;1:51, http://dx.doi.org/10.1186/1750-1172-1-51. Canga Y, Ozcan KS, Emre A, Kul S, Guvenc TS, Durmus G, et al. Coronary artery fistula: review of 54 cases from single center experience. Cardiology J. 2012;19(3):278-86, http://dx.doi.org/10.5603/CJ.2012.0050. Vavuranakis M, Bush CA, Boudoulas H. Coronary artery fistulas in adults; incidence, angiographic characteristics,natural history. Cathet Cardiovasc Diagn. 1995;35(2):116-20, http://dx.doi.org/10.1002/ccd.1810350207. Kasravi B, Reid CL, Allen BJ. Coronary artery fistula presenting as bacterial endocarditis. J Am Soc Echocardiogr. 2004;17(12):1315-6. Chamberlain MH, Henry R, Brann S, Angelini GD. Surgical management of a gigantic circumflex coronary artery aneurysm with fistulous connection to the coronary sinus. Eur J Cardiothorac Surg. 2001; 20(6):1255-7, http://dx.doi.org/10.1016/S1010-7940(01)00998-8. Mastroroberto P, Olivito S. Giant congenital fistula of the circumflex coronary artery with drainage into right atrium. Heart Lung Circ. In press 2012. Rajs J, Brodin LA, Hertzfeld I, Larsen FF. Death related to coronary artery fistula after rupture of an aneurysm to the coronary sinus. Am J Forensic Med Pathol. 2001;22(1):58-61, http://dx.doi.org/10.1097/00000433-200103000-00011.


CLINICS 2012;67(12):1527-1528

DOI:10.6061/clinics/2012(12)31

CASE REPORT

Successful treatment of thrombotic thrombocytopenic purpura associated with mitral valve replacement Yong Liu,I Jian Zhu,I,II Er-Ping Xi,I Wei Jiang,I Feng Xia,III Shui-Bo ZhuI,II I Wuhan General Hospital of Guangzhou Command, Department of Thoracic Cardiovascular Surgery, Wuhan, People’s Republic of China. II Southern Medical University, Guangzhou, People’s Republic of China. III Liaocheng People’s Hospital, Department of Cardiovascular Surgery, Liaocheng, People’s Republic of China.

Email: zhudandan2008@163.com Tel.: 86-13871161122

Thrombotic thrombocytopenic purpura (TTP) is a rare hematologic syndrome. It is a multisystem disorder characterized by microangiopathic hemolytic anemia and thrombocytopenia and may be accompanied by fever and renal or neurologic abnormalities (1,2). Most adult patients acquire (3) the disease as a result of autoantibodies against ADAMTS13 (i.e., idiopathic TTP), or it is observed in association with collagen vascular disease, bone marrow transplantation, systemic sclerosis, infections, polymyositis, pregnancy, or drug treatment, including cyclosporine, tacrolimus, ticlopidine, and antineoplastic agents (i.e., nonidiopathic TTP) (1,4). However, TTP associated with mitral valve replacement surgery has not been reported. In this paper, we describe a case of a 41-year-old female who developed acute TTP after mitral valve replacement surgery. The patient was treated with plasma exchange (PE) and recovered following an uneventful course.

symptoms and subcutaneous ecchymosis. A blood biochemical analysis revealed a serum lactate dehydrogenase of 821 U/L and 7.7 mmol/L total bilirubin (direct bilirubin, 2.1 mmol/L; indirect bilirubin, 5.6 mmol/L). Her urine exam was positive for protein (3+), leukocytes (normal), hemoglobin (2+), and bilirubin (1+). A direct Coombs test was negative, and PT and aPTT were within the normal range. Liver and renal function tests were normal. Physical examinations of the cardiovascular and respiratory systems and abdomen were normal. Abdominal ultrasonography, computed tomography, and magnetic resonance imaging of the brain revealed no abnormalities. The patient was diagnosed with acquired TTP and received a bolus of dexamethasone (10 mg), an infusion of a high volume of fresh frozen plasma, and a red blood cell transfusion. However, the patient did not respond adequately. Until eight hours after one-volume (2000 ml fresh frozen plasma) PE was treated, the patient’s body temperature began to normalize, and her neurologic and respiratory parameters improved. The patient was discharged after an additional eight days of hospitalization without plasma exchange.

CASE DESCRIPTION

DISCUSSION

A 41-year-old woman with a four-year history of shortness of breath and chest tightness was admitted to our hospital for evaluation. Echocardiography revealed mitral valve stenosis (mitral valve area 1.06 cm2) as a result of rheumatic heart disease. The patient underwent mitral valve replacement via a right atriotomy with a mechanical mitral valve (tissue annulus diameter of 27 mm; St. Jude Medical, St. Paul, Minnesota USA). Cardiopulmonary bypass lasted for 115 minutes, and the aortic clamp time was 43 minutes. Postoperative echocardiography revealed satisfactory valve function and confirmed the successful removal of the intracavitary lesion. Cefoxitin (3 g/day) was initiated on postoperative day 1 for prophylaxis. A complete blood count (CBC) was obtained daily and revealed a low platelet count (Table 2) on day 2. Platelets (400 ml) were transfused, but the patient developed fever (Table 1), and the platelet count dropped continuously (Table 2). In addition, the patient developed neurological

TTP is a rare hematologic emergency with an abrupt onset, and it is potentially fatal. It is clinically characterized by five typical symptoms: hemolytic anemia, thrombocytopenia, renal impairment, fever, and neurological disorders. Although TTP is associated with a severe deficiency of ADAMTS13 activity (5), measurements of ADAMTS13 activity are not required to conclusively confirm the diagnosis (6). Therefore, the diagnosis of TTP is based on the presenting clinical features (1). As in this case, there is typically insufficient time to evaluate ADAMTS13 activity, and a prompt diagnosis of TTP is critical for initiating treatment and reducing patient mortality. Mitral valve replacement is one of the best treatments for mitral stenosis. It has been performed successfully with excellent results and limited complications. The major complications of mitral valve replacement surgery include postoperative bleeding, ischemic stroke, myocardial infarction and death. This is the first case report of TTP as a complication of mitral valve replacement surgery. Although ADAMTS13 activity was not measured in our case, the clinical course was not affected by PC transfusion, and the rapid response to plasma exchange supports the TTP diagnosis. We will follow up this case and evaluate plasma ADAMTS13 activity and autoantibodies to exclude the

INTRODUCTION

Copyright ß 2012 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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Table 1 - Patient body temperature over the course of treatment.

Table 2 - Patient platelet counts over the course of treatment.

AUTHOR CONTRIBUTIONS

possibility of hereditary ADAMTS13 deficiency, which is extremely rare, or acquired autoantibodies against ADAMTS13, which often persist despite clinical resolution or remission (7). Daily plasma exchange remains the mainstay of treatment for acquired TTP regardless of its etiology (1). However, plasma infusion once every two weeks appears to be sufficient for hereditary TTP (8). Our patient recovered with one plasma volume and showed a good clinical outcome, with a normal temperature and improvement of neurological and respiratory parameters eight hours later. The course over the next eight days of hospitalization was uneventful and did not require a second plasma exchange. Although the occurrence of TTP associated with mitral valve replacement surgery is extremely rare, it is important to recognize TTP as a cause of thrombocytopenia and hemolytic anemia in any patient with mitral valve replacement surgery. The prompt initiation of treatment with plasma exchange remains the cornerstone of TTP treatment. Despite the prognosis of TTP associated with mitral valve replacement surgery, the schedule for PE remains poor, and further studies are necessary to improve the management of this disease.

Zhu J and Xi EP wrote the manuscript first draft. Zhu SB, Liu Y, Jiang W and Xia F contributed to the patient treatment and took part in the care of the patient. Liu Y participated in data collection and analysis. Zhu J conceived the study and performed the literature search. Xi EP revised the manuscript. Zhu SB was the first surgeon who provided clinical expertise, reviewed the manuscript and approved the manuscript final version.

REFERENCES 1. Allford SL, Hunt BJ, Rose P, Machin SJ. Guidelines on the diagnosis and management of the thrombotic microangiopathic haemolytic anaemias. Br J Haematol. 2003;120(4):556-73, http://dx.doi.org/10.1046/j.1365-2141. 2003.04049.x. 2. George JN. How I treat patients with thrombotic thrombocytopenic purpura-hemolytic uremic syndrome. Blood. 2000;96(4):1223-9. 3. Souto Filho JT, de Barros PV, da Silva AM, Barbosa FA, Ribas GF. Thrombotic thrombocytopenic purpura associated with mixed connective tissue disease: a case report. Case Report Med. 2011;2011:953890. 4. Franchini M. Thrombotic microangiopathies: an update. Hematology. 2006;11(3):139-46, http://dx.doi.org/10.1080/10245330600667583. 5. Zheng XL, Sadler JE. Pathogenesis of thrombotic microangiopathies. Annu Rev Pathol. 2008;3:249-77, http://dx.doi.org/10.1146/annurev. pathmechdis.3.121806.154311. 6. Tsai HM, Lian EC. Antibodies to von Willebrand factor-cleaving protease in acute thrombotic thrombocytopenic purpura. N Engl J Med. 1998;339(22):1585-94. 7. Zheng XL, Kaufman RM, Goodnough LT, Sadler JE. Effect of plasma exchange on plasma ADAMTS13 metalloprotease activity, inhibitor level, and clinical outcome in patients with idiopathic and nonidiopathic thrombotic thrombocytopenic purpura. Blood. 2004;103(11):4043-9, http://dx.doi.org/10.1182/blood-2003-11-4035. 8. Shepard KV, Fishleder A, Lucas FV, Goormastic M, Bukowski RM. Thrombotic thrombocytopenic purpura treated with plasma exchange or exchange transfusions. West J Med. 1991;154(4):410-3.

ACKNOWLEDGMENTS The authors thank X. Long Zheng at The Children’s Hospital of Philadelphia and University of Pennsylvania for his careful revision of the manuscript.

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CLINICS 2012;67(12):1529-1532

DOI:10.6061/clinics/2012(12)32

CASE REPORT

Respiratory failure after lung transplantation: extracorporeal membrane oxygenation as a rescue treatment Paulo Manuel Peˆgo-Fernandes,I Ludhmila Abraha˜o Hajjar,II Filomena Regina Barbosa Gomes Galas,II Marcos Naoyuki Samano,I Alexandre Kazantzi Fonseca Ribeiro,I Marcelo Park,III Rodolfo Soares,II Eduardo Osawa,II Fabio Biscegli JateneI I

Thoracic Surgery Division, Heart Institute (InCor) do Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo. II Anesthesiology Division, Heart Institute (InCor) do Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo. III Intensive Care Unit, Instituto Central do Hospital das Clı´nicas da Faculdade de Medicina da Universidade de Sa˜o Paulo. Email: paulo.fernandes@incor.usp.br / paulopego@incor.usp.br Tel.: 55 11 2661-5248

University of Sa˜o Paulo. During the previous six months, the patient was hospitalized four times due to worsening dyspnea and hypoxemia. Just before lung transplantation, the patient presented with pneumonia and right lung atelectasis, with an increased need for oxygen and noninvasive ventilation (Figure 1). Bilateral lung transplantation (LTx) was performed without complications. The ischemic time of the left graft was 660 minutes and that of the right graft was 415 minutes. The patient was not exposed to allogeneic blood transfusion, and fluid resuscitation was carried out with lactated Ringer’s solution and albumin. At the end of the surgery, the patient had a lactate level of 6 mmol/L and a mixed venous saturation (ScVO2) of 75%, and the cardiac output was 4.8 L/min. After a 16-hour procedure, the patient was brought to the ICU using mechanical ventilation (MV), intubated and received norepinephrine (0.15 mg/Kg/min). The patient presented no complications during the immediate postoperative period and was weaned from MV 18 hours after ICU arrival. However, on the third day after surgery, the patient developed respiratory failure due to severe hypoxemia (PO2/FiO2 of 130 mmHg), with normal filling pressures (a central venous pressure of 7 mmHg and a wedge pressure of 12 mmHg). A chest X-ray revealed diffuse bilateral patchy opacities (Figure 2). After approximately three hours of non-invasive mechanical ventilation and forced diuresis, respiratory function and gas exchange worsened (PO2/FiO2 of 100 mmHg and PaCO2 of 124 mmHg), and the hemodynamics of the patient progressively deteriorated. She presented a mean blood pressure of 50 mmHg, profuse sweating, and delayed peripheral perfusion. The patient was then placed under assisted pressure-controlled mechanical ventilation with an inspired oxygen fraction (FiO2) of 1.0, a positive endexpiratory pressure (PEEP) of 14 cmH2O, an inspiratory pressure of 26 cmH2O (12 cmH2O driving pressure), an inspiratory time of 0.80 seconds and a respiratory rate of 30. Applying these parameters, the arterial blood gas presented a PaO2 of 54 mmHg, a PaCO2 of 118 mmHg, a pH of 7.12 and an oxygen saturation of 80%. Subsequent tests revealed a progressive worsening of the physiological parameters, with a ScVO2 of 48% and lactate of 8 mmol/L. Hypoxemia and hypercapnia were persistent and refractory to recruitment

INTRODUCTION Hypoxemia is a frequent finding after lung transplantation (LTx) (1-2). The underlying mechanisms include alveoli collapse, diffuse alveolar damage, ventilation-perfusion mismatch, and alveolar-capillary membrane damage (3-5). Primary graft dysfunction (PGD) represents a multifactorial injury to the transplanted lung that develops in 15-25% of patients during the first days after transplantation; it is variously referred to as "ischemia-reperfusion injury" and "early graft dysfunction" (6). PGD is characterized by severe hypoxemia, lung edema, and the radiographic appearance of diffuse pulmonary opacities in the absence of another identifiable cause (7). Despite significant advances in organ preservation, surgical technique, and perioperative care, PGD is responsible for significant morbidity and mortality after lung transplantation (8-9). Most patients recover with intensive care unit (ICU) support that includes non-invasive and invasive ventilation, negative fluid balance, and nitric oxide. However, some patients with severe PGD develop refractory hypoxemia, resulting in shock, multiorgan failure, and mortality in 60% of cases (10-12). During the past few years, highlighted by the influenza-A H1N1 epidemic, gas exchange support using an extracorporeal membrane oxygenator (ECMO) has been used as life-saving therapy in severe cases of respiratory failure (13-15). We report the case of a patient with a severe form of PGD after lung transplantation who was successfully supported using veno-venous ECMO until respiratory recovery.

CASE DESCRIPTION A 20-year-old female patient with cystic fibrosis underwent bilateral lung transplantation without cardiopulmonary bypass at InCor of Hospital das Clı´nicas of the

Copyright ß 2012 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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maneuvers, with PEEP values of 20-40 cmH2O. A transesophageal echocardiogram was performed and showed no abnormality at the pulmonary vein anastomoses. Given the imminent risk of death from refractory hypoxemia, six hours after invasive mechanical ventilation, the ECMO team at our institution began veno-venous ECMO support as a rescue procedure. Using the Seldinger technique, 20-Fr draining cannulae were inserted into the left common femoral vein, and a return cannula was placed into the right jugular vein. The location was guided using ultrasound. A centrifuge magnetic pump with a polymethylpentene oxygenation membrane (Rotaflow/Jostra Quadrox, Maquet Cardiopulmonary AG, Hirrlinger, Germany) was used. Initially, the blood flow was maintained at 500 mL per minute until the system was filled with

blood. The blood flow and sweeper (gas) flow were subsequently increased to 2,000 mL per minute. The blood flow and sweeper flow were then manipulated to target a peripheral oxygen saturation of at least 90%. Anticoagulation with heparin was started with 15 U/Kg of heparin per hour, with the aim of reaching an activated partial thromboplastin time ratio of 1.5–2.0. After two hours of veno-venous ECMO support, gas analysis revealed increasing of PO2/FiO2 to 220, decreased levels of PaCO2 to 45 mmHg, and improvement of the physiologic parameters (lactate 2.5 mmol/L, ScVO2 of 75%) and weaning of norepinephrine. Mechanical ventilation was adjusted to achieve a positive end-expiratory pressure (PEEP) of 10 cmH2O, an inspired fraction of oxygen (FiO2) of 0.4, a driving pressure lower than 10 cmH2O, and a respiratory rate of 10 breaths per minute (5). The parameters that were checked daily included arterial blood gases, clots in the system that were visible through transillumination, pump campanula auscultation, and flowmeter lubrification to maintain a good signal quality. The ECMO blood flow was adjusted to maintain the PaO2 above 55 mmHg, and the sweeper flow was adjusted to maintain the pH$7.3 (through PaCO2 modulation). Fentanyl was used as an analgesic and sedative to reach a Richmond agitation sedation scale (RASS) score of zero and no pain. The body temperature was kept between 36 and 37 degrees Celsius using an external apparatus adapted to the ECMO system (Figure 3). A weaning (autonomy) test from ECMO support was carried out daily. Five days after treatment, the patient presented a PO2/FiO2 of 230 mmHg, and the FiO2 set was adjusted to 0.6 in the ECMO. The sedation was interrupted, and as patient maintained adequate arterial saturation and a respiratory rate of 20 breaths per minute while in spontaneous mode in mechanical ventilator with FiO2 of 0.30, she was successfully weaned from invasive ventilation. The patient stayed in ECMO during the next two days in an awake and cooperative state with no pain, at which point she was considered able to have the ECMO support removed. The decannulation was performed at the bedside without complications. The patient was discharged from the ICU after recovering lung function without complications.

Figure 2 - Chest X-ray immediately after orotracheal intubation showing diffuse bilateral opacities that are compatible with primary graft dysfunction after lung transplantation

Figure 3 - A patient in intensive care receiving mechanical ventilation and ECMO therapy.

Figure 1 - Chest X-ray showing a diffuse opacity of the right lung that is compatible with atelectasis.

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ECMO for PDG after lung transplantation Peˆgo-Fernandes PM et al.

transplantation who develop severe hypoxemia. The main challenges of this treatment are addressing the clear indications, costs, system availability, and team training. The Hospital das Clı´nicas ECMO team was innovative in Brazil and has become a referral center with physicians, nurses, and physiotherapists to assist patients and train individuals to administer ECMO. In a recent paper from this team, a 40% survival rate was described when using ECMO as a respiratory and/or cardiovascular support (30). The importance of this discussion is to call attention to the need to develop experience and perform more studies in patients treated with ECMO to better study the outcomes, determine the optimum treatment strategies, and optimize the patient and device selection, thus improving the outcomes of patients who require this unique therapy.

DISCUSSION Respiratory support with extracorporeal membrane oxygenation (ECMO) has been used since 1971, with varied results (16-19). In 2009, the influenza-A epidemic renewed the interest in this therapy, which had shown efficacy in treating refractory hypoxemia in many patients worldwide (20-21). The CESAR trial revealed a reduction in mortality at six months with ECMO compared with conventional protective mechanical ventilation in severe ARDS patients (22). In conjunction with lung transplantation, ECMO may be useful as a temporary support for respiratory failure while patients are waiting for the organ and after transplantation in cases of refractory hypoxemia (23-25). Most cases of severe hypoxemia after LTx are due to PGD and result in high rates of mortality. A few single-center experiences have been reported, with relatively few cases of ECMO after LTx (26-27). The Extracorporeal Life Support Organization (ELSO) registry, which was established to improve the quality and outcome of extracorporeal life support (ECLS) in patients treated with ECMO, currently includes 151 postLTx patients with PGD (28). The mean age is 35¡18 years. Indications for LTx included acute respiratory distress syndrome, (15%), cystic fibrosis (15%), idiopathic pulmonary fibrosis (8%), primary pulmonary hypertension, (10%), emphysema (15%), acute lung failure (11%), other (23%), and unknown (3%). The ECMO run time was 140¡212 hours. Veno-venous ECMO was used in 25 patients, venoarterial in 89 patients, and other modes in 15 patients (unknown in 22 patients). ECMO was discontinued in 93 patients because of lung recovery. It was also discontinued in 29 patients with multiorgan failure, 22 patients who died with no further specification, and seven patients for other reasons. In total, 63 (42%) of the patients survived the hospital stay. The major complications during ECMO included hemorrhage (52%), hemodialysis (42%), neurologic complications (12%), cardiac complications (28%), inotropic support (77%), and sepsis (15%) (28). In our patient, ECMO was placed on the third day after LTx due to refractory hypoxemia and hypercapnic acidosis. The patient presented no complications of the treatment, and the duration spent in ECMO was 168 hours. The V-V ECMO allowed adequate ventilatory support and resulted in a reversion of the acidosis and shock. The choice of ECMO modality (veno-venous or veno-arterial) depends on the hemodynamic stability and need for cardiac support (29). The veno-venous system is usually preferred in stable patients because it involves an easier implant technique and fewer bleeding and thrombotic complications. In our patient, veno-venous ECMO was used once the patient presented a normal ejection fraction without right ventricle dysfunction, and the shock was interpreted as a consequence of hypoxemia and acidosis. After a few hours of treatment and the recovery of oxygenation, the shock reversed, highlighting the right indication of the system. Although the ELSO registry was not primarily established to study ECMO in LTx, it provides valuable insights and evidence that there is indeed an appreciable salvage rate with the use of ECMO for PGD after LTx (28). Clearly, this is a high-risk patient population, and no single center can accumulate a large volume of ECMO experience for this specific indication. The case under discussion underscores the importance of ECMO as a rescue therapy in patients undergoing lung

AUTHOR CONTRIBUTIONS Peˆgo-Fernandes PM, Hajjar LA, Galas FR, Samano MN and Park M took part in the care of the patient and contributed equally in carrying out the manuscript preparation and revision. Ribeiro AK, Soares R and Osawa E were responsible for the medical literature search and preparation of the manuscript. Jatene FB was responsible for the final revision of the manuscript. All the authors have approved the final version of the manuscript.

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